Treatment of Nonalcoholic Fatty Liver Disease with Total ... fileResearchArticle Treatment of Nonalcoholic Fatty Liver Disease with Total Alkaloids in Rubus aleaefolius Poir through

Research ArticleTreatment of Nonalcoholic Fatty LiverDisease with Total Alkaloids in Rubus aleaefolius Poir throughRegulation of Fat Metabolism

Ying Li12 Jinyan Zhao34 Haiyin Zheng5 Xiaoyong Zhong34

Jianheng Zhou5 and Zhenfeng Hong34

1 Xiamen Hospital of Traditional Chinese Medicine Xiamen Fujian 361009 China2 Jinshan Street Community Health Service Xiamen Fujian 361000 China3 Academy of Integrative Medicine Fujian University of Traditional Chinese Medicine Fuzhou Fujian 350122 China4 Fujian Key Laboratory of Integrative Medicine on Geriatrics Fujian University of Traditional Chinese MedicineFuzhou Fujian 350122 China

5 Department of Integrative Medicine Fuzhou Fujian 350122 China

Correspondence should be addressed to Zhenfeng Hong zhenfenghongyahoocom

Received 5 May 2014 Accepted 7 August 2014 Published 14 October 2014

Academic Editor I-Min Liu

Copyright copy 2014 Ying Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Total alkaloids inRubus aleaefolius Poir (TARAP) is a folkmedicinal herb that has been used clinically inChina to treat nonalcoholicfatty liver disease (NAFLD) for many years However the mechanism of its anti-NAFLD effect is largely unknown In this studywe developed a NAFLD rat model by supplying a modified high-fat diet (mHFD) ad libitum for 8 weeks and evaluated thetherapeutic effect of TARAP inNAFLD rats as well as the underlyingmolecularmechanismWe found that TARAP could reduce theserum triglycerides (TG) total cholesterol (TC) and low-density lipoprotein (LDL-C) levels and increase the serum high-densitylipoprotein (HDL-C) level in NAFLD rats In addition TARAP treatment reduced expression of fatty acid synthetase (FAS) andacetyl-CoA carboxylase (ACC) and upregulated the expression of carnitine palmitoyltransferase (CPT) Our results suggest thatregulation of lipid metabolism may be a mechanism by which TARAP treats NAFLD

1 Introduction

Nonalcoholic fatty liver disease (NAFLD) is increasing inprevalence worldwide and is currently the most commonlydiagnosed liver disorder in western countries [1ndash4] It isestimated that NAFLD occurs in 10ndash24 of the population[5ndash7] Despite the elevated prevalence the pathogenesisof NAFLD remains poorly understood Abnormal lipidmetabolism and specifically excessive lipid accumulationare critical to NAFLD pathogenesis [8] Insulin resistanceoxidative stress and local microcirculation dysfunction mayalso contribute to development of NAFLD [9] Previousstudies demonstrated that the expression of genes encodingacetyl-CoA carboxylase (ACC) fatty acid synthetase (FAS)and carnitine palmitoyltransferase (CPT) was significantly

altered in NAFLD [10] further linking the occurrence ofNAFLD with disorders of hepatic lipid metabolism

Due to the key role of lipid accumulation in NAFLDprogression inhibiting lipid accumulation is a major focusof anti-NAFLD drug development A variety of anti-NAFLDagents are currently in preclinical development with somebeing currently used in clinical trials Unfortunately wideuse of these agents produces adverse outcomes consequentlythe most effective agents with minimal side effects are highlydesired

Natural products including traditional Chinesemedicine(TCM) exhibit relatively few side effects and have been inclinical use for thousands of years making such therapiesimportant alternative remedies for various diseases [11]Although the mechanism of action is unknown herbal

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 768540 7 pageshttpdxdoiorg1011552014768540

2 Evidence-Based Complementary and Alternative Medicine

remedies such as hawthorn Radix bupleuri and dried tan-gerine peel [12] have long been used to successfully treatNAFLD

Rubus (Rubus L) belonging to the Rosaceae family isa medicinal herb widely distributed throughout the worldRubus aleaefolius Poir (R aleaefolius) is one Rubus speciesused for heat-clearing arresting bleeding promotion ofblood circulation and removal of blood stasis It has beenused to treat various types of hepatitis in Anxi County ofFujian Province China and has shown significant thera-peutic effects on NAFLD Our previous studies in a carbontetrachloride-induced acute liver injurymodel in rats showedthat total alkaloids inRubus aleaefolius Poir (TARAP) amelio-rated adipose degeneration suggesting that TARAP could bean effective therapy for NAFLD [13] nonetheless the mech-anism of its anti-NAFLD activity still remains unknown Tofurther understand the mechanisms by which TARAP exertsanti-NAFLD effects we developed a rat NAFLD model andevaluated the effect of TARAP therapy on the expression ofgenes associated with lipid metabolism

