白细胞介素6通过抑制自噬促进人脐静脉内皮细胞凋亡

Interleukin 6 induces apoptosis of human umbilical vein endothelial cells by inhibiting autophagy

目的探讨白细胞介素6(IL-6)对人脐静脉内皮细胞(HUVECs)自噬和凋亡的影响,以及自噬与凋亡的相互作用。方法用5、10、20、50 ng/m L IL-6诱导体外培养的HUVECs 24 h。CCK-8法检测细胞存活率,流式细胞术检测细胞凋亡变化,单丹磺酰戊二胺(MDC)染色观察细胞嗜酸性自噬泡情况,透射电镜观察细胞超微结构的变化,蛋白质印迹法检测凋亡相关蛋白cleaved caspase-3和自噬相关蛋白微管相关蛋白轻链3(LC3)、p62的表达水平。采用IL-6分别联合自噬抑制剂3-甲基腺嘌呤(3-MA)或自噬诱导剂西罗莫司(SRL)处理HUVECs,观察细胞增殖、凋亡情况及相关蛋白的表达变化。结果与对照组相比,IL-6浓度依赖性降低HUVECs的存活率和LC3Ⅱ/LC3Ⅰ比值(P<0.05),增加细胞凋亡和cleaved caspase-3、p62的表达(P<0.05),细胞内自噬泡减少(P<0.05),且自噬小体明显减少。与IL-6刺激组相比,IL-6联合应用3-MA诱导HUVECs可进一步抑制HUVECs的自噬水平,细胞内自噬泡和自噬小体数目减少(P<0.05),LC3Ⅱ/LC3Ⅰ比值降低(P<0.05),p62表达增加(P<0.05),细胞cleaved caspase-3表达和凋亡增加(P<0.05)。与IL-6刺激组相比,IL-6联合应用SRL处理HUVECs可以抑制IL-6诱导的细胞自噬水平下降,细胞内自噬泡和自噬小体数目增多(P<0.05),细胞内LC3Ⅱ/LC3Ⅰ比值升高(P<0.05),p62表达下降(P<0.05),cleaved caspase-3表达和细胞凋亡水平下降(P<0.05)。结论 IL-6可通过抑制自噬促进HUVECs的凋亡。

Objective To investigate the effect of interleukin 6(IL-6)on autophagy and apoptosis of human umbilical vein endothelial cells(HUVECs)and the interaction between autophagy and apoptosis.Methods HUVECs were cultured in vitro and stimulated with 5,10,20,50 ng/L IL-6 for 24 h.The cell proliferation was detected by cell counting Kit-8(CCK-8)assay,the apoptosis was detected by flow cytometry,the acidic vesicle organelles were observed by monodansylcadaverine staining,the ultra-microstructural cellular structure was observed by transmission electron microscope,and the expressions of apoptosis-related protein cleaved caspase-3 and autophagy-related proteins,including microtubule-associated protein 1 light chain 3(LC3)and p62,were detected by Western blotting.HUVECs were stimulated with IL-6 combined with autophagy inhibitor 3-methyladenine(3-MA)or autophagy inducer sirolimus(SRL),and then the changes of cell proliferation,cell apoptosis and expressions of the above proteins were analyzed.Results Compared with the control group,the cell proliferation and the ratio of LC3Ⅱ/LC3Ⅰin HUVECs in the IL-6 groups were significantly decreased(P<0.05),the apoptosis and the expressions of cleaved caspase-3 and p62 were significantly increased(P<0.05),the apoptotic vacuole number was significantly decreased(P<0.05),and the autophagy was reduced.The above effects were concentration-dependent(P<0.05).Compared with the IL-6 group,the autophagy of HUVECs in the IL-6 combined with 3-MA group was inhibited,the numbers of autophagosome and apoptotic vacuole were significantly decreased(P<0.05),LC3Ⅱ/LC3Ⅰratio was significantly decreased(P<0.05),and the cell apoptosis and the expression of cleaved caspase-3 and p62 were significantly increased(P<0.05).Compared with the IL-6 group,the autophagy of HUVECs in the IL-6 combined with SRL group was promoted,the numbers of autophagosome and apoptotic vacuole were significantly increased(P<0.05),LC3Ⅱ/LC3Ⅰratio was significantly increased(P<0.05),and the cell apoptosis and the expression of cleaved caspase-3 and p62 were significantly decreased(P<0.05).Conclusion IL-6 can induce the apoptosis of HUVECs by inhibiting autophagy