| BackgroundRheumatoid arthritis(RA)is a chronic autoimmune disease characterized by aggressive arthritis of unknown etiology.It happens at any age.It is characterized by synovial hyperplasia and inflammatory cell filtration,leading to chronic inflammation of the joints and subsequent erosion of the cartilage and the bone.Fibroblast-like synoviocytes(FLS)in RA is a specialized cell type located in synovial joints,which increase in number and become a prominent component of the destructive pannus.Furthermore,RA-FLS play crucial roles in the damage,destruction and deformation of cartilage and joints.The activity of RA-FLS is regulated by variety of signaling pathways.In recent years,several lines of evidence have shown that the Wnt signaling pathway significantly participates in the RA pathogenesis,and revealed its important role in development of RA.Zinc and Ring Finger 3(ZNRF3)has found to be a kind of negative feedback regulator of Wnt signaling.ZNRF3 inhibit Wnt/?-catenin signaling through promoting ubiquitination and subsequent internalization and degradation of Wnt receptor FZD.However,the studies of ZNRF3 are mostly focused on tumors,and there are no related studies in RA.Therefore,this study focused on the expression of ZNRF3 in RA synovial tissue and RA-FLS,its effects on biological characteristics of RA-FLS and CIA mice.Finally,we explore the relevant molecular mechanism.ObjectivesPart1: To investigate the expression of ZNRF3 in RA synovial tissue and RA-FLS.Part2: To study the effects of ZNRF3 on the proliferation,apoptosis,cycle,migration,invasion and inflammatory in RA-FLS.Part3: To study the effects of ZNRF3 on joint swelling,synovial inflammation,apoptosis of synovial cells,destruction of cartilage and bone,and the secretion of inflammatory cytokine in collagen-induced arthritis mouse.Part4: To explore the mechanism of ZNRF3 in RA.MethodsPart1:(1)FLS were isolated by type 2 collagenase from synovial tissue of RA,OAand Trauma patients.(2)The synovial tissues of the three groups were compared by HE staining.(3)The expression of ZNRF3 were detected of the three groups by immunohistochemical method.(4)The cells were identified by flow cytometry and immunofluorescence.(5)The expression of ZNRF3 were observed by immunofluorescence in FLS among the three group.(6)The expressions of ZNRF3 in synovial tissue and FLS of RA,OA and trauma groups were compared by qPCR and Western Blot.Part2:(1)We constructed humanized ZNRF3 knockdown and overexpression lentivirus vector,and packed them into lentivirus.(2)The RA-FLS were infected by lentivirus and then selected by puromycin.(3)The transfection effect was measured by qPCR and Western Blot.(4)The proliferation of RA-FLS was compared after infection by the CCK8 assay.(5)Flow cytometry was used to detect the apoptosis rate of RA-FLS after infection,and Western blot was used to detect the expression of apoptosis-related proteins.(6)The cell cycle of RA-FLS after infection was detected by flow cytometry.(7)The ability of migration was determined by wound healing assay and Transwell migration assay.(8)The ability of invasion was compared by Transwell invasion assay.(9)The expression of IL-1?,IL-6 and IL-8 were measured by qRT-PCR and ELSIA.Part3:(1)We constructed murine ZNRF3 knockdown and overexpression lentivirus vector,and packed them into lentivirus.(2)Mouse model of collagen-induced arthritis were constructed.(3)The lentivirus was injected into the knee joint of CIA mouse,and the effects were detected by the vivo imaging cytometry.(4)Clinical onset and progression of arthritis was scored.(5)Mice were sacrificed after eyeball extraction.(6)The expression of TNF-?,IL-1?,and IL-6 were measured by multi-analyte flow assay kit.(7)The knee-joint of mice were collected,fixed,decalcified and stained by HE staining to observe and scored the pathological changes.(8)The damage of knee cartilage in mice was observed and sored by Saffron O-fast green assay.(9)TUNEL assay was used to detect the apoptosis of synovial cells.Part4:(1)The related proteins of Wnt/?-catenin signaling pathway and NF-?B signaling pathway were detected by Western blot.(2)ZNRF3 overexpressed plasmid and siRNA interference sequences were constructed and then transfected by Lipo2000.The effects of transfection were detected by Western blot.(3)RA-FLS were transfected with luciferase reporter plasmid and ZNRF3 overexpressed plasmid or siRNA interference sequences.The luciferase activity was determined by a luciferase reporter assay kit.ResultsPart1:(1)FLS were successfully isolated and cultured.The cells of the third passage were identified by Flow cytometry and the results showed that the positive rates of CD90 and CD29 were above 90%.The immunofluorescence results showed that most of the cells were positive for Vimentin protein and negative for CD68.(2)The HE results of synovial tissue showed that the synovium was thicker and more inflammatory cell infiltration in RA patients than in OA and Trauma patients.(3)The results of immunohistochemistry,qPCR and Western blot have shown that the expression of ZNRF3 in synovial tissue of RA patients was significantly higher than that of Trauma patients,but the difference was not statistically significant compared with OA patients.(4)Immunofluorescence showed that ZNRF3 was localized in the cytoplasm and plasma membrane of RA-FLS.(5)The results of qPCR and Western blot have shown that the expression of ZNRF3 in RA-FLS was significantly higher compared with Trauma-FLS,but the difference was not statistically significant compared with OA-FLS.Part2:(1)When the MOI was 10 in Sh-ctr group and LV-ctr,30 in Sh-ZNRF3 group,and 40 in LV-ZNRF3 group,the infection efficiency can meet the experimental requirements.