| BackgroundRheumatoid arthritis(RA)is a chronic systemic autoimmune disease characterized by symmetrical,erosive and destructive arthritis.Epidemiological data suggests that it is a result of the complex interaction among gene,environment,hormone,and the immune systems.Alteration in the biological characteristics and functional abnormalities of joint fibroblast-1ike synoviocyte(FLS)in RA patients are considered to be closely related to the pathogenesis of RA.The molecular mechanisms that lead to the "tumor-like proliferation" phenotype of RA-FLS are still poorly understood.It is speculated these features are imprinted upon onset of RA which is attribute to pathways activated by subsequently constant exposure of synovial cells to a complex inflammatory cytokine and cell-cell milieu.The effector molecules in this inflammatory environment include not only the widely recognized serum biomarkers such as RF and CCP antibodies,but also some new biomarkers discovered in the latest research,such as exosome which is also considered to have potential clinical diagnosis and treatment effects.Our previous analysis of mRNAdifferences in serum exosomes of 4 rheumatoid arthritis patients and 4 healthy volunteers have found a variety of significantly different mRNAs,including those that have been increasingly drawn attention in autoimmune diseases in recent years.A variety of molecules in the Wnt signaling pathway are valued,such as Dvl3,a subtype of Dvl that plays a role as a branchpoint in the Wnt canonical and non-canonical signaling pathways.By two decades of exploration,significant advances have been made in uncovering the mysterious role and the significance of being "activated" of Dvl including Dvl1、Dvl2and Dvl3 in mounts of respects including participation in the various developmental processes of cells in which were regarded as the key integrator of a variety of complex signals.Here,combined with one of the results of our previous research that Dvl3 mRNA is highly expressed in serum exosomes of RA patients,we speculate that Dvl3 may hold great promise in comprehensive grasp of the pathogenesis.Therefore,this study was performed to explore the expression pattern of Dvl3 in RA and investigate the function and mechanism of Dvl3 mRNA in RA by exosome intervention which will facilitate a better understanding of the pathogenesis of RA and the discovery of novel therapeutic targets in the future.ObjectivesPart 1: To clarify the expression pattern of Dvl3 in the synovial tissue and cells of RA patients and the synovial tissue of the collagen-induced arthritis mouse model(CIA).Part 2: To investigate the effect of exosome Dvl3 mRNA on the proliferation,apoptosis,migration,invasion and inflammatory response of fibroblast-like synovial cells in vitro.Part 3: To investigate the effects of exosome Dvl3 mRNA on the disease severity,pain degree,synovial inflammation and apoptosis,cartilage and bone destruction,and serum inflammatory factor levels in CIA models in vivo.Part 4: To explore the possible downstream mechanism of Dvl3 mRNA in exhibiting these effects.MethodsPart 1:(1)The knee joint synovial tissues of 6 patients with RA and Trauma respectively was collected to compare the pathology of the synovial tissues of the two groups by HE staining;(2)The expression level of Dvl3 was examined by immunohistochemistry(IF),WB and q PCR respectively in the synovial tissues of the two groups of patients;(3)Primary fibroblast-like synovial cells were harvested from the synovial tissues,and the expression levels of Dvl3 were compared by cellular IF,WB and q PCR;(4)CIA mouse model was constructed and the mouse knee joints were taken,fixed,embedded,and sectioned to further explore the expression levels of Dvl3 in the knee joints of disease model mice by immunohistochemistry(IHC),WB and q PCR.Part 2:(1)Dvl3 overexpression and interference lentivirus were constructed.Exploration and identification of the conditions of lentivirus infection to up-regulate or down-regulate Dvl3 in RA-FLS was performed;(2)We further explore the conditions at which exosomes obtained from the cell culturing supernatant by ultracentrifugation can stably alter the expression of Dvl3 level in the target RA-FLS,and the transfection effect was verified by q PCR and WB.