| Rheumatoid arthritis(RA)is a chronic,autoimmune disease dominated by synovitis.According to the pathogenesis characteristics of RA,obvious swelling and synoviocytes will occur in the joints of the patient,among which fibroblast-like synoviocytes(FLSs)plays an important role.FLSs is located in the synovial lining layer and is the main cell in synovial tissue.It has dual characteristics of inflammatory response and tumor-like proliferation.Activated FLSs can secrete TNF-α,IL-1β,IL-6 and other inflammatory factors,which aggravate the inflammatory response of RA,and it presents the characteristics of excessive proliferation of tumor-like cells,and can also lead to the formation of pannus.Meanwhile,FLSs can also secrete matrix metalloproteinases(MMPs)to destroy adjacent bone and cartilage.Up to now,the pathogenesis of RA is unknown,but a large number of studies have confirmed that the activation of FLSs and inflammation are closely related to the occurrence and development of RA.However,little is known about the molecular mechanisms involved.In order to explore the role of proteomics in the pathogenesis of RA,primary RA FLSS cells were extracted in this study,and Label-free experiment was conducted after 10ng/mL TNF-α stimulation.After screening,the procollagen C-proteinase enhancer protein(PCOLCE)was found to play an important role in RA.PCOLCE is rich in collagen and fibrotic tissues,participates in extracellular remodeling,and has anti-inflammatory and anti-angiogenic effects.Label-free experiment results showed that PCOLCE expression was down-regulated in RA FLSS after TNF-α stimulation,suggesting that PCOLCE may be involved in the occurrence and development of RA.However,the relevant mechanism has not been reported,so the following studies have been carried out in this subject:1.The expression of PCOLCE was decreased in RAFirst,synovial specimens of osteoarthritis(OA)and RA joints were collected to identify the synovial conditions of the collected patients with OA and RA by HE and IF,and then IHC was used to detect the expression of PCOLCE in synovial tissues.After extraction of primary RA FLSs,the expression of PCOLCE was detected by HE,IHC,IF,WB and q-PCR,and the results showed that the expression of PCOLCE was decreased in synovial tissue and FLSs of RA patients.2.PCOLCE inhibits RA FLSs inflammationWB and q-PCR results showed that TNF-α stimulated PCOLCE-RNAi could significantly reduce the protein and mRNA expressions of PCOLCE,and increase the protein and mRNA expressions of IL-6 and IL-17A.However,after the use of PCOLCE-PEX,the protein and mRNA expressions of PCOLCE were increased,and the protein and mRNA expressions of IL-6 and IL-17A were inhibited.ELISA assay showed that PCOLCE-RNAi could significantly promote the secretion of IL-1β and IL-8 in RA FLSs,while PCOLCE-PEX could inhibit the secretion of IL-1β and IL-8 in RA FLSs.3.PCOLCE inhibits the activation of RA FLSsAfter TNF-α stimulation,WB and Q-PCR results showed that PCOLCE-RNAi could significantly increase the protein expression and mRNA content of MMP-2 and MMP-3,and PCOLCE-PEX could significantly inhibit the protein expression and mRNA content of MMP-2 and MMP-3.Wound-Healing experiment and Transwell experiment showed that PCOLCE-RNAi could promote RA FLSs activation,while PCOLCE-PEX could inhibit RA FLSs activation.4.To explore the mechanism by which PCOLCE affects FLSs inflammation and activationAfter TNF-α stimulation,WB and Q-PCR results showed that PCOLCE-RNAi significantly increased the expression of NLRP3,IL-1β,and cleaved caspase-1 in FLFs,while overexpression of PCOLCE inhibited the expression of NLRP3,IL-1β,and cleaved caspase-1 in FLSs.WB results showed that the expression of NLRP3,IL-1β,IL-6,and IL-17A was significantly reduced after PCOLCE-RNAi was administered in combination with INF39,an inhibitor of the NLRP3 inflammasome pathway.Therefore,the results of this study showed that PCOLCE may inhibit the inflammation and activation of RA FLSs by inhibiting the NLRP3 inflammasome pathway. |
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