Effects Of NAD+ On AMPK/SIRT1 Signaling Pathway Of Experimental Autoimmune Encephalomyelitis Mice

Objective:Multiple sclerosis(MS)is a chronic inflammatory autoimmune demyelinating disease of the central nervous system(CNS).It is the most prevalent disabling neurological disease affecting young people.The etiology of MS remains unknown,however,it is believed to be caused by immune dysregulation triggered by genetic and environmental factors.The pathogenesis of MS is very complex but is widely thought to be a immune inflammatory injury mediated by autoreactive myelin-specific CD4+ T cells.Experimental autoimmune encephalomyelitis(EAE)is an internationally recognized animal model for MS.Recent studies suggest that Adenosine monophosphate-activated protein kinase/silent information regulator of transcription 1/(AMPK/SIRT1)pathway is pivotal in the regulation of immune homeostasis and inflammatory responses.SIRT1 is a member of the histone deacetylase class III family of proteins and is an NAD+-dependent histone and protein deacetylase.AMPK is an enzymatic complex that regulates the energetic metabolism,which has SIRT1-dependent anti-inflammatory activity.AMPK is activated in the form of p-AMPK.In this study,we used NAD+ as the drug intervention and observed the clinical symptoms,pathological change,expression of SIRT1,AMPK and p-AMPK in control,EAE,and NAD+ treatment groups to explore possible mechanisms of the protective effects on EAE of NAD+.Methods:1 24 Female wild-type C57BL/6 mice(8–10 weeks of ages,18–20g of body weight)were randomly divided into three groups: normal control group,EAE model group and NAD + treatment group,8 mice each group.Then EAE mice model was established with the MOG35-55 peptide,Complete Freund's Adjuvant,Mycobacterium tuberculosis H37 Ra and pertussis toxin.The mice in NAD+ treatment group were treated intraperitoneally with 250 mg/kg(body weight)NAD+ in phosphate-buffered saline(PBS)once daily from day 1(The day of immunization is day 0),another two groups were injected with the same amount of PBS once daily.All the groups of drug intervention began from day 1 to 30.Their weight and clinical score were recorded twice a day from day 1 to 30.The purpose of this part is to observe the disease course of EAE.2 24 Female wild-type C57BL/6 mice(8–10 weeks of ages,18–20g of body weight)were randomly divided into three groups: normal control group,EAE model group and NAD + treatment group,8 mice each group.Then EAE mice model was established.The mice in NAD+ treatment group were treated intraperitoneally with 250 mg/kg(body weight)NAD+ in PBS once a day from day 1(The day of immunization is day 0),Another two groups were injected with the same amount of PBS once daily.All the groups of drug intervention began from day 1 to day 25.Their weight and clinical score were recorded twice a day from day 1.On post-immunization day 25,three mice of each group were euthanized with perfusion,then the lumbar spinal cord were stained with Hematoxylin–eosin(HE)to assess the degrees of inflammatory infiltration.The other five mice of each group were euthanized freshly,the expression of SIRT1,AMPK and p-AMPK protein in the lumbar spinal cord and spleen was detected by Western blot technology.3 The experimental data were analyzed by SPSS21.0 software.The measurement data were presented as mean ± standard deviation.The onset rates of EAE between groups were analyzed by Fisher exact test.The difference in neurological scores was checked by the Mann-Whitney U test.After the data were tested by normality and homogeneity of variance,all the other statistical differences between groups were conducted by Student's t test or one-way analysis of variance(ANOVA)followed by the Student-Newman-Keuls(SNK)-q test for multiple comparisons.P<0.05 was considered statistically significant.Result:1 The disease course of mice in each group was compared: the mice in the normal control group did not develop disease.Compared to EAE group,the incidence,mean neurological score of the mice in NAD+ group were significantly lower and the mean onset time of the mice in NAD+ group was delayed(P<0.05).2 HE staining: At the peak of the onset(on post-immunization day 25),compared to the control group,the inflammatory cells in the spinal cord lumbar enlargement tissue of the mice in the EAE group and the NAD+ group were significantly higher(P<0.05).The inflammatory cells in the spinal cord lumbar enlargement tissue of the mice in the NAD+ group were significantly lower than in the EAE group(P<0.05).3 Western Blot(In the spinal cord): The expression level of SIRT1 and p-AMPK protein in the EAE group was lower than in the control group(P<0.05).Compared to the EAE group,The expression level of SIRT1 and p-AMPK protein in the NAD+ group was significantly higher(P<0.05).There is no statistical difference of the expression of AMPK in the three groups(P>0.05).4 Western Blot(In the spleen): The expression level of SIRT1 and p-AMPK protein in the EAE group was lower than in the control group(P<0.05).Compared to the EAE group,The expression level of SIRT1 and p-AMPK protein in the NAD+ group was significantly higher(P<0.05).There is no statistical difference of the expression of AMPK in the three groups(P>0.05).Conclusions:1 NAD+ can decrease the morbidity,delay the onset,alleviate clinical severity and suppress inflammatory infiltration of spinal cord in MOG-induced EAE in mice.2 The protective effect of NAD+ on EAE may be the result of activating the AMPK/SIRT1 pathway.