环孢素A对DSS结肠炎小鼠肠黏膜通透性影响及其机制的研究

目的探讨环孢素A(CsA)对葡聚糖硫酸钠(DSS)结肠炎小鼠肠黏膜通透性的影响及作用机制。方法 C57BL/6J小鼠(6～8周龄、体质量20 g±2 g、♂),实验分为正常对照组(饮用无菌蒸馏水+腹腔注射0.9%NS)、DSS模型组(DSS溶液+腹腔注射0.9%NS)、环孢素A组(DSS溶液+腹腔注射CsA)。小鼠自由饮用5%DSS溶液1周制备结肠炎模型,环孢素A(0.025 mg.g-1)腹腔注射给药,每天1次,共7 d。每日行DAI评分,实验结束后取结肠进行HE染色评分,结肠匀浆检测MPO活性、TNF-α、IFN-γ、IL-13和IL-17水平,另取小鼠小肠黏膜进行透射电镜检查和肌球蛋白轻链激酶(MLCK)活性测定,采用Evans blue和异硫氰酸荧光素-葡聚糖(FITC-D)方法检测小肠黏膜通透性。结果与正常对照组比较,模型对照组小鼠1周后出现明显体重减轻、便血和腹泻,DAI评分和HI评分增高,同时结肠黏膜MPO活性明显增高。电镜检查小鼠回肠黏膜上皮绒毛萎缩、排列不规则,细胞间连接复合体缩短、变宽,细胞间隙扩大,小肠黏膜通透性增高。小鼠结肠匀浆中TNF-α、IFN-γ、IL-13和IL-17水平均有不同程度增高,小肠黏膜中MLCK活性明显增高。与DSS模型组比较,环孢素A组小鼠DAI评分和HI评分明显降低,结肠黏膜MPO活性减低,回肠黏膜上皮细胞绒毛排列整齐,小肠黏膜通透性降低,小肠黏膜MLCK活性和结肠匀浆中TNF-α、IFN-γ、IL-13和IL-17水平均有不同程度降低。结论环孢素A具有明显的抗DSS小鼠结肠炎作用,机制可能与通过调控MLCK表达,改善肠黏膜通透性有关。

Effect of Cyclosporin A on the intestinal mucosal permeability of colitis in mice

Aim To investigate the mechanism and the effects of Cyclosporin A(CsA) on the intestinal mucosal permeability of dextran sulfate sodium(DSS)-induced colitis in mice.Methods Experimental colitis was induced in C57BL /6J mice by administration of 5% DSS CsA,Which was injected intraperitoneally at the dose of 0.025 mg.g-1 once a day from d 1 to d 7.The disease activity index(DAI) score was evaluated,and the colon tissue was collected for the assessment of histological changes.The activity of myeloperoxidase(MPO),and the levels of TNF-α,interferon-γ(IFNγ),interleukin-13(IL-13) and interleukin-17(IL-17) in the colon were determined.The small intestinal mucosa was ultrastructurally examined with transmission electron microscopy(TEM),the intestinal permeability was assayed using the FITC-dextran(FD-4) and Evans blue(EB) method,and the small intestine mucosa was collected to determine epithelial myosin light chain kinase(MLCK) enzymatic activity.Results CsA was found effective in reducing DAI score and histological index(HI) score,and decreasing the activity of MPO,the levels of TNF-α,IFN-γ,IL-13 and IL-17 in colitic mice.The small intestinal mucosal injury was ameliorated by CsA,and the amount of FD-4 permeating into blood and EB permeating into the intestinal wall were decreased by CsA in colitic mice.Moreover,the small intestinal epithelial MLCK enzymatic activity was downregulated by CsA.Conclusion CsA shows the obvious pharmacological effect of anti-colitis which might be associated with the inhibition of MLCK to ameliorate the intestinal mucosal permeability.