南蛇藤乙酸乙酯提取物通过活化caspase依赖的线粒体通路诱导HepG2细胞凋亡

Apoptosis induction of HepG2 cells by Acetoacetate extractive of Celastrus orbiculatus Thunb. via activating caspase-dependent mitochondrial pathway

目的通过检测南蛇藤乙酸乙酯提取物在诱导肝癌细胞HepG2凋亡过程中产生的凋亡蛋白表达量探讨其凋亡途径。方法应用Annexin V-FITC与碘化丙啶(promide iodine,PI)双染色法,通过流式细胞技术检测不同浓度(15μg/ml、30μg/ml、60μg/ml、120μg/ml和240μg/ml南蛇藤乙酸乙酯提取物)干预HepG2细胞24小时后的细胞凋亡情况;应用蛋白质印迹法检测不同浓度(30μg/ml、60μg/ml及120μg/ml)南蛇藤乙酸乙酯提取物作用HepG2细胞24小时及60μg/ml和120μg/ml南蛇藤乙酸乙酯提取物作用HepG2细胞48小时后细胞中细胞色素c、裂解的胱冬蛋白原9(cleaved caspase-9)和胱冬蛋白酶3(caspase-3)的表达。结果当南蛇藤乙酸乙酯提取物浓度为15μg/ml、30μg/ml和60μg/ml时,可诱导HepG2细胞出现细胞凋亡,且细胞色素c、裂解的胱冬蛋白原9和caspase-3的表达量增加;随南蛇藤提取物浓度的增加(30μg/ml、60μg/ml及120μg/ml),蛋白表达亦增强。结论南蛇藤乙酸乙酯提取物可能是通过促使膜间蛋白细胞色素c表达和释放活化caspase-9与caspase-3而诱导HepG2细胞的凋亡。

Objective To investigate the molecular mechanisms of acetoacetate extractive of Celastrus orbiculatus Thunb. inducing the apoptosis of HepG2 cells via detecting the expression of apoptosis proteins. Methods The apoptosis rates of HepG2 cells intervened by different concentrations of acetoacetate extractive of Celastrus orbicuIatus Thunb. （15 gg/ml, 30 gg/ml, 60 gg/ml, 120 gg/ml and 240 gg/ml） at 12 hours were detected by flow cytometry. The expression of Cytochrome-c, caspase 3 and cleaved caspase-9 after intervened by different concentrations of acetoacetate extractive of Celastrus orbiculatus Thunb. （30 9g/ml, 60 9g/ml and 120 gg/ml at 12 hours and 60 gg/ml and 120 gg/ml at 48 hours） were detected by Western blot. Results The acetoacetate extractive of Celastrus orbiculatus Thunb. （15 gg/ml, 30 pg/ml and 60 ~tg/ml） induced the apoptiosis of HepG2 cells, and facilitated the expression of apoptotic proteins such as Cytochrome-e, cleaved caspase-9 and caspase-3. With the increased concentration of acetoacetate extractive of Celastrus orbiculatus Thunb., the expression levels of apoptotic proteins improved. Conclusion The mechanism of the acetoacetate extractive of Celastrus orbiculatus Thunb. inducing the apoptosis of HepG2 cells may be the activation of the mitochondrial apoptotic pathway： leading to the release of mitochondrial mediators such as Cytochrome-c, cleaved caspase-9 and caspase-3.