XBP1调控TXNIP-NLRP3通路对小鼠肾小管上皮细胞缺氧复氧模型的影响及其作用机制  

XBP1 modulates hypoxia/reoxygenation injury in mouse renal tubular epithelial cells through TXNIP-NLRP3 signaling pathway

作　　者：倪海强 欧志宇 夏仁飞[2] 邓文锋[2] 粟大明 胡杨澄 徐健[2] 张季 宫念樵[3] 苗芸[2] Ni Haiqiang;Ou Zhiyu;Xia Renfei;Deng Wenfeng;Su Daming;Hu Yangcheng;Xu Jian;Zhang Ji;Gong Nianqiao;Miao Yun(The First Clinical Medical School,Southern Medical University,Guangzhou 510515,China;Organ Transplant Department,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China;Institute of Organ Transplantation,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Key Laboratory of Organ Transplantation,Ministry of Education,NHC Key Laboratory of Organ Transplantation,Key Laboratory of Organ Transplantation,Chinese Academy of Medical Sciences,Wuhan 430030,China)

机构地区：[1]南方医科大学第一临床医学院,广州510515 [2]南方医科大学南方医院器官移植科,广州510515 [3]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：《中华医学杂志》2020年第48期3863-3869,共7页National Medical Journal of China

基　　金：国家自然科学基金(82070770、81500573);广东省自然科学基金(2020A1515010674);广州市科技计划项目(201803010109);南方医院院长基金(2018B009、2018C003);国家级大学生创新创业训练计划项目(202012121046、X202012121239)。

摘　　要：目的探讨X盒结合蛋白1(XBP1)调控硫氧还蛋白互作蛋白-NOD样受体3(TXNIP-NLRP3)通路对小鼠肾小管上皮细胞(TCMK-1)缺氧复氧(H/R)模型的影响及其作用机制。方法根据干预的不同将细胞分为4组:si-NC组:转染小干扰RNA(siRNA)阴性对照(si-NC);si-XBP1组:转染靶向沉默XBP1的siRNA(si-XBP1);si-NC+H/R组:转染si-NC后H/R处理;si-XBP1+H/R组:转染si-XBP1后H/R处理。磷脂结合蛋白Ⅴ/碘化丙啶双标染色法检测细胞凋亡,JC-1探针染色法检测线粒体膜电位(MMP),MitoSOX^™探针染色法检测线粒体活性氧(mROS),Western印迹和实时定量PCR检测XBP1的蛋白质和mRNA水平以验证干扰效率,Western印迹检测TXNIP、NLRP3和白细胞介素-1β(IL-1β)的蛋白质水平变化,免疫荧光法检测线粒体和TXNIP共定位变化。使用独立样本t检验比较组间差异。结果与si-NC组相比,si-NC+H/R组细胞凋亡率较高,mROS产生较多,MMP较低;与si-NC+H/R组相比,si-XBP1+H/R组细胞凋亡率较低(12.08±0.51比19.01±1.80,P<0.05),mROS产生较少(34.63±0.64比48.17±1.84,P<0.01),MMP较高(1.03±0.11比0.45±0.08,P<0.05);在H/R时干扰XBP1U(蛋白质:1.31±0.18比0.23±0.02,P<0.01;mRNA:1.12±0.07比0.38±0.01,P<0.001)和XBP1S(蛋白质:1.13±0.17比0.28±0.07,P<0.01;mRNA:8.39±0.63比2.45±0.22,P<0.001)表达后,TXNIP(0.15±0.02比0.04±0.01,P<0.01)、NLRP3(1.13±0.12比0.51±0.12,P<0.05)、IL-1β(1.02±0.04比0.19±0.06,P<0.001)蛋白质的表达更低,同时,线粒体和TXNIP的共定位水平也更低。结论抑制XBP1表达能够减轻TCMK-1的H/R损伤,其机制可能是通过抑制TXNIP介导的NLRP3炎症小体的活化。Objective To investigate the role and regulation mechanism of X box binding protein 1(XBP1)for hypoxia/reoxygenation(H/R)injury in mouse renal tubular epithelial cells(TCMK-1)through thioredoxin interacting protein(TXNIP)-nucleotide-binding domain(NOD)-like receptor protein(TXNIP-NLRP3)signaling pathway.Methods The cells were divided into 4 groups:si-NC group transfected with negative control siRNA(si-NC),si-XBP1 group transfected with siRNA targeting XBP1(si-XBP1),si-NC+H/R group transfected with si-NC and exposed to H/R,and si-XBP1+H/R group transfected with si-XBP1 and exposed to H/R.The AnnexinⅤ/PI double-staining method was used to detect cell apoptosis;The mitochondrial membrane potential(MMP)was determined by using JC-1 dye;The mitochondrial reactive oxygen species(mROS)was assessed by using MitoSOX^™dye.The interference efficiency of XBP1 was tested by Western blotting and quantitative real-time polymerase chain reaction.The expression levels of TXNIP,NLRP3 and IL-1βprotein were detected by Western blotting.The colocalization of mitochondria and TXNIP was detected by double-labeling immunofluorescent staining.The intergroup difference was compared by using an independent samples t-test.Results Compared with the si-NC group,more mROS,apoptosis and lower MMP were observed in si-NC+H/R group.Compared with the si-NC+H/R group,less apoptosis(12.08±0.51 vs 19.01±1.80,P<0.05),mROS(34.63±0.64 vs 48.17±1.84,P<0.01)and higher MMP(1.03±0.11 vs 0.45±0.08,P<0.05)were observed in si-XBP1+H/R group.Down-regulation of XBP1U(protein:1.31±0.18 vs 0.23±0.02,P<0.01;mRNA:1.12±0.07 vs 0.38±0.01,P<0.001)and XBP1S(protein:1.13±0.17 vs 0.28±0.07,P<0.01;mRNA:8.39±0.63 vs 2.45±0.22,P<0.001)inhibited expression of TXNIP(0.15±0.02 vs 0.04±0.01,P<0.01),NLRP3(1.13±0.12 vs 0.51±0.12,P<0.05)and IL-1β(1.02±0.04 vs 0.19±0.06,P<0.001)during H/R.Meanwhile,TXNIP exhibited significantly much less colocalization with mitochondria in the si-XBP1+H/R group.Conclusion Supression of XBP1 expression can effectively alleviat

关 键 词：X盒结合蛋白1 硫氧还蛋白互作蛋白 NLRP3炎症小体 缺氧复氧 线粒体损伤 

分 类 号：R692[医药卫生—泌尿科学]