Mechanism Of Protective Effect Of Nicotinamide Adenine Dinucleotide On Optic Nerve And Retina In Experimental Autoimmune Encephalomyelitis Mice

Multiple sclerosis（MS）is an inflammatory demyelinating disease involving the central nervous system.The signs and symptoms are multiple in time and space.Microscopical pathological changes include perivascular inflammatory cell infiltration,myelin sheath loss and disintegration,axonal injury,and glial cell proliferation.According to statistics,about 40%of MS patients are accompanied by optic neuritis（ON）in the early stage of the disease,and as high as 80%in the progression of the disease.Acute optic neuritis can cause sharp loss of visual acuity,and more than half of the patients will leave varying degrees of permanent visual impairment,which seriously affects the ability to work,and is one of the important causes of neurological disability in young and middle-aged people.ON is an autoimmune mediated disease of acute optic nerve demyelination and retinal ganglion cell（RGCs）death.The pathological features of ON are inflammatory cell infiltration,axon loss,demyelination,and RGCs death.The causes of ON are very complex,such as oxidative stress,pathogen infection,malnutrition,environment and many other factors.Inflammation is thought to play a key role in the pathogenesis of ON.A growing body of evidence suggests that microglial cell activation,inflammatory cytokine secretion and inflammatory cell infiltration may be related to demyelination and axonal injury of ON.Current understanding of the pathogenesis of ON has benefited from the study of experimental autoimmune encephalomyelitis（EAE）.EAE-mediated mouse optic neuritis presents similarly to human disease and provides a living model for neuropathy.Nicotinamide adenine dinucleotide（NAD⁺）,also known as oxidative coenzyme I,plays an important role in the catabolism of fuel substrates in all cells.As an electron acceptor,NAD⁺cycles back and forth between the reduced state and the oxidation state,and its REDOX cycle is a key process in many metabolic pathways.A large number of studies have confirmed that NAD⁺has important effects on mitochondrial energy metabolism,calcium homeostasis,autophagy,cell differentiation,apoptosis,aging,gene expression,etc.Some researchers have found that NAD⁺can reduce the clinical symptoms and inflammatory cell infiltration in the brain and spinal cord of EAE mice,and protect against cell apoptosis.By activating AMPK/SIRT1 signaling pathway,NAD⁺alleviates the pro-inflammatory T cell response and reduces the degree of EAE.To our knowledge,the role of NAD⁺in the prevention and treatment of EAE mediated optic nerve and retinal injury has not been reported.Sirtuin 1（SIRT1）is an NAD⁺dependent deacetylase,which mainly exists in the nucleus and is widely involved in physiological processes such as oxidative stress,inflammation,growth and development,nutrient metabolism,cell differentiation,autophagy and apoptosis.Increasing evidence suggests that up-regulation of SIRT1 expression through transgenic overexpression or drug induction may have therapeutic value for a variety of neurodegenerative changes.Some research teams have found that SIRT1 overexpression can maintain the integrity of nerve axons and myelin sheath in the lumbar enlargement of EAE mice.However,the design of this study was limited to myelopathy and did not explore the effect of SIRT1 overexpression on the improvement of eye disease.In another study on experimental optic neuritis,resveratrol（SIRT1 activator）was shown to effectively reduce optic nerve inflammation,axonal damage,and maintain survival of RGCs in EAE and patho-mediated demyelinating disease.NAD⁺,as an essential key link in the deacetylation of SIRT1,can theoretically activate SIRT1 and up-regulate its expression level in cells.It was found that NAD⁺could up-regulate the expression level of SIRT1 in lumbar enlargement of EAE mice and reduce the m RNA transcription level of various pro-inflammatory cytokines in the spleen,which was consistent with the theoretical hypothesis.Therefore,this study speculated that NAD⁺may play a protective role on the inflammatory response and RGCs apoptosis induced by EAE by affecting the expression level of SIRT1.AKT（also known as protein kinase B）is a serine-threonine kinase that is a regulatory signal that mediates various cellular processes,such as cell growth,proliferation,apoptosis,metabolism,and angiogenesis.Abnormal AKT signaling pathway is involved in the development of MS disease.Some studies have found that reversible acetylation is a potential regulatory mechanism to control AKT activity.AKT’s p H domain lysine acetylation inhibits its binding to phosphatidylinositol 3,4,5-triphosphate（PIP3）,thus inactivating AKT.SIRT1 deacetylates the p H domain,which is a necessary process