The Identification And Mechanism Of Osteoarthritis Characterized By Synovial Lipid Metabolism Disorder

Background: Osteoarthritis is a multifactorial disease that affects the entire joint.Currently,conservative treatments for OA are mainly limited to reducing inflammation and relieving pain,and no precise treatment measures targeting the pathogenesis of OA have been clinically approved,which may be due to the existence of different dominant pathological mechanisms in the occurrence and development of OA.In view of the heterogeneity of OA,it is significant to explore specific dominant mechanisms from the transcriptomics datasets for the development of effective and precise OA treatment drugs.Objective: Based on the consensus clustering analysis of OA synovial transcriptomic data,we aimed to identify OA subtypes with different dominant pathological features and explore the pivotal pathogenesis of specific OA types based on patient surgical samples and rat models.Methods: 1.To identify OA subtypes with different dominated pathological mechanisms,unsupervised consensus clustering was performed based on the synovial transcriptomic data of 70 OA patients obtained from GEO database,and the characteristic pathways and key genes of each OA subcluster were identified with GO and KEGG pathway enrichment.2.To verify the characteristic pathway and key gene ADCY7 of lipid metabolism-related OA in transcriptome subcluster,knee tissue samples were obtained from patients undergoing total knee replacement,knee arthroscopy and amputation,the synovial ADCY7 expression level was detected by WB,which divided the samples into high ADCY7 group and low ADCY7 group.WB,RT-q PCR,immunohistochemistry,toluidine blue staining and microplate reader were used to evaluate the differences of lipolysis,inflammatory markers and MMP13 level between the two groups of synovium samples.The proliferative activity and migration ability of fibroblast-like synovial cells(FLS)were determined by MTT,CFU-F and Transwell methods.3.Further,the OA-rat model of lipid metabolism disorder was established by high fat diet(HFD),and the HFD+low-ADCY7 rat model was constructed with si ADCY7 by injection into the knee cavity.The cartilage destruction,synovial lipolysis and inflammation of knee joint in ND,HFD and HFD+si ADCY7 groups were evaluated by saffron solid green staining,OARSI score,immunohistochemistry,WB,toluidine blue staining and microplate reader.4.To investigate the effect of ADCY7 on synovial cells and the potential mechanism,FLS of HFD rats were extracted and transfected with si ADCY7 and p CDNA-ADCY7 to construct FLS with low and high ADCY7 expression.The proliferative activity,migration ability,inflammatory mediators and MMPs secreting ability of FLS in ND,HFD,HFD+si ADCY7 and HFD+p CDNA-ADCY7 groups were evaluated by WB,Ed U,CFU-F,Transwell and FITC-phalloidine staining and RT-q PCR.5.To explore the specific mechanism of ADCY7 promoting lipolysis and influencing the function of FLS,WB and RT-q PCR were used to detect the levels of PKA substrate,lipolysis related proteins and their phosphorylation levels in FLS among HFD,HFD+si ADCY7 and HFD+p CDNA-ADCY7 groups.Results: 1.The OA synovial transcriptome data were clustered into three subclusters,among which OA subcluster dominated by lipid metabolism disorder exist stably in training and verification cohort.The main KEGG pathway was "regulation of lipolysis in adipocytes",and ADCY7 was the most significant differentially highly expressed gene.2.Patients with high ADCY7 expression were mainly early-stage OA.In the early-stage OA patient group,the synovium of patients with higher ADCY7 expression exhibited higher inflammatory response,MMP13 level and lipolysis trend.The proliferation and migration abilities of FLS were enhanced in high ADCY7 group.3.The expression of ADCY7 was increased in the knee synovium of HFD rats,and the degree of synovial lipolysis,inflammation and cartilage destruction is increased.The synovial lipolysis,inflammation and cartilage destruction were inhibited in HFD+si ADCY7 group.4.After regulating the expression of ADCY7 in FLS,we found that ADCY7 can promote the proliferative activity,migration ability,inflammatory response and MMPs secretion ability of FLS by regulating the expression of ADCY7 in FLS.5.ADCY7 promotes PKA-mediated activation of PLIN1/HSL phosphorylation,thereby promoting intracellular lipolysis to influence FLS function.Conclusion: The ADCY7-PKA-PLIN1/HSL pathway could promote synovial inflammatory lipolysis and aggravate OA.This might be an important mechanism in the development of OA associated with lipid metabolism disorder.Intra-articular inhibition of ADCY7 expression to inhibit synovial inflammatory lipolysis and cartilage degeneration might be a valuable potential therapeutic strategy to delay the progression of this type of OA.