| Multiple sclerosis(MS)is an autoimmune disease involving the central nervous system(CNS).The clinical manifestations are characterized by multiple lesion symptoms and relapsing-remitting disease course.Multiple sclerosis is the common cause of permanent neurological dysfunction in young and middle-aged people.Experimental autoimmune encephalomyelitis(EAE)can simulate MS from a variety of aspects such as pathogenesis and pathological changes,and is widely used in the study of pathogenesis and treatment of MS.Although current research suggests that autoimmune imbalance,inflammation,apoptosis and oxidative stress were all involved,the exact mechanism of neurodegeneration in MS remains to be elucidated.Although current disease-modifying drug treatment can slow the progression of the disease to a certain extent and improve the subjective discomfort of patients,neurological deficits often last along the patient's entire life.Therefore,strengthening the exploration of new pathogenesis and treatment methods for multiple sclerosis has become an important issue.The normal function of the nervous system depends on the stability of immune function.The immune homeostasis is associated with multiple neurological autoimmune diseases such as multiple sclerosis.Immunomo-dulatory therapy is also an important method for the treatment of multiple sclerosis.The regulation of nervous system immune function involves multiple molecules or reaction pathways.Based on our recent research and literature search results,we proposed a new concept of immunoregulatory pathway:soluble CD83(sCD83)-indoleamine 2,3-dioxygenase(IDO)-tryptophan kynurenine pathway.sCD83 is an immunoregulatory molecule,indoleamine 2,3-dioxygenase and tryptophan kynurenine pathway products are involved in neuroimmunomodulation,CD83+T cells have immuno-modulatory functions.All aspects of this pathway are of great significance for MS treatment.Based on the above immunomodulatory pathway,in this study,we used an indoleamine-2,3-dioxygenase agonist(Tranilast),an antagonist(NLG919),and an antioxidant(Lipoic acid)as interfering agents,respectively,to treat a mouse model of experimental autoimmune encephalomyelitis induced in mice.From the aspects of myelin axon pathological changes,tryptophan kynurenine metabolism changes,inflammatory factor spectrum changes,apoptosis and oxidative stress damage,immune regulatory cell changes,we tried to explore the pathogenesis of multiple sclerosis and the implications of intervention agents for treatment.Part one The investigation of the effects of indoleamine-2,3-dioxygenase antagonist on experimental autoimmune encephalomyelitisObjective:To establish a mouse model of experimental autoimmune encephalomyelitis in C57BL/6 female mice as the study model of multiple sclerosis,indole-2,3-dioxygenase antagonist NLG919 was used for intervention,The incidence and symptoms of experimental miceand were observed.HE staining,electron microscopy,Western Blot,ELISA,flow cytometry were all applied to study the effects of NLG919 on EAE mice from the aspects of inflammatory pathological changes,apoptosis,oxidative stress,immune regulation.Methods:1.Establishment of a mouse model of experimental autoimmune encephalomyelitis.C57BL/6 female mice,about 8-10 weeks old,weighed about 18-20 g.The EAE model was induced in mice by subcutaneously injecting into the hindquarters with MOG35-55(250?g for four sites)emulsified in an equivalent volume of Complete Freund's Adjuvant,containing additional 4mg/ml of heat-killed mycobacterium tuberculosis H37Ra,immunized mice also received two intraperitoneal(i.p.)injections with 500ng of pertussis toxin.2.Grouping and intervention of experimental animals.The experimental mice were randomly divided into normal control group,EAE+vehicle group and NLG919 intervention group.A suspension of NLG919 was prepared with 1.5%sodium carboxymethylcellulose,and was administered daily for 7 days from the first day of onset.The NLG919 was administered orally at a dose of 50 mg/kg.The normal control group and the EAE+vehicle group mice were administered respectively with an equal amount of normal saline and 1.5%sodium carboxymethylcellulose daily for 7 consecutive days from the first day after the onset of the disease.3.Neurological function score.On the first day after immunization,the mice were scored and weighed twice by two laboratory assistants at two time points each morning and evening(8:00 and 16:00).The neurological function scores were recorded used Knoz 5 points method.4.Histopathological observation.The infiltration of inflammatory cells in the lumbar enlargement of the spinal cord of the experimental mice was observed by HE staining.The changes of myelin,axon and mitochondria in the lumbar spinal cord of the experimental mice were observed by transmission electron microscopy.5.The expression of amyloid precursor protein(APP)and the expression level of myelin basic protein(MBP)in the spinal cord of each group were detected by Western Blot.6.ELISA was used to evaluate the expression levels of inflammatory cytokines IFN-y,IL-10,IL-17A and TGF-?1,to evaluate the changes of oxidative stress factor 8-OHdG expression level,and to evaluate the changes of peripheral cell apoptosis factor caspase-8 expression levels.7.Flow cytometry was used to assess changes in the expression levels of peripheral immune-regulated cells(Tregs).8.Statistical analysis.Statistical analysis was performed using SPSS 21.0 statistical software(SPSS Inc.,Chicago,IL,USA).Measurement data are expressed