Effects Of DDN On TL1A/DR3 And Th9 Cell Subset In Collagen-induced Arthritis Mice

Rheumatoid arthritis(RA)is a refractory systemic autoimmune disease.The basic pathological manifestations are synovitis,synovial hyperplasia,pannus formation,and gradual destruction of articular cartilage and bone,eventually leading to joint deformity and loss of function.Although leflunomide,methotrexate and other drugs have been used to improve the condition of RA,the side effects are great and the patient’s tolerance is poor.Tumor necrosis factor-α(TNF-α)plays an important role in the occurrence and development of RA.Although biological antagonists such as TNF-αare effective for most patients,they are expensive,and nearly 30%of RA patients do not respond to the treatment.One possible explanation is the heterogeneity of the disease and the role of other TNF family members.TNF-like ligand 1A(TL1A),a member of TNF superfamily of proteins(TNFSF),was first discovered in 2002.TL1A and its receptor death receptor3(DR3,also known as TNFRSF25)play important roles in various autoimmune diseases such as RA,inflammatory bowel disease(IBD),systemic lupus erythematosus(SLE)and ankylosing spondylitis.A variety of cells and molecules are involved in the inflammatory response and tissue destruction of RA joints,and the activation of T cells is always considered to be the central link in the occurrence and development of RA.CD4~+T cells are the core and hub of cellular immunity.Th1,Th2,Th17,Treg and a new subset Th9 are closely related to the occurrence and development of RA.Th9 cell subset is a new CD4~+T cell subset first identified in 2008,which is characterized by the secretion of IL-9 cytokine.Studies have shown that Th9cell subsets and IL-9 are involved in the occurrence and development of autoimmune diseases such as RA,IBD and SLE.TL1A binds to its receptor DR3 and acts as a costimulatory signal to induce the activation and differentiation of T cells.In the presence of IL-4 and TGF-β,CD4~+T cells can differentiate into Th9 cell subsets,and TL1A can cooperate with TGF-βand IL-4 to promote the production of IL-9.Traditional Chinese medicine has the advantages of multiple targets,integrity and low toxic and side effects in the treatment of RA.It has been a research hotspot to find effective,low toxic and economic anti-RA drugs from TCM and explore their mechanism of action.The traditional Chinese medicine dioscorea nipponica has the functions of dispelling wind and removing dampness,promoting blood circulation and dredging collaterals.It is used to treat rheumatism,waist and leg pain,strain and sprain,and is considered as a spe CIAl medicine for arthralgia arthralgia.Dioscorea nipponica is effective in the treatment of RA with few adverse reactions,which is suitable for patients to take for a long time.A large number of previous experimental studies have confirmed that Pangolin has a positive effect on RA,and it can play a therapeutic role by regulating the balance of Th1/Th2 and Th17/Treg in CIA mice.CD4~+T cells are the core and hub of cellular immunity.Th1,Th2,Th17,Treg and a new subset Th9 are closely related to the occurrence and development of RA.On this basis,we further explored the regulatory effect of Dioscin of Dioscorea nipponica(DDN),the main medicinal component of Dioscorea nipponica,on Th9 cell subsets.Moreover,we investigated the effect of diosgenin on TL1A in vitro and found that diosgenin reduced TL1A protein expression in single cell suspension from lymph nodes of CIA mouse model in a dose-dependent manner.Therefore,we hypothesized that DDN may play a role in reducing inflammation and relieving RA symptoms by regulating TL1A,its receptor DR3and Th9 cell subsets differentiation-related factors.Objective:The purpose of this study was to establish a CIA mouse model treated with different doses of DDN,and then to detect the expression of TL1A and DR3.Th9 cell subsets and their specific transcription factors Purine-rich box 1(PU.1)and IL-9 expression,and Th9 cell differentiation related cytokines TGF-βand IRF-4 expression were investigated to explore the regulatory effect of DDN on TL1A/DR3 pathway and Th9 cell subsets.This study provides experimental basis for further revealing the pathogenesis of RA,searching for new therapeutic targets and elucidating the mechanism of traditional Chinese medicine pangolin in the treatment of RA.Methods:1.Establishment of CIA model and evaluation of therapeutic effectSixty male DBA1 mice aged 7-8 weeks were divided into control group,CIA model group,triptolide group and low,middle and high dose DDN group,with 10 mice in each group.The mice in CIA model group,triptolide group and DDN-low,DDN-mediumand,DDN-high dose groups were immunized by intradermally injection of 100μl complete Fred’s adjuvant and chicken typeⅡcollagen 1:1 mixed emulsion at the tail root,and the immunization was enhanced on the 21st day.The control group was injected with the