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Objective: To investigate the effects of nicotinamide adenine dinucleotide (NAD+) on the pathogenesis of the animal model for multiple sclerosis (MS)-experimental autoimmune encephalomyelitis (EAE)., Methods: EAE model was induced by myelin oligodendrocyte protein (MOG 35-55). Clinical scores of EAE were measured in mice with or without NAD+ treatment. Hematoxylin and Eosin (HE) and Luxol Fast Blue (LFB) staining were performed to assess inflammation and demyelination, respectively. Expressions of target proteins were measured by Western blot. The numbers of myeloid-derived suppressor cells (MDSCs) were measured by immunofluorescent staining and flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to measure the expressions of inflammatory cytokine in serum., Results: NAD+ treatment could decrease inflammatory cells and demyelination foci, attenuate the clinical scores of EAE and slightly delay disease onset. Western blot showed that NAD+ treatment up-regulated the expression of phosphorylated-STAT6 (p-STAT6) and SIRT1. Besides, NAD+ treatment up-regulated the expression of p-IκB and down-regulated the expression of p-NF-κB. In addition, NAD+ treatment could increase the numbers of CD11b+ gr-1+ MDSCs and the expression of Arginase-1. Moreover, NAD+ treatment up-regulated the expressions of IL-13 and down-regulated the expression of IFN-γ and IL-17., Conclusions: The present study demonstrated that NAD+ treatment may induce the CD11b+ gr-1+ MDSCs to attenuate EAE via activating the phosphorylation of STAT6 expression. Therefore, NAD+ should be considered as a potential novel therapeutic strategy for MS., (© 2020 The Author(s).)

Multiple sclerosis (MS) is characterized with multifocal demyelination resulting from activation and infiltration of inflammatory cells into the central nerve system. Recent reports suggest that p38 mitogen-activated protein kinase (MAPK) / serum- and glucocorticoid-inducible protein kinase 1 (SGK1) signaling pathway contributes to the pathology of MS through regulation of immunity. However, the role of this signaling pathway in MS-related macrophage activation and polarization has not been studied. Here, we used an experimental autoimmune encephalomyelitis (EAE) model for MS to study the role of p38MAPK/SGK1 signaling in the macrophage polarization and its effects on the development and severity of EAE. Here, we found that p38MAPK/SGK1 signaling is required for IL4-induced M2 macrophage polarization in vitro. Chitin-induced M2 macrophage polarization reduces the severity of EAE in mice. Generation of an adeno-associated virus (AAV) carrying sh-p38 or sh-SGK1 under the control of a CD68 promoter successfully knockdown p38 or SGK1 levels in vitro and in vivo. Treatment with AAV-sh-p38 or AAV-sh-SGK1 abolished the effects of Chitin on macrophage polarization and the severity of EAE. Thus, our data suggest that p38MAPK/SGK1 signaling induces M2 macrophage polarization, which reduces the severity of EAE, a model for MS.

Multiple sclerosis (MS) is a poorly understood disease mechanistically. MOG35-55 peptide induced experimental autoimmune encephalomyelitis (EAE) is a broadly used model to study MS. Using this model we have earlier shown that the antioxidant tempol or the small molecule inhibitor of p38 SB203580 can effectively prevent EAE progression. This effect was mediated by means of regulating immune inflammation, signaling by the p38MAPK-SGK1 pathway, and oxidative stress. However, there is a need to test drugs that can be used in pharmacological intervention of EAE. Given that nordihydroguaiaretic Acid (NDGA) has been shown to possess anti-oxidant activity and capacity of antagonizing autoimmune inflammation, we tested the effect of NDGA in ameliorating EAE in the current study. NDGA showed significant beneficial effect against EAE with both anti-inflammation and antioxidant activity. NDGA could weaken the immune inflammation at least partly by inhibiting the oxidant stress-p38MAPK-SGK1 pathway representing a target for putative pharmacological intervention. © 2018 IUBMB Life, 70(5):432-436, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

Background: Multiple sclerosis (MS), a complex disease associated with multifocal demyelination of the central nervous system and poorly understood etiology. It has been previously indicated that many factors, including oxidative stress and p38MAPK-SGK1 pathway, contribute to the pathogenesis of MS., Methods: This study, using an experimental autoimmune encephalomyelitis (EAE) model system, was aimed at investigating the molecular mechanisms determining interaction p38MAPK-SGK1 pathway and oxidative stress in MS pathogenesis. C57BL/6 mice was immunized with MOG35-55 peptide for EAE induction, which was followed by determination of the effect of treatment with classic p38 inhibitor SB203580 and antioxidant tempol on the development and progression of EAE., Results: Our experiments showed a dynamic change of immune inflammation, oxidative stress and p38MAPK-SGK1 pathway involvement in EAE demonstrating that p38MAPK-SGK1 pathway and oxidative stress contribute to the demyelination in central nerve system caused by Th17 inflammatory responses in a synergistic way. The administration of SB203580 and Tempol both markedly suppressed the progression of EAE. Furthermore, tempol showed a strong inhibiting effect to the p38MAPK-SGK1 pathway similar to SB203580 suggesting that oxidative stress exacerbates EAE via the activation of p38MAPK-SGK1 pathway., Conclusion: Cumulatively, our results show that oxidative stress p38MAPK-SGK1 signaling pathway may be a central player in EAE and even in MS.

