【作者】 柳辉；

【导师】 郭风劲；

【作者基本信息】 华中科技大学 ， 外科学（骨外科）， 2020， 博士

【摘要】 第一部分核受体亚家族1D组成员1(NR1D1)在体外对类风湿关节炎滑膜炎症的作用和机制研究目的研究NR1D1在体外调控类风湿关节炎滑膜纤维细胞(RAFLSs)炎症的作用及其作用机制。方法1.收集临床上骨性关节炎(Osteoarthritis,OA)患者和类风湿关节炎(Rheumatoid arthritis,RA)患者的滑膜组织并通过免疫组织化学分析NR1D1的表达量,同时从滑膜组织中分离培养RAFLSs,并通过流式细胞仪鉴定细胞纯度。2.不同炎症因子如IL-1β、TNF-α、LPS、IL-17体外刺激RAFLSs,通过蛋白印迹实验(Western blot)和实时定量PCR(RT-PCR)检测NR1D1的表达变化。3.通过流式细胞仪和CCK-8实验检测NR1D1激动剂SR9009对RAFLSs增殖和凋亡的影响。4.通过NR1D1激动剂SR9009,小干扰RNA(si RNA)和质粒转染调控RAFLSs内NR1D1的活性,然后再用IL-1β刺激,通过Western blot和RT-PCR检测调控NR1D1活性后,IL-6,COX-2,i NOS等炎症因子和基质金属蛋白酶(MMPs)的表达变化。5.通过NR1D1激动剂SR9009,si RNA和质粒转染调控RAFLSs内NR1D1的活性,然后通过免疫荧光、Western blot、RT-PCR检测NR1D1对活性氧(ROS)、Nrf2信号通路相关蛋白(HO-1、NQO-1、Nrf2、KEAP1)表达的影响。6.通过NR1D1激动剂SR9009和si RNA调控RAFLSs内NR1D1的活性,IL-1β刺激不同时间后通过免疫荧光、凝胶迁移实验(EMSA)和Western blot检测NR1D1活性对MAPK、NF-κB等信号通路的影响。结果1.免疫组化实验表明NR1D1在RA滑膜组织中的表达量比OA滑膜组织高,而体外Western blot和RT-PCR实验表明IL-1β和TNF-α刺激后RAFLSs中NR1D1的表达量下降。2.流式细胞仪和CCK-8结果均证实NR1D1激动剂SR9009对RAFLSs的增殖和凋亡没有明显的影响。3.SR9009药理激活NR1D1后会抑制IL-1β诱导RAFLSs产生IL-6、IL-8、COX-2、i NOS等炎症因子和MMP3、MMP13等基质金属蛋白酶的表达;在RAFLSs细胞用si RNA沉默NR1D1后,IL-1β诱导产生的IL-6、IL-8、COX-2、i NOS等炎症因子和MMP3、MMP13等基质金属蛋白酶的表达会增加,而用质粒过表达NR1D1后,上述炎症因子和MMPs表达则会下降。4.SR9009药理激活NR1D1后会抑制IL-1β诱导RAFLSs细胞内ROS的产生;此外,SR9009会促进Nrf2由胞浆转入胞核,激活Nrf2信号通路,提高抗氧化应激蛋白HO-1、NQO-1、Nrf2的表达;在RAFLSs细胞用si RNA沉默NR1D1后,抗氧化应激蛋白HO-1、NQO-1、Nrf2的表达表达会下降,而用质粒过表达NR1D1后,HO-1、NQO-1、Nrf2的表达表达会增加。5.免疫荧光实验表明,SR9009会抑制IL-1β刺激后RAFLSs细胞内P65的核转入;Western blot和EMSA实验证实SR9009会抑制MAPK、NF-κB信号通路的活性,而用si RNA沉默NR1D1后则会促进MAPK和NF-κB信号通路的活化。结论激活NR1D1可以在体外抑制RAFLSs细胞内炎症因子和MMPs的表达,促进Nrf2信号通路的活化,上述作用可能是通过抑制MAPK和NF-κB信号通路的活性实现的。第二部分NR1D1在体外对巨噬细胞极化和破骨细胞形成的作用和机制研究目的研究NR1D1在体外对巨噬细胞极化和破骨细胞分化及其功能的影响。方法1.体外分离培养小鼠骨髓单核巨噬细胞(bone marrow-derived myeloid lineage cells,BMMs),分别诱导其向M1型巨噬细胞(LPS(100ng/ml)+IFN-γ(10 ng/ml))和M2型巨噬细胞(IL-4(20 ng/ml))分化,然后加入不同浓度的SR9009,RT-PCR检测M1型巨噬细胞标志(IL-1、IL-6、TNF-α、i NOS)和M2型巨噬细胞标志(IL-10,Arginase-1)的表达,免疫荧光检测SR9009对BMMs向M1/M2细胞极化的影响。2.通过RT-PCR和Western blot检测BMMs向M1巨噬细胞和破骨细胞分化过程中NR1D1的表达变化。3.通过抗酒石酸磷酸酶(TRAP)染色、骨板吸收实验、骨架纤维形肌动蛋白(F-Actin)环染色、破骨细胞标志基因等实验检测NR1D1激动剂SR9009对破骨细胞分化和功能的影响。结果1.RT-PCR结果表明,SR9009抑制M1型巨噬细胞标志基因(IL-1、IL-6、TNF-α、i NOS)的表达,而促进M2型巨噬细胞标志基因(IL-10、Arginase-1)的表达;免疫荧光实验也证实,SR9009抑制M1型巨噬细胞的极化。2.在M1巨噬细胞和破骨细胞分化过程中,NR1D1的蛋白和m RNA表达量均下降。