same volume of saline as described above.The mice in the triptolide group were given triptolide tablet suspension(12μg/kg/d)by gavage from the first day after immunization,and those in the DDN-low,DDN-middle and DDN-high dose groups were given DDN suspension by gavage at the doses of 50,100 and 200mg/kg,respectively.The mice in the control group and the CIA model group were given equal volume of vehicle.Body weight,arthritis index,foot thickness and other indicators were also observed and recorded.Samples were taken on day 15 after reimmunization.2.To verify the regulatory effect of DDN on TL1A and DR3RT-q PCR was used to detect the m RNA expression levels of TL1A and DR3 in lymph node,spleen and knee synovial membrane.Western blot was used to detect the protein expression levels of TL1A and DR3 in lymph nodes,spleen and knee synovium.ELISA was used to detect the expression level of TL1A in serum.Flow cytometry was used to detect the expression of TL1A and DR3 in immune cells in lymph nodes and spleen.Immunofluorescence was used to detect the co-localized expression of TL1A and DR3 in the synovial tissue of the knee joint.3.To verify the regulatory effect of DDN on Th9 cell subsetsRT-q PCR was used to detect the m RNA expression levels of PU.1 in lymph node,spleen and knee synovial membrane.Western blot was used to detect the protein expression of PU.1 in lymph node,spleen and knee synovial membrane.ELISA was used to detect the expression of IL-9 in serum.Flow cytometry was used to detect the changes in the number of Th9 cell subsets in lymph nodes and spleen.4.To verify the regulatory effect of DDN on the differentiation related factors of Th9 cell subsetsRT-q PCR was used to detect the m RNA expression levels of TGF-βand IRF-4 in lymph nodes and knee joint synovitis.Western blotting was used to detect the protein expression of TGF-βand IRF-4 in lymph nodes and knee synovial membrane.Results:1.Assessment of modelingBody weight change:there was no significant difference among control group,CIA model group,triptolide group and DDN low,medium and high dose groups(P>0.05).Arthritis index score:compared with the control group,the CIA model group had increased scores(P<0.05).Compared with the CIA model group,the contrast scores of the triptolide group and the DDN group were decreased.The difference of paw thickness:compared with the control group,the difference of the CIA model group was increased(P<0.05).Compared with the CIA model group,the difference of paw thickness was reduced in the triptolide group and the DDN group.The results of pathological sections showed that the CIA model group had the symptoms of immune cell infiltration,bone erosion,cartilage destruction and synovial hyperplasia compared with the control group.Triptolide group and low,middle and high dose DDN groups had different degrees of therapeutic effect on CIA model,which could reduce or prevent the symptoms.2.DDN inhibited the expression of TL1A and DR3Compared with the control group,the expression of TL1A and DR3increased in CIA model group(P<0.05).After diosgenin treatment,m RNA and protein expression levels of TL1A and DR3 in lymph nodes,spleen and synovium of knee were decreased(P<0.05).Serum expression level of soluble TL1A was decreased(P<0.05).The co-localization of immunofluorescence showed that TL1A and DR3 were expressed on the surface of synovial tissue cells,and there was partial overlap,suggesting that TL1A and DR3 were expressed on different cell surfaces.The number of antigen-presenting cells and macrophages expressing TL1A on the surface of lymph nodes and spleen decreased(P<0.05)and the number of CD4~+T cells expressing DR3 decreased(P<0.05).3.DDN inhibited the expression of Th9 cell subsetsCompared with the control group,the expression of Th9 cell subsets was increased in the CIA model group(P<0.05).After diosgenin treatment,m RNA and protein expression of PU.1 in lymph nodes,spleen and synovial membrane of knee joint were decreased(P<0.05).The expression level of soluble IL-9 in serum decreased(P<0.05).Immunofluorescence showed that PU.1 was expressed in the synovial tissue cells,indicating Th9 cell infiltration in the synovial tissue.The number of Th9 cell subsets in lymph nodes and spleen was decreased(P<0.05).4.DDN inhibited the expression of differentiation related factors of Th9cell subsetsCompared with the control group,the expression of TGF-βand IRF-4 in the CIA model group increased(P<0.05).After DDN treatment,the m RNA and protein expression levels of TGF-βand IRF-4 in the lymph node and knee synovial membrane were decreased(P<0.05).Summary:1.DDN inhibited the expression of TL1A and DR3 in CIA mice.2.DDN inhibited the expression of Th9 cell-specific transcription factor PU.1,Th9 cell number and IL-9 secretion in CIA mice.3.DDN can inhibit the expression of TGF-βand IRF-4 in Th9 cell subsets.It may inhibit the differentiation of Th9 cells by inhibiting the TL1A/DR3pathway.