The Activation Of Synovial Fibroblasts Regulated By Notch Signal In Rheumatoid Arthritis

Rheumatoid arthritis(RA)is an autoimmune disease characterized by the proliferation of synovial tissue and the formation of pannus,leading to the destruction of cartilage and ossification and the infiltration of inflammatory cells.These pathological features are all related to the activation of fibroblast-like synoviocytes(FLS).The current antibody-based drugs in the market use inflammatory factors as drug targets;however,they only relieve symptoms without treating the root cause.There is no hope for complete cure,and 30%-40% of patients are resistant to previous therapies.Accumulating evidence suggests that the future of RA treatment lies in targeting synovial tissue,especially the matrix microenvironment.Therefore,it is particularly important to study the molecular mechanism of RA-FLS activation.In this study,we investigated whether hypoxic microenvironment is associated with RA-FLS activation.The main research content of this paper includes the following three parts.Part 1: The expression of Notch-1 and Notch-3 in RA synovium and RA-FLS.This part first comprised obtaining the synovial tissue of RA patients for investigation.Immunohistochemistry and fluorescence microscopy were used to detect the expression of Notch signaling protein.The active fragment of Notch-1(N1ICD)and the active fragment of Notch-3(N3ICD)were mainly expressed in synovial tissue.N1 ICD was mainly expressed in synovial tissue in the lining layer and around the pannus,while N3 ICD was mainly expressed in the sublining layer and around the pannus.An in vitro study using primary RA-FLS revealed an increased expression of Notch-1 and Notch-3 in the hypoxic environment,which could be inhibited by the HIF-1? small interfering(siHIF-1?)RNA.Moreover,chromosome immunoprecipitation assay showed that the expression of HIF-1? bound to the promoter regions of Notch-1and Notch-3 genes increased significantly under hypoxic conditions,indicating that HIF-1? directly regulates the expression of Notch-1 and Notch-3.In addition,the common ligands of the Notch signaling pathway DLL4 and Jagged1 and their downstream signaling molecules Hes1 and Hey1 showed an increased expression in the hypoxic microenvironment.These data indicate that Notch-1 and Notch-3 signaling molecules are activated in the RA synovial tissue and RA-FLS under hypoxic conditions.Part 2: RA-FLS activation via Notch-1 and Notch-3 signaling in a hypoxic microenvironment.Cartilage invasion is an important feature of RA pathology.The invasion mainly involves the angiogenesis,invasion,and migration of RA-FLS.Primary RA-FLS and vascular endothelial cells(HUVECs)were used for this study.Notch-1 or Notch-3expression was inhibited using small interfering RNA and N1 ICD or N3 ICD was overexpressed using an adenovirus vector.First,in order to investigate the effect of RA-FLS on angiogenesis in a hypoxic environment,RA-FLS and HUVECs were co-cultured in Matrigel.The formation of HUVEC tube network can be promoted in a hypoxic environment.While siNotch-1 and siNotch-3 inhibited the formation of HUVEC tube network,N1 ICD and N3 ICD overexpression promoted its formation in RA-FLS.In order to further verify the molecular mechanism of RA-FLS in promoting angiogenesis,we detected the expression of VEGF in RA-FLS under hypoxic conditions using western blotting(WB).VEGF expression increased under hypoxic conditions and could be significantly inhibited under the interference of siNotch-1 and siNotch-3.However,N1 ICD and N3 ICD overexpression in normoxia promoted VEGF expression.In order to detect the invasion of RA-FLS in a hypoxic environment,we performed invasion assays using Transwell.RA-FLS invasion increased under hypoxic conditions,which could be inhibited by siNotch-1 and siNotch-3.However,N1 ICD and N3 ICD overexpression significantly increased the invasion of RA-FLS.In order to further investigate the molecular mechanism of invasion,we used WB to detect the expression of matrix metalloproteinase 2(MMP2),MMP3,MMP9,and MMP13.The expression