Effects Of Dishevelled2 On Apoptosis And Inflammatory Cytokines In Rheumatoid Arthritis Fibroblast-like Synoviocytes Through Crosstalk With The NF-κB Pathway

The main pathological changes of rheumatoid arthritis(RA)is a synovitis,which characterized by a loss of synovial cell apoptosis,tumor-like growth and more secretion of inflammatory mediators.Synovitis can cause articular cartilage destruction and bone erosion,eventually leading to irreversible damage to the joints and thus disability.Fibroblast-like synoviocyte(FLS)is an important part of synovial membrane,and also is an important participant in the above pathological process.The activation of RA-FLS is associated with multiple signaling pathways,and the most classical signaling pathway is the NF-κB signaling pathway.In RA-FLS,the NF-κB pathway is activated and the expression of downstream target genes(such as cIAP1,cIAP2,IL-1,IL-6)increases the anti-apoptotic ability of RA-FLS as well as the inflammatory factors,adhesion molecules,and the secretion of matrix degrading enzymes.In addition to NF-κB signaling pathway,the role of Wnt signaling pathway in RA is also a hotspot in recent years.Evidence suggests that Wnt signaling pathway in RA is also actived,involved in RA-FLS activation.Disheveling(Dvl)is a key signaling protein molecule in the middle of the Wnt signaling pathway and is a very important and interesting molecule.Dvls has three isoforms(Dvl1,DVL2,Dvl3)and four highly conserved domains(DIX domain,PDZ domain,DEP domain,and DSV).Dvls is not only the splitter of a classic and non-classical Wnt path,but also with other signal path on the dozens of protein interaction,and it is also the Wnt pathway can access a variety of intersection with BMP,mTOR and so on.Although there are some studies on Dvl protein in the tumor,there are no studies on the expression and function of Dvl protein in RA.Therefore,this study focused on the expression of DVL2 in RA synovial tissue and RA-FLS,and its effect on RA-FLS apoptosis and inflammatory factor secretion and related molecular mechanisms.Ⅰ The expression of DVL2 is higher in RA synovial tissue and RA-FLSFirst,we collaborated with arthrodesis to obtain synovial tissue in patients with RA,osteoarthritis(OA),and normal trauma(Trauma).The expression of DVL2 protein was detected by immunohistochemistry(IHC)and Westblot.The results showed that although the expression of DVL2 protein was not significantly different between RA and OA,the expression of DVL2 protein in RA synovial tissue was significantly higher than that in Trauma patients’,which suggesting that DVL2 may participate in the proliferation of RA synovial through the Wnt pathway.In addition,FLS was also extracted from the synovial tissue and tested.The results showed that the expression of DVL2 in RA-FLS was higher than that in Trauma-FLS.Immunofluorescence assay of RA-FLS and IHC of synovial tissue showed that DVL2 was expressed in the cytoplasm and nucleus of FLS,suggesting that different cell localization of DVL2 might play different functions.Ⅱ DVL2 in vivo can promote RA-FLS apoptosis and inhibit the secretion of inflammatory factorsAfter knowing that DVL2 was highly expressed in RA synovial tissue and RA-FLS,in order to further study its function,we established the collagen-induced arthritis rat(CIA)model.And we then constructed humanized DVL2 overexpression lentivirus vector(LV-DVL2),packed into lentivirus and injected into the knee joint of CIA rats.After the rats overexpressing DVL2,synovium,cartilage,and changes in subchondral bone and articular synovial cell apoptosis and inflammatory cytokine secretion were detected.Interestingly,overexpressing DVL2 in knee of CIA rats not only did not further increase the synovial proliferation,but to a certain extent inhibited synovitis and the joint destruction.Histopathological examination of synovial membrane and total knee joint revealed that DVL2 could reduce synovitis in CIA rats.The expression of IL-1β,IL-6 and IL-8 in the knee joint of CIA rats was decreased by ELISA analysis of synovial fluid.Micro-CT test results show that DVL2 can reduce the destruction of subchondral bone in CIA rats.However,DVL2 did not significantly reduce the destruction of articular cartilage in CIA rats.Further TUNEL test results showed that DVL2 could significantly increase the apoptosis of synovial membrane of CIA rats,suggesting that DVL2 could regulate theapoptosis of synovial cells in vivo,except that it may be involved in the proliferation of RA synovium.