Effect Of Traditional Chinese Medicine Huaier Aqueos Extract On The Subsets Of Peripheral CD4~+T Cells In Patients With Idiopathic Thrombocytopenic Purpura

Objective:Idiopathic thrombocytopenic purpura(ITP)is a kind of acquired autoimmunity disease characterized by excessive destruction of platelet and weakened production of platelet.Though the etiology of ITP is complicated,the immune-mediated mechanism is the crucial factor in the pathogenesis of ITP.Accumulating evidences have shown that CD4~+T cells play an important part in the pathogenesis of ITP.Huaier is a kind of traditional Chinese medicine(TCM),whose active ingredient is proteoglycan.As is shown by various studies,huaier has the functions of anti-tumor,immunity enhancement and anti-inflammation.This study is aimed to investigate the effect of huaier aqueos extract on the subsets of CD4~+T cells in ITP patients.Methods:(1)Suspended Jurkat cell line and treated them with the stimulation of anti-CD3/CD28 antibody(anti-CD3 antibody:3?g/ml;anti-CD28 antibody:2?g/ml)with/without various concentrations of huaier aqueos extract in 96 well plate for 24,48,72 hours.Check the cell viability of Jurkat cell line by cell counting kit-8(CCK8).Wright’s-Giemsa staining was performed to observe the morphological of anti-CD3/CD28 antibody activated Jurkat cell line treated with/without various concentrations of huaier aqueos extract for 72h.The appropriate concentration of huaier aqueos extract was determined for subsequent experiments.Peripheral blood mononuclear cells(PBMC)in patients with ITP stimulated by anti-CD3/CD28 antibody were treated with/without 3mg/ml huaier aqueos extract for 72h before the apoptosis assay.The effect of huaier aqueos extract on the apoptosis of ITP PBMC was detected by flow cytometry.(2)PBMC extracted from ITP patients stimulated by anti-CD3/CD28 antibody were incubated with/without 3mg/ml huaier aqueos extract for72h.Then the flow cytometry methods were used to detect the expression of activation marker ICOS、CD38、CD25 and PD-1 on CD4~+T cells.(3)PBMC from ITP patients and health controls were directly stimulated by appropriate concentrations of phorbol12-Myristate 13 Acetate(PMA),Ionomycin,Brefeldin A(BFA)for 4 hours and then were collected to detect the ratio of Th1 and Th2 cells by flow cytometry.(4)PBMC from ITP patients stimulated by anti-CD3/CD28 antibody were cultured with or without3mg/ml huaier aqueos extract for 72h.Then the cells were collected to measure the percent of Th1,Th2,Th17,Treg cells by flow cytometry.(5)The supernatant collected in the previous step was collected to detect the cytokines such as IFN-γ,TNF-α,IL-2,IL-4,IL-6,IL-10 and so on by ELISA and CBA.Results:(1)Compared with control group,the cell viability of huaier-treated(1mg/ml-9mg/ml)Jurkat cell line was inhibited by different concentrations of huaier aqueos extract in a dose-dependent manner after 24,48 and 72 hours for stimulation.When the concentration of huaier aqueos extract comes to 3mg/ml,the cell viability of Jurkat cells line is approximately 50 percent.Wright’s-Giemsa staining showed that the morphology of untreated cells appear in a regular of shape of lymphocyte,complete membrane and no marginal destruction,while treated cells showed enlarged size,crenated membrane and cytoplasmic vacuoles with the increase of the concentration of huaier.In order to further verify the safety of huaier aqueos extract concentration,we used flow cytometry to detect the effect of 3 mg/ml huaier aqueos extract on the apoptosis of PBMC in peripheral blood of ITP patients.Flow cytometry methods(FCM)has shown that there was no significant difference in the apoptosis rate of ITP PBMC between the control group and 3mg/ml huaier group,indicating that this concentration could effectively inhibit cell viability but had no significant difference in the effect of apoptosis.(2)Flow cytometry results showed that 3mg/ml-huaier-stimulated PBMC from ITP patients had a higher level of activated molecules ICOS,CD25,CD38(P<0.01,P<0.01,P<0.01)than the untreated cells.The level of PD-1 on CD4~+T cells treated with huaier has no significant differences with untreated cells.(3)Compared with that in PBMC of healthy people,higher levels of Th1 cells(ns)and lower levels of Th2 cells(ns)in PBMC from ITP patients were proved by flow cytometry.The ratio of Th1/Th2 in ITP patients is higher than that in health control(P<0.05),indicating Th1/Th2 cell polarization imbalance in ITP patients.Flow cytometry results have shown that the rates of Th2(P<0.01)in ITP PBMC were up-regulated by huaier aqueos extract,while the rates of Th1(P<0.01)were down-regulated.Furthermore,CBA results also indicated that Th1-related cytokines in the supernatant of huaier-treated group decreased significantly,while Th2-related factors increased in varying degrees.(4)Flow cytometry results have shown that the rates of Th17 cells(P<0.05)were up-regulated by huaier aqueos extract,while the rates of Treg(P<0.01)were down-regulated in patients with ITP.There was no significant difference in the cytokines such as IL-17,IL-23,IL-1β,TGF-βbetween the two groups.Conclusions:Huaier aqueos extract could suppress the proliferation of T cells.The rates of activation molecules ICOS,CD25,CD38 on CD4~+T cells in ITP patients were down-regulated by huaier aqueos extract.Huaier aqueos extract could correct the unbalanced Th1/Th2 polarization and inhibit the secretion of pro-inflammatory factors such as IL-2,TNF,IFN-γin PBMC of ITP patients.Our experimental study focuses on the effect of huaier aqueos extract on the subsets of CD4~+T cells in the peripheral blood of ITP,providing theoretical support and experimental basis for the application of huaier aqueos extract in ITP treatment.