Curtis DobrowolskiDepartment of Biology and Marine

ScienceJacksonville University

Rate of Phagocytosis in Macrophages Stimulated by Astragalus membranaceus

IntroductionMacrophages are the first line of defense in

the human immune systemPhagocytoseImportant antigen presenting cells, required

for adaptive immune response

IntroductionAdvantages of increased phagocytosis

Removal of pathogenMore T cell interaction, faster adaptive immune

responseMore memoryMore Antibody production

More cytokines released increased inflammation and response from other immune

cells

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IntroductionAstragalus membranaceus

Shrub native to east Asia, primarily ChinaA. membranaceus is a common medicinal herb

used in eastern medicineOne of the 50 fundamental Chinese medicinal herbs

Huáng qí- root teaUsed primarily when symptoms of an infection first

ariseShown to increase antibody production in mice (1) Increased cytokine production (2)

http://nccam.nih.gov/health/astragalus/

50 Fundamental Chinese Medicinal Herbs1 Agastache rugosa 26 Gentiana loureiroi2 Alangium chininese 27 Gleditsia sinensis3 Anemone chininese 28 Glycyrrhiza uralensis4 Anisodus tanguticus 29 Hydnocarpus anthelmintica5 Aridisia j aponica 30 Ilex purpurea6 Aster tataricus 31 Leonurus japonicus7 Astragalus membranaceus 32 Ligusticum wallichii8 Camillia sinesis- Traditional Black Tea 33 Lobella chinensis9 Cannabis sativa- Marijuana 34 Phellodendron amurense

10 Cathamus tinctorius 35 Platycladus orientalis11 Cinnamomum cassia- Cinnamon 36 Pseudolarix amabilis12 Cissampelous pareira 37 Psilopeganum sinense13 Coptis chinensis 38 Pueraria lobata14 Corydalis ambigua 39 Rauwolfia serpentina15 Croton tiglium 40 Rehmannia glutenosa16 Daphne genkwa 41 Rheum officinale17 Datura metel 42 Rhododendron tsinghaiense18 Datura tatula 43 Saussurea costus19 Dendrobium nobile -Orchid 44 Schisandrac hinensis20 Dichroa febrifuga 45 Scutellaria baicalensis21 Ephedra sinica 46 Stemona tuberosa22 Eucommia ulmoides 47 Stephania tetrandra23 Euphorbia pekinensis 48 Styphnolobium japonicum24 Flueggea suffruticosa 49 Trichosanthes kirilowii25 Forsythia suspensa 50 Wiksotroemia indica

HypothesisIn vitro phagocytosis in macrophages increases when subjected to A. membranaceus

MethodsIC-21 macrophages were cultured in tissue culturing

flasksMacrophages grown RPMI 1640 media supplemented

with 10% fetal bovine serum and 1% pen/strep antibiotic

Incubated at 37°C with 5% CO2 until cells were fully confluent

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MethodsMacrophages were lifted with Ca2+ depleted PBS

SolutionPBS= phosphate buffered saline

PBS/macrophage solution was stained with trypan blue vital dye and counted using a hemocytometer

Using (Q1+Q2+Q3+Q4)*104=cells/ml equation concentrations were determined

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MethodsUsing the count from the hemocytometer we

placed 300,000 cells in each micro-chamberEqual amount of RPMI 1640 media were added

to each micro-chamberIncubated for minimum of 3 hours

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MethodsMedia/PBS solution was removedMacrophages were incubated for a minimum

of 24 hours to allow the medias to have its effects on the macrophages Media alone0.3% EtOH Control1% astragalus

Control

1%A.M.

1%A.M.

EtOH Control

Control

1%A.M.

EtOH Control

1%A.M.

MethodsMedia was removed1μ diameter fluorescently tagged latex

beads were layered on top of macrophages, at a concentration of 20 beads per cell

Media alone, EtOH and astragalus media were then added to the respective wells

MethodsMedia and beads were removed and wells

were washed 3 timesTo remove beads that have not been

phagocytosedWells were removed and slides were

stained with protocol hema 3 stainIncreasing phagocytosis

More cells phagocytosingSubset of cells phagocytosing more

Methods

Data collectedTotal number of cells (75-175 cells counted

per experimental well)Number of cells that phagocytosed any

beadsNumber of cells that phagocytosed >1 bead

Rate of phagocytosis calculated

Results

0.00%

0.50%

1.00%

1.50%

2.00%

2.50%

3.00%

Rate

of P

hago

cyto

sis

Rate of Phagocytosis of Any Number of Beads

Media Alone

EtOH Control

1% A.M. Media for 60 Min

1% A.M. Media for 90 Min

P Value 0.396169

P Value 0.000463

P Value 0.000538

N=9

Results

-0.10%

0.00%

0.10%

0.20%

0.30%

0.40%

0.50%

0.60%Ra

te o

f Pha

gocy

tosi

s of M

ore

than

One

Bea

d

Rate of Phagocytosis of More than One Bead

Media Alone

EtOH Control

1% A.M. Media for 60 Min

1% A.M. Media for 90 Min

P value 0.3867711

P value 0.445056

P value 0.0167912

N=9

Conclusions The rate of in vitro phagocytosis in

macrophages increases when subjected to A. membranaceus

Could be beneficial in helping immuno-compromised patientsHIV/AIDS patientsGenetic disorders

More research is need to fully understand A. membranaceus’ immuno-stimulating properties and benefits

Acknowledgements I would like to thank Dr. Karen Jackson PhD,

for all of her help in culturing macrophages in a less than ideal environment

Also I would like to thank Sally and Judy in the Science and Math division office, for signing me out a lab key nearly everyday of the year

References1. Zhao, K S, Mancini, C, et al.,

“Enhancement of the immune response in mice by Astragalus membranaceus extracts.”, Immunopharmacology, 1990 Nov-Dec; 20(2): 225-233

2. Bedir E, Pugh N, et al., “Immunostimulatory effects of cycloartane-type triterpene glycosides from astragalus species.”, Biol Pharm Bull, 2000 Jul; 23(7): 834-7