线粒体钙离子单向转运蛋白在Caco-2细胞缺氧复氧损伤中的作用及机制
Role and mechanism of mitochondrial calcium uniporter in Caco-2 cell hypoxia and reoxygenation injury
图拉妮萨·喀迪尔 1罗杰 1廖师师 1景怡馨 1张贻帼 1陈榕 1孟庆涛1
作者信息
- 1. 武汉大学人民医院麻醉科 湖北 武汉 430060
- 折叠
摘要
目的:探讨线粒体钙离子单向转运蛋白(MCU)在Caco-2细胞缺氧复氧损伤中的作用机制.方法:取正常对数期生长的Caco-2细胞,随机分为4组:对照组(CTL)(n=6)、对照+Ru360组(CTL+Ru360)(n=6)、缺氧/复氧组(H/R组)(n=6)、缺氧/复氧+Ru360组(H/R+Ru360组)(n=6).培养Caco-2细胞,采用缺氧12 h、复氧2 h的方法进行缺氧/复氧建立肠缺血再灌注损伤细胞模型.缺氧/复氧刺激前1h予以MCU抑制剂Ru360干预.采用CCK-8法检测细胞活力,TUNEL法检测细胞凋亡,乳酸脱氢酶(LDH)检测细胞损伤情况,活性氧(ROS)检测试剂盒测定线粒体ROS水平,用JC-1标记的荧光探针检测线粒体膜电位水平,ATP检测试剂盒检测ATP含量,用Rhod-2 AM检测线粒体钙离子含量,Western Blot法检测细胞MCU蛋白表达水平.结果:与CTL组比较,H/R组细胞活力降低,LDH水平升高,细胞凋亡增加,线粒体膜电位降低,ATP含量降低,线粒体ROS产生增加,MCU蛋白水平增高,线粒体钙离子水平增加(P＜0.05);经Ru360处理后,与H/R组比较,H/R+Ru360组细胞活力升高,LDH水平降低,细胞凋亡减少,线粒体膜电位升高,ATP含量升高,线粒体ROS产生减少,MCU蛋白水平降低,线粒体钙离子水平减少(P＜0.05);CTL组与CTL+Ru360组比较上述指标差异无统计学意义(P＞0.05).结论:Ru360抑制MCU激活,维持线粒体钙稳态,减轻线粒体功能障碍,从而保护Caco-2细胞H/R诱导的损伤.
Abstract
Objective:To investigate the role of mitochondrial calcium uniporter(MCU)in hypoxia/reoxygen-ation(H/R)injury in Caco-2 cells.Methods:Human intestinal epithelial cells Caco-2 cells grown at nor-mal logarithmic phase were randomly divided into four groups:control group(CTL group)(n=6),con-trol+Ru360 group(CTL+Ru360)(n=6),hypoxia/reoxygenation group(H/R group)(n=6),and H/R+Ru360 group(H/R+Ru360 group)(n=6).Caco-2 cells were cultured,and the cell model of intesti-nal ischemia-reperfusion injury was established by hypoxia for 12 h and reoxygenation for 2 h.The MCU inhibitor Ru360 was administered 1 h before the H/R stimulation.Cell viability was detected by the CCK-8 method;apoptosis was assayed by the TUNEL method;lactate dehydrogenase(LDH)was detected for cell damage;mitochondrial ROS levels were measured by ROS detection kit,and mi-tochondrial membrane potential level was assayed by JC-1labelingg method;labeled fluorescent probe ATP assay kit was adopted to detect ATP content;mitochondrial calcium content was detected with Rhod-2 AM labeling method,and MCU was detected by Western Blot.Results:Compared with the CTL group,the H/R group showed decreased cell activity,increased LDH levels,increased apopto-sis,decreased mitochondrial membrane potential,decreased ATP content,increased mitochondrial ROS production,increased MCU,and increased mitochondrial calcium ion levels(P＜0.05);after Ru360 treatment,compared with the H/R group,the H/R+Ru360 group showed increased cell activi-ty,decreased LDH levels,decreased apoptosis,increased mitochondrial membrane potential,and in-creased ATP content,decreased mitochondrial ROS production,decreased MCU and decreased mito-chondrial calcium ion levels(P＜0.05);there was no statistical difference in the above indexes between the CTL group and the CTL+Ru360 group(P＞0.05).Conclusion:Ru360 may protect Caco-2 cells from H/R-induced injury by inhibiting MCU activation,maintaining mitochondrial calcium homeosta-sis,and reducing mitochondrial dysfunction.
关键词
缺血再灌注/线粒体钙单向转运蛋白/缺氧/复氧损伤/线粒体/线粒体功能Key words
Ischemia Reperfusion/Mitochondrial Calcium Uniporter/Hypoxia/Reoxygen-ation/Mitochondria/Mitochondrial Function引用本文复制引用
基金项目
国家自然科学基金面上项目(82172155)
湖北省重点实验室开放基金(2021KEY032)
出版年
2024
NETL
NSTL
万方数据