龙牙楤木总皂苷通过调控AMPK/Nrf2信号抑制缺氧/复氧诱导的心肌细胞铁死亡

Inhibitory Effect of Total Saponins of Aralia elata on Hypoxia/Reoxygenation-Induced Iron Death of Cardiomyocytes by Regulating AMPK/Nrf2 Signaling

目的:探究龙牙楤木总皂苷对AMPK/Nrf2信号通路的影响。方法:将人心肌细胞AC16进行体外培养,分为空白对照组、缺氧/复氧模型组、铁离子螯合剂对照组,铁离子螯合剂组、龙牙楤木总皂苷低(0.02μg/mL)浓度组、龙牙楤木总皂苷中(0.1μg/mL)浓度组、龙牙楤木总皂苷高(0.5μg/mL)浓度组,丹参滴丸(50μg/mL)阳性对照组,AMPK抑制剂Compound C(5μg/mL)+龙牙楤木总皂苷(0.5μg/mL)组,Nrf2抑制剂ML385(2μg/mL)+龙牙楤木总皂苷(0.5μg/mL)组。MTT法检测细胞存活率;试剂盒检测细胞内Fe^(2+)、ROS水平;免疫荧光法检测脂质过氧化物及GPX4、ACSL4蛋白表达;qRT-PCR检测GPX4、ACSL4 mRNA表达,Western Blot检测GPX4、ACSL4、p-AMPK、Nrf2蛋白表达;结果:与空白对照组比较,模型组细胞存活率、GPX4 mRNA表达显著降低,Fe^(2+)含量、ROS和脂质过氧化水平、ACSL4 mRNA表达显著升高,p-AMPK、Nrf2蛋白表达显著降低(P<0.01)。与模型组比较,铁离子螯合剂组细胞存活率、GPX4 mRNA表达显著升高,Fe^(2+)、ROS、脂质过氧化水平及ACSL4 mRNA表达显著降低(P<0.01);龙牙楤木总皂苷各组和丹参滴丸组细胞存活率及GPX4、p-AMPK、Nrf2蛋白表达显著升高,心肌细胞中Fe^(2+)、ROS、脂质过氧化水平及ACSL4蛋白表达显著降低(P<0.05或P<0.01)。与龙牙楤木总皂苷组比较,Compound C+龙牙楤木总皂苷组和ML385+龙牙楤木总皂苷组细胞存活率显著降低,Fe^(2+)含量显著升高,Nrf2蛋白表达显著降低,Compound C+龙牙楤木总皂苷组ROS和脂质过氧化水平显著升高,p-AMPK蛋白表达显著降低(P<0.05或P<0.01)。结论:龙牙楤木总皂苷可通过激活AMPK/Nrf2信号通路,抑制H/R诱导的心肌细胞铁死亡。

Objective:To investigate the effect of total saponins of Aralia elata on AMPK/Nrf2 signaling pathway.Methods:Human cardiomyocytes AC16 were cultured in vitro and divided into blank control group,hypoxia/reoxygenation model group,iron ion chelating agent control group,iron ion chelating agent group,low(0.02μg/mL),medium(0.1μg/mL),high(0.5μg/mL)concentration groups of total saponins of Aralia elata,Danshen dropping pills(50μg/mL)positive control group,AMPK inhibitor Compound C(5μg/mL)with total saponins of Aralia elata(0.5μg/mL)group and Nrf2 inhibitor ML385(2μg/mL)with total saponins of Aralia elata(0.5μg/mL)group.Cell survival rate was detected by MTT assay.The kits were used to detect Fe^(2+)and ROS levels in cells.The level of lipid peroxides and protein expressions of GPX4,ACSL4 were detected by immunofluorescence.The mRNA expressions of GPX4 and ACSL4 were detected by qRT-PCR,and the protein expressions of GPX4,ACSL4,p-AMPK and Nrf2 were detected by Western Blot.Results:Compared with blank control group,cell survival rate and GPX4 mRNA expression in model group were significantly decreased,Fe^(2+)content,ROS and lipid peroxidation levels,ACSL4 mRNA expression were significantly increased,and protein expressions of p-AMPK,Nrf2 were significantly decreased(P<0.01).Compared with model group,cell survival rate and GPX4 mRNA expression in iron ion chelating agent group,while Fe^(2+),ROS and lipid peroxidation levels and ACSL4 mRNA expression were significantly decreased(P<0.01).The cell survival rate and GPX4,p-AMPK and Nrf2 protein expressions of total saponins of Aralia elata groups and Danshen dropping pills group were significantly increased were significantly increased,while Fe^(2+),ROS,lipid peroxidation levels and ACSL4 protein expression in cardiomyocytes were significantly decreased(P<0.05 or P<0.01).Compared with the total saponins of Aralia elata groups,the cell survival rate of Compound C with total saponins of Aralia elata(0.5μg/mL)group and ML385 with total saponins of Aralia elata(0.5μg/mL)group was significantly decreased,the Fe^(2+)content was significantly increased,the protein expression of Nrf2 was significantly decreased,ROS and lipid peroxidation levels in Compound C with total saponins of Aralia elata group were significantly increased,the p-AMPK protein expression was significantly decreased(P<0.05 or P<0.01).Conclusion:Total saponins of Aralia elata can inhibit H/R-induced iron death of cardiomyocytes by activating AMPK/Nrf2 signaling pathway.