Central Administration of 5Z-7-Oxozeaenol Protects Experimental Autoimmune Encephalomyelitis Mice by Inhibiting Microglia Activation

Transforming growth factor β-activated kinase 1 (TAK1), a vital upstream integrator of multiple pro-inflammatory signaling pathways, mediates the production of pro-inflammatory cytokines, chemokines, and adhesion molecules. Investigations targeting TAK1 provide new therapeutic options for chronic inflammatory disorders, autoimmune diseases, and cancer. However, the role and mechanism of the TAK1 inhibitor 5Z-7-oxozeaenol in treating autoimmune demyelinating diseases remain unclear. This work aimed to identify whether 5Z-7-oxozeaenol exerts neuroprotective effects on experimental autoimmune encephalomyelitis (EAE) in mice. Here, we demonstrate that 5Z-7-oxozeaenol efficiently alleviates the symptoms of EAE by decreasing the levels of pro-inflammatory cytokines in splenocytes and central nervous system, diminishing the number of activated microglia and inhibiting the p38MAPK, JNK, and ERK signaling pathways. Furthermore, we demonstrate that administration during the symptomatic time window is required for 5Z-7-oxozeaenol efficacy. These results suggest that TAK1 inhibition may provide a potent approach toward treating autoimmune demyelinating diseases.


INTRODUCTION
Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease of the central nervous system (CNS) that is characterized by demyelination and axonal destruction. Although the etiopathogenesis of MS has not been well elucidated, a cascade of pathological events is known to be involved in its pathology, including the disruption of the blood-brain-barrier (BBB), the activation of microglia and the infiltration of lymphocytes into the CNS, eventually giving rise to demyelination and degeneration of axonal structures (Lassmann et al., 2012;Martín et al., 2012;Goldmann and Prinz, 2013;Ciccarelli et al., 2014).
The activation of microglia has been described in both acute and chronic stages of MS and in its mouse model, experimental autoimmune encephalomyelitis (EAE). In fact, activated microglia nodules are the hallmark of MS lesion formation (Singh et al., 2013). Microglia are innate immune cells in the CNS that belong to the mononuclear phagocytic family and serve to patrol and monitor the microenvironment of the parenchyma. The repertoire of functions of microglia in MS is nuanced, complex and not well understood, though it has been well established that microglia contribute significantly to the overall disease severity of MS. Overactive microglia are responsible for detrimental effects leading to profound neurologic impairments, accounted for, in part, by their roles in producing pro-inflammatory cytokines, nitrogen species, reactive oxygen, and proteolytic enzymes (Merson et al., 2010). They are the main sources of cytokines and chemokines during CNS autoimmune inflammation (Hanisch, 2002). They also upregulate major histocompatibility complex class II molecules and present self-antigens, which function to drive autoimmunity (Correale, 2014).
Transforming growth factor β-activated kinase 1 (TAK1), also known as the mitogen-activated kinase kinase kinase (MAP3K), is a common upstream integrator of NF-κB, p38MAPK, JNK, and ERK and can be activated by transforming growth factor-β, TNF-α, IL-1, LPS, and BCR/TCR (Ajibade et al., 2013). Activated TAK1 strongly elicits the expression of pro-inflammatory cytokines, chemokines, and adhesion molecules. Given the role of TAK1 as a vital regulator of inflammatory and immune signaling pathways, the suppression of TAK1 production has been explored as a therapeutic strategy for chronic inflammatory disorders, including autoimmune diseases and cancer (Sakurai, 2012). Selective TAK1 knock out in microglia has been reported to prevent the release of pro-inflammatory mediators and effector molecules in the EAE model, leading to significantly ameliorated CNS inflammation and reduced tissue damage . However, the use of a pharmacological TAK1 inhibitor in treating EAE has not been described. 5Z-7-oxozeaenol, a resorcylic acid lactone derived from fungus, is a powerful inhibitor of TAK1 (Ninomiya-Tsuji et al., 2003). Selective inhibition of TAK1 with 5Z-7-oxozeaenol is known to block pro-inflammatory signaling (Wu et al., 2013); however, the impact of 5Z-7-oxozeaenol on EAE has not been fully delineated. Herein, we assessed the effect and mechanism of 5Z-7-oxozeaenol on EAE mice. Our results demonstrate that 5Z-7-oxozeaenol can effectively alleviate the symptoms of EAE, reduce the inflammatory response and demyelination in the CNS, decrease the levels of IL-17A, IFN-γ, TNF-α, and IL-6, and reduce the number of activated microglia in the CNS by inhibiting the p38MAPK, JNK, and ERK signaling pathways. These results suggest that TAK1 inhibition may provide a potent approach toward treating autoimmune demyelinating diseases.