of MMP2,MMP3,MMP9,and MMP13 increased in a hypoxic environment.siNotch-1 inhibited the expression of MMP2 and MMP9,while N1 ICD overexpression promoted their expression in a hypoxic environment.siNotch-3 inhibited the expression of MMP3 and MMP13,while N3 ICD overexpression promoted the expression of MMP3 and MMP13 in a hypoxic environment.Therefore,Notch-1 and Notch-3regulate the expression of different MMPs in a hypoxic environment.In order to investigate the migration of RA-FLS in a hypoxic environment,we used a live cell imaging.Hypoxia accelerated the migration rate of RA-FLS,while siNotch-1 inhibited their migration.N1 ICD overexpression significantly increased the migration rate of RA-FLS.In tumor cells,hypoxia-induced cell migration first occurs through epithelial-mesenchymal transition.In order to investigate whether RA-FLS migrates under hypoxic conditions through epithelial-mesenchymal transition,we determined the expression of the epithelial-mesenchymal transition markers Snail,Vimentin,and E-cadherin in RA-FLS.Hypoxia induced the expression of Snail and Vimentin,while inhibited the expression of E-cadherin in RA-FLS.However,these trends could be reversed by siNotch-1.In addition,N1 ICD overexpression in RA-FLS significantly upregulated the expression of Snail and Vimentin and downregulated the expression of E-cadherin.These results indicate that hypoxia-induced migration occurs via the Notch-1 signaling pathway.The hyperplasia of synovial tissue is mainly related to RA-FLS proliferation,which is associated with RA-FLS apoptosis and autophagy.In order to investigate the molecular mechanism of RA-FLS apoptosis under the hypoxic environment,we used an apoptosis detection kit.Hypoxia could inhibit RA-FLS apoptosis,while siNotch-3could promote apoptosis.N3 ICD overexpression promoted RA-FLS apoptosis.Further studies on the molecular mechanism of apoptosis showed that the expression of cleaved caspase3 and cleaved PARP decreased in a hypoxic environment,while siNotch-3 could promote their expression.N3 ICD overexpression can inhibit the expression of cleaved caspase3 and cleaved PARP.In order to investigate the molecular mechanism of RA-FLS autophagy under a hypoxic environment,we used an autophagy kit to detect the formation of RA-FLS autophagosomes under hypoxic conditions.Hypoxia could promote the formation of RA-FLS autophagosomes,while siNotch-3 inhibited their production.Similarly,N3 ICD overexpression promoted the production of autophagosomes.In order to further study the molecular mechanism of RA-FLS autophagy,we determined the expression level of the autophagy marker protein LC-3B.Hypoxia promoted LC-3B expression,which was inhibited by siNotch-3.Similarly,N3 ICD overexpression also promoted LC-3B expression.These results show that RA-FLS apoptosis and autophagy are regulated by Notch-3.Part 3: Role of Notch signaling inhibitors in the treatment of collagen-induced arthritis in rats.This part of the research mainly used collagen-induced arthritis rats(CIA)and applied imaging and histology tests to verify the efficacy of a Notch signaling inhibitor(LY411575).Histological and imaging results showed that the joints of rats were significantly improved 15 days and 28 days after the administration of LY411575(5mg/kg and 10 mg/kg,respectively).Serum levels of the inflammatory factors TNF-?,IL-6,and VEGF decreased significantly after 15 days and 28 days of treatment.In summary,this study provides evidence for the functional relationship between HIF-1? and Notch-1 and Notch-3 signaling in the regulation of the RA-FLS activation mechanism in RA.In the hypoxic microenvironment,Notch-1 and Notch-3 exert an independent as well as a combined effect on the activation of RA-FLS.They jointly regulate angiogenesis and invasion;while Notch-1 regulates the migration and interstitial transformation of RA-FLS,Notch-3 regulates the apoptosis and autophagy of RA-FLS,further confirming that Notch signals are involved in the pathogenesis of RA.Furthermore,the CIA animal model confirmed the feasibility of Notch as a drug target for RA treatment.