The Role And Mechanism Of The Interaction Between Nuclear Factor NR4A1 And NF-κB Pathway In OA Pathogenesis

Osteoarthritis(OA),featured with cartilage degeneration and joint dysfunction,is a leading cause of disability in elder populations.Local and systematic inflammation were proved to play central roles in OA pathogenesis,along with the chronic activation of NF-κB signal pathway.Targeting the inhibitory regulation of inflammation and NF-κB signal pathway is an effective approach to maintain cartilage catabolic homeostasis and ameliorate OA progression.Previous study revealed that,NR4A1(Nuclear receptor subfamily 4,group A-1)is a key regulator of inflammation and NF-κB signal pathway.NR4A1 can reduce the binding ability of 65 and repress it’s down-stream genes expression through trans-repression.Apart from the direct protein-protein interaction,NR4A1 also inhibit p65 nuclear translocation by inducing the expression of NF-κB inhibitors like IκBa at transcriptional level.In other inflammation-related diseases,re-activation of NR4A1 was demonstrated to repress local inflammation significantly and prevent disease progression,exhibiting the therapeutic potential.However,the role and mechanism of the interaction between NR4A1 and NF-κB pathway on OA pathogenesis was poorly understood.In the present study,we detected the expression of NR4A1 in human OA cartilage tissues and investigated the influence of NR4A1 in chondrocytes’ inflammation and NF-κB activation in vitro.Meanwhile,we explored the expression and phosphorylated inhibition of NR4A1 under continuous IL-1β stimulation and possible mechanism.Re-activation of NR4A1 by cytosporone B and its role in OA prevention were also verified in vivo and in vitro.In addition,the effect of NR4A1 on the pathological changes of cartilage progenitor cells(CPCs)under inflammatory micro-environment were preliminarily investigated.This study consists three parts:1.The expression of NR4A1 in OA cartilage tissue and in vitro OA model;2.The role and mechanism of the interaction between NR4A1 and NF-κB pathway on chondrocytes and osteoarthritis;3.The effects of NR4A1 to the proliferation and differentiation of cartilage progenitor cellsChapter I The expression of NR4A1 in OA cartilage tissue and in vitro OA modelObjective:To explore the expression of NR4A1 in OA cartilage tissue and in vitro OA model.Methods:Articular cartilage samples were collected from patients who received the total hip arthroplasty surgery in the second affiliated hospital of Zhejiang university,whose diagnosis were hip OA or femoral neck fracture(10 samples for each group).Through real-time PCR(RT-PCR),western-blot and immunofluorescence(IF),we detected the expression level and tissue location of NR4A1,chondrocyte inflammatory and matrix-degrading genes,and p65,a key effector of NF-κB signal pathway.In vitro,an OA model was conducted in cultured rat chondrocytes though a stimulation of IL-1β.The expression of NR4A1 was then investigated in the in vitro OA model with or without NF-κB signal inhibitor JSH23.Results:The mRNA level of chondrocyte inflammatory and matrix-degrading genes iNOS,COX-2 and matrix metalloproteinase(MMP)3,9,13 were significantly higher in OA cartilage tissues than the normal cartilage tissues.Both NR4A1 and p65 were significantly up-regulated and co-located in the inflamed chondrocytes in the erosive area of human OA cartilage.In vitro,the expression of NR4A1 was induced by IL-1β in cultured rat chondrocytes,which was significantly blocked by NF-κB inhibitor JSH23 in a concentration-dependent manner(P<0.05).Conclusion:NR4A1 is up-regulated in OA cartilage tissue and in vitro OA model through the activation of NF-κB signal pathway.Chapter Ⅱ The mutual regulation of NR4A1 and NF-κB signaling pathway in osteoarthritisObjective:To clarify the effect and mechanism of NR4A1 on the inflammatory response of chondrocytes,explore the effect and mechanism of long-term inflammatory stimulation on the expression and activity of NR4A1 in chondrocytes,and verify the protective effect of NR4A1 specific agonist cytosporone B on cartilage degeneration in vivo and in vitro.Methods:In rat chondrocytes,overexpression and knockdown of NR4A1 expression was achieved by overexpressing plasmid and siRNA,respectively.The expression of IL-1β induced inflammatory genes and matrix-degrading genes and the activation ofNF-κB signaling pathway’ were detected by RT-PCR,Western-blot,immunofluorescence,and luciferase reporter gene detection.At different time points of IL-1β stimulation in rat chondrocytes,the dynamic changes of NR4A1 expression,acetylation of histone H3(H3 Lys27)and H4(H4 lys8),and the expression of histone deacetylase(HDAC1,2,3,4,5,6,7,9,10)were measured by RT-PCR and Western-blot.The expression of NR4A1 after IL-1β and HD AC inhibitor SAHA co-stimulation for 72 hours was also detected by Western-blot.The phosphorylation level of NR4A1 under the stimulation of IL-1β with or without p38 inhibitor SB203580,JNK inhibitor SP600125 and ERK inhibitor FR180204 were detected by Western-blot.The influence of different concentrations of cytosporone B on the expression of IL-1β induced inflammatory genes and matrix-degrading genes,and the expression and phosphorylation of NR4A1 was detected by RT-PCR and Western-blot.The rat OA model of the knee joint was made by medial meniscus excision.After the operation,1 M cytosporone B or equal amount of solvent