Role of nuclear factor κB in multiple sclerosis and experimental autoimmune encephalomyelitis

The transcription factor nuclear factor κB（NF-κB） plays major roles in inflammatory diseases through regulation of inflammation and cell viability.Multiple sclerosis（MS） is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system（CNS）.It has been shown that NF-κB is activated in multiple cell types in the CNS of MS patients,including T cells,microglia/macrophages,astrocytes,oligodendrocytes,and neurons.Interestingly,data from animal model studies,particularly studies of experimental autoimmune encephalomyelitis,have suggested that NF-κB activation in these individual cell types has distinct effects on the development of MS.In this review,we will cover the current literature on NF-κB and the evidence for its role in the development of MS and its animal model experimental autoimmune encephalomyelitis.

MicroRNAs as disease progression biomarkers and therapeutic targets in experimental autoimmune encephalomyelitis model of multiple sclerosis

Multiple sclerosis is an autoimmune neurodegenerative disease of the central nervous system characterized by pronounced inflammatory infiltrates entering the brain,spinal cord and optic nerve leading to demyelination.Focal demyelination is associated with relapsing-remitting multiple sclerosis,while progressive forms of the disease show axonal degeneration and neuronal loss.The tests currently used in the clinical diagnosis and management of multiple sclerosis have limitations due to specificity and sensitivity.MicroRNAs(miRNAs)are dysregulated in many diseases and disorders including demyelinating and neuroinflammatory diseases.A review of recent studies with the experimental autoimmune encephalomyelitis animal model(mostly female mice 6–12 weeks of age)has confirmed miRNAs as biomarkers of experimental autoimmune encephalomyelitis disease and importantly at the pre-onset(asymptomatic)stage when assessed in blood plasma and urine exosomes,and spinal cord tissue.The expression of certain miRNAs was also dysregulated at the onset and peak of disease in blood plasma and urine exosomes,brain and spinal cord tissue,and at the post-peak(chronic)stage of experimental autoimmune encephalomyelitis disease in spinal cord tissue.Therapies using miRNA mimics or inhibitors were found to delay the induction and alleviate the severity of experimental autoimmune encephalomyelitis disease.Interestingly,experimental autoimmune encephalomyelitis disease severity was reduced by overexpression of miR-146a,miR-23b,miR-497,miR-26a,and miR-20b,or by suppression of miR-182,miR-181c,miR-223,miR-155,and miR-873.Further studies are warranted on determining more fully miRNA profiles in blood plasma and urine exosomes of experimental autoimmune encephalomyelitis animals since they could serve as biomarkers of asymptomatic multiple sclerosis and disease course.Additionally,studies should be performed with male mice of a similar age,and with aged male and female mice.

Variation in experimental autoimmune encephalomyelitis scores in a mouse model of multiple sclerosis

Multiple sclerosis(MS) is a common demyelinating central nervous system disease associated with progressive physical impairment. To study the mechanism underlying disease pathogenesis and develop potential treatments, experimental autoimmune encephalomyelitis(EAE) is often used as an animal model. EAE can be induced in various species by introducing specific antigens, which ultimately result in motor dysfunction. Although the severity of the paralysis is indicated using the EAE score, there is no standard scoring system for EAE signs, and there is variability between research groups with regard to the exact EAE scoring system utilized. Here, we describe the criteria used for EAE scoring systems in various laboratories and suggest combining EAE score with another quantitative index to evaluate paralysis, such as the traveled distance, with the goal of facilitating the study of the mechanisms and treatment of MS.

Fasudil-modified macrophages reduce inflammation and regulate the immune response in experimental autoimmune encephalomyelitis

