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Suppression of Collagen Induced Arthritis by NA-2
and Rutin through Regulation of RANKL Pathway
A DISSERTATION SUBMITTED FOR THE PARTIAL FULFILMENT FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY
BY
ANUM GUL
2017
Dr. Panjwani Center for Molecular Medicine and Drug Research,
International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan
CERTIFICATE
It is certified that the thesis entitled, “Suppression of Collagen Induced Arthritis by NA-
2 and Rutin through Regulation of RANKL Pathway”, submitted by Ms. Anum Gul,
fulfills the requirement for the award of Doctor of Philosophy in Molecular Medicine. To
the best of my knowledge, no part of the work has been submitted for another degree or
qualification in any other institute.
Dr. Shabana U. Simjee
Research Supervisor
Dedicated to
My Beloved Mother, Father
&
Siblings
CONTENTS
Page. No
Acknowledgements...................................................................................................................... I
List of Tables ............................................................................................................................... II
List of Figures ........................................................................................................................... III
List of Abbreviation ................................................................................................................... V
Summary ................................................................................................................................... IX
صہ ال X ............................................................................................................................................ خ
CHAPTER 1
INTRODUCTION ....................................................................................................................... 1
1.1. Rheumatoid Arthritis and Its Prevalence ........................................................................ 2
1.2. Clinical Manifestation ......................................................................................................... 2
1.3. Risk factors and Pathogenesis of Rheumatoid Arthritis ............................................... 3
1.4. Reactive Oxygen and Nitrogen Species (ROS & RNS) .................................................. 5
1.4.1. Nitric Oxide ................................................................................................................. 7
1.4.2. Hydrogen Peroxide...................................................................................................... 8
1.5. Antioxidant Defense Mechanisms ..................................................................................... 8
1.5.1. Glutathione (GSH) .................................................................................................. 9
1.6. Cytokines in the Pathogenesis of Rheumatoid Arthritis................................................ 9
1.6.1. Pro-Inflammatory Cytokine: TNF-α ........................................................................ 10
1.6.2. Pro-inflammatory Cytokine: IL-1β .......................................................................... 11
1.7. RANK-RANKL-OPG in Bone Diseases ......................................................................... 11
1.7.1. RANK/RANKL Pathway in Rheumatoid Arthritis ................................................ 12
1.7.2. RANK/RANKL Pathway and the Immune Response ............................................ 13
1.8. Overview of Treatment Strategies................................................................................... 14
1.8.1. Non-Biological Therapies for Rheumatoid Arthritis .............................................. 16
1.8.1.1. Non-Steroidal Anti-Inflammatory Drugs (NSAIDS)....................................... 16
1.8.1.2. Glucocorticoids (GCs) ....................................................................................... 17
1.8.1.3. Conventional DMARDs .................................................................................... 17
1.8.2. Biologics in the Treatment of RA ............................................................................ 19
1.8.2.1. Overview of Current Biologic Therapies ......................................................... 19
1.8.3. Non-Pharmacological Treatments for RA ............................................................... 20
1.9. Collagen Induced Arthritis (CIA) Rat Model ............................................................... 22
1.10. Compounds Used in the Current Study ....................................................................... 23
1.10.1. N-(2-Hydroxyphenyl) Acetamide (NA-2) ............................................................. 23
1.10.2. Rutin ......................................................................................................................... 24
1.10.3. Rutin Gold Nanoparticles (Rutin-GNPs) ............................................................... 24
1.11. Objectives .......................................................................................................................... 26
CHAPTER 2
METHODOLOGY.................................................................................................................... 27
2.1. Animal Care ........................................................................................................................ 28
2.2. Chemicals ............................................................................................................................ 28
2.3. Collagen Suspension Preparation.................................................................................... 28
2.4. Induction of Arthritis ........................................................................................................ 28
2.5. Treatment Strategy ............................................................................................................ 28
2.6. Clinical Assessment of Arthritis ...................................................................................... 30
2.6.1. Arthritic Score ........................................................................................................... 30
2.6.2. Hind Paw Volume Measurement ............................................................................. 30
2.6.3. Body Weight Measurement ...................................................................................... 30
2.7. Histopathological Examination........................................................................................ 32
2.7.1. Fixation ...................................................................................................................... 32
2.7.2. Decalcification........................................................................................................... 32
2.7.3. Processing .................................................................................................................. 32
2.7.3.1. Dehydration ........................................................................................................ 32
2.7.3.2. Embedding .......................................................................................................... 32
2.7.3.3. Microtomy .......................................................................................................... 33
2.7.3.4. Staining ............................................................................................................... 33
2.8. Inflammatory Markers Analysis ..................................................................................... 33
2.8.1. Reactive Oxygen Species (ROS) and Antioxidant Measurement .......................... 33
2.8.1.1 Nitric Oxide Determination (NO) ...................................................................... 33
2.8.1.2. Peroxide Determination (PO) ............................................................................ 34
2.8.1.3. Glutathione Quantification (GSH) .................................................................... 34
2.9. Pro-Inflammatory Cytokines IL-1β and TNF-α Analysis ........................................... 35
2.9.1. IL-1β Measurement ................................................................................................... 35
2.9.2. TNF-α Measurement ................................................................................................. 35
2.10. Real time Reverse Transcriptase Polymerase Chain Reaction (Real Time RT-
PCR) ............................................................................................................................................ 36
2.10.1. RNA Isolation and Quantification ......................................................................... 36
2.10.2 The cDNA Synthesis................................................................................................ 36
2.10.3. Real time Reverse Transcriptase-PCR Amplification of cDNA .......................... 37
2.11. Immunohistochemistry for c-Fos, iNOS and pAkt in Spleen Tissue ....................... 37
2.11.1. Image Analysis ........................................................................................................ 41
2.12. Statistical Analysis ........................................................................................................... 41
CHAPTER 3
RESULTS ................................................................................................................................... 43
3.1. Clinical Parameters of Collagen Induced Arthritis...................................................... 44
3.1.1. Effect of NA-2 on Clinical Parameters .................................................................... 44
3.1.1.1. Arthritic Score ................................................................................................... 44
3.1.1.2. Body Weight ...................................................................................................... 45
3.1.1.3. Paw Oedema ....................................................................................................... 45
3.1.2. Effect of Rutin on Clinical Parameters .................................................................... 48
3.1.2.1. Arthritic Score .................................................................................................... 48
3.1.2.2. Body Weight ...................................................................................................... 48
3.1.2.3. Paw Oedema ...................................................................................................... 50
3.1.3. Effect of Rutin-GNPs on Clinical Parameters of RA ............................................. 52
3.1.3.1. Arthritic Score .................................................................................................. 52
3.1.3.2. Body Weight ...................................................................................................... 52
3.1.3.3. Paw Oedema ...................................................................................................... 52
3.2. Histological Analysis of Knee Joints ............................................................................... 56
3.2.1. NA-2 Treatment ........................................................................................................ 56
3.2.2. Rutin Treatment......................................................................................................... 56
3.2.3. Rutin-GNPs Treatment ............................................................................................. 56
3.3. Measurement of Oxidative Stress Markers in Serum .................................................. 61
3.3.1. Effect of NA-2 Treatment ......................................................................................... 61
3.3.1.1. Serum Nitric Oxide (NO) .................................................................................. 61
3.3.1.2. Serum Peroxide (PO) ......................................................................................... 61
3.3.1.3. Glutathione (GSH) ............................................................................................. 61
3.3.2. Effect of Rutin on Oxidative Stress Parameters ...................................................... 63
3.3.2.1. Serum Nitric Oxide (NO) ................................................................................. 63
3.3.2.2. Serum Peroxide (PO) ........................................................................................ 63
3.3.2.3. Serum Glutathione (GSH) ................................................................................. 63
3.3.3. Effect of Rutin-GNPs on Oxidative Stress Parameters .......................................... 64
3.3.3.1. Serum Nitric Oxide (NO) .................................................................................. 64
3.3.3.2. Serum Peroxide (PO) ......................................................................................... 64
3.3.3.3. Effect of Rutin-GNPs on Serum Glutathione (GSH) ....................................... 64
3.4. Measurement of Pro-Inflammatory Cytokines in Serum............................................ 67
3.4.1. Effect of NA-2 on Serum IL-1β and TNF-α Concentration ................................... 67
3.4.2. Effect of Rutin on IL-1β and TNF-α Concentration ............................................... 67
3.4.3. Effect of Rutin-GNPs on IL-1β and TNF-α Concentration .................................... 70
3.5. Gene Expression Studies Using Quantitative Real time RT-PCR ............................. 70
3.5.1. Effect of NA-2 Treatment ......................................................................................... 70
3.5.2. Effect of Rutin Treatment ......................................................................................... 72
3.5.3. Effect of Rutin-GNPs Treatment.............................................................................. 73
3.6. Immunohistochemistry of c-Fos, pAkt and iNOS in Spleen Tissue ........................... 76
3.6.1. Effect of NA-2 on c-Fos, pAkt and iNOS Protein Expression ............................... 76
3.6.2. Effect of Rutin on c-Fos, pAkt, and iNOS Protein Expression .............................. 81
3.6.3. Effect of Rutin-GNPs on c-Fos, pAkt, and iNOS Protein Expression................... 86
CHAPTER 4
DISCUSSION............................................................................................................................. 91
CONCLUSION ........................................................................................................................ 100
FUTURE IMPLICATIONS .................................................................................................. 101
REFERENCES ........................................................................................................................ 102
PERSONAL INTRODUCTION ........................................................................................... 126
GLOSSARY ............................................................................................................................. 127
APPENDIX I ............................................................................................................................ 130
APPENDIX II .......................................................................................................................... 133
LIST OF PUBLICATIONS ……………………………………......................................134
I
Acknowledgements
First, I am thankful to Almighty Allah, who has bestowed upon me His blessings that I was
able to complete my research successfully.
My heartfelt gratitude to my supervisor Dr. Shabana U. Simjee whose constant support,
expertise, and encouragement has made me able to contribute in the field of Molecular
Medicine. Without her devoted supervision, I could never finish this work. I am also grateful
to my former supervisor Dr. Talat Makhmoor. I cannot forget her initial training my whole
life. She always believed in me, and stood with me in my hard times.
I am very much thankful to Ms. Nadira Panjwani (Chairperson, Dr. Panjwani Memorial Trust)
who has established this international research center and provided us the opportunities for
research of international level and showing our strengths to the rest of the world. Moreover, I
am indebted to Prof. Dr. Atta-ur-Rahman F.R.S., N.I., H.I., S.I., T.I. (Patron in Chief) and Prof. Dr. M.
Iqbal Choudhary H.I., S.I., T.I. (Director, PCMD) for running such a remarkable institution and
for providing us the best working environment.
I am very thankful to Prof. Dr. Shaheen Faizi S.I., Dr. Raza Shah T.I., all my teachers, and PCMD
faculty members.
I am grateful to all my friends who provided me support in hard times, especially Areeba
Anwar and Maryam Mazhar.
I am very much thankful to my loving mother Saeeda Haider, father Ghulam Haider and
siblings Meisha Gul and Ali Shayan Haider for their unconditional support throughout my
studies.
II
List of Tables
Table No. Title Page No.
1.1 Approved biologic therapies of rheumatoid arthritis………………...21
2.1 Treatment regime used for the testing of compounds
for anti-arthritic activity in collagen induced arthritis
model in rats…………………………………………………………29
2.2 Scoring system used to assess severity of paw
inflammation………………………………………………………...31
2.3 Composition of RNA/Primer mixture……………… ……................38
2.4 Composition of cDNA synthesis mixture…………………...............38
2.5 Real time RT- PCR reaction mixture recipe………...........................39
2.6 Steps involved in real time RT-PCR reaction……………..............39
2.7 Primer sequences, annealing temperature and expected
product size……………………………………….............................40
2.8 List of antibodies for immunohistochemistry……………….............42
3.1 Histological scoring system adopted to monitor the histological
changes in arthritic and non-arthritic rats…………………………...57
III
List of Figures
Figure 1.1. Factors involved in the progression of rheumatoid arthritis .............................. 4
Figure 3.1. Effect of NA-2 on mean arthritic severity score in rats induced with collagen
induced arthritis .................................................................................................. 46
Figure 3.2. Effect of NA-2 on mean change in body weight .............................................. 46
Figure 3.3. Effect of NA-2 on mean change in hind paw volume after arthritis induction.
...................................................................................................................................................... 47
Figure 3.4. Effect of rutin on mean arthritic score ............................................................... 49
Figure 3.5. Mean change in body weight of arthritic and non-arthritic animals................ 49
Figure 3.6. Mean change in paw volume following treatment with rutin .......................... 51
Figure 3.7. Effect of rutin-GNPs on arthritic score.. ........................................................... 53
Figure 3.8. Effect of rutin-GNPs on mean change in body weight..................................... 54
Figure 3.9. The mean change in paw volumes following treatment with rutin-GNPs ...... 55
Figure 3.10. Effect of NA-2 treatment on joint destruction in CIA rats………………….58
Figure 3.11. Effect of rutin treatment on Knee joints sections from CIA rats……………59
Figure 3.12. Effect of rutin-GNPs treatment on Knee joints sections from CIA rats…….60
Figure 3.13. Effect of NA-2 on serum NO, PO and GSH level in arthritic and non-arthritic
rats ....................................................................................................................... 62
Figure 3.14. The NO, PO and GSH concentration measured following rutin treatment..... 65
Figure 3.15. Serum NO, PO and GSH concentration measured after treatment of rutin-
GNPs.. ................................................................................................................. 66
Figure 3.16. Effect of NA-2 on IL-β and TNF-α concentration.. ......................................... 68
Figure 3.17. Effect of rutin on IL1-β and TNF- α concentration .......................................... 69
Figure No. Title Page No.
IV
Figure 3.18. Effect of rutin-GNPs on IL-1β and TNF-α concentration ................................. 71
Figure 3.19. The effect of NA-2 on fold change in RANK, RANKL, NF-κB, c-fos, c-jun
and Akt gene expression .................................................................................... 74
Figure 3.20. Effect of rutin on RANK, RANKL, NF-κB, c-fos, c-jun and Akt gene
expression............................................................................................................ 74
Figure 3.21. Expression of RANK, RANKL, NF-κB, c-fos, c-jun and Akt genes following
rutin-GNPs treatment. ........................................................................................ 75
Figure 3.22. Effect of NA-2 on c-Fos, pAkt and iNOS Protein Expression ........................ 77
Figure 3.23. Effect of NA-2 on immunohistochemistry of c-Fos in spleen tissue. ............. 78
Figure 3.24. Effect of NA-2 on Immunohistochemical analysis of pAkt in arthritic and
non-arthritic rats.. ............................................................................................... 79
Figure 3.25. Immunohistochemical analysis of iNOS expression after treatment with NA-2
............................................................................................................................. 80
Figure 3.26. Effect of rutin on c-Fos, pAkt and iNOS protein expression. .......................... 82
Figure 3.27. Immunohistochemistry of c-Fos in rutin treated animals…………………...83
Figure 3.28. Immunohistochemical analysis of pAkt in rutin treated group……………..84
Figure 3.29. Photomicrograph of iNOS expression in rutin treated animals……………..85
Figure 3.30. Effect of rutin-GNPs on c-Fos, pAkt and iNOS expression………………...87
Figure 3.31. Effect of rutin-GNPs on Immunohistochemical analysis of c-Fos .................. 88
Figure 3.32. Immunohistochemistry of pAkt on rutin-GNPs treated rats ............................ 89
Figure 3.33. Immunohistochemistry of iNOS in spleen tissue of rutin-GNPs treated group
...................................................................................................................................................... 90
V
List of Abbreviation
ACPA Anti-citrullinated protein antibody
AIA Adjuvant-induced arthritis
ANOVA Analysis of variance
AP1 Activator protein 1
ASA Acetylsalicylic acid
ATF Activating transcription factor 3
CAT Catalase
CD Cluster of differentiation
cDNA Complementary DNA
CFA Complete Freund’s adjuvant
CIA Collagen induced arthritis
COX Cyclo-oxygenase
CP Ceruloplasmin
CSF1R Colony Stimulating Factor 1 Receptor
c-Src Proto-oncogene tyrosine-protein kinase Src
CTGF Connective tissue growth factor
CTLA Cytotoxic T lymphocyte antigen
CyA Cyclosporine A
DMARDs Disease modifying anti-rheumatic drugs
DNTB 5, 5’-dithiobis 2-nitrobenzoic acid
EBV Epstein-Barr virus
VI
eNOS Endothelial nitric oxide synthase
ERK Extracellular signal regulated kinase
Fab Fragment of antigen binding
Fc Fragment of crystallization
GAB2 GRB2 Associated Binding Protein 2
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GC Glucocorticoid
GNPs Gold nanoparticles
GSH-Px Glutathione peroxidase
GST Glutathione S-transferase
H & E Hematoxylin and eosin
H2O2 Hydrogen peroxide
HCQ Hydroxychloroquine
HLA Human leukocyte antigen
HLA-DRB1 Human leukocyte antigen- DR beta 1
IBD Inflammatory bowel disease
IFA Incomplete Freund’s adjuvant
IFN-γ Interferon gamma
IKK2 Inhibitor of nuclear factor-kappaB kinase subunit beta
IL-1β Interlukin-1-beta
iNOS Inducible NOS
JNK c-Jun N-terminal kinase
LPS Lipopolysaccharide
VII
MAF Macrophage activating factor
MAPK Mitogen activated protein kinase
M-CSF Macrophage colony-stimulating factor
MK2 MAP Kinase-Activated Protein Kinase 2
MMPs Matrix metalloproteinases
MTX Methotrexate
NA-2 N-(2-hydroxyl phenyl) acetamide
NF-κB Nuclear factor-κB
nNOS Neuronal NOS
NO Nitric oxide
NOSs NO synthases
NSAIDs Non-steroidal anti-inflammatory drugs
OPG Osteoprotegerin
p38 Protein 38
PAD Peptidylarginine deiminase
PBS Phosphate buffered saline
PCR Polymerase Chain Reaction
PGE2 Prostaglandin E2
PI3K Phosphotidyl-inositol-3- phosphate
PsA Psoriatic arthritis
RA Rheumatoid arthritis
RANK Receptor activator of NF-κB
RANKL Receptor activator of NF-κB ligand
VIII
RF Rheumatoid Factor
RNS Reactive nitrogen species
ROS Reactive oxygen species
S.E.M Standard error of mean
SAARDs Slow acting anti-rheumatic drugs
SE Shared epitope
SOD Superoxide dismutase
SPSS Statistical package for social sciences
SSZ Sulfasalazine
STAT3 Signal transducer and activator of transcription 3
TMB 3,3',5,5'-Tetramethylbenzidine
TNFR Tumor necrosis factor receptor
TNF-α Tumor necrosis factor alpha
TRAF TNF receptor associated factor
Treg cells Regulatory T cells
IX
Summary
Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease of unknown
etiology that results in inflammation in the synovial joint, and irreversible bone and cartilage
destruction. It includes a complex series of events involving environmental and genetic factors
that leads to the pathogenesis of RA. Various drugs have been used for RA treatment but none
is able to prevent the bone destruction that is the most severe outcome of RA progression.
RANK and RANKL are involved in the maintenance of normal bone physiology and any
alteration in the level of these molecules is responsible for bone pathologies. In osteoclast
research, RANKL is becoming an important therapeutic target since it binds to mature
osteoclasts and causes induction of bone resorbing activity. Beside this, RANKL and RANK
are also key players of the inflammatory cascade in human and animal models of arthritis. In
the current study, we explored the effect of N-(2-hydroxyl phenyl) acetamide (NA-2), rutin,
indomethacin + rutin, and rutin-GNPs on the attenuation of RANK, RANKL, and other pro-
inflammatory mediators downstream of RANKL pathway to understand the possible
mechanism behind the anti-arthritic effect of these compounds in collagen induced arthritis
(CIA) model in rats. The progression of arthritis was analyzed by clinical parameters such as,
body weight, paw volume and arthritic score that showed marked improvement after treatment
with the compounds. Similarly, the level of inflammatory markers in serum such as, TNF-α,
IL-1β, NO and PO after compounds treatment were also reduced with parallel increase in the
GSH level. The gene expression analysis of RANK, RANKL, c-fos, c-jun, Akt, and NF-κB
was done in spleen tissue by real time PCR and the compounds treatment successfully
downregulated the expressions of these genes. Immunohistochemical analysis of iNOS, c-Fos,
and pAkt in spleen tissue also showed marked suppression in the protein expression in arthritic
rats, treated with the above cited compounds. These findings suggest that NA-2, rutin (both
as a single agent and in combination with indomethacin or gold nanoparticles) are potent anti-
rheumatic agents and can inhibit the inflammatory response and destruction of bone and
cartilage possibly by modulation of RANK/RANKL pathway.
X
صہ ال خ
ڈیر وائ ٹس ا وماٹ ھرائ م کیآرت ے۔ اس مرض م یدائ سم م ںیمرض ہ اعث ج ے ب لوم وجہ ک امع ںین
ہ نے ٹھوں ک یاپ عمال جوڑوں اور پ ت س ا ا عت ک وت مداف الف ق س یخ ل واپ اب اق سوزش اور ن ڈ ی وںیہ
مزور یک ہ یک با ے۔ یاور ت ا ہ وت اعث ہ ا ب ہیک لوں ک دہیچیمرض پ لہ وار مرح س ل س صورت م ی ںی
ے اور اس عمل م ا ہ وت ر ہ صہ ل یاتیاور ماحول یاتینیج ںیظاہ ےیعوامل ح ڈی۔ رںیہ ت وائ وماٹ
شمار ادو ے الف ب ے خ ٹس ک ھرائ وت اتیآرت عمال ہ ت س ہ یا وئ ںیہ یآر گر ک ھ یم ڈ یب یک وںیدوا ہ
مزور ہ یک ے، دور ن لو ہ ہ اک پ سے خطرن سب ا ہ اس مرض ک سک ںیجو ک ر کی۔ ریک کیاور ر ن ن
سےیدو ا لیا زیکیمال ہ ول ڈ ںی ز یک وںیجو ہ ارمل ف وجین ہ یال تے ھ رار رک رق و ب اور ان ںیک
عمول ںیم بد یم ڈ یلیت نم د وںیماریب یک وںیہ و ج کی۔ رںیہ یتیک ڈ لیا ن ے وںیہ ے امراض ک ک
الج م ڈ کیا ںیع ے جو ہ دف ہ م ہ ے خل اںیاہ ے وال رن مزور ک سٹ وںیک سٹ،یاو ال ھ مل وک سات ے ک
ر ھڈ الوہ ر اںیک ے ع ے۔ اس ک ا ہ رت مزور ک کیک کیاور ر ن وں م لیا ن سان ے ںیان وروں ک اور جان
ٹس ماڈل م ھرائ سوزش پ ںیآرت ے م دای رن ہ ںیک ے رت ردار ادا ک م ک ئے حالںیاہ ہی۔ اس ل حق قیت
و، ر نیا ںیم ن،یاے ٹ نیر وٹ ڈوم وٹ ھ ان سات ے سنیک ھا نیاور ر ت ڈ ن وٹ ول ویگ ارٹ ن لزیپ ک
ول و ک چا گ جنیک الف جان ے خ ٹس ک ھرائ ے آرت ے وال ون سے ہ و ر ای بات ک ہ ان مرک اک ک،یت ن
کیر کیاور ر لیا ن ھوے م لیا ن ات سوزش پ دیمز ںیپ ے مال دای ے وال رن زیکیک ے ول ٹان ھ ر گ پ
لوم ک و مع نہ عمل ک ک ر اور مم ے اث و طب ایک نے ک ڑھ ے ب ے درد ک کے۔ جوڑوں ک س یجا
رزیرامیپ سایج ٹ ٹھ نجا حجم اور گ ے وزن، پ سم ک ہ ج کور ک ایک س لوم ک یا سے مع ایگ ایمدد
ست ے ا بات ک عد ان پ عمالاور ان مرک ے ب رزیرامیک تر ںیم ٹ ہ ھید یب ئ یک طرح ان ی۔ اسیگ
ے س وں ک ٹس زدہ چوہ ھرائ سے آرت عمال ت س ے ا بات ک وں م رمیمرک مون سوزش پ ںین ے دای رن ک
ے مال زیکیوال سایج ول ہ آئ ا،یون ب لیا یک فا،یا فیا نیا یٹ ٹ سائ ل ٹرک آک ائ ر ڈین اور پ
سائ ے م یک ڈیآک ٹان ھ و گ سطحوں ک ھا ںی ات لوٹ ے م یون کیاور گ ڑھان و ب سطح ک ام ںی ابیک
ے۔ ج وئ ت ہ اب ر ر یاتینیث سطح پ ک،ی کیر ن وس، س یس ل،یا ن ے ٹ یف نیاور ا ،یجون، اے ک
ا ب فیا اپ ل یک ہ ت ھے، ر یجو ک ئے ت ئے گ صل ک سے حا لی م پ ئ ائ سے یآر ک یس یٹ مدد
ے م ے جان ر ل سطح پ م و ک بات ان ک ئے اور مرک چے گ ام ںیجان ے۔ ام ابیک ٹوکیرہ س وہ لیمیون ک
سٹیٹ ل سے ت وس، س یآئ ںیم ی وس اور پ ین ے ٹ یف روٹ یاے ک زیپ ا ن وا پ ٹا ہ ھ و گ ۔ ان ایک
سے ج تائ ہین ہ ا ے ک ا ہ وت ر ہ و نیظاہ ن،یر اے ٹ نیر وٹ ڈوم وٹ ھ ان سات ے سنیک ھا نیاور ر ت وٹ
ڈ ن ول ویگ ارٹ ن لزیپ و ک الف ق ے خ ٹس ک ھرائ ہ یآرت تے ھ ر رک ک،یاور ر ںیاث کیر ن اور لیا ن
سے پ ردہ د دایان سرےک سوزش و ڈ ی سوزش، ہ ر و ک داز ہ ر ان ر اث ل وںیعوامل پ ارٹ یک جیاور ک
ہ با ہ یک یت ے رت ھام ک ۔ںیروک ت
1
CHAPTER 1
INTRODUCTION
2
1.1. Rheumatoid Arthritis and Its Prevalence
Rheumatoid arthritis (RA) is an autoimmune disorder that leads to disability, bone and
cartilage damage ultimately leading to pulmonary and cardiovascular disorders (Picerno,
Ferro, Adinolfi, Valentini, Tani, and Alunno, 2015). The prevalence of RA is 0.5–1% in
Western adult populations and is comparatively similar across Asia, South Africa, North
America and Europe (Jacobsson, Hanson, Knowler, Pillemer, Pettitt, Mccance, and Bennett,
1994). Annually, the incidence of RA is between 0.15 and 0.88 per 1000 and females are
affected two to three times more as compared to male population. The disease can occur at
any stage of life but individuals with 45 to 65 years of age are more prone to develop this
condition (Scott and Wolfe, 2010).
