目的 探讨自噬诱导剂能否通过激活自噬减少高糖高脂诱导的小鼠胰岛 β 细胞铁死亡.方法 选取小鼠胰岛瘤 MIN6 细胞,通过 25 mmol·L-1高糖联合 200 μmol·L-1棕榈酸钠(high Glucose and high Sodium Palmitate,GP)干预,建立胰岛 β细胞损伤模型,在此基础上给予雷帕霉素(rapamycin,RAPA)及铁死亡诱导剂(erastin,Era)干预 24 h后,用 CCK8 检测细胞活力;用试剂盒检测细胞亚铁离子、谷胱甘肽(glutathione,GSH)、超氧化物歧化酶(Superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)及活性氧(reactive oxygen species,ROS)水平;Western blot检测铁死亡相关蛋白谷胱甘肽过氧化物酶 4(Glutathione Peroxidase 4,GPX4)和酰基辅酶A 合成酶长链家族成员 4(acyl-CoA synthetase long-chain family member 4,ACSL4)、铁蛋白(Ferritin,FE)及自噬相关蛋白微管相关蛋白 l轻链 3Ⅱ(Mi-crotubule—associated protein1 light chain3,LC3Ⅱ)、P62 水平.结果 与对照组相比,GP 组及 Era组 MIN6 细胞活力、GSH含量、SOD活力均降低,ROS、MDA及亚铁离子增多,GPX4 蛋白表达水平降低,ACSL4、LC3Ⅱ、P62、FE 蛋白表达升高(P<0.05).与 GP 相比,RAPA干预后细胞活力增加,RAPA使亚铁离子、MDA 及 ROS 水平降低,GSH、SOD含量、GPX4、LC3Ⅱ蛋白表达水平升高,ACSL4、P62、FE 蛋白表达降低(P<0.05).结论 铁死亡参与 GP 诱导的MIN6 细胞损伤,其机制可能与自噬流阻滞有关.雷帕霉素可改善 β细胞铁死亡.
Objective To investigate whether autophagy inducer can reduce ferroptosis of mouse pancreatic β-cells induced by high-glu-cose and high-lipid through activation of autophagy.Methods The pancreatic β-cell injury model was established by treating high glu-cose(25 mmol·L-1)and sodium palmitate(200 μmol·L-1)(GP)in MIN6 cell.And on this basis,treated with rapamycin(RAPA)and erastin(Era).And the cell viability was detected by CCK8 assay.Furthermore,ferrous ion,the concentration of reactive oxygen species(ROS),malondialdehyde(MDA),glutathione(GSH)and the activity of superoxide dismutase(SOD)were detected by com-mercial kit.Glutathione Peroxidase 4(GPX4)and acyl-CoA synthetase long-chain family member 4(ACSL4),ferritin(Ferritin,FE)and Microtubule-associated protein 1 light chain 3Ⅱ(LC3Ⅱ)and P62 were detected by Western blot.Results GP and Era inhibited MIN6 cell viability,GSH content,SOD activity,increased ROS level,MDA and ferrous ion content,decreased GPX4 protein expres-sion level and increased ACSL4,LC3II,P62,FE protein expression level(P<0.05).RAPA intervention increased cell viability,de-creased ferrous ions,MDA and ROS levels,increased GSH,SOD content,increased GPX4,LC3Ⅱ protein expression,and decreased ACSL4,P62,and FE protein expression(P<0.05).Conclusion Ferroptosis is involved in GP-induced MIN6 cell damage,and the mechanism may be related to autophagic flux blockage.Rapamycin may improve β-cell ferroptosis.