Investigating Protection Effects And Mechanisms Of NAD~+ In Experimental Autoimmune Encephalomyelitis(EAE)

Multiple sclerosis(MS)is an inflammatory and demyelinating disease caused by autoimmune inflammation of central nervous system(CNS),primarily affecting young people worldwide.The characteristic pathogenesis of the disease is determined by demyelination,axonal injury,neuronal damage,oligodendrocyte loss and glial scar formation.At present,most drug therapies,such as glatiramer acetate,interferon(IFN)-β and fingolimod,focus on mediating immunity.Nevertheless,these agents have only partial effects.Therefore,new effective treatments of MS are urgently needed.Previous studies have shown that the immune system imbalance mainly in the adaptive immune response involving T cells is a causative factor of MS.And the imbalanced responses of anti-inflammatory T cell subsets,including T helper type2(Th2)and regulatory T cell(Treg),and pro-inflammatory T cell subsets,including T helper type1(Th1)and T helper type17(Th17),play vital roles in the MS pathogenesis.Therefore,it is believed that new agents that can decrease pro-inflammatory Th1 and Th17 subsets and increase anti-inflammatory Th2 and Treg subsets are needed for the treatment of MS.Sirtuins(SIRTs)belongs to a family of histone deacetylases and NAD~+ is required for deacetylation.AMP-activated protein kinase(AMPK)is a vital kinase in modulation of the cellular energy metabolism,and it also has SIRT1-dependent anti-inflammatory activity.Studies have shown that AMPK activates SIRT1 through promoting the generation of SIRT1 activator NAD~+.And SIRT1 modulates the immunoinflammatory responses through suppressing the nuclear factor kappa B(NF-κB)expression.Moreover,SIRT1 can also serve as the key molecule that activates AMPK.Therefore,both AMPK and SIRT1 are crucial in maintaining the inflammatory responses and show a synergistic effect on cellular energy metabolism.These studies suggest that the AMPK-SIRT1 pathway might become a new target for the treatment of MS.A great amount of researches have demonstrated that NAD~+ has an important effect on mitochondrial functions,gene expression,calcium homeostasis,energy metabolism and aging.Study has also suggested that NAD~+ intervention can reduce death of neurons and astrocytes caused by oxidative stress and modulate CD4+cell differentiation.In our study,we established an EAE model and focused on the possible mechanisms of NAD~+ in ameliorating clinical manifestation and severity of EAE.Our results suggested that NAD~+ administration mitigated the severity of EAE through activating the AMPK/SIRT1 pathway and alleviating pro-inflammatory T cell responses.Part one The pathological study and effect of NAD~+ on prevention of experimental autoimmune encephalomyelitisObjective:After the establishment of animal model of multiple sclerosis,we use NAD~+ for drug intervention to assess the effect on the EAE mice by HE staining,Weil staining and immunohistochemistry staining.Methods: 1All the experimental protocols were approved by the institutional animal care and use committee and the local experimental ethics committee.Female wild-type C57BL/6 mice(8-10 weeks of ages,18-20g)were brought from Vital River Laboratories(Beijing,China).Food and water were available to paralyzed mice.And they were housed under specific sterile circumstances.2 The EAE mice were subcutaneously injected at four flank sites with 250 μg of MOG35-55(Lysine Bio-system,Xi’an,China)emulsified in an equivalent volume of Complete Freund’s Adjuvant(CFA,Sigma,St Louis,MO,USA)containing 4 mg/ml of heat-killed mycobacterium tuberculosis H37Ra(Difco Laboratories,Detroit,MI,USA).The mice were also received intraperitoneal injection with 500 ng of pertussis toxin(Alexis,San Diego,CA,USA)on the same day and 2 days later.3 The mice were randomly placed into three groups of 8 mice in each group.One group of EAE mice was treated intraperitoneally with 250 mg/kg(body weight)NAD~+ in PBS once daily and another group of vehicle-treated EAE mice was treated with phosphate-buffered saline(PBS)once daily.And the third group of non-EAE healthy mice was injected with PBS at the same time as the control.All the groups of drug intervention began from day 0 to day 25.Each day,the mice were weighted and the Weaver’s score was used to evaluate the neurological function.The evaluation can further assess the disease progression by distinguishing individual limbs,not by assessing the disabilities of both fore limbs and hind limbs together.The total score(from 0 to 15)is the sum of the scores(from 0 to 2)for the tail and the scores(from 0 to 3)for four limbs.The tail was