1.2. Clinical Manifestation
The symptoms of RA progression vary but some features are same in all individuals like
morning joint stiffness, pain and movement impairment. Typically, the symptoms include
generalized inflammation, the individual experience weight loss, fatigue, low grade fever and
exhaustion. The most commonly affected area in the body as a result of arthritis progression
are joints especially small joints of hand and feet along with some larger joints and their
inflammation leads to swelling of the synovium.
In RA, the damage to articular cartilage results in anatomical changes in joints over the course
of time. The blend of these constant changes prompts some clinically recognized patterns of
deformation, for example, swan-neck and boutonnière disfigurements of the fingers, hammer-
toe distortions in the feet, the ulnar deviation of the hands, and others. RA patients with severe
joint disease develop extra articular complications like lung and cardiac involvement,
vasculitis and in some RA patients the increase in mortality rate is because of high occurrence
of cardiovascular disease (Wållberg-Jonsson, Johansson, Ohman, and Rantapää-Dahlqvist,
1999; Watson, Rhodes, and Guess, 2003).
3
1.3. Risk factors and Pathogenesis of Rheumatoid Arthritis
Despite intensive research, the pathogenesis of RA is poorly understood and a complex
interaction among genetic and environmental factors and continuous activation of both innate
and adaptive immunity results in breakdown of immunological tolerance, antigen specific B
and T cell activation and abnormal presentation of autoantigens. All these events eventually
lead to bone destruction and synovial hyperplasia resulting in deformity and swelling of joint
and ultimately systemic inflammation (Fig. 1.1) (MacGregor, Snieder, Rigby, Koskenvuo,
Kaprio, Aho, and Silman, 2000; McInnes and Schett, 2011; Picerno, Ferro, Adinolfi,
Valentini, Tani, and Alunno, 2015).
The most common genetic risk factor is associated with human leukocyte antigen (HLA)
DRB1 locus that contain a conserved sequence of amino acid QKRAA called the shared
epitope (SE), which is associated with RA susceptibility (Gregersen, Silver, and Winchester,
1987). On the other side, environmental factors such as smoking, infectious agents and
gastrointestinal microbiome also participates in the RA pathogenesis. The interactions
between HLA-DRB1 alleles and smoking synergistically increase the risk of RA (Klareskog,
Rönnelid, Lundberg, Padyukov, and Alfredsson, 2008; Scher and Abramson, 2011).
Mechanistically, smoking causes the induction of peptidylarginine deiminase (PAD) enzymes
that cause post translational modification of protein resulting in citrullination of protein that
elicits anti-citrullinated protein antibody (ACPA) response against citrullinated proteins
(Vincent, De Keyser, Masson-Bessière, Sebbag, Veys, and Serre, 1999; De Rycke, Peene,
Hoffman, Kruithof, Union, Meheus, Lebeer, Wyns, Vincent, and Mielants, 2004). Infectious
agents such as E. coli, cytomegalovirus, Epstein-Barr virus (EBV) and proteus species and
their products (e.g. heat shock proteins) have been suspected as potential triggers of RA but
the mechanisms of pathogenesis are not known completely. It is postulated that some form of
molecular mimicry might be involved (Auger and Roudier, 1997; Kamphuis, Kuis, de Jager,
Teklenburg, Massa, Gordon, Boerhof, Rijkers, Uiterwaal, and Otten, 2005). In RA
pathogenesis, pro-inflammatory cytokines and chemokines produced by synovial cells are
also key players (Kirkham, Lassere, Edmonds, Juhasz, Bird, Lee, Shnier, and Portek, 2006).
In addition to this, macrophages, the innate effector cells, with antigen presentation and
phagocytic ability also serve as central effectors of synovitis. The infiltration and activation
4
Figure 1.1. Factors involved in the progression of rheumatoid arthritis. Environmental
and genetic factors lead to breakdown of tolerance to self-proteins having citrulline residues
generated by post-translational modification. In bone marrow or secondary lymphoid tissues,
this anti-citrulline protein response is generated in B and T cell. After these events,
inflammatory response is observed in the joints that may involve neurologic, microvascular,
biomechanical and other pathways. Synovitis occurs as a result of positive feedback loops that
promote systemic disorders that ultimately lead to RA. RF denotes rheumatoid factor and
ACPA denotes anti–citrullinated protein antibody (adapted from McInnes and Schett, 2011).
5
of macrophages and T cells in the synovium results in the production of various growth factors
and cytokines like TNF-α (tumor necrosis factor-α), TGF- β (transforming growth factor β),
interleukins i.e. IL-1, IL- 2, IL- 6, IL-8, IL-10, IL-17, insulin like growth factor and platelet
derived growth factors. All these effector molecules are involved in cartilage and bone
destruction, synovitis and systemic complications (Stolt, Bengtsson, Nordmark, Lindblad,
Lundberg, Klareskog, and Alfredsson, 2003; Bresnihan, and Tak, 2005; Haringman, Gerlag,
Zwinderman, Smeets, Kraan, Baeten, McInnes, Maruotti, Cantatore, Crivellato, Vacca, and
Ribatti, 2007; Cornish, Campbell, McKenzie, Chatfield, and Wicks, 2009). In the synovium,
neo-angiogenesis which is the growth of new capillary blood vessels is also observed in the
persistence of inflammation in patients of RA. The release of different enzymes and
metalloproteinases that are damaging to bone and cartilage are also released by the cells of
synovium and articular cartilage (Smith and Haynes, 2002).
B cells are also key players in the progression of RA. Synovium infiltration with B cells results
in its differentiation into plasma cells that produces Rheumatoid Factor (RF). In addition,
activation of synovial fibroblast leads to the destruction of matrix tissues by the action of
enzymes like collagenases and metalloproteinases. The overall result of all these activities is
cartilage and bone erosion, pannus formation and joint swelling with destruction of
periarticular structures (Smith and Haynes, 2002; Stolt, Bengtsson, Nordmark, Lindblad,
Lundberg, Klareskog, and Alfredsson, 2003).
1.4. Reactive Oxygen and Nitrogen Species (ROS & RNS)
Reactive oxygen species (ROS) are important mediators in the cellular signaling cascades
(Irani, 2000). Appropriate levels of ROS are required for several physiological processes and
have beneficial effects such as in tissue regeneration, wound healing and protection from
pathogens (Bhattacharyya, Chattopadhyay, Mitra, and Crowe, 2014; Datta, Kundu, Ghosh,
De, Ghosh, and Chatterjee, 2014). Deleteriously higher levels of ROS lead to disease
development and progression especially neurodegenerative diseases, cardiovascular diseases,
cancer and chronic inflammation (Halliwell and Cross, 1994; Bhattacharyya, Chattopadhyay,
Mitra, and Crowe, 2014). Some of the factors which trigger increased production of ROS
include exposure to UV radiation, alcohol consumption, cigarette smoking, some drugs,
6
inflammatory disorders and ischemia–reperfusion injury (Halliwell and Cross, 1994; Iuchi,
Akaike, Mitsui, Ohshima, Shintani, Azuma, and Matsumoto, 2003; Mallick, Yang, Winslet,
and Seifalian, 2004; Roessner, Kuester, Malfertheiner, and Schneider-Stock, 2008; Wu and
Cederbaum, 2009; Jia, Koonce, Griffin, Jackson, and Corry, 2010; Deavall, Martin, Horner,
and Roberts, 2012)
The family of ROS is comprised of peroxide, superoxide and hydroxyl radicals which are
involved in oxidation of proteins, nucleotides, polysaccharides, DNA and lipids by the
unpaired free radicals. In inflammation, ROS are produced that promotes release of variety of
potentially harmful enzymes, neutrophils degranulation and the destruction of bone and
cartilage (Stamp, Khalilova, Tarr, Senthilmohan, Turner, Haigh, Winyard, and Kettle, 2012).
In patients with RA several inflammatory products mediated by ROS have been identified in
the peripheral blood and synovial fluid (Hadjigogos, 2003; Filippin, Vercelino, Marroni, and
Xavier, 2008; Winyard, Ryan, Eggleton, Nissim, Taylor, Faro, Burkholz, Szabó-Taylor, Fox,
Viner, Haigh, Benjamin, Jones, Whiteman, 2011).
ROS and RNS are highly reactive entities which are very difficult to be determined directly
in vivo. However, measurement of resulting effects of ROS and RNS on various cellular and
extracellular components is more practical (Vasanthi, Nalini, and Rajasekhar, 2009). Still
there is no clear proof that whether ROS are involved in initiation of autoimmunity or
ameliorate chronic inflammation in RA. Under physiological conditions, T cell functions are
regulated by NO, but overproduction of NO may lead to functional changes in T lymphocytes
(Nagy, Koncz, Telarico, Fernandez, Érsek, Buzás, and Perl, 2010). Some researchers have
identified the relationship between the RA disease and the level of serum nitrite (Onur, Akıncı,
Akbıyık, and Ünsal, 2001; Choi, 2003).
There are various antioxidant systems present in cells required for defense against free radical
damage. Red blood cells in human circulation scavenge H2O2 and O2- that are produced by
activated neutrophils as a result of redox reaction utilizing superoxide dismutase (SOD),
catalase (CAT) and glutathione peroxidase (GSH-Px) dependent mechanisms. There is a
controversy regarding antioxidant status in erythrocytes and serum of RA patients, since few
studies have shown decreased while others have reported unaltered activity of antioxidant
7
enzymes (Cimen, Çimen, Kacmaz, Öztürk, Yorgancioğlu, and Durak, 2000; Nivsarkar, 2000;
Afonso, Champy, Mitrovic, Collin, and Lomri, 2007). Oxidative stress mediates inflammatory
responses in the arthritic joint (Henrotin, Bruckner, and Pujol, 2003; Datta, Kundu, Ghosh,
De, Ghosh, and Chatterjee, 2014). Hydrolysis of collagen and degradation of the extracellular
matrix in cartilage are also attributed to the intra-articular ROS which activates
metalloproteinase resulting in the development of osteoarthritis. (Goldring, 2003; Henrotin,
Bruckner, and Pujol, 2003; Yamazaki, Fukuda, Matsukawa, Hara, Matsushita, Yamamoto,
Yoshida, Munakata, and Hamanishi, 2003; Yamazaki, Fukuda, Matsukawa, Hara, Yoshida,
Akagi, Munakata, and Hamanishi, 2003; Fay, Varoga, Wruck, Kurz, Goldring, and Pufe,
2006; Regan, Bowler, and Crapo, 2008; Yu, Yi, Yuh, jin Park, Kim, Bae, Ji, Kim, Park, and
Kim, 2012).
1.4.1. Nitric Oxide
Nitric oxide (NO) is a molecule with a very short half-life. In various physiological functions,
NO plays an important role such as mitochondrial functions, regulation of inflammation,
apoptosis and blood vessel tone (Brown, 1999; Beltrán, Mathur, Duchen, Erusalimsky, and
Moncada, 2000). It is synthesized from L-arginine by NO synthases (NOSs) such as neuronal
NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) (Chiavaroli, Giannini, De
Marco, Chiarelli, and Mohn, 2011). NO is produced in different cell types such as phagocytic
neutrophils, leukocytes, chondrocytes, and synoviocytes. NO is mainly involved in regulation
of immune system, neurotransmission, and vasodilatation (Labunskyy and Gladyshev, 2013).
Furchgott and Zawadzki, first identified NO as endothelium derived relaxant factor (Furchgott
and Zawadzki, 1980). They observed the importance of intact endothelium for acetyl choline-
induced blood vessel relaxation. This observation led to the discovery of endothelium-derived
relaxant factor, identified as NO (Palmer, Ferrige, and Moncada, 1987). In rodent
macrophages, LPS and inflammatory cytokines like IL-1-β, IFN-γ and TNF-α induces the
expression of iNOS in vitro that leads to long term production of NO in large amount (Brown,
1999). Several studies have reported higher levels of endogenous NO synthesis in patients
with RA, suggesting the stimulating role of NO in the progression of RA (Farrell, Blake,
Palmer, and Moncada, 1992; Pham, Rahman, Tobin, Khraishi, Hamilton, Alderdice, and
8
Richardson, 2003). The regulation of iNOS expression occurs at transcriptional level whereas
intracellular Ca2+ regulates eNOS and nNOS. In the inflamed joint, different cells such as
macrophages, osteoblasts, fibroblasts, neutrophils, osteoclasts and endothelial cells produces
NO in the synovium of RA patients (Van'T Hof and Ralston, 2001; Nagy, Clark, Buzás,
Gorman, and Cope, 2007; Firestein, Budd, Gabriel, McInnes, and O'Dell, 2012). It has been
reported that NOS inhibition contribute to decrease in disease progression in experimental RA
(McCartney-Francis, Allen, Mizel, Albina, Xie, Nathan, and Wahl, 1993).
1.4.2. Hydrogen Peroxide
Hydrogen peroxide is a member of reactive oxygen species (ROS) family that are produced
as a result of oxidative metabolism (Bedard and Krause, 2007). H2O2 is also involved in
regulation of many cellular events including cell proliferation, transcription factors activation,
and apoptosis (Rojkind, Dominguez-Rosales, Nieto, and Greenwel, 2002). H2O2 modulate
many different signaling pathways including p38, ERK, MAPK, JNK and PI3K/Akt (Gabbita,
Robinson, Stewart, Floyd, and Hensley, 2000; Hensley, Robinson, Gabbita, Salsman, and
Floyd, 2000). H2O2 can stimulate the macrophages to release high mobility group 1 protein
resulting in augmentation of pro-inflammatory stimuli or modify expression of leukocyte
endothelial adhesion and leukocyte adhesion molecule (Tang, Shi, Kang, Li, Xiao, Wang, and
Xiao, 2007; Sen and Roy, 2008). Hydrogen peroxide and superoxide radical induces the
release of TNF-α and interleukins from T lymphocytes. It induces endothelial cells that results
in production of adhesive molecules, growth factors and inflammatory cytokines by immune
cells thereby aggravating inflammation and tissue destruction (Los, Dröge, Stricker, Baeuerle,
and Schulze‐ Osthoff, 1995; Feldmann, Brennan, and Maini, 1996).
1.5. Antioxidant Defense Mechanisms
There are multiple defense systems in our body collectively called antioxidants. Their function
is to fight against free radical-induced oxidative stress. In humans, these antioxidants are
present in erythrocytes, plasma as well as serum. Serum antioxidants include ascorbate,
transferrin and ceruloplasmin (CP). Erythrocytes have glutathione S-transferase (GST),
superoxide dismutases (SODs), glutathione peroxidase (GPx), glutathione (GSH) and catalase
(Cat) that work in co-ordination to handle intracellular oxidative stress (McCord and Edeas,
9
2005; Valko, Leibfritz, Moncol, Cronin, Mazur, and Telser, 2007). Imbalance in oxidant to
antioxidant ratio in the body leads to potentially destructive effects on cellular functions and
activities (Soneja, Drews, and Malinski, 2005; Blair, 2006).
1.5.1. Glutathione (GSH)
Glutathione (L-γ-glutamyl-L-cisteinylglycine) is an important part of antioxidant defense
mechanism. In prokaryotes and eukaryotes, it is present in cells as a tripeptide, a non-protein
sulfhydryl molecule (Pastore, Federici, Bertini, and Piemonte, 2003). In normal metabolic
process hydrogen peroxides are produced, GSH play central role in the protection of cells
from harmful effects of peroxide (PO) (Dröge, Schulze-Osthoff, Mihm, Galter, Schenk, Eck,
Roth, and Gmünder, 1994). In the cell, the enzyme glutathione reductase converts the inactive
oxidized form of glutathione (GSSG) into active reduced form GSH. Glutathione reductase is
constitutively active but in oxidative stress, the level of GSH is high (Pastore, Federici,
Bertini, and Piemonte, 2003). Reduced levels of GSH may contribute to
inflammatory/immunomediated diseases, inflammation, cellular injury in RA, vascular injury
and tissue damage (Collard, Agah, and Stahl, 1998).
1.6. Cytokines in the Pathogenesis of Rheumatoid Arthritis
Cytokines are small proteins from diverse group, which includes chemokines, interferon,
interleukins, monokines, colony stimulating factors and lymphokines that are synthesized
during immune response. These cytokines bind to their respective receptors and perform their
function that is characterized by pleiotropy and redundancy. Cytokine receptors are either
heterodimeric or heterotrimeric grouped into families with sharing of one receptor subunit by
each member of the receptor family. Imbalance or uncontrolled production of these cytokines
leads to inflammatory response and may lead to autoimmunity. In case of rheumatoid arthritis,
the secretion of cytokines in synovium alters with the disease progression. In the early stage
of RA, Th cells in the synovium express IL-2, IL-4, IL-13, IL-15 and IL-17. Whereas in the
synovium of full blown RA, Th cells express IFN-γ, TNFα and IL-10. However, expression
of IL-2, IL-4, IL-5 and IL-13 cytokines by Th cells is either very low or completely absent.
The underlying mechanism of fluctuating cytokine profile with the progression of disease is
still not completely understood (Cope, 2008).
10
1.6.1. Pro-Inflammatory Cytokine: TNF-α
TNF-α is one of the most important primary cytokines of the cascade involved during the
course of RA and regulates the secretion of other pro-inflammatory cytokines present in the
synovial tissue. TNF-α is largely produced by immune cells such as neutrophils, natural killer
cells, endothelial cells, activated lymphocytes and macrophages (Monaco, Nanchahal, Taylor,
and Feldmann, 2015). TNF-α stimulates synovial fibroblasts and subsequent production of
CTGF (connective tissue growth factor), that leads to the osteoclasts activation resulting in
the disturbances of cartilage homeostasis and ultimately destruction of the joint (Monaco,
Nanchahal, Taylor, and Feldmann, 2015). Furthermore, the functions of Treg cells are
inhibited by TNF-α by inducing resistance in effector T cells against Treg-mediated
suppression (Komatsu and Takayanagi, 2015).
TNF-α mediates clearly distinguished range of RA elements. Beginning the course of disease
through activation of osteoclasts and chondrocytes to cause articular destruction and bone
resorption thereafter, induction of endothelial cells and amplification of chemokines resulting
in pannus formation along with stimulation of PGE2 synthesis and nociceptors sensitization
to induce pain and fever (Brennan and McInnes, 2008). Since a number of TNF-α inhibitors
have shown a great deal of success for the treatment of RA, therefore TNF-α is a center of
attention for immunologists and rheumatologists. TNF-α inhibitors decrease inflammation
and offers bone protection in patients of RA (Scott and Kingsley, 2006). It has been found
that inhibition of TNF-α reduces bone resorption in postmenopausal osteoporosis in vivo
(Charatcharoenwitthaya, Khosla, Atkinson, McCready, and Riggs, 2007). TNF-α induces
bone destruction in a manner very similar to other inflammatory cytokines, including IL-1 and
M-CSF (Kudo, Fujikawa, Itonaga, Sabokbar, Torisu, and Athanasou, 2002; Kitaura, Zhou,
Kim, Novack, Ross, and Teitelbaum, 2005; Wei, Kitaura, Zhou, Ross, and Teitelbaum, 2005).
It acts by stimulating the RANKL production and M-CSF (macrophage colony-stimulating
factor) from stromal cells and osteoblasts and amplify differentiation into osteoclasts
independently of RANK-RANKL signaling (Kawai, Stein, Perrien, and Griffin, 2012).
Moreover, the expression of RANK upregulates by synergistic effect of TNF-α and RANKL
(Komine, Kukita, Kukita, Ogata, Hotokebuchi, and Kohashi, 2001).
11
1.6.2. Pro-inflammatory Cytokine: IL-1β
IL-1 is an inflammatory cytokine which is highly expressed in inflamed synovium in RA
(Symons, McDowell, Di Giovine, Wood, Capper, and Duff, 1988). Like TNF-α, it is produced
by several immune cells mainly by monocytes and macrophages (Chizzolini, Dayer, and
Miossec, 2009). IL-1 is more potent than TNF-α in causing cartilage destruction
(Vervoordeldonk and Tak, 2002). Previous studies have shown the important role of IL-1 on
bone metabolism by observing noticeable protection against structural damage offered by IL-
1 inhibition in an arthritis model (Joosten, Helsen, Saxne, van de Loo, Heinegård, and van den
Berg, 1999). Moreover, various animal models with IL-1 signaling deficiency have shown
increased bone density, cortical thickness and trabecular bone mass with reduced
osteoclastogenesis (Lee, Fujikado, Manaka, Yasuda, and Iwakura, 2010; Simsa-Maziel,
Zaretsky, Reich, Koren, Shahar, and Monsonego-Ornan, 2013).
1.7. RANK-RANKL-OPG in Bone Diseases
The RANK-RANKL-OPG axis regulates normal physiology of bone. Changes in interaction
and ratio of RANK-RANKL-OPG has significant role in bone diseases. Imbalance in the
coordination between activity and level of these molecules causes osteoclast formation and
activity to increase or decrease resulting in abnormal bone phenotypes such as osteoporotic or
osteopetrotic. Typical examples of such bone loss are associated with inflammatory diseases
such as periodontal disease, RA, and periprosthetic bone loss. Increased levels of RANKL as
compared to OPG are found to be observed in gingival tissue, prosthetic loosening, RA,
synovial-like soft tissue and soft tissues surrounding the damaging bone in cases of active
periodontal disease ( Haynes, Crotti, Potter, Loric, Atkins, Howie, and Findlay, 2001; Crotti,
Smith, Weedon, Ahern, Findlay, Kraan, Tak, and Haynes, 2002; Crotti, Smith, Hirsch,
Soukoulis, Weedon, Capone, Ahern, and Haynes, 2003; Holding, Findlay, Stamenkov, Neale,
Lucas, Dharmapatni, Callary, Shrestha, Atkins, and Howie, 2006; Haynes, Crotti, Weedon,
Slavotinek, Au, Coleman, Roberts‐ Thomson, Ahern, and Smith, 2008; Jiang, Li, Quan, Xiao,
Zhao, Wang, Liu, Gou, An, and Huang, 2015). Moreover, the increases RANKL expression
compared to OPG is associated with stimulated activity and differentiation of osteoclasts
(bone resorbing cells) (Haynes, Crotti, Potter, Loric, Atkins, Howie, and Findlay, 2001; Crotti,
12
Smith, Hirsch, Soukoulis, Weedon, Capone, Ahern, and Haynes, 2003; Crotti, Smith, Findlay,
Zreiqat, Ahern, Weedon, Hatzinikolous, Capone, Holding, and Haynes, 2004; Holding,
Findlay, Stamenkov, Neale, Lucas, Dharmapatni, Callary, Shrestha, Atkins, and Howie, 2006;
Haynes, Crotti, Weedon, Slavotinek, Au, Coleman, Roberts‐ Thomson, Ahern, and Smith,
2008; Van Tuyl, Voskuyl, Boers, Geusens, Landewé, Dijkmans, and Lems, 2010) .
