TRPV4在心肌细胞缺氧损伤中的作用及机制研究  

Role and mechanism of TRPV4 in myocardial hypoxia injury

作　　者：陈卓 秦昆 何玥颖 魏朋 谢高倩 苗路伟 陈克明 CHEN Zhuo;QIN Kun;HE Yue-ying;WEI Peng;XIE Gao-qian;MIAO Lu-wei;CHEN Ke-ming(Basic Medical Laboratory of the 940th Hospital of Joint Logistics Support Force of Chinese People's Liberation Army,Lanzhou 730050,China;College of Life Science and Engineering,Lanzhou University of Tenchnology,Lanzhou 730050,China)

机构地区：[1]解放军联勤保障部队第940医院基础医学实验室,甘肃兰州730050 [2]兰州理工大学生命科学与工程学院,甘肃兰州730050

出　　处：《中国病理生理杂志》2022年第7期1194-1200,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金面上项目(No.81770879);联勤保障部队第九四〇医院实验室培育项目(No.2021yxky081)。

摘　　要：目的:探究瞬时受体电位阳离子通道V亚家族成员4(transient receptor potential cation channel subfamily V member 4,TRPV4)在H9C2细胞中缺氧环境下的作用机制。方法:采用H9C2细胞建立缺氧损伤模型,检测缺氧不同时间细胞内乳酸脱氢酶(lactate dehydrogenase,LDH)释放率、丙二醛(malondialdehyde,MDA)含量、活性氧(reactive oxygen species,ROS)水平及过氧化氢酶(catalase,CAT)和超氧化物歧化酶(superoxide dismutase,SOD)活性。将细胞分为常氧组(0 h)、HC-067047(TRPV4抑制剂)预处理+常氧(0 h)组、缺氧(24 h)组和HC-067047预处理+缺氧(24 h)组,使用Fluo-4 AM检测细胞内Ca^(2+)含量,JC-1检测线粒体膜电位,Western blot检测TRPV4蛋白水平,RT-qPCR检测TRPV4的mRNA水平,DCFH-DA检测ROS含量,酶标仪检测CAT和SOD活性,CCK-8法检测细胞活力。结果:与0 h组相比,随着缺氧时间的延长,LDH释放率提高(P<0.05),细胞内ROS和MDA含量上升(P<0.05),CAT和SOD活性增强(P<0.05),氧化损伤逐渐加重并在24 h达到顶峰。此外,缺氧后细胞出现钙超载现象,线粒体膜电位下降,TRPV4被激活。使用TRPV4抑制剂HC-067047预处理后,H9C2的氧化损伤明显被抑制,细胞活力上升(P<0.05),并且钙超载现象和线粒体膜电位下降均有所逆转。结论:在心肌细胞缺氧后,TRPV4被激活,Ca^(2+)内流,线粒体膜电位受损,ROS增多,氧化损伤加重,抗氧化系统被激活,CAT和SOD活性上升,进而调控氧化与抗氧化平衡。AIM:To explore the mechanism of transient receptor potential cation channel subfamily V member 4(TRPV4)in H9C2 rat cardiomyocytes under hypoxia.METHODS:The release of lactate dehydrogenase(LDH),the content of malondialdehyde(MDA),the level of reactive oxygen species(ROS)and the activity of catalase(CAT)and superoxide dismutase(SOD)in H9C2 cells were determined.The cells were divided into normoxia(0 h)group,HC-067047(TRPV4 inhibitor)pretreatment plus normoxia(0 h)group,hypoxia(24 h)group,and HC-067047 pretreatment plus hypoxia(24 h)group.Fluo-4 AM were used to detect intracellular Ca^(2+)content,and JC-1 was used to evaluate mitochondrial membrane potential.Western blot was used to detect TRPV4 protein level,and RT-qPCR was used to detect TRPV4 mRNA level.DCFH-DA was used to detect ROS content.CAT and SOD activity was detected by ELISA,and CCK-8 assay was used to detect cell viability.RESULTS:Hypoxia stimulated the LDH release in a time-dependent manner(P<0.05).Hypoxia increased intracellular ROS and MDA content as well as CAT and SOD activity.The oxidative damage was increased and reached the peak at 24 h after oxygen deprivation.In addition,calcium overload was observed in the cells after hypoxia.Mitochondrial membrane potential was decreased,while TRPV4 was activated.HC-067047,a TRPV4 inhibitor,inhibited the oxidative damage of H9C2 cells,and increased the cell viability.Calcium overload and mitochondrial membrane potential decline were reversed after treatment with HC-067047.CONCLUSION:The Ca^(2+)influx,loss of mitochondrial membrane potential,and increased TRPV4,ROS,CAT and SOD are involved in oxidative stress after myocardial hypoxia.

关 键 词：瞬时受体电位阳离子通道V亚家族成员4 心肌细胞 氧化应激 

分 类 号：R542.2[医药卫生—心血管疾病] R363.2[医药卫生—内科学]