【摘要】 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。更多还原

【Abstract】 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 p r o l i f e r a t i o n a n d t h e r a t i o o f L C 3 Ⅱ/L C 3 Ⅰ i n H U V E C s i n t h e I L-6 g r o u p s w e r e s i g n i f i c a n t l y d e c r e a s e d(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.更多还原