Mechanism Study Of Syndecan-4 On Fibroblast-like Synoviocytes Of Rheumatoid Arthritis

Objective: Rheumatoid arthritis(RA)is a chronic,systemic autoimmune disease of unknown etiology.The main pathological changes of RA are mainly chronic and persistent synovial inflammation of the symmetrical multiple joints throughout the body,synovial tissue hyperplasia,and destruction of articular cartilage.In the late stage,joint stiffness or deformity can lead to loss of joint function.Patients with a long course of RA may also be associated with involvement of the circulatory,respiratory and urinary systems.Due to the long course,recurrence and incurability,RA has a high disability rate in the late stage,which has brought heavy burden to both patients and society.Although the research on the molecular mechanism of RA pathology has made great progress,and a series of bio-targeted drugs had been developed to inhibit the progress of RA to a certain extent,our current treatments still cannot cure RA.Therefore,it is still the research hotspot and direction of today’s scholars to deeply explore the molecular mechanisms of RA pathology and to screen the new drug targets.The maintenance of microenvironment in the joint mainly depends on the synovial tissue.And the pathological changes of synovial tissue are considered the initiating factors for the development of RA.Fibroblast-like synoviocytes(FLSs)are the main cell types of synovium tissue.Therefore,the pathological changes or abnormal biological behaviors of FLSs are the main causes of synovium inflammation and pathological hyperplasia,mainly manifested as persistent inflammation,continuous proliferation and apoptosis inhibition.At the same time,inflammatory factors in the joint microenvironment also promote the proliferation and apoptosis inhibition of FLSs.The proliferation of the inflammatory FLSs will further increase the levels of inflammatory factors in the joint microenvironment,forming a vicious cycle of inflammation-proliferationinflammation.Therefore,a systematic study of FLSs inflammation-related molecular mechanisms will be of great significance for elucidating the pathogenesis of RA and the development of new drugs in the future.As a cell membrane protein,syndecan-4(SDC4)is a receptor for a variety of cytokines or chemokines,and can regulate cell functions by mediating a series of intracellular signal transductions.According to reports,the abnormal expression of SDC4 appears in many inflammatory diseases and could participate in the regulation of the body’s immune system.Therefore,we speculated that SDC4 was abnormally expressed in rheumatoid arthritis fibroblast synovial cells(RA-FLSs)and could regulate the inflammation level of RA-FLSs through its downstream signal transduction.Method:(1)Gene chip datasets GSE55457,GSE55584,and GSE55235 were downloaded from the Gene Expression Omnibus(GEO)database,including33 RA and 20 normal synovial membrane samples.After normalizing the data,the differently expressed genes(DEGs)were screened.The key gene and signal pathway were screened with GO and KEGG signaling pathway enrichment analysis and protein network interaction analysis.(2)Synovial membranes from RA and knee trauma patients were collected,respectively.And the human primary FLSs were extracted by mechanical crushing.The expression of FLSs-specific protein Vimentin was detected by immunofluorescence.Hematoxylin-eosin staining(HE)experiment observed the morphology of FLSs.Quantitative Real-time PCR(qRT-PCR)was performed to verify the expression of part DEGs(CXCL9,CCR5,CCL18,CSF1 R,MMP1)and key gene(SDC4)in both groups.SDC4 shRNA plasmid vector was constructed and transfected into the third generation RA-FLSs to silence SDC4.qRT-PCR and Western-Blot(WB)experiments to detect the transfection efficiency.Enzymelinked immunosorbent assay(ELISA)experiment was used to detect the inflammatory factors IL-1β,IL-6 and TNF-α levels in RA-FLSs infected with plasmid.RA-FLSs apoptosis rate after infection was examined by flow cytometry and the apoptosis-related proteins like P53,cleaved caspase-3,Bcl-2 and Bax were detected with WB.(3)The SDC4 shRNA plasmid vector was constructed and transfected with the third generation RA-FLSs to silence SDC4.The ROS level of RA-FLSs after infection was detected by flow cytometry and the levels of oxidative stress related proteins eNOS and Nrf2 of RA-FLSs were detected by WB.Results:(1)Based on the threshold of Fold Change(FC)≥2,P<0.05,there were a total of 207 down-regulated genes and 329 up-regulated genes between RA and normal synovial membranes.GO enrichment analysis showed that the DEGs were mainly involved in the biological processes such as plasma membrane,immune response and chemokine activation.KEGG signal pathways enrichment results suggested that the DEGs were mainly enriched in related pathways such as cytokine receptor interaction.The results of protein network interactions revealed that membrane protein SDC4 was the key gene in the DEGs network.With the KEGG website prediction,we found that SDC4 could regulate the cellular inflammation by mediating oxidative stress-related pathways.(2)FLSs were successfully isolated from human synovial tissues,and cell identification was performed in the third-passage FLSs.Immunofluorescence results showed that Vimentin protein was positive in both groups.HE staining showed that the cells were long spindle shaped with large and oval nuclei.The cells were arranged in a polar and radial pattern.qRT-PCR results showed that the expression levels of CXCL9,CCR5,CCL18,CSF1 R,MMP1 and key genes SDC4 were significantly higher in the RA than in the normal group,which verified the reliability of the bioinformatic analysis results.After the RA-FLSs being transfected with SDC4 shRNA plasmid and control plasmid,the experiment was divided into four groups: Normal group(Normal),RA-FLSs group(RA),RAFLSs transfected with control plasmid group(RA+sh NC),RA-FLSs transfected with SDC4 shRNA plasmid group(RA+shSDC4).48 hours after transfection,the ELISA test results showed that the levels of inflammatory factors IL-1β,IL-6 and TNF-α in RA+shSDC4 group were significantly lower than those in the RA group.Flow cytometry apoptosis detection showed the apoptosis level of RA+shSDC4 group was significantly increased compared with RA group.The results of WB indicated that the expression of pro-apoptotic proteins P53,cleaved caspase-3 and Bax in RA+shSDC4 group were significantly increased compared with RA group,while the expression of anti-apoptotic protein Bcl2 in RA+shSDC4 group was significantly decreased compared with RA group.(4)Similarly,the ROS level in RA+shSDC4 group was significantly lower than that in RA group.WB results indicated that the expression level of eNOS protein in RA+shSDC4 group was significantly lower than that in RA group,while the expression level of antioxidant protein Nrf2 in RA+shSDC4 group was significantly higher than that in RA group.Conclusion: This subject verified that SDC4 as a cell membrane protein was significantly overexpressed in RA-FLSs through bioinformatics technology and cell experiments.Abnormally overexpressed SDC4 could increase the inflammation level of RA-FLSs and inhibit the apoptosis of RA-FLSs.At the same time,SDC4 can regulate the inflammation and apoptosis levels of RA-FLSs by mediating the oxidative stress signaling pathway.Inhibiting the expression of SDC4 not only reduces the level of oxidative stress,but also alleviates the inflammatory level of RA-FLSs and promotes its apoptosis.Therefore,SDC4 might be a new direction for basic research of RA or a new target for future drug design.