Effects Of High Concentrations Of Insulin On The Biological Behavior Of Fibroblast-like Synovial Cells In Vitro And Its Mechanism

Background:Recent years,a growing number of new reports on the relationship between diabetes and osteoarthritis,suggest that diabetes should be probably an independent risk factor for osteoarthritis.Fibroblast-like synovial cells(FLS)lies inside the synovium lining.Not only do they maintain the intra-articular environment,but can also release inflammatory factors into the articular cavity.So changes of synoviocytes’ related functions would cause or aggravate articular cartilage degeneration.With deeper understanding of insulin resistance,hyperinsulinemia is widely considered as one of common pathophysiologic changes at the early onset of diabetes,which has gotten the attention of the researchers.As an important hormone in the body,insulin can regulate energy metabolism and has other important effects in vivo.As we all know,autophagy plays an important role in the maintenance of endocellular and extracellular homeostasis and involves in many known diseases like diabetes and osteoarthritis as well.However,the relevant regulatory mechanisms of autophagy are still under continuous study.At present,there is no related reports about how high concentrations of insulin affect the basic biological function of synovial fibroblasts such as proliferation and autophagy,the release of macrophage-derived chemokines as well as inflammatory response to IL-1β in vitro.Therefore,this study is expect to find the potential mechanism linking between diabetes and osteoarthritis,indeep the understanding of KOA and provide more theoretical basis for the clinical management of osteoarthritis.Objective:To study the effects of high-concentration insulin on FLS including the proliferation,autophagy,the release of macrophage-derived chemokines and inflammatory response in vitro,and to explore the possible molecular mechanisms.Methods:Fibroblast-like synovial cells were isolated and cultured from the KOA patients during the total knee replacement by the two-step enzyme digestion method.The effect of different concentrations of insulin on FLS proliferation was measured by CCK-8 technique at the different time points.Then we took the advantage of the chemotaxis of macrophage in the transwell migration assays to reflect the indirect level of chemotactic factors for macrophage in the supernatant of these groups.In one group,FLSs were treated with insulin for 24h;as a contrast,FLSs were cultured in normal medium for 24h in another group.And the effects of high concentration of insulin(1000nmol/L)on AKT pathway and autophagy-related protein LC3-Ⅱ in FLSs were detected by Western blotting.Rapamycin can inhibit mTOR,so western blotting was used to detect the content of LC3 protein to study whether the inhibiting effect of rapamycin counteracts the negative effect of high concentrations of insulin on autophagy.We tried to use recombinant human IL-1β(10ng/mL)to trigger FLSs’inflammation response.Then the expressions of IL-1β,IL-6 and MMP9 in FLSs were detected by RT-qPCR after FLS was induced by recombinant human IL-1β(10ng/mL)for 24h in order to testify if inflammation model was established.We also used Western blotting to study the effect of IL-1β induction alone and the one of IL-1βtogether with high-concentration insulin on autophagy in FLSs cells.Meanwhile the effects of large-dose insulin on cell inflammatory response were studied by RT-qPCR,which detected the relative expression of IL-1β,IL-6 and MMP9 at RNA level.Results:A large number of purified fibroblast-like synovial cells were obtained by two-step enzyme digestion.Insulin could remarkably promote FLS cells proliferation,which was dependent on the concentration.However,with the insulin increased to a certain concentration,this stimulus effect is no longer increased(p<0.05).After counting the cells on the bottom of the membrane,the number of that in INS + FLS group was obviously higher than that in INS group and FLS group(p<0.01).We detected relative content of objective proteins in FLSs at 2h、6h、24h by Western blotting.The results revealed that while the relative content of pAKT gradually elevated,the relative content of LC3-Ⅱ significantly lowered compared with the control group(p<0.05).It was also observed in FLSs rapamycin could improve LC3-Ⅱ.Nevertheless,in the group added high-concentration insulin and rapamycin at the same time,LC3-Ⅱ was lower than that in rapamycin group but higher than that in insulin group(p<0.05).As known as an inflammatory cytokines,IL-1β was confirmed to increase LC3-Ⅱ in our study.Specifically,LC3-Ⅱ in IL-1 added INS groupwas lower than that in IL-1β group but higher than that in insulin group(p<0.05).Besides,we used RT-qPCR to show that high-concentrations of insulin could significantly reduce the expression of IL-1β,IL-6 and MMP9 in IL-1β-induced FLS(p<0.05).Conclusion:Insulin could promote the proliferation of fibroblast-like synovial cells through AKT/mTOR pathway and inhibit autophagy.In addition,high concentrations of insulin could decrease both autophagy and inflammatory activity induced by IL-1β and enhance the FLSs’ chemotaxis of macrophage.This study could offer a new idea of the gradual treatment program for osteoarthritis patients especially with diabetes mellitus.