for the binding of AKT to PIP3 as well as the localization and activation of AKT membrane.Meanwhile,SIRT1-dependent deacetylation also promotes PIP3 binding and membrane localization of PDK1.Therefore,we propose the possibility that SIRT1 may slow the progression of EAE mediated optic neuritis through deacetylation-dependent activation of AKT.Currently,the relationship between NAD⁺/SIRT1 and inflammation and apoptosis in the retina/optic nerve in MS/EAE has not been reported at home and abroad.Therefore,in our experimental study,we used myelin oligoendrocyte glycoprotein(MOG_35-55)to immunize EAE mouse model,to observe whether NAD⁺has effective protective effect on EAE mediated optic neuritis,and to investigate whether NAD⁺regulates the immune response of EAE by upregulating the expression level of SIRT1 and affecting the SIRT1/AKT pathway.Part One Analysis and pathological study of the protective effect of nicotinamide adenine dinucleotide on visual inflammation in mice with experimental autoimmune encephalomyelitisObjective:After the successful establishment of experimental autoimmune encephalomyelitis animal model,NAD⁺drug intervention was given,and the onset time and symptoms of each group were recorded.Peak in the disease in mice optic nerve tissue HE dyeing,LFB staining and Bielschowsky silver,from the perspective of pathology identification of NAD⁺optic neuritis of experimental autoimmune encephalomyelitis mediated the effects of the relevant pathological change,studying whether NAD⁺can alleviate the inflammation of mice optic neuritis,loss of myelin and axon damage.Methods:1.Select experimental animals.SPF grade inbred female C57BL/6 mice were used,aged 8 to 10 weeks and weighing 18 g to 20 g.The mice were kept in an environment with indoor temperature of 24±2℃and relative humidity of50%-60%.The mice were given 12-hour alternating cycle illumination to ensure sufficient feed and clean water.The mice could eat and drink freely.2.EAE animal model was established.MOG_35-55 peptides were diluted with normal saline and prepared Ten milligrams per milliliter.Equal volume of complete Freund’s adjuvant（CFA）and a certain amount of tuberculin were added to make the final concentration of Mycobacterium tuberculosis H37RA to be 4 mg/m L.The mixture was fully emulsified to form oil-in-water emulsion,and each mouse was injected subcutaneously at any four points on both sides of the spinal column on the back of the mouse at a dose of 0.1 m L.0.5 ml of pertussis toxin（PTX）was injected intraperitoneally on day 0（0h）and day 2（48h）,respectively.3.Animal grouping and drug intervention.C57BL/6 mice were randomly divided into blank control group,EAE model group,EAE+NAD⁺prevention group and EAE+NAD⁺treatment group.NAD⁺was dissolved and diluted with phosphate buffered saline（PBS）and prepared and used on a daily basis.The immunization day was recorded as day 0,and mice in the EAE+NAD⁺prevention group were intraperitoneally injected with 250 mg/kg NAD⁺every day from day 1 of self-immunization.EAE+NAD⁺treatment group from the onset of symptoms（Weaver score≥1）,250 mg/kg NAD⁺was injected intraperitoneally to each animal daily.The blank control group and the EAE model group were given the same amount of PBS from the first day of immunization,and continued to the onset peak.4.Neurological function was scored using the Weaver 15 scale.He staining showed the pathological changes of optic nerve tissue in each group.LFB staining was used to evaluate the demyelination degree of optic nerve,and Bielschowsky silver staining was used to observe the defect of optic nerve axons.Score:0=no inflammatory cell infiltration;1 point=mild cell infiltration of optic nerve or optic nerve sheath;2 points=moderate infiltration;3 points=severe infiltration;4 points=lots of penetration.Any nerve with detectable inflammation（score 1 to 4）is considered to be optic neuritis.LFB staining demyelination score:0=no demyelination;1 point=mild demyelination;2 points=moderate demyelination;3 points=severe demyelination.5.All data were analyzed using Graph Pad Prism 7（USA）.All results were expressed as mean±standard deviation（SD）.One-way analysis of variance（ANOVA）was used for statistical difference analysis between groups.Clinical neurological function was evaluated by Mann-Whitney U test.A statistically significant difference was considered when P<0.05.Results:1.NAD⁺intervention can delay the onset time of EAE mice,improve symptoms and reduce neurological function scores.2.The results of HE staining showed that the optic nerve structure in the EAE model group was significantly changed,with obvious vacuolar degeneration,disordered arrangement of nerve fibers,discontinuous fracture,and a large number of inflammatory cells infiltration.3.The results of LFB staining showed that there were white vacuolar