Metabolomic And Proteomic Analysis Of The Role And Mechanism Of The Infrapatellar Fat Pad In Knee Osteoarthritis

BackgroundKnee osteoarthritis(OA)is one of the most common bone and joint diseases in clinic.The main symptoms are pain and limited joint movement,and joint deformity may occur in advanced stages of the disease.The etiology and risk factors of OA mainly include trauma,age,obesity,gender and heredity.In the progression of OA,the different constituent structures throughout the joint are affected,and the pathology is characterized by cartilage degeneration,osteophyte formation,subchondral osteosclerosis,synovial and infrapatellar fat pad(IPFP)degeneration.The treatment of OA mainly includes oral non-steroidal drugs in the early stage and joint replacement surgery in the late stage.However,patients with OA continue to suffer a significant burden due to the lack of cause-specific therapeutic drugs,the longevity of joint prostheses,and the surgical complications.IPFP is an important component in the knee joint.In recent years,more and more studies have paid attention to its role in OA.Therefore,exploring the role and mechanism of IPFP in OA is of great clinical significance for a comprehensive understanding of OA and finding new therapeutic targets.ObjectivesMetabolomic and proteomic analyses of IPFP in OA and control groups were performed to identify Differentially Expressed Metabolites(DEMs)and Differentially Expressed Proteins(DEPs),and bioinformatics analyses were performed to identify the pathogenesis of IPFP in OA,and finally to identify important pathways in which both DEMs and DEPs are enriched and explore their effects on the development of OA.Methods1.Part I:In this study,patients who underwent Total Knee Arthroplasty(TKA)for OA and Meniscoplasty for meniscus injury were selected from Hennan Hospital between January 2022 and October2022.IPFP samples were obtained intraoperatively.The age,gender and BMI information of the patients were collected,and the IPFP metabolic profiles of the OA group(n=28)and the control group(n=17)were compared by Liquid Chromatograph Mass Spectrometer(LC-MS)technology.The Orthogonal Partial Least Squares-Discriminant Analysis(OPLS-DA)model was used to screen the DEMs in IPFP,and the KEGG pathway analysis was performed for the DEMs.2.Part II: The sample collection method was the same as the first part.Label-free quantitative proteomics technology was used to compare the IPFP protein profiles of patients in OA group(n=8)and control group(n=8),and DEPs were screened out.GO and KEGG enrichment analysis,Protein protein interaction(PPI)and Hub node analysis were also conducted.3.Part 3:Sample collection method is the same as the first part.We looked for important pathways where both DEMs and DEPs are enriched,and finally selected the glycolysis pathway for study.The expression of key proteins in glycolysis pathway was verified by western blot.The change of IPFP lactate value between OA group(n=8)and control group(n=8)was compared with lactic acid kit to verify the difference of glycolysis process between groups.Finally,after the chondrocytes were treated with lactic acid,the changes of Collagen II,Collagen X and MMP13 markers of cartilage degeneration were detected by western blot test.Results1.147 DEMs were identified in this study,and the results of KEGG pathway analysis revealed that DEMs were mainly enriched in pathways related to carbon metabolism,insulin signaling pathway,amino acid anabolism,gluconeogenesis and other substance metabolism in cancer.2.In this study,516 DEPs were identified.GO enrichment analysis showed that DEPs significantly enriched complement activation,classical pathway and daunorubicin metabolic process in Biological processes(BP).In Cel Glular Component(CC),DEPs significantly enriched blood particles and extracellular space.In Molecular Function(MF),DEPs significantly enriched alditol:NADP+1-oxidoreductase activity,D-threo-aldose 1-dehydrogenase activity,antigen-binding activity.KEGG analysis indicated that DEPs mainly enriched metabolic pathways,Systemic lupus erythematosus,and Primary immunodeficiency.In addition,DEPs are also enriched in pathways related to the metabolism of sugars,amino acids and lipids.Protein interaction network(PPI)and HUB node analysis identified the top10 proteins(ENO1,CAT,APOA1,HSPE1,FBL,UQCRC1,PPIF,TTR,TARDBP and C1QBP)as key proteins of IPFP in OA.3.The glycolytic pathway was selected for study by joint analysis of DEPs and DEMs.The four DEPs,PGM1,PGK1,PGAM1 and EN01,existed in the glycolytic pathway network in direct action with DEMs;Western blot results found that PGM1,PGAM1 and EN01 proteins were highly expressed in the IPFP of OA group;the results of lactate value measured by lactate kit suggested that the IPFP of OA group contained higher lactate value;Using lactic acid treatment of chondrocytes,Western blot results found that Collagen II and Aggrecan expression was decreased,and Collagen X and MMP13 expression was increased.Conclusions1.Based on metabonomics and proteomic analysis,we identified the DEMs and DEPs existing in IPFP during the development of OA,and conducted bioinformatics analysis to identify some key proteins and signaling pathways related to the lesions of OA.2.The glycolytic pathway is enhanced in the IPFP of OA,and the acidic environment caused by it can cause cartilage degeneration.We also found three key proteins in the glycolytic pathway,namely PGM1,PGAM1 and EN01.The disorder of glycolysis helps to understand the mechanism of OA from a new perspective.