1.7.1. RANK/RANKL Pathway in Rheumatoid Arthritis
In the area of osteoclast research, identification of the receptor activator of nuclear factor- κB
(NF-κB) RANK and its ligand RANKL led to remarkable development (Kong, Feige, Sarosi,
Bolon, Tafuri, Morony, Capparelli, Li, Elliott, and McCabe, 1999). RANKL belongs to TNF
superfamily of cytokines and it was originally identified as a dendritic cell survival factor
produced by T cells activation (Hofbauer, Khosla, Dunstan, Lacey, Boyle, and Riggs, 2000).
In the presence of M-CSF, osteoclasts are produced after differentiation from hematopoietic
precursor cells in vitro after stimulation by RANKL (Fata, Kong, Li, Sasaki, Irie-Sasaki,
Moorehead, Elliott, Scully, Voura, and Lacey, 2000). RANKL leads to the activation of the
bone-resorbing activity of mature osteoclasts. RANKL binds to its transmembrane receptor
RANK that belongs to the TNF receptor superfamily. These receptors are expressed by mature
osteoclasts and dendritic cells as well as by osteoclast precursor cells of monocyte-
macrophage lineage (Neumann, Gay, and Müller‐ Ladner, 2005). These Osteoclast precursors
have been shown to exist in spleen in wild-type mice (Takahashi, Akatsu, Udagawa, Sasaki,
Yamaguchi, Moseley, Martin, and Suda, 1988). RANK stimulation results in interaction with
TRAF6 and GAB2 that leads to further activation of series of downstream intracellular
signals, finally stimulates activation of NF-κB and AP1 transcription factors (Grigoriadis,
Wang, Cecchini, Hofstetter, Felix, Fleisch, and Wagner, 1994; Gravallese, Manning, Tsay,
Naito, Pan, Amento, and Goldring, 2000; Lam, Takeshita, Barker, Kanagawa, Ross, and
Teitelbaum, 2000; Ruocco, Maeda, Park, Lawrence, Hsu, Cao, Schett, Wagner, and Karin,
2005; Wei, Kitaura, Zhou, Ross, and Teitelbaum, 2005) (Fig. 1.2). Osteoprotegerin (OPG) is
another important factor in this system. It is a soluble receptor of RANKL and related to TNF
receptor superfamily. Interaction of RANKL and OPG inhibits the RANKL binding to its
receptor RANK, thereby inhibiting RANKL activity. Kong et al reported that CD4+ T cells
activation can promote osteoclast differentiation through production of soluble or surface-
13
bound RANKL. The study also demonstrated RANKL expression in the synovial tissue of
adjuvant-induced arthritic (AIA) rats by activated T cells. These studies revealed the central
role of RANKL produced by activated T lymphocytes and synovial fibroblasts in joint and
bone destruction in RA (Kong, Feige, Sarosi, Bolon, Tafuri, Morony, Capparelli, Li, Elliott,
and McCabe, 1999; Wong, Besser, Kim, Arron, Vologodskaia, Hanafusa, and Choi, 1999;
Hofbauer, Khosla, Dunstan, Lacey, Boyle, and Riggs, 2000).
1.7.2. RANK/RANKL Pathway and the Immune Response
RANKL has immunoregulatory functions and it is produced by spleen, thymus, activated T
cells, lymph nodes and lymphoid patches in the intestine. RANK is expressed in
hematopoietic precursors, mature T cells and surface of dendritic cells. As a result of RANKL
and RANK interaction, T lymphocytes after activation by dendritic cells undergo
proliferation. RANKL treatment stimulates the antigen specific primary T cells response and
it might be important for memory response of T lymphocytes. In addition to this, RANK-
RANKL interaction is also responsible for expression of CD-40 and IL-12 production in
dendritic cells (Anderson, Maraskovsky, Billingsley, Dougall, Tometsko, Roux, Teepe,
DuBose, Cosman, and Galibert, 1997; Wong, Rho, Arron, Robinson, Orlinick, Chao,
Kalachikov, Cayani, Bartlett, and Frankel, 1997; Kong, Feige, Sarosi, Bolon, Tafuri, Morony,
Capparelli, Li, Elliott, and McCabe, 1999; Josien, Li, Ingulli, Sarma, Wong, Vologodskaia,
Steinman, and Choi, 2000). In the light of these reports, RANKL/RANK system appears to
be important in the development of lymphoid tissue as well as key player in B and T
lymphocytes precursors’ maturation in bone marrow (Josien, Wong, Li, Steinman, and Choi,
1999; Kong, Yoshida, Sarosi, Tan, Timms, Capparelli, Morony, Oliveira-dos-Santos, Van,
and Itie, 1999). Study on human volunteers have reported the role of RANKL in the formation
of osteoclast like cells in the peripheral blood mononuclear cells (PBMCs) (Kotake, Udagawa,
Hakoda, Mogi, Yano, Tsuda, Takahashi, Furuya, Ishiyama, and Kim, 2001). Another study
showed that RANKL expression by activated T cells is able to control bone remodeling and
osteoclastogenesis both in vitro and in vivo (Josien, Li, Ingulli, Sarma, Wong, Vologodskaia,
Steinman, and Choi, 2000). Therefore, RANKL/RANK system might be responsible for bone
loss as a result of systemic activation of T cells. Under normal homeostasis, these T cells are
probably not required because studies on mice model lacking T cells showed normal
14
development of bone (Nakashima and Penninger, 2003). Whereas, remodeling of bone
through RANKL system can be disturbed by chronic T cell activation (Vidal, Brandstrom,
Jonsson, and Ohlsson, 1998).
1.8. Overview of Treatment Strategies
Till late 1980s, the available options for the treatment of RA were limited. Most of these
agents were slow in action and some of them such as gold salts and penicillamine were
associated with serious toxicities. These agents were generally described as ‘go low, go slow’.
In the beginning, non-steroidal anti-inflammatory drugs (NSAIDs) were recommended for the
treatment continued for longer duration (1–2 years). If this treatment was not beneficial,
specific therapy with anti-rheumatic agents in smaller doses were initially recommended and
after a long trial period at each step advancement in the treatment regime was suggested. Anti-
rheumatic medications were never administered as combination therapies but only as single
therapies. This strategy of treatment is called pyramid approach in which the base consisted
of approaches that was applicable for all RA patients e.g. simple analgesics, physical therapy,
rehabilitation and above the basal treatment approach stands the NSAIDs; moving upward in
the pyramid is the treatment strategy that was appropriate only to a limited number of RA
patients including the use of DMARDs and finally at the top of the pyramid comes the
experimental therapies for RA such as plasmapheresis. Other than these treatment modalities,
Glucocorticoid (GC) injections and oral dose and rheumatologic surgeries are also included if
needed. This pyramid treatment strategy was revised in 1980s based on several key
observations. It was recognized that early stages of RA are characterized by irreversible
damage to the articular structures which can be decreased to some extent using DMARDs.
The main ideas in remodeling or inverting of pyramid treatment strategy were the employment
of DMARDs at earlier stages of RA, optimum dosing, escalating therapies and consideration
of combination of DMARDs (Wilske and Healey, 1989; Gremillion and Van Vollenhoven,
1998). In early 1990s, several studies reported the use of combination therapy. In this context,
15
Figure 1.2. RANKL stimulates RANK-dependent signaling cascade. Interaction of RANK
and RANKL results in association of GAB2 and TRAF6 adaptor molecules. This complex
further leads to activation of p38 (MAPK)-MK2 pathway first, then it results in IKK2
phosphorylation causing dissociation of IκB from NF-κB. Then other family of transcription
factors c-fos and c-jun are activated, finally followed by c-src/PI3K/Akt signaling cascade.
CSF1R binds to its receptor MCSF and ERK activation are other key events in the
development of osteoclast. CD44 is a negative regulator that interfere directly with these
pathways (adapted from Schett, Hayer, Zwerina, Redlich, and Smolen, 2005).
16
study reported that combination of hydroxychloroquine (HCQ), methotrexate (MTX) and
sulfasalazine (SSZ) were more effective than treatment with only methotrexate and the
combined effect of two other drugs (O'dell, Leff, Paulsen, Haire, Mallek, Eckhoff, Fernandez,
Blakely, Wees, and Stoner, 2002).
The main concern regarding the use of combined DMARDs was associated with toxicities
that need to be managed. In another study, the combination of cyclosporine A (CyA) and
MTX and was found to be superior to MTX alone tested in a randomized trial (Tugwell,
Pincus, Yocum, Stein, Gluck, Kraag, McKendry, Tesser, Baker, and Wells, 1995). However,
it is noteworthy that this strategy of combining drug effect was not found to be successful in
all the combination therapy trials. One such randomized trial reported that MTX + AZA was
less effective compared to MTX only and was also more toxic (Willkens, Urowitz, Stablein,
Mckendry, Berger, Box, Fiechtner, Fudman, Paul Hudson, and Marks, 1992). Some
uncontrolled observational experiments also demonstrated that combination therapies
including cytotoxic drugs were in some cases even more aggressive and are associated with
considerable toxicities (Wollenhaupt and Zeidler, 1997; McCarty and Carrera, 1982; Walters
and Cawley, 1988). Later on, liver toxicity was observed as a result of this practice (Weinblatt,
Dixon, and Falchuk, 2000). Therefore, close monitoring is required, if combination therapy is
used.
1.8.1. Non-Biological Therapies for Rheumatoid Arthritis
The non-biological treatment modalities mainly include NSAIDs, glucocorticoids (GCs), and
DMARDs.
1.8.1.1. Non-Steroidal Anti-Inflammatory Drugs (NSAIDS)
NSAIDs belong to a diverse group of medications that share a common mode of action but
are different structurally. They act by blocking the rate limiting enzyme cyclo-oxygenase
(COX) involved in the prostaglandins production. They are primarily used for the treatment
of pain, temporary aches and musculoskeletal conditions such as tendonitis and bursitis.
Sometimes these agents are used to treat gout and spondyloarthropathies as a long-term
medication. These are commonly used for the treatment of osteoarthritis but also used in the
RA treatment. As a main therapy, NSAIDs can only be used in mild cases of RA. However,
17
these agents could be added in appropriate anti-rheumatic treatment to provide symptomatic
relief. The main side effects of NSAIDs are gastric problems that can aggravate to cause peptic
ulcers, gastritis, perforations and bleeding. Therefore, histamine 2-antagonists, proton-pump
antagonists, or misoprostol are usually given with NSAIDs. In order to avoid gastrointestinal
toxicity related to NSAIDs, COX-2 specific inhibitors were developed. Unfortunately, the use
of this class of drug was reduced due to unexpected high risk of cardiovascular pathologies.
1.8.1.2. Glucocorticoids (GCs)
The efficacy of GCs (corticosteroids) has been reported for suppression of inflammation in
many diseases including RA. In inflammatory condition, the long term use of glucocorticoids
at effective dosage is associated with many side-effects. Therefore, its use is restricted to
specific cases, for example, in life threatening conditions of RA. High-dose of GCs is used in
combination with strong immunosuppressive agents. In special circumstances, moderate
doses of GCs for short duration can be used as a bridging therapy while slow acting DMARDs
start their action. Low doses of GCs in combination with DMARDs therapy do not show an
effective anti-inflammatory response, however, two randomized clinical trials have reported
enhanced efficacy of DMARDs when given in combination with low-dose GCs and to some
extent showed mild protective effect in radiological damage (Svensson, Boonen, Albertsson,
Van der Heijde, Keller, and Hafström, 2005; Wassenberg, Rau, Steinfeld, and Zeidler, 2005).
In rheumatologic practice, intra-articular GC injections are commonly used and can be very
effective and safe if used appropriately. In Denmark, two clinical trials in RA patients were
conducted using multiple intra-articular GC injections that showed excellent recovery
(Hetland, Stengaard-Pedersen, Junker, Lottenburger, Hansen, Andersen, Tarp, Svendsen,
Pedersen, and Skjødt, 2008; Hørslev-Petersen, Hetland, Junker, Pødenphant, Ellingsen,
Ahlquist, Lindegaard, Linauskas, Schlemmer, and Dam, 2013).
1.8.1.3. Conventional DMARDs
Conventional DMARDs are diverse group of pharmacological agents with anti-rheumatic
properties. Its mode of action is still not completely clear. Some common features shared by
these include low cost, relieves symptoms, reasonable tolerability, but it takes long time to
show its effect usually weeks to months that’s why these are commonly referred as SAARDs
18
(slow acting anti-rheumatic drugs) and these drugs are also associated with toxic side effects.
Therefore, careful monitoring is required during treatment.
In DMARDs, one of the most common and effective drug is methotrexate (MTX) and it is
used as a first-line therapy for RA. MTX was initially synthesized for the treatment of cancer.
It is an anti-metabolite and folate antagonist. In chronic conditions like psoriatic arthritis
(PsA), psoriasis and RA, it has shown efficacy and safety at low dosage. Later on, a study
suggested that the efficacy of MTX in RA is not due to its anti-metabolic property, but it
specifically enhances adenosine production which is an anti-inflammatory endogenous
mediator (Cronstein, Eberle, Gruber, and Levin, 1991; Cronstein, Naime, and Ostad, 1995,
1993). The side effects associated with MTX are mainly hepatic dysfunction, mouth ulcers,
myelosuppression, gastrointestinal symptoms and teratogenicity.
Sulphasalazine (SSZ) is another member of DMARD family of drugs, developed initially for
the RA treatment. It was assumed that RA developed because of some bacteria present in the
gut that are causing inflammatory changes. In 1940s, Karolinska Institute (Stockholm), Nanna
Svartz designed a molecule possessing anti-inflammatory and antibacterial properties. The
efficacy of SSZ was not recognized until post-world war chaos. It was rediscovered in the
1960s and found to be effective for RA and inflammatory bowel disease (IBD) treatment. SSZ
is recommended as a substitute to MTX in RA. In order to achieve greater efficacy, SSZ can
also be given in combination with MTX. SSZ is associated with several side effects such as
hepatic dysfunction, allergic reactions (sulfa allergy), myelosuppression and gastrointestinal
symptoms.
Hydroxychloroquine (HCQ) was originally discovered as an anti-malarial agent. However, it
was serendipitously found to possess anti-rheumatic properties as well. It is rarely used as
monotherapy because considered to be a weaker agent. HCQ in combination with SSZ and
MTX can be used as a triple therapy treatment strategy initiated by O’Dell et al (O'dell, Leff,
Paulsen, Haire, Mallek, Eckhoff, Fernandez, Blakely, Wees, and Stoner, 2002). HCQ is well-
tolerated with small chances of retinopathy.
Leflunomide is a well-known pyrimidine synthesis antagonist. Its risks, efficacy and side
effects are comparably similar to MTX (Weinblatt, Kremer, Coblyn, Maier, Helfgott, Morrell,
19
Byrne, Kaymakcian, and Strand, 1999). It can be used as an alternative of MTX. Combined
treatment with leflunomide and MTX increases efficacy but can cause severe toxicity problem
(Kremer, Genovese, Cannon, Caldwell, Cush, Furst, Luggen, Keystone, Weisman, and
Bensen, 2002).
1.8.2. Biologics in the Treatment of RA
In 1975, the discovery of monoclonal antibodies by Kohler and Milstein (Köhler and Milstein,
1975) suggested a new field in the area of therapeutics. This development in the field of
medicine helped in the designing of new drugs with targeted biological effect for the relevant
pathology. Soon after its discovery, it entered the field of transplantation and oncology.
Biological therapies in RA first targeted T lymphocytes, but these treatments were found to
be either toxic (anti-CD52) or ineffective (anti-CD4) (Moreland, Pratt, Mayes, Postlethwaite,
Weisman, Schnitzer, Lightfoot, Calabrese, Zelinger, and Woody, 1995; Van Der Lubbe,
Dijkmans, Markusse, Nässander, and Breedveld, 1995; Isaacs, Manna, Rapson, Bulpitt,
Hazleman, Matteson, Clair, Schnitzer, and Johnston, 1996). In the meantime, TNF antagonists
were used for the first time in septic shock but it was also not successful ( Cohen and Carlet,
1996; Fisher Jr, Agosti, Opal, Lowry, Balk, Sadoff, Abraham, Schein, and Benjamin, 1996;
Reinhart, Wiegand-Lohnert, Grimminger, Kaul, Withington, Treacher, Eckart, Willatts,
Bouza, and Krausch, 1996; Clark, Plank, Connolly, Streat, Hill, Gupta, Monk, Shenkin, and
Hill, 1998). However, in vitro study done by Kennedy Institute in London suggested that TNF
antagonism would be effective for the RA patients treatment and they were successful in
convincing the company that developed first anti-TNF monoclonal antibodies for allowing
them to do trial (Brennan, Jackson, Chantry, Maini, and Feldmann, 1989). The trial ended up
with unexpected results and that led to the start of new era in the treatment strategy of RA
(Van Vollenhoven, 2015).
1.8.2.1. Overview of Current Biologic Therapies
Currently, nine biological agents have been approved for the RA treatment. Among these nine
agents, five are TNF antagonist and the rest have different mode of action. These biological
agents are summarized in table 1.1 (McInnes and Schett, 2011). Generally, they are called
DMARDs but they are differentiated from conventional DMARDs and referred as biological
20
DMARDs. The biologics especially anti-TNF agents have fast action. Some RA patients on
anti-TNF therapy often reports improvement in their condition on the day of treatment or
afterwards. Such results were only seen previously with the use of very high dose of
glucocorticoids (GCs) or after two to three months of therapy with methotrexate (MTX). The
biologics are relatively safe and well tolerated but the long-term use of these blocking agents
were developing concerns regarding serious infections and other malignancies as a potential
consequence of their use for such a long period. It became clear that long term use of anti-
TNF therapy leads to the reactivation of latent tuberculosis in RA patients and increase in the
risk of skin cancer was also reported (Van Vollenhoven, 2015).
The use of these biologic DMARDs has brought revolution in the RA management, but there
are concerns related to its toxic effects. Inhibition of TNF blocks the signaling of over
expressed protein in RA but in this process it also inhibits signaling protein that is important
for the normal immune response that leads to increased risk of bacterial and fungal infections
and other opportunistic pathogens (Saag, Teng, Patkar, Anuntiyo, Finney, Curtis, Paulus,
Mudano, Pisu, and Elkins‐ Melton, 2008). Etanercept and infliximab were the first two anti-
TNF agents that were used in clinical practice (Van Vollenhoven, 2015). After these two
adalimumab, cetrolizumab and golimumab were used (Doran, Crowson, Pond, O'Fallon, and
Gabriel, 2002). Most of the clinical trials with the TNF-blockers have shown risk of serious
infections (Ruderman, 2012).
1.8.3. Non-Pharmacological Treatments for RA
Beside the use of pharmacological treatments, non-pharmacological mode of treatment
strategies play significant role in the management of RA. During the entire period of RA
treatment, physiotherapy is suggested for medical rehabilitation of RA patients. The goal of
non-pharmacological interventions is to alleviate the patient’s sufferings, to control pain and
maintain general well-being (Stenström and Minor, 2003). Adjustments in daily life at the
work place and in the home can provide benefits to the patients by occupational therapy.
Nutritional improvement can also serve benefits; however, the practice is rather limited.
Gluten-free and vegan diet is found to provide some improvement to the patients in one small
randomized study (Hafström, Ringertz, Spångberg, Von Zweigbergk, Brannemark, Nylander,
21
Table 1.1. Approved biologic therapies of rheumatoid arthritis.
Name Class Target Structure
Adalimumab Cytokine inhibitor TNF-α Human monoclonal antibody
Certolizumab
pegol
Cytokine inhibitor TNF-α Pegylated humanized Fab
fragment of an anti–TNF-α
monoclonal antibody
Etanercept Cytokine inhibitor TNF-α TNF-α receptor–Fc fusion
Golimumab Cytokine inhibitor TNF-α Human monoclonal antibody
Infliximab Cytokine inhibitor TNF-α Chimeric monoclonal
antibody
Tocilizumab Cytokine inhibitor Interleukin-6
receptor
Humanized monoclonal
antibody
Anakinra Cytokine inhibitor Interleukin-1 Interleukin-1 receptor
antagonist
Rituximab Cell-depleting agent CD20 Chimeric monoclonal
antibody
Abatacept Cell-depleting agent CD80 and
CD86
CTLA4–Ig fusion protein
22
Rönnelid, Laasonen, and Klareskog, 2001). In another study, the consumption of
‘Mediterranean diet’ showed considerable benefits to patients (Sköldstam, Hagfors, and
Johansson, 2003) and was generally acceptable for the long duration. Significant
cardiovascular health benefits were also observed. At some point during the disease period,
most patients require psychosocial support that can be provided by a psychologist or social
worker, support groups organized through patient associations’ clinics and hospitals. The
rheumatologist can also play the role of a psychologist to provide emotional support to the
patient (Van Vollenhoven, 2015).
1.9. Collagen Induced Arthritis (CIA) Rat Model
One of the most extensively studied animal models of RA is collagen induced arthritis (CIA).
The important feature of CIA model is that it mimics human arthritis with both immunological
and pathological features. CIA is basically an autoimmune disease affecting joints and require
immune function of both T and B cell to autologous type II collagen (CII) (Andersson and
Holmdahl, 1990). RA is not limited to the joint but also involves multiple tissues and complex
pathways. Therefore, it is important to choose an animal model that has similarity to the
human condition of RA so that it can be translatable to the human studies (McNamee,
Williams, and Seed, 2015). Collagen can be obtained from various sources including chick,
bovine, human and porcine. The overall response varies considerably with the strain and
conditions of injection (Andersson and Holmdahl, 1990). In a model of CIA, collagen is
responsible for the induction of immune response. It is useful for determining the efficacy of
drug with anti-arthritic potential in a controlled manner. This kind of model is also helpful for
exploring the role of specific genes involved in RA, through the use of knockout mice
(Vincent, Williams, Maciewicz, Silman, Garside, and Group, 2012). Strains of mice and rats
that are susceptible for CIA development are used for induction of immunity with type II
collagen emulsified either in Freund's complete adjuvant (CFA) or Freund's incomplete
adjuvant (IFA) (Trentham, 1982; Holmdahl, Andersson, Goldschmidt, Jansson, Karlsson,
Malmstrom, and Mo, 1989).
The histology of CIA bone and cartilage resembles that of RA in terms of bone and cartilage
erosion and infiltrating cells in synovial tissue (Nabozny, Bull, Hanson, Griffiths, Luthra, and
23
David, 1994). Chronic pain is a noticeable feature associated with RA and providing pain
relief to RA patient is an important task. Animal models of arthritis are also used to assess
pain and for the investigation of action of analgesics. When evoked pain response is tested,
onset of CIA is characterized by increased sensitivity to mechanical and thermal stimuli,
making it a suitable model to test analgesics (Inglis, Notley, Essex, Wilson, Feldmann, Anand,
and Williams, 2007).
1.10. Compounds Used in the Current Study
In the present study, we have used N-(2-hydroxyphenyl) acetamide (NA-2), rutin and its gold
nanoparticles.
1.10.1. N-(2-Hydroxyphenyl) Acetamide (NA-2)
NA-2 is a derivative of acetaminophen (2-Acetamidophenol), which has been known as
antipyretic and analgesic agent. Acetaminophen has been used widely as an alternative to
aspirin because of less side effects but it has been reported to have nephrotoxic and
hepatotoxic effects due to conversion of drug by cytochrome P-450 dependent oxidase system
into reactive intermediate (Potter, Davis, Mitchell, Jollow, Gillette, and Brodie, 1973;
Mohandas, Duggin, Horvath, and Tiller, 1981; Patten, Thomas, Guy, Lee, Gonzalez,
Guengerich, and Yang, 1993). Classical NSAIDs have been used since long for the treatment
of RA but they only provide symptomatic relief and they also don’t protect the joint
destruction process (Santana-Sabagun and Weisman, 2001). Therefore, drugs with minimal
side effects over long term use and targeted mode of action are required. NA-2 is available
commercially and commonly known as 2-hydroxyacetamide, 2-acetamidophenol, O-
acetaminophenol, 2-acetylaminophenol and O-hydroxyacetanilide. After extensive literature
survey, we were not able to find much work on this compound, however in 2005, study by
Saeed and Saeed suggested the use of NA-2 as anti-platelet aggregating and anti-inflammatory
agent and they also reported that NA-2 is less toxic in comparison to aspirin or paracetamol
(Saeed and Saeed, 1975). NA-2 has been reported as potent anti-inflammatory and anti-
arthritic agent by our research group (Perveen, Hanif, Jawed, Jamall, and Simjee, 2014; Jawed,
Shah, Jamall, and Simjee, 2010; Jawed, Jamall, Shah, Perveen, Hanif, and Simjee, 2014) but
the possible mechanism behind its anti-arthritic potential is not clear yet. In the current study,
24
we aim to investigate the mechanism of NA-2 anti-arthritic activity with specific emphasis
on its role in regulation of RANK-RANKL pathway.