myelin deletion areas in the EAE model group,and the area of myelin demyelination was large.4.Bielschowsky silver staining results showed that axons in the EAE model group were light brown to dark brown,with axon fracture and cavity change.After NAD⁺intervention,inflammatory cell infiltration,loss of myelin sheath,and axon damage were reduced.Conclusions:1.NAD⁺intervention can delay the onset time of EAE mice and reduce the neurological function score of EAE mice.2.NAD⁺intervention can alleviate the inflammatory response,demyelination and axon injury of the optic nerve in EAE mice,and has protective effects on the optic nerve prevention and treatment.Part Two Nicotinamide adenine dinucleotide has a protective effect on experimental autoimmune encephalomyelitis mediated optic neuritis by reducing immune inflammatory response and apoptosisObjective:Various inflammatory factors and pro-inflammatory cells play an important role in the pathogenesis of EAE mediated optic neuritis,leading to demyelination,axon defect and loss of RGCs.We used MOG_35-55 to induce C57BL/6 mice to form an animal model of EAE mediated optic neuritis,and administered NAD⁺drugs for intervention.The expressions of immune cells（microglia,astrocytes）,pro-inflammatory cytokines TNF-αand IL-6,and the apoptosis of cells were observed in each group.To provide further experimental evidence of the protective effect of NAD⁺on optic neuritis.Methods:1.Select experimental animals.SPF grade inbred female C57BL/6 mice were used,aged 8 to 10 weeks and weighing 18 g to 20 g.The mice were kept in an environment with indoor temperature of 24±2℃and relative humidity of50%-60%.The mice were given 12-hour alternating cycle illumination to ensure sufficient feed and clean water.The mice could eat and drink freely.2.EAE animal model was established.MOG_35-55 peptides were diluted with normal saline and prepared Ten milligrams per milliliter.Equal volume of complete Freund’s adjuvant（CFA）and a certain amount of tuberculin were added to make the final concentration of Mycobacterium tuberculosis H37RA to be 4 mg/m L.The mixture was fully emulsified to form oil-in-water emulsion,and each mouse was injected subcutaneously at any four points on both sides of the spinal column on the back of the mouse at a dose of 0.1 m L.0.5 ml of pertussis toxin（PTX）was injected intraperitoneally on day 0（0h）and day 2（48h）,respectively.3.Animal grouping and drug intervention.C57BL/6 mice were randomly divided into blank control group,EAE model group,EAE+NAD⁺prevention group and EAE+NAD⁺treatment group.NAD⁺was dissolved and diluted with phosphate buffered saline（PBS）and prepared and used on a daily basis.The immunization day was recorded as day 0,and mice in the EAE+NAD⁺prevention group were intraperitoneally injected with 250 mg/kg NAD⁺every day from day 1 of self-immunization.EAE+NAD⁺treatment group from the onset of symptoms（Weaver score≥1）,250 mg/kg NAD⁺was injected intraperitoneally to each animal daily.The blank control group and the EAE model group were given the same amount of PBS from the first day of immunization,and continued to the onset peak.4.Immunofluorescence was used to detect the expression of IBA1（microglia）and GFAP（astrocytes）in optic nerve tissue.The transcriptional levels of pro-inflammatory cytokines TNF-αand IL-6 m RNA were detected by real-time fluorescence quantitative PCR in fresh optic nerve and retina tissues.5.TUNEL and Brn3a immunofluorescence staining were used to detect the number and apoptosis of RGCS cells in the retina.At the protein expression level,the changes of cleaved-Caspase 3 and cleaved-PARP were observed by western blot.6.All data were analyzed using Graph Pad Prism 7（USA）.All results were expressed as mean±standard deviation（SD）.One-way analysis of variance（ANOVA）was used for statistical difference analysis between groups.A statistically significant difference was considered when P<0.05.Results:1.Immunofluorescence staining showed that the fluorescence intensity of IBA1 and GFAP in the optic nerve tissue of the EAE model group was significantly increased compared with that of the blank control group,and the fluorescence intensity of the two biomarkers in the NAD⁺prevention group and the treatment group was significantly decreased.2.Fluorescence quantitative PCR indicated that the secretion of TNF-αand IL-6 in the optic nerve and retinal tissue of the EAE model group was significantly increased compared with that of the blank control group,while the secretion of TNF-αand IL-6 was decreased after NAD⁺intervention.3.The number of RGCs in the Brn3a-labeled EAE model group was the lowest,while the apoptosis rate of RGCs in the TUNEL labeled EAE model group was the highest.After NAD⁺intervention,the apoptotic cells decreased compared with RGCs.Conclusions:1.NAD⁺can reduce the