Celastrol Inhibits Invasion Of Rheumatoid Fibroblast-like Synoviocyte Through Suppression Of TLR4/MMP-9Signal Pathway

IntroductionRheumatoid arthritis (RA) is a progressive inflammatory autoimmune disease mainly affecting the joints, characterized by synovial hyperplasia and inflammatory cells infiltration, leading to tissue destruction and functional disability. Among the inflammatory cell populations that might participate in RA pathogenesis, fibroblast-like synoviocytes (FLSs) are crucial in initiating and driving RA in concert with inflammatory cells, and contribute to the destruction of cartilage and bone by secreting metalloproteinases (MMPs) into the synovial fluid and by direct invasion into extracellular matrix (ECM), and further exacerbates joint damage. The control of RA-FLS invasion represents an important therapeutic target. MMPs are involved in the development and processes of RA, and MMPs are responsible for the invasion properties of many cell lines. MMPs are mainly induced in RA-FLSs by pro-inflammatory cytokines, through activation of Toll-like receptor4(TLR4)/nuclear factor-KB (NF-κB) inflammatory signal pathway. Lipopolysaccharide (LPS), a cell wall constituent of gram-negative bacteria, is released during lysis of bacteria and exerts direct effects on tumor cell proliferation, invasion and metastasis in vitro and in vivo. TLR4is the receptor of LPS, which also expressed in FLS. So far, the role and mechanism of TLR4on the FLS invasion is still not clear.Celastrol, a pentacyclic-triterpene extract from Celastrus orbiculatus Thunb, is used in traditional Chinese medicine as an anti-tumor agent. Plant-derived herbal products are generally less toxic and better tolerated than many conventional drugs in the treatment of RA. However, the mechanisms of action of many potentially anti-arthritic plant products are not fully defined. Recent reports suggest that Celastrus extract and its bioactive celastrol have beneficial anti-arthritic effects in an adjuvant-induced arthritis (AIA) model. Studies to define the therapeutic mechanism of celastrol in RA showed that it suppressed pro-inflammation cytokines, synovial immune cell infiltration and proliferation, modulated immune responses rather than induced immunosuppression, and inhibited bone damage in an AIA model. However, the effect and molecular mechanisms underlying the effect of celastrol on LPS induced RA-FLS invasion are not yet fully understood.In the present study, we treated human RA-FLSs with celastrol in vitro and examined the effect of celastrol on LPS-induced cell migration and invasion. Furthermore, we investigated the mechanisms involved in and evaluated the therapeutic efficacy of celastrol in an in vivo collagen-induced arthritis (CIA) rat model. The research will be described in four parts as follows:Part I Effect of celastrol on LPS-induced RA-FLS migration and invasionObjectives:To examine the effect of celastrol on Lipopolysaccharide (LPS)-induced rheumatoid arthritis fibroblast-like synoviocyte (RA-FLS) migration and invasion.Methods:FLSs were obtained from the synovium of active RA patients, Isolated RA-FLSs were identified by flow cytometry as a homogeneous population (CD14and CD68are macrophage markers and CD90is a fibroblast marker). FLSs were treated with various concentrations of celastrol in serum-containing medium for24h, and cell viability was determined using the MTT assay. Whether celastrol could inhibit LPS-induced cell migration and invasion was analyzed using a Transwell chamber.Results:RA-FLSs were identified by flow cytometry as phenotype:<1%CD14, CD68and>98%CD90, cells used for experiments were at the third to sixth passage. Treatment with0.05to0.2μM celastrol had no significant effect on cell viability at24h and48h. However,0.4μM and0.8μM celastrol decreased cell viability by approximately1and2-fold, respectively, in comparison with the control group. The in vitro migration ability of FLSs was increased3.05-fold when stimulated with LPS for24h. Similarly, data obtained from the invasion assay showed that LPS increased cell invasion7.02-fold in comparison with the control group. However, LPS-induced cell migration and invasion were inhibited by celastrol in a dose-dependent manner.Conclusion:These results suggest that non-toxic concentration celastrol ranging from0.05μM to0.2μM could inhibit the RA-FLS migration and invasion induced by LPS in vitro, which showed that celastrol might provide benefit in the treatment of RA by inhibiting LPS-induced FLS motility.Part Ⅱ Celastrol inhibits LPS-induced RA-FLS migration and invasion by suppression of MMP-9activityObjectives:To examine whether celastrol inhibits LPS-induced RA-FLS migration and invasion by suppression of metalloproteinases (MMPs) activity.Methods:The effect of celastrol on MMP-1, MMP-2, MMP-3and MMP-9gene and protein expression was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The cell supernatants were collected and assayed for the secretion and proteolytic activity of MMP-9protein, by Enzyme-linked immuno sorbent assay (ELISA) and gelatin zymography respectively. Furthermore, to identify the role of MMP-9in the invasiveness induced by LPS, MMP-9siRNA was used to inhibit MMP-9.Results:LPS induced MMP-1, MMP-2and MMP-9mRNA expression in RA-FLSs, whereas treatment with celastrol suppressed LPS-induced MMP-9expression in a dose-dependent manner; however, MMP-1, MMP-2and