TPOL triggers apoptosis with mitochondrial injury through activating a ROS-dependent p53/p21/p27/Rb/Bax/Cyto C/caspase-mediated signaling  

TPOL通过激活ROS依赖的p53/p21/p27/Rb/Bax/Cyto C/caspase信号引发伴随线粒体损伤的细胞凋亡

作　　者：CHENG Zongwei ZENG Boning XING Feiyue 程宗伟;曾博宁;邢飞跃(暨南大学免疫生物学系,组织移植与免疫研究中心,广东广州510632)

机构地区：[1]Department of Immunobiology,Institute of Tissue Transplantation and Immunology,Jinan University,Guangzhou 510632,China

出　　处：《中国病理生理杂志》2024年第8期1488-1496,共9页Chinese Journal of Pathophysiology

基　　金：Supported by the National Natural Science Foundation of China(No.81172824)。

摘　　要：AIM:To explore the influence of ethyl(2,4,6-trimethylbenzoyl)phenylphosphinate(TPOL)on cell apoptosis and its potential mechanism.METHODS:HEK293T cells sensitive to TPOL were treated with different concentrations of TPOL with or without exposure to light radiation,before treatment with various inhibitors,N-acetyl-Lcysteine(NAC),pifithrin-αand Z-DVED-FMK.Cell viability was measured by CCK-8 assay.Annexin V/propidium iodide staining was used to count the number of apoptotic cells.DCFH-DA staining was used to detect reactive oxygen species(ROS)levels,and JC-1 staining was used to assess mitochondrial membrane potential by flow cytometry.The expression of apoptosis-related proteins and cell cycle-regulated molecules was measured by Western blot.RESULTS:TPOL enhanced the apoptosis of HEK293T cells in a dose-dependent manner(P<0.05),with a decrease in Bcl-2 and increases in Bax and cytochrome C(Cyto C),followed by up-regulation of activated caspase-9 and caspase-3,and the cleavage of PARP(P<0.05).The TPOL-enhanced cleavage of caspase-3 and PARP was rescued by Z-DVED-FMK(P<0.01).TPOL also led to a rapid increase in ROS,a reduction in mitochondrial membrane potential,and the release of Cyto C(P<0.01),all of which could be reversed by the ROS scavenger NAC.Moreover,the TPOL-caused alterations in p21,p27,Rb,and CDK2 were also recovered by the p53 inhibitor pifithrin-α(P<0.05).The TPOL-induced changes in Bax,Bcl-2,cleaved caspase-9,activated caspase-3,and cleaved PARP were subsequently rescued by pretreatment with pifithrin-α(P<0.05).CONCLUSION:TPOL can induce cellular apoptosis with ROS-mediated mitochondrial membrane damage through the activation of a ROS-dependent p53/p21/p27/Rb/Bax/Cyto C/caspase-mediated signal axis.目的:探讨2,4,6-三甲基苯甲酰基苯基膦酸乙酯(TPOL)对细胞凋亡的影响及其潜在机制。方法:在光照或非光照条件下用不同浓度的TPOL处理TPOL敏感的HEK293T细胞,或在TPOL处理前加入不同的抑制剂N-乙酰半胱氨酸(NAC)、pifithrin-α和Z-DVED-FMK。CCK-8法测定细胞活力;膜联蛋白V/碘化丙啶染色定量凋亡细胞数;DCFH-DA染色结合流式细胞术测量活性氧(ROS)水平;JC-1染色评估线粒体膜电位;Western blot分析凋亡相关蛋白和细胞周期调节分子的表达。结果:TPOL以剂量依赖的方式增强HEK293T细胞的凋亡(P<0.05),与Bcl-2的减少、Bax和细胞色素C(Cyto C)的增加、激活的caspase-9和caspase-3的上调以及PARP的裂解有关(P<0.05)。TPOL增强的caspase-3切割和PARP裂解可以被Z-DVED-FMK挽救(P<0.01)。TPOL处理会导致细胞内ROS迅速增加、线粒体膜电位降低和Cyto C的释放(P<0.01),而ROS清除剂NAC可逆转这一现象(P<0.01)。TPOL诱导的p21、p27、Rb和细胞周期蛋白依赖性激酶2的变化也可以被p53抑制剂pifithrin-α恢复(P<0.05)。pifithrin-α预处理能不同程度恢复TPOL诱导的Bax、Bcl-2、切割的caspase-9、活化的caspase-3和裂解的PARP的变化(P<0.05)。结论:TPOL可通过激活ROS依赖的p53/p21/p27/Rb/Bax/Cyto C/caspase信号诱导细胞凋亡,伴随ROS介导的线粒体膜损伤。

关 键 词：ethyl(2 4 6-trimethylbenzoyl)phenylphosphinate reactive oxygen species MITOCHONDRIA APOPTOSIS 

分 类 号：Q255[生物学—细胞生物学] R363[医药卫生—病理学] Q731[医药卫生—基础医学]