NADPH氧化酶Nox-4在DSS诱导的小鼠肠炎中的表达及作用

Role of NADPH Oxidase Nox-4 on DSS Induced Mice Inflammatory Bowel Diseases Model

目的研究Nox-4及抗氧化物在葡聚糖硫酸钠(dextran sulphate sodium,DSS)诱导的小鼠炎症性肠病(inflammatory bowel disease,IBD)模型中的表达水平,探讨氧化应激及NADPH氧化酶在IBD发病机制中的作用。方法将16只SPF级雄昆明小鼠随机分为正常组和DSS组,每组8只,正常组正常饮水,DSS组饮用2. 5%DSS溶液,7天建立小鼠肠炎模型。通过结肠长度、HE染色组织病理学评分、疾病活动指数(disease activity index,DAI)评估肠道炎症程度;免疫组织化学法检测结肠组织中Nox4蛋白含量,并分别采用实时定量PCR与生化方法检测组织中Nox-4的mRNA表达水平及血清浓度,评价机体氧化应激程度;利用实时定量PCR法分析小鼠肠道组织中Mn-SOD、GSH和CAT mRNA的表达情况,评价机体抗氧化功能;实时定量PCR法检测结肠组织中MCP-1、IL-1β、TNF-α的mRNA表达水平、ELISA法检测上述3种炎性因子血清浓度以反应机体炎症程度。结果与正常组相比,DSS组小鼠体质量明显下降(P <0. 01),DAI评分明显增高(P=0. 000),结肠长度明显缩短(P <0. 01),肠道呈现中至重度炎症状态; DSS组结肠组织中Nox-4蛋白表达明显上调,Nox-4 mRNA含量显著升高(P=0. 000),血清Nox-4浓度在DSS组较正常组更高(P <0. 01);结肠组织中抗氧化物Mn-SOD、GSH和CAT mRNA的表达在DSS组中均明显增高(P=0. 000);比较机体炎症指标,DSS组小鼠血清中MCP-1、IL-1β、TNF-α浓度较正常组均呈不同程度上升(P均=0. 000),结肠组织中3种炎性因子的mRNA相对表达量较正常组显著升高(P均=0. 000)。结论 Nox-4通过氧化应激途径参与了肠道黏膜炎性反应的进程,并可能在IBD的转归中发挥着更为重要的作用。

Objective To investigate the role of NADPH oxidase in inflammatory bowel disease(IBD)by researching on theNox-4 expression in a dextran sulphate sodium(DSS)induced IBD model on mice.Methods Sixteen specific pathogen free(SPF)male Kunming mice were randomly and equally divided into normal and DSS groups.The DSS group was fed on 2.5%(v/w)DSS for 7 days while clean water for normal group.The extent of intestinal inflammation was assessed by the colon length,the histopathological score of HE staining and the disease activity index(DAI).We used immunohistochemical method to detect colon Nox4 protein,real-time quantitative PCR(real-time Q-PCR)for Nox-4 mRNA expression and biochemical detection for Nox4 serum concentration.The evaluation of colonic antioxidant function by Mn-SOD,GSH and CAT mRNA was performed by Real-time Q-PCR,so were the inflammatory indicators MCP-1,IL-1βand TNF-αmRNA in colon.ELISA was also used to detect the serum concentration of the three inflammatory indicators.Results We observed considerable decrease in body mass(P<0.01)and the length of colon(P<0.01)in DSS group.The DAI score and inflammatory degree were significantly high in DSS group(P=0.000).The expression of Nox-4,Mn-SOD,GSH,CAT and inflammatory indicators MCP-1,IL-1β,TNF-αin both colon and serum were remarkablly higher in DSS group when compared with those in normal controls.Conclusion Nox-4 participates in the process of inflammatory intestinal mucosa through mediating oxidative stress,and may play a more important role in the prognosis of IBD.