PLCL1 Regulates Fibroblast-like Synoviocytes Inflammation Via NLRP3 Inflammasomes In Rheumatoid Arthritis

Rheumatoid Arthritis（RA）is a systemic,chronic,autoimmune,inflammatory disease that affects both large and small synovial joints in a symmetrical manner.RA starts with joint pain,inflammatory cells gradually erode and destroy the joint,and further deterioration of the joint state will cause permanent and irreversible damage to the joint,and eventually cause deformity and disability.There are approximately 1.2 million cases of rheumatoid arthritis worldwide.Although there are various treatments and medications for rheumatoid arthritis,none of them guarantees a complete cure.Fibroblast-like synoviocytes（FLS）are lining cells in joints,and their epigenetic changes make them invasive and play an important role in the pathological process of RA.In RA,FLS,in addition to its role as an extracellular matrix and joint lubricant,also produces pathogenic mediators such as cytokines and proteases that promote disease pathogenesis.In the study of the pathological mechanism of RA,the study of cytokines is the most extensive.There are roughly two cytokines that play a pathogenic role in RA,one is chemokine and the other is inflammatory factor.Chemokines are responsible for recruiting various immune cells such as macrophages,T cells and B cells.Inflammatory factors are responsible for the interaction between immune cells and various primitive cells in the joint cavity.This creates a cycle of chronic inflammation that exacerbates the disease process.Based on the previous GWAS sequencing of the Anhui Provincial Laboratory of Inflammatory and Immune Diseases,this topic further analyzed the five RA susceptibility loci screened by them,namely IL12RB2,PLCL1,CCR2,TCF7 and IQGAP1.In this study,the synovial tissue of osteoarthritis（OA）patients was used as a control,and it was found that the m RNA expression of Phospholipase C-Like 1（PLCL1）in the synovial tissue of RA patients was increased and the most obvious.After experiments,it was found that PLCL1 protein plays an important role in RA.PLCL1 is abundant in proliferative cells,and has the effect of regulating lipid metabolism and promoting inflammatory response.Pre-experiments in this study showed that PLCL1 was highly expressed in RA tissue,suggesting that PLCL1 may be involved in the pathological mechanism of RA.The preliminary exploration of PLCL1 in this topic resulted in the following results:1.PLCL1 elevated expression in K/BxN miceIn this study,K/BxN mice,a model of spontaneous arthritis,were selected,and their synovial tissues were collected from their paternal KRN mice as controls.The success of the K/BxN mouse model was identified by HE staining.At the same time,the expression of PLCL1 protein in the synovial tissue of K/BxN mice was detected by IHC staining,and the resu Lts showed that PLCL1 was highly expressed in K/BxN mice.2.PLCL1 is highly expressed in RAThis project collected synovial tissue from OA patients and RA patients.The pathological tissue was identified by HE staining,and the expression of PLCL1 in RA synovial tissue was also detected by IHC.After primary RA FLS was extracted,the way of stimulating FLS with TNF-α was screened from IL-1β,IL-6,CXCL8,IL-17 A,TNF-α,IFN-γ and LPS by RT-qPCR.The stimulation effect of 5 concentrations of TNF-α was detected by RT-qPCR and WB,and 10ng/m L of TNF-α was finally selected to stimulate FLS to simulate the chronic inflammatory environment of RA in vivo.Finally,IF double staining was used to detect PLCL1 and Vimentin proteins in FLS before and after TNF-α stimulation.To verify that PLCL1 was up-regulated after 10ng/m L TNF-α stimulated FLS,the synovial cytoskeleton protein was simultaneously stained.3.PLCL1 promotes the inflammatory response in RA FLSThe expression of PLCL1 in RA FLS was specifically silenced by PLCL1-RNAi,and the expression of PLCL1 was significantly down-regulated after silencing detected by WB.The inflammatory response of FLS was detected by RT-qPCR,WB and Elisa.The results showed that the m RNA and protein expressions of IL-1β,IL-6 and IL-8 were inhibited but not completely counteracted after silencing PLCL1;6 and IL-8 m RNA and protein expression were significantly increased4.To explore the mechanism of PLCL1 affecting the inflammatory response of FLSWhen PLCL1 was silenced,we found that the expressions of NLRP3 and cleavedCaspase-1 in FLS also decreased,and the content of IL-1β in the supernatant of FLS detected by Elisa was also significantly decreased.After PLCL1 was overexpressed with PLCL1-PEX,FLS The expressions of NLRP3 and cleaved-Caspase-1 in the FLS were increased,and the content of IL-1β in the FLS supernatant detected by Elisa was also significantly increased.After expressing PLCL1,the NLRP3 inflammasome pathwayspecific inhibitor INF39 was used,and the WB results showed that the expressions of NLRP3,IL-1β,IL-6 and cleaved-Caspase-1 were inhibited.Therefore,the results of this study suggest that PLCL1 may mediate the inflammatory response of RA FLS by regulating the activation of the NLRP3 inflammasome.