Effects Of Metformin On Proliferation And Osteogenesis Of FLSs In Interfacial Membrane Through AMPK/m-TOR Pathway

Background:Aseptic loosening(AL)is one of the main reasons for the loosening of the joint prosthesis.A certain degree of micro-motion at the interface between the prosthesis and the bone will inhibit the growth of the bone around the prosthesis,forming a certain gap.And fill the membrane.The limiting membrane mainly contains macrophages,synovial fibroblasts(FLSs),foreign body giant cells,osteoclasts,and lymphatic cells,among which fibroblasts account for 30%-70%.Synovial fibroblasts upregulate the ratio of RANKL/OPG induced by wear particles,promote osteoclast differentiation and maturation,and promote osteolytic progression.On the other hand,metformin exerts anti-inflammatory effects on multiple tissues and organs through AMPK channels,such as inhibiting osteolytic inflammation of macrophages such as L-6,iNOS,and OPG induced by lipopolysaccharide(LPS)and wear particles.The expression of factors.At the same time it can promote osteoblast osteogenesis,compound AMPK inhibitor compound C inhibit osteoblasts extracellular osteogenesis,synovial fibroblasts and osteoblasts have some homology,with osteogenic potential.In addition,metformin can inhibit a variety of tumor cell proliferation,promote its apoptosis or inhibit its cycle,but also can inhibit fibroblast proliferation.Synovial fibroblasts up-regulate the ratio of RANKL/OPG and synergize with macrophage osteolysis.The proliferative formed boundary membrane provides a good microenvironment for osteolysis.Therefore,this study focuses on the effect of metformin on proliferation and osteogenesis of synovial fibroblasts.Exploring the role of metformin in inhibiting the proliferation of synovial fibroblasts and promoting synovial fibroblast osteogenicity provides a new idea for the prevention and treatment of aseptic loosening of prosthesis,and is beneficial to the new use of old drugs.Objective:To investigate the effect and mechanism of Metformin on the proliferation inhibition and osteogenesis of synovial fibroblasts.METHODS:The synovial membranes of patients undergoing revision and arthroscopic surgery were reconstructed.The primary synovial fibroblasts were extracted by tissue adherence method and digestion method.The proliferation of loose synovial membrane was detected by HE and histochemical staining,and cytochemical staining was detected.Synovial fibroblasts are of mesenchymal origin.MTT and EdU detected the effect of metformin on the proliferation of synovial fibroblasts.Apoptosis and cycle-flow assays were used to determine the specific mechanism of inhibition of proliferation.Western blotting was used to detect the cycle-related and apoptosis-related proteins and to detect the pathway proteins.Alkaline phosphatase staining and alizarin red staining detected metformin to promote alkaline phosphatase synthesis and osteogenic efficiency in synovial fibroblasts.Results:Metformin inhibited the proliferation of synovial fibroblasts in a concentration-and time-dependent manner.Metformin could induce synovial fibroblasts to arrest in the G2/M phase through the AMPK/m-TOR pathway,but its apoptosis was absent.Influence;Simultaneous inhibition of phosphorylation of p70s6k by the AMPK/m-TOR pathway,and promotion of phosphorylation of 4E-BP1;Further found that metformin promotes alkaline phosphatase and extracellular calcium nodules of synovial fibroblasts through AMPK channels.Formation promotes synovial fibroblast osteogenesis.Conclusion:Metformin can induce synovial fibroblasts arrest in G2/M phase through the AMPK/m-TOR pathway,inhibit the phosphorylation of p70s6k,and promote the phosphorylation of 4E-BP1 and inhibit the proliferation of synovial fibroblasts.And promote synovial fibroblast osteogenesis.This provides the basis and new drug treatment target for the clinical research of metformin in the prevention and treatment of aseptic loosening in the later period.