二甲双胍对血管内皮细胞自噬的作用

Effect of metformin on autophagy in human umbilical vein endothelial cells

目的观察二甲双胍对内皮细胞自噬的影响,并探讨其可能的调控机制。方法以不同浓度二甲双胍(0、2.5、5、10、20、40 mmol/L)处理人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)24 h,并以5 mmol/L二甲双胍分别处理细胞24、48、72 h,用CCK-8法检测细胞存活率;药物处理细胞分4组:对照组(ctl)、二甲双胍组(Met)、TGFβ1组(TGF)、二甲双胍+TGFβ1处理组(M+T),透射电镜观察各组细胞中自噬体的形成;免疫荧光和Western Blot检测自噬相关分子LC3Ⅱ、Beclin-1的表达。在原分组基础上进一步加入Hippo信号通路抑制剂XMU-MP-1和Hippo信号转录因子YAP阻断剂维替泊芬处理,包括XMU-MP-1处理组(XMU),二甲双胍+XMU-MP-1处理组(M+X)、维替泊芬组(Ver)、维替泊芬+TGFβ1共处理组(V+T)、维替泊芬+XMU-MP-1共处理组(V+X),Western Blot检测LC3Ⅱ、YAP、pYAP及其靶蛋白CTGF的表达。结果CCK-8结果表明,与0 mmol/L组相比,10、20、40 mmol/L二甲双胍处理24 h,细胞存活率显著降低(P<0.01),因此后续实验采用5 mmol/L二甲双胍处理24 h;电镜、免疫荧光及Western blot结果表明,与ctl组比较,TGF组细胞内自噬体形成增多,LC3Ⅱ、Beclin-1的表达上调(P<0.05),YAP磷酸化水平降低(P<0.01),CTGF表达上调(P<0.01),加入二甲双胍共处理的M+T组中上述变化明显被阻断;与TGF组比较,V+T组LC3Ⅱ和CTGF表达均下调(P<0.01)。此外,与ctl组比较,XMU组LC3Ⅱ表达增强(P<0.01)、YAP磷酸化水平降低(P<0.01)、CTGF表达(P<0.01)显著上调,与TGF组表达趋势一致;经二甲双胍处理(M+X组),同样逆转上述变化,与M+T组表达趋势一致;与XMU组比较,加入维替泊芬的V+X组LC3Ⅱ和CTGF的表达均下调(P<0.01)。结论二甲双胍通过阻断YAP信号分子的激活抑制了TGFβ1诱导的内皮细胞自噬,从而可能对血管起到保护作用。

Objective To observe the effects of metformin on the autophagy of endothelial cells and explore the underlying mechanism.Methods Human umbilical vein endothelial cells(HUVECs)were treated with 0,2.5,5,10,20 and 40 mmol/L metformin for 24 h or with 5 mmol/L metformin for 24,48 and 72 h,and cell survival after the treatment was assessed using CCK-8 assay.The formation of autophagosomes in HUVECs treated with metformin and transforming growth factor-β1(TGF-β1),alone or in combination,was observed with transmission electron microscopy,and the expression levels of autophagy-related molecules LC3 II and Beclin-1 were detected using immunofluorescence assay and Western blotting.The effects of XMU-MP-1(an inhibitor of Hippo signaling pathway)and verteporfin(a blocker of Hippo signal transcription factor YAP)in the absence or presence of metformin or TGF-β1 on the expression levels of LC3 II,YAP,pYAP and CTGF(the target protein of pYAP)were detected using Western blotting.Results CCK-8 assay showed that compared with the control cells,HUVECs treated with 10,20 and 40 mmol/L metformin for 24 h exhibited significantly reduced cell survival rates(all P<0.01),and we therefore used 5 mmol/L metformin for 24 h in the subsequent experiments.The results of electron microscopy,immunofluorescence assay and Western blotting showed that treatment with TGF-β1 significantly increased the number of autophagosomes,enhanced the expression of LC3 II and Beclin-1(P<0.05),lowered the expression of pYAP(P<0.01),and up-regulated CTGF expression in HUVECs(P<0.01).These changes were significantly blocked by metformin.The expression of levels LC3 II and CTGF were also inhibited in the cells by treatment with verteporfin and TGF-β1(P<0.01).XMU-MP-1 showed similar effects to TGF-β1 and significantly up-regulated the expression of LC3 II(P<0.01),decreased the expression of pYAP(P<0.01),and enhanced the expression of CTGF(P<0.01);such effects of XMU-MP-1 were also significantly blocked by metformin(all P<0.05).The expression levels of CTGF and LC3 II were obviously down-regulated in the cells treated with both verteporfin and XMU-MP-1(P<0.01).Conclusion Metformin may protect the veins by inhibiting TGF-β1-induced autophagy and blocking the activation of YAP signaling in the endothelial cells.