Animals
C57BL/6 female mice (19-22 g) were purchased from the Experimental Animal Center of Guangdong Province, China. All mice were fed in pathogen-free conditions with standard laboratory chow and water ad libitum. All experiments were approved and conducted in accordance with the guidelines of the Animal Ethics Committee of the Nanfang Hospital. Efforts were made to minimize the suffering of mice during experiments.

EAE Induction
For delivery of 5Z-7-oxozeaenol, 8-to 9-week-old female mice were subjected to lateral ventricle puncture and catheterized with tubes at 1 week before induction. To induce EAE, 9-to 10-week-old female mice were subcutaneously injected in the groin and axilla with 200 µg MOG 35−55 in phosphate-buffered saline (PBS) emulsified in an equal volume of complete Freund's adjuvant (CFA) containing 0.5 mg of M. tuberculosis H37RA. As a control, mice were immunized with PBS emulsified in an equal volume of CFA containing same amount of H37RA. All mice were intraperitoneally injected with 400 ng pertussis toxin at the time of immunization and 48 h later.

Neurological Deficit Evaluation
Mice were weighed and scored blindly by a trained observer every day starting at the day after immunization as follows : 0, no detectable symptoms of EAE; 0.5 distal paralyzed tail; 1.0, completely paralyzed tail; 1.5, paralyzed tail and hind limb weakness; 2, unilateral partial hind limb paralysis; 2.5, bilateral partial hind limb paralysis; 3, complete bilateral hind limb paralysis; 3.5, complete hind limb paralysis and unilateral forelimb paralysis; 4, total paralysis of fore-and hind limbs.

Histology and Immunohistochemistry
Mice were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer on day 21 after immunization. The spinal cords were removed, post-fixed in the same fixative and paraffin embedded. Four-micrometer-thick paraffin-embedded sections of lumbar segment of the spinal cord were stained with hematoxylin and eosin (H&E), Luxol fast blue (LFB) and immunohistochemistry to visualize leukocyte infiltration, demyelination, or microglia activation. Iba-1, a marker of microglia, was used to detect microglia activation in the spinal cord.
The images were observed and photographed under a DM4000 + LED Leica fluorescent microscope. For quantification, five randomly chosen fields from each section were evaluated. Cells were counted in designated areas using Image-J.

T Cell Recall Assay and ELISA
On day 21 after immunization, splenocytes were mechanically dissociated into single cell suspensions. To prepare a homogeneous cell suspension, dispersed cells were passed through a 70 µm nylon sieve and centrifuged for 5 min at 1000 rpm. The cell pellet was incubated with 5 ml red blood cell lysis buffer for 5 min, and then 10 ml complete media was added to stop the reaction and the lysate was centrifuged for 5 min at 1000 rpm. Splenic cells (2.5 × 10 6 /ml) were plated in 24-well culture plates in RPMI 1640 (Gibco, Grand Island, NY, United States) supplemented with 10% FBS (Gibco), penicillinstreptomycin solution (penicillin 100 U/ml, streptomycin 100 µg/ml; Beyotime Biotechnology, China) and 20 µg/ml MOG 35−55 were added to the suspension. Splenocytes were cultured at 37 • C in a humidified atmosphere of 5% CO 2 . Supernatants were harvested after 48 h, and IL-17A, IFN-γ, TNF-α, and IL-6 concentrations were determined by ELISA (Biolegend, San Diego, CA, United States) according to the manufacturer's instructions.

