Study On The Mechanism Of Modified Duhuo Jisheng Mixture In Regulating Intestinal Barrier And Microbiota Metabolism In KOA Mic

Objective: To explore the content of "gut-joint axis" from the aspects of gut barrier and gut microbiota metabolism,and to study the mechanism of Jiawei Duhuo Jisheng mixture on knee osteoarthritis(KOA)in mice by improving gut barrier and regulating gut microbiota metabolism.Methods: The KOA model was induced by medial meniscus instability surgery(DMM)on the right knee joint.Eight-week-old C57BL/6 mice were randomly divided into Sham group(n=6),Model group(n=6),Jiawei Duhuo Jisheng Mixture group(n=6)and positive drug group(Mobic group,n=6).One week after surgery,the corresponding intervention drugs were intragastrically administered.The sham operation group and the model group were given the same amount of normal saline every day.Eight weeks after operation,the tissues of knee joint,colon,serum and cecum of mice were taken for the following studies:(1)The joints were scanned by Micro-CT,and histomorphology of the knee joint was stained with safranin-O/fast green staining to observe the condition of articular cartilage,synovial membrane and osteophyte.The content of type II collagen in articular cartilage and positive expression of matrix metalloproteinase 13(MMP13)were detected by immunohistochemistry.(2)The morphology of the colon tissue was observed by HE staining,the positive expressions of tight junction proteins ZO-1 and Occludin in the colon tissue was observed by immunohistochemistry,and the level of serum lipopolysaccharide(LPS)and interleukin-1β(IL-1β)were detected by ELISA to assess the gut permeability and inflammation.(3)The cecal contents of mice were subjected to LC/MS non-targeted metabolomics detection,followed by data processing,differential metabolite screening and metabolic pathway enrichment analysis.Results:(1)The joints of KOA mice modeled by DMM surgery degenerated,including reduction of cartilage matrix content,rough and defective cartilage surface,reduction of type II collagen content in cartilage,increase of positive expression of MMP13,synovitis and osteophyte formation;Jiawei Duhuo Jisheng Mixture significantly improved the joint degeneration of KOA mice,alleviated the destruction of joint cartilage,synovitis and osteophyte formation,increased the content of type II collagen of joint cartilage,and reduced the expression of MMP13.(2)The gut barrier structure of KOA mice was damaged,including morphological damage to the colon tissue,shortening or even missing of the colon crypts,and significant reduction in the positive expression of gut tight junction proteins ZO-1 and Occludin;Jiawei Duhuo Jisheng Mixture significantly improved the colon pathology of KOA mice and increased the positive expressions of ZO-1 and Occludin.The expressions of serum LPS and IL-1β in KOA mice were significantly increased,and the levels of LPS and IL-1β were significantly decreased with Jiawei Duhuo Jisheng Mixture.(3)Compared with the sham group,the fecal metabolic characteristics of KOA mice were changed,and 228 metabolites were significantly changed;Jiawei Duhuo Jisheng Mixture regulated the feces metabolites,up-regulating 69 metabolites and down-regulating 70 metabolites,including dopamine and others.The results of enrichment analysis of metabolic pathways showed that the intervention of Jiawei Duhuo Jisheng Mixture on KOA might be related to sphingolipid metabolism and linoleic acid metabolism.Conclusion:(1)Jiawei Duhuo Jisheng Mixture can alleviate joint degeneration in KOA mice.(2)Gut barrier may play a role in the process of KOA disease,and Jiawei Duhuo Jisheng mixture may improve the function of gut barrier to alleviate KOA inflammation in mice.(3)KOA may lead to the change of fecal metabolic characteristics,and the intervention of Jiawei Duhuo Jisheng mixture on KOA may be related to its regulation of fecal metabolites.