乌司他丁对肠道上皮细胞屏障损伤的保护作用分析

Protective effect of ulinastatin on intestinal epithelial cell barrier

目的探讨乌司他丁(UTI)对肠道上皮细胞屏障损伤的保护作用及其机制。方法培养Caco-2细胞,并建立肿瘤坏死因子α(TNF-α)诱导的单层上皮细胞模型,随机分为空白对照组、TNF-α模型组、UTI低剂量组(5万U/kg)、UTI中剂量组(10万U/kg)、UTI高剂量组(20万U/kg)。采用Millicell电阻仪和多功能读板仪分别测定各组上皮细胞电阻(TER)和异硫氰酸荧光素标记的葡聚糖(FITC-dextran),酶联免疫吸附测定法(ELISA)测定白细胞介素6(IL-6)、IL-10水平,流式细胞仪测定细胞凋亡率,免疫蛋白质印记(Western blot)技术测定紧密连接蛋白闭合蛋白(occludin)和紧密黏连蛋白1(ZO-1)表达水平。结果与空白对照组相比,TNF-α模型组TER、FITC-dextran、occludin和ZO-1表达水平均明显下降,IL-6、IL-10、细胞凋亡率明显升高(P<0.05);与TNF-α模型组比较,经UTI处理后,TER、FITC-dextran、occludin和ZO-1表达水平升高,IL-6、IL-10、细胞凋亡率降低(P<0.05),其中以UTI高剂量组变化最显著(P<0.05)。结论 UTI可能通过抑制炎性介质释放,调节紧密连接相关蛋白表达,抑制TNF-α诱导的肠道上皮细胞屏障功能损伤。

Objective To invcstigatc thc protcctivc effect and mechanism of ulinastatin （UTI.） on intestinalepithelial cell barrier. Methods Caco-2 cells were cultured,epithelial cells monolayer models induced by tumor necrosis factor-α（TNF-α ） were established and were randomly divided into blank control group,TNF-a model group, low-dose UTI group （50 000 U/kg ）, middle-dose UTI group （100 000 U/kg） and high-dose UTI group （200 000U/kg.）. The transcpithclial electrical resistance （TER） and fluoresce in isothiocyanate markeall groups were determined by Milliccll electrical resistance meter and multifunctional plate reader. Interleukin 6 （IL-6） and IL-10 were determined by enzyme-linked immunosorbent assay （EUSA）. T＇he apoptosis rate was measured by flow cytometry, and the expression of tight junction protein occludin and ZO-1 were measured by Western blThe expression of TER,FITC-dextran,occludin and ZO-1 were significantly lower in TNF-a model group than thosein blank control group, while IL-6, IL-10 and apoptosis rate were significantly higher （P 〈 0.05）. After UTItreatment, TER,FITC-dextran, occludin and ZO-1 were significantly higher than those in TNF-a model group, while IL-6,IL-10 and apoptosis rate were significantly lower （ P 〈 0.05）. UTI change in high-dose group was the mostsignificant （ P〈 0. 05）. Conclusion UTI may protect the intestinal epithelial cell barrier injury induced by TNF-α through inhibiting the release of inflammatory mediators and regulating the expression of tight junction associatedproteins.