| Part 1 The effect and mechanism of Nuclear receptor subfamily 1 group D member 1(NR1D1)on rheumatoid arthritis synovial inflammation in vitroObjective To investigate the role and mechanism of NR1D1 in regulating inflammation of synovial fibroblasts(RAFLSs)in rheumatoid arthritis in vitro.Methods 1.Synovial tissues of patients with clinical OA and RA were collected and the expression level of NR1D1 was analyzed by immunohistochemistry.Meanwhile,RAFLSs were isolated from synovial tissues and cultured,and cells purity was identified by flow cytometry.2.RAFLSs were stimulated by different inflammatory factors such as IL-1?,TNF-?,LPS,and IL-17 in vitro,and the expression changes of NR1D1 were detected by Western blot and real-time quantitative PCR(RT-PCR).3.The effects of NR1D1 agonist SR9009 on the proliferation and apoptosis of RAFLSs were detected by flow cytometry and CCK-8 experiments.4.NR1D1 agonist SR9009,small interfering RNA(si RNA)and plasmid transfection were used to regulate NR1D1 activity in RAFLSs,and then stimulated by IL-1?.Expression changes of inflammatory factors such as IL-6,COX-2,i NOS and matrix metalloproteinase(MMPs)were detected by Western blot and RT-PCR after regulation of NR1D1 activity.5.NR1D1 activity in RAFLSs was regulated by NR1D1 agonist SR9009,si RNA and plasmid,and then the effects of NR1D1 on reactive oxygen species(ROS)and Nrf2 signaling pathway related proteins(HO-1,NQO-1,Nrf2,and KEAP1)were detected by immunofluorescences,Western blot and RT-PCR.6.NR1D1 activity in RAFLSs was regulated by NR1D1 agonist SR9009 and si RNA,and the effects of NR1D1 activity on MAPK and NF-?B signaling pathway were detected by immunofluorescences,EMSA,Western blot after IL-1? stimulation at different time.Results 1.Immunohistochemical experiments showed that the expression level of NR1D1 in the synovial tissue of RA was higher than that of OA,while in vitro Western blot and RT-PCR experiments showed that the expression of NR1D1 in RAFLSs decreased after stimulation with IL-1? and TNF-?.2.Flow cytometry and CCK-8 experiments confirmed that NR1D1 agonist SR9009 had no significant effect on the proliferation and apoptosis of RAFLSs.3.After pharmacological activation of NR1D1 by SR9009,the expression of IL-6,IL-8,COX-2,i NOS and matrix metalloproteinases(MMP3 and MMP13)were inhibited in RAFLSs induced by IL-1?;RAFLSs were silenced with si RNA and then stimulated with IL-1?,the expression inflammatory factors such as IL-6,IL-8,COX-2,i NOS and matrix metalloproteinases such as MMP3 and MMP13 increased,while expression of above-mentioned inflammatory factors and MMPs decreased after overexpression of NR1D1 by plasmid.4.SR9009 pharmacologically activates NR1D1 to inhibit IL-1?-induced ROS production in RAFLSs cells.In addition,SR9009 promoted the transfer of Nrf2 from the cytoplasm to the nucleus,activated the Nrf2 signaling pathway,and improved the antioxidant stress proteins HO-1,NQO-1 and Nrf2 expression;after RAFLSs silenced NR1D1 with si RNA,the expression of anti-oxidative stress proteins HO-1,NQO-1,and Nrf2 decreased,and after overexpression of NR1D1 with plasmid,HO-1,NQO-1,Nrf2 expression increased.5.Immunofluorescence experiments showed that SR9009 inhibited the nuclear transfer of P65 in RAFLSs cells after IL-1? stimulation;Western blot and EMSA experiments confirmed that SR9009 inhibited the activity of MAPK and NF-?B signaling pathways,but after NR1D1 was silenced by si RNA promoted the activation of MAPK and NF-?B signaling pathway.Conclusions Activation of NR1D1 can inhibit the expression of inflammatory factors and MMPs in RAFLSs cells in vitro and promote the activation of Nrf2 signaling pathway.The above effects may be achieved by inhibiting the activity of MAPK and NF-?B signaling pathway.Part 2 The effect and mechanism of NR1D1 on macrophage polarization and osteoclastogenesis in vitroObjective To study the effects of NR1D1 on macrophage polarization and osteoclastogenesis and function in vitro.Methods 1.Bone marrow-derived myeloid lineage cells(BMMs)were isolated and cultured in vitro,and induced to differentiate into M1 type macrophages(LPS(100ng/ml)+IFN-?