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

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 by directly regulating ITPR transcription and translation.Down-regulating XBP1s may inhibit ITPR expression and mitigate mitochondrial damage.