没食子儿茶素没食子酸酯拮抗百草枯诱导SK-N-SH细胞凋亡的机制

Mechanism of epigallocatechin-3-gallate against apoptosis of SK-N-SH cells induced by paraquat

目的探讨茶多酚中主要活性成分没食子儿茶素没食子酸酯(EGCG)拮抗百草枯诱导人神经母细胞瘤细胞株SK-N-SH细胞凋亡的作用机制。方法培养SK-N-SH细胞并给予400μmol.L-1百草枯作用72 h诱导细胞凋亡,建立细胞凋亡模型。实验分为6组:空白对照组、百草枯模型组、维生素E(10μmol.L-1)组和EGCG(10、5、1μmol.L-1)3个剂量组。药物处理细胞2 h后,加入百草枯,72 h后检测细胞内超氧化物歧化酶(SOD)活性及丙二醛(MDA)、活性氧(ROS)的含量,罗丹明123染色及流式细胞术测线粒体膜电位,RT-PCR检测细胞bcl-2 mRNA与bax mRNA表达情况。结果与空白对照组比较,百草枯模型组细胞SOD活性降低,MDA和ROS含量增加,线粒体膜电位下降,bcl-2 mRNA表达减弱,bax mRNA表达增强,bcl-2/bax比值降低(均P<0.01)。对百草枯损伤的SK-N-SH细胞,维生素E和EGCG可明显提高SOD活性,降低MDA和ROS的含量,恢复线粒体膜电位,增强bcl-2 mRNA表达,减弱bax mRNA表达,升高bcl-2/bax比值(P<0.05或P<0.01)。结论 EGCG拮抗百草枯诱导SK-N-SH细胞凋亡的机制可能与提高细胞抗氧化能力、保护线粒体结构和功能的完整性。

AIM To approach the mechanism of epigallocatechin-3 gallate（EGCG） on apoptosis of human neuroblastoma（SK-N-SH） cells induced by paraquat（PQ）.METHODS SK-N-SH cells were cultured and added with 400 μmol.L-1 PQ for 72 hours to set up the model of cell apoptosis.The cells were randomly divided into 6 groups： blank control group,PQ model group,vitamin E（VE,10 μmol.L-1） group,and 3 EGCG（10,5 and 1 μmol.L-1） groups.After treatment of SK-N-SH cells with VE or different concentration of EGCG for 2 hours,PQ（400 μmol.L-1） was added to the cultures for 72 hours,respectively.The activity of SOD and the contents of MDA and ROS were assayed.Rhodamine 123 staining and FCM was used to detect the mitochondrial membrane potential（MMP）.The expression of apoptotic gene bcl-2 mRNA and bax mRNA were detected by RT-PCR method.RESULTS Compared with the blank control group,there were lower activity of SOD,higher levels of MDA and ROS,lower amplitude of MMP,weaker expression of bcl-2 mRNA,stronger expression of bax mRNA,and lower ratio of bcl-2 / bax in the PQ model group（P 0.01）.For the damage of the SK-N-SH cells induced by PQ,VE and EGCG could obviously increase the activity of SOD,reduce the contents of MDA and ROS,regain amplitude of MMP,enhance the expression of bcl-2 mRNA,reduce the expression in bax mRNA,and improve the ratio of bcl-2 mRNA to bax mRNA（P 0.05 or P 0.01）significantly.CONCLUSION The mechanism of EGCG antagonizing the apoptosis of SK-N-SH cells induced by PQ may be concerned with enhancing cell antioxidant capacity,protecting the integrity of mitochondrial structure and function,and suppressing the over expression of apoptosis gene in mitochondria.