[XBP1通过TXNIP-NLRP3信号通路调节小鼠肾小管上皮细胞的缺氧/复氧损伤]

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

作者信息

Ni H Q, Ou Z Y, Xia R F, Deng W F, Su D M, Hu Y C, Xu J, Zhang J, Gong N Q, Miao Y

机构信息

The First Clinical Medical School, Southern Medical University, Guangzhou 510515, China.

Organ Transplant Department, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2020 Dec 29;100(48):3863-3869. doi: 10.3760/cma.j.cn112137-20201102-02996.

DOI:10.3760/cma.j.cn112137-20201102-02996

PMID:33371632

Abstract

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. 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 Annexin Ⅴ/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-1β protein 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 -test. 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, 0.05), mROS (34.63±0.64 vs 48.17±1.84, 0.01) and higher MMP (1.03±0.11 vs 0.45±0.08, 0.05) were observed in si-XBP1+H/R group. Down-regulation of XBP1U (protein: 1.31±0.18 vs 0.23±0.02, 0.01; mRNA: 1.12±0.07 vs 0.38±0.01, 0.001) and XBP1S (protein: 1.13±0.17 vs 0.28±0.07, 0.01; mRNA: 8.39±0.63 vs 2.45±0.22, 0.001) inhibited expression of TXNIP (0.15±0.02 vs 0.04±0.01, 0.01), NLRP3 (1.13±0.12 vs 0.51±0.12, 0.05) and IL-1β (1.02±0.04 vs 0.19±0.06, 0.001) during H/R. Meanwhile, TXNIP exhibited significantly much less colocalization with mitochondria in the si-XBP1+H/R group. Supression of XBP1 expression can effectively alleviate H/R-induced TCMK-1 cells injury, whose mechanism may be inhibition of TXNIP-induced NLRP3 inflammasome activation.

摘要

通过硫氧还蛋白相互作用蛋白（TXNIP）-核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）信号通路，研究X盒结合蛋白1（XBP1）在小鼠肾小管上皮细胞（TCMK-1）缺氧/复氧（H/R）损伤中的作用及调控机制。将细胞分为4组：转染阴性对照小干扰RNA（si-NC）的si-NC组、转染靶向XBP1的小干扰RNA（si-XBP1）的si-XBP1组、转染si-NC并暴露于H/R的si-NC+H/R组、转染si-XBP1并暴露于H/R的si-XBP1+H/R组。采用膜联蛋白Ⅴ/碘化丙啶（Annexin Ⅴ/PI）双染色法检测细胞凋亡；使用JC-1染料检测线粒体膜电位（MMP）；使用MitoSOX™染料评估线粒体活性氧（mROS）。通过蛋白质印迹法和定量实时聚合酶链反应检测XBP1的干扰效率。采用蛋白质印迹法检测TXNIP、NLRP3和白细胞介素-1β（IL-1β）蛋白的表达水平。通过双标免疫荧光染色检测线粒体与TXNIP的共定位。采用独立样本t检验比较组间差异。与si-NC组相比，si-NC+H/R组观察到更多的mROS、细胞凋亡，且MMP更低。与si-NC+H/R组相比，si-XBP1+H/R组观察到细胞凋亡减少（12.08±0.5l比19.01±1.80，P<0.05）、mROS减少（34.63±0.64比48.17±1.84，P<0.01），且MMP更高（1.03±0.11比0.45±0.08，P<0.05）。在H/R期间，XBP1U（蛋白：1.31±0.18比0.23±0.02，P<0.01；信使核糖核酸：1.12±0.07比0.38±0.01，P<0.001）和XBP1S（蛋白：1.13±0.17比0.28±0.07，P<0.01；信使核糖核酸：8.39±0.63比2.45±0.22，P<0.001）的下调抑制了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）和IL-1β（1.02±0.04比0.1l9±0.06，P<0.001）的表达。同时，在si-XBP1+H/R组中，TXNIP与线粒体的共定位显著减少。抑制XBP1表达可有效减轻H/R诱导的TCMK-1细胞损伤，其机制可能是抑制TXNIP诱导的NLRP3炎性小体激活。