1.10.2. Rutin
Rutin belongs to flavonoids group of compounds. Flavonoids are present in almost all foods
of plant origin (Hollman and Katan, 1997) and have shown numerous biological activities e.g.
anti-hypertensive, anti-oxidant, anti-platelet, anti-inflammatory, anti-neoplastic anti-
microbial, anti-viral etc. in both in vitro and in vivo models (Formica and Regelson, 1995). In
inflammatory animal models flavonoids have shown potent anti-inflammatory response (Di
Carlo, Mascolo, Izzo, and Capasso, 1999; Guardia, Rotelli, Juarez, and Pelzer, 2001).
Likewise, rutin has also shown potent therapeutic effects like anti-inflammatory, anti-oxidant,
anti-carcinogenic in various animal studies (La Casa, Villegas, De La Lastra, Motilva, and
Calero, 2000; Kamalakkannan and Prince, 2006; Bishnoi, Chopra, and Kulkarni, 2007; Khan,
Ahmad, Ishrat, Khuwaja, Srivastawa, Khan, Raza, Javed, Vaibhav, and Khan, 2009). In the
treatment of diseases like inflammatory bowel disease, osteoarthritis and rheumatoid arthritis,
the potential therapeutic role of rutin has been supported by various animal and human studies.
Study by Ostrakhovitch and Afanas’ev showed effective inhibitory effect of rutin on oxygen
radical overproduction in RA patients (Ostrakhovitch and Afanas’ev, 2001; Kauss, Moynet,
Rambert, Al-Kharrat, Brajot, Thiolat, Ennemany, Fawaz, and Mossalayi, 2008).
1.10.3. Rutin Gold Nanoparticles (Rutin-GNPs)
Nanoparticles are 1-100 nm size particles. There is recent interest in the use of these
nanoparticles in the treatment and diagnosis of different diseases, since the size of these
nanoparticles is similar to the biological molecules present in the human system like
nucleic acid and proteins and also similar to the virus structure (Wiesenthal, Hunter,
Wang, Wickliffe, and Wilkerson, 2011). In medicine, one of the major advantages of using
engineered nanoparticles is the targeted delivery to specific site of action. Drugs like
celecoxib, ibuprofen, and diclofenac have been investigated for osteoarthritis and have
shown significantly reduced level of inflammatory cytokines TNF-α and IL-6 in the RA
patients synovial fluid and higher doses of these drugs showed more promising anti-
inflammatory response in RA patients thus improving the quality of life (Gallelli, Galasso,
25
Falcone, Southworth, Greco, Ventura, Romualdi, Corigliano, Terracciano, and Savino,
2013). Therefore, these drugs can be used in the form of nanoparticles. The nanodrugs
when given to the patients, release slowly towards their target site and low doses of
these nanodrugs are enough to produce its effect, therefore its use can prevent the
harmful side effects associated with high doses (Radad, Al-Shraim, Moldzio, and Rausch,
2012). These nanoparticles are also suitable for those drugs that are not soluble in water
and they can be designed to specifically inhibit or enhance immune response and even
avoid recognition by immune system (Cooper, 2010; Zolnik, Gonzalez-Fernandez,
Sadrieh, and Dobrovolskaia, 2010). Recent advances in technology has enabled
researchers to use these engineered nanoparticles for imaging, diagnosis of disease,
targeted delivery of drug and treatment at the same time (Sanvicens and Marco, 2008).
Limited research has been done in the area of targeted drug delivery in RA. Few examples
include the targeted MTX delivery using poly (lactic-co-glycolic acid) nanoparticles with CD-
64 antibody in vitro in RAW 264.7 cells (Das Neves, 2014). In comparison to free drug, the
MTX nanoparticles were found to be more effective (Zhou, Yan, Hu, Springer, Yang,
Wickline, Pan, Lanza, and Pham, 2014). In one study, IL-6 monoclonal antibody has been
investigated in CIA mice in which the antibody was targeted with tocilizumab loaded
hyaluronate gold nanoparticles (Lee, Lee, Bhang, Kim, Kim, Ju, Kim, and Hahn, 2014). It
was also reported that in CIA mice, small dose of MTX nanoparticles were far better in
therapeutic response than the high doses (Scheinman, Trivedi, Vermillion, and Kompella,
2011). Therefore, we aim to investigate the role of rutin-GNPs (rutin-GNPs) on RA
progression and the possible mechanism of action.
26
1.11. Objectives
The current treatment strategies for RA involve lifelong treatment with serious side effects.
There is unmet need for the development of therapeutic agent that suppresses and cures the
disease process without exerting toxic effects. In the light of aforementioned role of pro-
inflammatory cytokines and RANKL/RANK pathway in arthritis, we planned to investigate:
1. The effect of NA-2, rutin, indomethacin + rutin and rutin-GNPs on the development of
arthritis in CIA rats.
2. The effect of these compounds on pro-inflammatory and oxidative stress markers i.e. IL-
1β, TNF-α, NO, PO and GSH in serum samples.
3. The role of these compounds in regulation of RANK/RANKL signaling cascade by
determining change in gene expression of RANKL, RANK and its downstream mediators
i.e. NF-κB, c-fos, c-jun and Akt by quantitative RT-PCR.
4. The change in protein expression of c-Fos, pAkt and iNOS in spleen tissue by
immunohistochemistry.
27
CHAPTER 2
METHODOLOGY
28
2.1. Animal Care
Female Wistar rats 160-200 gms were housed in the animal resource facility under pathogen
free conditions at controlled temperature (21ºC ± 2ºC) and humidity (55ºC ± 5ºC) and light-
dark cycle of 12:12 hour with free access of standard diet and drinking water. The ethical
guidelines set by the International Center for Chemical and Biological Sciences (ICCBS),
Scientific Advisory Committee on Animal Care, Use, and Standards, were followed.
2.2. Chemicals
Bovine type II Collagen (lyophilized) was purchased from Chondrex (Redmond, WA, USA).
Freund’s complete adjuvant, NA-2 and indomethacin were purchased from Sigma (St. Louis,
MO, USA). The Freund’s incomplete adjuvant was purchased from MP Biochemicals
(Aurora, OH, USA). Rutin and its gold nanoparticles were provided by our collaborators
(ICCBS, University of Karachi, Pakistan).
2.3. Collagen Suspension Preparation
Lyophilized bovine type II collagen (CII) was mixed with Freund’s complete adjuvant (CFA)
in 1:1 ratio to make final concentration of 2 mg/mL for initial dose and incomplete Freund’s
adjuvant (IFA) for booster dose.
2.4. Induction of Arthritis
A volume of 0.2 ml (0.2 mg) collagen CII suspension was injected subcutaneously (s.c.) at
the tail base. On the seventh day after injection of CFA-CII, 0.1 ml of suspension containing
CII-IFA was re-injected at the same site. Animals were kept under observation until symptoms
of arthritis were prominent. Compounds treatment was started on the day of arthritis induction.
2.5. Treatment Strategy
Animals were divided into groups as outlined in table 2.1. Indomethacin was used as the
reference drug. Doses of NA-2, Rutin and its gold nanoparticles were selected after
preliminary dose finding studies on acute carrageenan-induced inflammatory rat model.
29
Table 2.1. Treatment regime used for the testing of compounds for anti-arthritic activity
in collagen induced arthritis model in rats.
Groups Doses Route of administration
Normal Control - -
Vehicle Control 0.5 mL/animal Intraperitoneal
Gold Nanoparticles only 0.5 mL/animal Intraperitoneal
NA-2 only 5 mg/kg Intraperitoneal
Rutin only 50 mg/kg Intraperitoneal
Arthritic Control - -
Arthritic Vehicle 0.5 mL/animal Intraperitoneal
Arthritic + Gold Nanoparticles 0.5 mL/animal Intraperitoneal
Indomethacin + Arthritis 5 mg/kg Intraperitoneal
NA-2 + Arthritis 5 mg/kg Intraperitoneal
Rutin-25 + Arthritis 25 mg/kg Intraperitoneal
Rutin-50 + Arthritis 50 mg/kg Intraperitoneal
Indomethacin + Rutin + Arthritis Indomethacin 2 mg/kg
and Rutin 25 mg/kg
Intraperitoneal
Rutin-GNPs +Arthritis 12.5mg/kg Intraperitoneal
30
2.6. Clinical Assessment of Arthritis
During the course of in-vivo experiment, animals were observed for changes in body weight,
paw oedema and arthritic score.
2.6.1. Arthritic Score
Animals were kept under careful observation for monitoring the symptoms of arthritis. All the
immunized rats developed CIA, apparent by inflamed paw, after approximately 18-22 days.
The severity of arthritis in each paw was scored on a scale of 0-4 as shown in table 2.2
(Bakharevski, Stein-Oakley, Thomson and Ryan, 1998). The arthritic index was calculated as
a summated index derived for each rat yielding a maximum score of 16/ animal.
2.6.2. Hind Paw Volume Measurement
The severity of arthritis was analyzed by measurement of change in paw volume using
Plethysmometer (model 7140, Ugo Basile, Italy) on alternate days starting from day 0 till the
termination of the experiment. It considers three dimensional changes in the pattern of
swelling of individual limbs in comparison to diameter measurements of tibiotarsal joints.
2.6.3. Body Weight Measurement
Before the induction of arthritis (day 0) till the termination of the study (day 22), change in
body weight of all the animals was measured every alternate day by limiting its movement in
a restrainer of an appropriate size to determine the progression of disease. The average change
in body weight was determined by subtracting the individual weight recording on alternate
days from weight of day 0.
31
Table 2.2. Scoring system used to assess severity of paw inflammation.
Score Condition
0 Normal, no signs of arthritis
1 swelling or redness of the paw or 1 digit
2 two joints involved
3 more than two joints involved
4 severe arthritis of the entire paw and all digits
32
2.7. Histopathological Examination
On day 22, histological examination of specimens from the knee joints was done. The steps
involve in histology are mentioned below:
2.7.1. Fixation
The joint samples were immersed in 10% neutral buffered formalin overnight for fixation.
2.7.2. Decalcification
After fixation, the joints were immersed in decalcifying solution for almost four weeks in 10%
EDTA solution and then re-immersed in fixative to stop further decalcification.
2.7.3. Processing
Tissue processing involves multiple steps that are mentioned below:
2.7.3.1. Dehydration
Dehydration involves use of increasing strength of dehydrating agent (alcohol) that causes
slow removal of water thus causing minimal damage to the tissue. The joint samples were
immersed step by step in each solution for one hour at 42ºC starting from 70% alcohol, 90%
and finally the samples were kept in 100% alcohol for 60 min (three times). The samples were
then kept in 100% alcohol overnight at 42ºC.
2.7.3.2. Embedding
After dehydration the samples were immersed in melted paraffin at 66ºC in oven and paraffin
was changed three times and finally the samples were kept overnight in paraffin in the oven
at 66ºC. Next day the samples were taken out of the paraffin and transferred to the tissue
embedding mold and then melted paraffin was poured on the mold and the cassette was put
on top of the mold and allowed to cool down on cooling pad of the tissue embedding system
and finally after cooling and setting of the paraffin, the sample was taken out of the mold and
cassette was removed and kept at room temperature.
33
2.7.3.3. Microtomy
The tissue sections were cut with 4 μm thickness using microtome. The sections were then
transferred into hot water bath (45ºC), and then taken on the slides and kept on hot plate at
45ºC overnight for drying.
2.7.3.4. Staining
The slides containing tissue sections were immersed in xylene for 1-2 minute and then
transferred to decreasing % of alcohol i.e., from 100%, 90% and 70% for 2 minutes each and
finally in water. The sections were then stained with hematoxylin for 1 minute then washed
with water and then stained with eosin for 1 minute and then again washed with water and
dehydrated with increasing concentration of alcohol (70%, 90%, 100%) and slides were
mounted and bone sections were then observed under microscope (CK X41, Olympus, Tokyo,
Japan). Images were captured and visualized using image analysis software (NIS element AR
3.0).
2.8. Inflammatory Markers Analysis
2.8.1. Reactive Oxygen Species (ROS) and Antioxidant Measurement
Reactive oxygen species, nitric oxide (NO), peroxide (PO) and antioxidant glutathione (GSH)
levels in serum samples of arthritic, normal and treatment groups were determined
colorimetrically by specific assay kits.
2.8.1.1 Nitric Oxide Determination (NO)
NO was measured using Quantichrome Nitric Oxide assay kit (BioAssay Systems, Hayward,
USA) by following the manufacturer’s protocol. The kit measures NO by reduction of nitrate
to nitrite. Briefly, the standard dilutions were prepared from 0-50 μM concentration. The
deproteination of serum samples was done by addition of 75 mM ZnSO4 and centrifuging at
14000 rpm for 5 mins. Supernatant was further treated with 55 mM NaOH and then glycine
buffer was added and incubation was done at room temperature for 15 mins with activated
cadmium granules (3 granules/sample) with intermittent shaking. Finally, test samples (100
μL) and standards were added into the 96-wells plate in triplicate. Reagent A and reagent B
34
(50 μL/each) were added in each well and incubated for 5 mins at room temperature and then
absorbance was taken at 540 nm. The NO concentration was measured using standard curve.
Where n=dilution factor.
2.8.1.2. Peroxide Determination (PO)
Peroxide production was measured by QuantichromeTM peroxide assay kit (BioAssay systems,
Hayward, USA). Briefly, standard dilutions from 0-100 μM concentration were prepared.
Standards and blank (20 μL each) in duplicate and samples in quadruplicate were transferred
to 96-well plates. Working reagent A (WRa; 100 μL) was added to the standard wells, blank
and first two wells of each sample and in the last two wells, working solution B (WRb) was
added and then the plate was incubated for 30 mins at room temperature and OD was taken at
585 nm (ELISA reader, Spectra Max 340). The sample OD was compared with calibration
curve.
2.8.1.3. Glutathione Quantification (GSH)
Serum concentration of GSH in samples of arthritic, normal and test groups was measured by
5, 5’-dithiobis 2-nitrobenzoic acid (DNTB) method using QuantichromeTM Glutathione assay
kit (Bioassay Systems, Hayward, USA). Briefly, serum sample and reagent A (120 μL each)
were mixed together and centrifuged for 2 minutes at 14,000 rpm. Clear supernatant in a
volume of 200 µL and 100 µL of reagent B were added in duplicate into 96-wells plate. In the
blank wells 100 µL of distilled water and 100 µL of GSH standard (100 μM) were added into
the respective wells of standard. Final volume of 300 µL in each well was adjusted with
distilled water followed by incubation at room temperature for 25 mins. The OD was taken at
412 nm (ELISA reader, Spectra Max 340).
The concentration of GSH was calculated by standard curve as follows:
Concentration of NO (μM) = OD Sample – OD Blank x n
Slope
Conc of GSH (μM) = OD Sample – OD Blank 100 n
OD Standard – OD Blank
35
2.9. Pro-Inflammatory Cytokines IL-1β and TNF-α Analysis
The Quantification of IL-1β and TNF-α was performed with commercially available ELISA
kits and all the steps were performed following manufacturer’s guidelines. The concentration
of cytokines was determined by plotting the standard curve on MicroCal origin program
(Micro Cal Inc. USA).
2.9.1. IL-1β Measurement
All the standard dilutions, samples and other reagents were prepared according to kit manual
(R&D systems, Minneapolis, USA). Briefly, the pre-coated microplate was used and assay
diluent (50 µL) was added to each well of microplate and then sample, control and standard
were added (50 µL each) into the plate. The plate was mixed gently followed by incubation at
room temperature for 2 hours. The samples were then aspirated and washed five times with
the wash buffer (400 µL/well) and then rat IL-1β conjugate (100 µL) was added to all the
wells and the plate was sealed and incubated for 2 hours at room temperature. 100 μL substrate
solution was added in all wells after plate washing followed by incubation for 30 mins in dark.
At the end, 100 µL stop solution was added. OD was taken at 450 nm.
2.9.2. TNF-α Measurement
The preparation of the reagents and samples was done following the manufacturer’s
instructions (Thermo Scientific, Waltham, USA). Briefly 50 µL of pretreatment buffer was
added to pre-coated 96 wells microplate. Serum samples and standards (50 µL each) were
added to each well in duplicate followed by incubation at room temperature for 1 hour. The
wells were washed with wash buffer three times with squirt bottle then biotinylated antibody
reagent was added to all the wells and plate was sealed and incubated for further 1 hour. The
plate was rewashed thrice and then Streptavidin-HRP reagent (100 µL) was added to each
well followed by incubation at room temperature for 30 minutes. Following incubation, the
plate was washed and then TMB substrate solution (100 µL) was added to all the wells and
incubated in dark at room temperature for 10 minutes. Finally, 100 µL stop solution was added
and OD was taken at 450nm.
36
2.10. Real time Reverse Transcriptase Polymerase Chain
Reaction (Real Time RT-PCR)
At the end of each experiment, spleen tissues were extracted from all the rats and stored at -
80oC for gene expression studies.
2.10.1. RNA Isolation and Quantification
The total RNA isolation from spleen tissue was done by Trizol RNA extraction method, a
modified method originally developed in 1987 and proved to be reliable method for RNA
isolation form tissues (Chomczynski & Sacchi, 1987). The frozen tissues (10-20 mg) were
placed in clean DNase/RNase free tubes containing Trizol (1 mL) (Invitrogen, CA, USA) and
homogenized using Ultra-Turrax homogenizer (Ultra-Turrax T8; IKA, Germany). The tubes
were incubated at room temperature for 5 mins and chloroform (200 μL) was added to all the
tubes and after shaking incubated for 3 mins at room temperature and then tubes were
centrifuged at 4ºC for 15 minutes at 12000 ×g. After centrifugation, total RNA present in the
upper aqueous phase was carefully shifted to a new tube and 500 μL isopropanol was added
into the tube. The tubes were incubated for 10 minutes followed by centrifugation at 4oC at
12,000 ×g for 10 mins. The supernatant was discarded and RNA pellets were mixed with 1
ml ethanol (75%) and centrifuged at 7500 ×g for 5 mins at 4oC. Supernatant was discarded
and pellet was air dried for 10 mins. Finally, the pellet was re-suspended in RNase free water
and quantification and purity of RNA was analyzed using nanodrop 2000 UV-Visible
spectrophotometer (Thermo Scientific Inc.) and then stored at -70ºC for further use.
2.10.2 The cDNA Synthesis
The cDNA was synthesized by reverse transcription of total RNA using Revert Aid TM First
strand cDNA Synthesis Kit (Thermo Scientific, Waltham, USA). The cDNA synthesis was
done using manufacturer’s protocol. Briefly, 1μg of RNA was used to make cDNA. The RNA
and primer mixture was made by adding the components in 0.5 mL tube in amounts shown in
the table 2.3. The mixture was incubated for 5 mins at 65ºC. The cDNA synthesis mix (Table
2.4) was added to the RNA/Primer mixture. The mixture (random hexamer primer) was then
37
incubated for 5 mins at 25ºC and then for 60 mins at 42ºC. The reaction was stopped by
incubation at 70ºC for 5 mins and the cDNA was stored at -70ºC.
2.10.3. Real time Reverse Transcriptase-PCR Amplification of cDNA
The cDNA was then amplified according to manufacturer’s protocol using Maxima SYBR
green master mix (2X) (Table 2.5). Briefly, 2 μL of diluted cDNA (1:10) was used as a
template for real time PCR. Negative control without reverse transcriptase enzyme was used
to confirm the absence of genomic DNA contamination. GAPDH (glyceraldehyde-3-
phosphate dehydrogenase) gene was used as a reference housekeeping gene. The final reaction
mixture was placed in preheated real time PCR and the reaction details are mentioned in table
2.6. The list of primer sequences for the corresponding genes, primer concentration and
annealing temperature is mentioned in table 2.7.
2.11. Immunohistochemistry for c-Fos, iNOS and pAkt in Spleen
Tissue
The antibodies used for immunohistochemical analysis of c-Fos, iNOS and pAkt, in Spleen
tissues are given in table 2.8. After the development of full blown arthritis, the animals were
sacrificed. The spleen tissues were perfused with chilled PBS and fixed with 10% formalin
for 24 hrs. The samples were then dehydrated with increasing concentration of alcohol and
embedded in paraffin and tissue sectioning was done using microtome and the sections were
transferred to properly labeled poly-L-lysine coated slides. For immunostaining, the labeled
slides were rehydrated by transferring the slides step by step in xylene 100% (20-30 mins) →
100% alcohol (10 mins) → 90% alcohol (5 mins) → 70% alcohol (10 mins) → H2O (10 mins).
After that PBS was poured on top of sections and the slides were incubated at 42ºC in oven
for 15 mins in humid chamber. After incubation, the tissues were soaked with Roti-
immunoblock working solution (Carl Roth, Karlsruhe, Germany) and the slides were
incubated in oven at 42ºC for 30 mins. After blocking the tissue sections were incubated with
primary antibodies (1:100 dilution) of respective markers using blocking solution and
incubated at 42ºC for 1 hour. After incubation, the slides were washed with PBS (three times)
followed by incubation with secondary antibody (1:200 dilutions) at 42ºC for 45 mins-1 hour.
38
Table 2.3. Composition of RNA/Primer mixture.
Component Volume
Total RNA (1 μg) 2 μL
Random Hexamer Primer 1 μL
Nuclease Free Water 9 μL
Total 12 μL
Table 2.4. Composition of cDNA synthesis mixture.
Component Volume
5X Reaction Buffer 4 μL
Ribolock RNase Inhibitor (20U/μL) 1 μL
10mM dNTP Mix 2 μL
Revert Aid M-MuLV RT (200U/μL) 1 μL
39
Table 2.5. Real time RT- PCR reaction mixture recipe.
Component Volume (μL)
Maxima SYBR Green (2X) 12.5 μL
Forward primer (10μM) 0.5 μL (0.5 μM)
Reverse Primer (10μM) 0.5 μL (0.5 μM)
Template DNA 2 μL
Nuclease free water to 25 μL
Total 25 μL
Table 2.6. Steps involved in real time RT-PCR reaction.
Step Temperature (ºC) Time No. of Cycles
Initial denaturation 95 2 mins 1
Denaturation 95 15 secs
Annealing 60 30 secs 40
Extension 72 30 secs
40
Table 2.7. Primer sequences, annealing temperature and expected product size.
Genes Accession.
No
Primer Sequence
(Forward/Reverse Primer)
Annealing
Temperature
(Tm)
Product
Size (bp)
RANK NM_001
271235.1
5’-A A C T C C A A C T C A A C G G A T G G-3’ 59.97 ºC 124
5’-T G G G A A G G C C T A T G C T G T A G-3’ 60.23 ºC
RANKL NM_057
149.1
5’-A C A G C G C T T C T C A G G A G T T C-3’ 59.75 ºC 136
5’-G A C T T T A T G G G A A C C C G A T G-3’ 59.24 ºC
c-Fos NM_022197.2
5’-C C T G C C T C T T C T C A A T G A C C-3’ 59.80 ºC 111
5’-G A T G C C G G A A A C A A G A A G T C-3’ 59.68 ºC
c-Jun NM_021
835.3
5’-C C G G A G A A G A A G C T C A C A A G-3’ 60.13 ºC 111
5’-C T C T G G G T C A G G A A A G T T G C-3’ 59.84 ºC
NF-κB
p65
NM_199
267.2
5’-G A A C T T G T G G G G A A G G A C T G-3’ 58.10 ºC 128
5’-C T A A T G G C T T G C T C C A G G T C-3’ 58.33 ºC
Akt1 NM_033
230.2
5’-A C T C A T T C C A G A C C C A C G A C-3’ 59.97 ºC 169
5’-A C C A C G T T C T T C T C G G A G T G-3’ 60.30 ºC
GAPDH NM_017
008.4
5’-A T G A C T C T A C C C A C G G C A A G-3’ 60.13 ºC 136
5’-T A C T C A G C A C C A G C A T C A C C-3’ 59.86 ºC
41
The slides were washed and stained with DAPI (1:10000 dilutions in PBS) and incubated for
10 mins at room temperature for nuclear staining. Finally, the slides were washed and mounted
with mounting media and protected from light. Negative controls were also processed by
simply elimination of the primary antibody from the procedure in order to determine the
specificity of primary antibody. Upright multichannel fluorescent microscope (Nikon Eclipse
90i, Nikon Corporation, Japan) was used for the visualization of sections and pictures were
taken using camera (Nikon, DXM1200C) and the images were analyzed using image analysis
software (NIS Element AR 3.0).
2.11.1. Image Analysis
The analysis of images was done using ImageJ software (National Institutes of Health, USA).
The Data was obtained from three sections per rat and presented as means ± S.E.M.
2.12. Statistical Analysis
The SPSS version 22 was used for the analysis of data. All the data was analysed using one-
way ANOVA and Bonferroni’s Post Hoc test was used to determine the mean differences
between different groups. The values equal or less than the level of P < 0.05 were considered
statistically significant.
42
Table 2.8. List of antibodies for immunohistochemistry.
