HIF对乙醇诱导肠上皮细胞屏障功能损伤的影响

Effect of Hypoxia-induced Factor 1α and 2α in Ethanol-induced Intestinal Epithelium Cellular Barrier Dysfunction

目的 探究低氧诱导因子(hypoxia-inducible factor, HIF)-1α(HIF-1α)和2α(HIF-2α)对乙醇诱导的结肠腺癌细胞(colorectal adenocarcinoma cell, Caco-2)单层膜肠上皮屏障功能损伤的作用及机制。方法 利用Caco-2细胞建立单层膜肠上皮细胞屏障模型,用不同浓度乙醇处理,MTT法检测细胞的增殖活性。选取合适的乙醇作用浓度和时间刺激Caco-2细胞,ELISA检测炎性细胞因子白介素-1β(interleukin 1β,IL-1β)和白介素-6(interleukin 6,IL-6)水平,Western blot法和RT-PCR检测HIF-1α、HIF-2α、人质膜型唾液酸酶(Neu3)蛋白及mRNA的表达,采用跨上皮电阻(transepithelial electrical resistance, TEER)评估细胞单层膜的通透性。转染小分子干扰RNA(small interfering RNA, siRNA)敲低HIF-1α、HIF-2α水平后,乙醇处理Caco-2细胞,检测细胞的增殖活性、炎性因子水平、TEER数值和Neu3的表达。最后,敲低Neu3的表达,乙醇处理细胞,检测细胞单层膜的TEER。结果当乙醇浓度>5%时,对Caco-2细胞增殖活性的抑制呈现浓度依赖性。用5%浓度的乙醇刺激Caco-2细胞1h,与对照组比较,发现其炎性细胞因子IL-1β和IL-6的分泌增加,HIF-1α、HIF-2α、Neu3的表达升高,TEER水平下降(P<0.05)。分别敲低Caco-2细胞的HIF-1α和HIF-2α表达后,与阴性序列对照组比较,其细胞增殖活性减弱,均促进了IL-1β和IL-6的分泌,两者TEER数值也进一步降低(P<0.05)。并且,敲低HIF-2α抑制了细胞中Neu3的表达(P<0.05)。最后,敲低Neu3使乙醇诱导的细胞单层膜TEER值下降(P<0.05)。结论 一定浓度和时间的乙醇暴露可引起肠上皮细胞增殖活性减弱和炎性细胞因子释放,使肠上皮细胞屏障功能受损,HIF-1α和HIF-2α参与了该过程的调节。敲低HIF-1α或HIF-2α可进一步加重乙醇诱导的肠上皮细胞屏障功能的损伤,具体机制可能是肠上皮细胞中HIF-2α,而不是HIF-1α通过调控Neu3来实现。

Objective To investigate the effect of hypoxia-induced factor 1αand 2α(HIF-1αand HIF-2α)on ethanol-induced intestinal epithelium cellular barrier dysfunction of colorectal adenocarcinoma cell(Caco-2)monolayers and its mechanism.Methods Intestinal epithelium monolayer cellular barrier model was obtained by Caco-2 in vitro and induced by ethanol with different concentrations.The cell viability was measured by MTT method.Caco-2 cells were treated with or without ethanol according to the optimum concentration and time.The secretions of interleukin 1β(IL-1β)and interleukin 6(IL-6)were detected by ELISA.The expressions of HIF-1α,HIF-2α,and Neu3 were analyzed by Western blot and RT-PCR.The permeability of Caco-2 cell monolayers was evaluated by transepithelial electrical resistance(TEER).Next,Caco-2 cells were transfected with small interfering RNA(siRNA)to knock down the expression of HIF-1αand HIF-2α.The cell viability,the levels of IL-1βand IL-6,and the expression of Neu3 and TEER in each group were detected,respectively.Then,the expression of Neu3 was inhibited with siRNA to assess the permeability of Caco-2 cell monolayers by TEER.Results The inhibitory effect of ethanol on Caco-2 cells was dose-dependent when the concentration of ethanol was higher than 5%.When Caco-2 cells were treated with ethanol with a concentration of 5%for one hour,the levels of proinflammatory cytokines IL-1βand IL-6 were promoted.Compared to the control group,ethanol also induced the expression of HIF-1α,HIF-2α,Neu3 and reduced TEER values(P<0.05).After HIF-1αor HIF-2αsiRNA interference in ethanol-induced Caco-2 cells,the cell viability was inhibited,the secretions of IL-1βand IL-6 were significantly elevated and the values of TEER were decreased(P<0.05).Interestingly,the expression of Neu3 was inhibited when HIF-2αwas deleted but not HIF-1α(P<0.05).Furthermore,TEER analysis showed an increased cellular permeability when the expression of Neu3 was inhibited(P<0.05).Conclusion The HIF-1αor HIF-2αsignal pathway may be involved regulation process of ethanol-induced increased permeability of the intestinal epithelium cellular barrier.In this process,Neu3 may be mediated by HIF-2αbut not HIF-1α,contributing to intestinal epithelium cellular barrier dysfunction in ethanol-induced Caco-2.