2 Materials and Methods

21 Preparation of Total Alkaloids from Rubus aleaefolius Poir(TARAP) TARAPwas prepared as previously described [14]Briefly the root of R alceifolius Poir was collected fromAnxi of Fujian Province and the alkaloids were extractedaccording to the following procedure The herb powder (1 g)was extracted with 50mL chloroform methanol ammoniasolution (15 4 3) for 2 hours in an ice bath followed by30min of sonication and filtration The filtered solution wascollected and dried The resultant residue was dissolved in2mL of 2 sulfuric acid solution and filteredThe filter paperand residue were rewashed with 2mL of 2 sulfuric acidsolution and buffer solution (pH 36) Buffer was then addedto a final volume of 50mL and the solution was saved forfuture use Acid dye colorimetry was used to measure totalalkaloid content Total alkaloid yield was 081mg of alkaloidper gram of initial herb powder

22 Reagents Polyene phosphatidylcholine (PP) was pur-chased from Sanofi-Aventis Ukraine Limited Liability Com-pany Trizol kit M-MLV first strand cDNA synthesis kit andTaqDNApolymerase were purchased fromLife Technologies(Carlsbad CA USA) SuperScript II reverse transcriptasewas purchased from Promega (Madison WI USA) Biow-est Agarose gel was purchased from Spain Assay kits formeasuring triglycerides (TG) total cholesterol (TC) high-density lipoprotein (HDL-C) and low-density lipoprotein(LDL-C) activity were obtained from the Jiancheng Instituteof Biotechnology (Nanjing China) All other chemicalsunless otherwise stated were obtained from Sigma-AldrichChemicals (St Louis MO USA)

23 Development of NAFLDAnimal Model Male 8-week-oldSprage-Dawley (SD) rats (Slike Co Ltd Shanghai China)weighing 180sim200 g were housed five per cage in an envi-ronmentally controlled roomwith a temperature of 22 plusmn 1∘C

relative humidity of 40ndash60 air ventilation of 12ndash18 timeshand a 1212 h artificial lightdark cycle of 150ndash300 lux Therats were provided with food and water ad libitum Allanimal studies were approved by the Fujian Institute of Tradi-tional Chinese Medicine Animal Ethics Committee (FuzhouChina) Experimental procedures were in accordance withtheGuidelines for Animal Experimentation of FujianUniver-sity of Traditional Chinese Medicine (Fuzhou China)

To establish the animal model 48 rats were randomlydivided into four groups (normal group model group and2 treatment groups) with 12 rats in each group after adaptivebreeding The rats in the model and treatment groups werefed with a modified high-fat diet (mHFD) ad libitum for 8weeks while rats in the normal group received a normal fatdietThemHFD recipe used to develop theNAFLD ratmodelwas composed of 873 basal fodder 10 lard 2 cholesteroland 07 swine bile salt After 8 weeks on the mHFD therats in the treatment group were randomly divided into high-dose TARAP group (144 gkg bodyweightday) and low-doseTARAP group (072 gkg bodyweightday) The rats in thenormal group and model group were orally treated withdistilled water (10mLkg) Body weights and food uptakewere measured once a week After 28 days the animals wereweighed and anaesthetized by intraperitoneal (ip) injectionof 1 ketamine-diazepam after 4-hour food deprivation andblood samples were obtained aseptically from the abdominalaorta for measuring TG TC HDL-C and LDL-C levelsLivers were rapidly dissected and part of each liver wascut and fixed in 4 formaldehyde in normal saline forhistological analysis The rest of the liver was snap-frozen inliquid nitrogen and stored at minus70∘C until being used

24 Histological Examination Small pieces of liver tissuewere collected from the same position of each rat andfixed with 10 formalin for 24 h Samples were paraffinembedded and then sections of 4-5mm were prepared Thesections were subsequently stained with hematoxylin andeosin (HampE) Histologic evaluation was performed twiceby a pathologist unaware of the treatments on 2 separateoccasions A semiquantitative scoring system was used toassess the severity of hepatic steatosis and inflammatory cellinfiltration in 10 microscopic fields as described previously[15] In brief the following criteria were used for scoringhepatic steatosis grade 0 (minus) no fat grade 1 (+) fattyhepatocytes occupying 33 of the hepatic parenchyma grade2 (++) fatty hepatocytes occupying 33ndash66 of the hepaticparenchyma and grade 3 (+++) fatty hepatocytes occupyinggt66 of the hepatic parenchyma

25 Biochemical Assays Blood-containing tubeswere allowedto stand at room temperature for 2 h Serums were obtainedby centrifugation at 3000timesg for 20min in 4∘C and stored inminus20∘C Serums TG TC HDL-C and LDL-C levels weremea-sured by the kits according to the manufacturerrsquos instruction

