利拉鲁肽对大鼠心肌微血管内皮细胞缺氧复氧损伤的影响

李丹丹; 张颖; 陈韵岱

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武警医学 ›› 2018, Vol. 29 ›› Issue (9) : 843-849.

论著

利拉鲁肽对大鼠心肌微血管内皮细胞缺氧复氧损伤的影响

李丹丹, 张颖, 陈韵岱

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Liraglutide protects cardiac microvascular endothelial cells against hypoxia/reoxygenation injury

LI Dandan, ZHANG Ying, and CHEN Yundai.

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摘要

目的探究胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)类似物利拉鲁肽(GLP-1 analogue,Liraglutide)对大鼠心肌微血管内皮细胞(cardiac microvascular endothelial cells, CMEC)缺氧复氧损伤的影响。方法双酶消化法体外分离培养SD大鼠CMEC,实验的细胞分为正常对照组、Liraglutide(0~100 nM)组、H/R组、H/R+Liraglutide组。建立缺氧复氧模型(hypoxia/reoxygenation model,H/R)诱导的细胞氧化应激凋亡模型,流式细胞学检测细胞凋亡率,DCFHDA荧光探针检测胞内ROS的变化免疫荧光染色和Western Blot检测XO、caspase3等蛋白分子的表达情况,通过比较各组中XO、ROS以及凋亡相关蛋白caspase3的表达情况阐明Liraglutide在抗细胞氧化应激过程中的保护作用机制。结果与正常对照组细胞相比,H/R模型条件诱导细胞中XO表达增加,生成过量ROS及凋亡蛋白的表达,最终导致凋亡细胞数量显著提高至20.66%±1.30%。Liraglutide预处理12 h抑制了H/R诱导的XO的激活,最终降低胞浆过量ROS并将细胞的凋亡数量降低至8.36±1.19%。结论 H/R模型导致XO-ROS激活,生成过量胞浆ROS,最终介导了CMEC的线粒体凋亡通路激活,而予以Liraglutide预处理可以逆转上述过程。

Abstract

Objective To explore the effects of liraglutide, a GLP-1 analogue, on CMECs and the mechanisms by which liraglutide reduces the oxidative stress apoptosis of CMECs under a hypoxia/reoxygenation (H/R) model.Methods In vitro cultured CMECs of SD rats were purified with the differential adhesion method and identified immunocytochemically using CD31 antibody. GLP-1R and CD31 were assessed by co-location immunohistochemistry. MTT assay was performed to assess the proliferation of the first-generation cells exposed to different concentrations (0-100 nM) of liraglutide. An H/R model was used to induce cellular apoptosis, and the apoptotic rate was assessed by low cytometry. DCFHDA was used to evaluate the ROS contents. Western blot and immunohistochemistry were used to assess the expressions of XO and caspase3. The expressions of XO, ROS and caspase3 in each group were compared to illustrate the liraglutide-mediated anti-apoptotic functions.Results The H/R model induced a higher expression of XO, and consequently generated excessive ROS which was responsible for cellular apoptosis (20.66%±1.30%). Lirglautide pretreatment for 12 h could suppress the XO activation, ROS outburst and cellular apoptosis (8.36%±1.19%).Conclusions This study has confirmed that H/R can induce CMECs’ oxidative damage through the XO-ROS injury signals, and that liraglutide pretreatment may suppress such damage to CMECs.

导出引用

李丹丹, 张颖, 陈韵岱. 利拉鲁肽对大鼠心肌微血管内皮细胞缺氧复氧损伤的影响[J]. 武警医学. 2018, 29(9): 843-849

LI Dandan, ZHANG Ying, and CHEN Yundai.. Liraglutide protects cardiac microvascular endothelial cells against hypoxia/reoxygenation injury[J]. Medical Journal of the Chinese People Armed Police Forces. 2018, 29(9): 843-849

中图分类号： R542

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