| Osteoarthritis is a common degenerative joint disease,which is one of the main causes of joint dysfunction and low quality of life in the elderly.A large number of studies have shown that the development of osteoarthritis is the result of multiple environmental and genetic factors.NF-?B pathway is one of the main pathways involved in inflammatory reaction in osteoarthritis(OA).Reducing the activation of NF-?B in osteoarthritis plays an important role in inhibiting cartilage inflammation and slowing down the progress of OA.Previous studies have shown that orphan receptor NOR1(also known as NR4A3)can regulate extracellular matrix of smooth muscle cells and regulate the activity of NF-?B pathway by directly regulating the synthesis of IKK and level of IKB in dendritic cells.But the role of NOR1 in OA is not yet clear.Therefore,the purpose of this study was to study the role of NOR1 in the pathogenesis and development of OA.Firstly,we studied the expression of NOR1 in OA.Then we used lentivirus and siRNA tools to study the effect of over expression or knock down of NOR1 on chondrocytes inflammatory reaction and related inflammatory pathways in OA,and further our understanding of OA We hope to find new targets for OA therapy.This study is consisted of three parts:1.The expression of NOR 1 in the OA cartilage from patients and IL-1?-stimulated rat chondrocytes in vitro.2.The pro-inflammatory effect of NOR1 in osteoarthritis in vitro and rat model of OA3.Mechanism study in vitro.Chapter ?the expression of NOR1 in the OA cartilage from patients and IL-1?-stimulated rat chondrocytes in vitro.Objective:To investigate the expression of NOR1 in OA cartilage from patients and IL-1?-stimulated rat chondrocytes in vitro.Methods:Femoral heads were obtained from 10 patients with osteoarthritis and 10 patients with femoral neck fractures who underwent THA in our facility.The expression of NOR1 and OA markers(cox2 and co12)in human OA cartilage and normal cartilage were detected by RT-PCR,Western-blot,and immunofluorescence.Rat chondrocytes in vitro were stimulated by IL-1?,and the expression of MMP3/9,iNOS,COX2,COL2 and SOX9 at mRNA and protein levels were detected by RT-PCR and Western blot to verify the validity of IL-1? stimulation.Results:The mRNA and protein levels of NOR1 in human OA cartilage were higher than those in age and sex-matched patients with femoral neck fracture(P<0.05).And results of immunofluorescence showed that the high expression of NOR1 was co-located with the high expression of COX2 and the low expression of collagen II in OA cartilage.In vitro results showed that the expression of these pro-inflammatory factors such as MMP3/9,iNOS,COX2 increased significantly at the mRNA and protein levels after 24h of IL-1 ? stimulation,while the expression of COL2 and SOX9 decreased.It accords with the pathological changes of OA in human body.Then,we detected the expression of NOR1 in chondrocytes stimulated by IL-1? at different time points.Data showed that the expression of NOR1 increased significantly in the early and late stages of IL-1?stimulation(P.<0.05).Conclusion:NOR1 is highly expressed in the OA cartilage from patients and IL-1?-stimulated rat chondrocytes in vitro.Chapter ? role of NOR1 on IL-1?-induced inflammation in vitro and cartilage degeradation in vivoObjective:To investigate the role of NOR1 on IL-1?-induced inflammation in vitro and cartilage degeradation in vivoMethods:siRNA or lentiviral packaging of NOR1 overexpression plasmid were used to transfect rat chondrocytes in vitro to interfere(knock-down)or over-express the expression of NOR1.NOR1 overexpression or interference efficiency in chondrocytes were detected by RT-PCR,Western blot,and immunofluorescence assay.Expression of MMP3/9,iNOS,and COX2 in presence or absence of IL-1 ? were also detected by RT-PCR and Western blot.In the rats' experimental part,DMM was used to induce OA model in rats.All the rats were divided into 5 groups:lentivirus-overexpressing NR4A3 particles were injected intra-articularly in OA rats every 2 weeks in the Lenti-OE group