as "mean ± standard deviation'(x ± s).The data was tested for normality and homogeneity of variance.Statistical analysis of differences between the two groups was performed using Student's t test,and statistical differences between the three or more groups were analyzed by one-way ANOVA and Newman-Keuls multiple comparison test.The difference was statistically significant at P<0.05.Results:1.The intervention of NLG919 on EAE mice worsened the clinical symptoms and increased the neurological score of EAE mice;2 NLG919 intervention in EAE mice can aggravate the infiltration of inflammatory cells in the central nervous system(lumbar enlargement);3.After intervention of ELG mice in EAE mice,it was observed by transmission electron microscopy that NLG919 can aggravate the degree of destruction of the spinal cord lumbar myelin,axons and mitochondria;4.Intervention of NLG919 in EAE mice reduced cytokine TGF-?1 expression,increased IL-10,significantly increased IFN-y expression,and IL-17A expression showed a similar trend to IFN-y;5.Intervention of NLG919 in EAE mice resulted in a significant increase in 8-OHdG production;6.Intervention of NLG919 drugs in EAE mice reduced caspase-8 expression;7.Intervention of NLG919 drugs in EAE mice resulted in a significant reduction in Tregs levels.Part two The investigation of the effects of indoleamine-2,3-dioxygenase agonist on experimental autoimmune encephalomyelitisObjective:To establish a mouse model of experimental autoimmune encephalomyelitis in C57BL/6 female mice as the study model of multiple sclerosis,indole-2,3-dioxygenase agonist Tranilast was used for intervention.The incidence and symptoms of experimental miceand were observed.HE staining,electron microscopy,Western Blot,ELISA,RT-PCR,flow cytometry were all applied to study the effects of Tranilast on EAE mice from the aspects of inflammatory pathological changes,apoptosis,oxidative stress,immune regulation.Methods:1.Establishment of a mouse model of experimental autoimmune encephalomyelitis.C57BL/6 female mice,about 8-10 weeks old,weighed about 18-20 g.The EAE model was induced in mice by subcutaneously injecting into the hindquarters with MOG35-55(250?g for four sites)emulsified in an equivalent volume of Complete Freund's Adjuvant,containing additional 4mg/ml of heat-killed mycobacterium tuberculosis H37Ra,immunized mice also received two intraperitoneal(i.p.)injections with 500ng of pertussis toxin.2.Grouping and intervention of experimental animals.The experimental mice were randomly divided into normal control group,EAE+vehicle group and Tranilast intervention group.A suspension of Tranilast was prepared with 1.5%sodium carboxymethylcellulose,and was administered daily for 7 days from the first day of onset.The Tranilast was administered orally at a dose of 200 mg/kg.The normal control group and the EAE+vehicle group mice were administered respectively with an equal amount of normal saline and 1.5%sodium carboxymethylcellulose daily for 7 consecutive days from the first day after the onset of the disease.3.Neurological function score.On the first day after immunization,the mice were scored and weighed twice by two laboratory assistants at two time points each morning and evening(8:00 and 16:00).The neurological function scores were recorded used Knoz 5 points method.4.Histopathological observation.The infiltration of inflammatory cells in the lumbar enlargement of the spinal cord of the experimental mice was observed by HE staining.The changes of myelin,axon and mitochondria in the lumbar spinal cord of the experimental mice were observed by transmission electron microscopy.5.Western Blot was used to assess the changes of sCD83 levels in serum.6.RT-PCR and ELISA were used to evaluate the changes of KYN and indoleamine 2,3-dioxygenase(IDO)expression levels.7.The levels of inflammatory related cytokines IFN-y,IL-10,IL-17A,and TGF-?1 were evaluated by ELISA.8.The change in the expression level of oxidative stress factor 8-OHdG was evaluated by ELISA.9.The change in the expression level of apoptotic factor Caspase-8 was evaluated by ELISA.10.Flow cytometry was used to assess changes in the expression levels of peripheral immune-regulated cells(Tregs).11.Statistical analysis.Statistical analysis was performed using SPSS 21.0 statistical software(SPSS Inc.,Chicago,IL,USA).Measurement data are expressed as "mean±standard deviation"(x±s).The data was tested for normality and homogeneity of variance.Statistical analysis of differences between the two groups was performed using Student's t test,and statistical differences between the three or more groups were analyzed by one-way ANOVA and Newman-Keuls multiple comparison test.The difference was statistically significant at P<0.05.Results:1.Tranilast treatment significantly reduced the mean maximum neurological score in EAE mice,and the course of the tranilast-treated mice was shortened and recovery accelerated.2.The HE staining of the pathological tissue sections of the EAE vehicle group showed diffuse inflammatory cells,and the inflammatory cells of the EAE+Tranilast group were significantly reduced.Lighter myelin destruction,axonal degeneration,and mildly damaged mitochondria were observed in the EAE-Tranilast group of mice.3.Western blot results showed that the level of sCD83 in the EAE-Tranilast group was particularly high,and the difference was statistically significant.4.RT-PCR and ELISA results showed that IDO mRNA and KYN expression were significantly increased in the Tranilast treated group compared with