基于miR-21、miR-150和miR-200b研究参苓白术散对克罗恩病大鼠肠黏膜屏障的干预机制

The intervention mechanism of Shenling Baizhu San on miR-21,miR-150 and miR-200b in intestinal mucosal barrier of rats with Crohn’s disease

目的:探讨参苓白术散对克罗恩病大鼠肠黏膜屏障中miR-21、miR-150和miR-200b的作用机制,深入分析参苓白术散治疗克罗恩病的相关作用机制。方法:将66只SD大鼠随机分为正常对照组(予0.9%的氯化钠溶液20 mL/kg)、模型组(予0.9%的氯化钠溶液20 mL/kg)、阳性对照组(予6%美沙拉嗪缓释颗粒悬浊液0.2 g/kg)、低剂量参苓白术散组(予参苓白术散5 g/kg),中剂量参苓白术散组(予参苓白术散10 g/kg),大剂量参苓白术散组(予参苓白术散20 g/kg),每组11只。除正常对照组外,其余大鼠建立克罗恩病模型。治疗3周后,观察不同剂量参苓白术散对结肠组织病理形态的影响;Western blot法(WB)检测肠黏膜组织中Claudin-1蛋白的含量,实时荧光定量聚合酶链式反应(qRT-PCR)检测肠黏膜组织中miR-21、miR-150和miR-200b微小核糖核酸(Micro RNA,miRNA)的表达量。结果:与正常对照组比较,模型组大鼠结肠组织中Claudin-1蛋白和miR-200b的miRNA表达量明显降低(P<0.01),而miR-21、miR-150的miRNA表达量明显升高(P<0.01)。与模型组比较,阳性对照组和三个参苓白术散组大鼠结肠组织中Claudin-1蛋白、miR-200b的miRNA表达量显著升高(P<0.05或P<0.01);而阳性对照组和参苓白术散中、高剂量组miR-21、miR-150的miRNA表达量减少(P<0.05或P<0.01),参苓白术散中、高剂量组中Claudin-1蛋白表达量与正常对照组相比无显著差异(P>0.05)。结论:参苓白术散具有明显改善肠黏膜屏障的作用,其作用机制之一可能是通过调控miR-21、miR-150和miR-200b的表达量,从而调节Claudin-1蛋白的表达,达到改善克罗恩病大鼠肠黏膜屏障功能的作用。并且本研究结果提示在克罗恩病模型大鼠结肠组织中Claudin-1蛋白的表达与miR-21、miR-150呈负相关,与miR-200b呈正相关。

Objective:To investigate the mechanism of action of Shenling Baizhu San(参苓白术散)on miR-21,miR-150 and miR-200b in intestinal mucosal barrier of Crohn’s disease rats.The relevant mechanism of action of Shenling Baizhu San in the treatment of Crohn’s disease was revealed.Methods:66 SD rats were randomly divided into 6 groups,namely the normal control group(normal saline,20 mL/kg),model group(normal saline,20 mL/kg),positive control group(6%mesalazine sustained-release granule suspension,0.2 g/kg),low-dose Shenling Baizhu San group(5 g/kg),medium-dose Shenling Baizhu San group(10 g/kg),and high-dose Shenling Baizhu San group(20 g/kg).Crohn’s disease was modeled in all rats except the normal group.After 3 weeks of treatment,the pathological changes of colonic tissue mediated by different doses of Shenling Baizhu San were observed.The content of claudin-1 protein in intestinal mucosa was detected by Western blot(WB).qRT-PCR was used to detect the transcription level of miR-21,miR-150 and miR-200b in intestinal mucosa.Results:Compared with the normal control group,the expressions of claudin-1 protein and miR-200b miRNA in the colon tissue of the model group were significantly reduced(P<0.01),while the expressions of miR-21 and miR-150 were significantly increased(P<0.01).Compared with the model group,the expressions of claudin-1 protein and miR-200b miRNA in the colon tissue of the positive control group and the three Shenling Baizhu San groups were significantly increased(P<0.05 or P<0.01).The expression levels of miR-21 and miR-150 in the medium-dose and high-dose Shenling Baizhu San groups were reduced(P<0.05 or P<0.01).There was no significant difference in the expression of claudin-1 protein in the medium-dose and high-dose Shenling Baizhu San groups compared with the normal control group(P>0.05).Conclusion:Shenling Baizhu San can significantly improve the intestinal mucosal barrier.One of its mechanisms may be to regulate the expressions of miR-21,miR-150 and miR-200b,thereby regulating the expression of claudin-1 protein and improving the intestinal mucosal barrier function in Crohn’s disease rats.And the results of this study suggest that the expression of claudin-1 protein in colon tissue of CD model rats is negatively correlated with miR-21,miR-150,and positively correlated with miR-200b.