The Effect And Regulation Mechanism Of BMP3 On Fibroblast-like Synoviocytes In Rheumatoid Arthritis

Rheumatoid arthritis(rheumatoid arthritis,RA)is a chronic autoimmune disease characterized by tumor-like hyperplasia of synovial tissue,persistent synovial inflammation,bone erosion,and progressive joint destruction.T lymphocytes,B lymphocytes,macrophages and fibroblast-like synoviocytes(FLSs)are involved in the pathogenesis of RA.Activated FLSs stimulate and recruit inflammatory cells into synovial tissue by secreting chemokines,matrix metalloproteinases,and inflammatory factors.These recruited inflammatory cells secrete more inflammatory factors to stimulate the proliferation and migration of synovial cells and exacerbate their inflammatory response.Therefore,FLSs are a key factor in the course of RA disease.Although the exact pathogenesis of RA has not yet been fully clarified,the proliferation and migration of synovial tissue and FLSs are undoubtedly closely related to the inflammatory response of RA pathogenesis.Therefore,studying how to inhibit the proliferation and migration of FLSs and the inflammatory response in RA has positive significance for elucidating the disease mechanism and treatment.Bone morphogenetic proteins(BMPs),which are members of the transforming growth factor ?(TGF-?)superfamily.They are named for their ability to induce ectopic formation of bone marrow cartilage and bone tissue.At present,it is clear that BMPs not only have osteogenesis,but also participate in embryonic development,organ formation,and the occurrence and development of various diseases.Studies have shown that BMP3 also plays an important inhibitory role in diseases such as tumors.It not only inhibits the proliferation of colorectal cancer and bile duct cancer cells and promotes apoptosis,but also inhibits the proliferation and activation of primary fibroblasts of mouse pulmonary fibrosis induced by bleomycin.More importantly,there is evidence that BMP3 m RNA expression is lower in the synovium of CIA mice than in normal mice.However,the role and mechanism of BMP3 in RA FLSs have not been reported.In order to explore the regulatory effect and mechanism of BMP3 on the proliferation and migration of FLSs and the secretion of inflammatory factors during the pathogenesis of RA.The following researches have been carried out in this subject:1.The expression of BMP3 is down-regulated in knee joint synovial and fibroblast-like synovial cells from RA patients and AIA ratsIn order to study whether BMP3 plays a regulatory role in RA,knee synovial samples were collected from patients with osteoarthritis(OA)and RA patients.Hematoxylin and eosin(HE)and western blot(WB)experiments verified the RA histopathology and high expression of inflammatory factors in the samples.Using immunohistochemistry(IHC),immunofluorescence(IF),WB,and quantitative real-time PCR(q-PCR)and other experimental methods found that the expression of BMP3 in the synovium and FLSs of RA patients was down-regulated compared with OA patients.This study also established an adjuvant-induced arthritis(AIA)model by injecting CFA into rats.The rat AIA model was established successfully by measuring rat paw swelling and arthritis scores,enzyme linked immunosorbent assay(ELISA),HE staining,WB and q-PCR.IHC,IF,WB and q-PCR were also used to detect that BMP3 expression in synovial tissue and FLSs of AIA rats was down-regulated compared with normal rats.2.BMP3-RNAi promotes the proliferation of AIA FLSs and the migration of AIA and RA FLSsWB and q-PCR results showed that BMP3-RNAi transfection significantly reduced them RNA and protein expression levels of BMP3 in AIA FLSs,and significantly increased the m RNA and protein expression levels of C-Myc and Cyclin D1.This project also found that BMP3-RNAi transfection resulted in a significant increase in the number of AIA FLSs cells in the S + G2 / M phase through cell cycle experiments.CCK8 and Ed U cell proliferation experiments showed that BMP3-RNAi transfection significantly promoted the proliferation of AIA FLSs.WB and q-PCR results showed that BMP3-RNAi transfection significantly up-regulated m RNA and protein expression levels of MMP-3 and MMP-9 in AIA FLSs,while TIMP-1 m RNA and protein expression were significantly down-regulated.Wound-healing and Transwell experiments also found that BMP3-RNAi transfection increased the migration and invasion ability of AIA FLSs.In addition,in this study,after we stimulated AIA FLSs and RA FLSs with 10 ng / m L TNF-?,we found that BMP3-RNAi transfection also increased MMP-3 and MMP-9 m RNA and protein expression levels in TNF-?