| Objective: Osteoarthritis is a global common chronic joint degenerative disease,which occurs frequently in middle-aged and elderly people and women,and the incidence rate is increasing year by year,which brings a burden to personal life and social medical security.The large number of gut microbes contains a large amount of genetic information,and has become the second gene pool of the body.Gut microbes dysbiosis and alterations in its metabolites lead to a range of diseases,including chronic inflammation and bone disease.In recent years,more and more studies have shown that the gut microbes and its metabolites may be related to the pathogenesis and development of osteoarthritis,and the symptoms of the disease can be alleviated by interfering with the gut microbes.Clostridium butyricum is an intestinal probiotic which provides energy for intestinal epithelial cells by producing short-chain fatty acids,promotes the proliferation of intestinal epithelial cells,assists in the repair of the intestinal barrier,and inhibits pathogenic bacteria proliferation in the intestinal tract by changing the intestinal microenvironment.MIYAIRI(MY)is one type of Clostridium butyricum tablet that has been used to treat and improve gastrointestinal symptoms caused by intestinal flora disorder caused by various reasons.At present,there have been some discussions on MIYAIRI in the treatment of intestinal diseases and depression,However,there are few studies on its role and mechanism in osteoarthritis.The proposed study aims to investigate the effect of Clostridium butyricum on osteoarthritis,and to preliminarily explore the mechanism of Clostridium butyricum by immunohistochemistry,16 S r DNA sequencing technology and metabolomic analysis.Methods: In the current study,osteoarthritis model rats were constructed by surgical operation,and Clostridium butyricum was administered to them after the model was constructed.After 4 weeks of Clostridium butyricum administration,the knee joint and tibia muscle were isolated for immunohistochemical experiments and quantitative detection of succinate dehydrogenase(SDH)and muscle glycogen(MG).Feces were collected,and 16 S r DNA sequencing was performed.Based on the data,α and βdiversity were analyzed to determine the changes of intestinal flora diversity and the changes of bacterial phyla and genera under different treatments,and to analyze the functions of differential flora.Metabolomics detection of rat feces was performed to compare the differences of metabolites under different treatments,and enrichment analysis of related pathways was carried out.Combining sequencing data with omics data,Pearson correlation analysis and redundancy analysis were performed at the phylum and genus levels to explore the correlation between gut microbiota composition and differential metabolites.Finally,the expressions of energy metabolism-related genes,myogenesis-related genes,neuromuscular junction-related genes and inflammatory cytokines were detected by RT-q PCR and western blotting.Results: Immunohistochemical results showed that Clostridium butyricum administration to rats could alleviate the symptoms of knee joint OA in rats,and increase the contents of succinate dehydrogenase and muscle glycogen in the tibia muscle of rats.Data from 16 S r DNA sequencing showed that compared with control group,the diversity of gut microbiota in rats with osteoarthritis decreased,while Clostridium butyricum treatment increased the evenness values of Chao1,Shannon and Pielou,and increased the diversity of gut microbiota in OA rats.The abundances of Prevotella,Ruminococcus,Desulfovibrio,Shigella,Helicobacter and Streptococcus were higher in OA rats,while Lactobacillus,Oscillobacter,Clostridium and Faecalibacterium were more abundant in the Clostridium butyricum treatment group.A total of 395 differential metabolites were identified in the fecal samples of rats in the OA group and OA+MY group by metabolomics,including 204 down-regulated metabolites and 191 up-regulated metabolites.These metabolites with significantly different contents were mainly enriched in the intestinal immune network related to Ig A production,steroid hormone biosynthesis,vitamin digestion and absorption,and arachidonic acid metabolism.Clostridium butyricum treatment increases the abundance of beneficial bacteria(Lactobacillus,Oscillospira,Clostridium and Faecalis)in the gut and reduces pathogenic microorganisms(Prevotella,Ruminococcus,Desulfovibrio,Shigella,Helicobacter and Streptococcus)in the intestinal tract,thereby enhancing the joint repair effect of OA model rats and relieving OA.Subsequently,the expression levels of genes involved in muscle fatty acid metabolism,glucose metabolism and energy metabolism,and protein ubiquitination and degradation were quantitatively detected in different treatment groups.Compared with the control group,the m RNA levels of AMPK,Tfam,Mruf1,Myod,Ldh,Chrna1,Chrnd,Rapsyn and Agrin were significantly decreased in the OA group,while the m RNA expressions of Lcad and Mcad were increased.With treatment of Clostridium butyricum,the m RNA levels of AMPK,Tfam,Myod,Ldh,Chrna1,Chrnd,Rapsyn and Agrin were significantly increased in rats,but the expressions of Mruf1,Lcad and Mcad were still decreased.The m RNA expression of IL-1β in the OA group was significantly higher than that in the control group,while Clostridium butyricum treatment decreased the expression of IL-β.Conclusion: In conclusion,the existence of the gut-muscle-joint axis is suspected,because Clostridium butyricum may promote joint damage repair and relieve OA by regulating the expression of gut flora diversity,metabolite-related pathways,and muscle function-related protein molecules. |
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