| Multiple sclerosis(MS)is a chronic autoimmune disease of the central nervous system(CNS)with worldwide distribution.Neuroinflammation and demyelination maintained by lymphocytes,activated microglia and macrophages,which often lead to permanent disability.The etiology and pathogenesis of MS are still unclear,which may be the result of multiple factors such as genetics,inflammation,immunity,neuronal degeneration and environmental geography.At present,the recognized pathogenesis are abnormal immune attack and combined action of various immune cells and cytokines,causing inflammation,myelin sheath loss,axonal injury and cell death.Current therapies such as anti-inflammatory,immunomodulatory or immunosuppression and new-available drugs are partially effective in the early stages of the disease,but have limited benefits for MS patients of progression.As a result,it is urgent to search for the new drugs for MS patients.As a classical energy molecule,nicotinamide adenine dinucleotide(NAD+)not only plays an important role in energy metabolism and mitochondrial function,but also in aging,gene expression,calcium homeostasis and cell death.NAD+is a vital cofactor in many metabolic pathways,which plays a role in neurodegeneration and dysfunction caused by NAD+damage in the pathogenesis of MS.It is significant for autophagy to control inflammation and immunity by phagocytosis of dysfunctional organelles and proteins.Alterations of autophagy-related genes or defects of autophagy pathways have a specific meaning in neurodegenerative diseases,providing protection against neurological diseases by promoting the differentiation of mesenchymal stem cells(MSCs)into nerve cells.However,at present,whether NAD+is involved in autophagy in MS remains unclear.Neuroinflammation is a significant factor in the pathogenesis of neurodegenerative diseases.Some scholars have hypothesized that the high energy consumption of inflammatory response leaded to the impaired mitochondrial function,which leaded to the disorder of energy metabolism of neurons,resulted the failure to maintain the function of neurons and axons completely,and neuronal death and/or axonal degeneration.Inflammation may induce oxidative stress-related mitochondrial DNA deletions in neurons,which may lead to neurodegenerative changes in MS.The inflammatory response involves a multi-molecule complex called inflammasome,leading to the cleavage and secretion of inflammatory cytokines.In recent years,NLRP3inflammasome has got more attention in MS and its animal model of experimental autoimmune encephalomyelitis(EAE).The severity of MS patients was strongly correlated with IL-1?,IL-18,and caspase-1 levels,and patients with NLRP3 gain mutations showed a high degree of comorbidities with MS.In the animal model of EAE,mice with NLRP3 gene deficiency showed a delayed course of disease,reduced disease severity and demyelination,and decreased Th1 and Th17 cells in peripheral lymphoid tissue and spinal cord.The above provide a theoretical basis for us to explore the role of NLRP3 inflammasome in EAE.In our experiment,we established the EAE animal model with MOG35-55in C57BL/6 mice to observe the potential therapeutic efficacy of NAD+against EAE and the contributions of autophagy and the NLRP3inflammasome.We speculated that NAD+would inhibit the production of NLRP3 and inflammatory cytokines through activation of autophagy and thereby attenuate EAE-associated white matter inflammation,axonal damage,and neurological dysfunction.And we analyzed the dynamic changes of NLRP3 inflammasome in serum of patients with MOG antibody-related diseases at acute and remission stages.These findings highlight NAD+mediated modulation of autophagy and neuroinflammation as a potential therapeutic strategy for the treatment of inflammatory demyelinating diseases in the central nervous system.Part One The neuroprotective and anti-inflammatory effects of NAD+ in experimental autoimmune encephalomyelitis miceObjective:After the establishment of animal model of experimental autoimmune encephalomyelitis(EAE)with MOG35-55,we evaluated the nerve function score,pathology analysis with HE staining and LFB staining of lumbar intumescence spinal cord,PCR analysis between EAE