[1]李冰玉,刘威远,林鑫,等.二甲双胍对血管内皮细胞自噬的作用[J].陆军军医大学学报(原第三军医大学学报),2020,42(08):783-789.
 LI Bingyu,LIU Weiyuan,LIN Xin,et al.Effect of metformin on autophagy in human umbilical vein endothelial cells[J].J Amry Med Univ (J Third Mil Med Univ),2020,42(08):783-789.
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二甲双胍对血管内皮细胞自噬的作用(/HTML )
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陆军军医大学学报(原第三军医大学学报)[ISSN:1000-5404/CN:51-1095/R]

卷:
42卷
期数:
2020年第08期
页码:
783-789
栏目:
基础医学
出版日期:
2020-04-30

文章信息/Info

Title:
Effect of metformin on autophagy in human umbilical vein endothelial cells
作者:
李冰玉刘威远林鑫赵祉颖王志刘杨东王韵
400016 重庆,重庆医科大学附属第一医院血管外科1,400038 重庆,陆军军医大学(第三军医大学)基础医学院细胞生物学教研室2
Author(s):
LI Bingyu1 LIU Weiyuan2 LIN Xin2 ZHAO Zhiying2 WANG Zhi2 LIU Yangdong1 WANG Yun2

1Department of Vascular Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016; 2Department of Cell Biology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing, 400038, China

关键词:
血管损伤内皮细胞自噬TGF&beta1二甲双胍YAP
Keywords:
vascular injury endothelial cells autophagy transforming growth factor-&beta1 metformin yes-associated protein
分类号:
R322.12;R966;R977.15
文献标志码:
A
摘要:

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。

Abstract:

ObjectiveTo observe the effects of metformin on the autophagy of endothelial cells and explore the underlying mechanism. MethodsHuman 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 LC3II 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 LC3II, YAP, pYAP and CTGF (the target protein of pYAP) were detected using Western blotting. ResultsCCK-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 LC3II 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 LC3II 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 LC3II (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 LC3II were obviously down-regulated in the cells treated with both verteporfin and XMU-MP-1 (P<0.01). ConclusionMetformin may protect the veins by inhibiting TGF-β1-induced autophagy and blocking the activation of YAP signaling in the endothelial cells.

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更新日期/Last Update: 2020-04-23