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

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

Abstract

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 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 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 alleviate H/R-induced TCMK-1 cells injury, whose mechanism may be inhibition of TXNIP-induced NLRP3 inflammasome activation.

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。.

Keywords: Hypoxia/reoxygenation; Mitochondrial injury; NLRP3 inflammasome; Thioredoxin interacting protein; X box binding protein 1.

MeSH terms

  • Animals
  • Carrier Proteins
  • Epithelial Cells / metabolism
  • Hypoxia
  • Inflammasomes* / metabolism
  • Mice
  • NLR Family, Pyrin Domain-Containing 3 Protein* / genetics
  • NLR Family, Pyrin Domain-Containing 3 Protein* / metabolism
  • Reactive Oxygen Species / metabolism
  • Signal Transduction
  • Thioredoxins / metabolism
  • X-Box Binding Protein 1 / genetics

Substances

  • Carrier Proteins
  • Inflammasomes
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • Nlrp3 protein, mouse
  • Reactive Oxygen Species
  • Txnip protein, mouse
  • X-Box Binding Protein 1
  • Xbp1 protein, mouse
  • Thioredoxins