Effects Of Teriflunomide Combined With Total Glucosides Of Paeony On Balance Of Th17/Treg Cells In Mice With Experimental Autoimmune Encephalomyelitis

Objective: To explore effects of Teriflunomide(TIF)combined with Total glucosides of paeony(TGP)on the ratio of Th17,Treg cells and the expression of cytokines IL-17 and IL-10 from the spleen tissue of experimental autoimmune encephalomyelitis(EAE)mice,in order to investigate the therapeutic effect of combination therapy on EAE mice,and so we could provide a theoretical basis for the combination therapy to multiple sclerosis in the clinical work.Methods: 75 female C57BL/6 mice(18g to 20g)which were 8 to 10 weeks old,were randomly divided into 5 different groups,15 for Control,EAE,TIF,TGP and TIF+TGP group separately.The EAE animal model was established with MOG35-55,supplemented by Complete Freund's Adjuvant,Mycobacterium tuberculosis,and Pertussis Toxin,while the Control group was not granted immunity.The day on which the mice were immunized was recorded as Day 0.From Day 1,the TIF group mice were orally given TIF 15mg/kg;the TGP group mice were orally given TGP 500mg/kg;the TIF+TGP group mice were orally given TIF 15 mg/kg and TGP 500mg/kg.The Control group and EAE group mice were orally given the same amount of solvent.From the beginning of immunization,two observers observed the incidence of mice twice a day respectively and recorded weight changes and the neurological scores by using Weaver 15 points.Mice were sacrificed on day26 after immunization.The spleen cells were removed from the spleen tissue of the mice under sterile conditions for culture.The ratio of Th17 and Treg cells in each group was determined by flow cytometry.The m RNA expression levels of cytokines IL-17 and IL-10 of the spleen tissue were detected by real-time quantitative PCR.The levels of IL-17 and IL-10 in culturedsupernatants of spleen cells were detected by ELISA.Results:1 The incidence of mice in each group: There was no significant difference about morbidity among the EAE group,the TIF group and the TGP group(P> 0.05),and the TIF+TGP group reduced significantly compared with the EAE group,the TIF group and the TGP group(P<0.05),however no mice in the Control group got sick.The mean clinical scores of mice in the TIF group,the TGP group and the TIF+TGP group reduced significantly compared with the EAE group(P<0.05),and the TIF+TGP group reduced significantly compared with the TIF group and the TGP group(P<0.05),while there was no significant difference between the TIF group and the TGP group(P> 0.05).2 The ratio of Th17 and Treg cells in each group: comparing to the Control group,the ratio of Th17 cells were significantly increased(P<0.05)and Treg cells were significantly decreased(P<0.05)in the EAE group;comparing to the EAE group,the ratio of Th17 cells were significantly decreased in the TIF group,the TGP group and the TIF+TGP group(P<0.05),and the ratio of Treg cells were significantly increased in the TGP group and the TIF+TGP group(P<0.05),but there was no significant difference in the TIF group(P> 0.05);the ratio of Th17 cells were significantly decreased(P<0.05)and Treg cells were significantly increased(P<0.05)in the TIF+TGP group comparing to the TIF group and the TGP group;there was no significant difference between the TIF group and the TGP group(P> 0.05).3 The m RNA expression levels of IL-17 and IL-10 of the spleen tissue:comparing to the Control group,the m RNA expression levels of IL-17 were significantly increased(P<0.05)and IL-10 were significantly decreased(P<0.05)in the EAE group;comparing to the EAE group,the m RNA expression levels of IL-17 were significantly decreased(P<0.05)and IL-10 were significantly increased(P<0.05)in the TIF group,the TGP group and the TIF+TGP group;the m RNA expression levels of IL-17 were significantly decreased(P<0.05)and IL-10 were significantly increased(P<0.05)in the TIF+TGP group comparing to the TIF group and the TGP group;there was nosignificant difference between the TIF group and the TGP group about the m RNA expression levels of IL-17 and IL-10(P> 0.05).4 The concentration of IL-17 and IL-10 in cultured supernatants of spleen cells: comparing to the Control group,the concentration of IL-17 were significantly increased(P<0.05)and IL-10 were significantly decreased(P<0.05)in the EAE group;comparing to the EAE group,the concentration of IL-17 were significantly decreased(P<0.05)and IL-10 were significantly increased(P<0.05)in the TIF group,the TGP group and the TIF+TGP group;the concentration of IL-17 were significantly decreased(P<0.05)and IL-10 were significantly increased(P<0.05)in the TIF+TGP group comparing to the TIF group and the TGP group;there was no significant difference between the TIF group and the TGP group about the concentration of IL-17 and IL-10(P>0.05).Conclusions:1 TIF and TGP could reduce the neurological scores of EAE,and combination drug therapy had a more obvious effect and could reduce the morbidity significantly.2 TIF and TGP could decrease the ratio of Th17 cells and the m RNA and protein expression levels of IL-17.TIF could increase the m RNA and protein expression levels of IL-10.TGP could increase the ratio of Treg cells and the m RNA and protein expression levels of IL-10.Combination drug therapy had a more obvious effect.3 TIF combined with TGP may protect against EAE mice by modulating Th17/Treg cells balance.