Markers Primary Ab Company Secondary Ab Company
c-Fos c-Fos Rabbit
Polyclonal IgG
(sc-52)
Santa Cruz
Biotechnology
Cy3 goat antibody
IgG (H+L)
(A10520)
Invitrogen
pAkt Affinity Purified
Rabbit IgG
(AF887)
Invitrogen Cy3 goat antibody
IgG (H+L)
(A10520)
Invitrogen
iNOS iNOS Rabbit
polyclonal
(Ab15323)
Abcam Cy3 goat antibody
IgG (H+L)
(A10520)
Invitrogen
43
CHAPTER 3
RESULTS
44
3.1. Clinical Parameters of Collagen Induced Arthritis
Animals in different treatment groups were carefully monitored on alternate days for severity
of arthritis using standard arthritic scoring criteria (Table 2.2), change in body weight and paw
volume. The effects of test compounds on clinical signs of arthritis are discussed in the
subsequent sections.
3.1.1. Effect of NA-2 on Clinical Parameters
3.1.1.1. Arthritic Score
The severity of arthritis was measured by analysing the mean arthritis severity score. Which
was determined by adding the score of individual paws of each rat. The animals in all the
groups showed no change in the mean arthritic score till day 10 (Fig. 3.1). However, from day
10 onward, inflammation was observed in arthritic control and NA-2 treated animals either in
one or both hind paws. In contrast, the reference drug indomethacin did not show any sign of
inflammation till day 12. On day 14, a significantly increased mean arthritic score (p < 0.0001)
was found in the untreated arthritic group compared to the non-arthritic animals and this
increase was persistent till the end of the study (day 22).
Next, we compared the NA-2 treated animals with the untreated arthritic group and observed
that it was able to reduce the arthritic score from day 14. This reduction in arthritic score of
the treated group was observed till the end of the study (p < 0.0001). The post hoc test revealed
a significant reduction in the mean arthritic score of indomethacin treated animals (p < 0.05),
on day 14, in comparison to NA-2 treatment, however this difference was not observed from
day 16 onwards.
Since the compound NA-2 was soluble in 5% DMSO, therefore we also set a vehicle control
group for arthritic and non-arthritic animals. The statistical analysis of the data gathered from
this control group did not reveal any significant difference from normal control or arthritic
control groups.
45
3.1.1.2. Body Weight
The mean change in the body weight of the animals in various groups is shown in figure 3.2.
A gradual increase in the body weights of the animals in all the groups was observed until day
8. However, on day 10 onwards, the arthritic control animals demonstrated decrease in their
mean change in body weight and from day 12 onwards, this decrease was significant (p <
0.05) compared to the non-arthritic group. Both NA-2 (5 mg/kg) and indomethacin (5 mg/kg)
treatment groups showed increase in body weight from day18 onwards but compared to
untreated arthritic group, only indomethacin exhibited significant increase (p < 0.05) in mean
change in body weight on day 22 but NA-2 group remain statistically non-significant. The
Bonferroni’s post hoc test showed non-significant difference within the non-arthritic control
groups, arthritic control groups and between NA-2 and indomethacin treatment groups.
3.1.1.3. Paw Oedema
The change in the volume of both right and left paw was determined on day 0 till day 22 (Fig.
3.3). The non-arthritic control group exhibited only slight change in paw volume till the end
of the study because of increase in body weight but this increment was non-significant. In
comparison to the normal control animals, the untreated arthritic animals revealed significant
increase in both right and left paw volume (p < 0.0001 and p < 0.05 for right and left paw)
from day 12 and remain significant till the end of the study (p < 0.0001). From day 12
onwards, in comparison to the arthritic control group, indomethacin group started to show
significant reduction in both right paw (p < 0.0001) and left paw volume (p < 0.05) and this
difference remain significant till the end of the study (p < 0.0001). Similarly, from day 12,
NA-2 treatment group showed significant decrease in the right paw volume (p < 0.001) and
from day 14 in the left paw volume (p < 0.05). This mean change in paw volume of both right
and left paw remains significant in comparison to the arthritic control rats till day 22 (p <
0.0001). The statistical analysis demonstrated non-significant difference between the
treatment groups.
46
Figure 3.1. Effect of NA-2 on mean arthritic severity score in rats induced with
collagen induced arthritis. All values are shown as mean ± S.E.M. (12 animals/group).
The untreated arthritic animals demonstrated significant increase in the mean arthritic
score from day 14 onwards (#p < 0.0001) as compared to the normal control rats.
However, in comparison to the arthritic control rats, NA-2 and indomethacin treated
animals exhibited significant decrease in the mean score from day 14 (*p < 0.0001).
Figure 3.2. Effect of NA-2 on mean change in body weight. The values are mean ±
S.E.M. post induction of arthritis (12 animals/group). The arthritic control rats exhibited
significant reduction in body weight compared to the normal control animals from day
12 onwards (#p < 0.05). Whereas, on day 22, the control drug indomethacin treated
animals demonstrated significant increase in body weight compared to the arthritic
control animals (*p < 0.05).
#
#
* *
**
0
2
4
6
8
10
12
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
Mea
n
Art
hri
tic
Sco
re
Days after Immunization
Normal Control Vehicle Control
NA-2 Only Arthritic Control
Arthritic + Vehicle Arthritic + Indomethacin (5 mg/kg)
Arthritic + NA-2 (5 mg/kg)
#
#
*
-20
-15
-10
-5
0
5
10
15
20
25
30
35
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
Mea
n C
han
ge
in B
od
y W
eig
ht
(gm
s)
Days after Immunization
Normal Control Vehicle Control
NA-2 Only Arthritic Control
Arthritic + Vehicle Arthritic + Indomethacin (5 mg/kg)
Arthritic + NA-2 (5 mg/kg)
47
Figure 3.3. Effect of NA-2 on mean change in hind paw volume after arthritis
induction. Each value represents mean ± S.E.M. (12 animals/group). Compared to the
normal control animals, the untreated arthritic control animals exhibited significant
increase in both right (#p < 0.0001) and left paw volume (#p < 0.05) from day 12 onwards
and remain significant till day 22 (#p < 0.0001 for right paw and ## p < 0.0001 for left paw).
From day 12 onwards, compared to the arthritic control, indomethacin (5mg/kg) treated
group showed significant decrease in both right paw volume (***p < 0.0001) and left paw
volume (*p < 0.05) and remain significantly less till day 22 (***p < 0.0001). In contrast
NA-2 group showed significant reduction in right paw volume on day 12 (**p < 0.001,)
and from day 14 left paw volume (*p < 0.05) and the significance persist till day 22 for
both right and left paw compared to the arthritic control group (***p < 0.0001).
#
#
*** ***** ***
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
Ch
an
ge
in
Rig
ht
Paw
Volu
me
(mL
)
Days after Immunization
Normal Control Vehicle Control
NA-2 Only Arthritic Control
Arthritic + Vehicle Arthritic + Indomethacin (5 mg/kg)
Arthritic + NA-2 (5 mg/kg)
#
##
* ***
* ***
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
Ch
an
ge
in L
eft
Paw
Volu
me
(mL
)
Days after Immunization
Normal Control Vehicle Control
NA-2 Only Arthritic Control
Arthritic + Vehicle Arthritic + Indomethacin (5 mg/kg)
Arthritic + NA-2 (5 mg/kg)
B
A
48
3.1.2. Effect of Rutin on Clinical Parameters
3.1.2.1. Arthritic Score
Figure 3.4 demonstrates the mean arthritic severity score of non-arthritic and arthritic animals.
All the animals look normal with no prominent change in mean arthritic severity score till day
10. On day 12, compared to the normal control rats, the untreated arthritic animals exhibited
increase in score that was significantly higher on day 14 onwards (p < 0.0001). From day 14
onwards, in comparison to the untreated arthritic control animals, the score of indomethacin
(5 mg/kg) treatment group exhibited marked reduction (p < 0.0001). Treatment of arthritic
rats with rutin (25 mg/kg) showed gradual increase in the arthritic score from day 12 till day
20, and on day 22 the score decreased slightly in comparison to the untreated arthritic animals.
A gradual increase in arthritic score of the rutin (25 mg/kg) treated arthritic group was lower
than the arthritic control animals and on day 14, it was significantly less than the arthritic
control rats (p < 0.01). On day 14 onwards, arthritic score of Indomethacin + rutin (2 mg/kg
+ 25 mg/kg) treated groups demonstrated a significantly reduced mean arthritic score (p <
0.0001). The arthritic animals treated with rutin (50 mg/kg) also demonstrated a significant
decrease in the arthritic score from day 16 (p < 0.05) and the score continue to decrease
significantly till day 22 (p < 0.001). Next the statistical analysis was done to determine the
differences within the treatment groups and it showed that in comparison to rutin (25 mg/kg),
from day 14 onwards, indomethacin treatment group (p < 0.0001) and day 18 onwards
indomethacin + rutin treatment group (p < 0.05) showed significant decrease in the mean
arthritic severity score. Rutin (50 mg/kg) also demonstrated reduced arthritic score but it was
statistically non-significant in comparison to rutin (25 mg/kg) group. The difference within
the non-arthritic control animals was non-significant.
3.1.2.2. Body Weight
The animals showed increment in the average body weight till day 8 (Fig. 3.5). When
compared to the non-arthritic control rats, the untreated arthritic animals exhibited significant
reduction (p < 0.05) in mean change in their body weights from day 12 onwards and it
continued to decrease significantly till day 22 (p < 0.0001). The indomethacin treated group
demonstrated significant increase (p < 0.05) in average change in body weight in comparison
49
Figure 3.4. Effect of rutin on mean arthritic score. The values represent mean ± S.E.M.
of 12 animals/group. Day 14 onwards, the arthritic control rats showed significantly
increased arthritic score compared to normal control animals (#p < 0.0001). The
indomethacin treatment reduced the paw oedema (τp < 0.0001) from day 14 onward. On
day 14, the rutin (25 mg/kg) exhibited significantly less (**p < 0.01) arthritic score as
compared to the arthritic control rats, day 16 onwards non-significant difference was
observed. On day 14 onwards, indomethacin + rutin (2 mg/kg + 25 mg/kg) showed
significant decrease in score (**p < 0.01 and τp < 0.0001). From day 16 onwards, rutin (50
mg/kg) score continue to decrease significantly till the end of the study (***p < 0.001).
Figure 3.5. Mean change in body weight of arthritic and non-arthritic animals. The
values are mean ± S.E.M. (12 animals/group). Day 12 onwards, the untreated arthritic
animals showed significantly reduced body weight (#p < 0.05) compared to the normal
animals and continue to decrease significantly till day 22 (##p < 0.0001). The indomethacin
treated group showed significant increase in body weight compared to the arthritic control
group on day 22 (*p < 0.05). Rutin (50 mg/kg) and indomethacin + rutin (2 mg/kg + 25
mg/kg) exhibited non-significant increase in average body weight. Whereas, rutin (25
mg/kg) showed decrease in body weight compared to arthritic control animals.
#
#
ττ
**
*
*****
τ0
5
10
15
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
Mea
n A
rth
riti
c S
core
Days After Immunization
Normal Control Rutin Only
Arthritic Control Arthritic + Indomethacin (5 mg/kg)
Arthritic + Rutin (25 mg/kg) Arthritic + Rutin (50 mg/kg)
Arthritic + Indomethacin + Rutin (2 mg/kg + 25 mg/kg)
# ##
*
-20
0
20
40
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
Mea
n c
han
ge
in b
od
y w
eig
ht
(gm
s)
Days after immunization
Normal Control Rutin Only
Arthritic Control Arthritic + Indomethacin (5 mg/kg)
Arthritic + Rutin (25 mg/kg) Arthritic + Rutin (50 mg/kg)
Arthritic + Indomethacin + Rutin (2 mg/kg + 25 mg/kg)
50
to the arthritic control rats on day 22. Day 16 onwards, rutin (50 mg/kg) and day 18 onwards
indomethacin + rutin (2 mg/kg + 25 mg/kg) treated animals showed gradual but non-
significant increase in body weight till the end of the study. Rutin (25 mg/kg) group showed
gradual decrease in body weight from day 10 till day 22. Bonferroni’s post hoc test determined
the differences between the treatments groups and it showed that in comparison to the
indomethacin group, the rutin (25 mg/kg) group showed significant decrease (p < 0.01) in
mean change in body weight. There was no significant difference within the normal and
arthritic control animals.
3.1.2.3. Paw Oedema
The mean change in paw volume of both right and left paw is shown in figure 3.6. From day
12, clear inflammation was observed in the arthritic control rats which was significantly higher
in comparison to the normal control group in both right paw (p < 0.0001) and left paw (p <
0.05). It was observed that the indomethacin (5 mg/kg) and indomethacin + rutin (2 mg/kg +
25 mg/kg) treated groups exhibited reduced paw volume from day 12 onwards. When
compared with the arthritic control animals, this reduction was significantly different both in
right paw volume (p < 0.0001) and left paw volume (p < 0.05). The arthritic animals treated
with 50 mg/kg of rutin demonstrated a gradual increase in their paw volume from day 12 till
day 16, however this increase was markedly less than arthritic control group. From day 16
paw volumes begin to reduce gradually and on day 22 the reduction was significant in
comparison to the untreated arthritic animals (p < 0.001). The comparison of treatment groups
showed that from day 12 till the end of the study, the indomethacin (5 mg/kg) and
indomethacin + rutin group (2 mg/kg + 25 mg/kg) showed significant reduction in their right
(p < 0.0001) and left paw volumes (p < 0.01) compared to rutin (25 mg/kg). On day 12, rutin
(50 mg/kg) in comparison to the rutin (25 mg/kg), exhibited marked decrease in the right paw
volume (p < 0.0001) and remain significant till day 22 (p < 0.01) treatment group.
51
Figure 3.6. Mean change in paw volume following treatment with rutin. All values are
mentioned as mean ± S.E.M. of n=12/group. A significant increase in both right and left
hind paw volume of arthritic control rats was detected from day 12 onwards (##p < 0.0001
and #p < 0.05) compared to normal control group. Day 12 till the end of the study,
indomethacin and indomethacin + rutin treatment group showed significant decrease in
both right (***p < 0.0001) and left paw volume (*p < 0.05). Rutin (50 mg/kg) also showed
significant decrease in right paw volume on day 12 and 22 (***p < 0.0001 and **p < 0.001)
and left paw volume from day 20 onwards (*p < 0.05 and **p < 0.001).
##
##
*** ******
**
***
***
0
0.5
1
1.5
2
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
Ch
an
ge
in R
igh
t P
aw
Volu
me
(mL
)
Days after Immunization
Normal Control Rutin Only
Arthritic Control Arthritic + Indomethacin (5 mg/kg)
Arthritic + Rutin (25 mg/kg) Arthritic + Rutin (50 mg/kg)
Arthritic + Indomethacin + Rutin (2 mg/kg + 25 mg/kg)
#
##
* ***
***
* ***
0
0.5
1
1.5
2
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
Ch
an
ge
in L
eft
Paw
Volu
me
(mL
)
Days after Immunization
Normal Control Rutin Only
Arthritic Control Arthritic + Indomethacin (5 mg/kg)
Arthritic + Rutin (25 mg/kg) Arthritic + Rutin (50 mg/kg)
Arthritic + Indomethacin + Rutin (2 mg/kg + 25 mg/kg)
B
A
52
3.1.3. Effect of Rutin-GNPs on Clinical Parameters of RA
3.1.3.1. Arthritic Score
For initial 10 days, all the animals showed score of 0. However, on day 12, except
indomethacin (5 mg/kg) group, the arthritic control group and rutin-GNPs treated groups (12.5
mg/kg) showed slight increase in their mean arthritic score which was significantly high (p <
0.0001, Fig. 3.7) from day 14 onwards compared to the non-arthritic control rats. The score
of the arthritic control group gradually increase with time and remain significantly higher (p
< 0.0001) till the end of the study. In comparison to the arthritic control group, both
indomethacin (p < 0.0001) and rutin-GNPs (p < 0.001) treatment exhibited significantly
reduced mean arthritic score on day 14 and remain significant till the end of the study (p <
0.0001 and p < 0.01 for indomethacin and rutin-GNPs respectively). The indomethacin score
in comparison to the rutin-GNPs treatment group, was significantly less from day 14 till day
16 (p < 0.0001; p < 0.05) and then the difference became non-significant till the end of the
study. There was also no significant difference between controls included in this study.
3.1.3.2. Body Weight
Figure 3.8 depicts the mean change in body weight with S.E.M. The mean change in body
weight of all the animals showed increasing trend till day 8. Compared to normal control, the
untreated arthritic animals, started to show significant reduction (p < 0.05) in body weight
from day 12 onwards and this pattern was consistent till day 22 (p < 0.0001). Indomethacin
demonstrated significant increase in body weight on day 22 (p < 0.05) but rutin-GNPs
treatment group showed no significant increase compared to the arthritic control animals. The
statistical analysis to find the difference between treatment groups showed no change and non-
significant difference was found within the normal and arthritic control animals.
3.1.3.3. Paw Oedema
The mean change in hind paws volume of all the groups is shown in figure 3.9. In comparison
to the normal control group, the arthritic control group showed significant increase in the right
and left paw volume from day 12 onwards (p < 0.0001 and p < 0.05 for right and left paw
respectively). In contrast, from day 12 onwards, compared to the arthritic control group,
53
indomethacin (5 mg/kg) treatment group, showed significant reduction in mean change in
volume of both hind paws (p < 0.0001 and p < 0.05 for right and left paw respectively) and
this pattern of significance continued till the end of the experiment (p < 0.0001). Whereas,
rutin-GNPs (12.5 mg/kg) treatment group showed significant decrease in right paw volume
on day 12 and 22 (p < 0.0001 and p < 0.05 respectively) and from day 14 onwards, in the left
paw volume (p < 0.05 and p < 0.0001). The post hoc analysis also determined the difference
between the treatment groups and it showed that the indomethacin (5 mg/kg) treatment group
demonstrated significant reduction in mean change in right paw volume from day 20 onwards
(p < 0.05). Whereas, the left paw volume showed no significant difference compared to rutin-
GNPs (12.5 mg/kg) treatment group. The difference within non-arthritic control groups was
statistically non-significant.
Figure 3.7. Effect of rutin-GNPs on arthritic score. All the values are shown as mean ±
S.E.M. (12 animals/group). In comparison to the normal control animals, on day14
onwards, the untreated arthritic rats exhibited significant increase in mean arthritic severity
score (#p < 0.0001). Whereas, indomethacin and rutin-GNPs treatment group showed
significant reduction in mean arthritic score from day 14 (***p < 0.0001 and **p < 0.001)
and remain significant till day 22 (***p < 0.0001 and *p < 0.01).
#
#
******
***
0
2
4
6
8
10
12
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
Mea
n A
rth
riti
c S
core
Days after Immunization
Normal Control Gold Nanoparticles Only
Arthritic Control Arthritic + Gold Nanoparticles
Arthritic + Indomethacin (5 mg/kg) Arthritic + Rutin-GNPs (12.5 mg/kg)
54
Figure 3.8. Effect of rutin-GNPs on mean change in body weight. The values are
mentioned as mean ± S.E.M. (12 animals/group). In comparison to the normal control rats,
the untreated arthritic control animals exhibited significant reduction in body weight from
day 12 onwards (#p < 0.05) and increase in significance till day 22 (##p < 0.0001). The
indomethacin treatment group showed significant increase in weight on day 22 compared
to the arthritic control group (*p < 0.05).
# ##
*
-20
-15
-10
-5
0
5
10
15
20
25
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
Mea
n C
han
ge
in B
od
y W
eig
ht
(gm
s)
Days after Immunization
Normal Control Gold Nanoparticles Only
Arthritic Control Arthritic + Gold Nanoparticles
Arthritic + Indomethacin (5 mg/kg) Arthritic + Rutin-GNPs (12.5 mg/kg)
55
Figure 3.9. The mean change in paw volumes following treatment with rutin-GNPs.
The values are mentioned as mean ± S.E.M. The animals in the arthritic control
demonstrated significant increase in mean change in both right and left paw volume from
day 12 onwards compared to the normal control group (#p < 0.05 and ##p < 0.0001). In
contrast, indomethacin (5 mg/kg) treatment showed significant reduction in paw volume
from day 12 onwards compared to the arthritic control group (**p < 0.0001 and *p < 0.05
for right and left paw). Whereas, rutin-GNPs group showed significant reduction in right
paw volume on day 12 and 22 (**p < 0.0001 and *p < 0.05) and from day 14 onwards in
the left paw (*p < 0.05 and **p < 0.0001) respectively.
## ##
****
**
*
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
Ch
an
ge
in R
igh
t P
aw
Volu
me
(mL
)
Days after Immunization
Normal Control Gold Nanoparticles Only
Arthritic Control Arthritic + Gold Nanoparticles
Arthritic + Indomethacin (5 mg/kg) Arthritic + Rutin-GNPs (12.5 mg/kg)
# ##
* ***
**
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
Ch
an
ge
in L
eft
Paw
Volu
me
(mL
)
Days after Immnunization
Normal Control Gold Nanoparticles Only
Arthritic Control Arthritic + Gold Nanoparticles
Arthritic + Indomethacin (5 mg/kg) Arthritic + Rutin-GNPs (12.5 mg/kg)
B
A
56
3.2. Histological Analysis of Knee Joints
When the experiment was terminated (day 22), knee joint samples were collected from all the
included groups and H & E staining was performed for analysis of changes in joint
architecture. The histological characteristics associated with the pathogenesis of RA included
granulation tissue proliferation, synovial infiltration with lymphocytes and other
inflammatory cells and collapse of articular surface and pannus formation. The histological
scores were also determined for arthritic and non-arthritic animals indicating 4 as the
maximum score for the highest degree of inflammation and joint erosion (Table 3.1). The
representative joint histology and their respective scores of all the arthritic and non-arthritic
animals treated with the studied compounds is mentioned in the subsequent section.
3.2.1. NA-2 Treatment
An intact articular cartilage with negligible synovial infiltration can be seen in H&E stained
knee joint sections of the normal control animals (Fig. 3.10). In contrast, arthritic control
group showed prominent synovial infiltration and damaged articular cartilage. The
indomethacin and NA-2 treatments were able to reduce the infiltration in the synovium as well
as reduced the further destruction of articular cartilage in comparison to that observed in
arthritic group receiving no other treatment.
3.2.2. Rutin Treatment
In contrast to the arthritic control group which showed destruction of articular cartilage and
highly infiltrated synovial membrane, the treatment groups, i.e. the rutin (50 mg/kg),
indomethacin and indomethacin + rutin (2 mg/kg + 25 mg/kg) demonstrated less synovial
infiltration and almost negligible tissue damage (Fig. 3.11). In contrast, the treatment of
arthritic animals with rutin (25 mg/kg) was unable to reverse the inflammation and almost
same tissue damage was seen which was prominent in untreated arthritic animals.
3.2.3. Rutin-GNPs Treatment
The knee joints histopathological analysis of rutin-GNPs (12.5 mg/kg) treated group showed
less infiltration of synovium and intact cartilage (Fig. 3.12).
57
Table 3.1. Histological scoring system adopted to monitor the histological changes
in arthritic and non-arthritic rats.
Group Knee Joint
Inflammation Erosion
Normal Control 0 0
Arthritic Control 4 4
Arthritis + Indomethacin 2 1
Arthritis + NA-2 2 1
Arthritis + Rutin (25 mg/kg) 4 3
Arthritis + Rutin (50 mg/kg) 2 1
Arthritis + Indomethacin + rutin (2
mg/kg + 25 mg/kg)
2 1
Arthritis + Rutin-GNPs 2 1
The score was determined on 0-4 scale. Where 0 score indicates absence of inflammation
or erosion in the joints. Whereas, score of 2-3 indicated mild to moderate tissue erosion
and inflammation, score 4 demonstrate severe inflammation, extensive articular destruction
and severe bone erosion.
A
58
Figure 3.10. Effect of NA-2 treatment on joint destruction in CIA rats. (A) Normal
control; lack of lymphocyte proliferation and intact articular cartilage can be observed. (B)
Arthritic control; pronounced proliferation of lymphocytes (arrow 2) and erosion of articular
cartilage (arrow 1) as compared to the normal control can be seen. (C and D) Indomethacin
and NA-2 treated group; prominent decrease in inflammatory changes can be observed as
compared to the arthritic control. Where AC: articular cartilage; SM: synovial membrane; P: periarticular tissue.
A B
C D
1 2
P P
AC AC
AC
SM
AC
59
Figure 3.11. Effect of rutin treatment on Knee joints sections from CIA rats. (A)
Normal control; negligible synovial infiltration with lymphocytes and intact articular
cartilage can be observed. (B) Arthritic control; prominent lymphocytic proliferation
(arrow 2) and collapse of articular surface (arrow 1) as compared to the normal control can
be seen. (C) Indomethacin treated group; reduced inflammatory response can be observed
compared to arthritic control. (D) rutin (25 mg/kg) treatment group showed almost similar
inflammatory changes compared to the arthritic control rats. (E and F) The rutin (50 mg/kg)
and Indomethacin + rutin treatment group showed normal joint architecture with no visible
signs of cartilage erosion and less synovial infiltration can be seen compared to the
untreated arthritic control group. Where AC: articular cartilage; SM: synovial membrane;
P: periarticular tissue.