were injected each week in the joint cavity.After 6 and 12 weeks,the animals were sacrificed for safranin O staining.Results:Overexpression of NR4A1 significantly inhibited the IL-1β induced expression of inflammatory genes and matrix-degrading genes COX-2,iNOS,MMP3,9,and 13 at mRNA and protein levels(P<0.05).,while the expression of these genes increased significantly in the NR4A1 knockdown group(P<0.05).Immunofluorescence showed that IL-1β induced p65 nuclear translocation decreased significantly in NR4A1 overexpressing group,but increased in NR4A1 knockdown group.Overexpression of NR4A1 significantly inhibited the activity of the luciferase reporter gene activity of the NF-κB signaling pathway(P<0.05),which was significantly increased by the knockdown of NR4A1(P<0.05)in contrast.IL-1β treatment increased the expression of NR4A1 in rat chondrocytes,but the expression in long-term treatment was relatively inhibited.The acetylation of histone H3 Lys27 and H4 lys8 decreased under the long-term treatment of IL-1β.The expression of HDAC1-6 and HD AC10 were unregulated at different time points after IL-1β treatment.HDAC inhibitor SAHA treatment could promote the expression of NR4A1 in chondrocytes after 72 hours of.The phosphorylation of NR4A1 increased under IL-1β treatment and could be inhibited by p38 inhibitor SB203580,JNK inhibitor SP600125 and.ERK inhibitor FR180204.Cytosporone B could repress the high expression of COX2,iNOS,MMP3,MMP9 and MMP13 genes in rat chondrocytes induced by IL-1β in a concentration dependent manner.In addition,cytosporone B could promote NR4A1 gene expression and significantly reduce IL-1β induced NR4A1 phosphorylation in a concentration dependent manner.In rat OA model,safranin O staining revealed that the cartilage structure was more complete and the staining pigment was less in the cytosporone B group.Conclusion:NR4A1 could reduce chondrocyte inflammation and degradation through the inhibition of NF-κB signaling pathway.Long term inflammation inactivates NR4A1 through the HD AC-dependent transcription inhibition and MAPK-dependent phosphorylation.Reactivation of NR4A1 by cytosporone B ameliorated chondrocytes inflammation and osteoarthritis in vivo and in vitro.Chapter Ⅲ Effect and mechanism of NR4A1 on cartilage progenitor cells in inflammatory microenvironmentObjective:To explore the effect and mechanism of NR4A1 on cartilage progenitor cells(CPCs)in inflammatory microenvironment.Methods:Cartilage progenitor cells were isolated through extremely low density planting,and the chondrogenic,osteogenic and adipogenic differentiatial abilities of the isolated cells were tested.Surface makers(CD29,CD34,CD45 and CD90)of the isolated cells were detected by flow cytometry.The influence of NR4A1 specific agonist cytosporone B on the proliferation ability and migration ability of cartilage progenitor cells under IL-1β treatment was detected by CCK-8 test and scratch test respectively.The expression of cleaved Caspase3 was detected by Western-blot.Safranin O staining and RT-PCR analysis of of cartilage related gene collagen Ⅱ,Aggrecan and SOX9 were used to evaluate the effect of cytosporone B on the chondrogenic ability of CPCs under under IL-1β treatment.The the expression of Smad3,phosphorylated Smad3,beta-catenin and activation state beta-catenin were also detected by Western-blot.In vivo,The newborn C57 mice were injected with BdrU(50mg/kg)intraperitoneally once a day for 3 days,and then naturally grew to adult(8 weeks).The OA model of the primed adult mice were developed by anterior cruciate ligament transection(ACLT).After operation,cytosporone B(10mg/kg)or equal amount of solvent were injected intraperitoneally every other day.7 days after operation,the animals were sacrificed and the knee joint were sampled for immunofluorescence against BrdU and Ki67.Results:The isolated rat CPCs were demonstrated with the chondrogenic,osteogenic and adipogenic abilities,and the surface makers characteristics were CD29+CD34-CD45-CD90+.The CCK-8 assay and scratch test showed that the proliferation and migration abilities of cartilage progenitor cells decreased with the treatment of IL-1β,and the relative OD value was significantly lower than that of the control group(P<0.05)at 72 hour timepoint.Cytosporone B could reverse the decline of CPCs proliferation and migration and inhibit the cleaved Caspase3 expression induced by IL-1β in a concentration dependent manner(P<0.05).After chondrogenic induction,Safranin O staining and the mRNA level of collagen II,Aggrecan and SOX9 in IL-1β treatment group were significantly lower than that of the control group(P<0.05),which could be significantly restored by 1μM cytosporone B(P<0.05).Cytosporone B could also reverse the IL-1β induced inhibition of phosphorylation of Smad3 and activatedβ-catenin.The results of BrdU labeling experiment showed that there was a group of BrdU positive Ki67 negative cells on the surface of articular cartilage in normal adult mice,whereas a part of which came into Ki67 positive at 7 days after OA modeling operation.The proportion of BrdU and Ki67 double positive cells in cytosporone B treatment group was significantly higher than that in the group(P<0.05).Conclusion:NR4A1 agonist cytosporone B can improve the proliferation,migration ability and chondrogenic differentiation ability of cartilage progenitor cells in inflammatory microenvironment in vivo and in vitro.