原代培养大鼠心肌细胞低氧损伤时琥珀酸G蛋白偶联受体通路的变化

doi:10.3969/j.issn.1004-616x.2020.05.006

癌变·畸变·突变 ›› 2020, Vol. 32 ›› Issue (5): 363-368.doi: 10.3969/j.issn.1004-616x.2020.05.006

原代培养大鼠心肌细胞低氧损伤时琥珀酸G蛋白偶联受体通路的变化

张方圆, 齐越, 原美茹, 任建平, 刘永学

军事医学研究院辐射医学研究所, 北京 100850

收稿日期:2019-09-19 修回日期:2020-06-17 出版日期:2020-10-01 发布日期:2020-10-12
通讯作者: 刘永学,E-mail:liuyongxue2008@sina.com E-mail:liuyongxue2008@sina.com
作者简介:张方圆,E-mail:506467403@qq.com。

Changes of the succinate receptor pathway in primary rat cardiomyocyte cultures under hypoxic conditions

ZHANG Fangyuan, QI Yue, YUAN Meiru, REN Jianping, LIU Yongxue

Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China

Received:2019-09-19 Revised:2020-06-17 Online:2020-10-01 Published:2020-10-12

摘要/Abstract

摘要： 目的：探讨琥珀酸G蛋白偶联受体（GPR91）通路与低氧环境心肌细胞损伤之间的关系。方法：体外培养乳鼠原代心肌细胞，建立低氧模型，再利用siRNA干扰技术降低细胞内GPR91的表达，随机分为阴性对照组（CTL）、CTL+siGPR91组、琥珀酸盐组、琥珀酸盐+siGPR91组、低氧CTL组、低氧CTL+siGPR91组、低氧琥珀酸盐组和低氧琥珀酸盐+siGPR91组。10%氧含量下培养5 d后，CCK-8法检测心肌细胞的存活率、原代心肌细胞低氧模型的细胞ATP含量，Western blot法检测心肌损伤相关蛋白钙调素依赖蛋白激酶Ⅱ（CaMK Ⅱ）、B型尿钠肽（BNP）、GPR91以及PI3K/Akt蛋白通路Akt及P-Akt的表达变化。结果：与CTL组比较，低氧环境下可见心肌细胞存活率降低，ATP含量减少，BNP及CaMK Ⅱ表达上调（均为P < 0.05）；siRNA干扰组心肌细胞GPR91的表达水平下调（P < 0.05），心肌细胞存活率上升、ATP含量增多、BNP及CaMK Ⅱ蛋白表达下调、PI3K及下游信号分子Akt磷酸化水平降低（均为P < 0.05）。结论：GPR91可能通过调控PI3K/Akt通路的磷酸化介入心肌缺血损伤的病理过程。

关键词: 原代心肌细胞, 心肌缺氧, 琥珀酸G蛋白偶联受体91, PI3K/Akt通路

Abstract: OBJECTIVE: To investigate relationships between expression in the succinate receptor pathway and cardiomyocyte injury under a hypoxic environment. METHODS: Primary cardiomyocytes of suckling mice were cultured in vitro under different hypoxic environment. The siRNA interference technology was used to reduce expression of intracellular succinate receptor G protein-coupled receptor 91 (GPR91). The different cultures were randomly divided into the control (CTL),CTL+siGPR91 group,succinate group,succinate+siGPR91 group,hypoxic CTL group,hypoxic CTL+siGPR91 group,hypoxic succinate group and hypoxic Succinate+siGPR91 group. After 5 days of cultivation under 10% oxygen content,CCK-8 was used to detect the survival rate of cardiomyocytes. In addition,cellular ATP contents of primary cardiomyocyte hypoxia model were determined. Changes in expression of Akt and P-Akt in the PI3K/Akt protein pathway were determined using the Western blot. RESULTS: Compared with the CTL group,the survival rate and the content of ATP of myocardial cells were reduced,and expressions of BNP and CaMK Ⅱ were up-regulated in the hypoxic environment (P < 0.05). Compared with the CTL group,expression levels of GPR91 in the siRNA interference group were suppressed (P < 0.05). At the same time,myocardial cell survival rates and ATP contents were increased, BNP and CaMK Ⅱ protein expressions were down-regulated,and phosphorylation levels of PI3K and levels of downstream signaling molecule Akt were reduced (P < 0.05). CONCLUSION: Our data show that GPR91 was involved in the pathological process of myocardial ischemic injury by regulating the phosphorylation of PI3K/Akt pathway.

Key words: primary cardiomyocyte, myocardial hypoxia, GPR91, PI3K/Akt pathway

中图分类号:

R364.4

张方圆, 齐越, 原美茹, 任建平, 刘永学. 原代培养大鼠心肌细胞低氧损伤时琥珀酸G蛋白偶联受体通路的变化[J]. 癌变·畸变·突变, 2020, 32(5): 363-368.

ZHANG Fangyuan, QI Yue, YUAN Meiru, REN Jianping, LIU Yongxue. Changes of the succinate receptor pathway in primary rat cardiomyocyte cultures under hypoxic conditions[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2020, 32(5): 363-368.

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原代培养大鼠心肌细胞低氧损伤时琥珀酸G蛋白偶联受体通路的变化

Changes of the succinate receptor pathway in primary rat cardiomyocyte cultures under hypoxic conditions

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