The Role Of Trail-DR5 By IL13Rα1/STAT6 In Apoptosis Induction Of Fibroblast-like Synoviocytes Of Rheumatoid Arthritis

Background:Rheumatoid arthritis(RA)is a chronic autoimmune disease that can occur at any age.The main clinical manifestation is erosive arthritis,which eventually leads to joint deformity and loss of function.At present,although more rheumatoid arthritis related factors have been discovered,the molecular mechanism of its occurrence is not very clear so far,and domestic and foreign scholars are conducting in-depth research from multiple signaling pathways.Among them,some scholars found that the number of fibroblast-like synoviocytes(FLS)in rheumatoid arthritis showed a pathological increase,and their apoptosis showed a downward trend.The activation pathway of fibroblast-like synovial cells regulated by multiple signaling pathways plays a very important role in the occurrence and development of RA.It has been found that a variety of stress-inducing factors such as inflammatory cells can induce endoplasmic reticulum stress response,and endoplasmic reticulum stress can promote the proliferation of fibroblast-like synovial cells,and also promote the secretion of various cytokines by intra-articular cells,which were involved in the progression of rheumatoid arthritis.Therefore,the results of the study show that endoplasmic reticulum stress is closely related to the pathological progression of rheumatoid arthritis,and studying how to overcome the apoptosis resistance of the above endoplasmic reticulum stress may provide new clinical insights for rheumatoid arthritis.treatment strategy.Among numerous endoplasmic reticulum stress-related cytokines and their signaling pathways,IL-13 is one of the cytokines with protective effects,and can be used as a serum marker and therapeutic target for rheumatoid arthritis.Second,the tumor necrosis factor-related apoptosis-inducing ligand Trail is a known apoptosis-related gene,and its proteins have agonistic receptors DR4 and DR5.DR5-type receptor(Trail-R2)is highly expressed in synovial tissue and fibroblasts of rheumatoid arthritis,and the specific activation pathway of Trail-DR5 signaling can effectively improve the arthritis phenotype of rheumatoid arthritis.However,the exact roles of IL-13 and its receptors in the cellular response to ER stress during the occurrence and development of RA remain unclear,and the signaling pathways downstream of IL13Rα1 under ER stress also need to be further explored.In particular,whether IL13Rα1 is involved in the pathological process of rheumatoid arthritis fibroblast-like synovial cells through STAT6 activation of the Trail-DR5 signaling pathway has not been reported so far,which deserves further research.Objective:In this study,we aimed to determine the role of IL13Rα in the resistance of fibroblast-like synoviocytes in rheumatoid arthritis,and assess the possibility of IL13Rα as a target in rheumatoid arthritis therapy.Methods:IL13Rα1 expression was assessed in the synovial tissue by RT-qPCR,immunohistology,and Western blot.Gain or loss of functional analysis was applied to evaluate the biological roles of IL13Rα1 in RA FLSss.Cell viability and apoptosis were assessed by MTS,Western blot,and flow cytometry.The therapeutic effects of IL13Ral on the severity of type Ⅱ collagen-induced arthritis(CIA)in DBA-/1J mouse model were evaluated by scoring synovitis,hyperplasia,cartilage degradation,and bone destruction.Results:1.The expression of IL13Rα1 in synovial tissue and fibroblasts of patients with rheumatoid arthritis is decreased:Compared with patients with osteoarthritis,in the synovial tissue of patients with rheumatoid arthritis,whether it is IL13Rα1 mRNA expression level,or Its protein level was lower than that of IL13Rα1 in synovial tissue of patients with osteoarthritis.2.Endoplasmic reticulum stress inducers reduce the expression level of IL13Rα1 in fibroblast-like synoviocytes:After detection by immunofluorescence,it was found that IL13Rα1 was mainly located in the cytoplasm of fibroblast-like synoviocytes.IL13Rα1 protein was significantly decreased under treatment conditions with the inducer cobalt chloride(CoC12),which mimics hypoxia,or the classical endoplasmic reticulum stress inducers,tunicamycin(Tm)and thapsigargin(Tg),indicating that IL13Rα1 May be involved in the cellular response of fibroblast-like synoviocytes to endoplasmic reticulum stress.3.IL13Rα1 inhibits the viability of rheumatoid arthritis fibroblast-like synoviocytes(RA FLSss):Compared with the negative control siCtrl,when IL13Rα1 is silenced,the cell viability of RA FLSss is increased.This cell viability was decreased after Tm,Tg and CoCl2 treatment,while IL13Rα1 silencing partially restored cell viability.On the contrary,the cell viability of the cells with high expression of IL13Rα1 after Tm,Tg and CoC12 treatment was