| IntroductionRheumatoid arthritis?RA?is a complex systemic autoimmune disease and the exact pathogenesis is still unknown.Although researchers have studied RA for many years,no drug has been found that can continuously relieve or effectively treat RA.The main clinical manifestations of RA are synovial inflammation of the hands and knuckles,and fibroblast-like synoviocyte?FLS?abnormal proliferation plays an important role in the pathogenesis of RA.As a nuclear transcription factor activated by ligands,aryl hydrocarbon receptor?Ahr?is found in many cells and tissues and is considered to be a key factor in regulating the immune response.At present,Ahr agonistic-mediated immune regulation mechanisms have been reported in a variety of autoimmune diseases,such as autoimmune arthritis,experimental autoimmune encephalomyelitis,and experimental colitis.Studies have shown that the expression of Ahr in synovial tissue and synovial fluid of RA patients is significantly increased,suggesting that excessive activation of Ahr may lead to abnormal proliferation of FLS,which in turn leads to malignant transformation of RA and bone destruction.In animal model experiments,higher expressions of Ahr in inguinal lymph nodes and FLS cells were detected in CIA mice,and the degree of joint synovial damage in Ahr-/-CIA mice was significantly lower than in CIA mice.Further research found that Ahr in the T cells of the specific knockoutmice caused a decrease in Th17 and a significant reduction in CIA arthritis.Studies have reported that when FLS cell lines and RA synovial cells of RA patients are stimulated by Ahr ligand,they will up-regulate the secretion of pro-inflammatory cytokines such as IL-1?and IL-6,suggesting that Ahr is involved in the function of synovial cells.Regulation,excessive activation of the Ahr signal plays an important role in the development of RA.Paeoniflorin-6'-O-benzene sulfonate?code:CP-25?,a new compound independently developed by our institute,is based on paeoniflorin?Pae?structurally modified new reactive monomers.The previous research of the research group found that CP-25 can significantly inhibit the systemic and local joint synovial inflammation of adjuvant arthritis?AA?rats by regulating FLS function and the expression of immune cells.Therefore,this subject takes FLS as the research object,and establishes an AA rat model in vivo and gives CP-25 treatment at the same time.In vivo experiments study the effect of CP-25 on Ahr expression.In vitro,MH7A cell line was used to study the effect of Ahr activation on MH7A function,the regulation of CP-25 on Ahr expression and the interaction between Ahr and GRK2 and some mechanisms.Objectives1. To clarify the role of CP-25 in the treatment of autoimmune arthritis by regulating the expression of Ahr.2.To clarify that CP-25 can inhibit the expression of Ahr in synovial cells and regulate the interaction between Ahr and GRK2,which is one of the mechanisms of its therapeutic effect.Methods1. Wistar rats were used to establish AA model,which was randomly divided intomodel group,CP-25 group?50 mg/kg/day?,paroxetine group?15 mg/kg/day?,CP-25group?50 mg/kg/day?and paroxetine group?15 mg/kg/day?and normal control group.Each dose was calculated according to the body weight of the rats and administered by gavage once a day.The administration time was 16 days.Normal group and AA group were given normal saline for parallel control.2. Measure and evaluate the body weight,arthritis index,number of joint swelling,globalassessment and paw valume every 3 days,calculate thymus and spleen index,CCK-8detect the proliferation of thymus T cells and spleen B cells of rats,the staining of spleen and joint tissues,pathological changes and related indexes were observed,and the expression of IL-1?in serum of rats in each group was detected by ELISA.3.The FLS cells of normal group,AA group and each administration group were isolated and cultured.The expression of Ahr was detected by Western blot and laser confocal method,the proliferation ability of FLS was detected by CCK-8 method,the expression of IL-1?in the supernatant of FLS culture was detected by ELISA.4.In vitro experiment:Western blot,Laser Confocal method,CCK-8 method and Transwell plate method were used to detect the effect of CP-25 on the expression and function of Ahr in Kyn stimulated MH7A cells,CCK-8 method and Transwell method were used to detect the effects of MH7A cell proliferation and migration after Ahr interference.Co-Immunoprecipitation?Co-IP?and Laser Confocal methods