Study On Mechanism Of Yiyi Fuzi Baijiang Formula Treating TNBS-induced Ulcerative Colitis Based On Metabolomics And Th17/treg Cell Balance

Ulcerative colitis(UC)is an inflammatory bowel disease(IBD).The pathogenesis of UC is related to the inappropriate immune response of genetically susceptible individuals to intestinal symbiotic microorganisms caused by exposure to environmental risk factors.UC is characterized by persistent inflammation of the large intestine involving the rectum and may continue to be retrogradely distributed to the cecum.The global incidence of UC has been on an upward trend in recent years,with huge medical and social costs.At present,the drugs used in the treatment of UC,such as mesalazine,corticosteroids and biological agents,can effectively alleviate the symptoms of UC,but they have the disadvantages of long medication cycle,many adverse reactions and easy recurrence after cure.Yiyi Fuzi Baijiang formula(YFB)is a classic prescription for the treatment of intestinal carbuncle,which is from the ’Jingui Yaolue’ of Zhang Zhongjing,a famous medical scientist in the Eastern Han Dynasty.The formula was composed of Coix seed,Radix Aconiti Lateralis and Patrinia villosa.Yiyi Fuzi Baijiang formula is mainly used in the treatment of inflammatory diseases,especially ulcerative colitis.However,the mechanism of Yiyi Fuzi Baijiang formula in the treatment of UC has not been elucidated.In this paper,UPLC-MS was used to analyze its related metabolic pathways,and its mechanism of regulating inflammasome pathway and Th17/Treg cell balance was further analyzed.By detecting the contents of various metabolites in liver tissue and serum of rats with ulcerative colitis,to study whether Yiyi Fuzi Baijiang formula plays a therapeutic role in UC by affecting metabolism,and to detect the expression of FOXp3,RORγt and inflammasome pathway proteins in colon tissue of UC rats,the levels of Th17/Treg related cytokines and inflammasome pathway related inflammatory factors in serum and colon tissue were detected to elucidate its mechanism of action.UPLC-QQQ-MS/MS was used to determine the content of six main alkaloids in Yiyi Fuzi Baijiang formula.After that,48 rats were randomly divided into 6 groups(n= 8)including normal group,model group,positive group,YFB(0.682 g/kg)group,YFB(1.364 g/kg)group and YFB(2.046 g/kg)group.Except for the normal group,the other groups were constructed by enema with 100 mg/kg TNBS/50% ethanol to establish a rat model of ulcerative colitis.The rats were treated for 14 consecutive days,and the body weight changes of the rats were detected every day.At the same time,the fecal traits and occult blood of the rats were recorded for disease activity index(DAI)score.After 14 days of continuous administration,the rats were dissected after anesthesia,the blood was taken from the abdominal aorta,the colon tissue was taken and its length was measured,and the pathological changes of the colon tissue were observed by HE staining to evaluate the efficacy of Yiyi Fuzi Baijiang formula.UPLCQTrap-MS was used to detect the contents of 83 metabolites in rat serum and liver tissues,respectively.Combined with single factor statistical analysis(Mann-Whitney U test)and multivariate statistical analysis(PLS-DA,OPLS-DA),the differential biomarkers were screened and the related metabolic pathways were analyzed.The levels of IL-17 A,IL-21,IL-22,IL-6,TNF-α,IL-1β and IL-18 in serum and colon homogenate were detected by enzyme linked immunosorbent assay(ELISA).Immunohistochemistry(IHC)and Western blot were used to detect the expression levels of NLRP1,NLRP3,NLRC4,ASC,pro-caspase1,Caspase-1,RORγt and FOXp3 proteins in rat colon tissues,and the mechanism of Yiyi Fuzi Baijiang formula in the treatment of ulcerative colitis rats was analyzed.The experimental results are as follows:In the pharmacodynamics of Yiyi Fuzi Baijiang formula on TNBS-induced UC rat model:(1)Compared with the normal group,the rats in the model group were in poor condition,with dull fur,loose stool,obvious bloody stool,significantly reduced body weight,significantly shortened colon length.The DAI score was significantly increased compared with the normal group(P < 0.01).The state of rats in the positive drug group and each dose of YFB group was significantly improved.Compared with the model group,the shortening degree of colon tissue was reduced,the weight loss was reduced,and the DAI score was significantly decreased,with statistically significant differences(P < 0.01).