scored as follows: 0 = normal;1 = partial paralysis,2 = complete paralysis.The hind limbs and fore limbs were evaluated respectively as follows: 0 = normal;1 = weak or altered gait;2 = partial paralysis;3 = completely paralyzed limb.Therefore,score 14 represents fully quadriplegic mice,while score 15 represents died ones.4 The lumbar spinal cords were carefully dissected from mice(n = 3 mice/per group)and embedded in paraffin 25 days post-immunization.The sections(5-μm thick)of the spinal cords were stained with WEIL,hematoxylin & eosin(H&E)to evaluate the degrees of demyelination and inflammatory injuries.And at least 3-6 sections from each mouse were semi-quantitatively analyzed by light microscopy.The assessment of inflammation was scored as the following standard: 0,normal;1,mild inflammation = lymphocyte infiltrates partially around meninges and blood vessels;2,moderate inflammation = 1–10 lymphocyte infiltrates in the spinal cord;3,severe inflammation = 11–100 lymphocyte infiltrates in the spinal cord;and 4,massive inflammation,= over 100 lymphocyte infiltrates in the spinal cord.We used the following standard to assess demyelination: 0 = normal;1= myelin sheath injuries in rare areas;2 = myelin sheath injuries in a few of areas;and 3 = massive myelin sheath injuries.5 The Myelin basic protein(MBP)and neurofilament(NF)200 expression levels in the lumbar spinal cords were analyzed through immunohistochemistry staining.Anti-MBP(1:200,Proteintech,USA)or anti-NF200(1:200,Proteintech,USA)antibodies were used to incubate sections at 4°C overnight.And biotinylated secondary antibodies and an ABC kit were used to check the bound antibodies and diaminobenzidine were used to visualize sections.The immunostained sections were checked by a light microscope.6 Statistical analyses were conducted using statistical package for the social sciences(SPSS)19.0 software.All the results were expressed as the mean ± standard deviation(SD).Statistical differences in the scores of clinical signs were checked by the Mann-Whitney U test.After data were tested for 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 or Dunnett’s for multiple comparisons.P-values of < 0.05 were considered statistically significant.Results: 1 NAD~+ treatment alleviated clinical severity of MOG-induced EAE in mice.The clinical signs and scores of mice from different groups were observed and calculated longitudinally.The scores of clinical signs in the vehicle-treated EAE mice were obviously higher than those in the NAD~+-treated mice.2 NAD~+ treatment suppressed inflammation,demyelination and axonal damage in EAE mice.The stained sections were examined using a light microscope.In the healthy control mice,no inflammatory infiltrate was detected in the CNS,and myelin sheaths and axons were regularly arrayed in the spinal cord.On the contrary,numerous inflammatory cells,massive demyelination and axonal injuries in the spinal cords of the vehicle-treated EAE mice were observed.However,the number of inflammatory cells in the spinal cords of mice was significantly reduced in NAD~+ treatment group and fewer signs of demyelination and axonal damage were detected.And the pathological scores of the vehicle-treated EAE mice by semi-quantitative analysis were obviously higher than those of the NAD~+-treated mice.Part two Treatment with NAD~+ inhibited the development of experimental autoimmune encephalomyelitis by modulating Th1/Th17 immune responses in miceObjective:To explore the immunomodulatory effect of NAD~+,we assess its effect on the differentiation and function of Th1 cells and Th17 cells.Methods: 1All the experimental protocols were approved by the institutional animal care and use committee and the local experimental ethics committee.Female wild-type C57BL/6 mice(8-10 weeks of ages,18-20g)were brought from Vital River Laboratories(Beijing,China).Food and water were available to paralyzed mice.And they were housed under specific sterile circumstances.2 The EAE mice were subcutaneously injected at four flank sites with 250 μg of MOG35-55(Lysine Bio-system,Xi’an,China)emulsified in an equivalent volume of Complete Freund’s Adjuvant(CFA,Sigma,St Louis,MO,USA)containing 4 mg/ml of heat-killed mycobacterium tuberculosis H37Ra(Difco Laboratories,Detroit,MI,USA).The mice were also received intraperitoneal injection with 500 ng of pertussis toxin(Alexis,San Diego,CA,USA)on the same day and 2 days later.The mice were randomly placed into three groups of 8 mice in each group.One group of EAE mice was treated intraperitoneally with 250 mg/kg(body weight)NAD~+ in PBS once daily and another group of vehicle-treated EAE mice was treated with phosphate-buffered