-stimulated FLSs,and down-regulated TIMP-1 m RNA and protein expression levels.At the same time,Wound-healing test showed that BMP3-RNAi transfection also promoted the migration of TNF-? stimulated FLSs.3.BMP3 overexpression plasmid inhibits the proliferation of AIA FLSs and the migration of AIA and RA FLSsThe WB and q-PCR results in this experiment showed that BMP3 overexpression plasmid(BMP3-PEX)transfected with AIA FLSs significantly increased the m RNA and protein expression levels of BMP3,and significantly down-regulated the m RNA and protein expression levels of C-Myc and Cyclin D1.In addition,cell cycle experiments found that BMP3-PEX transfection of AIA FLSs significantly inhibited the increase in the number of S+G2/M phase cells,and CCK8 and Ed U cell proliferation experiments detected that BMP3-PEX transfection significantly inhibited the proliferation of AIA FLSs.WB and q-PCR results showed that BMP3-PEX transfection of AIA FLSs significantly reduced the m RNA and protein expression levels of MMP-3 and MMP-9,but upregulated the expression levels of TIMP-1.Wound-healing and Transwell experiments also found that BMP3-PEX transfection inhibited the migration and invasion of AIA FLSs.In addition,in this study,we also stimulated AIA FLSs and RA FLSs with 10 ng / m L TNF-?,and found that transfection of BMP3 overexpression plasmids(BMP3-PEX and BMP3-pc DNA3.1)also down-regulated the m RNA and protein expression of MMP-3 and MMP-9 in FLSs stimulated by TNF-?,also up-regulated m RNA and protein expression of TIMP-1.At the same time,Wound-healing experiments found that BMP3-PEX and BMP3-pc DNA3.1 transfection also inhibited the migration of TNF-? stimulated AIA and RA FLSs.4.BMP3-RNAi silencing BMP3 promotes the expression of inflammatory factors in FLSsWB and q-PCR experiments showed that BMP3-RNAi transfection down-regulated the expression of BMP3,which significantly increased the expression of IL-6,IL-1?,IL-17 A and TNF-? protein and m RNA in AIA FLSs.The ELISA kit detected that BMP3-RNAi also increased the levels of IL-6,IL-1? and TNF-? in the supernatant of AIA FLSs medium.Similarly,q-PCR experiments revealed that BMP3-RNAi transfection significantly increased the expression of adhesion factors VCAM-1 and chemokines CCL-2,CCL-3 in AIA FLSs.We also stimulated AIA FLSs and RA FLSs with 10 ng / m L TNF-? in this phase of the study.WB and q-PCR results showed that BMP3-RNAi also increased the expression of IL-6,IL-1?,IL-17 A and TNF-? proteins and m RNA in AIA FLSs and RA FLSs with TNF-? stimulation.q-PCR results showed that BMP3-RNAi up-regulated the expression of VCAM-1,CCL-2 and CCL-3 m RNA in AIA FLSs and RA FLSs with TNF-? stimulation.5.Overexpression plasmid up-regulating BMP3 inhibits the expression of inflammatory factors in FLSsWB and q-PCR experiments showed that BMP3 overexpression plasmid up-regulated the expression of BMP3 and significantly inhibited the expression of IL-6,IL-1?,IL-17 A,and TNF-? proteins and m RNA in AIA FLSs.The ELISA kit detected that the BMP3 overexpression plasmid also reduced the contents of IL-6,IL-1? and TNF-? in the supernatant of AIA FLSs medium.Similarly,q-PCR experiments revealed that BMP3 overexpression plasmid transfection significantly down-regulated the expression of adhesion factors VCAM-1 and chemokines CCL-2,CCL-3 in AIA FLSs.Similarly,WB and q-PCR results showed that BMP3 overexpression plasmids downregulated the expression of IL-6,IL-1?,IL-17 A,and TNF-? proteins and m RNA in AIA and RA FLSs stimulated by TNF-?.q-PCR results showed that BMP3 overexpression plasmid also reduced the expression of VCAM-1,CCL-2 and CCL-3 m RNA in AIA and RA FLSs with TNF-? stimulation.6.Regulation of BMP3 on FLSs may be related to TGF-?1/smad signaling pathwayWB experiment results showed that compared with FLSs in normal rats,the expression of p-Smad2 in AIA FLSs was significantly increased,and AIA FLSs transfected with BMP3-RNAi significantly increased in p-Smad2 expression compared with control groups.However,BMP3-PEX transfection significantly reduced the expression level of p-Smad2 protein in AIA FLSs.In subsequent experiments,we also stimulated AIA FLSs and RA FLSs with 10 ng / m L TNF-?,and found that the expression level of p-Smad2 protein was significantly increased compared with the non-stimulated group,and the expression of p-Smad2 protein was increased by BMP3-RNAi transfection.But the expression level p-Smad2 was reduced by BMP3 overexpression plasmid.These results suggest that BMP3 may regulate the proliferation and migration of FLSs through the TGF-?1 / smad signaling pathway.7.Effects of BMP3 overexpression adenovirus on AIA ratsIn the in vivo experimental part of the rat,we injected adenovirus ad-BMP3 into the knee joint cavity of AIA rats.After 2 weeks,the rat in vivo imaging experiment was carried out,and the results showed that the adenovirus successfully replicated in the rat.IF,IHC and WB experiments showed that BMP3 was successfully overexpressed in rat synovial tissue.Measurements of swelling of paw in rats,arthritis index score,and HE staining confirmed that ad-BMP3 reduced the severity of AIA and synovial tissue hyperplasia and inflammatory infiltration.At the same time,WB test also found that adBMP3 inhibited the expression of IL-6,IL-1?,IL-17 A and TNF-? in rat synovial tissue.The ELISA results showed that ad-BMP3 reduced the levels of IL-6,IL-1? and TNF-? in the serum of AIA rats.