| Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by joint inflammation,synovial hyperplasia,cartilage degeneration,bone erosion,and pannus,resulting in joint deformity and loss of function.In recent years,the role of synovial lesions in the pathogenesis of arthritis has been paid more and more attention while the systemic immunological mechanism of RA has been discussed.Synovial dysplasia is mainly related to abnormal proliferation of fibroblast-like synoviosytes(FLS)and abnormal secretion of inflammatory factors.Under inflammatory conditions,VEGF secreted by FLS can increase vascular permeability and promote the formation of new blood vessels,which transport inflammatory cells to the site of synovial inflammation and provide nutrients and oxygen to the synovial membrane,thus maintaining the state of chronic inflammation.Collagen triple helix repeat containing-1(CTHRC1)can repair vascular damage,reduce collagen deposition,promote cell migration,and participate in vascular remodeling and the formation of osteoblasts.In recent years,it has been reported that CTHRC1 is highly expressed in experimental arthritis and synovial tissues of RA in mice,and specifically expressed in the synovialmembrane and bone-pannus interface.The expression levels of WNT5 A and its receptor Frizzled 5(Fzd5)were higher in RA synovial tissue specimens,and CTHRC1,as a secretory regulator of WNT signaling,formed a complex with WNT-Fzd5-LRP5/6to activate extracellular regulated protein kinases(ERK).Phosphorylated ERK promotes the expression of hypoxia inducible factor-1(HIF-1?)by activating nuclear factor kappa-B(NF-?B),leading to high expression of VEGF and involvement in angiogenesis.Currently,the major therapeutic drugs for RA comprise steroidal anti-inflammatory drugs,non-steroidal anti-inflammatory drugs,disease-modifying drugs and biological agents.Methotrexate,as the preferred drug for the treatment of RA,has a clear therapeutic effect,but there are still adverse reactions such as liver damage,pulmonary lesions and bone marrow suppression,which are not conducive to long-term treatment.As a biological agent with obvious curative effect,etanercept can competitively block the binding of TNF-? to cell surface receptors,and inhibit the inflammatory process.However,there are still local adverse reactions in the skin,moreover,the treatment course is long and the cost is high,so etanercept is suitable for patients with refractory severe RA.Therefore,exploring new specific therapeutic targets is of great significance to further explore the pathogenesis of RA.Immunoglobulin D(Ig D)is an immunoglobulin discovered in 1965,comprising secreted Ig D(s Ig D)and membranigd(m Ig D).Although Ig D is extremely low in the body,it plays a crucial role in autoimmune diseases.The serum s Ig D level of patients with autoimmune diseases such as RA,systemic lupus erythematosus(SLE)and Sjogren's syndrome(SS)was higher,and the concentration of anti-Ig D autoantibody was elevated in many patients with RA.In the established Ig D transgenic mouse model,the mice were found to have skin ulcers and abnormal swelling of liver,spleen and kidney,which may be chiefly connected with the high expression of s Ig D in the serum.On account of the above findings,it is speculated that high levels of Ig D may lead to inflammation and immune tissue damage,so Ig D plays a vital role in autoimmunediseases.In 1980,immunoglobulin D receptor(Ig DR)was found on human peripheral blood T cells and non-T cells,and then Ig DR was also found on mouse T cells.SIg D takes an immunomodulatory role by specific binding with Ig DR.Our research group preliminary found that s Ig D and m Ig D levels in peripheral blood of RA patients were higher than those of healthy controls.Moreover,the high level of s Ig D in the serum of RA patients was positively correlated with the serum levels of human soluble nuclear factor ?B receptor activation factor ligand,rheumatoid factor and c-reactive protein,suggesting that s Ig D may be an effective biological targets for the treatment of RA.Based on the research background and results of Ig D,our research group constructed and purified Ig D-Fc-Ig fusion protein.The Ig D-Fc-Ig was synthesized by connecting the human Ig D-Fc segment and the human Ig G-Fc segment with Ig D/Ig DR as the target to specifically block the Ig D/Ig DR pathway and regulate the cellular function of Ig DR expression in order to treat RA.The previous study of the research group found that Ig D-Fc-Ig fusion protein could significantly inhibit the proliferation and differentiation of experimental arthritis T cells,and could play a therapeutic role in collagen induced arthritis(CIA)by regulating Ig D-Ig DR-Lck signaling pathway on T cells.In addition,specific membrane Ig DR was found on FLS in RA patients,and Ig D could promote FLS proliferation.Based on the above findings,the following hypothesis is proposed: Whether Ig D can bind to Ig DR on FLS and promote abnormal