Spinal Cord Cytokine Measurement and ELISA
Mouse spinal cord tissues were homogenized in RIPA buffer containing protease inhibitors and centrifuged at 14,000 rpm for 15 min. The protein in the supernatants was quantified by the BCA method. The supernatant was also used to measure the levels of IL-17A, IFN-γ, TNF-α, and IL-6 by the sandwich ELISA method (Biolegend, San Diego, CA, United States) according to the manufacturer's instructions. The concentrations of cytokines were expressed as picograms per 100 µg total protein (pg/100 µg total protein).

Cell Culture and Treatment
The mouse microglia cell line BV2 was cultured in DMEM supplemented with 6% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37 • C in humidified atmosphere of 5% CO 2 . After 24 h, the medium was exchanged with DMEM containing 5Z-7oxozeaenol (500 nM/ml) or DMSO (5 µl/ml). After 30 min, the medium was exchanged again with DMEM with or without LPS (1.0 µg/ml), and the cells were incubated for 6 h. Subsequently, the total protein of BV2 cells was extracted. The expression of TAK1, NF-κB, and MAPK-related proteins were detected by Western blotting.

Statistical Analyses
Statistical relevance was analyzed by SPSS 20.0 software. All data were presented as mean ± SEM. Daily mean clinical EAE scores among different time points and maximum clinical and histopathological scores between groups were assessed by the Kruskal-Wallis test followed by the Mann-Whitney U-test.
FIGURE 1 | The effect of different doses of 5Z-7-oxozeaenol treatment on the neurological deficit scores in EAE mice. Three doses of 5Z-7-oxozeaenol were used to treat EAE mice. Clinical scores of EAE were recorded from the day of immunization until 21 days after immunization. Each group included four mice. One mouse from the 5Z-7-oxozeaenol 3.2 µg-EAE group died during the experiment. Data are means ± SEM. * Adjusted P < 0.017 versus DMSO-EAE group (Mann-Whitney U-test). OZ, 5Z-7-oxozeaenol.
For other analyses with a single treatment factor, including immunohistochemistry, ELISA, and Western blot analysis, data were analyzed by one-way ANOVA. Comparisons between multiple groups were followed by least a significant difference post hoc comparison. GraphPad Prism software was utilized to plot the data, and P ≤ 0.05 was considered statistically different.
Delivery of 5Z-7-Oxozeaenol during the Effector Phase (12-21 d) and the Entire Phase (0-21 d) Alleviates the Severity of EAE To evaluate the efficacy of TAK1 inhibition in treating EAE mice, we applied 5Z-7-oxozeaenol at different phases and used clinical scoring to assess neurological deficits over a period of Frontiers in Pharmacology | www.frontiersin.org (B) Max clinical score of EAE in five groups. 5Z-7-oxozeaenol 1.6 µg (0→12 d)-EAE group, n = 8; the other groups, n = 9. Data are means ± SEM. * Adjusted P < 0.017, * * P < 0.01 and * * * P < 0.001 versus DMSO-EAE group (Mann-Whitney U-test). OZ, 5Z-7-oxozeaenol. 21 days after EAE induction with MOG 35−55 . The DMSO-CFA (negative control) group showed no visible neurobehavioral deficits during the entire period. In the DMSO-EAE (model control) group, neurological deficits began to appear on the 11th day, worsened gradually and reached a peak on the 18th day. Therefore, we used day 12 as a cutoff point for application of 5Z-7-oxozeaenol either prior to (0→12 d; induction phase), subsequent to (12→21 d; effector phase), or both prior and subsequent to (0→21 d; entire phase) disease onset. Compare to the DMSO-EAE group, both the 5Z-7-oxozeaenol 1.6 µg (12→21 d)-EAE and 5Z-7-oxozeaenol 1.6 µg (0→21 d)-EAE groups showed efficiently improved clinical outcomes from 18/19 day post-immunization (d.p.i), as assessed by the daily mean clinical EAE scores (Figure 2A). Both of these groups also had lower max clinical scores, which indicates that mice in these groups had attenuated symptoms ( Figure 2B). However, the 5Z-7-oxozeaenol 1.6 µg (0→12 d)-EAE group showed no significant effect on the daily mean clinical EAE scores and max clinical scores relative to the scores of the DMSO-EAE group (Figure 2). These results indicate that 5Z-7-oxozeaenol effectively attenuates the symptoms of EAE when applied during the effector phase or entire phase.