to detected the interaction between GRK2 and Ahr.The effect of CP-25 on the expression of CYP1A1 in MH7A cells stimulated by Kyn was detected by Q-PCR.Results1.The effect of CP-25 on the the clinical manifestation,pathological changes and inflammatory cytokines in AA ratsCP-25 can significantly relive the arthritis index,paw swelling number and paw swelling degree of AA rats,and the effect of CP-25 with paroxetine combination is more obvious than that of alone,CP-25 can significantly alleviate the pathological changes of ankle joint and spleen tissue of AA rats,the results of ELISA show that under the inflammatory state,the expression of IL-1?in serum and synovial cell culture supernatant is significantly increased,while CP-25 and paroxetine can inhibit the expression of IL-1?,there was no significant difference in the effect of CP-25 with paroxetine on the expression of IL-1?.2. Effect of CP-25 on the expression of Ahr and the activation of immune cells of AA ratsThe results of immunohistochemistry showed that the expression of Ahr in synovium of AA rats was significantly higher than that in the normal group,suggesting that Ahr was involved in the pathological changes of synovium,and CP-25 could down regulate the expression of Ahr.CCK-8 showed that CP-25 could significantly reduce the proliferation of thymus T cells and spleen B cells.3. Effect of CP-25 on the expression and proliferation of Ahr in FLS of AA ratsWestern blot method and confocal laser scanning microscope method showed that compared with the normal group,the total expression and nuclear expression of Ahr in FLS of AA rats were significantly increased,suggesting that the expression of Ahr in inflammatory environment was increased and there was significant intranuclear metastasis,CP-25 could down regulate the expression of Ahr,CCK-8 results showed that CP-25 could significantly inhibit the proliferation of FLS.4. Effect of CP-25 on Ahr expression and function of MH7A cells stimulated by KynCompared with the control group,Kyn?200?M?treatment significantly promoted the proliferation and migration of MH7A cells after 48 h,CP-25 treatment inhibited the proliferation and migration of MH7A cells in vitro.Western blot method and Confocal laser scanning microscope method showed that Kyn?200?M?could significantly increase the expression of Ahr in MH7A cells,and CP-25(10-5mol/L)could significantly reduce the expression of Ahr 48 hours later.Q-PCR results showed that Kyn?200?M?promoted the expression of CYP1A1 in MH7A cells,and CP-25(10-5mol/L)significantly reduced the expression of CYP1A1.It is suggested that the increased of Kyn level can activate Ahr,regulate the gene expression mediated by Ahr,and CP-25 can inhibit the transcriptional activation mediated by Ahr.5. Effect of CP-25 on the co-expression of Ahr and GRK2 in MH7A cells stimulated by KynThe results of confocal laser showed that after 48 hours of Kyn?200?M?stimulation,the expression of GRK2 was significantly higher than that of the control group.The results of Co-IP showed that after 48 hours of Kyn?200?M?stimulation,the expression of Ahr and GRK2 in MH7A cells was significantly increased.After CP-25(10-5mol/L)was administered in vitro,the interaction between Ahr and GRK2was significantly inhibited.6. Effect of Ahr si RNA transfection on MH7A functionThe results of the CCK-8 method showed that compared with the control group,the proliferation response of MH7A after Ahr gene interference was reduced,and the transwell chamber method detected that its migration capacity was significantly reduced.Conclusions1.Compared with the control group,the expression of Ahr in synovium tissue of RA patients and synovium of AA rats are increased,suggesting that Ahr participates in the pathogenesis of RA.2.CP-25 significantly improves the systemic and local arthritis of AA rats,inhibits the proliferation of thymus T cells and spleen B cells,and reduces the secretion of IL-1?in serum and FLS culture supernatant.CP-25 can inhibit the proliferation of FLS in AA rats by regulating the expression of Ahr.3.Kyn promotes the proliferation and migration of MH7A cells,indicating that Kyn activation of Ahr may be involved in chronic joint synovial inflammation.CP-25 can regulate the interaction between Ahr and GRK2,thereby inhibiting the nuclear translocation of Ahr and the activation of downstream pathways,and inhibiting the abnormal activation of FLS,which may be one of the mechanisms for its therapeutic effects. |
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