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

作　　者：图拉妮萨·喀迪尔 罗杰 廖师师 景怡馨 张贻帼 陈榕[1] 孟庆涛[1] TULANISA Kadier;LUO Jie;LIAO Shishi;JING Yixin;ZHANG Yiguo;CHEN Rong;MENG Qingtao(Dept.of Anesthesiology,Renmin Hospital of Wuhan University,Wuhan 430060,Hubei,China)

机构地区：[1]武汉大学人民医院麻醉科,湖北武汉430060

出　　处：《武汉大学学报（医学版）》2024年第5期515-519,共5页Medical Journal of Wuhan University

基　　金：国家自然科学基金面上项目(编号:82172155);湖北省重点实验室开放项目(编号:2021KEY032)。

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1 h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。Objective:To investigate the role of mitochondrial calcium uniporter(MCU)in hypoxia/reoxygenation(H/R)injury in Caco-2 cells.Methods:Human intestinal epithelial cells Caco-2 cells grown at normal logarithmic phase were randomly divided into four groups:control group(CTL group)(n=6),control+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 intestinal 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 mitochondrial 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 apoptosis,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 activity,decreased LDH levels,decreased apoptosis,increased mitochondrial membrane potential,and increased ATP content,decreased mitochondrial ROS production,decreased MCU and decreased mitochondrial 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 homeostasis,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 

分 类 号：R574[医药卫生—消化系统]