group and NAD+drug treatment group,and observed the inflammatory cell infiltration,myelin damage,and the change of inflammatory cytokines after NAD+treatment.Methods:1.Selected the experimental animals.Female C57BL/6 mice were purchased from Shanghai Xipu-Bikai Experimental Animal Company,which aged about 6-8 weeks and weighing about 18±2g.The mice were raised in a constant humidity and temperature environment,kept light and dark for 12hours alternately every day,and had sufficient food and water.2.Established the EAE animal model.MOG35-55 peptides were diluted with normal saline into 10mg/ml,and join the complete freund's adjuvant(CFA)and a certain amount of tuberculosis,then connect the syringe and whipped alternately,until fully mixed emulsifying,and make the concentration of H37Ra to 4 mg/ml.Both sides of the spine in each mouse were divided into four quadrants,each mouse was injected 0.1 ml of emulsion of each quadrant.The mice were also received intraperitoneal injection with0.5ml of pertussis toxin on the same day(0 h)and 2 days(48 h)later.3.After the establishment of the EAE animal model,the experimental animals were grouped and the disease was evaluated.Mice in the NAD+group were given intraperitoneal injection of NAD+250 mg/kg·d from the first day after immunization,and mice in the control group(CON)and EAE model groups were given intraperitoneal injection of the same amount of phosphate buffer every day.The mice were sacrificed until the end of observation.The Knoz 5 score was used to score the neurological function.4.Histopathological observation.The spinal cord enlargement of mice was treated with paraffin embedded treatment and sliced into sections with a thickness of 5?m.Under light microscopy,we observed mouse lumbar intumescence spinal inflammatory cells infiltration and myelin sheath depigmentation with HE and LFB staining.5.The expressions of IL-1?,IL-10,IFN-?,IL-2,IL-18 and IL-17A in spinal cord were detected by qPCR.6.SPSS 21.0 statistical software was used for statistical analysis.Measurement data were expressed as mean±standard deviation(x±s).The data were tested for normality and homogeneity of variance.Clinical neurological function scores were evaluated by Mann-Whitney U test.One-way-ANOVA test was used to compare the mean of multiple groups of measurement data,and SNK-Q test was used for pairwise comparison.P<0.05was considered to be statistically significant.Results:1.Drug intervention with NAD+could reduce the neurological function score,improve the clinical symptoms of EAE mice,and delay the onset time.2.NAD+could inhibit inflammatory cell infiltration in spinal cord tissue of EAE mice,protect myelin sheath and reduce axonal injury.3.The spinal cord tissue were collected in the peak period of mice onset and detected by PCR.Compared with the control group,the m RNA levels of NLRP3,IL-1?,IFN-?,IL-2,IL-18 and IL-17A were significantly increased in the EAE model group,while the m RNA levels of above in the NAD+treatment group were significantly decreased compared with the EAE group.Compared with the control group,the expression level of IL-10 m RNA in the EAE group was significantly decreased,while the expression level of IL-10m RNA in the NAD+treatment group was significantly increased compared with the EAE model group.Part Two Autophagy plays an important role in the pathogenesis of experimental autoimmune encephalomyelitisObjective:After finishing the EAE animal model of C57BL/6 mice with MOG35-55,we assessed the nerve function score in different groups of mice using NAD+and autophagy inhibitor(3-MA)intervention respectively,and observed the effect of blocking autophagy on EAE related lesions in mice,and whether inhibition of autophagy could affect the inflammatory response and the differentiation of Th1 and Th17 cells.Methods:1.Selected the experimental animals.Female C57BL/6 mice were purchased from Shanghai Xipu-Bikai Experimental Animal Company,which aged about 6-8 weeks and weighing about 18±2 g.The mice were raised in a constant humidity and temperature environment,kept light and dark for 12hours alternately every day,and had sufficient food and water.2.Established the EAE animal model.MOG35-55 peptides were diluted with normal