Delivery of 5Z-7-Oxozeaenol during the Effector Phase (12-21 d) and Entire Phase (0-21 d) Inhibits Inflammation and Demyelination in the Spinal Cords of EAE Mice To elucidate the basis for the beneficial effect of 5Z-7-oxozeaenol, we further examined the pathological changes in EAE mice upon 5Z-7-oxozeaenol treatment. On the 21 st day after immunization, we performed H&E staining to examine inflammatory cell infiltration. Consistent with the clinical findings, inflammatory infiltration was not apparent in the lumbar segment of the spinal cord for the DMSO-CFA group, but was greatly elevated in the parenchyma of the DMSO-EAE group. The infiltration was reduced in the 5Z-7-oxozeaenol 1.6 µg (12→21 d)-EAE and 5Z-7-oxozeaenol 1.6 µg (0→21 d)-EAE groups, for which inflammatory cell infiltration only was apparent in the perivascular areas and meninges (Figure 3). However, the infiltration in the 5Z-7-oxozeaenol 1.6 µg (0→12 d)-EAE group was not statistically reduced.
To evaluate the effects of 5Z-7-oxozeaenol on demyelination, we also performed LFB staining. A similar pattern was observed of statistical reduction in demyelination levels in EAE mice upon treatment with 5Z-7-oxozeaenol on days 12→21 and days 0→21, but not days 0→12 (Figure 4). Therefore, these results suggest that administration of 5Z-7-oxozeaenol during the effector phase and entire phase effectively reduces EAE severity by suppressing spinal cord inflammation and demyelination.
Delivery of 5Z-7-Oxozeaenol during the Effector Phase (12-21 d) and the Entire Phase (0-21 d) Is Associated with Decreased Microglia Activation in the Spinal Cord Microglia serve as one of the most important immunocompetent cells and a first line of defense in the CNS in response to tissue damage. Excessive or continuous activation of microglia leads to direct neurotoxicity, characterized by the production of pro-inflammatory cytokines (such as TNF-α, IL-6, and NO) and impaired neural cell function (Hanisch, 2002;Xu et al., 2016). Secreted cytokines and other harmful factors will promote the occurrence and development of inflammatory reaction. We wondered whether the neuroprotective effects of 5Z-7oxozeaenol in the EAE mouse model might be explained by 5Z-7oxozeaenol-induced inhibition of microglia activation. To assess this possibility, lumbar segments of spinal cord cross sections from CFA/EAE mice were subjected to immunohistochemistry for Iba-1, a marker for microglia activation. Few Iba-1-positive microglia were observed in the DMSO-CFA group, but a large number of Iba-1-positive microglia with more irregular cell body hypertrophy and shorten processes (active microglia) were observed in the DMSO-EAE group. The morphology and Iba-1-positivity in the 5Z-7-oxozeaenol 1.6 µg (0→12 d)-EAE group were similar to that of the DMSO-EAE group, but the 5Z-7-oxozeaenol 1.6 µg (12→21 d)-EAE and 5Z-7oxozeaenol 1.6 µg (0→21 d)-EAE groups displayed suppressed levels of microglia activation with improved morphology (Figure 7).
Application of 5Z-7-Oxozeanol during the Effector Phase (12-21 d) and the Entire Phase (0-21 d) Reduces the Level of Activated TAK1 in the EAE Model 5Z-7-oxozeaenol is known as an inhibitor that potently suppresses TAK1 activation. To confirm the influence of 5Z-7-oxozeaenol administration on TAK1 in the setting of EAE, we analyzed spinal cord homogenates by Western blotting of phosphorylated (activated) TAK1 and total TAK1. Compared to the phospho-TAK1 level in the DMSO-CFA group, the level in the DMSO-EAE group was increased. The 5Z-7-oxozeaenol 1.6 µg (12→21 d)-EAE and 5Z-7-oxozeaenol 1.6 µg (0→21 d)-EAE groups displayed reduced phospho-TAK1 levels compared to the level in the DMSO-EAE group, whereas the level in the 5Z-7-oxozeaenol 1.6 µg (0→12 d)-EAE group was not significantly FIGURE 7 | The effect of 5Z-7-oxozeaenol treatment on Iba-1-positive microglia in the spinal cords of EAE mice. Immunohistochemistry was used to evaluate the Iba-1-positive microglia in the lumbar intumescence spinal cord in each group. Counts of Iba-1-positive microglia were determined by quantitative method. Five fields from each mouse were analyzed. (A) Representative immunohistochemistry of Iba-1-positive microglia. (B) Quantification of Iba-1-positive microglia in the spinal cord. Each group included five mice, except for the 5Z-7-oxozeaenol 1.6 µg (0→12 d)-EAE group, which only had four mice because one died. * * * P < 0.001 versus DMSO-EAE group, ### P < 0.001 versus DMSO-CFA group (one-way ANOVA). Data are means ± SEM. OZ, 5Z-7-oxozeaenol. Scale bar = 50 µm for 20×, 100 µm for 40×. different (Figure 9). These finding verify the function of 5Z-7-oxozeaenol in suppressing TAK1 activation and support the role of TAK1 in mediating the neurodegenerative outcome of EAE.
(0→21 d) phase reduced ERK1/2 activation (Figures 10A,G). According to these results, we conclude that administration of 5Z-7-oxozeaenol inhibits the p38MAPK, JNK and ERK pathways, all of which may play important roles in mediating its effects in inactivating microglia and reducing neuroinflammation.