Ⅲ DVL2 also promotes RA-FLS apoptosis and inhibits the secretion of inflammatory factors in vitroIn order to further study the function and possible mechanism of DVL2 in RA,we extracted human RA-FLS cells and overexpressed the DVL2 gene,and then performed high-throughput m RNA sequencing.The results showed that NF-κB pathway and its TNF pathway has been significantly reduced.Therefore,we observed the effect of DVL2 on RA-FLS cell activity,apoptosis,cycle and secretion of inflammatory factors after overexpression in RA-FLS.The results showed that the activity of DVL2-expressing RA-FLS cells in vitro was significantly decreased,which was more obvious after the addition of TNF-α.Furthermore,the apoptosis and cell cycle of RA-FLS were detected.The results showed that DVL2 could significantly increase the apoptosis rate of RA-FLS and cause some influence on the cell cycle.Detection of cell supernatant by ELISA showed that DVL2 reduced IL-1β,IL-6 and IL-8 secreted by RA-FLS.And this phenomenon is also become more obvious after the stimulation TNF-α also.These phenomena suggest that DVL2 in the inflammatory environment can offset a part of the inflammatory response,to a certain extent,to curb the infinite proliferation of RA-FLS.Ⅳ DVL2 affects RA-FLS apoptosis and secretion of inflammatory factors by inhibiting NF-κB pathwayCombined with the above results,we hypothesized that DVL2 can inhibit the proliferation of RA-FLS and the secretion of inflammatory factors is likely to inhibit NF-κB pathway and inhibit its gene.Therefore,we first demonstrated the differential gene of mRNA sequencing by RT-PCR.It was found that DVL2 could inhibit the expression of anti-apoptotic genes A20,GADD45β and cIAP2 in RA-FLS,but there were no obvious effect on cIAP1.When administered TNF-α stimulation,RT-PCR showed DVL2 play asignificant inhibitory effect on the expression of A20,cIAP1 and cIAP2,but the expression of GADD45β has no significant change.The expression of IL-6 was significantly down-regulated in RA-FLS When DVL2 is overexpressed,while the expression of IL-1β and IL-8 did not change significantly.After administration of TNF-α,DVL2 can inhibit the IL-1β,IL-6 and IL-8 gene expression in RA-FLS.Because these anti-apoptotic genes and inflammatory factor gene can be NF-κB pathway regulating,so we infer DVL2 is to achieve the above functions by inhibiting NF-κB pathway.So how does DVL2 inhibit the NF-κB pathway? First of all,we found that DVL2 in RA-FLS can inhibit the P65 into the nucleus through the Flowsight.Through the IP detection found DVL2 in RA-FLS can also interact with P65,ChIP detection revealed that DVL2 interacts with P65 to inhibit the binding of P65 to the target gene promoter,thereby reducing the expression of anti-apoptotic genes and inflammatory cytokines.In summary,from the collection of clinical synovial samples to the culture of primary cells,from in vivo animal experiments to in vitro cell experiments to genetically modified,the expression of DVL2 in the synovial membrane of RA patients and the effect of DVL2 on the proliferation of RA-FLS and the secretion of inflammatory factors in vitro were studied systematically in this study.In addition to observing the phenomenon,we also use mRNA-seq,IP,Flowsight,ChIP and other methods on the depth mechanism study of DVL2.It was found that DVL2 could promote RA-FLS apoptosis and inhibit the secretion of inflammatory factors,although DVL2 was highly expressed in RA synovial membrane and may be involved in tumor-like growth of synovial membrane.And DVL2 has been able to play the role of this double-edged sword,mainly on the one hand is the Wnt signal pathway in the important positive regulatory protein,on the other hand DVL2 can also affect the P65 into the nucleus and its downstream regulatory genes promoter binding to inhibit NF-κB pathway.The discovery of DVL2 multifunctional function providing important information for understanding the functional dialogue between Wnt and NF-κB pathways in RA,and also providing an idea for understanding the development of RA and finding new therapeutic targets.