| Rheumatoid arthritis(RA)is a chronic autoimmune disease characterized by joint injury and synovial inflammation,and its pathogenesis remains unclear.They may be caused by excessive activation of fibroblast-like synoviocyte(FLS)of non-immune cells and immune cells such as peritoneal macrophage(p M),dendritic cells(DC),T cells and B cells.The metabolic pathway of L-tryptophan(Trp)plays an important role in the immune system.Trp is an essential amino acid in human body and a precursor of many signal molecules,which can regulate the adaptive immune response.The vast majority of Trp is metabolized through the L-kynurenine(Kyn)pathway.This pathway produces Kyn and its downstream products,which are involved in inflammatory and immune responses.In the normal body,Trp and Kyn maintain a dynamic balance,but when the concentration of Trp and Kyn in the body changes,RA and other autoimmune diseases,kidney diseases,viral infections or malignant tumors and other diseases will be induced.Due to the close relationship between Kyn metabolism and inflammatory response,Kyn has become a recognized participant in RA and other diseases.Indoleamine 2,3 dioxygenase 1(IDO1),Indoleamine 2,3 dioxygenase 2(IDO2)and tryptophan 2,3 dioxygenase(TDO2)are the first rate-limiting enzymes that catalyze the changes in Trp and Kyn contents caused by this metabolic pathway.Collagen induced arthritis of mice liver and Trp concentration in serum is reduced,Kyn concentration increases,IDO1 role in the immune system is more clear,in normal expressed in p M,DC cells in the body is low,and it has a lower substrate selectivity,and in such as RA that can be in the body of illness interferon-?,interleukin 1 rise significantly high expression of inflammatory factors such as IDO1 mainly affects the initial phase of the immune response.TDO2 is mainly expressed in mammalian liver under physiological conditions,with high substrate selectivity,and plays a dominant role in the metabolic pathway of Trp.In tumor immunity,TDO2 can promote the differentiation of Th1 and Th17.However,the role of TDO2 in the immune system is poorly understood.This subject through establishing an adjuvant-induced arthritis(AA)model in rats,studies the expression of TDO2 in different tissues and cells of AA rats through in vivo and in vitro experiments,and preliminarily discusses its functional mechanism.Objective: To detect the expression of TDO2 in different tissues and cells;to study the effects of TDO2 inhibitors on various cell functions,to elucidate the role of TDO2-mediated tryptophan pathway in the pathogenesis of RA,and to inhibit the effectiveness of TDO2 in improving RA;whether it can become a new target for the treatment of RA.Methods: The serum and liver tissues of AA rats were taken at various stages of AA rats by establishing an AA rat model.The concentrations of Trp and Kyn in serum and liver of AA rats were detected by HPLC.The normalization was detected by immunohistochemistry.And the expression of IDO1 and TDO2 in synovial joint tissues and liver tissues of AA rats,observed the expression changes of IDO1 and TDO2 in various stages of AA rats;isolated and cultured FLS,p M,T,B of normal and AA rats The expression of TDO2 in immune cells and non-immune cells was detected by western blot and laser confocal scanning microscope.The effect of Kyn and TDO2 inhibitors 680C91 on the proliferation of FLS and FLS transfected with TDO2 si RNA was detected by CCK-8 assay.Transwell plate method was used to detect the effects of Kyn and 680C91 on the migration and invasion of FLS and FLS transfected TDO2 si RNA.The effect of 680C91 on i NOS and Arg1 in p M cells was detected by western blot,the effect of 690C91 on p M M1 and M2 polarization was observed;the effect of Kyn and TDO2 inhibitors on the proliferation of T and B lymphocytes was detected by CCK-8 method.Results: 1.Changes of Trp,Kyn concentration and Kyn/Trp ratio in serum and liver of different stages of AA rats Compared with the normal group,the concentration of Trp in different inflammatory phases of AA rats decreased in serum and liver,Kyn concentration increased,and Kyn/Trp ratio increased.The Kyn/Trp ratio in the serum of AA rats was significantly increased in the early stage,while the Kyn/Trp ratio in the liver tissues was significantly increased in the peak period of AA rats.2.Expression changes of IDO1 and TDO2 in synovial tissue and liver tissue of AA rats at different stages of inflammation Compared with the normal group,the expression of IDO1 was increased in the synovial tissues of different stages of AA rats,and the expression was highest in the early stage.The expression of TDO2 in the synovial tissue and liver tissues of the AA rats increased at different stages of inflammation,the highest expression was observed in the peak phases of AA rats.3.Expression changes of TDO2 in FLS,p M,T,B lymphocytes Western blot showed that the expression of TDO2 in FLS,p M,T,B lymphocytes was significantly higher than that in the normal group.The results of laser confocal scanning microscope and western blot were consistent.TDO2 was expressed in the cytoplasm of FLS.Compared with the normal group,the expression in AA rats was increased,and it was expressed in the cytoplasm and nucleus of p M.It is also elevated compared to the normal group.4.Effect of Kyn and TDO2 inhibitor 680C91 on proliferation of T and B lymphocytes Compared with the control group,Kyn(50?M,100?M,200?M)also promoted the proliferation of T and B lymphocytes in normal and AA rats.680C91(2.5?M,5.0?M,10.0?M)was administered in a concentration-dependent manner.It inhibits the proliferation of T and B lymphocytes.5.Effect of TDO2 inhibitor 680C91 on p M polarization in AA rats 680C91 can reduce the expression level of i NOS and increase the expression level of Arg1,suggesting that 690C91 can inhibit the polarization of M1 and promote the polarization of M2.6.Effect of Kyn and TDO2 inhibitor 680C91 on FLS function in AA rats Compared with the control group,Kyn(50?M,100?M,200?M)significantly promoted the proliferative response,migration and invasion of FLS in normal and AA rats.In vitro administration of 680C91(2.5?M,5.0?M,10.0?M)inhibited the proliferation,migration and invasion of FLS abnormalities in AA rats.7.Effect of transfection of TDO2 si RNA on FLS function in AA rats Compared with the control group,the TDO2 gene interfered with FLS proliferation,migration and invasion was reduced,and the function and activation ability of FLS recovered after Kyn was added in vitro.Conclusions: 1.TDO2 expression and enzyme activity were higher in the peak stages of AA rats,while IDO1 expression and enzyme activity were higher in the early stage of AA rats,suggesting that both TDO1 and IDO1 are involved in the tryptophan pathway in RA,but TDO2 is involved in the peak phases of RA,and IDO1 plays a role in the early stages.2.The expression of TDO2 in non-immune cell FLS and immune cells p M,T cells and B cells of AA rats was significantly increased,suggesting that the high expression of TDO2 in each cell may be involved in the development of RA.3.Kyn can promote the proliferation,migration and invasion of normal and AA rats,and enhance the proliferation of T and B lymphocytes,indicating that Kyn may have a pro-inflammatory effect on normal rats,and can also aggravate the inflammation of AA rats.4.TDO2 inhibitor can inhibit the proliferation,migration and invasion of AA rats,reduce the proliferation of T and B lymphocytes,and inhibit the polarization of M1 in p M,and promote the polarization of M2,suggesting that if TDO2 is reduced Expression or activity can slow the onset or progression of inflammation.5.TDO2 plays an important role in the development of RA,so it is expected to become a new target for the treatment of RA by inhibiting the expression of TDO2 or its enzyme activity. |
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