炎调方通过PD-1/PD-L1通路抑制脓毒症大鼠T淋巴细胞衰竭的实验研究

Experimental study on inhibition of T lymphocyte failure by Yantiao Formula through the PD-1/PD-L1 pathway in septic rats

目的研究炎调方对脓毒症大鼠T淋巴细胞及T淋巴细胞表面表面程序性死亡蛋白(PD-1)、程序性死亡蛋白配体(PD-L1)表达的影响。方法SD大鼠(雄性)分为正常组、假手术组、模型组和炎调方组。盲肠结扎穿孔(CLP)制备脓毒症大鼠模型,于造模后12、24、48、72 h采集外周血处死大鼠(每个时间点各5只大鼠),采用流式细胞分析法测定CD3^(+)、CD4^(+)、CD8^(+)比例;双抗体夹心酶联免疫吸附法(ELISA)测定CD4^(+)及CD8^(+)表面PD-1、PD-L1表达水平。结果白介素-6(IL-6)在脓毒症模型组12 h分泌达到高峰后逐渐下降,降钙素原(PCT)在模型组24 h达到高峰后逐渐下降;炎调方组在12 h降低IL-6的水平(P<0.05),24 h时明显降低IL-6、PCT的水平(P<0.01)。模型组CD3^(+)、CD4^(+)、CD8^(+)比例早期分泌开始减少,48 h分泌达最低点(P<0.01);炎调方组在48 h时可升高CD3^(+)、CD4^(+)、CD8^(+)比例,提高CD4^(+)/CD8^(+)的比值而具体调节T细胞活化和增殖的功能。模型组CD4^(+)、CD8^(+)表面PD-1、PD-L1表达水平早期24 h即表达增多(P<0.01),72 h表达最多;炎调方组24 h时即可抑制PD-1、PD-L1的表达水平而具体改善T细胞衰竭的作用。CD4^(+)的比例与CD4^(+)表面PD-1、PD-L1的表达呈负相关(P<0.01);CD8^(+)比例与CD8^(+)表面PD-1、PD-L1的表达呈负相关(P<0.01)。结论脓毒症早期48 h内即出现了T细胞活化和增殖的功能衰竭引起的T细胞免疫抑制。炎调方能在脓毒症早期改善T细胞活化和增殖的功能,其机制可能与抑制PD-1/P-L1通路有关。

Objective To study the effect of Yantiao Formula on the expression levels of programmed cell death protein-1(PD-1)and programmed cell death ligand-1(PD-L1)in T lymphocytes and on T lymphocyte surface of septic rats.Methods Male Sprague-Dawley(SD)rats were divided into control group,sham operation group,model group and Yantiao Formula group.The cecal ligation and puncture(CLP)method was adopted to prepare the sepsis model in rats.After 12 h,24 h,48 h and 72 h of modeling,rats were subjected to the collection of peripheral blood samples and sacrificed.The proportions of CD3^(+),CD4^(+)and CD8^(+)T cells were measured by flow cytometry.Expression levels of PD1 and PD-L1 on the surface of CD4^(+)and CD8^(+)cells were measured by enzyme-linked immunosorbent assay(ELISA).Results We detected the expression levels of inflammatory factor interleukin 6(IL-6)and infection indicator procalcitonin(PCT)in rats.In model group,IL-6 level peaked at 12 h and gradually decreased,and PCT peaked at 24 h and gradually decreased.In Yantiao Formula group,IL-6 level was reduced from 12 h(P<0.05),and significantly reduced IL-6 and PCT levels were detected at 24 h(P<0.01).The proportions of CD3^(+),CD4^(+)and CD8^(+)T cells in the model group were reduced in the early stage,and bottomed at 48h(P<0.01).In Yantiao Formula group,the proportions of CD3^(+),CD4^(+)and CD8^(+)T cells were elevated at 48 h.Increasing CD4^(+)/CD8^(+)was capable of regulating the activation and proliferation of T cells.Expression levels of PD1 and PD-L1 on the surface of CD4^(+)and CD8^(+)T cells were upregulated as early as at 24 h in the model group,and peaked at 72 h(P<0.01).The treatment of Yantiao Formula effectively inhibited PD1 and PD-L1 on the surface of CD4^(+)and CD8^(+)T cells as early as at 24 h,thus alleviating T lymphocyte failure.The proportion of CD4^(+)T cells was negatively correlated with expression levels of PD1 and PD-L1 on the surface of CD4^(+)T cells(P<0.01),and the proportion of CD8^(+)T cells was negatively correlated with expression levels of PD1 and PD-L1 on the surface of CD8^(+)T cells(P<0.01).Conclusion T lymphocyte failure of the suppressed activation and proliferation occurs in the early stage at 48 h of sepsis.Yantiao Formula improves T cell activation and proliferation in the early stage of sepsis by inhibiting the PD-1/PD-L1 pathway.