Multiple sclerosis is characterized by demyelination and neuronal loss caused by inflammatory cell activation and infiltration into the central nervous system.Macrophage polarization plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis,a traditional experimental model of multiple sclerosis.This study investigated the effect of Fasudil on macrophages and examined the therapeutic potential of Fasudil-modified macrophages in experimental autoimmune encephalomyelitis.We found that Fasudil induced the conversion of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type,as shown by reduced expression of inducible nitric oxide synthase/nitric oxide,interleukin-12,and CD16/32 and increased expression of arginase-1,interleukin-10,CD14,and CD206,which was linked to inhibition of Rho kinase activity,decreased expression of toll-like receptors,nuclear factor-κB,and components of the mitogen-activated protein kinase signaling pathway,and generation of the pro-inflammatory cytokines tumor necrosis factor-α,interleukin-1β,and interleukin-6.Crucially,Fasudil-modified macrophages effectively decreased the impact of experimental autoimmune encephalomyelitis,resulting in later onset of disease,lower symptom scores,less weight loss,and reduced demyelination compared with unmodified macrophages.In addition,Fasudil-modified macrophages decreased interleukin-17 expression on CD4^(+)T cells and CD16/32,inducible nitric oxide synthase,and interleukin-12 expression on F4/80^(+)macrophages,as well as increasing interleukin-10 expression on CD4^(+)T cells and arginase-1,CD206,and interleukin-10 expression on F4/80^(+)macrophages,which improved immune regulation and reduced inflammation.These findings suggest that Fasudil-modified macrophages may help treat experimental autoimmune encephalomyelitis by inducing M2 macrophage polarization and inhibiting the inflammatory response,thereby providing new insight into cell immunotherapy for multiple sclerosis.

Emodin attenuates inflammation and demyelination in experimental autoimmune encephalomyelitis

Emodin,a substance extracted from herbs such as rhubarb,has a protective effect on the central nervous system.However,the potential therapeutic effect of emodin in the context of multiple sclerosis remains unknown.In this study,a rat model of experimental autoimmune encephalomyelitis was established by immune induction to simulate multiple sclerosis,and the rats were intraperitoneally injected with emodin(20 mg/kg/d)from the day of immune induction until they were sacrificed.In this model,the nucleotide-binding domain-like receptor family pyrin domain containing 3(NLRP3)inflammasome and the microglia exacerbated neuroinflammation,playing an important role in the development of multiple sclerosis.In addition,silent information regulator of transcription 1(SIRT1)/peroxisome proliferator-activated receptor-alpha coactivator(PGC-1α)was found to inhibit activation of the NLRP3 inflammasome,and SIRT1 activation reduced disease severity in experimental autoimmune encephalomyelitis.Furthermore,treatment with emodin decreased body weight loss and neurobehavioral deficits,alleviated inflammatory cell infiltration and demyelination,reduced the expression of inflammatory cytokines,inhibited microglial aggregation and activation,decreased the levels of NLRP3 signaling pathway molecules,and increased the expression of SIRT1 and PGC-1α.These findings suggest that emodin improves the symptoms of experimental autoimmune encephalomyelitis,possibly through regulating the SIRT1/PGC-1α/NLRP3 signaling pathway and inhibiting microglial inflammation.These findings provide experimental evidence for treatment of multiple sclerosis with emodin,enlarging the scope of clinical application for emodin.

Zuogui pills for myelinolysis in a rat model of experimental autoimmune encephalomyelitis

Zuogui pills have been shown to attenuate the inflammatory reaction in a rat model of experimental autoimmune encephalomyelitis （EAE）. The present study attempted to investigate the pathology underlying the influence of Zuogui pills on myelinolysis in EAE rats. Hematoxylin-eosin and Luxol fast blue staining showed that the myelinolysis foci in the cerebrum, cerebellum, brain stem, and the spinal cord of EAE rats were significantly decreased, along with serum myelin basic protein content following treatment with Zuogui pills.

Dynamic interplay of T helpercell subsets in experimental autoimmune encephalomyelitis

