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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(H_2O_2)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)、H_2O_2组(500μmol/L H_2O_2)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,H_2O_2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H_2O_2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H_2O_2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H_2O_2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。
BACKGROUND: The destruction of the tight junctions between intestinal epithelial cells and the impaired intestinal barrier function play an important role in the occurrence and development of a series of gastrointestinal diseases. Objective: To investigate the protective effect of ulinastatin on intestinal epithelial barrier injury induced by hydrogen peroxide (H_2O_2). Methods: The monolayer epithelial barrier model was prepared by Caco-2 cell culture in vitro. The model was divided into blank control group (without intervention), H 2 O 2 group (500 μmol / L H 2 O 2) and low concentration (500 U / mL) (3 000 U / mL) ulinastatin treatment group and appropriate treatment. The level of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were measured. The function of epithelial barrier was evaluated by the trans-epithelial cell resistance (TEER) and sodium fluorescein permeability. Western blotting and immunofluorescence were used to detect the expression of tight junction protein ZO-1, occludin expression and localization, transmission electron microscopy of tight junction ultrastructure. Results: Compared with the blank control group, the level of MDA and the permeability of sodium fluorescein in Caco-2 cells of H 2 O 2 group were significantly increased, and the expressions of SOD, TEER, ZO-1 and occludin were significantly decreased Significance (P <0.05). Transmission electron microscopy and immunofluorescence assay showed that the brush border of H 2 O 2 group was damaged and the intercellular junctions were blurred. The protein distribution of ZO-1 and occludin was discontinuous and the fluorescence intensity was low. The above indexes in ulinastatin group were significantly improved compared with those in H 2 O 2 group (P <0.05), and those in high-concentration group were more obvious. CONCLUSION: Ulinastatin may play a protective role in H 2 O 2 -induced intestinal epithelial barrier injury. Its mechanism may be related to its antioxidant activity and regulation of tight junction protein expression and distribution.