26 RNA Extraction and RT-PCR Analysis Total RNAwas isolated from fresh liver tissue using Trizol reagentOligo(dT)-primed RNA (1120583g) was reverse-transcribed with

Evidence-Based Complementary and Alternative Medicine 3

Control Model PP

TARAP-L TARAP-H

Figure 1 Effects of TARAP on hepatic morphology in mHFD rats Control ldquominusrdquo no fat model ldquo+++rdquo fatty hepatocytes occupying gt66 ofthe hepatic parenchyma PP ldquo+rdquo fatty hepatocytes occupying 33 of the hepatic parenchyma TARAP-L ldquo++rdquo fatty hepatocytes occupying33ndash66 of the hepatic parenchyma TARAP-H ldquo+rdquo fatty hepatocytes occupying 33 of the hepatic parenchyma representative images weretaken at a magnification times400

SuperScript II reverse transcriptase according to the manu-facturerrsquos instructionsThe obtained cDNAwas used for PCRGAPDH was used as an internal control The primers usedfor amplification of FAS ACC CPT and GAPDH genes wereas follows FAS F 51015840-CCT TAG TAC TGC GTG GTC GTAT-31015840 R3 51015840-CAG AGG GTG CTT GTT AGA AAG AT-31015840(301 BP) ACC F 51015840-TGA GGA GGA CCG CAT TTA TC-31015840 R 51015840-GAA GCT TCC TTC GTG ACC AG-31015840 (565 bp)CPT F 51015840-TAT GTG AGG ATG CTG CTT CC-31015840 R 51015840-CTCGGA GAG CTA AGC TTG TC-31015840(629 bp) GAPDH F 51015840-AGA TCC ACA ACG GAT-31015840 R 51015840-TCC CTC AAG ATTGTC AGC AA-31015840 (308 bp) Amplification of each gene wasperformed with a thermal cycler (GE9600 USA) using thefollowing cycling parameters denaturing 95∘C for 5min 30cycles of 95∘C for 30 s and annealing temperature for 30 s and72∘C for 1min followed by a final extension of 10min at 72∘CGene expression was determined for 6ndash8 samples randomlyselected from each group and each sample was replicated for3 timesThe PCR products were separated by electrophoresison a 15 agarose gel The DNA bands were examined usinga Gel Documentation System (BioRad Model Gel Doc 2000USA)

27 Immunohistochemical Assay A 05 cm times 05 cm times 01 cmblock of tissue was collected from liver tissue of each ratTissue blocks were rinsed with phosphate buffer solution(PBS) fixed with 10 formaldehyde for 12ndash24 h and subse-quently embedded in paraffin archived and then sectionedThe paraffin sections were used for FAS ACC and CPTimmunohistochemical stainingThe primary antibodies were

polyclonal rabbit anti-rat FAS ACC and CPT (all in 1 200dilution Santa Cruz Biotechnology) PBS was used to replacethe primary antibody as a negative control After washingwith PBS slides were incubated with biotinylated secondaryantibody followed by conjugated horseradish peroxidase(HRP) labelled streptavidin (Dako) and then washed withPBS Color was developed using DAB chromogen accord-ing to the manufacturerrsquos instructions After staining fivehigh-power fields (400x) were randomly selected in eachslide and the average proportions of positive cells in eachfield were counted using the true color multifunctional cellimage analysis management system (Image-Pro Plus MediaCybernetics USA)

28 Statistical Analyses All data are the means of three mea-surements and data were analyzed using the SPSS package forWindows (Version 115 USA) Statistical analysis of the datawas performed with Studentrsquos 119905-test and ANOVA with posthoc analysis of significance was determined Differences with119875 lt 005 were considered statistically significant

3 Results

31 TARAP Ameliorated Hepatic Steatosis in NAFLD RatsHistological examination is regarded as the ldquogold standardrdquofor determining the presence and severity of NAFLD [16]We therefore assessed the therapeutic efficacy of TARAP byexamining its effect on histological changes in the liver tissuesof NAFLD rats Compared with control group rats withnormal liver histology rats in the model group developed


00

02

04

TARAP-HTARAP-LPPModel

TG (m

mol

L)

Control00

12

24


TC (m

mol

L)

Control

lowast

lowast

(a)

00

06

12


HD

L-C

(mol

L)

Control00

06

12

TARAP-HTARAP-LModel

LDL-

C (m

olL

)

Control PP

lowast

lowast

(b)

