肾上腺髓质素对溃疡性结肠炎大鼠肠黏膜通透性的影响

Effect of adrenomedullin on intestinal permeability in rats with ulcerative colitis

目的:探讨肾上腺髓质素(adrenomedullin,AM)改善溃疡性结肠炎(ulcerative colitis,UC)大鼠模型肠黏膜通透性的作用机制.方法:清洁级♂SD大鼠24只(180-200 g),随机分成3组:AM组、模型组和正常组,每组8只.模型组及AM组均给予TNBS灌肠,以建立UC大鼠模型.正常组给予生理盐水灌肠.造模后注意观察大鼠毛发、活动及大便情况的变化,记录每组大鼠体质量.48 h后AM组给予1 mL AM灌肠,而模型组、正常组则给予1 mL生理盐水灌肠,连续灌肠7 d后,腹腔注射10%水合氯醛麻醉大鼠,留取大鼠血液,离心后分离上层血清于无菌EP管中冻存,留取大鼠结肠组织,部分立即用4%多聚甲醛固定,余下组织冻存于-80℃.HE染色评估大鼠结肠病理变化,Western blot测定肠道黏膜RhoA、Rho激酶1(Rho associated kinase 1,ROCK1)、肌球蛋白轻链激酶(myosin lightchain kinase,MLCK)、核因子-κB(nuclear factor-κB,NF-κB)p65及紧密连接(tight junction,TJ)蛋白Occludin的表达,酶联免疫吸附测定法(ELISA)试剂盒检测血清肿瘤坏死因子-α(tumor necrosis factorα,TNF-α),干扰素-γ(interferon-γ,IFN-γ)表达的水平.结果:TNBS造模后的大鼠出现腹泻及黏液脓血便,体质量明显减轻,肠管扩张,肠壁变薄,部分结肠黏膜可见散在溃疡,与正常组比较,模型组大鼠血清中TNF-α、IFN-γ的表达明显升高(P<0.05),结肠组织中RhoA、ROCK1、MLCK及NF-κB p65蛋白的表达显著增高(P<0.05),而TJ蛋白Occludin的表达较正常组明显降低(P<0.05),与模型组比较,给予AM治疗后的大鼠TNF-α、IFN-γ表达降低(P<0.05),结肠组织中RhoA、ROCK1、MLCK及NF-κB p65蛋白的表达显著下降(P<0.05),而TJ蛋白Occludin的表达明显升高(P<0.05)有统计学意义.结论:AM可能通过调节RhoA介导的NF-κB p65/MLCK信号通路,改善UC肠道黏膜屏障功能.

AIM：To investigate the possible mechanism of adrenomedullin（AM） to protect the intestinal epithelial barrier function in a rat model of ulcerative colitis（UC）.METHODS：Twenty-four male Sprague-Dawley rats were randomly divided into three groups：a normal control group,a model group and an AM group.Each group contained 8 rats.All the rats except those in the normal control group were administered with TNBS to induce colitis.Rats in the normal control group was given physiological saline instead.Forty-eight hours after inducing colitis,in the AM group,AM（1.0 μg of AM diluted in 1.0 mL of saline）was injected into the lumen of the colon.Rats in the normal control group and model group were given 1.0 mL of saline instead.All of them were treated for seven consecutive days.Blood samples were collected to measure serum tumor necrosis factor-α（TNF-α） and interferon-γ（IFN-γ） levels by ELISA.Colon tissues were taken to detect the protein expression of RhoA,Rho associated kinase 1（ROCK1）,MLCK,nuclear factor-κB（NF-κB） p65 and Occludin by Western blot analysis.RESULTS：All the rats induced with TNBS developed symptoms such as profound and sustained weight loss,bradykinesia and purulent and bloody stools.The levels of serum TNF-αand IFN-γ in model rats were significantly higher than those in the normal group（P〈0.05）,however,serum TNF-α and IFN-γ were significantly decreased after AM treatment（P〈0.05）.The expression levels of RhoA,ROCKI,MLCK and NF-κB p65 showed a significant increase in the model group compared with the normal group（P〈0.05）,while treatment with AM clearly reduced their expression.The expression of Occludin in model rats was significantly decreased（P〈0.05）,while in the AM group,the reduced Occludin expression appeared to resume（P〈0.05）.CONCLUSION：AM improves intestinal mucosal barrier function in UC possibly by regulating the NF-κB p65/MLCK signal pathway mediated by RhoA.