MFG-E8 Regulates Chondrocyte Senescence And Synovial Macrophage In Osteoarthritis

BackgroundOsteoarthritis is the most common chronic disabled disease.And its pathological features include cartilage destruction,subchondral bone sclerosis,synovial thickening,osteophyte formation and so on.The pathogenesis of osteoarthritis involves genetic factors,metabolic abnormalities,traumatic stress,cell senescence,synovitis and etc.,but the specific mechanism remains to be clarified.Previous studies have shown that milk fat globule epidermal growth factor 8(MFG-E8)is an important molecule that mediates the phagocytosis of apoptotic cells.in addition,MFG-E8 also plays an important role in inhibiting inflammation,regulating macrophage polarization,inhibiting apoptosis,regulating the balance between osteoclast and osteoblast.However,the role of MFG-E8 in the occurrence and development of osteoarthritis has not been reported.ObjectiveThe purpose of this study was to determine the expression of MFG-E8 in cartilage and serum under osteoarthritis conditions,and then to verify whether MFG-E8 plays a protective role in osteoarthritis.Finally,we try to identify the upstream and downstream mechanisms of MFG-E8 regulation to figure out whether miRNA and a certain pathway activation play important roles.MethodsTibial plateau cartilages were collected from patients who got severe osteoarthritis and were performed total knee arthroplasty,with the injured medial side of cartilage as the experimental group and the less damaged lateral side of cartilage as the control group.14-week-old mice were performed with DMM surgery,and the serum and joint were collected at 4 weeks and 8 weeks post operation.The primary chondrocytes were extracted from tibial plateau cartilage of 3-6 days old mice and treated with IL-1β.The expression of MFG-E8 was evaluated at the level of cell,mice,body fluid and human tissue by immunohistochemistry,WB,qPCR,and ELISA.DMM mice were intraarticularly injected with MFG-E8 recombinant protein or neutralizing antibody,the osteophyte formation was evaluated by micro-CT,and then the sections of joints were stained with Safranin O-fast green,H&E,and toluidine blue to determine the degree of cartilage damage and synovial inflammation.Immunohistochemistry,immunofluorescence,WB,qPCR,and β-galactosidase staining were used to detect the catabolic synthesis index(MMP13,COL2),the senescence hallmarks(p16,p21,p53,βgalactosidase),the polarization hallmarks of synovial macrophages(F4/80,iNOS,CD206),and the expression of inflammatory factors(IL-1β,IL-6,TNF-α)in vitro and in vivo after the MFG-E8 recombinant protein or neutralizing antibody administration.After MFG-E8 intervention,the p65 phosphorylation of cartilage and synovium was detected by immunofluorescence.MFG-E8 neutralization antibody and NF-κB pathway inhibitor JSH-23 were treated in primary cartilage and RAW264.7 cells at the same time.WB was used to verify the degree of p65 phosphorylation and the expression of the above hallmarks.Finally,we used bioinformatic analysis to predict the binding between miRNA and MFG-E8.qPCR and WB were applied to detect the expression level of miRNA and its effect on MFG-E8 expression in osteoarthritis cartilage,and whether MFG-E8 intervention could reverse the phenotypic changes caused by miRNA mimics and inhibitor transfection.ResultsThe expression of MFG-E8 was significantly down-regulated in human osteoarthritis cartilage,mouse DMM cartilage and serum,and in the supernatant of primary chondrocytes treated with IL-1β.Intra-articular injection of MFG-E8 recombinant protein reduced cartilage destruction,synovitis,and osteophyte formation,while MFG-E8 neutralization antibody injection further aggravated osteoarthritis.The supplement of recombinant MFG-E8 down-regulated the cartilage catabolic marker MMP-13,and up-regulated the anabolic index COL2,and inhibited the expression of cartilage senescence hallmarks(p16 p21,p53,β-galactosidase).Meanwhile,in synovial macrophages,the expression of M1 marker iNOS was down-regulated and M2 marker CD206 expression was up-regulated.While after MFG-E8 neutralization,cartilage decomposition and chondrocyte senescence were accelerated,and synovial macrophages inclined to M1 polarization.After treatment with MFG-E8 recombinant protein,the level of p65 phosphorylation was significantly inhibited,and the development of osteoarthritis induced by MFG-E8 neutralizing antibody could be partially reversed by NF-κB inhibitor JSH-23.The expression of miR-99b-5p is significantly up-regulated in osteoarthritis cartilage,while overexpression of miR-99b5P down-regulated the expression of MFG-E8.Overexpression of miR-99b-5p accelerated the decomposition and senescence of chondrocytes and promote the polarization of RAW264.7 cells to M1 subtype,while administration of recombinant MFG-E8 protein can partially reverse the destructive and pro-inflammatory effects of miR-99b-5p.ConclusionThe expression of MFG-E8 is significantly down-regulated under osteoarthritis conditions both locally and systematically.MFG-E8 plays a protective role in osteoarthritis by inhibiting the activation of NF-κB pathway,thus slowing down the catabolism and senescence of articular cartilage,promoting cartilage anabolism,regulating the polarization of synovial macrophages to M2 subtype which inhibits inflammation,and inhibiting the polarization of towards M1 subtype that promotes inflammation.The up-regulated miR-99b-5p in osteoarthritis significantly suppresses the expression of MFG-E8 and plays a destructive role in osteoarthritis.