since 1 week post-surgery.Lentivirus-control particles at the same dose were injected intra-articularly in OA rats in the Lenti-NC group.Lentivirus-shNR4A3 particles at the same dose were injected intra-articularly in OA rats in the Lenti-KD group Lentivirus-shcontrol particles at the same dose were injected intra-articularly in OA rats in the Lenti-shCon group.Rats were euthanized after 4 weeks of treatment,and the knees were preserved in 4%paraformaldehyde solution.All the samples were stained with SO.Tissue fluorescence was used to detect the expression of NOR1 inflammatory marker COX2.Results:In vitro data showed that the overexpression of NR4A3 could significantly increase the mRNA level of MMP-3,MMP-9,INOS,COX2 in chondrocytes that stimulated by IL-1? for 24 hours,but had no significant effect on the mRNA level of Collagen 2 and Sox 9.Overexpression of NR4A3 could increase the protein level of MMP-9,INOS,COX2 in chondrocytes that stimulated by IL-1? for 24 hours,but had no significant effect on the protein level of MMP-3.It could also enhance the IL-1?-induced degradation of Col2 and Sox-9 in chondrocytes.Knock down of NR4a3 could significantly decrease the mRNA level of MMP-3,MMP-9,INOS,COX2 in chondrocytes that stimulated by IL-1? for 24 hours.In addition,knock down of NR4A3 significantly decreased the protein level.of MMP-9,INOS,COX2 in chondrocytes that stimulated by IL-1? for 24 hours,and significantly alleviated IL-1?-induced degradation of Col2 and Sox-9.in vivo results showed that the degree of cartilage degeneration and the expression of COX2 in NR4A3 overexpressed knees were more severe than those of the Len-NC group.Degree of degeneration of the knee joint cartilage of rats intra-articularly injected with lentivirus-shNR4A3 particles was lighter than that of Lenti-shCon group,and the expression of COX2 was also decreased.Conclusion:NOR1(NR4A3)plays a pro-inflammatory role in osteoarthritis.Chapter ? the Mechanism study of NOR1 on OsteoarthritisObjective:To explore the mechanism of NOR1 effect on Osteoarthritis.Methods:siRNA or lentiviral packaging of NOR1 overexpression plasmid were used to transfect rat chondrocytes in vitro to interfere(knock-down)or over-express the expression of NOR1.The phosphorylation of P65 and the degradation of IKB? in the NF-?B pathway induced by IL-1? and the phosphorylation of p38,ERK and JNK in the MAPK pathway were detected by Western blot.The nuclear translocation of p65 induced by IL-1? was oberseved by immunofluorescence technique.The activation of NF-?B pathway was detected by luciferase reporter gene assay.We designed a rescue experiment additionally.After treated with JSH23,a specific inhibitor of NF-?B pathway,RT-PCR and Western blot techniques were used to detect the expression of iNOS,COX2 and MMP9 in NR4A3 overexpressed chondrocytes stimulated by IL-1?.Results:Phosphorylation level of JNK,erk and p38 did not change significantly compared with the positive control under the same IL-1? stimulation,but phosphorylation level of p65 and degradation level of IKB? induced by IL-1? were significantly increased.The increased nuclear translocation of p65 and the luciferase reporter gene data showed that the activity of NF-?B pathway in NR4A3-overexpressed chondrocytes was higher than control chondrocytes.After pre-treatment of JSH-23,the enhanced expression of MMP-9,INOS,COX2 induced by over-expression of NR4A3 could be alleviated,and there was no significant difference between the overexpression group receiving JSH-23 and JSH-23 treated control chondrocytes.In NR4A3 knock down chondrocytes,phosphorylation level of JNK,erk and p38 did not change significantly under the same IL-1? induction,but the phosphorylation level of p65 and the degradation level of IKBa were decreased obviously.The decreased nuclear translocation of p65 and luciferase reporter gene assay showed that the activity of NF-?B pathway in NR4 A3-knockdown chondrocytes was decreased after IL-1(3 induction.Conclusion:NOR1(NR4A3)can promote inflammation by enhauce activition of NF-?B pathway. |
PDF Full Text Request