肿瘤坏死因子α通过抑制线粒体呼吸链复合体Ⅲ诱导L929-A细胞发生RIP1激酶依赖性细胞凋亡

摘要

Objective To explore the mechanism by which tumor necrosis factor alpha(TNF-α) induces RIP1 kinase-dependented apoptosis in L929-A fibroblastoma cells.Methods The sub-mitochondrial localization of receptor-interacting protein 1(RIP1),caspase-8 and Bid proteins was detected by dose-gradient trypsin digestion and Western blotting.The levels of reactive oxygen species (ROS),intracellular calcium concentration,mitochondrial membrane potential (MMP),and cellular adenosine triphosphate(ATP) content were determined by fluorescent probe labeling and flow cytometry assay.The mitochondrial respiratory chain complex Ⅰ and Ⅲ activities were detected by commercial kits.Nec-1,A RIP1 kinase specific inhibitor,and RIP1-/-or Bid-/-L929-A cells were used to detect the roles of RIP1 kinase and Bid protein in cell death.Results RIP1,caspase-8 and Bid proteins were co-located in the outer membrane of mitochondrial.TNF-α exposure for 3 h could induce Bid cleavage,inhibit mitochondrial respiratory chain complex Ⅲ activity and reduce MMP.Following these changes and after TNF-α exposure for 6-12 h,the intracellular calcium concentration and ROS were increased,whereas the ATP concentration was decreased,and the cells were killed.Inhibiting RIP1 kinase or knockdown RIP1 or Bid protein could suppress all the cytotoxic effects of TNF-α.Conclusion TNF-α treatment can result in RIP1 kinase-mediated Bid cleavage and inhibit mitochondrial respiratory chains and cell energy metabolism,which ultimately leads to the death of L929-A cells.%目的 探讨肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)诱导L929-A细胞发生受体相互作用蛋白激酶1(receptor-interacting protein 1,RIP1)依赖性凋亡的分子机制.方法 通过胰蛋白酶浓度梯度消化及蛋白质印迹法检测RIP1、胱天蛋白酶8(caspase-8)和Bid蛋白的表达和线粒体定位;利用荧光探针标记法检测TNF-α处理后L929-A细胞内的活性氧(reactive oxygen species,ROS)水平、胞内钙离子浓度、线粒体膜电位(mitochondrial membrane potential,MMP)及三磷酸腺苷(adenosine triphosphate,ATP)浓度,应用试剂盒检测线粒体呼吸链复合体Ⅰ、Ⅲ的活性变化;采用RIP1激酶特异性抑制剂坏死抑素1(necrostatin-1,Nec-1)和Bid基因敲除的L929-A细胞评估RIP1激酶活性和Bid蛋白在介导细胞死亡中的作用.结果 RIP1、caspase-8和Bid蛋白均定位在线粒体外膜上;TNF-α处理后3 h即可诱导Bid剪切,伴随Bid剪切,线粒体呼吸链复合体功能检测显示复合体Ⅲ的活性受到抑制,MMP下降.TNF-α处理后6~12 h细胞内ROS升高、钙离子浓度上升、ATP浓度降低;抑制RIP1激酶活性或敲低Bid蛋白可完全拮抗TNF-α诱导的细胞毒性.结论 TNF-α通过诱导RIP1激酶活性依赖的Bid剪切,继而抑制线粒体呼吸链和细胞能量代谢,诱导细胞死亡.