A B
C D
E F
2 1 SM
AC
AC
AC
P P
AC
2
60
Figure 3.12. Effect of rutin-GNPs treatment on Knee joints sections from CIA rats .
(A) Normal control; lack of lymphocyte proliferation and damage to articular cartilage can
be clearly observed. (B) Arthritic control; pronounced lymphocytic proliferation (arrow 2)
and collapse of articular structure (arrow 1) can be seen compared to the normal control.
(C and D) Indomethacin and rutin-GNPs treated group showed no signs of bone damage
and less synovial infiltration can be seen as compared to the arthritic control group. Where AC: articular cartilage; SM: synovial membrane; P: periarticular tissue.
A B
C D
2
SM 1
P P
AC
AC
AC
61
3.3. Measurement of Oxidative Stress Markers in Serum
The oxidative stress parameters include nitric oxide (NO), peroxide (PO) that are pro-
inflammatory markers and glutathione (GSH), which is an endogenous antioxidant system
were determined from serum samples of all the animals at the end of in vivo study. The effect
of the compounds used in the study on these parameters is mentioned below.
3.3.1. Effect of NA-2 Treatment
3.3.1.1. Serum Nitric Oxide (NO)
The level of serum NO in the untreated arthritic control rats was significantly increased (p <
0.0001) as compared to the normal control rats (Fig. 3.13). The NA-2 and indomethacin
treatment significantly reduced serum NO concentration compared to the arthritic control
group (p < 0.0001). The statistical analysis revealed non-significant difference between the
treatment groups and also within the arthritic and non-arthritic control groups studied.
3.3.1.2. Serum Peroxide (PO)
The concentration of peroxide in serum sample of arthritic and non-arthritic animals is
mentioned in figure 3.13. The arthritic control rats exhibited significant increase (p < 0.0001)
in the PO concentration as compared to the normal control animals. The PO level was
significantly reduced in the treatment groups, when compared to the arthritic control animals
(p < 0.0001). However, difference between the treatment groups and similarly within the non-
arthritic and arthritic control animals was non-significant.
3.3.1.3. Glutathione (GSH)
The figure 3.13 shows the concentration of serum GSH. Statistical analysis demonstrated a
significantly decreased level of serum GSH in the untreated arthritic control animals as
compared to the normal control animals (p < 0.0001). A significant increase in the GSH level
was noted in the arthritic groups treated with NA-2 and indomethacin (p < 0.0001) as
compared to the arthritic control group.
62
Figure 3.13. Effect of NA-2 on serum NO, PO and GSH level in arthritic and non-
arthritic animals. Data is shown as mean ± S.E.M. for 12 animals/group. Animals in
the arthritic group demonstrated significantly higher level of NO, PO and significantly
low level of GSH concentration compared to the normal control animals (#p < 0.0001).
Indomethacin and NA-2 treatment groups showed significantly reduced level of NO and
PO and significantly high GSH level (*p < 0.0001) as compared to the untreated arthritic
control animals.
#
* *
0
2
4
6
8
10
12
14
16
18
20
Normal Control Vehicle Control NA-2 Only Arthritic
Control
Arthritic +
Vehicle
Arthritic +
Indomethacin (5
mg/kg)
Arthritic + NA-
2 (5 mg/kg)
NO
Con
cen
trati
on
(μ
g/m
L)
Treatment Groups
#
* *
0
5
10
15
20
25
30
Normal ControlVehicle Control NA-2 Only Arthritic
Control
Arthritic +
Vehicle
Arthritic +
Indomethacin
(5mg/kg)
Arthritic +NA-
2 (5 mg/kg)
PO
Con
cen
trati
on
(n
g/m
L)
Treatment GroupsB
#
*
*
0
1
2
3
4
5
6
7
8
Normal Control Vehicle Control NA-2 Only Arthritic
Control
Arthritic +
Vehicle
Arthritic +
Indomethacin (5
mg/kg)
Arthritic + NA-
2 (5 mg/kg)
GS
H C
on
cen
tra
tio
n (
mg
/dL
)
Treatment GroupsC
A
63
3.3.2. Effect of Rutin on Oxidative Stress Parameters
3.3.2.1. Serum Nitric Oxide (NO)
Compared to the normal control group, significantly increased serum NO level was detected
in the arthritic control rats (p < 0.0001; Fig. 3.14). Whereas, the serum level of NO in
indomethacin (5 mg/kg, p < 0.0001), rutin (50 mg/kg, p < 0.0001), and indomethacin + rutin
(2 mg/kg + 25 mg/kg, p < 0.001) treated groups was significantly reduced in comparison to
arthritic control animals. It was also noted that treatment of arthritic animals with rutin (25
mg/kg) had no effect on the measured NO concentration and it was almost same as that of the
arthritic control animals.
3.3.2.2. Serum Peroxide (PO)
The arthritic control group showed significantly high PO level in serum compared to the
normal control group (p < 0.0001; Fig. 3.14). The indomethacin (5 mg/kg), rutin (50 mg/kg)
and indomethacin + rutin (2 mg/kg + 25 mg/kg) showed significantly reduced PO level in
comparison to the arthritic control group (p < 0.0001). Whereas, rutin (25 mg/kg)
demonstrated non-significant difference in PO concentration as compared to the arthritic
control rats. The statistical analysis of treatment groups showed significant reduction in PO
concentration (p < 0.0001) in comparison to the rutin (25 mg/kg). The difference within the
normal and arthritic control animals was statistically non-significant.
3.3.2.3. Serum Glutathione (GSH)
A prominent decrease in the serum glutathione concentration was detected in the arthritic
control rats compared to the normal control animals (p < 0.0001; Fig. 3.14). All the treatment
groups except rutin (25 mg/kg), showed significantly high concentration of GSH in
comparison to the untreated arthritic animals (p < 0.0001 for indomethacin (5 mg/kg) and
rutin (50 mg/kg) and p < 0.001 for indomethacin + rutin (2 mg/kg + 25 mg/kg) treatment
group respectively). When treatment groups were compared, the indomethacin (5 mg/kg) and
rutin (50 mg/kg) treatment groups showed significantly high GSH level (p < 0.0001 and p <
0.05) in comparison to the rutin (25 mg/kg). The difference within the arthritic and normal
control groups was statistically non-significant.
64
3.3.3. Effect of Rutin-GNPs on Oxidative Stress Parameters
3.3.3.1. Serum Nitric Oxide (NO)
Figure 3.15, shows concentration of NO in the non-arthritic and arthritic rats. In comparison
to the normal control rats, the untreated arthritic control animals revealed significantly high
NO concentration (p < 0.0001). In contrast, indomethacin and rutin-GNPs showed significant
decrease in NO concentration in comparison to the arthritic control group (p < 0.0001). Non-
significant difference was found between the treatment groups.
3.3.3.2. Serum Peroxide (PO)
In the arthritic control rats, the concentration of PO was significantly high compared to the
non-arthritic control rat (p < 0.0001; Fig. 3.15). Whereas, in comparison to the arthritic control
group, the indomethacin and rutin-GNPs treatment group demonstrated significant reduction
in the PO level (p < 0.0001). The difference between the treatment groups was non-
significant. Similarly, no difference was found within the normal and arthritic control group.
3.3.3.3. Effect of Rutin-GNPs on Serum Glutathione (GSH)
Figure 3.15 demonstrates the level of GSH in the serum samples of untreated arthritic and
non-arthritic groups. The GSH concentration of arthritic control animals was found to be
significantly decreased as compared to the normal control rats (p < 0.0001). The indomethacin
(5 mg/kg) and rutin-GNPs (12.5 mg/kg) showed significantly high level of GSH as compared
to the arthritic control animals (p < 0.0001). The statistical analysis exhibited non-significant
difference between the treatment groups and within the arthritic and non-arthritic control
group.
65
Figure 3.14. The NO, PO and GSH concentration measured following rutin
treatment. The values are shown as mean ± S.E.M. for n=12/group. Significant
increase in NO, PO concentration and significant reduction in GSH level was found in
arthritic control animals compared to the normal control (#p < 0.0001). In contrast, the
indomethacin (5 mg/kg) and rutin (50 mg/kg) showed significant reduction in NO and
PO concentration and significant increase in GSH concentration (**p < 0.0001).
However, indomethacin + rutin (2 mg/kg + 25 mg/kg) treatment group also showed
significant reduction in NO and PO level and significant increase in GSH level
compared to the arthritic control animals (*P < 0.001 for NO and GSH and **p <
0.0001 for PO).
#
**
** *
0
2
4
6
8
10
12
14
16
18
20
Normal
Control
Rutin Only Arthritic
Control
Arthritic +
Indomethacin
(5 mg/kg)
Arthritic +
Rutin (25
mg/kg)
Arthritic +
Rutin (50
mg/kg)
Arthritic +
Indomethacin +
Rutin (2 mg/kg
+ 25 mg/kg)
NO
Con
cen
trati
on
(μ
g/m
L)
Treatment Groups
#
** ** **
0
5
10
15
20
25
30
Normal Control Rutin Only Arthritic
Control
Arthritic +
Indomethacin
(5 mg/kg)
Arthritic +
Rutin (25
mg/kg)
Arthritic +
Rutin (50
mg/kg)
Arthritic +
Indomethacin +
Rutin ( (2
mg/kg + 25
mg/kg)
PO
Con
cen
trati
on
(n
g/m
L)
Treatment GroupsB
#
****
*
0
1
2
3
4
5
6
7
8
Normal Control Rutin Only Arthritic
Control
Arthritic +
Indomethacin (5
mg/kg)
Arthritic +
Rutin (25
mg/kg)
Arthritic +
Rutin (50
mg/kg)
Arthritic +
Indomethacin +
Rutin (2 mg/kg
+ 25 mg/kg)
GS
H C
on
cen
trati
on
(m
g/d
L)
Treatment GroupsC
A
66
Figure 3.15. Serum NO, PO and GSH concentration measured after treatment of
rutin-GNPs. The values are mentioned as mean ± S.E.M. for 12 animals/group. A
significant increase in NO and PO level and significantly reduced GSH level was detected
in the untreated arthritic control rats compared to the normal control rats (#p < 0.0001).
However, the indomethacin and rutin-GNPs treatment showed significant reduction in the
NO and PO concentration and significant increase in GSH concentration in comparison
to the arthritic control animals (*p < 0.0001).
#
**
0
5
10
15
20
25
Normal Control Gold Nanoparticles
Only
Arthritic Control Arthritic +
Goldnanoparticles
Arthritic +
Indomethacin (5
mg/kg)
Arthritic + Rutin-
GNPs (12.5 mg/kg)
NO
Con
cen
trati
on
(μ
g/m
L)
Treatment GroupsA
#
* *
0
5
10
15
20
25
30
Normal Control Gold Nanoparticles
Only
Arthritic Control Arthritic + Gold
nanoparticles
Arthritic +
Indomethacin (5
mg/kg)
Arthritic + Rutin-
GNPs (12.5 mg/kg)
PO
Con
cen
trati
on
(n
g/m
L)
Treatment GroupsB
#
**
0
1
2
3
4
5
6
7
8
Normal Control Gold Nanoparticles
Only
Arthritic Control Arthritic + Gold
Nanoparticles
Arthritic +
Indomethacin (5
mg/kg)
Arthritic + Rutin-
GNPs (12.5 mg/kg)
GS
H C
on
cen
trati
on
(m
g/d
L)
Treatment GroupsC
67
3.4. Measurement of Pro-Inflammatory Cytokines in Serum
TNF-α and IL-1β concentration was determined in both arthritic and non-arthritic animals.
The subsequent sections demonstrate the concentration of both these cytokines following
treatment with test compounds.
3.4.1. Effect of NA-2 on Serum IL-1β and TNF-α Concentration
The concentration of both IL-1β and TNF-α in serum samples of arthritic and non-arthritic
rats are mentioned in figure 3.16. In comparison to the non-arthritic control animals, IL-1 β (p
< 0.0001) and TNF- α (p < 0.01) level was significantly increased in the untreated arthritic
control animals. However, indomethacin treatment (5 mg/kg) exhibited significant decrease
in IL-1β (p < 0.0001) and TNF-α (p < 0.001) concentration compared to the arthritic control
group. The NA-2 (5 mg/kg) treatment group also showed significant decrease in both the
cytokine level (p < 0.0001 for IL-1β and p < 0.01 for TNF- α) respectively. There was no
significant difference within the arthritic, non-arthritic and treatment groups.
3.4.2. Effect of Rutin on IL-1β and TNF-α Concentration
The arthritic control group demonstrated significantly high serum concentration of IL-1β (p
< 0.0001) and TNF-α (p < 0.01) compared to the normal control rats (Fig. 3.17). However,
indomethacin (5 mg/kg), rutin 50 (mg/kg) and indomethacin + rutin (2 mg/kg + 25 mg/kg)
demonstrated significantly reduced IL-1β concentration (p < 0.0001) and TNF-α
concentration (p < 0.001 for indomethacin and p < 0.01 for rutin-50 mg/kg and indomethacin
+ rutin treatment groups, respectively) in comparison to the arthritic control rats. IL-1β and
TNF-α concentration of rutin (25 mg/kg) treatment group was non-significant compared to
the arthritic control animals. The comparison of treatment groups showed that indomethacin
(5 mg/kg, p < 0.0001); rutin (50 mg/kg, p < 0.001) and indomethacin + rutin (2 mg/kg + 25
mg/kg, p < 0.0001) showed significantly reduced level of IL-1β compared to the rutin (25
mg/kg). Whereas, TNF- α concentration of only indomethacin (5 mg/kg) treatment group was
significantly reduced (p < 0.012). The difference within the normal control groups was non-
significant.
68
Figure 3.16. Effect of NA-2 on IL-β and TNF-α concentration. Each bar represents
mean ± S.E.M. (12 animals/group) A) A significantly high level of IL-1β was observed
in the arthritic control animals (#p < 0.0001) compared to the normal control animals.
Whereas, NA-2 and indomethacin treatment showed significantly reduced level as
compared to the arthritic control group (*p < 0.0001) B) The arthritic control rats
showed significantly high level of TNF-α in the serum samples compared to the normal
control rats (#p < 0.01). Whereas, the serum level of TNF-α in the indomethacin (**p <
0.001) and NA-2 (*p < 0.01) treated animals was significantly low in comparison to the
arthritic control animals.
#
**
0
20
40
60
80
100
120
Normal Control Vehicle Control NA-2 Only Arthritic
Control
Arthritic +
Vehicle
Arthritic +
Indomethacin (5
mg/kg)
Arthritic + NA-
2 (5 mg/kg)
IL-1
βC
on
cen
trati
on
(p
g/m
L)
Treatment Groups
#
** *
0
50
100
150
200
250
300
350
400
450
500
Normal Control Vehicle Control NA-2 Only Arthritic
Control
Arthritic +
Vehicle
Arthritic +
Indomethacin (5
mg/kg)
Arthritic +NA-
2 (5 mg/kg)
TN
F-α
Con
cen
trati
on
(p
g/m
L)
Treatment GroupsB
A
69
Figure 3.17. Effect of rutin on IL1-β and TNF-α concentration. A) The serum
concentration of IL-1β of all the rats is shown as mean ± S.E.M. (12 animals/group).
Compared to the normal control rats, the untreated arthritic control rats exhibited marked
increase in the level of IL-1β (#p < 0.0001), whereas the treatment groups except rutin (25
mg/kg) showed significantly decreased level of IL- β (*p < 0.0001) B) The arthritic control
group showed significant increase in the TNF-α concentration in comparison to the normal
control group (#p < 0.01). Compared to the arthritic control rats, except rutin (25 mg/kg)
that showed non-significant reduction in TNF-α level, all the treatment groups i.e.
indomethacin, rutin (50 mg/kg) and indomethacin + rutin (2 mg/kg + 25 mg/kg) showed
significantly decreased TNF-α level (**p < 0.001 and *p < 0.01).
#
*
* *
0
20
40
60
80
100
120
Normal Control Rutin Only Arthritic Control Arthritic +
Indomethacin (5
mg/kg)
Arthritic + Rutin
(25 mg/kg)
Arthritic + Rutin
(50 mg/kg)
Arthritic +
Indomethacin +
Rutin (2 mg/kg +
25 mg/kg)
IL-1
βC
on
cen
trati
on
(p
g/m
L)
Treatment GroupsA
#
*** *
0
50
100
150
200
250
300
350
400
450
500
Normal Control Rutin Only Arthritic Control Arthritic +
Indomethacin (5
mg/kg)
Arthritic + Rutin
(25 mg/kg)
Arthritic + Rutin
(50 mg/kg)
Arthritic +
Indomethacin +
Rutin (2 mg/kg
+ 25 mg/kg)
TN
F-α
Con
cen
trati
on
(p
g/m
l)
Treatment GroupsB
70
3.4.3. Effect of Rutin-GNPs on IL-1β and TNF-α Concentration
A significantly high level of IL-1β (p < 0.0001) and TNF-α (p < 0.01) in the untreated arthritic
rats was found as compared to the normal control rats (Fig. 3.18). In the treatment groups, i.e.
indomethacin (5 mg/kg) and rutin-GNPs (12.5 mg/kg), significant reduction in both IL1-β (p
< 0.0001) and TNF-α concentration (p < 0.001 and p < 0.01 for indomethacin and rutin-GNPs
treatment group, respectively) was found in comparison to the arthritic control rats. The
statistical analysis showed no difference within the normal control, arthritic control and the
treatment groups.
3.5. Gene Expression Studies Using Quantitative Real time RT-
PCR
The quantitative gene expression analysis of RANKL pathway i.e. RANK, RANKL, NF-κB,
c-fos, c-jun and Akt was done in the spleen tissue samples of the arthritic and non-arthritic
rats. The primer sequences of the genes studied is mentioned in table 2.7.
3.5.1. Effect of NA-2 Treatment
The change in the expression of RANK, RANKL, NF-κB, c-fos, c-jun and Akt genes
following treatment with NA-2 is mentioned in Figure 3.19. The arthritic control group
showed significantly high gene expression of all the aforementioned genes (p < 0.0001) in
comparison to the normal control animals. In comparison to the arthritic control rats, a
significant decrease in the fold change of RANK, RANKL, c-fos and c-jun gene expression
was detected in the indomethacin and NA-2 treatment groups (p < 0.0001). Similarly, a
significantly reduced expression of NF-κB was observed in the indomethacin (p < 0.001) and
NA-2 treatment groups (p < 0.0001). Significant reduction in the Akt gene expression was
also observed in the NA-2 (p < 0.01) and indomethacin (p < 0.0001) treatment groups. The
statistical analysis of all the genes mentioned above showed non-significant difference
between the treatment groups and similarly within the arthritic and non-arthritic control
groups.
71
Figure 3.18. Effect of rutin-GNPs on IL-1β and TNF-α concentration. The values are
shown as mean ± S.E.M. (12 animals/group). A) The serum IL-1β concentration of arthritic
control animals showed marked increase compared to the normal control animals (#p <
0.0001) and the indomethacin and rutin-GNPs treatment groups showed significantly
reduced IL-1β concentration (*p < 0.0001). B) The arthritic control rats showed
significantly high serum TNF-α concentration as compared to the normal control animals
(#p < 0.01). The indomethacin (5 mg/kg) and rutin-GNPs (12.5 mg/kg) treatment group
demonstrated significantly reduced level of TNF-α (**p < 0.001 and *p < 0.01)
respectively.
#
* *
0
20
40
60
80
100
120
Normal Control Gold Nanoparticles
Only
Arthritic Control Arthritic + Gold
Nanoparticles
Arthritic +
Indomethacin (5
mg/kg)
Arthritic + Rutin-
GNPs (12.5
mg/kg)
IL-1
βC
on
cen
trati
on
(p
g/m
l)
Treatment GroupsA
#
***
0
50
100
150
200
250
300
350
400
450
500
Normal Control Gold Nanoparticles
Only
Arthritic Control Arthritic + Gold
Nanoparticles
Arthritic +
Indomethacin (5
mg/kg)
Arthritic + Rutin-
GNPs (12.5 mg/kg)
TN
F-α
Co
nce
ntr
ati
on
(p
g/m
l)
Treatment GroupsB
72
3.5.2. Effect of Rutin Treatment
The effect of rutin on genes of RANKL pathway i.e. RANK, RANKL, NF-κB, c-fos, c-jun
and Akt genes is shown in figure 3.20. The arthritic control group as compared with the normal
control group showed significant increase in the expression of all the genes mentioned above
(p < 0.0001). Whereas the indomethacin (5 mg/kg), rutin (50 mg/kg) and indomethacin + rutin
(2 mg/kg + 25 mg/kg) treatment groups showed significant decrease in RANK gene
expression as compared to the untreated arthritic control animals (p < 0.0001). Rutin (25
mg/kg) showed non-significant change in comparison to the arthritic control group in all the
genes studied. The comparison of treatment groups showed a marked suppression in gene
expression in indomethacin, rutin (50 mg/kg) and indomethacin + rutin (2 mg/kg + 25 mg/kg)
treatment groups (p < 0.0001) compared to rutin (25 mg/kg). The difference within the normal
and arthritic control groups was statistically non-significant.
The expression of RANKL in indomethacin, rutin (50 mg/kg), and indomethacin + rutin (2
mg/kg + 25 mg/kg) treatment groups (p < 0.0001) was significantly down regulated as
compared to the arthritic control animals. Whereas, the comparison of treatment groups
revealed that in rutin (50 mg/kg) and indomethacin (5 mg/kg) group significant reduction in
the RANKL gene expression (p < 0.0001) was observed in comparison to rutin (25 mg/kg)
treatment group. Whereas, indomethacin + rutin (2 mg/kg + 25 mg/kg) also demonstrated
significant reduction in gene expression (p < 0.001) compared to rutin (25 mg/kg).
The NF-κB gene expression in the treatment groups except rutin (25 mg/kg) showed
significant reduction in comparison to the arthritic control group (p < 0.001 for indomethacin
and p < 0.0001 for rutin-50 mg/kg and indomethacin + rutin (2 mg/kg + 25 mg/kg) groups
respectively). The difference within the treatment groups was statistically non-significant.
The c-fos gene expression in all the treatment groups i.e. indomethacin, rutin (50 mg/kg) and
indomethacin + rutin (2 mg/kg + 25 mg/kg), showed significantly reduced expression in
comparison to the arthritic control group (p < 0.0001). Compared to the rutin (25 mg/kg)
treatment group, significant reduction in the gene expression was observed (p < 0.001 for
indomethacin and p < 0.0001 for rutin-50 mg/kg and indomethacin + rutin (2 mg/kg + 25
mg/kg treatment groups).
73
The c-jun gene expression in the indomethacin (5 mg/kg), rutin (50 mg/kg) and indomethacin
+ rutin (2 mg/kg + 25 mg/kg) treatment groups showed significant reduction (p < 0.0001) in
comparison to the arthritic control animals. Rutin (25 mg/kg) showed non-significant
difference in comparison to the arthritic control group. In comparison to rutin (25 mg/kg)
significant decrease in the indomethacin (p < 0.0001) and rutin (50 mg/kg p < 0.0001) and
indomethacin + rutin (2 mg/kg + 25 mg/kg, p < 0.01) treatment group was observed.
In the case of Akt gene expression, the indomethacin (p < 0.0001), rutin (50 mg/kg, p < 0.01)
and indomethacin + rutin (2 mg/kg + 25 mg/kg, p < 0.01) treated groups showed significantly
decreased expression as compared to the arthritic control animals. The rutin (50 mg/kg, p <
0.05) and indomethacin (5 mg/kg, p < 0.01) exhibited significant reduction in the expression
of Akt compared to the rutin (25 mg/kg) treatment group. The expression of Akt in
indomethacin + rutin (2 mg/kg + 25 mg/kg) was non-significant in comparison to the rutin
(25 mg/kg).
3.5.3. Effect of Rutin-GNPs Treatment
The effect of rutin-GNPs on the expression of RANK, RANKL, NF-κB, c-fos, c-jun and Akt
genes is shown in figure 3.21. The expression of all the genes in the arthritic control group
was significantly increased as compared to the normal control animals (p < 0.0001). Both the
treatment groups i.e. indomethacin and rutin-GNPs showed significantly reduced gene
expression of RANK, RANKL, c-fos and c-jun in comparison to the arthritic control group (p
< 0.0001).
The NF-κB and Akt expression in the indomethacin (p < 0.001 and p < 0.0001 for NF-κB and
Akt) treatment group significantly reduced in comparison to the arthritic control group and
similarly the rutin-GNPs treatment also showed significant reduction in gene expression (p <
0.0001 and p < 0.05 for NF-κB and Akt). The statistical analysis showed no significant
difference within the normal control, arthritic control, and treatment groups.
