摘要
目的探讨棕榈酸(PA)是否可诱导小鼠胰岛β细胞株MIN6细胞发生铁死亡。方法将小鼠胰岛β细胞株MIN6细胞分为正常对照组、PA组(1.0 mmol/L)、PA+凋亡抑制剂(Z-VAD-FMK)组(5μmol/L)、PA+铁死亡抑制剂(Fer-1)组(5μmol/L)、PA+坏死抑制剂(Nec-1)组(5μmol/L),每组设置3个平行组。采用CCK8法检测细胞存活率,电镜观察细胞超微结构改变,FerroOrange试剂盒检测细胞内Fe^(2+)水平,丙二醛(MDA)试剂盒检测MDA水平,流式法检测活性氧(ROS)水平,实时荧光定量PCR法检测凋亡基因半胱氨酸蛋白酶3(caspase3)、坏死基因受体结合丝氨酸苏氨酸激酶3(RIPK3)、铁死亡相关基因长链脂酰辅酶A合成酶4(ACSL4)、花生四烯酸-15-脂加氧酶(ALOX15)、前列腺素内过氧化物合酶2(Ptgs2)、铁蛋白重链(FTH)、铁蛋白轻链(FTL)和谷胱甘肽过氧化物酶4(GPX4)的mRNA表达水平,Western blotting法检测活化型caspase3(cleaved caspase3)、RIPK3、ACSL4、ALOX15和Ptgs2的蛋白表达水平。两组间比较采用t检验。结果与PA组相比,PA+Z-VAD-FMK组、PA+Fer-1组和PA+Nec-1组MIN6的存活率均更高(P<0.01)。PA组MIN6细胞电镜下可见凋亡、坏死、及铁死亡相关的形态学特征。与对照组相比,PA组MIN6细胞内Fe^(2+)相对荧光强度、MDA水平、总ROS均更高;caspase3、RIPK3、ACSL4、Ptgs2、ALOX15的mRNA相对表达量均更高,FTH和GPX4的mRNA相对表达量均更低;cleaved caspase3、RIPK3、ACSL4、ALOX15和Ptgs2的蛋白相对表达量均更高(均P<0.01)。与PA组相比,PA+Fer-1组Fe^(2+)相对荧光强度、MDA水平、总ROS均更低;ACSL4、Ptgs2、ALOX15的mRNA相对表达量均更低,FTH和GPX4的mRNA相对表达量均更高;ACSL4、ALOX15和Ptgs2的蛋白相对表达量均更低(均P<0.01)。结论PA可诱导小鼠胰岛β细胞株MIN6细胞发生铁死亡。
Objective To explore whether palmitic acid(PA)can induce ferroptosis in pancreatic beta-cell line MIN6 cells.Methods Pancreatic beta-cell line MIN6 cells were divided into normal group,PA group(1.0 mmol/L),PA+apoptosis inhibitor(Z-VAD-FMK)group(5μmol/L),PA+ferrostain-1(Fer-1)group(5μmol/L),PA+necrostatin-1(Nec-1)group(5μmol/L).Each group was set three parallels.Cell viability was detected by CCK8 method.Cellular ultrastructure was assessed with electron microscopy.FerroOrange was used to detect intracellular Fe^(2+).The lipid peroxidation assay kit was used to detect the concentration of malondialdehyde(MDA).Flow cytometry was used to detect intracellular reactive oxygen species(ROS)levels.Real-time quantitative polymerase chain reaction was used to detect the mRNA levels of caspase3,receptor-interacting protein kinase 3(RIPK3),acyl-CoA synthetase long-chain family member 4(ACSL4),prostaglandin endoperoxidase synthase 2(Ptgs2),arachidonate lipoxygenase 15(ALOX15),ferritin heavy chain(FTH),ferritin light chain(FTL),and glutathione peroxidase 4(GPX4).Western blotting was used to detect the proteins levels of cleaved caspase3,RIPK3,ACSL4,ALOX15 and Ptgs2.The t-test was used to compare two groups.Results Compared with the PA group,cell viability of MIN6 was higher in PA+Z-VAD-FMK group,PA+Fer-1 group or PA+Nec-1 group(P<0.01).MIN6 cells,which were treated with PA to exhibit the characteristic morphologic features,were associated with apoptosis,necrosis and ferroptosis under electron microscope.Compared with the control group,in PA group,the levels of the cellular intracellular Fe^(2+),MDA and total lipid ROS were increased.The mRNA relative expression levels of caspase3,RIPK3,ACSL4,Ptgs2 and ALOX15 were increased.The mRNA relative expression levels of FTH and GPX4 were decreased.The protein relative expression levels of cleaved caspase3,RIPK3,ACSL4,ALOX15 and Ptgs2 were increased(all P<0.01).Compared with the PA group,in PA+Fer-1 group,the levels of cellular intracellular Fe^(2+),MDA and total lipid ROS were decre
作者
秦璐
黄逸婷
李雨潇
秦瑶
石毓雯
许馨予
陈恒
张梅
Qin Lu;Huang Yiting;Li Yuxiao;Qin Yao;Shi Yuwen;Xu Xinyu;Chen Heng;Zhang Mei(Department of Endocrinology,First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China)
出处
《中华糖尿病杂志》
CAS
CSCD
北大核心
2022年第5期490-497,共8页
CHINESE JOURNAL OF DIABETES MELLITUS
基金
国家自然科学基金(81974103)。