the EAE-Veh group.5.ELISA results showed that compared with EAE-Veh,Tranilast treatment significantly reduced IFN-y expression,and IL-17A expression in different groups showed a similar trend to IFN-y.The expression of TGF-?1 in the Tranilast group was slightly lower than that in the EAE-Veh group.The level of IL-10 was most prominent in the Tranilast group,and the difference between the groups was statistically significant.6.The ELISA evaluation of oxidative stress factor showed that the 8-OHdG level in the EAE-Veh group was significantly higher than that in the control group and the Tranilast treatment group.7.The results of ELISA evaluation of apoptotic factors showed that the expression of caspase-8 in the EAE-Tranilast group was significantly increased compared with the EAE-Veh group and the control group.8.Flow cytometry evaluation of immunoregulatory cell expression showed that the number of Tregs in the Tranilast group was significantly higher than that in the control group and the EAE-Veh group.Part three The study of effects of lipoic acid on experimental autoim-mune encephalomyelitis miceObjective:To establish a mouse model of experimental autoimmune encephalomyelitis in C57BL/6 female mice as the study model of multiple sclerosis.based on soluble CD83-indoleamine-2,3-dioxygenase-tryptophan kynurenine pathway,lipoic acid was used for intervention.The incidence and symptoms of experimental mice were observed.Hematoxylin-eosin staining(HE staining),electron microscopy,Western Blot,ELISA,RT-PCR,flow cytometry were all applied to study the effects of lipoic acid on EAE mice from the aspects of inflammatory pathological changes,tryptophan metabolism,immune regulation.Methods:1.Establishment of a mouse model of experimental autoimmune encephalomyelitis.C57BL/6 female mice,about 8-10 weeks old,weighed about 18-20 g were used.The EAE model was induced in mice by subcutaneously injecting into the hindquarters with MOG35-55(250?g for four sites)emulsified in an equivalent volume of Complete Freund's Adjuvant,containing additional 4mg/ml of heat-killed mycobacterium tuberculosis H37Ra,immunized mice also received twice intraperitoneal(i.p.)injections with 500ng of pertussis toxin.2.Grouping and intervention of experimental animals.The experimental mice were randomly divided into normal control group,EAE+vehicle group and lipoic acid intervention group.Lipoic acid was dissolved in 100 mM Tris buffer of pH 7.4,and it was intraperitoneally injected into EAE mice daily at 100 mg/kg for 7 days from the first day after onset.The normal control group and the EAE+vehicle group mice were administered intraperitoneally an equal amount of normal saline and Tris buffer daily for 7 consecutive days from the first day after onset.3.Changes in the expression levels of indoleamine 2,3-dioxygenase(IDO)and KYN were evaluated by RT-PCR and ELISA,respectively.4.Changes in the levels of the inflammation related cytokines TNF-a,IL-10,and TGF-?1 were evaluated by ELISA.5.Serum sCD83 levels in EAE mice were detected by western-blot.6.The number of Tregs cells and the number of CD4+CD83+T cells in whole blood of each group of experimental mice were detected by flow cytometry.7.Statistical analysis.Statistical analysis was performed using SPSS 21.0 statistical software(SPSS Inc.,Chicago,IL,USA).Measurement data are expressed as "mean±standard deviation"(x±s).The data was tested for normality and homogeneity of variance.Statistical analysis of differences between the two groups was performed using Student's t test,and statistical differences between the three or more groups were analyzed by one-way ANOVA and Newman-Keuls multiple comparison test.The difference was statistically significant at P<0.05.Results:1.RT-PCR and ELISA results showed that the expression of IDO mRNA and KYN in the acute phase EAE vehicle group was significantly higher than that in the lipoic acid-treated group.2.The ELISA results showed that compared with the EAE vehicle group,the expression of TNF-a was decreased,the expression of TGF-?1 was increased,and the level of IL-10 was significantly increased.3.Serum sCD83 levels in EAE mice were detected by western-blot.The results showed that the level of sCD83 in the lipoic acid-treated group was slightly lower than that in the acute phase EAE+vehicle group.4.Flow cytometry was used to detect the number of Tregs and CD4+CD83+T cells in whole blood of the experimental mice.The results showed that the number of Tregs cells in the EAE+lipoic acid group was significantly increased compared with the EAE+vehicle group.The number of CD4+CD83+T cells in the lipoic acid treatment group was reduced compared to the acute phase EAE+vehicle group.Conclusion:1.Indoleamine-2,3-dioxygenase inhibitor NLG919 aggravates EAE symptoms and pathological damage.The possible mechanisms included promoting inflammatory reaction and oxidative stress,inhibiting peripheral cell apoptosis and reducing the production of immune regulatory cells.2.Indoleamine-2,3-dioxygenase agonist Tranilast improves EAE disease and alleviates pathological damage.The possible mechanisms included inhibition of inflammatory response and oxidative stress,promotion of peripheral cell apoptosis,and increase of immunoregulatory cells and molecules.3.Lipoic acid reduced the metabolism of kynurenine in tryptophan,inhibited inflammatory response,and promoted the relative expression of sCD83,an increase in the number of peripheral Tregs cells and CD4+CD83+T cells. |
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