The Effect Of AICD Of Regulatory T Cells In Septic Rats On Immunological Function Of Effector T Lymphocytes Induced By Xuebijing Injection

Objective:To observe the effect of apoptosis of CD4⁺CD25⁺ regulatory T cells in septic rats on immunological function of CD4⁺ T tymphocytes induced by Xuebijing injectionMethods:1):In vitro experiment:To separate the mononuclear cells of the spleen of rats asepsisly after sacrificed.1:CD4⁺T cells, CD4⁺CD25⁺ Treg and CD4⁺CD25 T cells were separated by immunomagnetic bead isolate system,CD25 was marked by fluorescein isothiocyante(FITC).The cell purity of CD4⁺CD25⁺ Treg was detected by 0.4%trypanblau and the survival rate was analyzed by Flow cytometry(FCM).2:The CD4⁺CD25⁺ Treg were divided into the control group,anti-CD3/CD28 stimulation group,anti-CD3/CD28+ lipopolysaccharide(LPS)stimulation group and anti-CD3/CD28+ LPS+Xuebijing stimulation groups(5mg/ml)group.The apoptotic rates of CD4⁺CD25⁺ Treg were analyzed by FCM used allophycocyanin (APC)-Annexin and 7-amino-actinomycin-D(7-AAD)at 3^thd.The geometric mean of fluorescence intensity of phycoerythrin(PE)-forkhead/winged helix transcription factor(Foxp3)and PE-cytotoxic T lymphocyte-associated antigen 4(CTLA-4)of CD4⁺CD25⁺ Treg was analysised by FCM.The interleukin(IL)-10 serected from CD4⁺CD25⁺ Treg was measured by enzyme-linked immunoadsorent assay(ELISA).3: Each group CD4⁺CD25⁺ Treg were co-cultured with CD4⁺CD25- T cells(1:1),T cell proliferation experiment was done by colorimetric method used thiazolyl blue(MTT).IL-2/sIL-2R,INFγ/IL-4/IL-17 were measured by ELISA.2):In vivo experiment:Replicated the cecal ligation and puncture(CLP)animal models,and seperated the.CD4⁺CD25⁺ Treg in vitro and cultured 24h.1:Rats were divilded into the control group, sham-operated group,CLP group and Xuebijing treatment group.Foxp3, CTLA-4,IL-10,apoptotic rates were tested by the above methods.2: Seperated CD4⁺CD25⁺ Treg cells,co-cultered with CD4⁺ T cells(1:1), MTT rooliferation experiment,IL-2/sIL-2R,INFγ/IL-4/IL-17,were measured.Results:1).In vitro experment 1:Apoptotic rate,empression of Foxp3 and CTLA-4 and secretion of IL-10:the apoptotic rate of the anti-CD3/CD28 group(25-40%)was higher than the control group obviously(P＜0.01).The mean fluorescence intensity of Foxp3 and CTLA-4 and secretion of IL-10were negatively correlated with the apoptosis rates.The apoptotic rate of anti-CD3/CD28 group was higher than the control group(P＜0.01).The group of anti-CD3/CD28 stimulated had the highest apoptotic rate,which had a obvious significance with other groups(P＜0.05 or P＜0.01).And the group of anti-CD3/ CD28+LPS+Xuebijing group was lower than the group of anti-CD3/CD28+LPS group.2:Effect on immunological function of CD4⁺ T lymphocytesâ‘ MTT rooliferation experiment:compared with control group,the Teff proliferative activity in response to ConA of septic group were significantly suppressed(P＜0.01),and compared with anti-CD3/CD28+LPS group,proliferative activity in anti-CD3/ CD28+LPS+Xuebijing group has a significantly improve(P＜0.01).â‘¡secretion of IL-2/slL-2Ra:IL-2:compared with control group,the level of IL-2 in the supermatant of septic group were significantly suppressed (P＜0.01),and compared with anti-CD3/CD28+LPS group,secretion of IL-2 in anti-CD3/CD28+LPS+Xuebijing group has a significantly improve(P＜0.01),sIL-2Ra:The secretion of sIL-2Ra of anti-CD3/ CD28+LPS group and anti-CD3/CD28+LPS+ Xuebjijing group had no significantly change,but both were higher than the control groups(P＜0.01).â‘¢Drift of Thl/Th2/Th17(IFNγ/IL-4/ IL-17):INFγsecreted in antiCD3/CD28+LPS+Xuebijing groups more than anti-CD3/CD28+LPS group(P＜0.05),IL-4 had an opposite tendency with INFγ.In anti-CD3/CD28+ Xuebijing injection group,IL-17 had significantly decreased compared with the group of anti-CD3/CD28(P＜0.05).2).In vivo experment 1:Apoptotic rate,empression of Foxp3 and CTLA-4 and secretion of IL-10:comperated with the sham-operated group,all indexs have no change with the control group(P＜0.05).In CLP group,the apoptotic rate was lower than the control group(P＜0.01),the empression of Foxp3 and CTLA-4 and the secretion of IL-10 were higher than the control group(P＜0.01).2:Effect on immunological function of CD4⁺ T lymphocytesâ‘ MTT rooliferation experiment:the Teff proliferative activity in response to ConA in CLP rats were significantly suppressed compared with control group(P＜0.01),The inhibition rate of Teff proliferative activity of Xuebijing treatment group was lower than CLP group,the secretion of IL-2 was higher than CLP groups.â‘¡secretion of IL-2/sIL-2Ra:the level of IL-2 of Teff in CLP rats were significantly suppressed compared with control group(P＜0.01),and secretion of sIL-2Ra in the supermatant was higher than the control groups.The level of sIL-2Ra of Xuebijing treatment group was lower than CLP group,the secretion of IL-2 was higher than CLP groups.â‘¢Drift of Th1/Th2/Th17 (IFNγ/IL-4/IL-17):their secretion of INFγ,IL-4 and ratio of INFγ/IL-4 had significantly increased compared with the control group(P＜0.05),and INFγ,IL-4,INFγ/IL-4 higher than the CLP group(P＜0.05).The secretion of IL-17 in each group was no change.Conclusion:1.CD4⁺CD25⁺ Treg shows apoptosis induced by anti-CD3/CD28 and the empression of Foxp3 and CTLA-4 and the secretion of IL-10 downregulated with the increase of apoptotic rate. 2.CD4⁺CD25⁺ regulatory T cells upregulated the suppression function on Teff in sepsis,and Xuebijing injection could down-regulate the suppression function effectively via reducing apoptosis of septic Treg.