| Backgrounds and AimsOsteoarthritis(OA),the most common joint degenerative disease,is increasing the economic burden of society due to the acceleration of aging process in China.It has become clear that OA not only occurs in articular cartilage,but also affects the whole joint.Local and systemic factors are intricate,working together to influence the clinical and pathological manifestations of OA and eventually leading to joint destruction.The pathogenesis of OA is still not fully understood.Drug therapy can alleviate clinical symptoms,but there is no drug to delay or prevent the progression of the disease.The pathogenesis of OA is related to a variety of signaling pathways,including transforming growth factor beta(TGF-beta)signaling pathway,NF-kappa B signaling pathway,Wnt/beta catenin signaling pathway,MAPK signaling pathway,etc.Latent transforming growth factor beta binding protein 1(LTBP-1)is an important component of extracellular matrix(ECM).It is expressed in many tissues and cells,participates in the activation of TGF-beta,and may regulate the activation of TGF-beta signaling pathway through protease and other factors.However,whether TGF-beta signaling pathway or LTBP-1 plays a regulatory role in OA synovial cells has not been reported.Previously,we detected the expression of LTBP-1 in human normal and OA fibroblast-like synoviosytes(FLSs)by gene chip assay,and found that the expression of LTBP-1 was increased in OA.Synovial inflammation promotes the progress of OA.A variety of pro-inflammatory factors have been found in synovial fluid,synovium and articular cartilage of OA.Interleukin-1 beta(IL-1beta)is an effective regulator of the catabolism of chondrocytes and synovial cells,which is also one of the most commonly used stimulators in establishing inflammation models.The purpose of this study was to investigate the difference of LTBP-1 expression between normal and OA fibroblast-like synovial cells and synovial tissue,and to explore the regulatory role of LTBP-1 in the TGF-beta signaling pathway of OA synovium.By stimulating OA-FLSs with IL-1beta to simulate the inflammatory environment of OA synovium in vivo,we explored the relationship between LTBP-1 and TGF-beta signaling pathway and synovial inflammation process,so as to provide a possible target for the preventio n and treatment of OA.Methods1.The synovial tissue of knee joint from patients undergoing leg amputation and total knee arthroplasty due to knee OA were isolated and cultured by enzymatic digestion in vitro.After 5 times of passages,the fibroblast-like synovial cells were purified and the expressions of vimentin and CD68 were identified by flow cytometry.CCK-8 assay was performed to detect the proliferative activity of human normal and OA-FLSs.2.Immunohistochemical staining and Western blot were used to detect the expression of LTBP-1 in human normal and OA synovial tissues.The expression of LTBP-1 in human normal and OA-FLSs was detected by real-time quantitative PCR,immunofluorescence staining and Western blot.3.Real-time quantitative PCR and Western blot were used to detect the activation of TGF-beta1,TGF-beta2,TGF-beta3,pSmad1/5/8,pSmad2 signaling pathway and the expression of OA-related effector molecule MMP-13 in normal and OA-FLSs.4.The knockdown of LTBP-1 was accquired through siRNA transfection with the efficiency verified by real-time quantitative PCR and Western blot.Real-time quantitative PCR and Western blot were used to detect the activation of TGF-beta1,TGF-beta2,TGF-beta3,pSmad1/5/8,pSmad2 signaling pathway and the expression of MMP-13 in human OA-FLSs.CCK-8 method was used to detect the proliferation activity of OA-FLSs.5.Stimulating OA-FLSs with IL-1beta was performed to simulate the inflammatory environment of OA synovium in vivo.Real-time quantitative PCR and Western blot were used to detect the activation of LTBP-1,TGF beta1,pSmad1/5/8,pSmad2 signaling pathway and the expression of MMP-13 in human OA-FLSs.CCK-8 method was used to detect the proliferation activity of human OA-FLSs after stimulating with IL-1beta.Result1.High purity of human normal and OA-FLSs could be obtained by passage culture after laboratory treatment of clinical synovial tissue.Flow cytometry results showed that the positive rate of vimentin was over 95%,in accordence with the characteristics of FLSs,and the purity of cells reached the requirements of the next experiment.CCK-8 assay results showed that the proliferation of human OA-FLSs was significantly higher than that of normal FLSs.2.The results of immunohistochemical staining and Western blot showed that the expression of LTBP-1 in synovial tissue of human OA was significantly higher than that in normal synovial tissue.The results of real-time quantitative PCR,immunofluorescence staining and Western blot stated clearly that LTBP-1 in human OA-FLSs was much more than that in normal FLSs.3.The results of real-time quantitative PCR and Western blot showed that compared with normal FLSs,the activity of TGF-beta1,TGF-beta3,pSmad1/5/8 and MMP-13 were up-regulated,while the activity of pSmad2 was down-regulated in human OA-FLSs.4.The results of real-time quantitative PCR and Western blot confirmed that knockdown of LTBP-1 by siRNA was feasible.The results of real-time quantitative PCR and Western blot showed that the expressions of TGF-beta1,TGF-beta3,pSmad1/5/8 and MMP-13 in human OA-FLS were down-regulated,whereas expression of pSmad2 was up-regulated after LTBP-1 was knocked down.Result of CCK-8 assay showed that the proliferative activity of human OA-FLSs decreased significantly after LTBP-1 was down-regulated.5.The results of real-time quantitative PCR and Western blot showed that LTBP-1,TGF-beta1,pSmad1/5/8 signal activity and MMP-13 were up-regulated and pSmad2 signal activity was down-regulated in human OA-FLSs after being stimulated with IL-1beta.CCK-8 assay showed that the proliferative activity of human OA-FLSs significantly decreased after being stimulated with IL-1beta.ConclusionThis study shows that LTBP-1 promotes the expression of OA-related effector molecules such as MMP-13 by regulating the TGF-beta signaling pathway of human OA fibroblast-like synovial cells,and then participates in the progress of OA synovial inflammation,and ultimately plays a promotive role in the process of OA.Therefore,LTBP-1 is expected to be a potential target for prevention and treatment of OA. |
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