MMP-3mRNA expression were not affected by celastrol treatment. The secretion and proteolytic activity of MMP-9in FLSs were induced when FLSs were cultured in serum-free medium with1μg/mL LPS for24h, treatment of FLSs with celastrol suppressed LPS-induced MMP-9secretion and activity in a dose-dependent manner. After siRNA knockdown of MMP-9and stimulation with LPS (1μg/mL), the number of cells migrating and invading the basal membrane decreased by70%compared with the control siRNA group.Conclusion:These results indicate that celastrol selectively inhibits LPS-induced MMP-9expression at both the gene and protein levels, and subsequently suppressed the enzymatic activity of MMP-9, furthermore, indicate the contribution of MMP-9to the migration and invasion induced by LPS.Part Ⅲ Celastrol inhibits the transcriptional activity of MMP-9by suppression of LPS stimulated TLR4/NF-κB signal pathwayObjectives:To examine whether celastrol inhibits the transcriptional activity of MMP-9by suppression of LPS stimulated Toll-like receptor4(TLR4)/nuclear factor-KB (NF-κB) signal pathway.Methods:RA-FLSs were transiently transfected with reporter genes that included the wild-type MMP-9promoter or a promoter with mutations in the NF-κB site or AP-1site. The effect of celastrol on the relative luciferase activity of MMP-9promoter was examined. Next, we examined the inhibitory effect of celastrol on the binding activity of NF-κB in the MMP-9promoter using electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay. Then, we examined whether celastrol regulated the TLR4/NF-κB signaling pathway. FLSs were pretreated with different concentrations of celastrol and stimulated with LPS for24h and then levels of TLR4, myeloid differentiation primary response gene88(MyD88), TIR domain containing adaptor inducing interferon β (TRIF), and inhibitor of nuclear factor-KB (IκBα) were measured by Western blot. Furthermore, the TLR4pathway inhibitor TAK-242and neutralizing antibody were used to examine the involvement of TLR4/NF-KB pathway in LPS-induced MMP-9activation.Results:Treatment with celastrol in the presence of LPS decreased the transcription activity of the reporter with the AP-1mutation, but had no effect on the reporter with NF-κB mutations. In vitro celastrol treatment suppressed LPS-induced NF-κB binding to MMP-9promoter on EMSA, in vivo ChIP showed that binding of NF-κB to the MMP-9promoter increased in response to LPS treatment, whereas LPS-induced NF-κB binding to the MMP-9promoter was significantly inhibited by celastrol. LPS stimulation induced significant phosphorylation of IκBα, and IκBα degradation, markedly inhibited the LPS-induced TLR4and MyD88expression, but celastrol could not inhibit the expression of TRIF. qRT-PCR showed that treatment of FLSs with TAK and anti-TLR4obviously decreased LPS-stimulated MMP-9mRNA expression, TAK and anti-TLR4inhibited LPS-induced MMP-9protein expression and activation, treatment of FLSs with TAK and anti-TLR4clearly decreased the LPS-induced MMP-9promoter luciferase activity.Conclusion:These results suggest that celastrol inhibits LPS-induced MMP-9activation by suppressing TLR4/MyD88/NF-κB pathway in RA-FLSs. Part IV Celastrol improves synovitis and joint destruction of CIA ratsObjectives:To investigate the anti-arthritic mechanisms involved in and evaluated the therapeutic efficacy of celastrol in vivo.Methods:Administration of celastrol (0.5mg/kg/d and1mg/kg/d, intraperitoneally) and methotrexate (MTX)(2mg/kg/w, intraperitoneally) for3weeks in a collagen-induced arthritis (CIA) rat model. Arthritic severity was assessed by articular index (AI) and measured every3days from day20to day40after primary immunization. On day42, the right hind limbs were subjected to radiographic analysis using a dental X-ray machine to assess joint damage. Then, the rats were sacrificed and the synovial membrane of right hind knee joints was prepared for histopathological analysis. The H&E-stained sections were used to determine the degree of synovial hyperplasia and inflammatory cells infiltration. Furthermore, the rats serum were collected and assayed for the activity of MMP-2and MMP-9protein by Western blot.Results:The onset of arthritis in ankle joints was visually determined between days18and21post immunization. Celastrol (0.5mg/kg) and celastrol (1mg/kg) treated groups showed reduced swelling in the soft tissue, less bone destruction, the joint space of high dose of celastrol-treated group and MTX-treated group was normal compared to the CIA group, and effect of high dose of celastrol-treated group and MTX-treated group was similar. An inhibitory effect on AI of the celastrol was observed from day22until the end of the observation period. By comparison, the effect of high dose of celastrol-treated group was better than that of the low dose-treated group. Celastrol (0.5mg/kg and1mg/kg) treatment groups had significantly ameliorated synovial hyperplasia and inflammatory cells infiltration compared to the CIA group, and the improvement of high dose of celastrol-treated group was slightly better than that of the low dose-treated group. Western blot results showed that celastrol (0.5mg/kg and1mg/kg) treatment could obviously inhibit the MMP-9activity rather than MMP-2in CIA rat serum.Conclusion:These results suggest that celastrol may improve synovitis and joint destruction of CIA rats by suppressing the MMP-9activity.