Figure 2 Effects of TARAP on mHFD-induced TG TC HDL-C and LDL-C in rats (a) TARAP significantly reduced serum TG and TC aswell as (b) LDL-C lowast119875 lt 005 (model versus control) 119875 lt 005 (high or low versus model) ANOVA and post hoc test

hepatic steatohepatitis characterized by the accumulation offat hepatocyte ballooning scattered lobular inflammatorycell infiltration and inflammatory foci (Figure 1) TARAPtreatment significantly ameliorated hepatic steatosis andinflammation in a dose-dependent manner

32 TARAP Altered the Levels of Serums TG TC HDL-Cand LDL-C in NAFLD Rats To further confirm the anti-NAFLD effect of TARAP we examined the levels of serumsTG TC HDL-C and LDL-C The rats in the model grouphad significantly higher serum TC TG and LDL-C levelsand lower HDL-C level compared with the normal rats (119875 lt005) (Figures 2(a) and 2(b)) The elevation of TC TG andLDL-C levels and reduction of HDL-C in NAFLD rats wereneutralized by TARAP treatment (119875 lt 005)

33 TARAP Suppressed Expression of ACC FAS and CPTin NAFLD Rats To explore the mechanism of anti-NAFLDactivity of TARAP we assessed the mRNA and proteinexpression of ACC FAS and CPT in liver tissues usingRT-PCR assay and immunohistochemistry analysis (IHC)respectively The results showed that the mRNA and proteinlevels of ACC and FAS were notably increased whereas CPTlevels were significantly decreased in the liver tissues of themodel group compared with the control group (119875 lt 005)(Figure 3) TARAP treatment significantly neutralized thealteration of ACC FAS and CPT mRNA and protein levelsin NAFLD rats (119875 lt 005) (Figures 4 and 5)

ACC

FAS

CPT

GAPDH

Model Control PP TARAP-L TARAP-H

Figure 3 Effects of TARAP on FAS ACC and CPT mRNA expres-sion in mHFD induced NAFLD rat GAPDH was used as internalcontrols for RT-PCR assays This experiment was performed intriplicate and similar results were obtained

4 Discussion

Nonalcoholic fatty liver disease (NAFLD) one of the mostcommon liver diseases in developed countries [17] is charac-terized by lipid accumulation in hepatocytes (gt5 of the liverweight) [18] NAFLD is one of the components of metabolicsyndrome (MS) brought on by obesity lack of physicalexercise and a high calorie diet [19 20] Pharmacotherapy isthe primary treatment for NAFLD however most medicinesexhibit some side effects As a result people are alwaysseeking natural products with less adverse effects for NAFLDtreatment


Con

trol

Mod

elPP

TARA

P-L

TARA

P-H

FAS ACC CPT

(a) (b) (c)

Figure 4 Protein expression and localization of hepatic FAS ACC and CPT in control model and different experimental groups Repre-sentative images are shown at 400x magnification


0

30

60


The g

ray

of A

CC

Control0

60

120


The g

ray

of F

AS

Control

0

60

120


The g

ray

of C

PT

Control

Group FAS ACC CPT

ControlModel

PPTARAP-LTARAP-H

2159 plusmn 544

9529 plusmn 978lowast

3965 plusmn 782

3286 plusmn 859

2535 plusmn 567

2238 plusmn 165


3265 plusmn 285

3121 plusmn 127

2928 plusmn 553

5358 plusmn 382


5655 plusmn 333

678 plusmn 305

7181 plusmn 467

Figure 5 Quantification of IHC assay is presented as gray value of positively stained cells lowast119875 lt 005 (model versus control) 119875 lt 005 (highor low versus model) ANOVA and post hoc test This experiment was performed in triplicate and similar results were obtained

Rubus aleaefolius Poir has relatively fewer side effects andhas been used clinically for thousands of years to treat variousdiseases including NAFLD As a well-known and importantChinese medicinal herb Rubus aleaefolius Poir has been usedas a major component in several TCM formulas for clinicaltreatment without apparent side effects Rubus aleaefoliusPoir is an important traditional heat-clearing and dampness-eliminating Chinese herb withmany reported pharmacologi-cal applications Recently ithas been demonstrated to imparthepatoprotection in carbon tetrachloride-induced acute liverinjury in rats Itsmechanismof anti-NAFLD activity is largelyunknown Therefore before Rubus aleaefolius Poir can befurther developed as an anti-NAFLD agent its underlyingmolecular mechanism should first be elucidated Here wereport for the first time that the total alkaloids in Rubusaleaefolius Poir (TARAP) exerts a beneficial effect on lipidmetabolism in NAFLD induced by a high-fat diet

Although the cause of NAFLD is not well understoodit is generally considered that excessive lipid accumulationwhich typically results from abnormal activation of lipidmetabolism plays a critical role in NAFLD development Inthe present study we demonstrated that TARAP significantlyreduced the serum levels of TC TG and LDL-C in NAFLDrats indicating that TARAP could modulate hyperlipidemia

