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目的观察tBHQ对内源性线粒体凋亡通路和凋亡相关蛋白Bcl-2等蛋白表达的影响,探讨tBHQ在无机砷诱导HaCaT细胞凋亡过程中的作用。方法采用分光光度法检测Caspase-3蛋白活力;Western blot法分析细胞内Caspase-3、CytC、Bcl-2和Bax蛋白表达水平。结果 NaAsO2单独作用于HaCaT细胞24 h,线粒体中Cyt C蛋白表达降低,而胞浆中Cyt C蛋白表达则随染砷剂量的增加而明显升高。tBHQ预处理12 h后再分别暴露于NaAsO2,线粒体中CytC蛋白表达明显恢复,胞浆中CytC蛋白表达则随tBHQ剂量的增加而明显回落。此外,NaAsO2单独作用于HaCaT细胞24 h,Procaspase-3蛋白表达降低,而Caspase-3活化程度均显著高于对照组(P<0.01),tBHQ预处理12 h后再暴露于NaAsO2,Procaspase-3蛋白表达均明显高于相同浓度砷单独作用组,而且Caspase-3酶活力得到明显抑制,差异均具有统计学意义(P<0.05)。NaAsO2单独作用于HaCaT细胞24 h,与对照组相比Bcl-2蛋白表达降低,而Bax蛋白表达明显升高。tBHQ预处理12 h后再分别暴露于NaAsO2,Bcl-2蛋白表达明显恢复,而Bax蛋白表达则随tBHQ剂量的增加而减少。结论 tBHQ能够影响线粒体凋亡途径拮抗NaAsO2诱导的人皮肤角质细胞凋亡;tBHQ能够诱导调控凋亡相关蛋白Bcl-2/Bax从而发挥抗凋亡作用。
Objective To investigate the effect of tBHQ on the expression of endogenous mitochondrial apoptosis pathway and apoptosis-related protein Bcl-2, and to explore the role of tBHQ in the apoptosis of HaCaT cells induced by inorganic arsenic. Methods The activity of Caspase-3 protein was detected by spectrophotometry. The protein expression of Caspase-3, CytC, Bcl-2 and Bax was analyzed by Western blot. Results After treated with NaAsO2 alone for 24 h, the expression of Cyt C in mitochondria decreased while the expression of Cyt C in cytoplasm increased with the increase of arsenic dose. After exposure to NaAsO2 for 12 h, the expression of CytC protein in mitochondria was significantly restored after tBHQ pretreatment, while the expression of CytC protein in cytoplasm decreased significantly with the increase of tBHQ dose. In addition, the NaAsO2 treated HaCaT cells for 24 h, Procaspase-3 protein expression decreased, while Caspase-3 activation was significantly higher than the control group (P <0.01), 12 h after tBHQ pretreatment exposure to NaAsO2, Procaspase-3 Protein expression were significantly higher than the same concentration of arsenic alone group, and Caspase-3 enzyme activity was significantly inhibited, the difference was statistically significant (P <0.05). NaAsO2 alone treated HaCaT cells for 24 h, compared with the control group, Bcl-2 protein expression decreased, while Bax protein expression was significantly increased. After pretreatment with tBHQ for 12 h, the expression of Bcl-2 protein was restored in NaAsO2, while the expression of Bax protein was decreased with the increase of tBHQ dose. Conclusion tBHQ can antagonize the mitochondrial apoptotic pathway to antagonize NaAsO2-induced apoptosis of human skin keratinocytes. TBHQ can induce the apoptosis-related protein Bcl-2 / Bax to exert anti-apoptotic effects.