activation of FLS? Whether abnormal activation FLS is related to the activation of the WNT signaling pathway? Whether the Ig D-Fc-Ig can block the binding of Ig D and Ig DR to regulate the WNT signaling pathway and inhibit the abnormal activation of FLS?In this study,rat FLS and human rheumatoid arthritis synovial fibroblast cell line(MH7A)were used as the subjects,and a CIA model was established in male Wistar rats to observe whether Ig D could regulate the activation of WNT-Fzd5-CTHRC1-NF-?B signaling pathway by combining with Ig DR in FLS and participate insynovitis,to elucidate that Ig D-Fc-Ig fusion protein may improve the synovial inflammation by inhibiting the WNT-Fzd5-CTHRC1-NF-?B signaling pathway,thereby playing a therapeutic role in CIA.In vitro,MH7 A cells were selected to study whether Ig D could promote the activation of WNT-Fzd5-CTHRC1-NF-?B signaling pathway through Ig DR signaling and the effect of Ig D-Fc-Ig fusion protein on this pathway.This study provides experimental basis for further revealing the pathogenesis of RA and clarifying the functional characteristics and mechanism of Ig D-Fc-Ig fusion protein in the treatment of RA.Objective:To investigate whether Ig D can regulate the activation of WNT-Fzd5-CTHRC1-NF-?B signaling pathway in FLS and participate in synovitis by binding with Ig DR,and to clarify that Ig D-Fc-Ig fusion protein may play a therapeutic role on CIA in rats by influencing synovitis,and that Ig D-Fc-Ig fusion protein may improve synovitis by inhibiting WNT-Fzd5-CTHRC1-NF-?B signaling pathway.Methods:1.Wistar male rats were selected to establish CIA model.The rats were randomly divided into 6 groups: normal group,model group,Ig D-Fc-Ig(1 mg/kg)group,Ig D-Fc-Ig(3 mg/kg)group,Ig D-Fc-Ig(9 mg/kg)group and etanercept(3 mg/kg)group,with 8 rats in each group.The dosage was calculated after weighing,and the dosage was given in the way of the tail vein once every 3 days for a total of 6 weeks.Normal group and model group were given normal saline for parallel control according to the weight of the rats.2.The rats were weighed every three days,and the volume of swelling of the toes was measured three times and recorded.The mean was taken to reduce the error,while the rats were evaluated by a fixed experimenter for overall indicators(body score,arthritis index and joint swelling number)and recorded.The joint tissues were stained with HE,the pathological changes were observed and the relevant indexes were scored.3.The level of CTHRC1 and VEGF in synovial tissues was deteceted by immunohistochemistry.4.Ig DR expression on FLS of rats was detected by immunofluorescence.5.Expression of CTHRC1,Fzd5,p-p65 and P65 in FLS of rats was detected by western blot.6.The co-localization of CTHRC1 and Fzd5 in FLS of rats was detected by immunofluorescence.7.In vitro experiments: human rheumatoid arthritis synovial fibroblast cell line MH7 A was selected as the study object in vitro.Ig DR expression on MH7 A was detected by immunofluorescence8.The optimal concentration and time of WNT stimulating MH7 A proliferation were determined by CCK-8.9.Expression of CTHRC1,Fzd5,p-P65,P65 and VEGF in MH7 A was detected by western blot.10.The co-localization of CTHRC1 and Fzd5 on MH7 A was detected by immunofluorescence.11.Enzyme-linked immunosorbent assay(ELISA)was used to detect VEGF-A in supernatant cultured with MH7 A.Results1.Ig D-Fc-Ig fusion protein has therapeutic effect on CIA ratsAnalysis and evaluation of paw inflammation indicators in CIA rats at different inflammatory periods showed that Ig D-Fc-Ig fusion protein significantly reduced arthritis index,the number of paw swelling,paw swelling volume and systemic score inCIA rats.2.Ig D-Fc-Ig fusion protein improved the pathological changes of CIA rats joint tissuesPathological changes of CIA rats were detected by HE staining.The results revealed that compared with the normal group,the synovial layers in the articular slices of the rats in the CIA model group were significantly increased and arranged in disorder,with abnormal proliferation of synovial cells,a large number of inflammatory cells infiltration and pannus formation,obvious erosion of cartilage tissue and inflammatory cell infiltration.Compared with the CIA model group,Ig D-Fc-Ig(9 mg/kg)and etanercept significantly inhibited the abnormal proliferation,cartilage erosion,inflammatory cell infiltration and pannus formation of synovial cells in CIA rats.3.Ig D-Fc-Ig fusion protein down-regulated the expression levels of CTHRC1 and VEGF in synovial tissues of CIA ratsThe expression of CTHRC1 and VEGF in synovial tissues of CIA rats was detect by immunohistochemistry.Image-J analysis system was used for analysis,the results showed that compared with the normal group,the expression of CTHRC1 and VEGF in synovial tissues of the CIA model group was significantly up-regulated.Compared with the CIA model group,Ig D-Fc-Ig(9 mg/kg)and etanercept significantly down-regulated the