Study Of Total Alkaloids From Sophora Alopecuroides On The Regulation Of Tfh Cells By TLRs-NF-κB Signal Pathway In CIA Mice

ObjectiveDBA/1J mice were immunized with type II collagen and following a second collagen immunization,the mice were treated with Total alkaloids of Sophora Alopecuroides(TASA)or dexamethasone(DEX)intraperitoneally once a day.The weight,arthritis index,arthritis score were evaluated.The percentages of T follicular helper cells(Tfh)and B cells were detected.The level of IL-21,IgG1,IgG2a and typeⅡcollagen-specific antibodies in serum were examined by Enzyme-linked immunosorbent assay,and the expression of TLR2,TLR4,TLR9,NF-κB p65,p-NF-κB p65,CXCR5,Bcl-6,ICOS in spleen follicular helper T cells and ankle joint were assessed to explore the protective effect of TASA on Tfh cells in CIA mice through the regulation of TLRs-NF-κB signaling pathway and its possible immunological mechanism,which may provide a theoretical basis for TASA in clinical treatment.MethodsThirty-six male DBA/1J mice were selected and divided into six groups(six in each group):collagen-induced arthritis(CIA),high dose group of TASA(OMT-H),medium dose group of TASA(OMT-M),low dose group of TASA(OMT-L),dexamethasone group(DEX),normal control group(NC).To mix Freund’s complete adjuvant with bovine type II collagen 1:1 and emulsify to a water-in-oil state.To take 100μl of the emulsion and inject it subcutaneously into the tail of the mice.The day of immunization is day 0,on the day of 21st,the emulsified mixture of Freund’s incomplete adjuvant and bovine type II collagen was subcutaneously immunized again in the tail roots of mice to construct a CIA model.During the modeling period,the body weight,arthritis index,and arthritis score of the mice were dynamically examined.On the day 15th,the mice were injected intraperitoneally with different concentrations of TASA,the normal and model groups were given the same amount of physiological saline,the positive drug control group was given dexamethasone injection.On the day 45th:(1)To evaluate the body weight,joint swelling degree and arthritis score every three days;(2)The spleen was weighed and the spleen index was calculated;(3)The number of germinal centers of the spleen and the joint histopathology were investigated by HE staining;(4)The expression of TLR2,TLR4,TLR9,NF-κB p65,p-NF-κB p65,CXCR5,Bcl-6,and ICOS in ankle joints were detected by immunohistochemistry and Western blot;(5)To detect the levels of IL-21,IgG1,IgG2a,and type II collagen-specific antibodies by ELISA.(6)Flow cytometry was used to assess the percentage of Tfh cells and B cells in spleen of CIA mice;(7)The spleen CD4~+T cells were sorted by immune magnetic beads and transplanted them into 24-well plates that pre-coated with CD3 and CD28.The CD4~+T cells were stimulated by IL-21 and IL-6 for 72h to obtain Tfh cells;(8)The mRNA transcription level and protein expression of TLR2,TLR4,TLR9,NF-κBp65,CXCR5,Bcl-6 and ICOS of spleen Tfh cells in CIA mice were detected by real-time fluorescent quantitative PCR and Western blot.Results(1)The CIA mouse model was established successfully;(2)Compared with the CIA model group,the joint swelling degree and arthritis score of the TASA high and middle dose groups were significantly reduced(p<0.05);(3)There are a large number of germinal centers in the spleen and the degree of destruction of the joint synovium in the model group mice was obvious,and a large number of inflammatory cells infiltrating with the formation of vascular crest.However,the synovium of the joints in high and medium doses groups had a little inflammatory cells infiltration,and no obvious formation of blood vessels were found.(4)Compared with the CIA model group,The expression of TLR2,TLR9,NF-κB p65,p-NF-κBp65,CXCR5,Bcl-6,and ICOS in the ankle joints of mice in the TASA high-dose group were significantly decreased(p<0.05);(5)High dose of TASA reduced the serum levels of IL-21,IgG1,IgG2a in the CIA group(p<0.05);(6)The spleen index and the number of germinal centers of mice in the high and middle dose of TASA groups were significantly reduced(p<0.05).(7)Compared with the CIA model group,the percentages of CD4~+CXCR5~+ICOS~+Tfh cells and B220~+GL-7~+B cells in TASA high and middle dose groups were significantly reduced(p<0.05);(8)High dose of TASA reduced the levels of TLR2,TLR4,TLR9,NF-κB p65,p-NF-κBp65,CXCR5,Bcl-6,and ICOS mRNA transcription and protein expression in CIA model group(p<0.05).ConclusionThe total alkaloids of sophora alopecuroides may down-regulate the frequency and function of Tfh cells and inhibit GCB cells via TLRs-NF-κB signal pathway to relieve the immune-pathological progression of CIA mice.