马钱苷调节AKT/AMPK/Nrf2通路改善氧葡萄糖剥夺/复氧诱导的神经元铁死亡的机制研究

Mechanism of AKT/AMPK/Nrf2 Regulated by Loganin to Improve Ferroptosis Induced by Oxygen Glucose Deprivation/Reoxygenation

目的:探讨马钱苷通过调节蛋白激酶B(AKT)/腺苷酸活化蛋白激酶(AMPK)/核因子-E2相关因子2(Nrf2)通路改善氧葡萄糖剥夺/复氧(OGD/R)诱导的神经元铁死亡的机制。方法:将神经元分为对照组、OGD/R组、OGD/R+L-马钱苷组、OGD/R+M-马钱苷组、OGD/R+H-马钱苷组、OGD/R+H-马钱苷+ML385组。透射电子显微镜观察神经元线粒体形态;检测铁含量、谷胱甘肽过氧化物酶4(GPX4)活性、4-羟基壬烯醛(4-HNE)、超氧化物歧化酶(SOD)、丙二醛(MDA)、还原型谷胱甘肽(GSH)/氧化型谷胱甘肽(GSSG),还原型辅酶Ⅱ(NADPH)/辅脱氢酶Ⅱ(NADP^(+))及乳酸脱氢酶(LDH)含量;使用CM-H2DCFDA、C11-BODIPY581/591分别检测细胞内和脂质活性氧(ROS)水平;四唑盐(MTT)试剂盒检测细胞活性;蛋白免疫印迹法(Western Blot)检测B细胞淋巴瘤2(Bcl-2)、Bcl相关X蛋白(Bax)、剪切的半胱天冬氨酸蛋白酶3(cleaved Caspase-3)、磷酸化的蛋白激酶B(p-AKT)/AKT、磷酸化的腺苷酸活化蛋白激酶(p-AMPK)/AMPK、Nrf2蛋白表达。结果:OGD/R组神经元线粒体出现碎片化现象,嵴减少,线粒体膜密度有所增加。与对照组比较,OGD/R组GSH/GSSG、NADPH/NADP^(+)、SOD、GPX4相对活性、细胞活力以及Bcl-2水平、p-AKT/AKT、p-AMPK/AMPK、Nrf2水平下降(P<0.05),Fe^(2+)含量、细胞内ROS水平、脂质ROS水平以及4-HNE、MDA水平、LDH释放量、Bax以及cleaved Caspase-3水平上升(P<0.05);马钱苷处理后神经元线粒体中的线粒体嵴变得较为完整,碎片化现象消失,OGD/R+L-马钱苷组、OGD/R+M-马钱苷组、OGD/R+H-马钱苷组较OGD/R组GSH/GSSG、NADPH/NADP^(+)、SOD、GPX4相对活性、细胞活力以及Bcl-2水平、p-AKT/AKT、p-AMPK/AMPK、Nrf2水平上升(P<0.05),Fe^(2+)含量、细胞内ROS水平、脂质ROS水平以及4-HNE、MDA水平、LDH释放量、Bax以及cleaved Caspase-3水平下降(P<0.05),且随着马钱苷剂量的增加,改善效果更显著;OGD/R+H-马钱苷+ML385组以上指标与OGD/R组趋势一致。结论:马钱苷可能通过调节AKT/AMPK/Nrf2通路改善OGD/R诱导的神经元铁死亡。

Objective:To investigate the mechanism of loganin improves the ferroptosis induced by oxygen glucose deprivation/reoxygenation(OGD/R)by regulating protein kinase B(AKT)/adenylate activated protein kinase(AMPK)/nuclear factor E2 associated factor 2(Nrf2)pathway.Methods:The neurons were divided into control group,OGD/R group,OGD/R+L-loganin group,OGD/R+M-loganin group,OGD/R+H-loganin group,OGD/R+H-loganin+ML385 group.The morphology of neuron mitochondria was observed by transmission electron microscope.Iron content,glutathione peroxidase 4(GPX4)activity,4-hydroxynonenal(4-HNE),superoxide dismutase(SOD),malondialdehyde(MDA),reduced glutathione(GSH)/oxidized glutathione(GSSG),reduced CoenzymeⅡ(NADPH)/CodehydrogenaseⅡ(NADP^(+)),and Lactate dehydrogenase(LDH)content were determined.Intracellular and lipid reactive oxygen species(ROS)levels were detected by CM-H2DCFDA and C11-BODIPY581/591,respectively.MTT assay was used to detect cell activity.B-cell lymphoma 2(Bcl-2),Bcl-associated X protein(Bax),cleaved Caspase-3,phosphorylated protein kinase B(p-AKT)/AKT,phosphorylated adenylate activated protein kinase(p-AMPK)/AMPK,Nrf2 protein expression were detected by Western Blot.Results:In OGD/R group,mitochondrial fragmentation occurred,ridge decreased,and mitochondrial membrane density increased.Compared with the control group,the relative activities of GSH/GSSG,NADPH/NADP^(+),SOD,GPX4,and the levels of Bcl-2,p-AKT/AKT,p-AMPK/AMPK and Nrf2 in OGD/R group decreased(P<0.05),Fe^(2+)content,intracellular ROS levels,lipid ROS levels,4-HNE,MDA levels,LDH release,Bax,and cleaved Caspase-3 levels increased(P<0.05).After treatment with loganin,the mitochondrial ridge in neuronal mitochondria became more complete and the fragmentation disappeared.The relative activity of GSH/GSSG,NADPH/NADP^(+),SOD,GPX4,Bcl-2 level,p-AKT/AKT,p-AMPK/AMPK,and Nrf2 in OGD/R+L-loganin group,OGD/R+M-loganin group and OGD/R+H-loganin group were higher than those in OGD/R group,Fe^(2+)content,intracellular ROS levels,lipid ROS levels,4-HNE,MDA levels,LDH release,Bax,and cleaved Caspase-3 levels were lower than those in OGD/R group(P<0.05).With the increase of the dose of loganin,the improvement effects were more significant.The above indexes of OGD/R+H-loganin+ML385 group were consistent with the trend of OGD/R group.Conclusion:loganin may ameliorate OGD/R-induced neuronal ferroptosis by regulating the AKT/AMPK/Nrf2 pathway.