(3)Tunel and flow cytometry detection were performed to determine the effect of different groups of exosomes on apoptosis after transfection of RA-FLS;CKK8 kit was used to determine cell viability;(4)Scratch test and Transwell migration test was performed to compare migration ability(5)Cell invasion ability was detected by Transwell invasion test and matrix metalloproteinases(MMPs)expression level;(6)Target cell RNA and supernatant was extracted and the cytokine expression levels was examined by q PCR and enzyme-linked immunosorbent assay(ELISA).Part 3:(1)The acquisition of exosomes was as same as that in part2.CIA mouse model was constructed,and exosomes were injected into the joint cavity of the mice once a week according to the pre-grouping;(2)The mouse foot is evaluated from the day after the second immunization.Mice were scored for disease severity by the degree of paw swelling;(3)The paw mechanical pain score was performed every four days and recorded the day after the second immunization;(4)Mice were sacrificed by cervical dislocation after the scoring were completed,and mice knee joints were taken,fixed,decalcified,and embedded in sections to evaluate the pathological changes of mouse knee joints by HE staining;(5)Synovial cell apoptosis was detected by Tunel staining;(6)Safranin O-fast green staining was used to evaluated synovium Cartilage destruction;(7)Blood from portal vein was collected and the expression level of inflammatory cytokines in serum was validated by ELISA kit.Part 4:(1)RNA from RA-FLS processed by exosomes from different groups was extracted,and the key signal molecules of Wnt signaling pathway was detected by q PCR to screen the possible downstream pathways of Dvl3;(2)WB verification;(3)Dvl3overexpression and interference plasmid was constructed followed by verification of transfection in RA-FLS.Dual luciferase reporter gene plasmid co-transfection experiment was performed to verify the activity of Dvl3 on TCF1 and ROCK2 promoter.ResultsPart 1:(1)A total of 6 cases of RA and traumatic knee joint synovium respectively were collected from which the primary FLS was successfully extracted for culturing.The specific surface marker Vimentin protein and the negative protein CD68 were used for cellular immunity Fluorescence was identified.(2)HE staining scores of the synovial membrane showed that compared with the trauma group,the synovial membrane of the RA group had significantly higher scores in terms of synovial hyperplasia,inflammatory cell infiltration and capillary hyperplasia.(3)The results of synovial immunohistochemistry(IHC),WB and q PCR showed that the expression level of Dvl3 in the synovium of RA patients was significantly increased.(4)The results of cellular immunofluorescence(IF),WB and q PCR showed that the expression of Dvl3 in RA-FLS was also significantly higher than that in the trauma group.(5)IHC,WB and q PCR of the knee joints of CIA mice indicated that the expression of Dvl3 in inflammatory arthritis was significantly up-regulated.Part 2:(1)The results of CKK8 assay showed that exosomes derived from the overexpression(OE)group can significantly promote cell proliferation activity,while exosomes derived from the interference group significantly inhibited FLS proliferation,but this effect was lagging behind the OE group.(2)The ratio of RA-FLS apoptosis was measured by Tunel method and flow cytometry.The results showed that the overexpression experimental group could significantly down-regulate cell apoptosis compared with the control group,while the interference group had the opposite effect of promoting cell apoptosis.(3)Migration and invasion experiments showed that overexpression of Dvl3 by exosomes can promote cell migration and invasion,and the expression level of matrix metalloproteinases was also increased.In the interference group,almost the opposite effect is observed.(4)Detection of cytokine mRNA levels by q PCR showed that Dvl3 overexpression exosomes significantly promoted the expression levels of TNF-α,IL-1β,IL-17,IL-21 and angiogenesis-related cytokines(VEGF),while in the interfere group,significant down-regulation of IL-17 and IL-21 were observed.These results were further verified by the ELISA kit to detect the level of each cytokine in the cell supernatant.Part 3:(1)The CIA mouse model was successfully constructed through the immunization of collagen.The disease score results showed that the arthritis index of mice in the overexpression exosome injection group increased more rapidly than the control group,and the peak of disease score was higher than that of the control group.There was a significant difference in the elevated levels of the two groups,and the mice in the interference exosome injection group showed a significant relief across the disease.