The Effect And Mechanism Of Sodium Butyrate On Osteoarthritis Based On M TOR Signaling Pathway

Objective: 1)The first was to analyze the pharmacological targets of sodium butyrate,a metabolite of intestinal flora,and the common genes and interaction mechanism in the pathological process of osteoarthritis through network pharmacology.To further investigate the effect of sodium butyrate on autophagy and m TOR signaling pathway in osteoarthritis chondrocytes;2)To further explore the effects of sodium butyrate induced chondrocyte autophagy on chondrocyte and OA mice articular cartilage catabolism,inflammatory response,apoptosis,reactive oxygen species,cell cycle and m TOR signaling pathway;3)Objective to explore the effect of sodium butyrate on autophagy and m TOR signaling pathway in osteoarthritis cartilage and subchondral bone by constructing osteoarthritis mouse model in vivo and giving different doses of sodium butyrate intervention.Methods: 1)Isolation,culture and identification of primary mouse chondrocytes.CCK-8 was used to evaluate the effect of sodium butyrate on the activity of normal and IL-1? treated chondrocytes.The effect of sodium butyrate on autophagy of chondrocytes in OA model was observed by Western blot,immunofluorescence and transmission electron microscope.The effect of sodium butyrate on m TOR signaling pathway was evaluated by Western blot and immunohistochemistry;2)The effects of sodium butyrate on chondrocytes matrix metabolism,inflammation,apoptosis,reactive oxygen species damage and cell cycle arrest of chondrocytes induced by IL-1 ? were investigated by Western blot,flow cytometry,immunofluorescence and immunohistochemistry;3)Six groups were set up in the pre experiment,namely sham operation group,ACLT + placebo group,ACLT + oral sodium butyrate treatment group.Cartilage degeneration was evaluated 60 days after operation to determine the optimal dosage.In the follow-up formal trial,three groups were set up,namely sham operation group,model group and sodium butyrate oral gavage treatment group.The calcification and proteoglycan of articular cartilage were evaluated by he and safranin green staining,and the degree of cartilage degeneration was evaluated by the cartilage tissue score of osteoarthritis in mice.The characteristic indexes of extracellular matrix metabolism and apoptosis were analyzed by immunohistochemistry.The expression of LC3,Beclin1 and p62 were detected by immunohistochemistry;4)The changes of subchondral bone microstructure induced by sodium butyrate were analyzed by micro CT;5)The effect of sodium butyrate on the proliferation of subchondral bone vessels was analyzed by micro CT angiography.The expression levels of angiogenesis related factors were determined by immunohistochemistry.Results: 1)Morphological observation showed that chondrocytes showed long spindle shape.Alcian blue staining and immunofluorescence showed the characteristic expression of collagen II.Chondrocytes of the second generation were selected for the experiment.CCK-8 results showed that sodium butyrate at a concentration of more than 500 ? m had a toxic effect on chondrocytes,and sodium butyrate at a concentration of 250 ? m had the most significant effect on enhancing the activity of chondrocytes intervened by IL-1?.Network pharmacology combined with bioinformatics analysis showed that sodium butyrate targeted genes were mainly related to growth factors,cell survival and immune or inflammatory response.The target genes of sodium butyrate were AKT1,TP53,PTEN,gsk3 b,MDM2,CDKN1 A,CDKN1B,Fox O1,Fox O3,m TOR and CDK4.Western blotting,immunofluorescence and transmission electron microscopy showed that sodium butyrate could effectively activate and promote the autophagy of chondrocytes and restore the blocked autophagy flow.Sodium butyrate can enhance the autophagy of chondrocytes by targeting PI3K/Akt/m TOR and MAPK/m TOR signaling pathways;2)Sodium butyrate can inhibit the expression of catabolism and inflammatory factors(IL-6,TNF-?,IL-10,COX-2,collagen II,aggrecan,MMP3,ADAMTS-5)and pro apoptotic factors(Bax,Cleared-caspase-3,Bcl-2)by promoting autophagy of chondrocytes.Flow cytometry analysis showed that sodium butyrate could inhibit IL-1?-induced abnormal ROS production,DNA damage and cell cycle arrest of chondrocytes by activating autophagy;3)Build OA model in vivo to explore the best dose of sodium butyrate is 150mg/kg.Formal experimental results showed that sodium butyrate could inhibit the degeneration of articular cartilage in experimental OA.Sodium butyrate can promote the expression of collagen II and inhibit MMP3.At the same time,it can inhibit the production of inflammatory factors and the abnormal expression of chondrocyte apoptosis index.In the experimental OA model,sodium butyrate treatment can also activate and promote the autophagy of chondrocytes and restore the normal autophagy flow;4)Micro CT examination showed that sodium butyrate could effectively inhibit the abnormal bone resorption and the formation of bone cyst in osteoarthritis;5)Micro CT angiography showed that sodium butyrate could effectively inhibit the number and volume of abnormal increase of blood vessels in subchondral bone.Conclusion: 1)Sodium butyrate can activate and promote autophagy of chondrocytes by inhibiting phosphorylation of m TOR signaling pathway in pathological state;2)Sodium butyrate can inhibit the catabolism,inflammatory reaction and apoptosis of Osteoarthritis Chondrocytes by enhancing the autophagy of chondrocytes;3)Sodium butyrate can reduce ROS and reduce the cycle arrest of chondrocytes by enhancing chondrocyte autophagy;4)Sodium butyrate can protect the integrity of cartilage and promote the synthesis of proteoglycans in OA mice.The mechanism of normal coupling between osteogenesis and osteoclasts was maintained;5)Sodium butyrate inhibited abnormal proliferation of the subchondral bone vessels by lowering the expression of angiogenic factors,and delayed the development of osteoarthritis.