The Mechanism Of NAD~+ Inhibiting Experimental Autoimmune Encephalomyelitis By Regulating The Activity Of Myelogenic Inhibitory Cells

Multiple sclerosis(MS)has long been considered as a complicated and refractory chronic demyelinating disease involving the central nervous system.Inflammatory cell infiltration,myelin loss and axonal injury are its main pathological features.The recurrent course and multiple lesions seriously affect the patient’s ability to work,which is one of the important causes of neurological disability in young and middle-aged people.There is abundant evidence that the pathogenesis of MS is T lymphocyte mediated immune response to myelin antigen.When activated,inflammatory T cells disrupt the blood-brain barrier and invade the central nervous system,attacking the myelin sheath and causing neuronal axon damage.The occurrence of autoimmune response in the central nervous system may be closely related to the dysfunction of immune tolerance caused by the change of peripheral T cell homeostasis.At present,the treatment of multiple sclerosis is mainly anti-inflammatory,and is only effective in the period of recurrence and remission.Therefore,the development of new treatments has become an urgent problem for multiple sclerosis patients.Experimental autoimmune encephalomyelitis(EAE)is an internationally recognized classic animal model of multiple sclerosis,widely used in the basic research of multiple sclerosis.It is mediated by innate immune system cells and autoimmune CD4+T cells.The histopathological and clinical similarities between EAE and MS allow findings from the EAE model to be extrapolated to patients with MS.Numerous studies have found in EAE animal models that bone-marrow specific CD4+T cells,helper T cells Thl and Th17 are all generated in peripheral lymphoid organs,activated there,and then released into peripheral blood circulation,which then enter the central nervous system through the blood-brain barrier to play a pathogenic role.Once entering the central nervous system,bone mare-specific CD4+T cells will be activated when they encounter specific antigen,triggering the recruitment of monocytes/macrophages and neutrophils in a large number of blood circulation in the central nervous system,forming an inflammatory response of the central nervous system.Bone marrow derived suppressor cells(MDSCs)are a heterogeneous group of cells with functional diversity and immune system origin.The most important feature of MDSCs is their ability to inhibit T cells.MDSCs in mice are usually identified by the co-expression of CDllb and gr-1,so MDSCs in mice are defined as CD11b+Gr-1+cells.Numerous experimental evidences confirmed that MDSCs could attenuate the pathogenesis of EAE.MDSCs use inducible nitric oxide synthase(NOS2)and Arginase-1 to control the response of inflammatory T cells.Th1 cytokines such as IFN-and TNF-induce NOS2,while Th2 cytokines such as il-4 and il-13 upregulate Arginase-1.Early studies have found that the immunosuppressive function of MDSCs is related to 1-arginine metabolism.L-arginine is the substrate of Arginase-1,which catalyzes 1-arginase to produce urea and 1-ornithine.Studies have shown that activated MDSCs express high levels of Arginase-1,which directly inhibits the function of CD4+T cells by consuming a large amount of 1-arginine.Nicotinamide adenine dinucleotide(NAD+)is a key coenzyme that transfers hydrogen ions in REDOX reaction.It is widely involved ina variety of physiological activities such as material metabolism,energy synthesis,gene expression,DNA repair and aging.Studies in the neurological fields of ischemia and trauma have also proved that NAD+is a neuroprotective agent with multiple targets and has potential therapeutic value for a variety of diseases.STAT6(The signal assay and activator of transcription6)is The main regulatory protein regulating The proliferation of MDSCs,and STAT6 is an important factor promoting The secretion of Arginase-1.However,the regulatory effect of STAT6 on MDSCs in EAE remains unclear.There is a clear link between the activation of SIRT1,a nicotinamide adenine dinucleotide(NAD+)-dependent protein deacetylase that catalyzes the removal of acetyl groups from a variety of protein substrates.Its physiological effects depend on NAD+.In this study,serum levels of Arginase-1 activity in peripheral blood of patients with multiple sclerosis were first analyzed,and the expression level of Arginase-1 activity was decreased compared with the normal control group.Arginase-1 is a very important cytokine for MDSCs to exert their inhibitory function on CD4+T cells.Let’s start from this direction,the use of NAD+to drug intervention of EAE mice,evaluate the occurrence status of the different groups of mice and observe the NAD+inflammation suppression effect of EAE mice and the pathological changes of myelin sheath depigmentation effect,NAD+is emphatically discussed whether can affect CD11b+ source sex Gr-1 the number of MDSCs and function and adjusting experimental autoimmune encephalomyelitis(EAE)the progress of the disease.In addition,we proposed a new molecular mechanism for NAD+to improve EAE,that is,NAD+treatment may induce MDSCs to weaken the course of EAE by activating the phosphorylation of SIRT6 expression,to investigate whether NAD+further protects EAE by regulating the SIRT1/STAT6/Arginase-1 pathway.Part one Study on the expression of argininase-1 in peripheral blood of active patients with multiple sclerosisObjective:Serum