(2)The results of HE staining of colon tissue showed that compared with the normal group,the colonic mucosa of the model group was seriously damaged and there was a large number of inflammatory cell infiltration.In the positive drug group and each dose of YFB group,the damage of colon tissue was improved,the structural damage was reduced,goblet cells appeared,and a small amount of inflammatory cell infiltration was observed.The effect of Yiyi Fuzi Baijiang formula on the metabolomics of TNBS-induced UC rat model:(1)The results of potential biomarker screening showed that 37 metabolites in serum of model group changed significantly compared with normal group,and 29 metabolites were significantly callback after administration by single factor statistical analysis of metabolites in serum and liver.In the liver,compared with the normal group,36 metabolites in the model group changed significantly,and 27 metabolites were significantly callback after administration.Combined with the results of multivariate statistical analysis and variable importance in the projection(VIP)value,25 potential biomarkers in serum samples and 24 potential biomarkers in liver samples were finally selected.(2)The results of metabolic pathway analysis showed that the pathogenesis of ulcerative colitis involved primary bile acid biosynthesis,glyoxylic acid and dicarboxylic acid metabolism,glycine,serine and threonine metabolism,and purine metabolism.The mechanism of Yiyi Fuzi Baijiang formula in the treatment of UC is related to the regulation of metabolic pathways such as primary bile acid biosynthesis,tricarboxylic acid cycle,fatty acid biosynthesis,cysteine and methionine metabolism.The effect of Yiyi Fuzi Baijiang formula on immune regulation of TNBS-induced UC rat model:(1)ELISA results of serum and colon tissue showed that compared with the normal group,IL-17 A,IL-21,IL-22,IL-6,TNF-α,IL-1β and IL-18 in the model group were significantly increased(P < 0.01),and the levels of IL-17 A,IL-21,IL-22,IL-6,TNF-α,IL-1β and IL-18 in the positive drug group and each dose of YFB group were lower than those in the model group(P< 0.05)in a dose-dependent manner.(2)The results of immunohistochemical detection of inflammasome pathway protein in colon tissue showed that the positive cells of NLRP1,NLRP3,NLRC4,ASC,pro-caspase1 and Caspase-1 in colon tissue of UC rats in model group were significantly higher than those in normal group(P < 0.01).Compared with the model group,the positive cells of NLRP1,NLRP3,NLRC4,ASC,pro-caspase1 and Caspase-1 in the positive drug group and each dose of YFB group were significantly reduced in a dose-dependent manner,and the results were statistically significant(P < 0.01).(3)The results of Western blotting of RORγt,FOXp3 and inflammasome pathway proteins in colon tissue showed that compared with the normal group,the expression of NLRP1,NLRP3,NLRC4,ASC,RORγt,pro-caspase1 and Caspase-1 protein in the colon tissue of the model group was significantly increased(P < 0.01),and the expression of FOXp3 protein was significantly decreased(P < 0.01).Compared with the model group,the expression of NLRP1,NLRP3,NLRC4,ASC,RORγt,pro-caspase1 and Caspase-1 protein in the positive drug group and each dose of YFB group were significantly decreased(P < 0.01),and the expression of FOXp3 protein was significantly increased(P < 0.01),with a dose-dependent relationship.The following conclusions can be drawn from the above researches:(1)Yiyi Fuzi Baijiang formula can improve the weight,DAI score and histopathology status of the colon in rats with ulcerative colitis,and has therapeutic effect on ulcerative colitis in a dose-dependent manner.(2)Yiyi Fuzi Baijiang formula can play a therapeutic role in ulcerative colitis by regulating metabolic pathways such as primary bile acid biosynthesis,tricarboxylic acid cycle,cysteine and methionine metabolism,and fatty acid biosynthesis.(3)Yiyi Fuzi Baijiang formula can inhibit the activation of inflammasome signaling pathways in colon tissue,regulate Th17/Treg cell balance,and the mechanism of action of Yiyi Fuzi Baijiang formula on ulcerative colitis is related to the regulation of intestinal immune balance in rats with ulcerative colitis.