TRAF6通过内源性凋亡通路促进卡介苗诱导的巨噬细胞凋亡

TRAF6 promotes Bacillus Calmette-Guérin-induced macrophage apoptosis through the intrinsic apoptosis pathway

目的探讨肿瘤坏死因子受体相关因子6(TRAF6)对卡介苗(BCG)诱导巨噬细胞凋亡的调控作用。方法Q-PCR检测活动性结核患者外周血中TRAF6的表达后利用不同感染复数的BCG感染RAW264.7细胞,在感染的不同时间通过Q-PCR和Westernblot检测巨噬细胞中Caspase 3和TRAF6的表达。时间梯度试验处理组分为5组:对照组(未感染,C)、BCG感染6 h组、12 h组、18 h组和24 h组;感染复数试验处理组分为5组:对照组(未感染,C)、5 MOI组、10 MOI组、15 MOI组和20 MOI组。随后,通过小干扰RNA技术敲减RAW264.7细胞中TRAF6的表达,并结合BCG感染建立TRAF6敲减模型。TRAF6敲减实验分为4组:阴性对照组(NC)、BCG单独感染组(BCG)、TRAF6小干扰RNA单独处理组(siRNA)和TRAF6小干扰RNA与BCG共处理组(siRNA+BCG)。利用平板涂布法检测巨噬细胞菌载量变化。通过流式细胞术检测巨噬细胞凋亡率和线粒体膜电位变化。利用Westernblot检测TRAF6、Caspase 3、PARP、Bcl-2,BAX的表达。使用免疫荧光技术检测TRAF6与Caspase 3的表达。结果与健康人群相比,活动性结核患者外周血中TRAF6高表达(P<0.001)。BCG感染显著上调RAW264.7中TRAF6与Caspase 3的表达,且当感染时间为18 h(P=0.03,P=0.04),感染复数为15时二者表达量最高(P<0.001,P<0.001)。敲减TRAF6上调BCG感染的巨噬细胞内的菌载量(P=0.05)。与BCG单独处理组相比,敲减TRAF6显著抑制BCG感染的巨噬细胞凋亡率(P<0.001),并显著下调Caspase 3(P=0.002)和PARP的表达(P<0.001)。同时,BCG感染RAW264.7细胞的线粒体膜电位上升(P<0.001),而敲减TRAF6抑制BCG感染的巨噬细胞线粒体膜电位(P<0.001)。敲减TRAF6显著下调BCG感染巨噬细胞中BAX表达(P=0.005)的同时上调Bcl-2的表达(P=0.04)。结论TRAF6通过内源性凋亡通路促进BCG感染的巨噬细胞凋亡。

Objective To investigate the role of tumor necrosis factor receptor-associated factor 6(TRAF6)in regulating Bacillus Calmette-Guérin(BCG)-induced macrophage apoptosis.Methods The expression of TRAF6 in peripheral blood samples of 50 patients with active tuberculosis(TB)and 50 healthy individuals were detected using quantitative real-time PCR(qPCR).RAW264.7 macrophages were infected with BCG at different MOI and for different lengths of time,and the changes in expressions of Caspase 3 and TRAF6 were detected with Western blotting and qPCR.In a RAW264.7 cell model of BCG infection with TRAF6 knockdown established using RNA interference technique,the bacterial load was measured and cell apoptotic rate and mitochondrial membrane potential(MMP)were determined with flow cytometry.The expression levels of TRAF6,Caspase 3,PARP,BAX and Bcl-2 in the cells were detected using Western blotting,and the expressions of TRAF6 and Caspase 3 were also examined with immunofluorescence assay.Results The expression of TRAF6 was significantly upregulated in the peripheral blood of patients with active TB as compared with healthy subjects(P<0.001).In RAW264.7 cells,BCG infection significantly increased the expressions of Caspase 3 and TRAF6,which were the highest in cells infected for 18 h and at the MOI of 15.TRAF6 knockdown caused a significant increase of bacterial load in BCG-infected macrophages(P=0.05),lowered the cell apoptotic rate(P<0.001)and reduced the expressions of Caspase 3(P=0.002)and PARP(P<0.001).BCG-infected RAW264.7 cells showed a significantly increased MMP(P<0.001),which was lowered by TRAF6 knockdown(P<0.001);the cells with both TRAF6 knockdown and BCG infection showed a lowered BAX expression(P=0.005)and an increased expression of Bcl-2(P=0.04).Conclusion TRAF6 promotes BCG-induced macrophage apoptosis by regulating the intrinsic apoptosis pathway.