accumulation of microglia and astrocytes in the optic nerve of EAE mice,and slow down the occurrence of inflammatory response.2.NAD⁺inhibited the expression of pro-inflammatory cytokines TNF-αand IL-6 in the optic nerve and retina of EAE mice.3.NAD⁺can protect the RGCs apoptosis in the retina of EAE mice.Part Three Nicotinamide adenine dinucleotide may exert a protective effect on experimental autoimmune encephalomyelitis mediated optic neuritis by activating the SIRT1/AKT pathwayObjective:The above conclusions suggested that NAD⁺could inhibit the apoptosis of retinal ganglion cells in EAE mice.To further verify,the expression levels of cleaved-Caspase 3 and cleaved-PARP in the optic nerve and retinal tissues of mice were observed in this part of the experiment from the protein expression level.Studies have shown that SIRT1 has anti-inflammatory and anti-apoptotic effects,and AKT is one of the main downstream factors of SIRT1.In EAE mediated optic neuritis,whether the anti-inflammatory and anti-apoptotic neuroprotective effects of NAD⁺are achieved by affecting SIRT1 expression and regulating the SIRT1/AKT pathway?Therefore,we observed the changes of SIRT1 and AKT by immunofluorescence and Western blot to provide more evidence for the regulation of the immune mechanism by NAD⁺.Methods:1.Select experimental animals.SPF grade inbred female C57BL/6 mice were used,aged 8 to 10 weeks and weighing 18 g to 20 g.The mice were kept in an environment with indoor temperature of 24±2℃and relative humidity of50%-60%.The mice were given 12-hour alternating cycle illumination to ensure sufficient feed and clean water.The mice could eat and drink freely.2.EAE animal model was established.MOG_35-55 peptides were diluted with normal saline and prepared Ten milligrams per milliliter.Equal volume of complete Freund’s adjuvant（CFA）and a certain amount of tuberculin were added to make the final concentration of Mycobacterium tuberculosis H37RA to be 4 mg/m L.The mixture was fully emulsified to form oil-in-water emulsion,and each mouse was injected subcutaneously at any four points on both sides of the spinal column on the back of the mouse at a dose of 0.1 m L.0.5 ml of pertussis toxin（PTX）was injected intraperitoneally on day 0（0h）and day 2（48h）,respectively.3.Animal grouping and drug intervention.C57BL/6 mice were randomly divided into blank control group,EAE model group,EAE+NAD⁺prevention group and EAE+NAD⁺treatment group.NAD⁺was dissolved and diluted with phosphate buffered saline（PBS）and prepared and used on a daily basis.The immunization day was recorded as day 0,and mice in the EAE+NAD⁺prevention group were intraperitoneally injected with 250 mg/kg NAD⁺every day from day 1 of self-immunization.EAE+NAD⁺treatment group from the onset of symptoms（Weaver score≥1）,250 mg/kg NAD⁺was injected intraperitoneally to each animal daily.The blank control group and the EAE model group were given the same amount of PBS from the first day of immunization,and continued to the onset peak.4.Brn3a-SIRT1 immunofluorescence double staining was used to detect the change of SIRT1 expression in retinal RGCs.5.Cleaved-Caspase 3,cleaved-PARP,SIRT1,AKT,p-AKT were detected by Western blotting.6.All data were analyzed using Graph Pad Prism 7（USA）.All results were expressed as mean±standard deviation（SD）.One-way analysis of variance（ANOVA）was used for statistical difference analysis between groups.A statistically significant difference was considered when P<0.05.Results:1.The results of double immunofluorescence staining showed that the number of RGCs was the lowest in Brn3a-SIRT1 labeled EAE model group.After NAD⁺intervention,the number of RGCs expressing SIRT1 in the retina of mice increased compared with the EAE model group.2.Western blotting results indicated that the cleaved-Caspase 3 and cleaved-PARP expression levels were the highest in the optic nerve and retina tissues of the EAE model group,and the expression levels were decreased after NAD⁺intervention.3.Western blotting indicated that the expression levels of SIRT1 and p-AKT in the optic nerve and retina were the lowest in the EAE model group.After NAD⁺intervention,the expression levels of both groups were significantly higher than those in the EAE model group.Conclusions:1.NAD⁺intervention can inhibit the expression of cleaved-Caspase 3 and cleaved-PARP in the optic nerve and retina,thus counteracting the apoptosis of EAE mediated optic neuritis.2.NAD⁺intervention can increase the number of SIRT1 expression cells in the retina of EAE mice,and has a protective effect on EAE mediated retinal ganglion cells（RGCs）injury.3.NAD⁺intervention can up-regulate the expression levels of SIRT1 and p-AKT in the optic nerve and retina tissues of EAE mice.NAD⁺may play an immunoregulatory role in EAE mediated optic neuritis through the SIRT1/AKT pathway.