Administration of 5Z-7-Oxozeaenol Interferes with LPS-Induced MAPK Signaling in BV2 Cells
To support the possibility that the in vivo effects of 5Z-7oxozeaenol on MAPK signaling may be mediated by direct effects on microglia, we examined the in vitro the ability of 5Z-7-oxozeaenol to alter MAPK signaling pathways in microglia. The mouse microglia cell line BV2 was pre-treated with DMSO or 500 nM 5Z-7-oxozeaenol for 30 min prior to activation of MAPK signaling by treatment with LPS (1 µg/ml). LPS increased the levels of p-TAK1, NF-κB, p-p38, p-c-Jun p-JNK, and p-ERK1/2. As expected, 5Z-7-oxozeaenol (500 nM) treatment blocked LPS-induced TAK1 phosphorylation at Ser192. 5Z-7oxozeaenol did not affect the inflammatory NF-κB signaling cascade, as evidenced by the expression levels of its inhibitor IκBα, which were almost the same with and without 5Z-7oxozeaenol. However, LPS-induced MAPK activation was largely inhibited by 5Z-7-oxozeaenol co-treatment (Figure 11). 5Z-7oxozeaenol led to a reduction in LPS-induced MAPK activation, whereas no effect on NF-κB activation was observed. These data demonstrate that 5Z-7-oxozeaenol has the potential to inhibit MAPK activation in a microglia cell line. Collectively, our data suggest that 5Z-7-oxozeaenol is a potent inhibitor of inflammatory cascades in microglial cells, which may contribute to its neuroprotective effects within the CNS parenchyma.

