Study On Intervention Mechanism Of Yi Medicine Yiyuanhuafu Decoction On T Lymphocyte Subsets In Ulcerative Colitis Model

Purpose:Yiyuan Huafu decoction is a prescription guided by Yi medicine theory"rotten intestine theory and yuan qi".It is often used in ulcerative colitis and anorectal diseases and has good clinical effect.The purpose of this study is to study the mechanism of Yi medicine Yiyuan Huafu decoction regulating T cell subsets in the treatment of ulcerative colitis,in order to provide scientific basis for clinical application of Yiyuan Huafu decoction in the treatment of ulcerative colitis,inherit and innovate Yi medicine.Method:1.We established the inflammatory cell model of mouse macrophage line Raw264.7induced by bacterial lipopolysaccharide（LPS）.The cytotoxicity of Yiyuanhuafu decoction extract on inflammatory cells was detected by MTT method,and the effect of Yiyuan Huafu decoction extract on NO production of inflammatory cell model was detected by Griss method.2.32 C57BL/6 healthy female mice were randomly divided with mass of body into four groups:vehicle group,model group,low dose of Yiyuanhuafu decoction(4.05g kg^-1)and high dose of Yiyuanhuafu decoction(8.1g kg^-1).Except for the normal group,the mice model of ulcerative colitis was induced by 3%sodium glucan sulfate（DSS）in the way of free drinking water,and the drinking water was changed every 2 days for 8 days.From the first day of drinking DSS,each group was given the corresponding dose by intragastric administration,once a day for 8 days.The mice were treated with Yiyuan Huafu decoction extract,the stool,activity and mass of the mice were observed;disease activity index（DAI）were evaluated the symptoms of ulcerative colitis;the morphological changes of colon were observed by hematoxylin-eosin staining（H&E）;and the relative expression levels of T cell subsets（IL-17A,RORγt,IFN-γ,Foxp3）in spleen and mesenteric lymph nodes were detected by fluorescence quantitative PCR（q PCR）.Flow cytometry was used to detect the expression of spleen and lymph node surface markers（CD4⁺,CD3⁺,CD44⁺,Ki67⁺,B220⁺）and intracellular markers Th1 cells(IFN-γ^（10）CD4⁺T cells),Th17 cells(IL-17^（10）CD4⁺T cells)and regulatory T cells（CD4⁺Foxp3⁺T cells）.Result:1.The effect of Yiyuanhuafu decoction on the activity of Raw264.7 cells and the inhibition on the release of NO.Yiyuanhuafu decoction at the dose of 125μg/ml、250μg/ml、500μg/ml significantly inhibited the production of NO（P<0.05 or P<0.01）.The IC50 of Yiyuanhuafu decoction was 202.6μg/ml,CC50>500μg/ml.2.The effect of Yiyuanhuafu decoction on the disease activity index of ulcerative colitis model mice.Compared with the model group,the mass of mice,colon length and colon mass in the Yiyuanhuafu decoction 4.05g·kg^-1and 8.1g·kg^-1groups,and the DAI score decreased significantly（P<0.05 or P<0.01）.3.Effect of Yiyuanhuafu decoction on pathological injury of colonic tissue in mice with ulcerative colitis.The colonic tissue of model group showed loss and exfoliation of large mucous layer and diffuse inflammatory cell infiltration,while the inflammatory cell infiltration and mucosal defect of colonic tissue and mucosal ulcer in 4.05g·kg^-1and 8.1g·kg^-1group were less than model group.4.The effect of Yiyuanhuafu decoction on lymphoid T cell subsets in mice with ulcerative colitis.（1）The results of flow cytometry showed that after intervention with Yiyuanhuafu decoction,the expression of splenic lymphocyteγδTCR was significantly down-regulated in 8.1g·kg^-1group（P<0.05）.However,Yiyuanhuafu decoction had no significant effect on the proportion and absolute number of CD4⁺,CD3⁺,CD44⁺,Ki67⁺and B220⁺in spleen and mesenteric lymph nodes.（2）The results of intracellular staining flow cytometry showed that compared with the model group,Yiyuanhuafu decoction at the dose of 4.05g·kg^-1and 8.1g·kg^-1inhibited the absolute number and proportion of Th1 cells(IFN-γ^（10）CD4⁺T cells)and Th17 cells(IL-17^（10）CD4⁺T cells)（P<0.05 or P<0.01）,but had no significant effect on the expression of Treg cells（CD4⁺Foxp3⁺T cells）.5.The effect of Yiyuanhuafu decoction on the expression of mRNA related factors of T cell subsets in the model of ulcerative colitis.The results of qPCR detection showed that compared with the model group,the expression of RORγτlymphocyte m RNA in mesenteric lymph node Th17 cells was significantly down-regulated in 8.1g·kg^-1group（P<0.05 or P<0.01）.For spleen,Yiyuanhuafu decoction 8.1g·kg^-1significantly down-regulated the expression of Th17cytokine IL-17A and transcription factor RORγτm RNA in UC mice（P<0.05）.Conclusion:Yiyuanhuafu decoction can improve the general conditions of ulcerative colitis model mice,such as body mass,colon mass,colon length and pathological score,and significantly down-regulate the expression ofγδTCR and Th17 cytokine IL-17A and transcription factor RORγτm RNA in ulcerative colitis model mice,and inhibit the absolute number and proportion of Th1 cells(IFN-γ^（10）CD4⁺T cells)and Th17 cells(IL-17^（10）CD4⁺T cells).Thus inhibit the inflammatory response induced by Th17 cells to mediate the treatment of ulcerative colitis.Give full play to the advantages of Yi medicine in the treatment of ulcerative colitis to provide aspects,multi-angle clinical needs for the treatment of ulcerative colitis,which can be used as a reference for workers in the study of ulcerative colitis.