74
Figure 3.19. The effect of NA-2 on fold change in RANK, RANKL, NF-κB, c-fos, c-
jun, and Akt gene expression. The values are mentioned as mean fold change in gene
expression ± S.E.M. The arthritic control group showed significantly high expression, as
compared to the normal group (#p < 0.0001). A marked suppression in the genes expression
of treatment groups i.e. indomethacin (***p < 0.0001 for RANK, RANKL, c-fos, c-jun, and
Akt, **p < 0.001 for NF-κB) and NA-2 (***p < 0.0001 for RANK, RANKL, NF-κB, c-fos,
and c-jun, *p < 0.01 for Akt) were observed.
Figure 3.20. Effect of rutin on RANK, RANKL, NF-κB, c-fos, c-jun, and Akt gene
expression. The bar graph represents mean fold change in expression ± S.E.M. In
comparison to the normal control group, significantly high gene expressions of the above-
mentioned genes were detected in the arthritic control group (#p < 0.0001). However, the
treatment groups, except rutin (25 mg/kg), exhibited significantly reduced gene
expressions, as compared to the arthritic control rats (*p < 0.01, **p < 0.001, ***p< 0.0001).
#
# ##
#
#
*** ***
**
***
***
********* ***
*** ***
*
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
RANK RANKL NF-κB c-fos c-jun AktFold
Ch
an
ge
in G
ene
Exp
ress
ion
Normal Control Vehicle Control
NA-2 Only Arthritic Control
Arthritic + Vehicle Arthritic + Indomethacin (5 mg/kg)
Arthritic + NA-2 (5 mg/kg)
#
# #
#
#
#
******
**
***
***
****** ***
***
***
***
*
******
***
***
****
0
0.5
1
1.5
2
2.5
3
3.5
4
RANK RANKL NF-kB c-fos c-jun Akt
Fold
Ch
an
ge
in G
ene
Ex
pre
ssio
n
Normal Control Rutin Only
Arthritic Control Arthritic + Indomethacin (5 mg/kg)
Arthritic + Rutin (25 mg/kg) Arthritic + Rutin (50 mg/kg)
Arthritic + Indomethacin + Rutin (2 mg/kg + 25 mg/kg)
75
Figure 3.21. Expression of RANK, RANKL, NF-κB, c-fos, c-jun, and Akt genes
following rutin-GNPs treatment. The values are mentioned as mean ± S.E.M. Compared
to the normal control rats, fold change in expression of all the genes studied in arthritic
control rats were significantly high (#p < 0.0001). Whereas, the expression of the above-
mentioned genes in rutin-GNPs (12.5 mg/kg) and indomethacin (5 mg/kg) treatment groups
reduced significantly, as compared to the arthritic control group (*p < 0.05, **p < 0.001, ***p < 0.0001).
#
# ##
#
#
*** ***
**
***
***
******
***
***
*** ***
*
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
RANK RANKL NF-κB c-fos c-jun Akt
Fo
ld C
ha
ng
e in
Gen
e E
xp
ress
ion
Normal Control Gold Nanoparticles Only
Arthritic Control Arthritic + Gold Nanoparticles
Arthritic + Indomethacin (5mg/kg) Arthritic + Rutin-GNPs (12.5mg/kg)
76
3.6. Immunohistochemistry of c-Fos, pAkt and iNOS in Spleen
Tissue
The spleen tissue was isolated from arthritic and non-arthritic rats at the end of in vivo study
and the expression of c-Fos, pAkt and iNOS was determined. The effect of compounds studied
on the expression of above-mentioned genes is shown below. The difference in protein
expression of the arthritic and non-arthritic control groups was non-significant. Therefore, in
microscopic images only normal and arthritic control group is shown and rest of the control
groups are shown in the bar graphs of the compounds.
3.6.1. Effect of NA-2 on c-Fos, pAkt and iNOS Protein Expression
Figure 3.22 shows the mean fluorescent intensity of c-Fos protein in the spleen tissue of
arthritic and non-arthritic animals. Immunohistochemical analysis showed a mild expression
of c-Fos in the normal control group (Fig. 3.23). A significantly high c-Fos expression in the
untreated arthritic control rats was observed in comparison to the normal group (p < 0.0001;
Fig 3.22). In contrast, the c-Fos expression was significantly decreased in the indomethacin
and NA-2 treated groups compared to the arthritic control animals (p < 0.0001). The post hoc
analysis revealed non-significant difference within control and treatment groups.
A baseline expression of pAkt was observed in the normal group (Fig. 3.24). The arthritic
control rats showed significantly high expression of pAkt in comparison to the normal control
group (p < 0.0001, Fig. 3.22). When the NA-2 and indomethacin treatment groups were
analysed for change in expression, a significantly reduced expression of pAkt was observed
compared to the arthritic control group (p < 0.0001). Whereas, change in protein expression
in the treatment groups found to be statistically non-significant.
The iNOS protein expression in spleen tissue samples of all the animals is mentioned in figure
3.25. The statistical analysis showed significantly upregulated iNOS expression in spleen
tissue in the arthritic control animals, compared to the normal control animals (p < 0.0001;
Fig. 3.22). In comparison to the arthritic control rats, NA-2 and indomethacin treatment
groups showed significantly reduced expression of iNOS (p < 0.0001). The comparison of
treatment groups showed non-significant difference.
77
Figure 3.22. Effect of NA-2 on c-Fos, pAkt and iNOS Protein Expression. Each bar
represents the mean fluorescent intensity of arthritic and non-arthritic rats (n=12
animals/group). A significantly high level of c-Fos expression was observed in the arthritic
control group (#p < 0.0001) as compared with the normal control animals. NA-2 and
indomethacin treatment group showed significant reduction in the expression of c-Fos, pAkt
and iNOS in comparison to the arthritic control group (*p < 0.0001).
# #
#
*
* **
**
0
5
10
15
20
25
C-Fos Akt iNOS
Mea
n F
luo
resc
ence
In
ten
sity
(Arb
itra
ry U
nit
)
Treatment Groups
Normal Control Vehicle Control
NA-2 Only Arthritic Control
Arthritic + Vehicle Arthritic + Indomethacin (5 mg/kg)
Arthritic + NA-2 (5 mg/kg)
78
Figure 3.23. Effect of NA-2 on immunohistochemistry of c-Fos in spleen tissue.
The figure shows the spleen tissue immunohistochemistry after treatment with NA-2
in arthritic and non-arthritic rats. The panel A and B are normal control and arthritic
control groups. Whereas, C and D are indomethacin and NA-2 treatment group. The
three columns represent DAPI, c-Fos and merged images for each sample.
Significantly high c-Fos expression can be seen in the arthritic control groups in
comparison to the normal groups that showed mild expression. The treatment groups
also showed significantly reduced expression of c-Fos in comparison to the arthritic
control groups. Scale bar corresponds to 25 μm.
79
Figure 3.24. Effect of NA-2 on Immunohistochemical analysis of pAkt in arthritic
and non-arthritic rats. The normal control group (panel A) has significantly low
pAkt expression compared to the arthritic control (panel B). Whereas, indomethacin
(panel C) and NA-2 (panel D) treatment group also have mild expression similar to
normal group. Scale bar corresponds to 25 μm.
80
Figure 3.25. Immunohistochemical analysis of iNOS expression after treatment
with NA-2. The figures represent the spleen tissue sections depicting the expression
of iNOS protein in the arthritic and non-arthritic rats after treatment with NA-2
compound. The arthritic control (panel B) represent significantly increased expression
of iNOS compared to the normal control group (panel A). Whereas, indomethacin
(panel C) and NA-2 (panel D) treatment showed significantly reduced expression, as
compared to the arthritic control. Scale bar corresponds to 25 μm.
81
3.6.2. Effect of Rutin on c-Fos, pAkt, and iNOS Protein Expression
The mean fluorescence intensity of c-Fos protein expression is mentioned in figure 3.26. A
prominent increase in the splenic c-Fos expression was detected in the untreated arthritic
control animals compared to the normal control rats (Fig. 3.27). The statistical analysis
demonstrated a marked increase in the mean fluorescence intensity of the c-Fos expression (p
< 0.0001) in the arthritic control rats. In comparison to arthritic control group, except rutin
(25 mg/kg), all the other treatment groups demonstrated a significantly reduced c-Fos
expression in comparison to the arthritic control group (p < 0.0001). The Bonferroni’s post
hoc test demonstrated that in comparison to 25 mg/kg dose of rutin, the indomethacin, rutin
(50 mg/kg) and indomethacin + rutin (2 mg/kg + 25 mg/kg) groups showed significant
decrease in the c-fos expression (p < 0.0001).
In the arthritic control animals, a marked increase in pAkt protein expression was detected
(Fig. 3.28), when compared with the normal control group and when analysed statistically,
found to be significant (p < 0.0001; Fig. 3.26). The immunohistochemical analysis of the
treatment groups showed that pAkt expression in rutin (25 mg/kg) was statistically non-
significant as compared to the arthritic control rats. Whereas, the rutin (50 mg/kg),
indomethacin (5 mg/kg) and indomethacin + rutin (2 mg/kg + 25 mg/kg) showed significantly
reduced expression (p < 0.0001). The comparison of treatment groups was also done to
determine differences between the treatment groups and significant decrease was observed in
the indomethacin (5 mg/kg, p < 0.001), rutin (50 mg/kg, p < 0.01) and indomethacin + rutin
(2.5 mg/kg + 25 mg/kg, p < 0.05) treatment groups in comparison to rutin (25 mg/kg).
The iNOS expression in the arthritic control exhibited significant increase compared to the
normal control (p < 0.0001, Fig. 3.26 and 3.29). The treatment groups depicted significant
suppression in the iNOS expression (p < 0.0001 for rutin (50 mg/kg), indomethacin (5 mg/kg)
and indomethacin + rutin treatment groups respectively), compared to the arthritic control.
Rutin (25 mg/kg) demonstrated non-significant difference compared to the arthritic control
group. The Post hoc analysis also revealed that all the treatment groups showed significant
reduction in iNOS protein expression compared to rutin (25 mg/kg) treatment group (p <
0.0001). However, non-significant difference was found within the control groups.
82
Figure 3.26. Effect of rutin on c-Fos, pAkt and iNOS protein expression. The bar graph
shows the mean fluorescent intensity ± S.E.M. of arthritic and non-arthritic rats (n=12
animals/group). The Immunohistochemical analysis of c-fos, pAkt and iNOS proteins
expression in the spleen tissue sample of untreated arthritic animals demonstrated significant
increase, compared to the normal control group (#p < 0.0001). Whereas, all the treatment
groups, except rutin (25 mg/kg) showed significantly reduced expression of afore-mentioned
proteins compared to the arthritic control group (*p < 0.0001).
##
#
*
* ***
** *
*
0
5
10
15
20
25
C-Fos Akt iNOS
Mea
n F
luo
resc
ence
In
ten
sity
(Arb
itra
ry U
nit
)
Treatment Groups
Normal Control Rutin Only
Arthritic Control Arthritic + Indomethacin (5 mg/kg)
Arthritic + Rutin (25 mg/kg) Arthritic + Rutin (50 mg/kg)
Arthritic + Indomethacin + Rutin (2 mg/kg + 25 mg/kg)
83
Figure 3.27. Immunohistochemistry of c-Fos in rutin treated animals. The
figures represented the c-Fos expression in the spleen tissue of rats. The normal
control (panel A) showed mild c-Fos expression compared to the arthritic control
animals (panel B). Whereas, treatment groups i.e. indomethacin (panel C), rutin
(50 mg/kg, panel E) and indomethacin + rutin (2.5 mg/kg + 25 mg/kg, panel F)
showed significantly reduced c-Fos expression. However, rutin (25 mg/kg, panel
D) showed significantly high expression compared to the normal control. Scale
bar corresponds to 25 μm.
84
Figure 3.28. Immunohistochemical analysis of pAkt in rutin treated group. The
arthritic control group (panel B) showed high expression of pAkt in the spleen tissue
as compared to the normal control rats (panel A). A prominent suppression in pAkt
expression was observed in the indomethacin (panel C), rutin (50 mg/kg, panel E)
and indomethacin + rutin (2 mg/kg + 25 mg/kg, panel F) treatment groups. The
rutin (25 mg/kg, panel D) group showed non-significant reduction in expression
compared to the arthritic control animals. Scale bar corresponds to 25 μm.
85
Figure 3.29. Photomicrograph of iNOS expression in rutin treated animals. The figures depicted mild expression of iNOS in the normal control (panel A).
Whereas, high level of iNOS expression was detected in the arthritic control
(panel B), compared to the normal control group. Similarly, rutin (25 mg/kg,
panel D) treatment group showed increased iNOS expression that was
significantly high compared to the rest of the treatment groups. The
indomethacin (panel C), rutin (50 mg/kg, panel E) and indomethacin + rutin
(2.5 mg/kg + 25 mg/kg, panel F) treatment groups showed significantly reduced
iNOS expression in comparison to the arthritic control group. Scale bar
corresponds to 25 μm.
86
3.6.3. Effect of Rutin-GNPs on c-Fos, pAkt, and iNOS Protein Expression
The immunohistochemical analysis of c-Fos expression in the spleen tissue of the arthritic and
non-arthritic rats is shown in figure 3.30 and 3.31. A prominent increase in the c-Fos
expression was observed in the arthritic control group as compared to the normal control
animals (p < 0.0001). Whereas, both the treatment groups i.e. indomethacin and rutin-GNPs
(12.5 mg/kg) showed low expression level compared to the arthritic control group and this
reduction was statistically significant (p < 0.0001). The Bonferroni’s post hoc test
demonstrated non-significant difference within the control and treated groups.
The protein expression of untreated arthritic control animals exhibited significant
upregulation, compared to the normal control animals (p < 0.0001, Fig. 3.30). The rutin-GNPs
(12.5 mg/kg) and indomethacin (5 mg/kg) treatment groups demonstrated significant
suppression of pAkt protein expression (Fig. 3.32), compared to the arthritic control group (p
< 0.0001). The post hoc analysis showed non-significant difference between the treatment
groups.
The protein expression of iNOS in the spleen tissue of arthritic rats was significantly high
compared to the normal control rats (p < 0.0001, figure 3.30 and 3.33). However, when
treatment groups i.e. indomethacin (5 mg/kg) and rutin-GNPs (12.5 mg/kg) were compared
with the arthritic control group, significant reduction in the protein expression was observed
(p < 0.0001). The difference within the arthritic and non-arthritic control groups and the
treatment groups was non-significant.
87
Figure 3.30. Effect of rutin-GNPs on c-Fos, pAkt and iNOS expression. The bar graph
represents values as mean ± S.E.M. (n= 12 animals/group). Significantly high c-Fos, pAkt
and iNOS protein expression was observed in the arthritic control animals compared to the
normal control animals (#p < 0.0001). In contrast, reduced expression was detected in the
treatment groups (*p < 0.0001 for both indomethacin and rutin-GNPs treatment group).
##
#
*
* ***
*
0
5
10
15
20
25
C-Fos Akt iNOS
Mea
n F
luo
resc
ence
In
ten
sity
(Arb
itra
ry U
nit
)
Treatment Groups
Normal Control Gold Nanoparticles Only
Arthritic Control Arthritic + Gold Nanoparticles
Arthritic + Indomethacin (5 mg/kg) Arthritic + Rutin-GNPs (12.5 mg/kg)
88
Figure 3.31. Effect of rutin-GNPs on Immunohistochemical analysis of c-Fos. The
arthritic control (panel B) showed significantly high level of c-Fos expression as
compared to the normal control animals (panel A). Whereas, the treatment groups, i.e.
indomethacin (panel C) and rutin-GNPs (panel D) showed significant reduction in c-
Fos expression. Scale bar corresponds to 25 μm.
89
Figure 3.32. Immunohistochemistry of pAkt on rutin-GNPs treated rats. The
normal control (panel A), showed low level of pAkt expression in comparison to the
arthritic control (panel B). Whereas, the arthritic groups treated with indomethacin
(panel C) and rutin-GNPs (panel D) showed significantly reduced pAkt expression
compared to the arthritic control group. Scale bar corresponds to 25 μm.
90
Figure 3.33. Immunohistochemistry of iNOS in spleen tissue of rutin-GNPs
treated group. In comparison to the arthritic control (panel B), the normal control
group (panel A) showed significantly reduced iNOS expression. Similarly, the
indomethacin (panel C) and rutin-GNPs (panel D) treatment group also showed
significantly reduced expression as compared to the arthritic control animals. Scale bar
corresponds to 25 μm.
91
CHAPTER 4
DISCUSSION
92
Rheumatoid arthritis is a an inflammatory autoimmune condition (Wong and Lord, 2004).
Continuous decrease in the disease severity is rarely achieved with the use of conventional
synthetic and biological DMARDs and patients often require lifelong treatment with serious
side effects. The precise molecular mechanism involved in the pathogenesis of RA is still
not clear due to which there is a lack of any effective treatment modality of RA. Therefore,
the identification of possible pathogenic mechanism causing the initiation and progression of
RA can result in the development of an effective treatment strategy (Goldring, 2003).
Members of the TNF receptor family and TNF ligand i.e. RANK and RANKL have been
identified and characterized that aid in the understanding of the molecular mechanism that
controls the formation of osteoclasts that are key players in the progression of RA (Lacey,
Timms, Tan, Kelley, Dunstan, Burgess, Elliott, Colombero, Elliott, and Scully, 1998; Myers,
Collier, Minkin, Wang, Holloway, Malakellis, and Nicholson, 1999; Cenci, Weitzmann,
Roggia, Namba, Novack, Woodring, and Pacifici, 2000; Shalhoub, Elliott, Chiu, Manoukian,
Kelley, Hawkins, Davy, Shimamoto, Beck, and Kaufman, 2000; Collin-Osdoby, Rothe,
Anderson, Nelson, Maloney, and Osdoby, 2001). Therefore, in the current study we focused
on the RANK/RANKL pathway to understand the molecular mechanism behind the anti-
arthritic activity of the test compounds studied i.e. NA-2, rutin, indomethacin + rutin and rutin
GNPs in CIA rats.
The test compounds were initially tested in acute carrageenan-induced inflammatory model
and after validating the anti-inflammatory effect, they were further tested in animal model of
arthritis i.e. collagen induced arthritis (CIA) with focus on RANK-RANKL pathway.
Trentham et al reported the use of CIA in rats for the first time after immunisation with type
II collagen (CII), which is one of the main components of articular cartilage. This model has
many similarities to the human arthritic condition in terms of histological, clinical and
immunological characteristics (Trentham, Townes, and Kang, 1977; Rosloniec, Cremer,
Kang, and Myers, 2001). Therefore, it has been used widely in studies involving the
preclinical evaluation of novel drugs for the treatment of RA (Billingham, 1983; Rosloniec,
Cremer, Kang, and Myers, 2001).
To evaluate the effect of NA-2, rutin, indomethacin + rutin and rutin-GNPs treatment on the
progression of CIA, the clinical parameters like change in body weight, paw volume and
93
arthritic score were examined during the entire study. The increase in paw volume is the result
of synovial infiltration of inflammatory mediators that include reactive oxygen species,
lymphocytes, prostaglandins and cytokines. Studies of inflammatory pain model reported that
the agents causing reduction of paw inflammation also involve in prevention of the
recruitment of cells involve in the inflammatory response towards the arthritic joints that
ultimately results in improvement in the mobility of animals and hence the animals can easily
access food and water (Ghirnikar, Lee, and Eng, 1998; McHugh and McHugh, 2000). Umar
et al reported the suppression of paw score and oedema in CIA rats after treatment with rutin
(Umar, Mishra, Pal, Sajad, Ansari, Ahmad, Katiyar, and Khan, 2012). Guardia et al also
demonstrated the protective effect of rutin on paw oedema and arthrogram score in AIA model
(Guardia, Rotelli, Juarez, and Pelzer, 2001). The observation in the current study is consistent
with the earlier findings. The untreated arthritic rats showed continuous decrease in the
average body weight and increase in paw volume and arthritic score till the end of the study,
whereas, the compounds NA-2, rutin (50 mg/kg), indomethacin + rutin (2 mg/kg + 25 mg/kg),
rutin-GNPs (12.5 mg/kg) and control drug indomethacin (5 mg/kg) treatment groups showed
increment in their body weight and significant reduction in the arthritic score and paw oedema.
Other studies from our research group have also reported the similar findings (Jawed, Shah,
Jamall, and Simjee, 2010; Jawed, Jamall, Shah, Perveen, Hanif, and Simjee, 2014; Perveen,
Hanif, Jawed, Jamall, and Simjee, 2014).
Besides the evaluation of the macroscopic parameters associated with the arthritis progression,
the CIA rats were also examined for the histological changes in the knee joints. We found that
the clinical parameters and histological score of arthritis were correlated well. The features
observed in the arthritic control rats were infiltration of synovial region with leukocytes,
cartilage and bone erosion and synovial cells proliferation. It has been reported in various
studies that the inflammatory response in arthritis leads to destruction of cartilage and
infiltration and proliferation of inflammatory immune cells in the inflamed tissues (Meyer,
Franssen, and Pap, 2006; Hayer, Pundt, Peters, Wunrau, Kühnel, Neugebauer, Strietholt,
Zwerina, Korb, and Penninger, 2009; Karmakar, Kay, and Gravallese, 2010). At the site of
tissue inflammation, activated fibroblast and macrophages leads to the secretion of
inflammatory molecules like prostaglandins, oxidative species, leukotrienes and proteinases
that triggers the process of cartilage destruction (Wieland, Michaelis, Kirschbaum, and
94
Rudolphi, 2005). The cartilage and bone damage results in the formation of cavity filled with
the synovial fibroblast and inflammatory cells (Ishikawa, Nishigaki, Miyata, Hirayama,
Minoura, Imanishi, Neya, Mizutani, Imamura, and Naritomi, 2005). The motor function in the
RA patients or arthritic animals is hindered due to pain in the joints and changes in the joints
architecture. The treatment strategy that can inhibit or retard the inflammatory response and
joint damage and improve the motor function can be considered therapeutically effective for
RA (Van der Leeden, Steultjens, Dekker, Prins, and Dekker, 2006; Hess, Axmann, Rech,
Finzel, Heindl, Kreitz, Sergeeva, Saake, Garcia, and Kollias, 2011). In our study, we also
observed cartilage damage and erosion in the knee joint tissues of the arthritic control animals
with extensive proliferation of inflammatory cells. However, the histological examination of
the arthritic rats treated with indomethacin and the test compounds (NA-2, rutin-50 mg/kg,
indomethacin + rutin and rutin-GNPs) demonstrated mild synovial infiltration and slight
cartilage damage as compared to the arthritic control rats. These histological analysis correlate
well with macroscopic observations of both arthritic control animals and arthritic animals
treated with the test compounds.
Reactive oxygen and nitrogen species (ROS and RNS) have been known for their role in the
pathophysiology of RA, since long as mediators of cartilage and bone damage. (Panasyuk,
Frati, Ribault, and Mitrovic, 1994; Nakamura, Shibakawa, Tanaka, Kato, and Nishioka, 2003).
The endogenous antioxidant system like GSH and SOD prevent this ROS induced damage. In
arthritic condition this balance of antioxidant and ROS system is disturbed and leads to the
cartilage and bone erosion (Grootveld, Henderson, Farrell, Blake, Parkes, and Haycock, 1991;
Hughes, Reynolds, Brown, Kelley, Thomson, Conn, Jonas, Westfall, Padilla, and Callahan,
2010). Study conducted by Umar et al reported that rutin treatment of arthritic rats leads to
significant suppression of ROS and significant increase in GSH level (Umar, Mishra, Pal,
Sajad, Ansari, Ahmad, Katiyar, and Khan, 2012). Kyung et al also reported that rutin inhibits
osteoclast formation by reducing the elevated ROS level in vitro in RANKL stimulated bone
marrow macrophages (Kyung, Lee, Shin, and Choi, 2008). Our observations were in accord
with these studies, since we have also observed similar findings. The untreated arthritic
animals exhibited significant rise in NO and PO concentration with parallel decrease in the
GSH level compared to the normal control animals. Whereas, the reference drug
indomethacin, NA-2, rutin (50 mg/kg), indomethacin + rutin (2 mg/kg + 25 mg/kg) and rutin-
95
GNPs (12.5 mg/kg) treatment groups showed significantly reduced NO and PO level with
parallel increase in GSH concentration, as compared to the untreated arthritic group. Several
studies have reported that the inhibitory activity of indomethacin treatment on generation of
ROS is through the inhibition of prostaglandin E2 and COX (Hrabák, Vercruysse, Kahán, and
Vray, 2001; Dalle-Donne, Rossi, Giustarini, Milzani, and Colombo, 2003). These findings
justify the use of indomethacin as a reference drug in the current study.
