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胰高血糖素样肽-1类似物利拉鲁肽对高糖环境下人脐静脉内皮细胞自噬的影响
作者:刘晓霞 刘晶 高晓芳 袁和菊 李娜 李兴 
单位:030001 太原 山西医科大学第二医院内分泌科(刘晓霞、刘晶、高晓芳、袁和菊、李娜、李兴) 
关键词:GLP-1类似物 利拉鲁肽 自噬 人脐静脉内皮细胞 AMPK/mTOR 高糖 
分类号:R587.2
出版年,卷(期):页码:2019,44(2):113-119
摘要:

[摘要]  目的  探讨胰高血糖素样肽-1(GLP-1)类似物利拉鲁肽(LIRA)对高糖环境下人脐静脉内皮细胞(HUVECs) 自噬的影响及其可能机制。方法  研究分3部分进行:(1)取体外培养的HUVECs作为研究对象,设对照组(A)和高糖组(B),分别用5.5mmol/L25mmol/L的正常及高糖培养基培养,培养时间为122448hRT-PCR测定细胞内自噬相关基因beclin-1LC3p62 mRNA的表达,选出高糖对自噬影响的最佳培养时间点;(2)HUVECs作为研究对象,予以低、中、高剂量(1050100nmol/L)LIRA进行干预(C1C2C3),按第一部分实验得出的最佳时间点培养细胞后,采用同样的方法检测上述指标,得出最适宜的LIRA干预浓度;(3)分别在最佳剂量LIRA干预高糖组(C)和单纯高糖组(B)的基础上,加入AMPK抑制剂ComC(10μmol/L)处理(D组和E),以激光共聚焦荧光显微镜观察LC3与绿色荧光蛋白(GFP)融合(GFP-LC3)的荧光斑点来监测自噬体的形成,Western blotting检测细胞内自噬标志物beclin-1p62蛋白的表达和LC3-/LC3-Ⅰ、p-AMPK/AMPKp-mTOR/mTOR比值的变化。结果  (1)A组相比,不同培养时间下Bbeclin-1LC3 mRNA表达减少,而p62 mRNA表达增加(P<0.05),且24h时这种变化最为显著,故选择24h作为最佳培养时间;(2)B组相比,C3beclin-1LC3 mRNA表达增加,p62 mRNA表达减少(P<0.05),且C1C2C3 LC3 mRNA表达上升(P<0.05),呈剂量依赖性,故将100nmol/L作为LIRA的最佳干预浓度;(3)B组相比,C组的beclin-1蛋白、LC3-/LC3-Ⅰ、p-AMPK/AMPK升高,p62蛋白、p-mTOR/mTOR降低(P<0.05)DLC3-/LC3-Ⅰ、 p-AMPK/AMPK比值低于C组,p-mTOR/mTOR比值高于C(P<0.05),而beclin-1p62表达与C组比较差异无统计学意义(P0.05)。与D组比较,ELC3-/LC3-Ⅰ降低,p62蛋白表达增多(P<0.05)。免疫荧光检测显示GFP-LC3自噬荧光点数量变化的趋势与蛋白水平变化趋势一致。结论  GLP-1类似物LIRA可能通过磷酸化激活AMPK/mTOR介导的信号通路,激活自噬的发生,对高糖培养的HUVECs产生保护作用。

[Abstract]  Objective  To explore the effect of glucagon-like peptide-1 (GLP-1) analogue liraglutide (LIRA) on autophagy of human umbilical vein endothelial cells (HUVECs) under high glucose condition, and its corresponding mechanism. Methods  (1) The HUVECs were cultured in vitro and assigned to control group (group A) and high glucose group (group B). Group A and B were cultured respectively in 5.5mmol/L and 25mmol/L glucose medium for 12, 24 and 48 hours. The expressions of autophagy-related genes (beclin-1, LC3 and p62 mRNA) were determined by RT-PCR, and then the optimal culture time of high glucose condition on autophagy was selected. (2) The high glucose group at the optimal culture time point was treated with low, medium and high dose (10, 50, 100nmol/L) of LIRA (C1, C2, C3 groups). The same method was used to detect the indicators above, and the optimal intervention concentrations of LIRA were obtained. (3) In addition to the treatment of LIRA at optimal dose under high glucose condition (group C) and high glucose culture alone (group B), the AMPK inhibitor ComC (10μmol/L) was introduced (group D and group E). The fluorescence spots of green fluorescent protein (GFP) conjugated LC3 (GFP-LC3) were observed by laser confocal fluorescence microscopy to monitor the formation of autophagosome. The expressions of cell autophagy marker beclin-1, p62 and the ratio change of LC3-/LC3-, p-AMPK/AMPK and p-mTOR/mTOR were measured by Western blotting. Results  (1) Compared with group A, the expressions of beclin-1 and LC3 mRNA in group B decreased, while of p62 mRNA increased at different culture time (P<0.05). The changes above were most significant at 24h, so the 24h was chosen as the optimal culture time. (2) Compared with group B, the expressions of beclin-1 and LC3 mRNA increased and of p62 mRNA decreased in C3 group (P<0.05), and the expressions of LC3 mRNA in C1, C2 and C3 groups all increased (P<0.05) in a dose-dependent pattern, so the optimal intervention concentration of LIRA was 100nmol/L. (3) The expressions of protein beclin-1, LC3-/LC3-and p-AMPK/AMPK increased, and of protein p62 and p-mTOR/mTOR decreased in group C compared with that in group B (P<0.05). The ratio of LC3-/LC3-and p-AMPK/AMPK was lower and of p-mTOR/mTOR was higher in group D than in group C (P<0.05). There was no significant difference in the expression level of protein beclin-1 and p62 between group C and D (P>0.05). Compared with group D, LC3-/LC3-ratio decreased and the expression of p62 protein increased in group E (P<0.05). The change trend of autophagy fluorescent spots GFP-LC3 was consistent with that of autophagy related protein. Conclusion  GLP-1 analogue liraglutide may protect HUVECs from high glucose condition by activating autophagy, which was achieved through AMPK-mediated signaling pathway.

基金项目:
山西省国际科技合作项目(2015081031);山西省卫生计生委科研项目(2014040)
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