Lipid metabolism is a complex process involving variousenzymes Some studies showed that expression of genesencoding lipid metabolism-related enzymes for exampleacetyl-CoA carboxylase (ACC) fatty acid synthetase (FAS)and carnitine palmitoyltransferase (CPT) was significantly

altered in NAFLD [21] further confirming a close correlationbetween the occurrence of NAFLD and disorders in hepaticlipid metabolism

ACC catalyzes the transformation of acetyl-CoA tomalonyl-CoA a key step in lipid metabolism Malonyl-CoAcan catalyze dietary-derived simple essential fatty acids intohigher polyunsaturated fatty acids [17] which is beneficialfor the body In addition malonyl-CoA binding inhibitsCPT thus preventing fatty acid oxidation [22] The resultis that the body obtains energy from mitochondrial fattyacid oxidation Although long-chain fatty acids provide theenergy by 120573-oxidation it also requires the CPT systemto enter liver mitochondria Under physiological conditionCPT is the primary regulator of fatty acid metabolism in theliver [23] Therefore disruption in mitochondrial fatty acidoxidation due to inhibition of the hepatic CPT system playsan important role in the pathogenesis of hepatic steatosis [24]Acetyl-CoA and malonyl-CoA can synthesize long-chainfatty acids by FAS which is important in fatty acid synthesisvia de novo lipogenesis FAS participates in the final step inthe fatty acid pathway by controlling the quantity of the fattyacid synthetase mRNA [25 26] Therefore it is considereda key enzyme of the lipid synthesis pathway Our resultsdemonstrated that TARAP could reduce the expression ofACC and FAS and increase the expression of CPT in livertissue of NAFLD rats

In summary we report that regulation of lipid metabo-lism may be one of the mechanisms by which TARAP treatsNAFLD


Conflict of Interests

The authors declared no potential conflict of interests withrespect to the authorship andor publication of this paper

Acknowledgments

This work was supported by the Nature Science Foundationof Fujian Province of China (nos 2010J01191 and 2010J01194)and the Project sponsored byMedical Originality Foundationof Fujian Province of China (no 2009-CX-18) The authorsthank Clarity Manuscript Consultants LLC for assistancewith editing the paper

References

[1] J M Clark F L Brancati and A M Diehl ldquoNonalcoholic fattyliver diseaserdquo Gastroenterology vol 122 no 6 pp 1649ndash16572002

[2] C A Matteoni Z M Younossi T Gramlich N Boparai andA J McCullough ldquoNonalcoholic fatty liver disease a spectrumof clinical and pathological severityrdquo Gastroenterology vol 116no 6 pp 1413ndash1419 1999

[3] E M Brunt C G Janney A M Di Bisceglie B A Neusch-wander-Tetri and B R Bacon ldquoNonalcoholic steatohepatitis aproposal for grading and staging the histological lesionsrdquo TheAmerican Journal of Gastroenterology vol 94 no 9 pp 2467ndash2474 1999

[4] N M W de Alwis and C P Day ldquoNon-alcoholic fatty liverdisease the mist gradually clearsrdquo Journal of Hepatology vol48 pp S104ndashS112 2008

[5] C-H ChenM-HHuang J-C Yang et al ldquoPrevalence and riskfactors of nonalcoholic fatty liver disease in an adult populationof Taiwan metabolic significance of nonalcoholic fatty liverdisease in nonobese adultsrdquo Journal of Clinical Gastroenterologyvol 40 no 8 pp 745ndash752 2006

[6] S Bellentani G Saccoccio F Masutti et al ldquoPrevalence of andrisk factors for hepatic steatosis in Northern Italyrdquo Annals ofInternal Medicine vol 132 no 2 pp 112ndash117 2000

[7] L Shen J-G Fan Y Shao et al ldquoPrevalence of nonalcoholicfatty liver among administrative officers in Shanghai an epi-demiological surveyrdquoWorld Journal of Gastroenterology vol 9no 5 pp 1106ndash1110 2003

[8] P Angulo ldquoNonalcoholic fatty liver diseaserdquo The New EnglandJournal of Medicine vol 346 pp 1221ndash1231 2002

[9] A J McCullough ldquoUpdate on nonalcoholic fatty liver diseaserdquoJournal of Clinical Gastroenterology vol 34 no 3 pp 255ndash2622002

[10] A K Saha and N B Ruderman ldquoMalonyl-CoA and AMP-activated protein kinase an expanding partnershiprdquoMolecularand Cellular Biochemistry vol 253 no 1-2 pp 65ndash70 2003

[11] H-F Ji X-J Li and H-Y Zhang ldquoNatural products and drugdiscoveryrdquo EMBO Reports vol 10 no 3 pp 194ndash200 2009

