The Influence Of T Lymphocyte Apoptosis In Spleen And Related Gene Expression On The Immune Function Of Sepsis

Objective To observe the relationship between T lymphocytic apoptosis inspleen and immunoparalysis in sepsis. The effect of glucocorticoid and AICD on theT lymphocyte apoptosis in spleen, apoptotic related gene mRNA levels and theactivity of caspase3 in T lymphocyte were observed. To investigate the mechanism ofthe T lymphocyte apoptosis in sepsis and its influence factors. To investigate themechanism of HuoXueHuaYu injection regulating the apoptosis of T lymphocyte andameliorating the immunoparalysis in sepsis.Methods In the 1st part, the sepsis models were established by cecal ligationand puncture (CLP),and the parameters of physiology and biochemical in modelswere detected. After that, 96 wistar rats were divided into four groups: control group(n=8), sham operation group (n=8), sepsis group (n=40), and HuoXueHuaYutherapeutic group (n=40). The sepsis group and HuoXueHuaYu therapeutic groupthen divided into 4h, 8h, 24h, 36h, 48h points. The rats were killed at the point afterthe operation and evaluated the IL-2, IL-10 levels in serum by the methods of ELISA,the apoptotic cells by TUNEL, the CD₃⁺ lymphocyte percent in spleen by flowcytometry, the activity of caspase3 in spleen by spectrophotometric method, and theexpression of Fas, FasL, Bcl-2, Bax proteins in spleen by immunohistochemistry. Inthe 2nd part, the T lymphocytes obtained from the spleens of BAB/C mice werecultured and induced apoptosis by dexamethasone and AICD. Apoptosis was assessedby flow cytometry at 6 h, 16 h after the stimulation of dexamethasone, and 18 hamong the AICD procesure. RT-PCR was used to evaluted the expression of Fas,FasL, Bcl-2, Bax mRNA, and the activity of caspase3 in T lymphocyte was detectedby spectrophotometric method at 6 h after the stimulation of dexamethasone, and 6 h,18 h among the AICD procesure. In the mean time the effect of HuoXueHuaYuinjection on those parameters was observed. In the 3rd part, the T lymphocytes wereobtained from the spleens of BAB/C mice and cultured with HuoXueHuaYu injectionThe MTT was measured to assessed the proliferation activity, and the natureapoptosis of T cells was investigated by flow cytometry. Dexamethasone and AICDwere used to induce apoptosis, and the IL-2 mRNA in T cells were dected by RT-PCR.Results In the 1 st part, the activity of caspase3 increased at 4h after the CLPoperation and there were more apoptosis appeared. Simultaneously the percent ofCD3⁺ lymphocyte in spleen decreased. At 36 h, 48 h after the CLP operation, the IL-2in serum decreased and IL- 10 increased obviously. The immunoparalysis appeared. Inthe mean time, the expression of Fas, FasL, Bax in spleen increased obviously inmodel animals, otherwise the Bcl-2 decreased. After the intervention withHuoXueHuaYu injection, the IL-2 level in serum increased and the IL-10 in serumdecreased. The intervention also lightened the activity of caspase3 and the apoptosis,and made the percent of CD3⁺ lymphocyte returned to normal level. The expressionof Fas, FasL, Bax reduced and Bax raised. In the 2nd part, the apoptosis of Tlymphocyte induced by dexamethasone was increased at 6 h, and there was little alivecells at 16 h. The expressions of Bax mRNA rised and the expression of Bcl-2 mRNAlessened at 6 h. There was no change in the expression of Fas mRNA, and theexpression of FasL mRNA was not detected. After the AICD, T lymphocyte apoptosisraised at 18 h. At 6 h after the AICD, there was no expression of FasL, Bax mRNA,and no change in the expression of Fas and Bcl-2 mRNA. At 18 h after the AICD, theexpressions of Fas, FasL, Bax mRNA rised and the expression of Bcl-2 mRNAlessened. The activity of caspase3 also ascended. HuoueHuaYu injection can cutdown the apoptosis induced by the two pro-apoptotic factors, make the expression ofFas, FasL, Bax mRNA decreased and Bcl-2 mRNA improved. The activity ofcaspase3 also fallen after the HuoueHuaYu injection treated. In the 3rd part,HuoXueHuaYu injection can improve the proliferation activity of T lymphocyte,lessen the nature apoptosis of T lymphocyte. HuoXueHuaYu injection can increasethe expressions of IL-2 mRNA in T lymphocyte induced by dexamethasone, promotethe expressions of IL-2 mRNA at early phase of AICD (6 h) and depress theexpressions at the late period (18 h).Conclusions There is a close relationship between T lymphocyte apoptosis inspleen and the immunoparalysis in sepsis models. The expression of apoptotic relatedproteins in spleen regulate the T lymphocyte apoptosis. T lymphocyte in spleen canbe induced to apoptotic by dexamethasone and AICD, and be regulated by the expression of apoptotic related gene. The apoptosis induced by dexamethasone wasregulated by the expression of Bax and Bcl-2 mRNA. The AICD of T lymophocytewas regulated by the change of Fas, FasL, Bcl-2, Bax mRNA expression.HuoueHuaYu injection can ameliorate the apoptosis through regulating the expressionof IL-2 mRNA which resulted in the improving the immune function of sepsis. It canimprove the proliferation activity of T lymphocyte, lessen the nature apoptosis of Tlymphocyte.