| Objective:Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS) that affects approximately one million people worldwide. Like most autoimmune diseases, it has a complex and poorly understood etiology, whereby environmental factors acting upon a susceptible genetic background culminate in disease. MS is considered a classical T cell-mediated disease and is characterized by inflammatory infiltration, demyelination, axonal damage, and progressive neurological dysfunction. Currently available disease modifying therapies are limited in their efficacy, and improved understanding of new pathways contributing to disease pathogenesis could reveal additional novel therapeutic targets. Experimental autoimmune encephalomyelitis (EAE) is the principal animal model of human MS. It is characterized by neuroinflammation, demyelination, axonal damage, and progressive neurological dysfunction similar to what is seen in MS.All of the currently approved MS disease modifying therapies are also efficacious in EAE, and several of these therapies were initially developed using EAE, reinforcing the relevance of this model.The etiology of MS has not yet been fully elucidated, but it is believed that inflammation induced by antigen-specific T cell immunity is the most important in disease initiation and progression. Both Thl and Th17 cells are currently thought to be involved in the pathogenesis of MS. Antigen-specific Th17 cells are major players in the pathogenesis of EAE. While CD4 T cells initiate the inflammatory cascade in CNS, secretion of reactive oxygen species by other immune and resident cells mediate the killing of oligodendrocytes. Damage to the oligodendrocyte-myelin-axon unit leads to impaired neural signal propagation, which in turn causes the neurological disability associated with MS. Notably, inflammation-related oxidative stress also contributes to the pathogenesis of MS and EAE. Oxidative stress results from high levels of reactive oxygen species (ROS), exhausting endogenous antioxidants. High levels of ROS can disrupt the blood-brain barrier (BBB), recruit leukocyte nigration and mediate myelin phagocytosis, and oligodendroglial and ixonal injury. In addition, oxidative stress can enhance autoimmune nflammation. Treatment with potent antioxidants may be promising in nhibiting the development and progression of MS. Although OS may ndeed worsen autoimmune disease, the data provided do not establish exactly which pathways OS works on to achieve this.However, the efficacy of either immune-modulating agents or anti-oxidants therapy is limited. In addition, the precise mechanisms underlying the crosstalk between inflammation and oxidative stress emain to be further investigated. Novel insights are needed to devise mproved treatments for this complex and heterogeneous disease.The p38 MAP kinase (MAPK)-SGKl signaling pathway is known to oe triggered by stress stimuli and to contribute to inflammatory responses. Activation p38 MAPK has been shown to be required for production of IL-17 by Th17 cells in vitro and in vivo. Importantly, a number of recent studies have identified this signaling pathway as a central player in MS and its principal animal model, experimental allergic encephalomyelitis. mproved understanding of the role p38 MAPK-SGK1 in MS and its nodels, is likely to lead to improved and personalized therapies for MS in the future.p38MAP kinase is involved in the development of demyelinating diseases. It is activated in response to oxidative stress, and plays a major role in the regulation of TH17 inflammatory responses. We infer p38MAPK-SGK1 may be the converging point of such pathogenic mechanisms. Oxidative stress contributes to the pathogenesis of MS and EAE via p38MAPK-SGK1 activation of central nervous system. Oxidative stress-p38MAPK-SGK1 axis may regulate key immunopathogenic mechanisms underlying EAE.Our previous study has showed treatment with NDGA inhibited EAE development and severity in mice by its anti-oxidant activity and antagonizing autoimmune inflammation. We hope to explore whether its efficacy partly comes from the suppression of above-mentioned axis.Methods:1 Induction and assessment of EAEMice were injected subcutaneously with 250μg MOG35-55 peptide emulsified in complete Freund’s adjuvant containing 4 mg/ml of heat-killed Mycobacterium tuberculosis. On Oh and 48h after immunization, the mice were injected intraperitoneally with 500 ng pertussis toxin.2 Assessment of EAE and treatmentSB203580 5mg/kg, TEMPOL 25mg/kg or NDGA 1Omg/kg i.p. injections were given once daily in each treatment groups. Control mice and EAE group received a daily injection (i.p.) of saline (vehicle)The mice were examined daily for clinical signs of EAE and scored as follows:The scale ranges from 0 to 15 and is the sum of the state of the tail and all of the four limbs. For the tail, a score of 0 reflects no signs, 1 represents a half paralyzed tail, while a score of 2 is given to a mouse with a fully paralyzed tail. For each of the hind-or forelimbs, each assessed separately,0 signifies no signs, a score of 1 is a weak or altered gait,2 represents paresis, while a score of 3 denotes a fully paralyzed limb. Thus, a fully paralyzed quadriplegic animal would attain a score of 14. Mortality equals a score of 15.3 Histology, immunohistochemistry and electron microscopyMice from each group were perfused through the left cardiac ventricle with saline, plus 0.5 M EDTA for 5-10 min followed by fixation with cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). Subsequently, spinal cords were carefully dissected out and post-fixed in 4% PFA for 3-4 h and processed for paraffin embedding. Quantification of neurological damage in EAE mice was performed via histological analysis of 5μm paraffin CNS sections of control, EAE, SB203580, Tempol, and NDGA EAE treated mice. Two different stains were used to detect inflammatory infiltrates (H&E) and demyelination (Luxol fast blue). Three spinal cord sections (5μm/each, interval of 100 μm) of individual mice were stained with hematoxylin & eosin(H&E) for routine evaluation of histology and inflammation, and were stained with Luxol Fast Blue (LFB) for the evaluation of demyelination. The degrees of inflammation and demyelination on three non-serial sections of each