saline(PBS)once daily.And the third group of non-EAE healthy mice was injected with PBS at the same time as the control.All the groups of drug intervention began from day 0 to day 25.3 On the post-immunization day 25,the splenocytes were isolated and then cultured in 24-well plates for 24 h.The cell samples were stimulated with 10 μg/ml of MOG35-55 peptide and exposed to Cell Stimulation Cocktail(e Bioscience,San Diego,CA,USA)during the last 15 h.The percentages of Treg cells were directly detected using splenic cells,not by applying the stimulation protocol mentioned above.To visualize intracellular cytokines,the cells stained with APC-labeled anti-CD25 and FITC-labeled anti-CD4 were fixed and permeabilized according to the manufacturer’s recommendations.And PE-anti-IFN-γ and APC-anti-IL-4 were used to detect Th1/Th2 cells.Moreover,cells were also stained with PE-anti-Foxp3 and PE-anti-IL-17 A for detecting Treg cells and Th17 cells(antibodies supplied by e Bioscience,San Diego,CA,USA).A FACS-Calibur flow cytometer(BD Biosciences)was then used for cell analysis.Cell samples were distinguished by the unique surface marker and gated by their characteristic side-scatter.Data were analyzed in a blinded fashion using Cell-Quest software.4 On post-immunization day 25,the splenocytes of mice from the three different groups were isolated and the cell samples were cultured in 24-well plates with 10 μg/ml of MOG35-55 peptide.Supernatants were collected two days later.And the production levels of IL-17 A,IL-10,TGF-β,IL-6 and IL-1β were examined using mouse cytokine-specific ELISA kits according to the manufacturer’s directions(Joyee Biotechnics,Shanghai,China).5 Five mice from the three groups were euthanized after immunization and NAD~+ treatment for 25 days and the splenocytes were isolated.Then,the relative m RNA levels of T-box expressed in T cell(T-bet),signal transducer and activator of transcription 3(STAT3),retinoid related orphan receptor gamma t(RORγt),forkhead box P(Foxp3),interleukin(IL)-1β,IL-6,IL-10,transforming growth factor(TGF)-β,IFN-γ,IL-2,IL-12 and IL-17 A were evaluated by q RT-PCR.Briefly,total RNA was first extracted from splenocytes with Trizol reagent(Life Technologies).Then,c DNA synthesis was performed using All-in-One? first-strand c DNA synthesis kit(Gene Copoeia,Rockville,USA)following the manufacturers’ instructions.The PCR amplification was performed by All-in-One? Mixture(Gene Copoeia,Rockville,USA),specific primers(Table 1)and the Roche Light Cycler 480 II system(Roche Diagnostics Gmb H,Mannheim,Germany).The PCR initial incubations were carried out at 95 °C for 10 min,followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s.The Sequence Detection Systems software was used to analyze data.The relative m RNA levels of each target gene were measured by 2-ΔΔCT method.6 Statistical analyses were conducted using statistical package for the social sciences(SPSS)19.0 software.All the results were expressed as the mean ± standard deviation(SD).After data were tested for 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 or Dunnett’s for multiple comparisons.P-values of < 0.05 were considered statistically significant.Results: 1 NAD~+ treatment modulated Th1 and Th17 cell differentiation.The results of flow cytometry showed the percentages of Th2 cells and Treg cells of mice from different groups have similar proportions.On the other hand,the vehicle-treated EAE mice had higher percentage of Th1 and Th17 cells than those in the healthy controls,whereas compared to the EAE mice,fewer Th1 and Th17 cells were observed in the NAD~+-treated mice.NAD~+ treatment efficaciously reduced the EAE-associated increased m RNA levels of T-bet,RORγ t and STAT3 and there were no notable differences of the relative m RNA levels of Foxp3 compared to the vehicle treatment.2 NAD~+ treatment regulated inflammatory response of Th1 and Th17 cells.We also characterized inflammatory cytokine expression in the spleen.The results of the inflammatory cytokines in the splenic supernatants by Elisa f proved the immune regulating effect of NAD~+.There were higher concentrations of IL-10 and TGF-β and lower level IL-1β,IL-6,IFN-γ and IL-17 A in NAD~+-treated mice than those in the vehicle-treated EAE mice.3 Comparing with those of the β-actin control,the m RNA levels of IL-1β,IL-6,IFN-γ,IL-2,IL-12 and IL-17 A in the NAD~+-treated mice were much lower than those in the vehicle-treated EAE mice.In addition,the relative m RNA levels of IL-10 and TGF-β in the NAD~+-treated mice were notably higher than those in the vehicle-treated EAE micePart three Treatment with NAD~+ inhibited the development of experimental