3.TRAP染色实验、骨板吸收实验、F-actin实验表明SR9009可以抑制破骨细胞分化和骨吸收功能,同时也抑制了破骨细胞标志基因如TRAP、CTSK、MMP-9、c-FOS和NFATc1等蛋白的表达。结论激活NR1D1抑制M1型巨噬细胞的极化,同时也抑制破骨细胞的分化和骨吸收功能。第三部分NR1D1对类风湿关节炎滑膜炎症和骨质破坏的体内研究目的通过胶原蛋白诱发的关节炎(Collagen-induced arthritis,CIA)构建体内RA模型,研究NR1D1激动剂SR9009对关节炎滑膜炎症和骨质破坏的影响。方法1.通过胶原蛋白成功在DBA-1小鼠诱导关节炎后,将小鼠分为4组:对照组、CIA组、CIA+SR9009(50mg/kg)、CIA+SR9009(100mg/kg);腹腔给药后,通过OARSI组织学评分、后肢肿胀厚度等方法评价SR9009对关节炎的影响。2.实验48天后处死DBA-1小鼠,收集小鼠血清和肢体标本,通过ELISA检测小鼠血清中IL-1β、IL-6、TNF-α、RANKL和OPG的含量变化;通过苏木精-伊红染色(Hematoxylin-Eosin staining,H&E)、番红固绿、甲苯胺蓝等组织学染色评估关节滑膜炎症和软骨破坏的程度;通过micro-CT和TRAP染色检测小鼠关节骨侵蚀的变化;通过免疫组化检测相关炎症指标和MMPs的变化。结果1.我们通过胶原蛋白成功构建了小鼠关节炎模型,同时发现给予NR1D1激动剂SR9009后,小鼠的关节红肿和肢体厚度症状减轻,OARSI评分和后肢爪厚度明显降低,尤其是在SR9009高剂量组。2.通过ELISA实验证实,SR9009抑制了CIA小鼠血清体内炎症因子IL-1β、IL-6、TNF-α和RANKL的表达;H&E染色、番红固绿和甲苯胺蓝染色证实SR9009抑制小鼠滑膜炎症和软骨破坏;micro-CT和TRAP染色结果表明SR9009抑制CIA小鼠的骨质破坏;免疫组化实验结果也表明SR9009抑制了滑膜内MMP3、MMP13、IL-1β、i NOS和COX2的表达。结论SR9009药理激活NR1D1抑制了CIA小鼠的滑膜炎症和骨质破坏。更多还原

【Abstract】 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-1β,TNF-α,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-1β.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-κB signaling pathway were detected by immunofluorescences,EMSA,Western blot after IL-1β 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-1β and TNF-α.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-1β;RAFLSs were silenced with si RNA and then stimulated with IL-1β,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-1β-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-1β stimulation;Western blot and EMSA experiments confirmed that SR9009 inhibited the activity of MAPK and NF-κB signaling pathways,but after NR1D1 was silenced by si RNA promoted the activation of MAPK and NF-κB 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-κB 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-γ(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-α,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-α,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-1β,IL-6,TNF-α,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-1β,IL-6,TNF-α 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-1β,i NOS and COX2 in the synovium.Conclusions SR9009 pharmacologically activated NR1D1 inhibited synovial inflammation and bone destruction in CIA mice.更多还原