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

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.