【摘要】 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。更多还原

【Abstract】 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-α and Z-DVED-FMK. Cell viability was measured by CCK-8 assay. Annexin V/propidium io-dide staining was used to count the number of apoptotic cells. DCFH-DA staining was used to detect reactive oxygen spe-cies(ROS) levels, and JC-1 staining was used to assess mitochondrial membrane potential by flow cytometry. The expres-sion 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.更多还原