[12] K Q Shi Y C Fan W Y Liu et al ldquoTraditional Chinesemedicines benefit to nonalcoholic fatty liver disease a system-atic review and meta-analysisrdquo Molecular Biology Reports vol39 no 10 pp 9715ndash9722 2012

[13] Z-F Hong T-J Li and J-Y Zhao ldquoEffect of total alkaloids ofRubus alceaefolius poiron on gene expressions of CYP2E1 andCYP3A1 in rats with acute liver injuryrdquo Zhongguo Zhong Xi YiJie He Za Zhi vol 29 no 8 pp 711ndash715 2009

[14] J Lin J Zhao T Li J Zhou J Hu and Z Hong ldquoHepatopro-tection in a rat model of acute liver damage through inhibitionof CY2E1 activity by total alkaloids extracted from Rubusalceifolius poirrdquo International Journal of Toxicology vol 30 no2 pp 237ndash243 2011

[15] H L Bonkovsky Q Jawaid K Tortorelli et al ldquoNon-alcoholicsteatohepatitis and iron increased prevalence of mutationsof the HFE gene in non-alcoholic steatohepatitisrdquo Journal ofHepatology vol 31 no 3 pp 421ndash429 1999

[16] E M Brunt ldquoSteatohepatitis pathologic features and differen-tial diagnosisrdquo Seminars in Diagnostic Pathology vol 22 no 4pp 330ndash338 2005

[17] C Postic and J Girard ldquoContribution of de novo fatty acid syn-thesis to hepatic steatosis and insulin resistance lessons fromgenetically engineered micerdquo Journal of Clinical Investigationvol 118 no 3 pp 829ndash838 2008

[18] P Angulo ldquoGI epidemiology nonalcoholic fatty liver diseaserdquoAlimentary Pharmacology amp Therapeutics vol 25 no 8 pp883ndash889 2007

[19] J B Dixon P S Bhathal and P E OrsquoBrien ldquoNonalcoholic fattyliver disease predictors of nonalcoholic steatohepatitis and liverfibrosis in the severely obeserdquo Gastroenterology vol 121 no 1pp 91ndash100 2001

[20] G Marchesini M Brizi G Blanchi et al ldquoNonalcoholic fattyliver disease a feature of themetabolic syndromerdquoDiabetes vol50 no 8 pp 1844ndash1850 2001

[21] A K Saha and N B Ruderman ldquoMalonyl-coa and AMP-activated protein kinase an expanding partnershiprdquoMolecularand Cellular Biochemistry vol 253 no 1-2 pp 65ndash70 2003

[22] N Ruderman and M Prentki ldquoAMP kinase and malonyl-CoAtargets for therapy of the metabolic syndromerdquo Nature ReviewsDrug Discovery vol 3 no 4 pp 340ndash351 2004

[23] K Bartlett and S Eaton ldquoMitochondrial120573-oxidationrdquoEuropeanJournal of Biochemistry vol 271 no 3 pp 462ndash469 2004

[24] W-N Cong R-Y Tao J-Y Tian G-T Liu and F Ye ldquoTheestablishment of a novel non-alcoholic steatohepatitis modelaccompanied with obesity and insulin resistance in micerdquo LifeSciences vol 82 no 19-20 pp 983ndash990 2008

[25] D Saggerson ldquoMalonyl-CoA a key signaling molecule inmammalian cellsrdquo Annual Review of Nutrition vol 28 pp 253ndash272 2008

[26] D L Cinti L Cook M N Nagi and S K Suneja ldquoThe fattyacid chain elongation system of mammalian endoplasmic retic-ulumrdquo Progress in Lipid Research vol 31 no 1 pp 1ndash51 1992


remedies such as hawthorn Radix bupleuri and dried tan-gerine peel [12] have long been used to successfully treatNAFLD

Rubus (Rubus L) belonging to the Rosaceae family isa medicinal herb widely distributed throughout the worldRubus aleaefolius Poir (R aleaefolius) is one Rubus speciesused for heat-clearing arresting bleeding promotion ofblood circulation and removal of blood stasis It has beenused to treat various types of hepatitis in Anxi County ofFujian Province China and has shown significant thera-peutic effects on NAFLD Our previous studies in a carbontetrachloride-induced acute liver injurymodel in rats showedthat total alkaloids inRubus aleaefolius Poir (TARAP) amelio-rated adipose degeneration suggesting that TARAP could bean effective therapy for NAFLD [13] nonetheless the mech-anism of its anti-NAFLD activity still remains unknown Tofurther understand the mechanisms by which TARAP exertsanti-NAFLD effects we developed a rat NAFLD model andevaluated the effect of TARAP therapy on the expression ofgenes associated with lipid metabolism

