双氧化酶2在5-羟色胺加重的小鼠结肠炎模型中的表达及作用

摘要

目的 探讨5-羟色胺(5-HT) 对双氧化酶2 (Duox2) 在小鼠结肠组织中的表达情况及其在炎症中的意义.方法 选取6~8周龄的健康清洁级C57BL/6J(B6)雄性小鼠,随机分为5组(n=8):对照组自由饮用无菌蒸馏水;葡聚糖硫酸钠(DSS)(1.0%、2.5%)组自由饮用相应浓度的DSS溶液;5-HT组采用直肠灌药方式(1.0 mg/kg)给药,每3 d 1次;DSS+5-HT组同时给予1.0% DSS和5-HT.于造模第6天处死小鼠取结肠组织.通过结肠长度、疾病活动指数(DAI)评分和HE染色等方法评估肠道炎症程度,采用实时定量PCR(RT-PCR)检测小鼠结肠组织中白介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α和 Duox2 mRNA表达水平,应用免疫组化检测结肠组织中Duox2蛋白的表达情况.结果 对照组和5-HT组小鼠无肠炎表现;DSS (1.0%) 组小鼠表现为轻度肠炎;DSS+5-HT组和DSS (2.5%) 组有严重急性肠炎表现.IL-1β、IL-6和TNF-α等表达量随炎症程度加重而增加.Duox2 mRNA在5-HT组和DSS (1.0%) 组表达增高(均P<0.05),在DSS+5-HT和DSS (2.5%) 组表达下调(均P<0.05).免疫组化结果显示Duox2蛋白主要表达于小鼠结肠上皮刷状缘及上皮细胞胞质中,与mRNA表达一致.结论 5-HT可能通过影响Duox2的表达在小鼠结肠炎的发生发展中发挥作用.%Objective To investigate the impact of 5-hydroxytryptamine (5-HT) on the expression of Duox2 in mouse colon and analyse its significance in pathogenesis of colitis.Methods Six-to-eight-week-old C57BL/6J(B6) male mice were randomly divided into 5 groups (n=8 each group): Mice in control group drank water freely.Mice in DSS (1.0%) groups and DSS (2.5%) were exposed to 1.0% dextran sulfate sodium (DSS) or 2.5% DSS.Mice in 5-HT groups were treated with 5-HT (1.0 mg/kg) by enema every 3 days.Mice in DSS+5-HT group were concurrently treated with 1.0% DSS and 1.0 mg/kg of 5-HT.At the end of 6th day of experiment, mice were scarified to collect colon tissue.The severity of colitis was evaluated by colon length, disease activity index (DAI) and pathological inflammatory scores of colon.Pro-inflammatory cytokine interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α) and Duox2 mRNAs in colon tissue were measured by quantitative real-time PCR (RT-PCR).The protein of Duox2 was evaluated by immunohistochemistry (IHC).Results Mice in control group and 5-HT group had no signs of inflammation.Mild colitis was found in mice treated with 1.0% DSS.Mice treated with 2.5% DSS suffered severe colitis.Mice receiving both DSS and 5-HT had a similar extent of colitis as those receiving 2.5% DSS.Furthermore, the expressions of IL-1β, IL-6 and TNF-α mRNA were increased corresponding to the severity of colitis.The expressions of Duox2 mRNA in DSS (1.0%) and 5-HT group were increased compared to the control group (both P<0.05).Duox2 mRNA was decreased in DSS+5-HT and DSS (2.5%) group (both P<0.05).IHC results showed that Duox2 protein was mainly expressed at the brush border membrane of colonic epithelium cell as well as in the cytoplasm;the expression of Duox2 protein was consistent with the gene expression.Conclusion 5-HT may be involved in the occurrence and development of colitis in mice by increasing Duox2 expression in colon.