mouse were assessed semi-quantitatively in a blinded manner.The levels of p38,p-p38,SGK1,IL-17,RORyt and HO-1 expression in the spinal cord tissue sections were characterized by immunohistochemistry. Briefly, the brain or spinal cord tissue sections (5 μm/each) were deparaffinized, rehydrated and treated with 3%(v/v) H2O2 in methanol for 30 min, followed by blocking with 5%(w/v) fat-free dry milk for 1h. The sections were incubated with anti-p38 anti-p-p38, anti-SGK1, anti-IL-17, anti-RORyt or anti-HO-1 at 4℃ overnight. After being washed, the bound antibodies were detected with biotinylated secondary antibodies and the ABC kit, and visualized using diaminobenzidine, followed by examination under a light microscope.The spinal cords of individual mice were dissected and the dorsal hone tissues in small blocks were pre-fixed with 4%(v/v) glutaraldehyde in 0.1 M PBS by immersion. After being washed with PBS four times, the dorsal hone tissues were post-fixed in 1%(w/v) buffered osmium tetroxide for 1 h, dehydrated through graded acetone, and embedded in epoxy resin Epon 812. The ultra-thin sections (70 nm) were stained with uranyl acetate and lead citrate, and examined with a electron microscope.4 Western blot analysis The spine tissues were dissected and frozen immediately in liquid nitrogen. The proteins were prepared according to the manufacturer’s instruction. The concentrations of proteins were measured using a BCA protein assay reagent kit. Equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electro-phoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes.Nonspecific reactivity was blocked by 5% non-fat dry milk in TBST buffer at room temperature for 1 h, followed by incubation with primary antibodies for p38MAPK, p-p38MAPK, SGK1, IL-17, RORyt and HO-1 at 4℃ for 12 h.After being washed with PBST for three times, the bound antibodies were detected with corresponding secondary antibodies. The relative levels of target protein to control expression were quantified by densitometric scanning.5 Measurement of malondialdehydeThe degrees of oxidative stress in the spines were assessed for the contents of malondialdehyde (MDA). Briefly, the brain tissues were collected post immunization and homogenized for assay. The concentrations of total proteins were measured using a BCA protein assay reagent kit, following the manufacturer’s instructions and the contents of MDA were determined using a MDA detection kit, according to the manufacturer’s instructions. The concentrations of MDA were calculated as nmol/mg proteins.6 Quantitative real-time PCR analysisSome mice from each group were sacrificed and their spinal cords were dissected. The relative levels of p38MAPK, SGK1, NFAT5 and HO-1 mRNA transcripts to the control β-actin were evaluated by quantitative RT-PCR. Briefly, total RNA was extracted from the spinal cords with Trizol reagent and after digesting with DNase 1, the RNA was reversely transcribed into cDNA using the PrimeScriptTM RT reagent Kit with gDNA Eraser, according to the manufacturers’instructions. The PCR amplification was carried out using SYBR(?) Premix Ex TaqTM II (Tli RNaseH Plus), ROX plus, and the specific primers on the Roche LightCycler 480IIsystem. The PCR reactions were performed at 95℃ for 30s and subjected to 45 cycles of 95℃ for 5 s and 60℃ for 40 s. Data were analyzed by Sequence Detection Systems software. The relative levels of each target gene mRNA transcripts to the control were calculated using the 2-ΔΔCT method.7 Statistical AnalysisData are presented as mean±SD. The onset rates of EAE between groups were analyzed by chi-square test. All other statistical comparisons among groups were examined using Student’s t test or ANOVA analysis followed by SNK-q test. P values less than 0.05 were considered statistically significant.Results:Our research showed a dynamic change of immune inflammation, oxidative stress and p38MAPK-SGK1 pathway in EAE. Along with the worsen of EAE clinical signs, the induction of Th17 cells was boosted, oxidative stress leveled up while expression of p38MAPK and SGK1 increased. Either p38MAPK-SGK1 pathway or oxidative stress showed consistency for EAE course, suggesting a strong correlation between them.Clearly, treatment with SB203580 or Tempol significantly reduced the clinical scores. The mean clinical scores in the mice from treatment groups were lower than that in the saline-treated control EAE mice throughout the observation period. Histological examination showed weakened inflammatory infiltration, reduced demyelinating area and lightened axonal injury in central nerve system. Result from Immunohistochemistry and western blot analysis suggested the Th17 mediated inflammation was much weakened. In addition, antioxidant therapy inhibited the p38MAPK-SGK1 pathway strongly. It suggested that oxidative stress exacerbated EAE via the activation of p38MAPK-SGK1 pathway. Oxidative stress-p38MAPK-SGK1 pathway may be a central player in MS and its principal animal model, EAE. To determine the potential effect of treatment with NDGA on the development and severity of EAE, we compared the EAE clinical score, histological injury and the expression of IL-17 and RORyt among different groups. NDGA relieved EAE clinical symptoms and impired the Thl7 inflammation similar to SB203580 and Tempol suggesting that both anti-inflammation and anti-oxidant activity contribute the protective effect of NDGA to EAE.Conclusion:In this study, we immunized with MOG35-55 peptide to induce EAE and tested the effect of treatment with SB203580, Tempol and NDGA on the development and progression of EAE in C57BL/6 mice. We report the dynamic change of oxidative stress and p38MAPK-SGK1 pathway as well as the correlation mechanism between them in EAE. We show the evidence from mice study supporting the role of Oxidative stress-p38MAPK-SGK1 axis in regulating key immunopathogenic mechanisms underlying autoimmune inflammatory disease of the central nervous system, and the potential of targeting p38MAPK pathway as a disease modifying therapy in MS.Furthermore, we determined the potential mechanisms underlying the action of NDGA in inhibiting EAE in mice. Treatment with NDGA inhibited the development and progression of EAE at least partly by inhibiting the p38MAPK-SGK1 pathway. |
