The Mechanism Of BMSCs-Exos Alleviating OA By Regulating Glutamine Metabolism Through Sox9

Osteoarthritis(OA),as a common degenerative disease,is an increasingly serious problem in an aging society,which will bring a high socioeconomic burden.The joint most commonly affected by OA is the knee joint,which is characterized by progressive destruction of articular cartilage,loss of extracellular matrix,and progressive inflammation.Sox9 is essential for cartilage homeostasis and affects cartilage homeostasis through glutamine metabolism,but there is no research on glutamine metabolism in OA.Therefore,this study aims to explore the use of Sox9 in bone marrow mesenchyme Stem cell-derived exosomes are related to the pathogenesis and progression of OA by regulating glutamine metabolism,providing a new strategy for the treatment of bone and joints.Objective: This study aim to explore the mechanism of BMSCs-Exos regulating glutamine metabolism through Sox9 on OA.First,to explore the differences between Sox9 expression and glutamine metabolism in OA patients and OA rats.Second,the effects of BMSCs-Exos on Sox9 expression and glutamine metabolism in OA chondrocytes were explored.Then,the effect and mechanism of BMSCs-Exos transporting mi R-23 a on OA chondrocytes were investigated by cell experiments.Finally,the effect of BMSCs-Exos transporting mi R-23 a on OA rats was verified by animal experiments.Methods: 1.Collect the basic information,imaging data,functional score and knee cartilage specimens of OA patients who meet the research standards,and detect the differences in Sox9,glutamine metabolism and chondrocyte function in knee cartilage of OA patients by q RT-PCR and WB.2.The OA rat model was constructed,the exercise ability was tested by treadmill,and the cartilage tissue damage was assessed by HE and Safranin O fast green staining,TUNEL fluorescence staining was used to detect chondrocyte apoptosis,and immunohistochemistry,q RT-PCR and WB were assessed.Sox9,glutamine metabolism,chondrocyte function and inflammatory response were further determined to further determine the effect of OA on Sox9 expression,glutamine metabolism and chondrocyte function.3.Bone marrow MSCs were isolated and cultured from rat femur and tibia,and exosomes were identified by flow cytometry,electron microscopy and immunofluorescence.4.Construct OA chondrocytes,exosome-treated cells and normal chondrocyte models,and detect Sox9 in chondrocytes by flow cytometry,CCK-8 colorimetry,immunofluorescence staining,biochemical kit detection,q RT-PCR and WB.expression,glutamine metabolism,apoptosis and proliferation,cell function and inflammatory response.5.To establish the c-MYC overexpressing cell line of rat chondrocytes,to detect the effect on glutamine metabolism and chondrocyte function in OA chondrocytes.6.The expression differences of different micro RNAs such as mi R-23 a in chondrocytes of OA rats were detected by q RT-PCR,and further verified in cartilage specimens of OA patients.7.By constructing blank control group,OA group,high expression and negative control group of mi R-23 a and Sox9,flow cytometry,CCK-8 colorimetry,immunofluorescence staining,biochemical kit detection,q RT-PCR,WB etc.detected the differences of Sox9 expression,glutamine metabolism,apoptosis and proliferation,cell function and inflammatory response of OA chondrocytes.8.The ACLT rat OA model was used to construct different group to evaluate the safety of exosome injection and the rats after exosome treatment.Exercise capacity,pathological detection of cartilage damage,immunohistochemistry,q RT-PCR and WB to evaluate Sox9 expression,glutamine metabolism,apoptosis and proliferation,cell function and inflammatory response.Results: 1.The expression of Sox9 in the damaged cartilage of OA patients decreased,the metabolism of glutamine increased,and the function of chondrocytes was inhibited.The same results were obtained in OA rats.It was also found that the exercise capacity of OA rats decreased,and the cartilage tissue was affected.damage,increased chondrocyte apoptosis,and aggravated inflammatory response.2.The exosomes derived from bone marrow mesenchymal stem cells were successfully isolated and cultured from rats.3.Treatment of OA chondrocytes with exosomes can up-regulate the expression of Sox9,reduce glutamine metabolism,reverse the increased apoptosis and decreased proliferation of OA chondrocytes,and promote the expression of cartilage characteristic proteins,and reduce the Inflammatory response of OA chondrocytes.4.High expression of c-MYC increases glutamine metabolism and reduces chondrocyte function.5.mi R-23 a has low expression in OA patients’ damaged articular cartilage and OA rat chondrocytes.6.BMSCs-Exos can deliver mi R-23 a to OA chondrocytes.7.Transporting mi R-23 a exosomes upregulates Sox9 expression in OA chondrocytes,reduces glutamine metabolism,improves chondrocyte function,reverses increased chondrocyte apoptosis and decreased proliferation,and reduces inflammation,while silencing Sox9 expression.These changes are then reversed.8.The exercise capacity of OA rats was improved after receiving mi R-23a-transported exosome treatment,and no adverse reactions occurred.The damaged articular cartilage of OA rats was repaired,glutamine metabolism was reduced,and chondrocytes were reversed.Increased apoptosis and decreased proliferation improved OA chondrocyte function and reduced inflammatory responses to alleviate OA progression.Conclusion : 1.There are differential expressions of Sox9 and glutamine in OA cartilage.2.BMSCs-Exos can improve the OA chondrocyte phenotype,while c-MYC hinders the therapeutic effect of exosomes on OA.3.mi R-23 a is differentially expressed in OA cartilage.BMSCs-Exos transporting mi R-23 a can delay OA progression by up-regulating Sox9 expression and reducing glutamine metabolism,improving OA chondrocyte function.4.The in vivo injection of exosomes is safe,and the treatment of exosomes transporting mi R-23 a can improve the exercise capacity of OA rats,reduce glutamine metabolism,promote cartilage repair and delay the progression of OA.