脱氧胆酸通过ROS/NF-κB通路对Barrett食管细胞氧化应激的影响

Effects of deoxycholic acid on oxidative stress in Barrett’soesophagus cells via ROS/NF-κB pathway

目的 探讨脱氧胆酸(DCA)对人BAR-T细胞氧化应激的影响及机制。方法 体外培养人Barrett上皮细胞株BAR-T,采用不同浓度的DCA(100、200、300μmol/L)和不同作用时间(30 min、60 min、3 h、6 h)干预BAR-T细胞。采用Real-time PCR法和Western blotting法检测环氧酶-2(COX-2)的mRNA和蛋白表达;采用显微镜观察和流式细胞仪检测细胞内活性氧(reactive oxygen species, ROS)含量,并与200μmol/L DCA+5 mmol/L ROS清除剂N-乙酰半胱胺酸(NAC)组进行比较;采用细胞免疫荧光法检测细胞p65蛋白入核情况,并与200μmol/L DCA+100μmol/L NF-κB通路抑制剂吡咯烷二硫代甲酸铵(PDTC)组进行比较。结果 与对照组相比,DCA可以显著升高BAR-T细胞中ROS的含量,呈剂量依赖性,5 mmol/L NAC明显抑制DCA诱导的ROS释放。与对照组相比,相同干预时间下,DCA(200、300μmol/L组)均可以显著升高COX-2 mRNA表达。与1 h组相比,200、300μmol/L DCA 6 h组均可以显著升高COX-2 mRNA表达。与对照组比较,200μmol/L DCA可以显著升高COX-2蛋白表达。同时,200μmol/L DCA可以促进p65蛋白的入核,PDTC可以抑制DCA的作用。结论 DCA可能通过升高细胞内ROS水平,促进p65蛋白入核以激活NF-κB信号通路,进而上调COX-2的表达。

Objective To explore the effects and mechanisms of deoxycholic acid(DCA)on oxidative stress in human BAR-T cells.Methods The human Barrett epithelial cell line BAR-T was cultured in vitro,and the BAR-T cells were intervened with different concentrations of DCA(100,200,and 300μmol/L)and different action time(30 min,60 min,3 h and 6 h).The mRNA and protein expression of COX-2 were detected by Real-time PCR and Western blotting assay.The intracellular reactive oxygen species(ROS)content was detected by direct microscope observation and flow cytometry,and was compared with that of 200μmol/L DCA+5 mmol/L NAC(ROS scavenger N-acetylcysteine)group;cellular immunofluorescence was used to detect the entry of cellular p65 protein into the nucleus,which was then compared with 200μmol/L DCA+PDTC(100μmol/L,NF-κB pathway inhibitor)group.Results Compared to the control group,DCA increased ROS content in BAR-T cells in a dose-dependent manner.Additionally,5 mmol/L NAC significantly inhibited DCA-induced ROS release.Furthermore,both 200μmol/L and 300μmol/L DCA significantly increased COX-2 mRNA expression compared to the control group at the same intervention time.The 6 h groups for both 200μmol/L and 300μmol/L DCA showed a significant increase in COX-2 mRNA expression compared to the 1 h group.Compared to the control group,treatment with 200μmol/L DCA significantly increased the expression of COX-2 protein.Meanwhile,200μmol/L DCA promoted the nucleation of p65 protein,which was inhibited by PDTC.Conclusion DCA activates the NF-κB signaling pathway and upregulates the expression of COX-2 by elevating the intracellular ROS expression and promoting the entry of p65 protein into the nucleus.