levels of arginase-1 in the peripheral blood of normal control group and active multiple sclerosis patients were detected by ELISA.The expression and changes of Arginase-1 in peripheral blood during the active phase of MS patients were described.Methods:1.From February 2019 to January 2020,10 patients with primary and recurrent multiple sclerosis who were admitted to the department of neurology of the second hospital of hebei medical university were selected,and the diagnostic criteria were all in line with McDonald’s revised multiple sclerosis diagnostic criteria in 2010.The normal control group included 10 patients,all from the physical examination center of the second hospital of hebei medical university,whose age and gender were matched with the multiple sclerosis group.Peripheral blood samples were collected before the patient was admitted to hospital in the acute active phase and did not begin receiving any treatment such as glucocorticoids or immunosuppressants.2.Serum levels of arginase-1 in peripheral blood of normal control group and multiple sclerosis group were detected by ELISA.3.All the experimental data were analyzed by SPSS22.0 software,and the measurement data were expressed as x ±s.The t test was used for both groups of data,and P<0.05 was considered statistically significant.Results:The results showed that the expression level of arginase-1 in serum of EAE group was significantly lower than that of normal control group.Part two Mechanism of NAD+ inhibiting experimental autoimmune encephalomyelitis by regulating CDllb+Gr-1+ derived MDSCs activityObjective:Use NAD+ drug intervention on the EAE mice,evaluate the occurrence status of the different groups of mice and observe the NAD+inflammation suppression of EAE mice and the pathological changes of myelin sheath depigmentation effect,NAD+is emphatically discussed whether can affect CD11b+source sex Gr-1 the number of MDSCs and function and adjusting experimental autoimmune encephalomyelitis(EAE)the progress of the disease.Methods:1.Select experimental animals.Female C57BL/6 mice aged 6-8 weeks were selected and their body weight was 20±2g.They were purchased from Beijing weitong lihua laboratory animal technology co.,LTD.The mice were raised in an SPF level environment with indoor temperature of 24±2℃ and relative humidity of 50%-60%.The light and dark environment was 12 hours each day.2.Establish EAE animal model.MOG35-55 peptides diluted with normal saline into 10 mg/ml,and join the isometric complete freund’s adjuvant(CFA)and a certain amount of tuberculin,then fully mixed emulsifying,eventually make the concentration of n/med tuberculosis bacili H37Ra to 4 mg/ml,on both sides of the spine in mice points respectively at 4 subcutaneous injection of 0.1 ml of emulsion,and then the immune 0 h and 48 h by intraperitoneal injection of two 0.5 ml pertussis toxin(PTX).3.Neurological function scores of mice were evaluated,and pathological changes of spinal cord were evaluated by HE and LFB staining.4.The quantitative changes of CD11b+Gr-1+derived MDSCs in spleen tissues of mice in each group were determined by flow cytometry and immunofluorescence staining.5.Expression levels of CD11b and Arginase-1 in spinal cord and spleen tissues were observed by immunofluorescence double staining.6.The levels of inflammatory cytokines of Th1 and Th2 and the expression level of Arginase-1 in serum of mice in each group were detected by ELISA7.All the experimental data were analyzed by SPSS22.0 software,and the measurement data were expressed as x ±s.The t test was used for both groups of data.Results:1.After NAD+intervention,the incidence rate of mice in the NAD+group was significantly lower than that in the EAE group.The neurological impairment score of mice in the NAD+group was significantly lower than that in the EAE group.2.NAD+intervention reduced myelin loss,improved inflammatory response in EAE mice,and reduced the number of inflammatory cells.Through observation of LFB staining pathological sections of mice at the peak of onset,the myelin sheaths in the anterior and lateral white matter of the spinal cord of the blank control group were well preserved and the edges were neat.However,pathological sections of EAE mice showed the absence of myelin sheath in white vacuoles in the shape of dots or sheets.However,the extent and extent of spinal white matter myelin loss in the NAD+ group were significantly reduced compared with the EAE group.Through HE staining of mice at the peak of the disease for pathological evaluation,we observed that the pathological sections of spinal cord lumbar enlargement of mice in the EAE model group showed diffuse inflammatory cells mainly composed of lymphocytes,and even showed "vascular sleeve"-like pathological manifestations.The number of inflammatory cells in NAD+treatment group was significantly reduced,the infiltration range was smaller,and the infiltration degree was also significantly reduced.The results of immunohistochemistry and fluorescence staining showed that a large number of macrophages were clustered in the spinal cord of mice in the EAE group,while macrophages were rarely clustered in the normal control group.A large number of CD4+T lymphocytes were present in the spinal cord of mice in the EAE group,while few CD4+T lymphocytes were present in mice in the normal control group.NAD+intervention significantly reduced the number of macrophages and CD4+T lymphocytes in EAE model mice.3.NAD+ intervention