To Explore The Interventional Effect Of Huan Kui Le Suspension On Ulcerative Colitis In Rats Based On The CD4~+T Cell Subsets And Metabonomics

Objective:Based on the balance of CD4~+T cell subsets and metabonomics,analyzed the disorders of CD4~+T cell subsets Th1/Th2,Thl7/Treg cells and metabonomics,so as to explore the pathogenesis of UC and the mechanism of HKL.Methods:(1)Selected Fifty male Wistar SPF degree rats,of which 12 rats were randomly set as the normal group,and38 of them were established UC model by TNBS/ethanol composite method,after identifying the model,they were randomly divided into negative control group,5-amino salicylic acid intervention group(5-ASA group)and HKL group.Each groups after 14days of drug intervention,the general condition,disease activity index(DAI),colon mucosal injury index(CMDI)and histopathological score(HIS)of rats in each group were observed;The expression levels of IFN-?,IL-12,IL-4,IL-13,IL-6,IL-17,TGF-?1,IL-10mRNA and protein in colon tissue were detected by RT-qPCR and ELISA respectively.The ratio of CD3~+CD4~+/CD3~+lymphocytes,Th1,Th2,Th17 and Treg cells in peripheral blood were analyzed by flow cytometry.(2)The differential metabolites and metabolic pathways were analyzed by ~1H-NMR in serum of rats,While the phosphorylation levels of mTOR and mTOR,p70S6K,4E-BP1 protein expression were detected by Western blotting.Results:(1)Compared with the normal group,the DAI,CMDI and HIS scores,the expression levels of IL-12,IFN-?,IL-6,IL-17,IL-10 mRNA and protein in colon tissue,the content of Th2,Th17 cells in peripheral blood and the ratio of Th17/Treg cells were significantly higher in the negative control group(P<0.05);and the body weight,the expression level of TGF-?1 protein in colon tissue and the content of Treg cells in peripheral blood were significantly lower(P<0.05);There was no significant difference in the ratio of CD3~+CD4~+/CD3~+,Th1 cell content and Th1/Th2 ratio(P>0.05).Compared with the negative control group,The DAI,CMDI and HIS scores,IL-6,IL-17,IL-10 mRNA and protein,Th2,Th17 cell content,Th17/Treg cell ratio in 5-ASA and HKL groups were significantly decredced(P<0.05);while the body weight,the expression of TGF-?1 mRNA and protein in colon significantly increased(P<0.05);There was no significant difference in the ratio of CD3~+CD4~+/CD3~+,Th1 cell content,Th1/Th2(P>0.05).(2)Compared with the normal group,the contents of lactate,VLDL,isoleucine,valine and glycine in the serum of the negative control group increased,while the contents of?-glucose,?-glucose,pyruvate,taurine,carnitine,alanine and glycoprotein decreased,the levels of p-mTOR and p70S6K protein expression in colon tissue were significantly increased(P<0.05),there was no significant difference in mTOR and 4-EBP1(P>0.05);Compared with the negative control group,The contents of lactate,VLDL and glycine in the 5-ASA group and HKL group decreased,?-glucose,?-glucose,carnitine and taurine increased,The expression levels of p70S6K and 4-EBP1 protein were significantly decreased(P<0.05),While there was no significant difference in mTOR and p-mTOR(P>0.05).Conclusion:(1)HKL inhibits the colonic and systemic abnormal immune response in UC rats by regulating colonic inflammatory factors expression and the peripheral blood Th17/Treg cells'balance disorders.(2)In this study,there were abnormalities in amino acid metabolism,energy metabolism,and glycolytic pathway in UC rats.Phosphorylation levels of mTOR were significantly increased in colon tissues,which may further affect the differentiation and function of Th17/Treg cell subsets,Further research and discussion to be carried out.