TNF-α and IL-1β are key players in the progression of RA (Kudo, Fujikawa, Itonaga,
Sabokbar, Torisu, and Athanasou, 2002; Kitaura, Zhou, Kim, Novack, Ross, and Teitelbaum,
2005; Wei, Kitaura, Zhou, Ross, and Teitelbaum, 2005). IL-1β and TNF-α are produced by
various immune cells, mainly activated T cells and macrophages (Jawed, Shah, Jamall, and
Simjee, 2010). TNF-α in combination with RANKL causes upregulation of RANK expression
(Komine, Kukita, Kukita, Ogata, Hotokebuchi, and Kohashi, 2001). It has been reported that
treatment with TNF-α inhibitors reduces inflammation and prevents bone resorption in RA
patients (Scott and Kingsley, 2006). Like TNF-α, studies have also reported protective effects
of IL-1β inhibition on structural damage to bones in animal model of RA (Joosten, Helsen,
Saxne, van de Loo, Heinegård, and van den Berg, 1999). Kyung et al also showed reduction
in TNF-α mRNA expression after treatment of RANKL stimulated bone marrow derived
macrophages with rutin (Kyung, Lee, Shin, and Choi, 2008). Similarly, rutin stabilized silver
nanoparticles also reported significant decrease in TNF-α in animal model of arthritis (Rao,
2015).
Extensive studies have been done on the effect of IL-1β and TNF-α on the progression of
arthritis. All these studies reported elevated level of these cytokines in the arthritic patients
and in vivo models of arthritis (Glenn and Gray, 1965; Ghirnikar, Lee, and Eng, 1998;
McHugh and McHugh, 2000; Coulthard, Pleuvry, Brewster, Wilson, and Macfarlane, 2002;
Laferrère, García-Lorda, Russell, and Pi-Sunyer, 2004; Van der Heijden, Oerlemans, Lems,
Scheper, Dijkmans, and Jansen, 2009). In correlation to the above mentioned findings, we
have also observed high TNF-α and IL-1β concentration in the arthritic animals. Following
treatment with NA-2, rutin (50 mg/kg) and indomethacin + rutin (2 mg/kg + 25 mg/kg), rutin-
GNPs (12.5 mg/kg) and indomethacin, a significantly decreased level of these cytokines was
observed.
96
Inducible nitric oxide synthase (iNOS) is an isoform of nitric oxide synthase (NOS), that is
expressed in the macrophages after exposure of bacterial endotoxin and pro-inflammatory
cytokine INF-γ, TNF-α and IL-1β (Nathan, 1992; Moncada and Higgs, 1993; McNeill,
Crabtree, Sahgal, Patel, Chuaiphichai, Iqbal, Hale, Greaves, and Channon, 2015). The
inflammatory cytokines induce iNOS production that leads to the increased production of NO
for long period in the affected tissues of RA patients (Aktan, 2004). Tozatto et al reported
increased iNOS protein expression in kidney and cardiac tissue in CIA mice (Palma Zochio
Tozzato, Taipeiro, Spadella, Marabini Filho, de Assis, Carlos, Girol, and Chies, 2015).
Cannon and Tanaka et al also reported upregulation of iNOS mRNA expression in spleen
tissue of CIA and AIA rats as compared to normal rats (Cannon, Openshaw, Hibbs, Hoidal,
Huecksteadt, and Griffiths, 1996; Tanaka, Matsui, Murakami, Ishizuka, Sugiura, Kawashima,
and Sugita, 1998). In light of the above findings, we also observed prominent reduction in
protein expression of iNOS in NA-2, rutin-50 mg/kg, indomethacin + rutin (2 mg/kg + 25
mg/kg), rutin-GNPs (12.5 mg/kg) and indomethacin treatment groups in comparison to the
arthritic control animals that exhibited high level of iNOS expression in spleen tissue.
Extensive studies have been done to determine the role of the RANK/RANKL/OPG signalling
pathway in RA (Neumann, Gay, and Müller‐ Ladner, 2005). RANKL interacts with its
cognate receptor RANK that leads to the further activation of series of downstream signalling
cascade that results in activation of downstream transcription factors NF-κB, c-fos and c-jun
followed by the activation of Akt (Grigoriadis, Wang, Cecchini, Hofstetter, Felix, Fleisch, and
Wagner, 1994; Gravallese, Manning, Tsay, Naito, Pan, Amento, and Goldring, 2000; Lam,
Takeshita, Barker, Kanagawa, Ross, and Teitelbaum, 2000; Ruocco, Maeda, Park, Lawrence,
Hsu, Cao, Schett, Wagner, and Karin, 2005; Wei, Kitaura, Zhou, Ross, and Teitelbaum, 2005).
Animal studies of arthritis have reported that animals genetically deficient in RANK or
RANKL or inhibition of RANKL- RANK interaction leads to protection of cartilage and bone
erosion regardless of the presence of joint inflammation (Simonet, Lacey, Dunstan, Kelley,
Chang, Lüthy, Nguyen, Wooden, Bennett, and Boone, 1997; Kong, Feige, Sarosi, Bolon,
Tafuri, Morony, Capparelli, Li, Elliott, and McCabe, 1999; Pettit, Ji, Von Stechow, Müller,
Goldring, Choi, Benoist, and Gravallese, 2001; Redlich, Hayer, Maier, Dunstan, Tohidast‐
Akrad, Lang, Türk, Pietschmann, Woloszczuk, and Haralambous, 2002; Romas, Sims, Hards,
97
Lindsay, Quinn, Ryan, Dunstan, Martin, and Gillespie, 2002; Lubberts, van den Bersselaar,
Oppers-Walgreen, Schwarzenberger, Coenen-de Roo, Kolls, Joosten, and van den Berg, 2003;
Li, Schwarz, O'Keefe, Ma, Looney, Ritchlin, Boyce, and Xing, 2004). Tsubaki et al reported
over expression of RANKL mRNA in the spleen and thymus samples of CIA mice (Tsubaki,
Takeda, Kino, Itoh, Imano, Tanabe, Muraoka, Satou, and Nishida, 2015). Wei et al also
reported that the animals with adjuvant induced arthritis showed upregulation of RANKL
(Wei, Tong, Xia, Lu, Chou, Wang, and Dai, 2013).
In the light of above findings, the present study also demonstrated increased mRNA and
protein expression of RANK and RANKL in the spleen samples of CIA rats. However, the
administration of NA-2, rutin (50 mg/kg), indomethacin + rutin (2 mg/kg + 25 mg/kg), rutin-
GNPs (12.5 mg/kg) significantly suppressed the CIA progression by downregulation of
RANK and RANKL that clearly shows the role of RANK/RANKL in the progression of
arthritis.
In addition to RANK and RANKL, some other markers that are downstream mediators of
RANK/RANKL signalling cascade were also studied. In the pathogenesis of RA, NF-κB plays
central role. The role of NF-κB in the regulation of different pro-inflammatory pathways is
well known. NF-κB regulates T and B lymphocytes activation and survival in spleen and
thymus that ultimately leads to the development of arthritis (Brown, Claudio, and Siebenlist,
2008). High activity of NF-κB has been reported in a number of studies involving
experimental arthritis (Tsao, Suzuki, Totsuka, Murata, Takagi, Ohmachi, Fujimura, and
Takata, 1997; Miagkov, Kovalenko, Brown, Didsbury, Cogswell, Stimpson, Baldwin, and
Makarov, 1998; Tak and Firestein, 2001). Activation of the inflammatory mediators involved
in the progression of RA like IL-1β, IL-6, TNF-α and RANKL at the transcriptional level is
dependent on the activation of NF-κB (Tracey, Klareskog, Sasso, Salfeld, and Tak, 2008).
Kyung et al also demonstrated inhibition of NF-κB activation by rutin treatment in RANKL
stimulated bone marrow derived macrophages (Kyung, Lee, Shin, and Choi, 2008). Inhibition
of NF-κB has been reported to suppress the progression of arthritis in various animal models
(Wakamatsu, Nanki, Miyasaka, Umezawa, and Kubota, 2005; Izmailova, Paz, Alencar, Chun,
Schopf, Hepperle, Lane, Harriman, Xu, and Ocain, 2007; Okamoto, Yoshio, Kaneko, and
Yamanaka, 2010). Our findings are also consistent with the above mentioned reports. In the
98
current study we observed upregulation of NF-κB in the arthritic control rats and the treatment
of NA-2, rutin-50 mg/kg, indomethacin + rutin (2 mg/kg + 25 mg/kg), rutin-GNPs (12.5
mg/kg) and indomethacin significantly suppressed the expression of NF-κB.
Activator protein 1 (AP-1) belongs to the transcription factor family that comprises of homo
and heterodimers of Fos, Jun, ATF and MAF family of proteins (Hai, Liu, Allegretto, Karin,
and Green, 1988; Angel and Karin, 1991). AP-1 is involved in different processes e.g. cell
migration, differentiation, apoptosis, proliferation etc. Beside this, it is also an important
player in inflammatory disorders like psoriasis, asthma and rheumatoid arthritis (Palanki,
2002; Shaulian and Karin, 2002; Hess, Angel, and Schorpp-Kistner, 2004; Wagner and
Eferl, 2005). The activation of P38/MAPKs leads to regulation of downstream mediators
that causes c-fos and c-jun transcriptional activation and finally upregulation of AP-1
complex expression takes place (Eferl and Wagner, 2003). AP-1 activation is an
important step in the process of joint damage (Shiozawa, Shimizu, Tanaka, and Hino,
1997). The transcription factors c-jun and c-fos are key players in the process of bone
resorption and blockade of any of these factors can prevent osteoclastogenesis
(Teitelbaum, 2004; Takayanagi, 2005). It has been reported that blockade of c-fos and c-
jun has prevented the RANKL induced osteoclastogenesis (Ikeda, Nishimura, Matsubara,
Tanaka, Inoue, Reddy, Hata, Yamashita, Hiraga, and Watanabe, 2004; Lee, Jin, Shim, Kim,
Ha, and Lee, 2010). Zhang et al demonstrated upregulated expression of c-fos and c-jun in
RA patients synovial tissue and peripheral blood compared to healthy individuals (Zhang, Yu,
Deng, Lv, Liu, Xiao, Yang, Zhang, and Li, 2015). Hashiramoto et al also reported upregulated
c-Fos protein expression in spleen of CIA rats (Hashiramoto, Yamane, Tsumiyama, Yoshida,
Komai, Yamada, Yamazaki, Doi, Okamura, and Shiozawa, 2010). Liu et al reported increased
mRNA expression of c-jun in LPS induced splenocytes isolated from CFA induced arthritic
rats (Liu, Li, He, Yang, Ruan, and Sun, 2016). Similarly, the present study also showed
upregulation of both c-fos and c-jun at mRNA and protein level in arthritic animals as
compared to the normal healthy rats and this upregulation was suppressed significantly in NA-
2, rutin-50 mg/kg, indomethacin + rutin (2 mg/kg + 25 mg/kg), rutin-GNPs (12.5 mg/kg), and
indomethacin treated groups.
99
Another downstream mediator of inflammatory cascade is Akt which is also known as protein
kinase B, belong to serine/threonine kinase family of proteins (King, Kobayashi, Cejas, Kim,
Yoon, Kim, Chiffoleau, Hickman, Walsh, and Turka, 2006). It is expressed mainly in the cells
of the innate immunity e.g. macrophages, neutrophils and dendritic cells. It is a key player in
the activation of different signalling cascades involved in apoptosis, cell growth etc. (Huang
and Lin, 2007; Fischer, Jacoby, Pape, Ward, Kuwertz-Broeking, Renken, Nizze, Querfeld,
Rudolph, and Mueller-Wiefel, 2009; Emamian, 2012). Studies have identified its role in
autoimmune diseases. AKT signalling pathway is involved in the regulation of inflammation.
Various Studies have reported the role of PI3K/AKT pathway in the inflammatory disorders
such as multiple sclerosis, asthma, psoriasis, RA and atherosclerosis (Bozinovski, Jones,
Vlahos, Hamilton, and Anderson, 2002; Chang, Sukhova, Libby, Schvartz, Lichtenstein,
Field, Kennedy, Madhavarapu, Luo, and Wu, 2007; Mortaz, Chiu, Lin, Chen, Huang, Tong,
Tzeng, Lee, Hsu, and Tang, 2009; Lazar, Koenderman, Kraneveld, Nijkamp, and Folkerts,
2009; Ma, Dela Cruz, Hartl, Kang, Takyar, Homer, Lee, and Elias, 2011; Lai, Li, Li,
Muehleisen, Radek, Park, Jiang, Li, Lei, and Quan, 2012). Tsubaki et al reported increased
activation of Akt in CIA mice (Tsubaki, Takeda, Kino, Itoh, Imano, Tanabe, Muraoka, Satou,
and Nishida, 2015). Wei et al also reported significant increase in phospho-Akt expression in
AIA rats (Wei, Tong, Xia, Lu, Chou, Wang, and Dai, 2013). In the current study, we also
found increased expression of pAkt in arthritic rats that decreased significantly, when the
animals were given NA-2, rutin-50 mg/kg, indomethacin + rutin (2 mg/kg + 25 mg/kg), rutin-
GNPs (12.5 mg/kg) and indomethacin treatment.
100
CONCLUSION
The present study has demonstrated that N-(2-hydroxyl phenyl) acetamide (NA-2), rutin,
indomethacin + rutin and rutin GNPs not only prevented the clinical signs of arthritis, ROS
and pro-inflammatory cytokines production in CIA model but for the first time demonstrated
that these compounds have significantly downregulated RANKL and its receptor RANK
expression and successfully suppressed the expression of other pro-inflammatory mediators
downstream of RANKL signalling cascade such as c-Fos, c-Jun, NF-κB and Akt in CIA
model. The compounds significantly reduced the arthritic score, paw oedema and the
inflammatory markers level in CIA rats. Our data suggest that expression of mediators of
RANKL pathway, along with ROS and circulating pro-inflammatory cytokines, can be used
to monitor the efficacy of treatment in animal model of RA. In our study, the expression of all
these markers of inflammation correlate well with the clinical symptoms in the untreated
arthritic group and the test compounds treatment successfully downregulated the level of these
markers. Thus, based on our data analysis, we found that RANKL and its receptor RANK
may be used as markers of disease induction. In this regard activation of RANKL can be a
useful tool after nociceptive stimulation to identify the effectiveness of different analgesics
used in the RA treatment. Taken together, it can be concluded that NA-2, rutin, and rutin-
GNPs might be potential candidates for RA treatment.
101
FUTURE IMPLICATIONS
In the current study, NA-2, rutin and rutin-GNPs showed promising anti-arthritic and anti-
inflammatory activity in CIA model of rat. We investigated the potential role of these
compounds in the regulation of RANK-RANKL pathway and to some extent we were able to
identify the key mediators downstream of RANKL signalling cascade but in the progression
of RA crosstalk of multiple pathways is involved and to get the complete information of RA
progression, other signalling pathways also need to be investigated. Therefore, the future
targets will be:
1. To study IL-6/STAT3 signalling pathway. It is constitutively activated in monocytes and
T lymphocytes circulating T cells of RA patients. Limited information is available on the
role of STAT3 pathway in the progression of RA. Investigation of effect of NA-2, rutin
and its gold nanoparticles on modulation of STAT3 pathway will be beneficial for the
development of targeted therapies for RA patients.
2. TNF-α and IL-1β production stimulates the expression of matrix metalloproteinases in RA
patients. These enzymes are involved in the destruction of extracellular matrix. Research
is going on to develop potent inhibitors of MMPs to prevent the cartilage and tissue
damage in RA patients. Therefore, future studies can include the determination of NA-2,
rutin and its gold nanoparticles effect on the expression of MMPs.
3. To determine the role of these compounds in the modulation of MEK/ERK signalling
pathway in CIA rats. Activation of MEK/ERK signalling cascade is responsible for the
production of various cytokines like IL-6, IL-1β, TNF-α and RANKL that are main players
responsible for the inflammatory response in RA.
4. Rheumatoid arthritis is a systemic disease and it affects multiple systems of human body.
Therefore, besides immune system which is mainly involved in autoimmune response
regulation, the role of studied compounds in modulation of RANKL pathway can also be
determined in other organ systems like bone, cartilage and cardiovascular system.
102
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PERSONAL INTRODUCTION
Anum Gul
E.mail: [email protected]
I was born in Karachi and completed my secondary school certificate from Khatoon-e-
Pakistan Govt. Girls Secondary School, Karachi in 2004. After matriculation, I completed my
higher secondary school certificate from Khatoon-e-Pakistan Govt. Degree College for
Women, Karachi in 2006. I completed B.Sc. (Hons) in Biotechnology in First class, First
Position from University of Karachi in 2009. In 2010, I received Gold medal for getting First
class “First Position” in my Master’s degree in Biotechnology from University of Karachi.
Since my childhood, whenever, I was asked about my future profession, I always said that I
want to be a scientist and during my masters, I was sure that I will do higher studies and will
do my best in the field of science. To continue my dream, I joined one of the best research
institute, Dr. Panjwani Center for Molecular Medicine and Drug Research, International
Center for Chemical and Biological Sciences, University of Karachi for getting Ph.D. in
molecular medicine in 2011.
Initially, I worked under the supervision of Dr. Talat Makhmoor till 2015 then I joined Dr.
Shabana U. Simjee and I was lucky to get training from both the supervisors. During my entire
stay in PCMD, I learned different molecular and cell biology techniques, attended and
participated as a volunteer in various seminars and international symposia held at my institute,
where I got experience to meet the world-renowned scientists. I also got opportunity to teach
microbiology in GRE preparatory classes held in PCMD. I thoroughly enjoyed every
academic and research activity during my Ph.D. studies.
127
GLOSSARY
Adjuvant: Any substance which enhance the efficacy of vaccine most commonly by
increasing their half-life.
Angiogenesis: the formation of new blood vessels, especially those that supply oxygen and
nutrients to cancerous tissues.
Anti-citrullinated protein antibodies: are autoantibodies (antibodies directed against one or
more of an individual’s own proteins) that are frequently detected in the blood of rheumatoid
arthritis patients. The main epitope for these antibodies is filaggrin.
Antigen: Any particular substance that provokes immune system to produce specific
antibodies against it.
B-cells: a lymphocyte derived from bone marrow that provides humoral immunity, it
recognizes free antigen molecules and matures into plasma cells that secrete antibodies, which
inactivate the antigens.
c-Fos: DNA-binding proteins encoded by the c-fos genes, involved in growth related
transcriptional control. It combines with c-jun to form c-fos/c-jun heterodimer (transcription
factor AP-1).
Chemokines: a large family of cytokines having chemokinetic and chemotactic properties,
known to stimulate leukocyte movement and attraction.
Chondrocytes: a connective tissue cell that occupies a lacuna (small cavities) within the
cartilage matrix also called cartilage cell.
128
Complement: a substance that is produced by a precursor protein or in response to the
presence of foreign material in the body that triggers or participates in a complement reaction.
c-Jun: is a protein encoded by proto oncogene c-jun that helps in cell differentiation, cell
growth and apoptosis.
Immunization: It’s an immunological procedure in which an organism’s immune system is
provoked against a particular antigen using immunogen.
Infection: It’s a condition that potentiates abnormal immune responses due to invasion of
microorganisms, resulting in serious pathological problems.
Leukotrienes: a class of small molecules produced by cells in response to allergen exposure,
they are the regulators of inflammatory and allergic reactions.
Mitogen: is a chemical substance usually protein that encourages a cell to commence cell
division, triggering mitosis it triggers signal transduction pathways in which mitogen activated
protein kinase is involved, leading to mitosis.
MMPs: enzymes related to tissue healing/remodeling and cancer cell metastasis.
Osteoblast: a cell from which bone develops or a bone-forming cell.
Osteoclast: a cell that absorbs bone.
Plasmin: an enzyme belongs to serine proteases, released as a zymogen called plasminogen
and converted into active plasmin by various enzymes. It is present in blood that degrades
many blood plasma proteins including fibrin, fibronectin, thrombospondin and laminin etc.
also known to activate collagenases, some mediators of complement system.
129
Prostaglandlins: any of a group of naturally occurring, chemically related fatty acids that
stimulate contractility of the uterine and other smooth muscle and have the ability to lower
blood pressure, regulate acid secretion of the stomach, body temperature, platelet aggregation,
and control inflammation and vascular permeability; they also affect the action of certain
hormones.
Proteoglycans: are proteins that are heavily glycosylated. The basic proteoglycan unit
consists of a "core protein" with one or more covalently attached glycosaminoglycan chains.
The chains are long, linear carbohydrate polymers that are negatively charged under
physiological conditions, due to the occurrence of sulfate and uronic acid groups.
Proteoglycans occur in the connective tissue.
T-cells: a small lymphocyte developed in the thymus; it orchestrates the immune system's
response to infected or malignant cells.
130
APPENDIX I
S.No. Name of Chemical Source Catalogue #
1. Chloroform Fischer Scientific C/4960/17
2. DAPI Wako 049-18801
3. DEPC Carl Roth K028.3
4. Dibasic Sodium Phosphate, Na2HPO4.2H2O Merck 1.065576
5. DMSO Wako 042-21765
6. DPX mounting medium Scharlau DP0050
7. Ethanol Fisher 64-17-5
8. Formalin Scharlau FO 0010
9. Isopropanol Tedia PR-1490
10. Monobasic Sodium Phosphate, NaH2PO4.2H2O Merck 1.06345
11. Paraffin Carl Roth Paraplast Plus
12. PEG-400 Scharlau PO0035
13. Roti-Histofix Carl Roth P087.1
14. Trizol Life Technologies 15596-026
15. Xylene Scharlau X10057
131
List of Staining Reagents
S.No. Name of Chemical Source Catalogue No.
1. 0.5% Eosin Y solution (Eosin G) Carl Roth X883.1
2. Hematoxylin Carl Roth T864.2
List of Instruments
S.No. Instrument Company
1. Centrifuge 5810R Eppendorf
2. Chemical Fume hood Lab Tech
3. Electronic Balance Sartorius, CP225D
4. Homogenizer Ultra-Turrax, T8, IKA, Germany
5. Hot plate drier for slides Medite
6. Incubator LabTech
7. Magnetic Hotplate stirrer Lab Tech
8. Master Cycler Pro S PCR Machine Eppendorf
9. Microtome Yidi
10. Nanodrop 2000
Spectrophotometer
ThermoScientific
11. Oven LabTech
132
12. Paraffin Embedding System Kedee
13. Periplastic Pump Eyela
14. pH meter Jenway
15. Real Time PCR Machine Agilent Technologies Stratagene Mx3000P
16. Surgical Instruments Local
17. Upright multichannel fluorescence
microscope
90i, Nikon Corporation, Japan
18. Vortex Mixer Lab Tech
19. Water bath LabTech
20. Water Purification System Millipore, Elix 5, Simplicity
133
APPENDIX II
1. Phosphate Buffer Saline
(a) 0.1 M Mono Basic Phosphate NaH2PO4.2H2O (Mol. Wt 156)
Dissolve 15.6gm in 1 L of 18.2 MΩ water to make 0.1M solution. Store this stock solution
at room temperature.
(b) 0.1M Di Basic Phosphate Na2HPO4.2H2O (Mol Wt. 178)
Dissolve 17.8gm in 1 L of 18.2 MΩ water to make 0.1 M solution. Put 1 L water in a beaker
and start stirring. Then add a small amount of Dibasic phosphate. Make sure to dissolve the
whole amount and only then add some more Dibasic phosphate and dissolve it. In this manner
add Dibasic phosphate gradually until the whole amount is dissolved. Store this stock solution
at room temperature. Never put the stock solution in the freeze as the Dibasic phosphate will
crash out of the solution.
(c) 0.1 M PB preparation
Take 500 mL 0.1 M Dibasic phosphate in a beaker. Put the beaker on the hotplate magnetic
stirrer. Put the electrode of the pH meter and the temperature sensitive probe in the solution
and start stirring at a medium speed. Add 0.1 M Mono basic phosphate until pH reaches 7.3.
Usually ~280 mL Mono is needed for 500 mL Di.
(d) 0.05 M PBS
Take 500 ml of 0.1 M PB and 500 ml of water 18.2 MΩ in 1L graduated cylinder. Add 9gm
of NaCl and stir through magnetic stirrer. Transfer in a clean and labeled 1L bottle and keep
at room temperature.
2. Formaline Fixative (3.7%) or Neutral Buffered Formaline (NBF)
1. Dissolve 200ml of Phosphate buffer (0.1 M), 40ml Formaline (37%) and 160ml of
simplicity water 18.2M in a beaker through magnetic stirrer.
2. Keep it at room temperature.
3. Decalcification Medium
Formalin (37%) 90 mL
Formic acid 10 mL
134
LIST OF PUBLICATIONS
1. Gul, A., Kunwar, B., Mazhar, M., Perveen, K., Simjee, S. U. (2017). N-(2-
Hydroxyphenyl) acetamide: a Novel Suppressor of RANK/RANKL Pathway in
Collagen-Induced Arthritis Model in Rats. Inflammation. doi: 10.1007/s10753-017-
0561-1.
2. Mazhar, M., Faizi, S., Gul, A., Kabir, N., Simjee, S. U. (2017). Effects of naturally
occurring flavonoids on ferroportin expression in the spleen in iron deficiency anemia
in vivo. RSC Advances. doi: 10.1039/c7ra02138k.
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