下调XBP1s通过抑制ITPR介导的线粒体功能障碍改善肾小管上皮细胞缺氧/复氧损伤  

Down-regulating XBP1s alleviates hypoxia/reoxygenation injury of renal tubular epithelial cells by inhibiting ITPR-mediated mitochondrial dysfunction

作　　者：倪海强 彭宣 顾世琦 宫念樵[1] Ni Haiqiang;Peng Xuan;Gu Shiqi;Gong Nianqiao(Institute of Organ Transplantation,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Key Laboratory of Organ Transplantation of Ministry of Education,Key Laboratory of Organ Transplantation of National Health Commission of China,Key Laboratory of Organ Transplantation of Chinese Academy of Medical Sciences,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属同济医院器官移植研究所、器官移植教育部重点实验室、国家卫生健康委员会、器官移植重点实验室、中国医学科学院器官移植重点实验室,武汉430030

出　　处：《器官移植》2024年第2期220-228,共9页Organ Transplantation

基　　金：国家自然科学基金(82170772);湖北陈孝平科技发展基金会青年科学专项基金(CXPJJH122001-2210)。

摘　　要：目的 探讨剪接型X盒结合蛋白1(XBP1s)对小鼠肾小管上皮细胞缺氧/复氧(H/R)损伤的影响及其作用机制。方法 将小鼠肾小管上皮细胞分为腺病毒阴性对照组(Ad-shNC组)、靶向沉默XBP1s腺病毒组(Ad-shXBP1s组)、Ad-shNC+H/R组、Ad-shXBP1s+H/R组。检测各组细胞凋亡水平、线粒体活性氧活性、线粒体膜电位及线粒体钙离子水平。使用染色质免疫共沉淀测序(ChIP-seq)分析XBP1s调控肌醇1,4,5-三磷酸受体(ITPR)家族的结合位点。检测各组XBP1s和ITPR家族信使RNA(mRNA)和蛋白表达水平。结果 与AdshNC组比较,Ad-shNC+H/R组细胞凋亡水平更高,线粒体活性氧水平升高,线粒体膜电位降低,线粒体钙离子水平升高;与Ad-shNC+H/R组比较,Ad-shXBP1s+H/R组细胞凋亡水平较低,线粒体活性氧水平下降,线粒体膜电位升高,线粒体钙离子水平降低(均为P<0.05)。与Ad-shNC组比较,Ad-shXBP1s组XBP1s、ITPR1、ITPR2和ITPR3 mRNA和蛋白相对表达量降低(均为P<0.05)。与Ad-shNC组相比,Ad-shNC+H/R组XBP1s、ITPR1、ITPR2和ITPR3蛋白相对表达量升高;与Ad-shNC+H/R组相比,Ad-shXBP1s+H/R组XBP1s、ITPR1、ITPR2和ITPR3蛋白相对表达量下降(均为P<0.05)。ChIP-seq结果显示,XBP1s能够结合ITPR1的启动子和外显子、ITPR2外显子和ITPR3外显子。结论 XBP1s可能通过直接调控ITPR转录和翻译而影响线粒体相关的内质网膜结构功能,下调XBP1s能够抑制ITPR表达,改善线粒体损伤。Objective To evaluate the effect of spliced X-box binding protein 1(XBP1s) on hypoxia/reoxygenation(H/R) injury of mouse renal tubular epithelial cells and unravel underlying mechanism.Methods Mouse renal tubular epithelial cells were divided into adenovirus negative control group(Ad-shNC group),targeted silencing XBP1s adenovirus group(Ad-shXBP1s group),Ad-shNC+H/R group and Ad-shXBP1s+H/R group.The apoptosis level,mitochondrial reactive oxygen activity,mitochondrial membrane potential and mitochondrial calcium ion level were detected in each group.Chromatin immunocoprecipitation followed by sequencing(ChIP-seq) was employed to analyze the binding sites of XBP1s in regulating the inositol 1,4,5-trisphosphate receptor(ITPR) family.The expression levels of XBP1s and ITPR family messenger RNA(mRNA) and protein were determined in each group.Results Compared with the Ad-shNC group,the apoptosis level was higher,mitochondrial reactive oxygen species level was increased,mitochondrial membrane potential was decreased and mitochondrial calcium ion level was elevated in the Ad-shNC+H/R group.Compared with the Ad-shNC+H/R group,the apoptosis level was lower,mitochondrial reactive oxygen species level was decreased,mitochondrial membrane potential was elevated,and mitochondrial calcium ion level was decreased in the Ad-shXBP1s+H/R group(all P<0.05).Compared with the Ad-shNC group,relative expression levels of XBP1s,ITPR1,ITPR2 and ITPR3 mRNAs and proteins were down-regulated in the Ad-shXBP1s group(all P<0.05).Compared with the Ad-shNC group,relative expression levels of XBP1s,ITPR1,ITPR2 and ITPR3 proteins were up-regulated in the Ad-shNC+H/R group.Compared with the Ad-shNC+H/R group,relative expression levels of XBP1s,ITPR1,ITPR2 and ITPR3 were down-regulated in the Ad-shXBP1s+H/R group(all P<0.05).ChIP-seq results showed that XBP1s could bind to the promoter and exon of ITPR1,the exon of ITPR2,and the exon of ITPR3.Conclusions XBP1s may affect mitochondria-associated endoplasmic reticulum membrane structure and function

关 键 词：器官移植 缺血-再灌注损伤 剪接型X盒结合蛋白1 肌醇1 4 5-三磷酸受体 线粒体损伤 内质网应激 线粒体相关的内质网膜 钙超载 

分 类 号：R617[医药卫生—外科学] R692[医药卫生—临床医学]