肠紧密连接ZO1蛋白测定对药物治疗溃疡性结肠炎大鼠疗效的评价

Detection of intestinal epithelial tight junction proteins ZO1 for evaluating efficacy of drug treating in rats with ulcerative colitis

目的 测定肠紧密连接ZO1蛋白,用其评价美沙拉嗪和蒙脱石散单独或联合治疗2-4-6三硝基苯磺酸/乙醇诱导的溃疡性结肠炎（UC）大鼠肠黏膜变化,探讨UC肠黏膜屏障损伤和修复的相关机制.方法将清洁级雄性SD大鼠随机分为正常组、模型组、美沙拉嗪组、蒙脱石散组、美沙拉嗪联合蒙脱石散组.适应性喂养3d予2-4-6三硝基苯磺酸液灌肠建模,建模满24h予每组大鼠相应的药物灌胃治疗.于灌胃治疗12 d时取大肠标本行病理组织学观察,同时使用免疫组织化学Elivision法及实时荧光定量PCR法检测肠紧密连接ZO1蛋白的表达.结果 造模24h,光镜及电镜下观察的大鼠结肠组织显示建模成功.灌胃治疗12 d时,各组间疾病活动指数评分比较显示,蒙脱石散组[（0.72±0.29）分]、美沙拉嗪组[（0.95±0.38）分]、美沙拉嗪联合蒙脱石散组[（0.60±0.00）分]均低于模型组[（1.18±0.53）分],组间比较差异有统计学意义（F=3.172,P=0.047）;各组间ZO1蛋白比较显示,蒙脱石散组（0.52 ±0.28）、美沙拉嗪组（0.35±0.17）、美沙拉嗪联合蒙脱石散组（0.78±0.13）均高于模型组（0.25±0.21）,组间比较差异有统计学意义（F =7.025,P=0.001）;各组间ZO1-mRNA比较显示,蒙脱石散组[（49.53±13.32）x10-4]、美沙拉嗪组[（43.50±18.60x10-4]、美沙拉嗪联合蒙脱石散组[（78.12±21.81）x10-4]均高于模型组[（16.60±23.23）x10-4],组间比较差异有统计学意义（F=10.660,P=0.000）.结论 测定肠紧密连接ZO1蛋白,有助于认识UC的发病机制,从而指导治疗.以美沙拉嗪联合蒙脱石散用于UC时,肠紧密连接ZO1蛋白表达升高更为明显.

Objective To detect the efficacy of intestinal epithelial tight junction protein ZO1 detection on evaluating the effects of single Mesalazine or Montmorillonite powder or both on treating rats with ulcerative colitis （UC） in order to explore potential mechanisms for the damages and the recovery of intestinal mucosa of rats with UC.Methods Male SD rats were randomized into 5 groups：normal control group, model control group, Mesalazine treatment group, Montmorillonite powder treatment group, and Mesalasine plus Montmorillonite powder treatment group.The acute colitis was induced in rats by 2-4-6 trinitrobenzene sulfonic acid/ethanol after adaptive feeding for 3 days.Then,the amount of drug was administered in each treatment group respectively.The expression of ZO1 proteins in intestinal mucosal epithelium in each group was detected by immunohistochemistry and real-time PCR（RT-PCR） on the 12th day after intervention respectively.Results Colon tissues by light microscope and electron microscope observation showed that models were made successfully 24 hours after 2-4-6 trinitrobenzene sulfonic acid was given to the mice by clystering.Disease activity index outcomes of Montmorillonite powder group [（0.72 ± 0.29） scores], Mesalazinc group [（0.95 ± 0.38） scores], Mesalazine plus Montmorillonite powder group [（0.60 ± 0.00） scores] were lower than those of the model control group[（1.18 ± 0.53）scores], and there was a statistical significance （F =3.172, P =0.047）.The expressions of ZO1 protein showed Montmorillonite powder group （0.52 ± 0.28）, Mesalazine group （0.35 ± 0.17）, Mesalazine plus Montmorillonite powder group （0.78 ± 0.13） were higher than model control group （0.25 ±0.21） , and there was a statistical significance（F =7.025 ,P =0.001）.The expressions of ZO1-mRNA showed Montmorillonite powder group [（49.53 ± 13.32）x10-4], Mesalazine group [（43.50 ± 18.60）x 10-4], Mesalazine plus Montmorillonite powder group [（78.12 ± 21.81） x 10-4] were higher than those of the model control group[（16.60 ± 23.23） x 10-4], and there was a statistical significance（F =10.660 ,P =0.000）.Conclusions Intestinal epithelial tight junction protein ZO1 is involved in the pathogenesis of UC.The expression of tight junction protein ZO1 in UC treated with Mesalazine plus Montmorillonite powder increased more.It is suggested that the Mesalazine plus Montmorillonite powder is helpful in improving the recovery of the damaged intestinal mucosa.