| Objective: This study aims to establish an animal model in line with the TCM syndrome of Wutou Decoction(WTD),known for its functions of "warming meridians,dispelling cold,removing dampness,and relieving pain," particularly for cold-damp bi syndrome.This model is based on collagen-induced arthritis(CIA)in rats with cold-damp bi syndrome.Using a combination of traditional Chinese medicine pharmacology and molecular biology research methods,we investigate the effects of WTD on joint lesions,intestinal barrier damage,and immune regulation in the model animals,conducting correlation analyses.We aim to study the therapeutic effects of WTD on rheumatoid arthritis(RA)through the "intestinal barrierjoint axis" and preliminarily identify the active ingredients of WTD that repair intestinal barrier damage.By analyzing gut microbiota and their metabolites using 16 SrRNA sequencing and metabolomics analysis,combined with previous research results,we elucidate the impact of WTD on the gut microenvironment of the model animals.Through fecal microbiota transplantation(FMT)and microbial metabolite intervention,we verify the effects of key microbiota and metabolites influenced by WTD on the "intestinal barrier-joint axis," thereby clarifying the mechanism by which WTD indirectly affects the "intestinal barrier-joint axis."Methods: A rheumatoid arthritis animal model(CIA rat model with cold-damp bi syndrome)conforming to TCM characteristics was established,and the animals were administered WTD decoction via gavage.First,the therapeutic effects on arthritis were evaluated by assessing joint swelling and pathological changes in the joints;cytokine levels(IFN-γ,TNF-α,IL-10,IL-1β,IL-13,IL-17,IL-22,and IL-23)in serum,organ indices of spleen and thymus,and distribution of immune cells(TH17/Treg)in the intestines,serum,and joints were measured;biochemical markers(DAO and LPS)indicative of increased serum translocation were detected,and pathological changes in intestinal tissues were observed.Based on these indicators,the efficacy of WTD in treating RA by regulating the "intestinal barrier-joint axis" was clarified.Additionally,immunofluorescence and Western blot methods were used to examine the expression and distribution of proteins related to tight junctions(ZO-1,Claudin-1,and Occludin)in different intestinal segments.Using molecular docking technology,the interactions between blood-borne components of WTD and ZO-1,Claudin-1,and Occludin were assessed to predict the active ingredients in WTD responsible for repairing the intestinal barrier.Subsequently,16 SrRNA sequencing technology was used to analyze the gut microbiota in the colonic contents of animals after WTD administration,evaluating the impact of WTD on the gut microbiota of model animals.UPLC-Q-TOF-MS/MS technology,combined with metabolomics research methods,was employed to detect and analyze endogenous metabolites in the colonic contents after WTD administration,aiming to identify major factors influenced by WTD in colonic metabolism.Fecal microbiota transplantation(FMT)was used to verify that WTD can intervene in the gut microenvironment,repair the intestinal barrier of model animals,and impact the "intestinal barrier-joint axis" to exert its therapeutic effects on RA.Results: WTD significantly improved the toe swelling and joint pathological changes in the CIA rat model with cold-damp bi syndrome,regulated the organ indices of the spleen and thymus;compared with the model group,WTD treatment significantly reduced the levels of IFN-γ,TNF-α,IL-1β,IL-13,IL-17,IL-22,and IL-23(P<0.05 and P<0.01)in CIA rats,and increased IL-10 levels(P<0.05 and P<0.01);WTD regulated the Th17/Treg cell ratio in the intestines,blood,and joints of CIA rats,reduced DAO and LPS levels in serum,decreased inflammation infiltration in the colon,repaired crypt loss,and improved pathological structure,indicating its role in repairing the intestinal barrier.These results suggest that WTD can repair intestinal barrier damage in model rats,regulate the distribution of immune cells in the intestine-blood-joint axis,lower pro-inflammatory cytokine levels,and thus improve joint damage in model animals,exerting therapeutic effects on RA.Immunofluorescence and Western blot results showed that WTD significantly increased the expression levels of Occludin,ZO-1,and Claudin-1 proteins in the colonic tissues of model animals,thereby repairing the intestinal barrier.Further analysis of the binding energy between blood-borne components of WTD and these target proteins revealed that Claudin-1had binding activities of-6.30 kcal/mol and-7.90 kcal/mol with benzoylmesaconine and glycyrrhizic acid,respectively,and-4.87 kcal/mol with ZO-1 and liquiritin,suggesting these components might be active ingredients in WTD that repair the intestinal barrier.16SrRNA sequencing results indicated no significant effect of WTD on gut microbiota diversity in rats but an increase in species abundance,adjusting the gut microbiota profile of model rats towards normal,mainly regulating Bacillus,Zurich rod bacteria,and Bifidobacteria.Fecal metabolomics identified 84 potential biomarkers related to WTD’s efficacy,closely associated with steroid biosynthesis,primary bile acid biosynthesis,vitamin B6 metabolism,phenylalanine metabolism,histidine metabolism,α-linolenic acid metabolism,and fatty acid metabolism.These metabolic pathways are related to the regulation of gut microbiota structure,suggesting that WTD may exert therapeutic effects by regulating the gut microenvironment in disease animals,thereby affecting the "intestinal barrier-joint axis."To verify this hypothesis,a fecal content transplantation experiment(FMT)was conducted after WTD intervention.The results showed that fecal contents from WTD-treated animals significantly improved toe swelling and joint pathological changes in CIA rats,regulated organ indices of spleen and thymus,improved the distribution of Th17 and Treg immune cells in the intestines,blood,and joints,and repaired intestinal barrier damage by increasing Claudin-1,ZO-1,and Occludin expression.The gut microbiota after fecal content intervention mainly included Firmicutes,Proteobacteria,and Bacteroidetes,consistent with results from WTD administration.These results further validate that WTD can regulate the gut microenvironment in model animals,impacting the "intestinal barrier-joint axis" and thereby exerting therapeutic effects on diseases.Conclusion: The results of this study indicate that WTD is a classical Chinese medicine prescription with definite efficacy for RA.It can exert therapeutic effects on RA by regulating the immune system,repairing the intestinal barrier,and adjusting the structure of gut microbiota and the level of endogenous metabolites in the intestine,thereby influencing the "intestinal barrier-joint axis." These findings provide an important theoretical and experimental basis for further research on the use of WTD in treating RA and other related immune diseases,offering new insights and methodological references for studying the mechanisms of traditional Chinese medicine in treating RA. |
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