autoimmune encephalomyelitis by by activating AMPK/SIRT1 signaling pathway in miceObjective : We use NAD~+ for treatment and explore its effect on modulating AMPK/SIRT1 signaling pathways in EAE mice.Moreover,we try to find a new drug to treat MS.Methods: 1All the experimental protocols were approved by the institutional animal care and use committee and the local experimental ethics committee.Female wild-type C57BL/6 mice(8-10 weeks of ages,18-20g)were brought from Vital River Laboratories(Beijing,China).Food and water were available to paralyzed mice.And they were housed under specific sterile circumstances.2 The EAE mice were subcutaneously injected at four flank sites with 250 μg of MOG35-55(Lysine Bio-system,Xi’an,China)emulsified in an equivalent volume of Complete Freund’s Adjuvant(CFA,Sigma,St Louis,MO,USA)containing 4 mg/ml of heat-killed mycobacterium tuberculosis H37Ra(Difco Laboratories,Detroit,MI,USA).The mice were also received intraperitoneal injection with 500 ng of pertussis toxin(Alexis,San Diego,CA,USA)on the same day and 2 days later.The mice were randomly placed into three groups of 8 mice in each group.One group of EAE mice was treated intraperitoneally with 250 mg/kg(body weight)NAD~+ in PBS once daily and another group of vehicle-treated EAE mice was treated with phosphate-buffered saline(PBS)once daily.And the third group of non-EAE healthy mice was injected with PBS at the same time as the control.All the groups of drug intervention began from day 0 to day 25. binding sites were blocked with 5% skimmed milk in TBST for an hour and the membranes subsequently were incubated overnight at 4 °C with primary antibodies against AMPK(1:500,Cell Signaling Technology),p-AMPK(1:500,Cell Signaling Technology),SIRT1(1:500,Abcam),P65(1:2000,Cell Signaling Technology),and p-P65(1:1000,Cell Signaling Technology),as well as glyceraldehyde-3-phosphate dehydrogenase(GAPDH)(1:5000,Bioworld)as a control.After three washes with TBST,the membranes were incubated with the appropriately conjugated secondary antibodies.The intensity of each band was measured quantitatively by an imaging densitometer(LI-COR Bioscience,Lincoln,Nebraska,USA).4 Statistical analyses were conducted using statistical package for the social sciences(SPSS)19.0 software.All the results were expressed as the mean ± standard deviation(SD).After data were tested for 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 or Dunnett’s for multiple comparisons.P-values of < 0.05 were considered statistically significant.3 On post-immunization day 25,five mice from the three groups were euthanized.Their spleen and the lumbar spinal cord was carefully removed and immediately frozen via submersion in liquid nitrogen.The nuclear-cytosol extraction kit(Applygen Technologies Inc.,Beijing,China)was used to extract the proteins,following manufacturer’s protocols.The BCA protein experiment reagent kit was used to measure protein concentrations(Novagen,Madison,WI,USA).Normalized proteins were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and then transferred to polyvinylidene fluoride membranes(PVDF,Millipore).Non specificResults: 1 NAD~+ treatment activated the AMPK/SIRT1 pathway.We investigated the expression levels of AMPK,p-AMPK,and SIRT1 in the spinal cords and spleens of mice from different groups at post-immunization day 25 by Western blot.These assays revealed that there were low expression levels of p-AMPK and SIRT1 in the vehicle-treated EAE mice and there were slightly higher expression levels of p-AMPK and SIRT1 in healthy controls.However,notably higher expression levels of p-AMPK and SIRT1 were detected in the NAD~+-treated mice.2 NAD~+ treatment suppressed NF-κB expression.We also evaluated whether NAD~+ treatment could inhibit NF-κB(P65)expression levels at post-immunization day 25 using Western blot.Lower levels of p-P65 expression were observed in the healthy controls,at the same time,there were higher p-P65 expression levels in the vehicle-treated EAE mice.However,p-P65 expression levels were dramatically decreased in the NAD~+-treated mice.Conclusion: 1 NAD~+ treatment suppressed the progression and development of EAE and alleviated inflammation through reducing infiltrating inflammatory cells,demyelination and axonal loss in mice.2 NAD~+ treatment could notably mitigate Th1/Th17 immune responses and promote IL-10 and TGF-β secretion in mice.3 NAD~+ treatment inhibited inflammatory responses by activating the AMPK/SIRT1 pathway and suppressing the activation of NF-κB.Obviously,NAD~+ may alleviate EAE through its potential immunomodulatory function.Treatment with NAD~+ could be a potential new therapy for MS.