2 Materials and Methods

21 Preparation of Total Alkaloids from Rubus aleaefolius Poir(TARAP) TARAPwas prepared as previously described [14]Briefly the root of R alceifolius Poir was collected fromAnxi of Fujian Province and the alkaloids were extractedaccording to the following procedure The herb powder (1 g)was extracted with 50mL chloroform methanol ammoniasolution (15 4 3) for 2 hours in an ice bath followed by30min of sonication and filtration The filtered solution wascollected and dried The resultant residue was dissolved in2mL of 2 sulfuric acid solution and filteredThe filter paperand residue were rewashed with 2mL of 2 sulfuric acidsolution and buffer solution (pH 36) Buffer was then addedto a final volume of 50mL and the solution was saved forfuture use Acid dye colorimetry was used to measure totalalkaloid content Total alkaloid yield was 081mg of alkaloidper gram of initial herb powder

22 Reagents Polyene phosphatidylcholine (PP) was pur-chased from Sanofi-Aventis Ukraine Limited Liability Com-pany Trizol kit M-MLV first strand cDNA synthesis kit andTaqDNApolymerase were purchased fromLife Technologies(Carlsbad CA USA) SuperScript II reverse transcriptasewas purchased from Promega (Madison WI USA) Biow-est Agarose gel was purchased from Spain Assay kits formeasuring triglycerides (TG) total cholesterol (TC) high-density lipoprotein (HDL-C) and low-density lipoprotein(LDL-C) activity were obtained from the Jiancheng Instituteof Biotechnology (Nanjing China) All other chemicalsunless otherwise stated were obtained from Sigma-AldrichChemicals (St Louis MO USA)

23 Development of NAFLDAnimal Model Male 8-week-oldSprage-Dawley (SD) rats (Slike Co Ltd Shanghai China)weighing 180sim200 g were housed five per cage in an envi-ronmentally controlled roomwith a temperature of 22 plusmn 1∘C

relative humidity of 40ndash60 air ventilation of 12ndash18 timeshand a 1212 h artificial lightdark cycle of 150ndash300 lux Therats were provided with food and water ad libitum Allanimal studies were approved by the Fujian Institute of Tradi-tional Chinese Medicine Animal Ethics Committee (FuzhouChina) Experimental procedures were in accordance withtheGuidelines for Animal Experimentation of FujianUniver-sity of Traditional Chinese Medicine (Fuzhou China)

To establish the animal model 48 rats were randomlydivided into four groups (normal group model group and2 treatment groups) with 12 rats in each group after adaptivebreeding The rats in the model and treatment groups werefed with a modified high-fat diet (mHFD) ad libitum for 8weeks while rats in the normal group received a normal fatdietThemHFD recipe used to develop theNAFLD ratmodelwas composed of 873 basal fodder 10 lard 2 cholesteroland 07 swine bile salt After 8 weeks on the mHFD therats in the treatment group were randomly divided into high-dose TARAP group (144 gkg bodyweightday) and low-doseTARAP group (072 gkg bodyweightday) The rats in thenormal group and model group were orally treated withdistilled water (10mLkg) Body weights and food uptakewere measured once a week After 28 days the animals wereweighed and anaesthetized by intraperitoneal (ip) injectionof 1 ketamine-diazepam after 4-hour food deprivation andblood samples were obtained aseptically from the abdominalaorta for measuring TG TC HDL-C and LDL-C levelsLivers were rapidly dissected and part of each liver wascut and fixed in 4 formaldehyde in normal saline forhistological analysis The rest of the liver was snap-frozen inliquid nitrogen and stored at minus70∘C until being used

24 Histological Examination Small pieces of liver tissuewere collected from the same position of each rat andfixed with 10 formalin for 24 h Samples were paraffinembedded and then sections of 4-5mm were prepared Thesections were subsequently stained with hematoxylin andeosin (HampE) Histologic evaluation was performed twiceby a pathologist unaware of the treatments on 2 separateoccasions A semiquantitative scoring system was used toassess the severity of hepatic steatosis and inflammatory cellinfiltration in 10 microscopic fields as described previously[15] In brief the following criteria were used for scoringhepatic steatosis grade 0 (minus) no fat grade 1 (+) fattyhepatocytes occupying 33 of the hepatic parenchyma grade2 (++) fatty hepatocytes occupying 33ndash66 of the hepaticparenchyma and grade 3 (+++) fatty hepatocytes occupyinggt66 of the hepatic parenchyma

25 Biochemical Assays Blood-containing tubeswere allowedto stand at room temperature for 2 h Serums were obtainedby centrifugation at 3000timesg for 20min in 4∘C and stored inminus20∘C Serums TG TC HDL-C and LDL-C levels weremea-sured by the kits according to the manufacturerrsquos instruction

