Preliminary Study On The Pathogenic Role And Mechanism Of Insulin In Synovial Inflammation Of Osteoarthritis

Background and Objective:Osteoarthritis(OA)is a common degenerative joint disease in the elderly.The main clinical manifestations of OA patients are joint pain and limited movement,accompanied by joint stiffness,swelling and other symptoms,which seriously affect the quality of life of patients.Due to the unknown etiology and pathogenesis of OA and the lack of effective treatments,most patients with OA eventually need artificial joint replacement to relieve their symptoms.In view of this,in-depth analysis of pathogenic factors and pathogenesis,finding therapeutic targets,so as to carry out early intervention treatment of OA is the current research focus.With the deepening of research in recent years,it has been found that metabolic syndrome(MS),especially type 2 diabetes mellitus(T2DM),can promote the development of OA.OA not only includes articular cartilage damage,but also involves a variety of joint soft tissues such as synovium and articular capsule A large number of studies have shown that synovial inflammation not only appears in the initial stage of OA,but also plays an important role in the entire disease progression of OA.Abnormal proliferation and activation of synovial cells and various inflammatory factors secreted by synovium are closely related to joint cartilage lesions and clinical symptoms(joint pain,swelling,etc.)of patients.Therefore,synovial inflammation can be considered as a new target for the treatment of OA.Hyperinsulinemia(HINS)is the common pathogenesis of MS and T2DM,and its role in OA remains unknown.Therefore,in this study,insulin was used to stimulate primary fibroblast-like synoviocytes(FLSs)in human knee joint,to observe whether it promotes synovial inflammatory response,and to explore its possible regulatory mechanism.Methods:1.FLSs were stimulated by insulin with different concentrations(0,100,200,500nmol/L)for three time periods(6,12,and 24h),CCK-8 method and Transwell method were used to detect the changes in cell proliferative activity and macrophage chemotaxis of FLSs,respectively.The gene expression and secretion of pro-inflammatory mediators such as interleukin(IL)-1β,IL-6,tumor necrosis factor(TNF)-α,matrix metalloprotein(MMP)-9,MMP-13 and the secretion of chemokines such as CXCL12,CCL2/MCP-1 and CCL5/RANTES were detected by reverse transcription-quantitative polymerase chain reaction(RT-qRCR)and enzyme-linked immunosorbent assay(ELISA)respectively.2.Western blot was used to detect the activation of PI3K/Akt,mTOR and NF-KB signaling in FLSs;Immunofluorescence(IF)was used to verify cellular localization of NF-KB signal subunit proteins.The effects of blocking the three signaling pathways on the changes of cell proliferation,pro-inflammatory mediators production and chemokines production by insulin in FLSs.3.FLSs were stimulated by insulin alone or the three signaling pathways in FLSs were pre-blocked,RT-qPCR was used to detect the common cellular receptors of IL-1β,IL-6 and TNF-α:interleukin-1 receptor(IL-1R)1,IL-1R3,Interleukin-6 receptor(IL-6R),glycoprotein 130(GP130),tumor necrosis factor receptor 1(TNFR1 or p55)and tumor necrosis factor 2 receptor 2,TNFR2 or p75)expression in FLSs;FLSs were stimulated by three pro-inflammatory cytokines of IL-1β,IL-6 and TNFa or were co-stimulated by insulin,RT-qPCR was used to detect the expression of MMP-9 and MMP-13,and Western Blot was used to detect the activation of PI3K/Akt,mTOR and NF-KB signaling in FLSs.Results:1.Insulin of certain concentration(0、100、200、500nmol/L)can significantly increase cell proliferative activity and inflammatory mediator production,and 500nM of insulin can enhance macrophage chemotaxis and promote chemokine production of FLSs.2.Insulin can activate the PI3K/mTOR/Akt/NF-KB signaling pathway in FLSs;Insulin-induced inflammatory responses are significantly reduced by pre-blocking three signaling pathways with specific pathway inhibitors in FLSs.3.500nM of insulin can upregulate cellular surface receptor levels of IL-1β,IL-6 and TNFa in FLSs,and PI3K/mTOR/Akt/NF-KB signaling inhibitors can reverse this process.Compared with the stimulation with three pro-inflammatory factors respectively,the expression levels of MMP-9 and MMP-13 as well as the activation levels of the three signaling pathways in FLSs were significantly increased by pre-stimulation with insulinConclusion:Insulin can do more than exacerbate synovial inflammation by activating three signaling pathways of NF-KB,PI3K/Akt,and mTOR at a certain concentration;it can also work synergistically with pro-inflammatory factors,amplify the synovial inflammation cascade.This study verifies that insulin is involved in synovial inflammation of OA and provides theoretical support for finding potential targets for clinical treatment of OA.