saline into 10mg/ml,and join the complete freund's adjuvant(CFA)and a certain amount of tuberculosis,then connect the syringe and whipped alternately,until fully mixed emulsifying,and make the concentration of H37Ra to 4 mg/ml.Both sides of the spine in each mouse were divided into four quadrants,each mouse was injected 0.1 ml of emulsion of each quadrant.The mice were also received intraperitoneal injection with0.5 ml of pertussis toxin on the same day(0 h)and 2 days(48 h)later.3.After finishing the EAE animal model,the experimental animals were grouped and the disease was evaluated.Mice in the NAD+group were given intraperitoneal injection of NAD+250 mg/kg·d from the first day after immunization,3-MA group mice were given intraperitoneal injection of 3-MA10 mg/kg·d and mice in the control group(CON)and EAE model groups were given intraperitoneal injection of the same amount of phosphate buffer every day.The Knoz 5 score was used to score the neurological function.4.Histopathological observation.The spinal cord enlargement of mice was treated with paraffin embedded treatment and sliced into sections with a thickness of 5?m.Under light microscopy,we observed mouse lumbar intumescence spinal inflammatory cells infiltration and myelin sheath depigmentation with HE and LFB staining.5.Detect the proportion of Th1 and Th17 cells in mice spleen cells using flow cytometry.6.SPSS 21.0 statistical software was used for statistical analysis.Measurement data were expressed as mean±standard deviation(x±s).The data were tested for normality and homogeneity of variance.Clinical neurological function scores were evaluated by Mann-Whitney U test.One-way-ANOVA test was used to compare the mean of multiple groups of measurement data,and SNK-q test was used for pairwise comparison.P<0.05was considered to be statistically significant.Results:1.Inhibition of autophagy aggravated the clinical symptoms of EAE,increased the clinical function score of mice,advanced the onset time,and delayed the spontaneous remission of EAE.2.Compared with the EAE model group,the infiltration of inflammatory cells in spinal lumbar enlargement of mice in the 3-MA group were significantly increased.3.Compared with the EAE model group,the proportions of Th1 cells and Th17 cells in the spleen of mice in the NAD+treatment group were significantly decreased,while the proportions of Th1 cells and Th17 cells in the spleen of mice in the 3-MA intervention group were significantly increased compared with the EAE group.Part Three Niacinamide adenine dinucleotide plays a neuroprotective role in experimental autoimmune encephalomyelitis mice by activating autophagy and regulating NLRP3 inflammasomeObjective:After finishing the EAE animal model of C57BL/6 mice with MOG35-55,we assessed the nerve function score in different groups of mice using NAD+,3-MA,and NAD+combined 3-MA intervention,and observed the effect of autophagy on EAE related lesions in mice,studied the changes of autophagy activity after drugs intervention,and provided experimental basis for autophagy and NLRP3 inflammatory corpuscle of EAE nerve protective effect.Methods:1.Selected the experimental animals.Female C57BL/6 mice were purchased from Shanghai Xipu-Bikai Experimental Animal Company,which aged about 6-8 weeks and weighing about 18±2g.The mice were raised in a constant humidity and temperature environment,kept light and dark for 12hours alternately every day,and had sufficient food and water.2.Established the EAE animal model.MOG35-55 peptides were diluted with normal saline into 10mg/ml,and join the complete freund's adjuvant(CFA)and a certain amount of tuberculosis,then connect the syringe and whipped alternately,until fully mixed emulsifying,and make the concentration of H37Ra to 4 mg/ml.Both sides of the spine in each mouse were divided into four quadrants,each mouse was injected 0.1 ml of emulsion of each quadrant.The mice were also received intraperitoneal injection with0.5 ml of pertussis toxin on the same day(0 h)and 2 days(48 h)later.3.After finishing EAE animal model,the mice were divided into EAE model group,NAD+treatment group,NAD+combined with 3-MA group,and normal mice without immunization were selected as normal control group(CON).NAD+and 