铁死亡抑制剂对心肌细胞缺氧/复氧损伤的干预作用及其机制  

Effect of iron death inhibitor on hypoxia/reoxygenation injury of cardiomyocytes and its mechanism

作　　者：韩月 李凤翔 杨国宏 袁辉[1] 张绪东[1] 孙健[1] HAN Yue;LI Feng-xiang;YANG Guo-hong;YUAN Hui;ZHANG Xyu-dong;SUN Jian(Mudanjiang Medical University,Mudanjiang 157011,China)

机构地区：[1]牡丹江医学院基础医学院,黑龙江牡丹江157011

出　　处：《中国应用生理学杂志》2022年第5期515-519,583,共6页Chinese Journal of Applied Physiology

基　　金：牡丹江医学院博士科研启动基金(2021-MYBSKY-023);黑龙江省省属高等学校基本科研业务费项目(2020-KYYWFMY-0039);黑龙江省卫生健康委科研项目(2020-416)。

摘　　要：目的:验证心肌细胞缺氧/复氧(H/R)损伤中是否有铁死亡的发生,以及探讨铁死亡抑制剂Ferrostatin-1(Fer-1)对心肌细胞H/R损伤的作用及其机制。方法:新生1~3 d SD乳鼠,提取原代心肌细胞,随机分为正常对照组(Control)、H/R组和H/R+Fer-1组。H/R组细胞培养52 h后,加入4 mmol/L Na 2 S 2 O 4溶液,缺氧1 h后,用含10%小牛血清的DMEM培养液复氧培养3 h。H/R+Fer-1组经Fer-1(2μmol/L)预处理24 h后再进行缺氧复氧处理。各组细胞应用紫外分光光度法检测乳酸脱氢酶(LDH)释放率,CCK-8法检测细胞存活率,黄嘌呤氧化酶法检测超氧化物歧化酶(SOD),化学比色法检测丙二醛(MDA),免疫荧光观测线粒体膜电位、活性氧(ROS)改变,Western blot检测铁死亡关键蛋白ACSL4、GPX4的表达。结果:与control组比较,H/R组细胞活性、SOD释放量和MMP水平均显著降低(P<0.05),LDH、MDA、ROS释放量均显著增加(P<0.05),ACSL4蛋白表达显著升高(P<0.05)、GPX4蛋白表达显著下降(P<0.05)。与H/R组比较,H/R+Fer-1组细胞活性、SOD释放量和MMP水平均显著升高(P<0.05),LDH、MDA、ROS释放量均显著降低(P<0.05),ACSL4蛋白表达显著下降(P<0.05)、GPX4蛋白表达显著升高(P<0.05)。结论:心肌细胞H/R损伤中有铁死亡的发生,Fer-1可通过调控ACSL4和GPX4抑制细胞内ROS的产生,从而减轻铁死亡引起的原代心肌细胞缺氧复氧损伤。Objective:To investigate the effects of ferrostatin-1(Fer-1)on cardiomyocyte hypoxia/reoxygenation injury and its mechanisms.Methods:The original generation of myocardial cells were extracted from 1~3 d newborn SD rats,which were randomly divided into normal control group(control),hypoxia reoxygenation(H/R)group and hypoxia reoxygenation+iron death inhibitors group(H/R+Fer-1).After 52 h of culture,cells in H/R group were added with 4 mmol/L Na 2 S 2 O 4 solution.After 1 h of hypoxia,cells were reoxygenated with DMEM medium containing 10%calf serum for 3 h.The H/R+Fer-1 group was pretreated with Fer-1(2μmol/L)for 24 h and then subjected to hypoxia and reoxygenation.The release rate of lactate dehydrogenase(LDH)was measured by UV spectrophotometry,the cell survival rate was measured by CCK-8 method,SOD was measured by xanthine oxidase method,MDA was measured by chemical coloration,and the changes of mitochondrial membrane potential and reactive oxygen species(ROS)were observed by immunofluorescence.Western blot was used to detect the expressions of ACSL4 and GPX4.Results:Compared with the control group,the cell activity,SOD release and MMP level were decreased(P<0.05),the levels of LDH,MDA and ROS were increased(P<0.05),the protein expression of ACSL4 was increased(P<0.05),and the protein expression of GPX4 was decreased(P<0.05)in H/R group.Compared with the H/R group,the cell activity,SOD release and MMP level were increased(P<0.05),the level of LDH,MDA and ROS were decreased(P<0.05),the protein expression of ACSL4 was decreased(P<0.05),and the protein expression of GPX4 was increased(P<0.05)in H/R+Fer-1 group.Conclusion:Fer-1 can inhibit the production of intracellular reactive oxygen species by regulating ACSL4 and GPX4,thereby alleviating the hypoxia and reoxygenation injury of primary cardiomyocytes caused by iron death.

关 键 词：SD乳鼠 铁死亡 Ferrostatin-1 ACSL4 GPX4 心肌缺氧复氧损伤 

分 类 号：R122[医药卫生—环境卫生学]