(2)The evaluation results of the mechanical pain threshold in mice showed that the pain threshold of mice in the interference group decreased significantly retard compared with the control group,while the overexpression group showed a rapid decrease in pain threshold,which was significantly different from the control group.(3)After scoring,samples were taken from the synovium of the mice knee joint.WB and q PCR tests showed that the injection of exosomes successfully altered the expression level of Dvl3 in the mouse knee joint.(4)Obvious synovial hyperplasia accompanied by a large number of inflammatory cell infiltration was observed under the microscope in the HE staining of the mouse knee joint from Lv-Dvl3 mRNA exosome injection group,and the pathological score was significantly higher than that of the control group;While the Dvl3 interference exosomes injection group effectively alleviated the inflammation of the joint,and the score was significantly lower than the control group.(5)The results of safranine O-fast green staining indicated that the overexpression Lv-Dvl3 mRNA exosomes intervention group had a significantly higher score than the control group,while the interference experiment group tended to show a protective effect on articular cartilage.The band is slightly wider than the control group,but the difference in scores between the two groups has no yet statistical significance.The detection of matrix metalloproteinases in mouse knee joints also further confirms the above results.Overexpression of Dvl3 exosomes may have the effect of up-regulating the expression of MMPs Promote erosion and destruction of joint synovium.(6)Tunel experiment indicated that the Sh-Dvl3 mRNA exosome injection group showed strong pro-apoptosis results,while the overexpression group showed a significant inhibitory effect on cell apoptosis.(7)The results of ELISA detected of serum inflammatory cytokines in mice showed that the increased expression of Dvl3 in the knee joints can significantly up-regulate the levels of TNF-α,IL-17 and IL-21 in the serum,while the interference group remarkable inhibited TNF-α and IL-17 expression.Part 4:(1)The mRNA levels of key molecules downstream of the Wnt signal in RA-FLS after exosome intervention was detected by q PCR.The results showed that,compared with the control group,the overexpression of Dvl3 by exosomes was accompanied by a significant increase in β-catenin and ROCK2,while no significant changes were observed in the key molecular of Rac pathway of JNK and the protein involved in intercellular connectivity of PRK.However,in the Dvl3 interference exosomes group,β-catenin and Rho A were significantly decreased compared with the control group.(2)WB was performed to further verify the expression levels of β-catenin and Rho A which turned out that both molecules are positively correlated with Dvl3.(3)WB and q PCR results successfully verified the transfection effect of Dvl3 plasmid.(4)The dual luciferase reporter gene detection system was used to examine the role of Dvl3 in the two Wnt pathways.The results suggested that Dvl3 was able to activate both classic and non-canonical Wnt signaling pathways.ConclusionThis study has conducted a detailed investigation on the expression of Dvl3 and the function of exosome Dvl3 mRNA through four parts: clinical and animal tissue sample expression,primary cell culture experimental research in vitro,animal models research in vivo,and specific molecular mechanisms.The high expression of Dvl3 in RA patients and animal models of inflammatory arthritis,and its over-expression by exosomes intervention may promote cell proliferation,inhibit apoptosis,promote migration,invasion and secretion of pro-inflammatory cytokines,and then participate in mediating arthritis progresses has been fully demonstrated.Further mechanism exploration has shown that the downstream pathways that exerted the above effects may attribute to the activation of both classic and non-classical of Wnt pathways,including activation ofβ-catenin/TCF and Rho A/ROCK pathways at the same time,the protective results obtained by Dvl3 interference exosomes group against inflammatory arthritis provide the possibilities to develop novel strategies aimed at deletion of exosome Dvl3 as a potential target for the treatment of RA. |
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