乌司他丁对过氧化氢诱导的肠上皮屏障损伤的保护作用

摘要

Background: Disruption of tight junctions between intestinal epithelial cells followed by loss of barrier function is crucial for the pathogenesis and progression of a variety of gastrointestinal disorders.Aims: To investigate the protective effect of ulinastatin on hydrogen peroxide (H2O2)-induced intestinal epithelial barrier disruption.Methods: Model of intestinal epithelial monolayer barrier was established with Caco-2 cells in vitro,and then divided into four groups: blank control group (without any intervention),H2O2 group (500 μmol/L H2O2),low-dose (500 U/mL) and high-dose (3 000 U/mL) ulinastatin groups (ulinastatin + H2O2).Level of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were detected;transepithelial electrical resistance (TEER) and flux of sodium fluorescein were measured to assess the barrier function;expression and localization of two tight junction proteins,ZO-1 and occludin were evaluated by Western blotting and immunofluorescence;ultrastructure of tight junctions was observed by transmission electron microscopy (TEM).Results: Compared with the blank control group,treatment of Caco-2 cell monolayers with H2O2 resulted in increase in level of MDA,flux of sodium fluorescein and decrease in activity of SOD,TEER and expressions of ZO-1 and occludin (P all ＜0.05).TEM and immunofluorescence showed that the brusher border of Caco-2 cells in H2O2 group was destroyed,the cell-cell junction was vague and the localization of ZO-1 and occludin was discontinuous and the fluorescence intensity was extremely low.While in ulinastatin groups,especially the high-dose group,all the indices above-mentioned were significantly improved (P all ＜0.05).Conclusions: Ulinastatin protects intestinal epithelial monolayer barrier against H2O2-induced disruption at least partially by its antioxidant activity and modulating expression and localization of tight junction proteins.%背景:肠上皮细胞间紧密连接的破坏及其所致的肠黏膜屏障功能受损在一系列胃肠道疾病的发生、发展中起重要作用.目的:探讨乌司他丁对过氧化氢(H2O2)诱导的肠上皮屏障损伤的保护作用.方法:以Caco-2细胞体外培养制备肠单层上皮屏障模型,将其分为空白对照组(不予干预)、H2O2组(500 μmol/L H2O2)和低浓度(500 U/mL)、高浓度(3 000 U/mL)乌司他丁治疗组并予相应处理.检测丙二醛(MDA)水平、超氧化物歧化酶(SOD)活性,以跨上皮细胞电阻(TEER)和荧光素钠透过率评估上皮屏障功能,蛋白质印迹法和免疫荧光法检测紧密连接蛋白ZO-1、occludin的表达和定位,透射电镜观察紧密连接超微结构.结果:与空白对照组相比,H2O2组Caco-2细胞单层上皮MDA水平、荧光素钠透过率明显升高,SOD活性、TEER、ZO-1和occludin蛋白表达明显降低,差异均有统计学意义(P＜0.05).透射电镜观察和免疫荧光法检测显示H2O2组细胞刷状缘受损,细胞间连接模糊,ZO-1、occludin蛋白分布断续不完整,荧光强度低.乌司他丁治疗组上述指标均较H2O2组显著改善(P＜0.05),高浓度组改善更为明显.结论:乌司他丁对H2O2诱导的肠单层上皮屏障损伤有一定保护作用,其机制可能与其抗氧化活性以及调节紧密连接蛋白表达和分布有关.