DISCUSSION
TAK1 has been found to play a critical role in innate and adaptive immunity, DNA damage response and inflammatory signaling (Dai et al., 2012). However, the role of TAK1 in autoimmune diseases of the brain had not been well characterized until recently, when conditional depletion of TAK1 in microglia was shown to block p65, JNK, ERK1/2, and p38MAPKK to protect mice from EAE, potentially by impeding the infiltration of peripheral immune cells into the CNS and overall dampening of inflammatory immune response. Microglia-specific expression of TAK1 is essential for the pathogenesis of autoimmune inflammation of the CNS . In this context, we examined the effect of TAK1 inhibition with 5Z-7-oxozeaenol in ameliorating the symptoms and cellular and molecular effects of EAE. Consistent with the results of Goldmann, we demonstrated that 5Z-7-oxozeaenol administration attenuates the production of pro-inflammatory cytokines and the activation of microglia by interfering with p38MAPK, JNK, and ERK signaling pathways, whereas it had no effect on NF-κB activation. Here, for the first time, we identified a unique effective therapeutic time-window of 5Z-7-oxozeaenol in MOGinduced EAE. Our results demonstrate that 5Z-7-oxozeanol shows therapeutic efficacy when administered either 12→21 d.p.i., corresponding to the symptomatic stage, or 0→21 d.p.i., corresponding to the entire stage.
Pro-inflammatory cytokines play a prominent role in classical neuroinflammatory diseases, such as MS and encephalitides. We demonstrated that splenocytes extracted from 5Z-7-oxozeaenoltreated EAE mice display a significant decrease in IL-17A, IFN-γ, TNF-α, and IL-6 production in response to MOG restimulation. In EAE mice model, the BBB is compromised. Immune cells can invade to the CNS through a compromised BBB and vice versa. 5Z-7-oxozeaenol may cross the BBB to exert its effect on splenocytes. Similar results have been described for other methods of TAK1 inhibition. For example, in vivo RNAi-mediated silencing of TAK1 in the mouse significantly decreases IL-17A, IFN-γ, and TNF-α secretion produced by the spleen in collagen-induced arthritis (Courties et al., 2010). 5Z-7-oxozeaenol also inhibits pro-inflammatory cytokines in the CNS. However, there is some disagreement in regards to the therapeutic time-window of 5Z-7-oxozeaenol for ameliorating the neurological deficit score, EAE pathology and pro-inflammatory cytokine production, which cannot be simply explained by limiting T cell responses or pro-inflammatory cytokines. These incongruences are likely to be explained by the contribution of other cells that contribute to the pathogenesis of EAE, such as microglia.
Microglia serve as the first scavengers after CNS insult, and activated microglia are both major producers and targets of proinflammatory cytokines (Zhou et al., 2015). Therefore, we also evaluated whether the protection by 5Z-7-oxozeaenol of EAE pathology might due to anti-inflammatory effects on microglia that reside within the CNS. Our results demonstrate that the ameliorated EAE pathologic features and clinical severity after treatment of 5Z-7-oxozeaenol during the effector phase (12→21 d.p.i.) and the entire phase (0→21 d.p.i.), accompanied by a marked reduction in CNS inflammation and demyelination, were coincident with decreased microglia activation in the CNS. This highlights the potent ability of 5Z-7-oxozeaenol to regulate microglia function, which would lead to a suppressed proinflammatory milieu in the CNS of EAE mice. Therapeutic approaches designed to modulate the activation of microglia are known to be beneficial to EAE. For example, 18 β-glycyrrhetinic acid suppresses the progression of EAE via inhibition of microglia activation (Zhou et al., 2015). Furthermore, pertussis toxin modulates the microglia and T cell profile to protect EAE (Yin et al., 2014). Finally, cannabidiol decreases spinal microglial activation and ameliorates symptoms of EAE in C57BL/6 mice (Kozela et al., 2011).