(10 ng/ml))and M2 type macrophages(IL-4(20 ng/ml)).Then different concentrations of SR9009 were added.RT-PCR was used to detect the expression of M1 macrophage markers(IL-1,IL-6,TNF-?,i NOS)and M2 macrophage markers(IL-10,Arginase-1).The effect of SR9009 on M1 / M2 cell polarization was detected by immunofluorescence experiments.2.Detection of NR1D1 expression during differentiation of BMMs into M1 macrophages and osteoclasts by RT-PCR and Western blot.3.Test the NR1D1 agonist SR9009 on the differentiation and function of osteoclasts by experiments such as anti-tartaric acid phosphatase(TRAP)staining,bone plate resorption experiment,cytoskeletal fibrillar actin loop staining,and osteoclast marker genes.Results 1.RT-PCR results showed that SR9009 inhibited the expression of M1 macrophage marker genes(IL-1,IL-6,TNF-?,i NOS)and promoted the expression of M2 macrophage marker genes(IL-10,Arginase-1);immunofluorescence experiments also confirmed that SR9009 inhibits the polarization of M1-type macrophages.2.During the differentiation of M1 macrophages and osteoclasts,the protein and m RNA expression of NR1D1 decreased.3.TRAP staining experiments,bone plate resorption experiments,and F-actin experiments showed that SR9009 can inhibit osteoclast differentiation and bone resorption,and also inhibited the expression of osteoclast marker genes such as TRAP,CTSK,MMP-9,c-FOS and NFATc1.Conclusions Activation of NR1D1 inhibits the polarization of M1 macrophages,and also inhibits osteoclast differentiation and bone resorption.Part 3 In vivo study of NR1D1 on rheumatoid arthritis synovial inflammation and bone destructionObjective The in vivo RA model was constructed by collagen-induced arthritis(CIA)to study the effects of NR1D1 agonist SR9009 on arthritis synovial inflammation and bone destruction.Methods 1.After collagen-induced arthritis was successfully induced in DBA-1 mice,the mice were divided into 4 groups: control group,CIA group,CIA + SR9009(50mg/kg),CIA + SR9009(100mg/kg);after intraperitoneal administration,the effects of SR9009 on arthritis were evaluated by OARSI histological score and hindlimb swelling thickness.2.DBA-1 mice were sacrificed 48 days after the experiment,mouse serum and limb samples were collected,and the levels of IL-1?,IL-6,TNF-?,RANKL and OPG in mouse serum were detected by ELISA;Hematoxylin-Eosin staining(H & E),Safranin O-fast green,toluidine blue and other histological staining to assess the degree of joint synovial inflammation and cartilage damage;micro-CT and TRAP staining to detect joint erosion in mice;The changes of synovial inflammation indexes and MMPs were detected by immunohistochemistry.Results 1.We successfully constructed a mouse arthritis model through collagen.At the same time,we found that after administration of the NR1D1 agonist SR9009,the joint swelling and limb thickness symptoms of the mice were reduced,and the OARSI score and hind paw thickness were significantly reduced,especially at high doses of SR9009 group.2.ELISA test confirmed that SR9009 inhibited the expression of inflammatory factors IL-1?,IL-6,TNF-? and RANKL in the serum of CIA mice;H & E staining,Safranin O-fast green and toluidine blue staining confirmed that SR9009 inhibited mouse synovium inflammation and cartilage destruction;micro-CT and TRAP staining results showed that SR9009 inhibited bone destruction in CIA mice;immunohistochemical results also showed that SR9009 inhibited the expression of MMP3,MMP13,IL-1?,i NOS and COX2 in the synovium.Conclusions SR9009 pharmacologically activated NR1D1 inhibited synovial inflammation and bone destruction in CIA mice. |
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