AIM： To investigate the temporal onset and dynamic interplay of CD4^＋ T helper cell subsets in experimental autoimmune encephalomyelitis （EAE）.METHODS： EAE was induced in C57BL/6 mice by im-munization with myelin oligodendrocyte glycoprotein peptide p35-55. The clinical signs were scored and the tissue samples and immune cells isolated for analysis at different phases of EAE. The expression levels of inflammatory cytokines and related transcription fac-tors were detected by quantitative reverse transcription polymerase chain reaction （PCR） and enzyme linked immunosorbant assay （ELISA）. The percentages of Th1, Th17, Th2, Treg and memory T cell subsets in EAE were analyzed by immunostaining and fow cytometry. The data were analyzed by statistical techniques.RESULTS： Quantitative real-time PCR analysis showed that EAE mice express elevated levels of Th1 [interferon gamma （ IFNγ ）, interleukin （ IL ） -12p40 ], Th17 [ IL-17 , related orphan receptor gamma （RORγ ）, IL-12p40] and Treg [ Foxp3, Epstein-Barr virus induced gene 3 （EBI3）, IL-10] genes in the central nervous system at the peak of the disease. Whereas, the expression of Th1 （ IFNγ , T-bet, IL-12p35, IL-12p40 ）, Th17 （RORγ, IL-12p40 ）, Th2 （ IL-4） and Treg （ Foxp3, EBI3） response genes was reduced in the spleen during pre-disease but gradually recovered at the later phases of EAE. ELISA and fow cytometry analyses showed an increase in Th17 re-sponse in the periphery, while Th1 response remained unchanged at the peak of disease. The mRNA levels of IFNγ, IL-17 and IL-12p40 in the brain were increased by 23 （P 〈 0.001）, 9 （P 〈 0.05） and 14 （P 〈 0.01） fold, respectively, on day 21 of EAE. Conversely, the mRNA expression of IL-10 was increased by 2 fold （P 〈 0.05） in the spleen on day 21. CD4^＋CD25^＋Foxp3＋Treg response was reduced at pre-disease but recovered to na？ve levels by disease onset. The percentage of CD25 Foxp3 regulatory T cells decreased from 7.7% in the na？ve to 3.2% （P 〈 0.05） on day 7 of EAE, which then increased to 8.4% by day 28. Moreover, the CD4＋CD127＋CD44high memory T cell response was increased during the onset and recovery phases of EAE. The memory and effector cells showed an in-verse relationship in EAE, where the memory T cells increased from 12.3% in nave to 20% by day 21, and the effector cells decreased from 32% in na？ve to 21% （P 〈 0.01） by day 21. The wild type C57BL/6 mice with EAE showed elevated levels of effector-memory T cells （TEM） with concomitant reduction in central-memory T cells （TCM）, but the EAE-resistant IL-7R defcient mice showed elevated TCM with no effect on TEM cells in EAE.CONCLUSION： Our fndings highlight the temporal on-set and dynamic interplay of effector, memory and regu-latory CD4^＋ T cell subsets and its signifcance to clinical outcome in EAE and other autoimmune diseases.

The next-generation sphingosine-1 receptor modulator BAF312(siponimod) improves cortical network functionality in focal autoimmune encephalomyelitis

Autoimmune diseases of the central nervous system(CNS) like multiple sclerosis(MS) are characterized by inflammation and demyelinated lesions in white and grey matter regions. While inflammation is present at all stages of MS, it is more pronounced in the relapsing forms of the disease, whereas progressive MS(PMS) shows significant neuroaxonal damage and grey and white matter atrophy. Hence, disease-modifying treatments beneficial in patients with relapsing MS have limited success in PMS. BAF312(siponimod) is a novel sphingosine-1-phosphate receptor modulator shown to delay progression in PMS. Besides reducing inflammation by sequestering lymphocytes in lymphoid tissues, BAF312 crosses the blood-brain barrier and binds its receptors on neurons, astrocytes and oligodendrocytes. To evaluate potential direct neuroprotective effects, BAF312 was systemically or locally administered in the CNS of experimental autoimmune encephalomyelitis mice with distinct grey-and white-matter lesions(focal experimental autoimmune encephalomyelitis using an osmotic mini-pump). Ex-vivo flow cytometry revealed that systemic but not local BAF312 administration lowered immune cell infiltration in animals with both grey and white matter lesions. Ex-vivo voltage-sensitive dye imaging of acute brain slices revealed an altered spatio-temporal pattern of activation in the lesioned cortex compared to controls in response to electrical stimulation of incoming white-matter fiber tracts. Here, BAF312 administration showed partial restore of cortical neuronal circuit function. The data suggest that BAF312 exerts a neuroprotective effect after crossing the blood-brain barrier independently of peripheral effects on immune cells. Experiments were carried out in accordance with German and EU animal protection law and approved by local authorities(Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen;87-51.04.2010.A331) on December 28, 2010.

