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

作　　者：张方圆 齐越 原美茹 任建平 刘永学 ZHANG Fangyuan;QI Yue;YUAN Meiru;REN Jianping;LIU Yongxue(Institute of Radiation Medicine,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]军事医学研究院辐射医学研究所,北京100850

出　　处：《癌变．畸变．突变》2020年第5期363-368,共6页Carcinogenesis,Teratogenesis & Mutagenesis

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(CaMKⅡ)、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CaMKⅡ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CaMKⅡ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。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 PI3 K/Akt pathway.

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 

分 类 号：R364.4[医药卫生—病理学]