脱氧胆酸对人食管腺癌细胞TNF-α、IL-8和IL-6表达的影响及其机制

Effects of deoxycholic acid on TNF-α,IL-8 and IL-6 levels in human esophageal adenocarcinoma cells

目的探讨脱氧胆酸(DCA)对人食管腺癌OE19细胞中炎症因子的影响及机制。方法 MTT法观察不同浓度DCA(50、100、150、200、250、300μmol/L)在不同的作用时间(1、3、6、12、24h)对OE19细胞活性的影响;采用Realtime PCR和ELISA方法分析DCA对炎症因子mRNA和蛋白水平的影响,使用流式细胞术检测各组DCA干预后细胞内活性氧(ROS)水平改变,并与200μmol/L DCA+5mmol/L NAC(ROS清除剂N-乙酰半胱胺酸)组进行比较。结果在OE19细胞系中,DCA具有明显的细胞毒性作用,呈剂量-时间依赖性,并且200μmol/L DCA作用6h是其发挥生物学作用的初始浓度及有效时间。与对照组相比,DCA组TNF-α、IL-8和IL-6mRNA和蛋白表达增加,呈剂量依赖性。DCA也增加了细胞内ROS水平,呈剂量依赖性。NAC明显抑制DCA诱导的TNF-α、IL-8和IL-6的表达及ROS的释放。结论 DCA对OE19细胞有明显的细胞毒性及促炎作用,其促炎机制可能与细胞内ROS水平升高有关。

ABSTRACT： Objective To investigate the esophageal adenocarcinoma cells. Methods effects of deoxycholic acid （DCA） on inflammatory factors in human The viability of OE19 cells exposed to different doses of DCA （50, 100, 150, 200, 250 and 300 μmol/L） for different time （1, 3, 6, 12 and 24 h） was measured by MTT assay. Inflammatory factorsTNF-α、IL-8and IL-6 mRNA and protein levels were assayed by real-time PCR and ELISA analysis. Flow cytometry was used to detect the level of intra-cellular reactive oxygen species （ROS） intervened by DCA, which was compared with that in DCA （200 μmol/L） plus N-acetylcysteine （NAC, 5 mmol/L） group. Results DCA induced cytotoxicity in OE19 cells in dose- and time-dependent manners. Significant cytotoxicity was found to be associated with 200μmol/L of DCA and the time of DCA exposure at 6 h. Treating OE19 cells with DCA significantly promoted the increase of TNF-α, IL-8 and IL-6 mRNA and protein levels and up-regulated intra- cellular ROS level in the dose-dependent manner. NAC, an ROS scavenger, significantly inhibited DCA-induced production of inflammatory factors and release of intra-cellular ROS. Conclusion DCA induces cytotoxicity and pro-inflammation in a dose-response manner probably through accumulation of intra-cellular ROS in OE19 cells.