Should mast cells be considered therapeutic targets in multiple sclerosis?

Mast cells are immune cells of the myeloid lineage that are found throughout the body,including the central nervous system.They perform many functions associated with innate and specific immunity,angiogenesis,and vascular homeostasis.Moreover,they have been implicated in a series of pathologies(e.g.,hypersensitivity reactions,tumors,and inflammatory disorders).In this review,we propose that this cell could be a relevant therapeutic target in multiple sclerosis,which is a central nervous system degenerative disease.To support this proposition,we describe the general biological properties of mast cells,their contribution to innate and specific immunity,and the participation of mast cells in the various stages of multiple sclerosis and experimental autoimmune encephalomyelitis development.The final part of this review is dedicated to an overview of the available mast cells immunomodulatory drugs and their activity on multiple sclerosis and experimental autoimmune encephalomyelitis,including our own experience related to the effect of ketotifen fumarate on experimental autoimmune encephalomyelitis evolution.

Multiple sclerosis and the role of immune cells

Multiple sclerosis(MS) is a complex disease with many different immune cells involved in its pathogenesis, and in particular T cells as the most recognized cell type. Recently, the innate immune system has also been researched for its effect on the disease. Hence, cells of the immune system play vital roles in either ameliorating or exacerbating the disease. The genetic and environmental factors, as well as the etiology and pathogenesis are of utmost importance for the development of MS. An insight into the roles play by T cells, B cells, natural killer cells, and dendritic cells in MS and the animal model experimental autoimmune encephalomyelitis, will be presented. Understanding the mechanisms of action for current therapeutic modalities should help developing new therapeutic tools to treat this disease and other autoimmune diseases.

Is there a window of opportunity for the therapeutic use of vitamin D in multiple sclerosis?

Multiple sclerosis is an autoimmune treatable but not curable disease.There are a multiplicity of medications for multiple sclerosis therapy,including a class entitled disease-modifying drugs that are mainly indicated to reduce the number and severity of disease relapses.Not all patients respond well to these therapies,and minor to severe adverse effects have been reported.Vitamin D,called sunshine vitamin,is being studied as a possible light at the end of the tunnel.In this review,we recapitulated the similar immunopathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis,the immunomodulatory and neuroprotective potential of vitamin D and the state-of-art concerning its supplementation to multiple sclerosis patients.Finally,based on our and other groups’experimental findings,we analyzed the need to consider the relevance of the route and the different time-point administration aspects for a more rational indication of this vitamin to multiple sclerosis patients.

NR4A1 agonist cytosporone B attenuates neuroinflammation in a mouse model of multiple sclerosis

Nuclear receptor subfamily 4 group A1(NR4A1)is an orphan nuclear receptor,which is expressed in the majority of cells.NR4A1 expression in peripheral blood mononuclear cells is low during the preclinical stage of multiple sclerosis.Knockout of the Nr4a1 gene in mice can aggravate the symptoms of experimental autoimmune encephalomyelitis(EAE),which is an animal model of multiple sclerosis.In this study,we intragastrically administered the NR4A1 agonist cytosporone B(Csn-B)to mice after inducing EAE.After treatment with Csn-B,the clinical symptoms in the EAE mice were substantially attenuated compared with that in PBS-treated control mice.The percentages of CD4+T cells and F4/80+cells in the central nervous system were decreased.In addition,interferon-γand interleukin-17 production by proinflammatory Th1/Th17 cells in the central nervous system and interferon-γlevels in splenocytes were decreased after Csn-B treatment.These findings suggest that the NR4A1 agonist Csn-B can alleviate nerve injury after EAE induction,and,therefore,may be useful as a potential treatment for multiple sclerosis.