expression of CTHRC1 and VEGF proteins in synovial tissues of CIA rats.4.Ig DR was found in FLS of CIA ratsImmunofluorescence was used to detect the expression of Ig DR on FLS of CIA rats,Ig DR was indirectly labeled by Ig D stimulation,and the results showed the presence of Ig DR on FLS of CIA rats.5.Ig D-Fc-Ig fusion protein significantly down-regulated the expression of CTHRC1,Fzd5 and p-P65 proteins in FLS of ratsThe expression of CTHRC1,Fzd5 and p-P65 and the role of Ig D-Fc-Ig fusion protein in FLS of rats were detected by Western blot.The results revealed that compared with the normal group,the expression of CTHRC1,Fzd5 and p-P65 protein in FLS of the CIA model group was significantly up-regulated.Compared with the CIA model group,Ig D-Fc-Ig(9 mg/kg)and etanercept significantly down-regulated the expression of CTHRC1,Fzd5 and p-P65 proteins in FLS of CIA rats.6.Ig D-Fc-Ig fusion protein significantly decreased the co-localization rate of CTHRC1 and Fzd5 in FLS of rats.The co-localization rate of CTHRC1 and Fzd5 in FLS of rats and the effect of Ig D-Fc-Ig fusion protein were detected by immunofluorescence.The results of laser confocal microscopy showed that the co-localization rate of CTHRC1 and Fzd5 in FLS of the CIA model group was significantly increased compared with the normal group.Compared with the CIA model group,Ig D-Fc-Ig(9 mg/kg)and etanercept significantly decreased the co-localization rate of CTHRC1 and Fzd5 in FLS of CIA rats.7.Ig DR is present on MH7AThe expression of Ig DR on MH7 A was detected by immunofluorescence,Ig DR was indirectly labeled by Ig D stimulation,and the results showed the presence of Ig DR on MH7 A.8.Ig D-Fc-Ig fusion protein significantly down-regulated the expression of CTHRC1,Fzd5,p-P65,P65 and VEGF in MH7 A stimulated by Ig D and WNTAfter stimulating MH7 A cells with Ig D(9 ?g/m L)and WNT5A(100 ng/m L)for48 hours,the expression of signaling pathway related proteins and the role of Ig D-Fc-Igfusion protein were detected by Western blot.Results show that compared with the control group,Ig D(9 ?g/m L)and WNT5A(100 ng/m L)stimulates MH7 A alone could significantly up-regulate the protein levels of CTHRC1,Fzd5 and VEGF.The combined stimuli of Ig D(9 ?g/m L)and WNT5A(100 ng/m L)significantly up-regulated the protein levels of CTHRC1,Fzd5,p-p65 and VEGF,and the up-regulated effect of Ig D(9 ?g/m L)and WNT5A(100 ng/m L)was more obvious than that of single stimulation group;compared with the combined stimuli group,Ig D-Fc-Ig(10 ?g/m L)and etanercept significantly down-regulated the expression of CTHRC1,Fzd5,p-P65 and VEGF protein expression.This suggests that Ig D-Fc-Ig fusion protein may down-regulate the expression level of VEGF by inhibiting the WNT-Fzd5-CTHRC1-NF-?B pathway.9.Ig D-Fc-Ig fusion protein significantly down-regulate the co-localization rate of CTHRC1 and Fzd5 in MH7 A stimulated by Ig D and WNTThe co-localization of CTHRC1 with Fzd5 and the role of Ig D-Fc-Ig fusion protein in MH7 A stimulated by Ig D(9 ?g/m L)and WNT5A(100 ng/m L)was detected by immunofluorescence.The results showed that,compared with the control group,Ig D(9 ?g/m L)and WNT5A(100 ng/m L)stimulates MH7 A alone can significantly increase the co-localization rate of CTHRC1 and Fzd5 in MH7 A.The combined stimuli of Ig D(9 ?g/m L)and WNT5A(100 ng/m L)had a more significant effect on the co-localization of CTHRC1 and Fzd5 in MH7 A than the stimulation alone;compared with the combined stimulus group,Ig D-Fc-Ig fusion protein(10 ?g/m L)and etanercept significantly reduced the co-localization of CTHRC1 and Fzd5 in MH7 A.10.Ig D-Fc-Ig fusion protein significantly down-regulate the level of VEGF-A in the supernatant of MH7 A culture stimulated by Ig D and WNTAfter stimulating MH7 A cells with Ig D(9 ?g/m L)and WNT5A(100 ng/m L)for48 hours,the changes of VEGF-A level in the supernatant of MH7 A culture in each group were detected by ELISA.The results showed that,compared with the control group,Ig D(9 ?g/m L)and WNT5A(100 ng/m L)stimulates MH7 A alone can significantly up-regulated VEGF-A levels in supernatant of MH7 A culture.The combined stimuli of Ig D(9 ?g/m L)and WNT5A(100 ng/m L)had a more significant effect on the up-regulation of VEGF-A than that of the stimulation alone group;compared with the combined stimulation group,Ig D-Fc-Ig(10 ?g/m L)and etanercept significantly reduced VEGF-A levels in the supernatant of MH7 A culture.Conclusion1.Ig D promotes the activation of FLS through the WNT-Fzd5-CTHRC1-NF-?B signaling pathway.2.Ig D may activate the WNT pathway through the Ig DR to promote the activation of FLS.3.Ig D-Fc-Ig may indirectly inhibit the formation of complex between CTHRC1 and the WNT receptor Fzd5 by blocking the binding of Ig D to Ig D receptor,further inhibit the nuclear transcription factor NF-?B,and down-regulate the synthesis and secretion of VEGF to achieve the goal of treating synovium inflammation. |
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