(2)Compared with the Sh-ctr group,the proliferation of RA-FLS in Sh-ZNRF3 was significantly inhibited.The cell activity of Sh-ZNRF3 group was further decreased after stimulated with TNF-?.Compared with LV-ctr,the proliferation of RA-FLS in LV-ZNRF3 was higher,and this effect remained in the presence of TNF-?.(3)The percentage of apoptotic cells measured by flow cytometry in Sh-ZNRF3 group was significantly higher than in Sh-ctr group,while the percentage of apoptotic cells in LV-ZNRF3 group was significantly lower than that in LV-ctr group.(4)The results of Western Blot showed that,compared with the blank control group and the Sh-ctr group,the expression of Bcl2 in the Sh-ZNRF3 group was significantly reduced,the expression of Bax was significantly increased,and the expression of cleaved caspase-3 and cleaved caspase-9 were significantly increased.Compared with the blank control group and LV-ctr group,the expression of Bcl2 in LV-ZNRF3 group was significantly increased,the expression of Bax was significantly decreased,and the expression of cleaved caspase-3and cleaved caspase-9 was significantly reduced.(5)Sh-ZNRF3 can block the G0/G1 phase of cell cycle in RA-FLS,while LV-ZNRF3 can promote the cell progression from G0/G1 phase to S phase in RA-FLS.(6)Compared with the Sh-ctr group,the migration rate of in the Sh-ZNRF3 group was significantly reduced.However,the migration rate inLV-ZNRF3 group was not significantly different from that in LV-ctr group.(7)The results of Transwell migration assay showed that the number of cells in the Sh-ZNRF3 group was significantly less than that in the Sh-ctr group.There was no significant difference between LV-ZNRF3 group and LV-ctr group.(8)The results of Transwell invasion assay indicated that the number of cells invaded in the Sh-ZNRF3 group was less than that in the Sh-ctr group.There was no statistically significant difference between LV-ZNRF3 group and LV-ctr group.(9)The results of qPCR and ELISA suggested that Sh-ZNRF3 could suppressed the expression of IL-1?,IL-6 and IL-8 in RA-FLS,while LV-ZNRF3 could increase the expression of IL-1?,IL-6 and IL-8 in RA-FLS.Part3:(1)The clinical arthritis score in the Sh-ctr group and Sh-ZNRF3 group was significantly higher than that in the normal control group.The clinical arthritis score in the Sh-ZNRF3 group was lower than the Sh-ctr group from the Day 35.There was no significant difference in the clinical arthritis score between the LV-ctr group and LV-ZNRF3 group,expect the Day 38.(2)The vivo imaging cytometry showed that the lentivirus injected into the knee joint was stably expressed in the mice.(3)The results of HE staining of knee joints showed that the scores of arthritis lesions in the Sh-ZNRF3 group was lower than that in the Sh-ctr group,while there was no significant difference between the LV-ZNRF3 group and LV-ctr group.(4)The plasma levels of TNF-?,IL-1?and IL-6 in Sh-ZNRF3 group were significantly lower than those in Sh-ctr group,while the plasma levels of TNF-?,IL-1? and IL-6 in LV-ZNRF3 group were significantly higher than those in LV-ctr group.(5)The cartilage erosion scores of Sh-ZNRF3 group was lower than Sh-ctr group,while there was no significant difference between LV-ZNRF3 group and LV-ctr group.(6)The effects of Sh-ZNRF3 and LV-ZNRF3 on the apoptosis of synovial cells is not significantly.Part4:(1)Compared with the Sh-ctr+TNF-? group,the expression of p-GSK3 was increased,but the expression of ?-catenin and c-myc was significantly decreased in the Sh-ZNRF3+TNF-? group.However,compared with LV-ctr+TNF-? group,the expression of p-GSK3 was decreased,but the expression of ?-catenin and c-myc was increased in LV-ZNRF3+TNF-? group.(2)The activity of TCF/ lef-1 reporter gene was detected by the Dual-luciferase reporter Assay.The result showed that the RLU1/RUL2 ratio of the Si-ctr+TNF-? group was significantly higher than the Si-ZNRF3 +TNF-? group.The RLU1/RUL2 ratio of Ctr+TNF-? group was significantly lower than ZNRF3+TNF-?group.(3)Western blot results showed that the expression of p-P65 and p-I?B weredecreased in Sh-ZNRF3+TNF-? group compared with Sh-ctr+TNF-? group.Compared with LV-ctr+TNF-? group,the expression of p-P65 and p-I?B in LV-ZNRF3+TNF-? group were increased.(4)The activity of NF/?B reporter gene was detected by the Dual-luciferase reporter assay.The results showed that the RLU1/RUL2 ratio of the Si-ctr+TNF-? group was significantly higher than the Si-ZNRF3+TNF-? group.The RLU1/RUL2 ratio of Ctr+TNF-? group was significantly lower than ZNRF3+TNF-?group.ConclusionThis study consists of four parts including clinical research,culture of primary cells,vivo study in mice,the exploration of mechanism.We found that the expression of ZNRF3 was higher in synovial tissue and FLS of RA patients.ZNRF3 could promote the proliferation and increase the production of inflammatory cytokines of both in vivo and in vitro,and aggravate the progress of the RA.To explore the underlying mechanism of ZNRF3,we found that the cross-talk between Wnt/?-catenin signaling pathway and the NF-?B signaling pathways may play an important role.ZNRF3 suppressed the phosphorylation of GSK3?and activated the NF-?B signaling pathway.The activated NF-?B signaling pathway increased the expression of IKK,and inhibited the degradation of ?-catenin,induced activation of ?-catenin/Tcf pathway.This provides a new potential target for RA treatment. |
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