Down-regulating XBP1s alleviates hypoxia/reoxygenation injury of renal tubular epithelial cells by inhibiting ITPR-mediated mitochondrial dysfunction
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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Ad-shNC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。 -
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
Abstract: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. -
图 2 各组细胞凋亡水平
注：与Ad-shNC组比较，aP>0.05；与Ad-shNC+H/R组比较，bP<0.05。
Figure 2. Apoptosis levels of cells in each group
图 3 各组细胞线粒体活性氧水平
注：与Ad-shNC组比较，aP<0.05；与Ad-shNC+H/R组比较，bP<0.05。
Figure 3. Mitochondrial reactive oxygen species levels of cells in each group
图 4 各组细胞线粒体膜电位水平
注：与Ad-shNC组比较，aP<0.05；与Ad-shNC+H/R组比较，bP<0.05。
Figure 4. Mitochondrial membrane potential levels of cells in each group
图 5 各组细胞线粒体钙离子水平
注：与Ad-shNC组比较，aP<0.05，与Ad-shNC+H/R组比较，bP<0.05。
Figure 5. Mitochondrial calcium levels of cells in each group
图 6 染色质免疫共沉淀测序峰图
Figure 6. Chromatin immunoprecipitation followed by sequencing peak maps
图 7 各组XBP1s和ITPR家族mRNA和蛋白表达水平
注：A图为各组mRNA表达水平；B、C图为各组蛋白表达水平。与Ad-shNC组比较，aP<0.05；与Ad-shNC+H/R组比较，bP<0.05。
Figure 7. The mRNA and protein expression levels of XBP1s and ITPR family in each group
表 1 引物序列
Table 1. Primer sequences
基因 序列（5’→ 3’） XBP1s 正义链 AAGAACACGCTTG GGAATGG 反义链 CTGCACCTGCTGCGGAC ITPR1 正义链 CTCTGTATGCGGAGGGATCTAC 反义链 CAGGATACTTAGCTATGAGGCG ITPR2 正义链 CTTCCTCTACATTGGGGACATC 反义链 GGGATACTTAGCTATGAGACGG ITPR3 正义链 CGACCTCATCAAGGCTCTCC 反义链 TCCCCATCTCGTTGTTCTGC β-actin 正义链 AGGCCAACCGTGAAAGATG 反义链 TGGCGTGAGGGAGAGCATAG 
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