胆红素对脂多糖诱导肠上皮损伤修复作用研究

The effect of bilirubin on repairing LPS-induced intestinal epithelial injury

目的探讨胆红素对脂多糖诱导的肠上皮细胞保护作用。方法利用脂多糖(LPS)刺激肠上皮细胞(IEC-6)诱导坏死性小肠结肠炎样(NEC-like)肠上皮损伤,分为对照(Control)组、LPS组、LPS+胆红素(LPS+Bilirubin)组。CCK-8实验检测不同浓度胆红素对LPS诱导IEC-6的影响;ELISA检测炎症因子TNF-α、IL-1β、IL-6和IL-10的表达水平;DCFH-DA检测细胞活性氧(ROS)表达,黄嘌呤氧化酶法测定超氧化物歧化酶(SOD),硫代巴比妥酸法测定丙二醛(MDA);TUNEL染色检测细胞凋亡;PCNA免疫荧光检测细胞增殖。结果与Control组比较,LPS组炎症因子TNF-α、IL-1β、IL-6、ROS、MDA表达水平以及TUNEL阳性细胞明显增加(P<0.05),IL-10、SOD表达水平与PCNA阳性细胞明显降低(P<0.05);与LPS组比较,LPS+Bilirubin组TNF-α、IL-1β、IL-6、ROS、MDA表达水平以及TUNEL阳性细胞显著下降,IL-10、SOD表达、PCNA阳性细胞显著升高(P<0.05)。结论胆红素通过降低炎症反应、氧化应激以及促进细胞增殖、抑制细胞凋亡,修复LPS诱导肠上皮细胞损伤,为坏死性小肠结肠炎治疗提供新策略。

Objective The aim of this study is to investigate the effect of bilirubin on LPS-induced intestinal epithelial injury.Methods LPS treated intestinal epithelial cell 6(IEC-6)was used as necrotizing enterocolitis-like(NEC-like)in vitro model.IEC-6 were divided into Control group,LPS group,and LPS+Bilirubin group.CCK-8 reagent was used to detect the effect of different concentrations of bilirubin on injured intestinal epithelium.The expression levels of inflammatory factors TNF-α,IL-1β,IL-6 and IL-10 were measured using ELISA.Intracellular reactive oxygen species(ROS)production was assessed by DCFH-DA staining.Superoxide dismutase(SOD)and malondialdehyde(MDA)activities were examined by SOD Assay Kit-WST and MDA assay kit-TBA.Apoptosis was evaluated by TUNEL staining and proliferating cell nuclear antigen(PCNA)were used to assessed proliferative capabilities of intestinal epithelial cells.Results Compared with the Control group,the expression levels of TNF-α,IL-1β,IL-6,ROS,MDA and TUNEL-positive cells were significantly increased in the LPS group(P<0.05),and the expression levels of IL-10,SOD and PCNA-positive cells were extremely decreased(P<0.05).In addition,the LPS+Bilirubin group showed markedly lower TNF-α,IL-1β,IL-6,ROS,MDA expression levels,TUNEL-positive cells,and considerably higher IL-10,SOD expression,PCNA-positive cells than LPS group(P<0.05).Conclusion Bilirubin was able to repair LPS-induced intestinal epithelial injury by reducing inflammatory response,decreasing oxidative stress,promoting cell proliferation and inhibiting cell apoptosis,which will be a promising therapeutic for NEC.