Early Growth Response Gene-1 Deficiency Interrupts TGFβ1 Signaling Activation and Aggravates Neurodegeneration in Experimental Autoimmune Encephalomyelitis Mice

Early growth response protein 1(Egr-1)triggers the transcription of many genes involved in cell growth,differentiation,synaptic plasticity,and neurogenesis.However,its mechanism in neuronal survival and degeneration is still poorly understood.This study demonstrated that Egr-1 was down-regulated at mRNA and protein levels in the central nervous system(CNS)of experimental autoimmune encephalomyelitis(EAE)mice.Egr-1 knockout exacerbated EAE progression in mice,as shown by increased disease severity and incidence;it also aggravated neuronal apoptosis,which was associated with weakened activation of the BDNF/TGFβ1/MAPK/Akt signaling pathways in the CNS of EAE mice.Consistently,Egr-1 siRNA promoted apoptosis but mitigated the activation of BDNF/TGFβ1/MAPK/Akt signaling in SH-SY5Y cells.Our results revealed that Egr-1 is a crucial regulator of neuronal survival in EAE by regulating TGFβ1-mediated signaling activation,implicating the important role of Egr-1 in the pathogenesis of multiple sclerosis as a potential novel therapy target.

Effect of Cordyceps sinensis on the Treatment of Experimental Autoimmune Encephalomyelitis： A Pilot Study on Mice Model

Background： As a traditional Chinese medicine, Cordyceps sinensis （CS） possesses a variety of immunoregulatory properties. This study aimed to explore the therapeutic potential of CS in a mice model of multiple sclerosis （MS）-experimental autoimmune encephalomyelitis （EAE）. Methods： Female C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein35–55 to induce EAE, followed by an instant intragastric feeding with a low dosage of CS （low CS group, n = 5）, high dosage of CS （high-CS group, n = 5）, or the same volume of normal saline （control group, n = 5）.All the mice were observed for clinical assessment. Over the 30 days of CS treatment, flow cytometry was used to detect the frequency of helper T-cell （Th） subsets, Th1 and Th17, and CD4＋ CD25＋ regulatory T cells in the spleen and lymph nodes. Meanwhile, pathological changes in brain were determined using both hematoxylin-eosin and luxol fast blue staining. Data were analyzed using the one-way analysis of variance （ANOVA）. Results： Over the 15 and 30 days of CS treatment, the clinical assessment for EAE demonstrated that both high-CS group （2.51 ± 0.31 and 2.26 ± 0.39 scores, respectively） and low-CS group （2.99 ± 0.40 and 2.69 ± 0.46, respectively） had lower disease severity scores than those of control group （3.57 ± 0.53 and 3.29 ± 0.53, all P 〈 0.01, respectively）. Meanwhile, after 15 and 30 days, the high-CS group （19.18 ± 1.34 g and 20.41 ± 1.56 g, respectively） and low-CS group （18.07 ± 1.18 g and 19.48 ± 1.69 g, respectively） had a lower body weight, as compared with control group （16.85 ± 1.15 g and 18.22 ± 1.63 g, all P 〈 0.01, respectively）.At 30 days post-CS treatment, there was a lower Th1 frequency in the lymph nodes （2.85 ± 1.54% and 2.77 ± 1.07% vs. 5.35 ± 1.34%, respectively; P 〈 0.05） and spleens （3.96 ± 1.09% and 3.09 ± 0.84% vs. 5.07 ± 1.50%, respectively; P 〈 0.05） and less inflammatory infiltration and demyelination in the brain of CS-treated mice than that of control group. Conclusions： Our preliminary study demonstrated that CS efficiently alleviated EAE severity and EAE-related pathology damage and decreased the number of Th1s in the periphery, indicating its effectiveness in the treatment of murine EAE. Thus, our findings strongly support the therapeutic potential of this agent as a new traditional Chinese medicine approach in MS treatment.

Characterization of myelin oligodendrocyte glycoprotein (MOG)35–55- specific CD8^+ T cells in experimental autoimmune encephalomyelitis

