| Objective: multiple sclerosis(MS) is a autoimmune disease,which is characterized with demyelinating lesions in the white matter of the central nervous system(CNS) and is susceptible to both individual and environmental factors. The etiology and mechanism is not completely clear, but we have found that some inflammatory cytokines is the main cause. Various cytokines in an autocrine or beside the secreted form, participate in the immune response and tissue repair process, and play a decisive role in the severity and development of multiple sclerosis disease. Experimental autoimmune encephalomyelitis(EAE) is a autoimmune disease,which is mainly mediated by special induced CD4 + T cell,and is characterized with mononuclear cells infiltration and demyelination around the small blood vessels in the central nervous system(CNS). EAE is the ideal animal model for human multiple sclerosis(MS) disease research and is significant to the study of clinical neuroimmunology. Fingolimod(FTY720) is a kind of new immunosuppressive agent which is extracted from Cordyceps sinensis-multiple shell bacteria element(myriocin, ISP-1) and is processed by removing the chiral center, modifying the side chain.W hen it enter in vivo, sphingosine kinase 2 act it into a form: single phosphate compounds(FTY720- P).Then FTY720 mainly play a role in activation of sphingosine-1-phosphate(sphingosine-1-phosphate(S1P) receptor.Meanwhile, unlike other agents for the treatment of MS, FTY720's unique lipophilic characteristic can make it easily enter the central nervous system. In this experiment, we use fingolimod as drug intervention. After the treatment,we observe the incidence rate, the severity of disease and the influence on the course of EAE.We study if Fingolimod can influence the proinflammatory and anti inflammatory cytokines including IL-17,TGF-?,ROR?t,FOXP3 and if it can release the degree of inflammatory infiltration.Then we explore the protective effect of EAE.Methods: 60 female C57BL/6 mice which is 8 to 10 weeks old and is weighted 18 g to 20 g were randomly divided into control group, EAE group and FTY720 group, each group were 20. were used as antigen to establish the animal model.After the model were established, 0.3mg/kg FTY720 were orally administered per day to the FTY720 group.While the control group and EAE group were orally given the same amount of saline. From the beginning of first days of immunization, two observers used Weaver 15 points twice a day to weight the neurological scores of mice until killed. On the twentieth day after immunization,which is equivalent to the peak incidence stage of course,the mice were killed. 15 mice were choosed from each group,and its neck were broken lead to death. The brain and spinal cord were quickly removed and were frozen in liquid nitrogen and under 80 degrees refrigerator. Then we used real-time quantitative PCR method to detect the level of IL-17,TGF-?,ROR?t,FOXP3 in spinal cord. 5 mice were choosed from each group and 4% paraformaldehyde were perfused into the vessels of the mice. After fixation, washing, dehydration, clearing, paraffin, embedding, slicing, sticky film, made paraffin sections, HE staining were used to observe the infiltration situation of inflammatory cell. The experimental data were analyzed by SPSS 13 software, the measurement data were meant by standard deviation( x±s), analysis of multiple sets of measurement data were compared using ANOVA variance, P < 0.05 was statistically significant. The measurement data were compared between groups using snk-q test, P < 0.05 was statistically significant. The percentage was expressed as the incidence rates,which were compared by using the chi square test, P < 0.05 was statistically significant.Result:1 The incidence of FTY720 group decreased significantly compared with EAE group(P<0.05).2 In twentieth days of onset, FTY720 group average neurological function scores were significantly lower than group EAE(P<0.05).3 The level of IL-17,TGF-?,ROR?t,FOXP3 expression in spinal cord tissues of mice: In twentieth days of onset, compared with the control group, the level of IL-17, ROR?t in EAE group on average increased significantly and statistically(P < 0.05). Compared with the EAE group, the level of IL-17, ROR?t in FTY720 groups on average decreased significantly and statistically(P < 0.05).In twentieth days of onset, compared with the control group, the level of TGF-?, FOXP3 in EAE group on average decreased significantly and statistically(P < 0.05). Compared with the EAE group, the level of TGF-?, FOXP3 in FTY720 groups on average increased significantly and statistically(P < 0.05).4 HE staining: In twentieth days of onset, compared with the control group,the infiltration of inflammatory cells in mice's spinal cord tissue in EAE group and FTY720 group increased significantly and statistically(P < 0.05); compared with the EAE group, the infiltration of inflammatory cells in mice's spinal cord tissue in FTY720 group decreased significantly and statistically(P < 0.05).Conclusion:1 FTY720 can reduce the incidence of EAE mice, delay the onset of EAE mice, relieve the severity of nerve injury and promote the recovery of neurological function,which has a protective effect on EAE mice.2 FTY720 can increase the level of anti-inflammatory cytokine including TGF-? and FOXP3 and decreased the level of proinflammatory cytokine including IL-17 and ROR?t in the spinal cord tissue of mice.3 FTY720 can reduce the inflammatory infiltration in the spinal cord tissue of EAE mice and reduce the degree of inflammatory reaction of EAE mice. |
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