Exploring The Immune Mechanism Of Experimental Autoimmune Encephalomyelitis Based On Tc17 Cells And The Intervention Effect Of Huangqi Guizhi Wuwu Decoctio

Objective:The primary aim of this study is to determine the similarities and differences between Th17 and IL-17 secreting CD8+cytotoxic T cells(Tc17)in MS/EAE.Based on the findings of primary aim,the secondary aim of this study is to understand the therapeutic effects of specific Tc17 cells when Huangqi Guizhi Wuwu decoction(HQGZWW)treat EAE,and hope to provide a theoretical and experimental basis for the new clinical treatment of MS.Methods:In this study,myelin oligodendrocyte glycoprotein peptides 35-55(MOG35-55),pertussis toxin(PTX),and complete Freund’s adjuvant(CFA)were used to induce female C57BL/6 mice to establish experimental autoimmune encephalomyelitis(EAE)animal model(n=20).Twelve to fourteen days after immunization,T cells were isolated from the spleen and purified into CD4+and CD8+T cells using CD4 and CD8 isolation kits,followed by flow cytometry analysis to determine purity and prepare MOG35-55 specific T cells.Interferon γ(IFN-γ)and interleukin(IL)-17 secretion in the supernatants of cultured CD4+and CD8+T cell are determined by enzyme-linked immunosorbent assay(ELISA).After adoptive transfer stimulation,Tc17 cells and Th17 T cells were tested for pathogenicity.The clinical course was measured by grade 0-5 EAE score,and the spinal cord was examined by hematoxylin and eosin(HE)and Luxol fast blue staining(LFB).Finished the primary aim,MOG35-55 EAE animal mice(n=32)were divided into 4 groups of 8 mice in each group.Two different doses(50g/kg in the high-dose group and 5g/kg in the low-dose group)of HQGZWW were used as the experimental group,prednisone(6mg/kg)as the positive control,and distilled water as the negative control.Starting 1 hour after immunization,two different doses of HQGZWW,prednisone and distilled water were gavaged for 14 days,the therapeutic efficacy was evaluated by EAE score,body weight,spinal cord histology and myelin staining,antigen-specific detection,and Elisa detection of IFN-γ and Il-17 secretion in supernatants of cultured TC17 and Th17 cells.Results:1.Purified CD8+CD3+and CD4+CD3+cells are respectively88.2%and 90.1%of the total CD3+cells after beads-purification,and these cells are in duced to Tc17 cells and Th17 T cells as described in methods.Tc17 cells and Th17 T cells were used MOG3 5-55 peptide for proliferation assays,an d it was shown that compared with Th17 T cells,Tc17 cells had less res ponse to MOG35-55,however this response was not depended on Th17 T c ells.Tc17 cells secreted less IFN-γ and IL-17 than Th17 T cells.2.In tEAE mouse models,similar EAE scores and slight inflammation an d demyelinating were found in Tc17 cells-tEAE mice compared to Th17 c ells-tEAE mice.3.Compared with low-dose HQGZWW and distilled water,EAE score and pathology were significantly improved in the high-dose HQGZWW group and prednisone group.Conlusion:1.Tc17 cells in EAE have independent pathogenicity,however the degree is lower than Th17 cells.The profiles of cytokine secreted by Tc17 cells after antigen stimulation is similar to those of Th17 cells.2.The effect of high-dose HQGZWW on EAE was similar to that of pred nisone,while low-dose HQGZWW did not.