TAK1 is highly expressed in the brain (Yamaguchi et al., 1995). However, it is currently unclear whether TAK1 is also activated in the brains of MS patients, while it has been shown that TAK1 and downstream NF-κB and MAPK signaling pathways are activated in EAE mice (Shin et al., 2003). Our results verify that the NF-κB, p38MAPK, JNK, and ERK pathways are activated by EAE induction. Furthermore, 5Z-7-oxozeaenol interfered with the p38MAPK, JNK, and ERK signaling cascades, but not the NF-κB signaling pathway. These results suggest that 5Z-7-oxozeaenol shows a level of selectivity in the down-stream signaling cascades that are inhibited. Similar to these findings, mice treated with 5Z-7-oxozeaenol show a significant reduction in the activity of p38MAPK after MCAO, but have no effect on JNK (Neubert et al., 2011). Though the NF-κB signaling pathway was not blocked by in vivo 5Z-7-oxozeaenol administration in our study, blockade of the NF-κB signaling pathway is known to enhance the capacity of immature dendritic cells to induce antigen-specific tolerance in EAE (Iruretagoyena et al., 2006). Furthermore, NF-κB inhibition has been shown to be an effective therapeutic approach for other autoimmune diseases. For example, transgenic inhibition of astroglial NF-κB protects experimental optic neuritis mice from optic nerve damage and retinal ganglion cell loss (Brambilla et al., 2012). Therefore, a synergistic approach could potentially enhance the effectiveness of 5Z-7-oxozeaenol.
However, even in the absence of NF-κB inhibition, the ability of 5Z-7-oxozeaenol to simultaneously inhibit each of the three MAPK pathways could explain its efficacy. In particular, p38MAPK plays a prominent role in multiple inflammatory diseases. Oral application of a highly specific p38 inhibitor, UR-5269, markedly reduces clinical symptoms of EAE (Namiki et al., 2012). Furthermore, inhibition of p38MAPK exhibits protective effects in animal models of rheumatoid arthritis (Brown et al., 2004). Inhibitors of p38MAPK have been the subject of clinical trials for the treatment of rheumatoid arthritis and psoriasis (Cohen, 2009); however, none of the inhibitors have progressed to phase III because of toxicity issues. Such side effects are thought to be due to the inhibition of p38MAPK feedback control loops, resulting in the hyperactivation of TAK1 and JNK kinases (Cohen, 2009). Therefore, TAK1 inhibition has an advantage of simultaneously inhibition of several of the pathways that are susceptible to feedback regulation.
To support the role of 5Z-7-oxozeaenol in microglia, we also assessed its in vitro ability to suppress NF-κB and MAPK signaling pathways in LPS-activated mouse microglia BV2 cells. Our results demonstrated that 5Z-7-oxozeaenol suppresses the activation of the MAPK pathways, but has no effect on NF-κB pathway. NF-κB can be activated through a canonical or non-canonical pathway. The canonical (or classical) NF-κB signaling pathway is induced through tumor necrosis factor receptor 1, Toll-like receptors, interleukin-1 receptor, or T and B cell receptors. Subsequently, TAK1 is phosphorylated, leading to downstream IκBα phosphorylation, which mediates NF-κB activation (Guire et al., 2013). Furthermore, analogous to the phenomenon observed with p38MAPK inhibitors (Cohen, 2009). Inhibition of NF-κB by 5Z-7-oxozeaenol could lead to hyperactivation of the NF-κB pathway by a feedback mechanism that occurs in the mouse model and in vitro.

CONCLUSION
The present study demonstrates that 5Z-7-oxozeaenol potently protects mice from EAE progression, decreases inflammatory responses and demyelination of the CNS, deactivates microglia and inhibits the secretion of pro-inflammatory cytokines by blocking the p38MAPK, JNK, and ERK signaling cascades. Our findings confirm that 5Z-7-oxozeaenol represents a potential novel drug that can be applied clinically to CNS autoimmune disorders. Other drugs targeting TAK1 might also provide a promising approach for modulating the course of the immune reaction in autoimmune demyelinating diseases and potentially other chronic inflammatory diseases.

AUTHOR CONTRIBUTIONS
SQ and PX conceived and designed all experiments in this study. LL established EAE mice model, performed H&E staining, LFB staining, immunohistochemistry, ELISA, and Western blots assays. XZ and WZ performed the behavioral experiment. HT carried out ELISA. SQ, LL, XZ, and PX analyzed the data. LL and SQ wrote the manuscript.