Aim: To observe the dynamic changes of heme oxygenase-1 (HO-1) mRNA and protein express in subfornical organ in rats with experimental allergic encephalomyelitis (EAE) to confirm that SFO is one of the sites for blood-bearing signaling molecules entering into brain., Methods: EAE was induced by CFA-GPSCH on Wistar rats, we observed the levels of HO-1 mRNA and its protein expression with immunohistochemistry and in situ hybridization technology on 1 d, 7 d, 14 d, and 21 d after EAE induction in SFO of rats. The relationship between HO-1 and symptoms of EAE was also investigated., Results: The expression levels of HO-1 mRNA and its protein expression were very low in the brains of the control group, whereas they were enhanced gradually with pathological course in the brain and onsets of symptoms, signs of EAE. On 1 d after induction of EAE, positive cells of HO-1 mRNA and its protein expression were observed at SFO, but the labeled cells were rarely seen in the other brain regions. On 7 d, the positive cells increased markedly. On 14 d the levels of HO-1 mRNA and its protein expression in the brains reached the peak, the positive cells of HO-1 were mainly located at the choroid plexuses and SFO, as well as the regions around "sleeve-like" lesion foci, all of which were coincident with the locations of lesions of EAE. The changes of incidence, symptom, reduction of the body weight, and pathology lesions of EAE in rat brains were the most significant. On 21 d, the levels of HO-1 mRNA and its protein expression reduced gradually, which was in parallel with remitted symptoms of EAE. When a specific inhibitor of HO-1, Snpp9, was applied, the symptoms and pathological lesions of EAE in brains were mitigated markedly., Conclusion: SFO may be one of the earliest sites for blood-bearing signaling molecules entering into brain. The dynamic changes of HO-1 mRNA and its protein expression are in parallel with the changes of symptoms and pathological lesions of EAE in the brains. Application of some inhibitors of HO-1 may be one of potential therapeutic methods for prevention and treatment of EAE.

In order to investigate the role of heme oxygenase-1 (HO-1) in the molecular mechanism of experimental allergic encephalomyelitis (EAE), which was induced by guinea pig spinal cord homogenate + complete freund adjuvant on Wistar rats, we observed the gene of HO-1 and its protein expression with reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistry 1, 7, 14, and 21 d after EAE induction in rats. The relationship between HO-1 and the symptoms of EAE was also observed. The results showed that the levels of HO-1 mRNA and its protein expression were very low in the brains of the control group, whereas they were enhanced gradually with pathological course in the brain and onsets of symptoms, signs of EAE. On day 7, the level of HO-1 mRNA reached the peak, but the expression level of HO-1 protein in the brains reached the peak on day 14. The immunoreactive cells of HO-1 were mainly located at the choroid plexuses and subfornical organ (SFO), as well as in regions around the "sleeve-like" lesion foci, all of which were coincident with the locations of lesions of EAE. The levels of HO-1 mRNA and its protein expression were lowered gradually on day 21, which were in parallel with the severities of symptoms and signs of EAE. After a specific inhibitor of HO-1, Snpp-9, was applied, both of the symptoms and pathological lesions of EAE in the rat brains were mitigated markedly. Therefore, these results may suggest that the dynamic changes of HO-1 mRNA and its protein expression are in parallel with the changes of symptoms and pathological lesions of EAE in the brain. In conclusion, the levels of HO-1 mRNA and its protein expression in brains may play an important role in the pathogenesis of EAE, and application of inhibitors of HO-1 may be one of the potential therapeutic ways for the prevention and treatment of EAE.

To investigate the role of activated nuclear factor-kappaB (NF-kappaB) in experimental allergic encephalomyelitis (EAE), the activity and protein expression of NF-kappaB p65 in rat brain tissues, which were extracted from EAE rats at 1, 7, 14 and 21 d respectively after EAE was induced by CFA-GPSCH, were measured with electrophoretic mobility shift assay and immunohistochemistry. The relationship between activated NF-kappaB and symptoms of EAE was also investigated. The results showed that protein expression level and the activity of NF-kappaB were very low in the brain of the control group. After EAE was induced, the activity of NF-kappaB and the level of the protein expression in the brains increased gradually with the development of symptoms and brain pathology of EAE. On d 14, both the activity and the level of protein expression in the brains reached a peak, the positive cells of NF-kappaB were mainly located at the choroid plexuses and subfornical organ, as well as around the regions of sleeve-like lesion foci, which were coincident with the locations of lesions of EAE. The incidence, symptoms, reduction of the body weight and pathology of EAE rats brains at the above locations were most significant. On d 21 the activity of NF-kappaB and level of the protein expression reduced gradually, which was in parallel with a gradual alleviation of the symptoms of EAE rats. After a specific inhibitor of NF-kappaB, PDTC was applied, the symptoms and pathological lesions of EAE rat brain were mitigated markedly. The above results indicate that the dynamic changes in the activity and protein expression of NF-kappaB were in parallel with the changes in symptoms and pathological lesion of EAE rat brains. In conclusion, the activated NF-kappaB in the brain may play a critical role in the pathogenesis of EAE, and application of some inhibitors of NF-kappaB, such as PDTC, may be one of the effective therapeutic methods for prevention and treatment of EAE.