could induce the expression of CD11b+Gr-1+ MDDSCs in the spleen.The contents of CDllb and gr-1 positive MDSCs in spleen cells of NAD+ group mice in EAE model group were detected by immunofluorescence double staining.Experimental results showed that the number of CD1b+Gr-1+MDSCs in NAD+group was significantly higher than that in EAE model group.Flow cytometry was used to detect the contents of CDllb and Gr-1 positive MDSCs in spleen cells of mice in the EAE model group and NAD+group,respectively.The results showed that the ratio of CDllb and Gr-1 positive MDSCs in spleen cells of mice in the NAD+group was higher than that in the EAE model group.4.NAD+ intervention can increase the expression of Arginase-1 in the spinal cord and spleen.The expression of Arginase-1 in the spinal cord and spleen of mice in each group was detected by immunofluorescence double staining with CDllb(a surface marker of MDSCs)and Arginase-1.The results showed that the expression of Arginase-1 in the spinal cord of the NAD+drug intervention group was significantly higher than that of the EAE group,and the expression of aArginase-1 in the spleen cells of the NAD+ drug intervention group was also significantly higher than that of the EAE group.5.Serum levels of Th1 inflammatory cytokines IFN-γ,Th2 inflammatory cytokines il-13 and Arginase-1 in each group were detected by ELISA.The results showed that the expression level of IFN-γ in serum of NAD+ drug intervention group was lower than that of EAE group.The expression level of il-13 in serum of NAD+drug intervention group was significantly higher than that of EAE group.The expression level of Arginase-1 in serum of NAD+ drug intervention group was significantly higher than that of EAE group.Part three NAD+ plays a protective role in experimental autoimmune encephalomyelitis by activating the SIRT1/STAT6/Aginase-1 pathwayObjective:The EAE animal model of C57BL/6 mice was established with mog35-55,and NAD+ was used for intervention.Western blot was used to detect SIRT1 protein expression,STAT6 phosphorylation and Aginase-1 protein expression in mice,to further elucidate the mechanism of NAD+ in treating EAE.Methods:1.Select experimental animals.Female C57BL/6 mice aged 6-8 weeks were selected and their body weight was 20±2g.They were purchased from Beijing weitong lihua laboratory animal technology co.,LTD.The mice were raised in an SPF level environment with indoor temperature of 24±2℃ and relative humidity of 50%-60%.The light and dark environment was 12 hours each day.2.Establish EAE animal model.MOG35-55 peptides diluted with normal saline into 10 mg/ml,and join the isometric complete freund’s adjuvant(CFA)and a certain amount of tuberculin,then fully mixed emulsifying,eventually make the concentration of n/med tuberculosis bacili H37Ra to 4 mg/ml,on both sides of the spine in mice points respectively at 4 subcutaneous injection of 0.1 ml of emulsion,and then the immune 0 h and 48 h by intraperitoneal injection of two 0.5 ml pertussis toxin(PTX).3.Protein expressions of SIRT1,p-STAT6,t-STAT6 and Arginase-1 were detected by Western blot.4.All the experimental data were analyzed by SPSS22.0 software,and the measurement data were expressed as x ±s.The t test was used for both groups of data.Results:1.Western Blot was used to evaluate the expression of SIRT1 in spinal cord tissue.Western Blot was used to detect SIRT1 protein expression in spinal cord tissues of the EAE model group and the NAD+treatment group:SIRT1 expression in spinal cord tissues of the NAD+treatment group was significantly increased compared with that of the EAE group.2.Expression of p-STAT6 and t-STAT6 in spinal cord was evaluated by Western Blot.Western Blot was used to detect the expression of p-STAT6 and t-STAT6 proteins in the spinal cord tissues of the EAE model group and the NAD+ treatment group.3.Expression of arginase-1 in spinal cord tissues was evaluated by Western Blot.Western Blot was used to detect the expression of Arginase-1 protein in the spinal cord tissues of the EAE model group and the NAD+treatment group.In the spinal cord tissues of mice,the expression of Arginase-1 protein in the NAD+treatment group was significantly increased compared with that in the EAE model group.Conclusion:1.By analyzing the peripheral blood serum of the active period of multiple sclerosis patients,it was found that the expression level of Arginase-1 activity was decreased compared with the normal control group.To find new ideas for the treatment of multiple sclerosis.2.The EAE animal model was successfully established with MOG 35-55.After NAD+intervention,the myelin sheath of EAE mice was absent,the inflammatory response of EAE mice was effectively controlled,and the number of inflammatory cells was significantly reduced.The results showed that NAD+promoted the secretion of arginase-1 by activating MDSCs,which led to the decrease of Th1 cytokine in serum and the increase of Th2 cytokine,and finally inhibited the aggregation of T lymphocytes and macrophages in the spinal cord of EAE mice and the inflammatory response,thus playing a protective role in EAE mice.3.NAD+can protect EAE mice by activating the SIRT1/STATE/Aginase-1 signaling pathway and thereby regulating the immune inflammatory response.Thus,we hypothesized that the activation of the SIRT1/STAT6/Aginase-1 signaling pathway using NAD+might be an effective therapeutic target for multiple sclerosis,and open up new ideas for the development of new clinical drugs.