苓术止泻口服液对脾虚泄泻大鼠结肠黏膜形态、通透性及紧密连接蛋白Claudin-1、Occludin和ZO-1 mRNA表达的影响

Effects of Lingzhu Zhixie Oral Liquid on Morphology,Permeability and mRNA Expression of Tight Junction Proteins Claudin-1,Occludin and ZO-1 in Colonic Mucosa of Rats with Splenasthenic Diarrhea

本试验旨在观察苓术止泻口服液对脾虚泄泻大鼠结肠黏膜结构、通透性及紧密连接蛋白Claudin-1、Occludin和ZO-1 mRNA表达的影响。将60只SD大鼠随机分为空白对照组、模型组、阳性药物组[20 g/(kg·bw)]、苓术止泻口服液高、中和低剂量组[40、20 g/(kg·bw)和10 g/(kg·bw)],采用“破气+苦下+饥饱失常”法复制脾虚泄泻大鼠模型,用苏木精-伊红(H.E.)染色法观察结肠黏膜组织病理学变化,采用酶联免疫法(ELISA)检测血清中二胺氧化酶(DAO)和D-乳酸(D-LA)的水平,以及荧光定量PCR法检测紧密连接蛋白Claudin-1、Occludin和ZO-1的mRNA表达水平。结果显示,苓术止泻口服液和阳性药物能不同程度修复大鼠受损结肠黏膜组织;与空白对照组相比,模型组大鼠血清中DAO和D-LA水平显著升高(P<0.05),受损结肠中的Claudin-1、Occludin和ZO-1蛋白基因的mRNA表达量显著降低(P<0.05);与模型组相比,苓术止泻口服液组和阳性药物组的大鼠血清中DAO和D-LA水平显著降低(P<0.05),结肠中Claudin-1、Occludin和ZO-1蛋白基因的mRNA表达量均显著升高(P<0.05);与阳性药物组相比,苓术止泻口服液中剂量组大鼠结肠中Claudin-1和ZO-1蛋白基因的mRNA表达量显著升高(P<0.05),高、中剂量组大鼠血清中DAO、D-LA水平和Occludin蛋白基因的mRNA表达量差异不显著(P>0.05)。试验结果表明,苓术止泻口服液能修复脾虚泄泻大鼠受损的结肠黏膜组织,降低结肠黏膜的通透性,这可能与结肠Claudin-1、Occludin和ZO-1紧密连接蛋白基因的mRNA表达有关。

The goal of this experiment was to investigate the effects of Lingzhu Zhixie oral liquid on the morphology and permeability of colonic mucosa as well as the mRNA expression of tight junction proteins,including Claudin-1,Occludin and ZO-1 in rats with spleen deficiency and diarrhea.Sixty SD rats were randomly divided into six groups,namely blank control group,model group,positive drug control group[20 g/(kg·bw)],Lingzhu Zhixie oral liquid high-dose,medium-dose and low-dose groups[40,20 g/(kg·bw)and 10 g/(kg·bw)],respectively.The histopathological changes of colonic mucosa were characterized by hematoxylin-eosin(H.E.)staining.The levels of diamine oxidase(DAO)and D-lactate(D-LA)in serum were detected by ELISA.The mRNA expression levels of tight junction proteins Claudin-1,Occludin and ZO-1 were probed by fluorescence quantitative PCR.The results demonstrated that both the Lingzhu Zhixie oral liquid and the positive drugs were able to repair the damaged colonic mucosal epithelial tissue structure.Compared with the blank control group,the serum levels of DAO and D-LA in the model group significantly increased(P<0.05).The mRNA expression levels of Claudin-1,Occludin and ZO-1 in damaged colon were significantly lower than those in the blank control group(P<0.05).Compared with the model group,the levels of DAO and D-LA in serum of rats in Lingzhu Zhixie oral liquid and positive drug treatment groups significantly decreased(P<0.05),whereas the mRNA expression levels of Claudin-1,Occludin and ZO-1 significantly increased(P<0.05).Compared to the positive drug control group,the mRNA expression levels of Claudin-1 and ZO-1 in the medium-dose group significantly increased(P<0.05),while the levels of DAO,D-LA and Occludin mRNA in the serum of the high-dose and medium-dose groups were not significantly different(P>0.05).These results suggested that Lingzhu Zhixie oral liquid could repair the damaged colon mucosal epithelial structure caused by spleen deficiency and diarrhea in rats,presumably due to the up-regulation of the expression of colon Claudin-1,Occludin and ZO-1 genes which could achieve the repair.