more obvious than that of the lentiviral transfection vector Lv-VCtrl.4.IL13Rα1 promotes the apoptosis of RA FLSss cells:The apoptosis of RA FLSss cells was further analyzed when the expression of IL13Rα1 was silenced or overexpressed.Compared with the control,the apoptotic proportion of cells decreased when IL13Rα1 expression was decreased,and the apoptotic proportion of cells increased when IL13Rα1 was overexpressed.Silencing IL13Rα1 can inhibit the apoptosis of cells due to CoCl2,Tm or Tg treatment,and overexpression of IL13Rα1 can promote apoptosis more obviously.Western Blot analysis showed that the Bax/Bcl-2 ratio in RA FLSss cells was significantly decreased when IL13Rα1 was knocked down,while the Bax/Bcl-2 ratio was significantly increased when IL13Rα1 was overexpressed.In the presence of its ligand IL-13,the rate of apoptosis was slightly increased.This increase in apoptosis was more pronounced when IL-13Rα1 was overexpressed and IL-13 stimulated RA FLSs,accompanied by an increase in the Bax/Bcl-2 ratio.However,no apparent increase in apoptosis was observed when IL13Rα1 was overexpressed in osteoarthritic fibroblast-like synoviocytes.5.IL13Rα1 promotes the apoptosis of RA FLSss by activating STAT6 to upregulate Trail:when IL13Rα1 is silenced,the expression of Trail mRNA in RA FLSss is decreased,and the expression of Trail mRNA is increased when IL13Rα1 is overexpressed.Still exist.The phosphorylation level of STAT6 was increased in RA FLSss when IL13Rα1 was overexpressed,and this increase was still evident when RA FLSss were stimulated with Tm or CoC12 simultaneously.After treatment of Lv-IL13Rα1-or Lv-VCtrl-transfected FLS cells with the STAT6 inhibitor AS1517499,the inhibitor treatment attenuated the increase in Trail caused by IL13Rα1 overexpression.In addition,the increase in apoptosis rate and Bax/Bcl ratio could be partially alleviated by AS1517499 treatment.The results showed that IL13Rα1 regulates apoptosis through STAT6-induced Trail expression.6.IL13Rα1 interacts with DR5 protein and stabilizes DR5 protein:Based on the identification of a series of proteins that may interact with IL13Rα1 by GST-pull down and mass spectrometry,we preliminarily determine the DR5 protein with potential interaction with it.Co-IP experiments showed that IL13Rα1 and DR5 interacted in RA FLSs,DR5 was present in the immunoprecipitates of IL13Rα1 antibody,and IL13Rα1 was also present in the immunoprecipitates of DR5,suggesting that there was an interaction between the two.And hypoxia and Tm treatment can increase this interaction,and confirm the co-localization of IL13Rα and DR5.The results further showed that high expression of IL13Rα1 could up-regulate the expression of DR5,while silencing IL13Rα1 down-regulated the expression level of DR5 protein in RA FLSs.Compared with the control,DR5 protein was degraded more slowly in RA FLSs overexpressing IL13Rα1 and faster in RA FLSs underexpressing IL13Rα1.7.DR5 is involved in IL13Rα1-induced apoptosis in RA FLSss:To further clarify the pro-apoptotic effect of DR5 involved in IL13Rα1,DR5-siRNA was transfected into IL13Rα1-overexpressing RA FLSs,and the silencing of DR5 was confirmed by Western blotting efficiency.The results showed that DR5-siRNA could reduce the apoptosis rate caused by IL13Rα1 overexpression.Therefore,IL13Rαl interacts with DR5 and enhances DR5 stability.DR5 is involved in the pro-apoptotic effect of IL13Rα1.8.IL13Rα1 can alleviate the symptoms of RA mouse animal model:after packaging the IL13Rα1 virus Lv-IL13Rα1 and the control virus Lv-Ctrl,we constructed a collagen-induced mouse rheumatoid arthritis model(CIA)Mice were treated with intra-articular injection of Lv-IL13Rα1 and Lv-Ctrl.21 days after injection,Western blot analysis of the expressions of DR5,Trail,Bax,Bcl-2,IL13Rα1 and GFP,etc.,found that in addition to the increase of IL13Rα1,DR5 and Trail,the Bax/Bcl ratio also increased in Lv-IL13Rα1-injected synovial tissue.A significant increase.Compared with the control group,the arthritis score and knee joint thickness were significantly improved in the intra-articular injection group of LvIL13Rα1.Histological analysis of joints showed that injection of Lv-IL13Rα1 reduced the degree of synovial hyperplasia,inflammation,cartilage destruction and bone erosion.At the same time,it was confirmed that the apoptosis of Lv-IL13Rα1 injection group was significantly increased in fresh FLS cells isolated from the CIA model.Conclusions:Together,The relatively low expression level of IL13Rα1 under normal steady-state conditions is one of the reasons for the apoptosis resistance of RA FLSss to ER stress.IL13Rα1 can promote the apoptosis of rheumatoid arthritis fibroblast-like synoviocytes by activating STAT6 to activate the Trail-DR5 signaling pathway.Targeting IL13Rα1 may become an innovative approach and strategy for RA treatment.