缺氧-复氧诱导HK2细胞HMGB1/TLR-4信号通路活化调控TNF-α、IL-1β表达的意义

摘要

Objective:To observe the expression of HMGB1,TLR-4,tumor necrosis factor α,interleukin 1β in the HK2 cell and the apoptosis ratio of human renal tubular epithelial cells (HK2) after anoxia/reoxygenation,investigating the role of anoxia/reoxygenation-simulated Renal ischemia reperfusion injury induced the activation of HMGB1/TLR-4 pathway on the regulation of late inflammatory reaction and apoptosis.Methods:The anoxia/reoxygenation models were established through exhausting the adenosine-triphosphate in the HK2 by the means of the administratiom of antimycin A to simulate the process of renal ischemia reperfusion injury.the HK2 cell were randomly divided into three groups:normal group,renal ischemia and reperfusion model group (I/R),treatment group with recombinant human High-mobility group box 1 (rHMGB1).At 12 h,24 h,48 h after reperfusion.apoptosis ratio of HK2 cell was detected by flow cytometry,the concentrations of HMGB1,TNF-α and IL-1β in cell culture supernatant were measured by Enzyme-linked immunosorbent assay (ELISA),and the expression of toll-like receptor 4 (TLR-4) protein were measured by means of immune confocal laser microscope and western blot.Results:anoxia/reoxygenation induced significant increases in the concentrations of HMGB1,TNF-α and IL-1β in cell culture supernatant,the up-regulation in the expression of TLR-4 protein at 12 h,reaching a peak at 24h after reperfusion.There is statistical significances at both time-points compared with normal group (P ＜ 0.01).rHMGB1 treatment could lead to decreases in the concentrations of HMGB1,TNF-α and IL-1β,down-regulating the protein expression of toll-like receptor 4(P ＜0.01 or P ＜0.05).Conclusion:Renal anoxia/reoxygenation induces the activation of HMGB1/TLR-4 pathway in the HK2 cell that is associated in the regulation of the expression of tumor necrosis factor α,interleukin 1β and the apoptosis of HK2 cell,mediating the development of acute kidney injury through the mechanism of late inflammatory reaction.%目的:观察缺氧-复氧诱导后HK2细胞的凋亡率、高迁移率蛋白B1(HMGB1)、Toll样受体4(TLR-4)、肿瘤坏死因子-α(TNF-α)、白介素1β(IL-1β)水平的改变,探讨缺氧-复氧模拟肾缺血-再灌注诱导HGMB1/TLR-4信号通路活化在调控晚期炎症反应和细胞凋亡的意义.方法:用抗霉素A处理HK2细胞(0.1μmol/L)耗竭ATP的方法建立缺氧-复氧HK2细胞模型以模拟缺血-再灌注损伤.将HK2细胞随机分成3组:normal组、I/R组和重组人HGMB1组(rHGMB1组),在复氧后12 h、24h、48 h3个时间点,用流式细胞术检测细胞的凋亡率,ELISA法检测细胞上清液HMGB1、TNF-α、IL-1β的水平,免疫激光共聚焦和western blot检测HK2细胞TLR-4蛋白的表达.结果:缺氧-复氧可诱导HK2细胞凋亡比率、细胞上清液重组人HMGB1、TNF-α、IL-1β水平及HK2细胞TLR-4蛋白12 h即明显增多,24h达高峰,与normal组相同时间点相比较,差异均有统计学意义(P＜0.01).经重组人HGMB1处理后,细胞的凋亡率、上清液HMGB1、TNF-α、IL-1β水平及HK2细胞TLR-4蛋白均明显降低,与I/R组相同时间点相比较差异均有统计学意义(P＜0.01或P＜0.05).结论:缺氧-复氧模拟肾缺血-再灌注可诱导HK2细胞HGMB1/TLR-4信号通路活化,参与调控TNF-α、IL-1β炎症介质的表达及HK2细胞的凋亡,通过晚期炎症和凋亡机制介导AKI的发生和发展.