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

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