26 RNA Extraction and RT-PCR Analysis Total RNAwas isolated from fresh liver tissue using Trizol reagentOligo(dT)-primed RNA (1120583g) was reverse-transcribed with


Control Model PP

TARAP-L TARAP-H






3 Results



00

02

04


TG (m

mol

L)

Control00

12

24


TC (m

mol

L)

Control

lowast

lowast

(a)

00

06

12


HD

L-C

(mol

L)

Control00

06

12

TARAP-HTARAP-LModel

LDL-

C (m

olL

)

Control PP

lowast

lowast

(b)





ACC

FAS

CPT

GAPDH



4 Discussion



Con

trol

Mod

elPP

TARA

P-L

TARA

P-H

FAS ACC CPT

(a) (b) (c)



0

30

60


The g

ray

of A

CC

Control0

60

120


The g

ray

of F

AS

Control

0

60

120


The g

ray

of C

PT

Control

Group FAS ACC CPT

ControlModel

PPTARAP-LTARAP-H

2159 plusmn 544


3965 plusmn 782

3286 plusmn 859

2535 plusmn 567

2238 plusmn 165


3265 plusmn 285

3121 plusmn 127

2928 plusmn 553

5358 plusmn 382


5655 plusmn 333

678 plusmn 305

7181 plusmn 467











Acknowledgments


References




























Control Model PP

TARAP-L TARAP-H






3 Results



00

02

04


TG (m

mol

L)

Control00

12

24


TC (m

mol

L)

Control

lowast

lowast

(a)

00

06

12


HD

L-C

(mol

L)

Control00

06

12

TARAP-HTARAP-LModel

LDL-

C (m

olL

)

Control PP

lowast

lowast

(b)





ACC

FAS

CPT

GAPDH



4 Discussion



Con

trol

Mod

elPP

TARA

P-L

TARA

P-H

FAS ACC CPT

(a) (b) (c)



0

30

60


The g

ray

of A

CC

Control0

60

120


The g

ray

of F

AS

Control

0

60

120


The g

ray

of C

PT

Control

Group FAS ACC CPT

ControlModel

PPTARAP-LTARAP-H

2159 plusmn 544


3965 plusmn 782

3286 plusmn 859

2535 plusmn 567

2238 plusmn 165


3265 plusmn 285

3121 plusmn 127

2928 plusmn 553

5358 plusmn 382


5655 plusmn 333

678 plusmn 305

7181 plusmn 467











Acknowledgments


References




























00

02

04


TG (m

mol

L)

Control00

12

24


TC (m

mol

L)

Control

lowast

lowast

(a)

00

06

12


HD

L-C

(mol

L)

Control00

06

12

TARAP-HTARAP-LModel

LDL-

C (m

olL

)

Control PP

lowast

lowast

(b)





ACC

FAS

CPT

GAPDH



4 Discussion



Con

trol

Mod

elPP

TARA

P-L

TARA

P-H

FAS ACC CPT

(a) (b) (c)



0

30

60


The g

ray

of A

CC

Control0

60

120


The g

ray

of F

AS

Control

0

60

120


The g

ray

of C

PT

Control

Group FAS ACC CPT

ControlModel

PPTARAP-LTARAP-H

2159 plusmn 544


3965 plusmn 782

3286 plusmn 859

2535 plusmn 567

2238 plusmn 165


3265 plusmn 285

3121 plusmn 127

2928 plusmn 553

5358 plusmn 382


5655 plusmn 333

678 plusmn 305

7181 plusmn 467











Acknowledgments


References




























Con

trol

Mod

elPP

TARA

P-L

TARA

P-H

FAS ACC CPT

(a) (b) (c)



0

30

60


The g

ray

of A

CC

Control0

60

120


The g

ray

of F

AS

Control

0

60

120


The g

ray

of C

PT

Control

Group FAS ACC CPT

ControlModel

PPTARAP-LTARAP-H

2159 plusmn 544


3965 plusmn 782

3286 plusmn 859

2535 plusmn 567

2238 plusmn 165


3265 plusmn 285

3121 plusmn 127

2928 plusmn 553

5358 plusmn 382


5655 plusmn 333

678 plusmn 305

7181 plusmn 467











Acknowledgments


References




























0

30

60


The g

ray

of A

CC

Control0

60

120


The g

ray

of F

AS

Control

0

60

120


The g

ray

of C

PT

Control

Group FAS ACC CPT

ControlModel

PPTARAP-LTARAP-H

2159 plusmn 544


3965 plusmn 782

3286 plusmn 859

2535 plusmn 567

2238 plusmn 165


3265 plusmn 285

3121 plusmn 127

2928 plusmn 553

5358 plusmn 382


5655 plusmn 333

678 plusmn 305

7181 plusmn 467











Acknowledgments


References






























Acknowledgments


References



























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