Effects Of NAD~+ On SIRT3/AMPK/mTOR Signaling Pathway Of Experimental Autoimmune Encephalomyelitis Mice

Objective:This study aimed to explore the effects of nicotinamide adenine dinucleotide?NAD⁺?on the expression of silent information regulator of transcription 3?SIRT3?from the brain tissue of experimental autoimmune encephalomyelitis?EAE?mice,and investigate the possible pathways and mechanisms of NAD⁺in the treatment of EAE mice,and find new ways to treat multiple sclerosis.Methods:1.C57BL/6 female mice?8-10 weeks old,body weight 18-20g?were chosen as experimental subjects.Then EAE mice model was induced by subcutaneously injecting into the hindquarters with the immunizing antigen MOG_35-55 peptide,emulsified in an equivalent volume of Complete Freund's Adjuvant,containing additional 4 mg/ml of heat-killed mycobacterium tuberculosis H37Ra,on the day of immunization?day 0 on the day of immunization?and the second day,immunized mice also received two intraperitoneal injections with 500ng of pertussis toxin?PTX?.2.The experimental mice were randomly divided into three groups:normal control group?Control group?,EAE model group and NAD⁺treatment group,with 8 mice in each group.From the first day after immunization,NAD⁺treatment group were intraperitoneally injected with 250 mg/kg NAD⁺in phosphate buffered saline?PBS?per day,another two groups were intraperitoneally injected with the same amount of PBS once daily.From day1 of immunization,the mice were observed by two observers twice daily and the body weight changes and neurological function scores according to Weaver's 15 method were recorded everyday.3.On the 25th day after immunization,four mice of each group were euthanized with perfusion.The lumbar spinal cord and brain of those mice were isolated and stained by HE and LFB,to observe the degree of inflammatory cell infiltration and demyelination.The expression of SIRT3 in each group also observed by immunohistochemical staining.The other four mice of each group were euthanized freshly,the expression of SIRT3,AMPK,p-AMPK,mTOR and p-mTOR protein in brain were detected by Western blot.4.SPSS 21.0 statistical software was used for statistical analysis.The measurement data were expressed as mean±standard deviation?Mean±SD?.When the comparison between the groups of measurement data is normal,the variance analysis of completely random design is adopted,the SNK-q test is applied when the variance is uniform,otherwise,the Dunnett's T3 test is applied,and the Kruskal-Wallis H test is applied when the variance is not normal.The disease course of mice in each group was expressed as a percentage and statistical analysis was performed using a chi-square test.Clinical neurological function scores were statistically analyzed using the Mann-Whitney U test.P<0.05 was considered as statistically significant.Results:1.NAD⁺treatment could reduce the incidence of EAE mice and alleviate the symptoms of neurological damage in EAE mice.2.Comparisons between with the Control group and the EAE group,showed that NAD⁺intervention can alleviate the inflammatory cells infiltration and demyelination of central nervous system,and increased the expression of SIRT3 in brain.3.NAD⁺treatment could increase the expression of SIRT3 and p-AMPK protein and decrease the expression of p-mTOR protein in brain of EAE mice.But there was no significant effect on the expression levels of AMPK and mTOR proteins.Conclusions:1.NAD⁺treatment could protect against experimental autoimmune encephalomyelitis?EAE?mice and increase the SIRT3 expression.2.NAD⁺might attenuates EAE by activating the SIRT3/AMPK/mTOR pathway.