白细胞介素6通过抑制自噬促进人脐静脉内皮细胞凋亡

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/mL 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 monodansyl cadaverine 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