3-MA were respectively dissolved in phosphate buffer solution and used on a daily.Mice in the 3-MA group received daily intraperitoneal injection of 3-MA 10 mg/kg,NAD+group received of NAD+250 mg/kg,and the combined group received of NAD+(250 mg/kg)and3-MA(10 mg/kg)from the second day after immunization.The CON and EAE group were intraperitoneally injected with the same amount of phosphate buffer every day.The Knoz 5 score was used to score the neurological function.4.The expression of IL-1?,NLRP3 and IL-17A in lumbar enlargement of spinal cord was detected by qPCR.5.Detect the expression of autophagy protein Beclin1,P62,LC3?/?in the spinal cord by western blot.6.SPSS 21.0 statistical software was used for statistical analysis.Measurement data were expressed as mean±standard deviation(x±s).The data were tested for normality and homogeneity of variance.Clinical neurological function scores were evaluated by Mann-Whitney U test.One-way-ANOVA test was used to compare the mean of multiple groups of measurement data,and SNK-q test was used for pairwise comparison.P<0.05was considered to be statistically significant.Results:1.Compared with the NAD+group,the neurological function score was increased in the combined group.2.Compared with the CON group,the transcription of IL-1?,NLRP3and IL-17A in EAE group were significantly increased,while the transcription in the NAD+group were decreased compared with the EAE group.Compared with the NAD+group,the transcription of IL-1?,NLRP3 and IL-17A of spinal cord in combined group were significantly increased,indicating that the anti-inflammatory effect of NAD+on EAE mice was weakened by the blocking of autophagy by 3-MA.3.Compared with mice in the EAE group,NAD+group could reduce the severity of the disease,enhance the expression of autophagy-related proteins LC3-II/I and Beclin1 in EAE mice,and reduce the expression of p62.Compared with the NAD+group,the protein expressions of LC3-II/I and Beclin1 were decreased in the combined group,while the expression of p62was increased.Part IV Transcription of NLRP3 in blood of MOG antibody-related diseases patients at the active and remission phasesObjective:To detect the transcription of NLRP3 in blood of MOGAD patients at the active and alleviative phases by qPCR.To describe the dynamic changes of NLRP3 in MOGAD patients at the active and remission phases.Methods:1.Selected experimental subjects.Twenty children diagnosed as MOGAD were admitted to the Department of Neurology of Hebei Children's Hospital from September 2020 to February 2021,whose diagnostic criteria were in conformity to the diagnostic criteria of Chinese experts in 2020.Peripheral blood samples were collected at acute active stage before any treatment such as glucocorticoids or immunosuppressants,and at follow-up during remission.2.Detected the transcription of NLRP3 in blood of MOGAD patients during the active and alleviative phases by PCR.3.SPSS 21.0 statistical software was used for statistical analysis.Measurement data were expressed as mean±standard deviation(x±s).The t test was used for the data of the two groups,and P<0.05 was considered statistically significant.Results:The transcription of NLRP3 in serum samples of MOGAD patients during active period was significantly higher than those in the alleviative period.Conclusion:1.NAD+could inhibit the progression of EAE,improve the clinical neurological function score,delay the onset time,improve the pathological changes,and reduce the proportion of Th1 and Th17 cells in spleen of mice.2.Autophagy plays a vital role in the pathogenesis of EAE mice.Blocking autophagy by 3-MA aggravated the clinical phenotype,delayed the spontaneous remission,increased the proportion of Th1 cells and Th17 cells in spleen.3.Autophagy was involved in NAD+mediated neuroprotective effect of EAE.NAD+may reduce the severity of EAE mice by inducing autophagy and regulating NLRP3 inflammasomes.4.The expression of NLRP3 during the active period of MOGAD patients was increased compared with the alleviative period,which may imply NLRP3may play a part in MOGAD and seek new ideas for the treatment of acquired inflammatory demyelinating diseases. |
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