Background:The pathogenesis of multiple sclerosis(MS)is mediated primarily by T cells,but most studies of MS and its animal model,experimental autoimmune encephalomyelitis(EAE),have focused on CD4^+ T cells.The aims of the current study were to determine the pathological interrelationship between CD4 and CD8 autoreactive T cells in MS/EAE.Methods:Female C57BL/6 mice(n=20)were induced by myelin oligodendrocyte glycoprotein(MOG)35-55 peptide.At 14 days after immunization,T cells were isolated from the spleen and purified as CD4^+ and CD8^+ T cells by using CD4 and CD8 isolation kits,and then the purity was determined by flow cytometric analysis.These cells were stimulated by MOG35–55 peptide and applied to proliferation assays.The interferon-gamma(IFN-γ)and interleukin(IL)-4 secretion of supernatant of cultured CD4^+ and CD8^+ T cells were measured by enzyme-linked immunosorbent assays(ELISA).For adoptive transfer,recipient mice were injected with MOG35–55-specific CD8^+ or CD4^+ T cells.EAE clinical course was measured by EAE score at 0–5 scale and spinal cord was examined by staining with hematoxylin and eosin and Luxol fast blue staining.Results:CD8^+ CD3^+ and CD4^+ CD3^+ cells were 86%and 94%pure of total CD3^+ cells after CD8/CD4 bead enrichment,respectively.These cells were stimulated by MOG35–55 peptide and applied to proliferation assays.Although the CD8^+ T cells had a generally lower response to MOG35–55 than CD4^+ T cells,the response of CD8^+ T cells was not always dependent on CD4.CD8^+ T cell secreted less IFN-γand IL-4 compared with CD4^+ T cells.EAE was induced in wildtype B6 na?ve mice by adoptive transfer of MOG35–55-specific T cells from B6 active-induced EAE(aEAE)mice.A similar EAE score and slight inflammation and demyelination were found in naive B6 mice after transferring of CD8^+ T cells from immunized B6 mice compared with transfer of CD4^+ T cells.Conclusion:Our data suggest that CD8^+ autoreactive T cells in EAE have a lower encephalitogenic function but are unique and independent on pathogenic of EAE rather than their CD4^+ counterparts.

Cornuside alleviates experimental autoimmune encephalomyelitis by inhibiting Th17 cell infiltration into the central nervous system

The present study was conducted to clarify the therapeutic effect of cornuside on experimental autoimmune encephalomyelitis(EAE)and its influence on T helper 17(Th17)cell and regulatory T(Treg)cell infiltration into the central nervous system.Rats were randomly placed into four treatment groups:control,EAE,EAE+cornuside,and EAE+prednisolone.The neurological function scores of rats were assessed daily.On the second day after EAE rats began to show neurological deficit symptoms,the four groups were treated with normal saline,normal saline,cornuside(150 mg/kg),and prednisolone(5 mg/kg),respectively.The treatment was discontinued after two weeks,and the spinal cord was obtained for hematoxylin and eosin(H&E)and luxol fast blue staining,as well as retinoic acid receptor-related orphan receptorγ(RORγ)and forkhead box protein P3(Foxp3)immunohistochemical staining.Blood was collected for Th17 and Treg cell flow cytometry testing,and the serum levels of interleukin(IL)-17A,IL-10,transforming growth factor-β(TGF-β),IL-6,IL-23,and IL-2 were measured via enzymelinked immunosorbent assay(ELISA).Compared with rats in the EAE group,rats in the EAE+cornuside and EAE+prednisolone groups began to recover from neurological deficits earlier,and had a greater degree of improvement of symptoms.Focal inflammation,demyelination,and RORγ-positive cell infiltration were reduced by cornuside or prednisolone treatment,whereas the Foxp3-positive cell numbers were not significantly different.Meanwhile,the number of Th17 cells and the IL-17A,IL-6,and IL-23 levels were lower in the blood after cornuside or prednisolone treatment,whereas the number of Treg cells or the levels of IL-10,TGF-β,and IL-2 were not markedly different.Cornuside can alleviate symptoms of EAE neurological deficits through its anti-inflammatory and immunosuppressive effects,and Th17 cells may be one of its therapeutic targets.

MiR-125a-5p Regulates Vitamin D Receptor Expression in a Mouse Model of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis(MS)is a chronic and incurable autoimmune neurodegenerative disease of the central nervous system.Although the symptoms of MS can be managed by vitamin D3 treatment alone,this condition cannot be completely eradicated.Thus,there might be unknown factors capable of regulating the vitamin D receptor(VDR).Genome-wide analysis showed that miRNAs were associated with VDRs.We sought to determine the role and mechanism of action of miRNA-125a-5p and VDRs in a model of MS,mice with experimental autoimmune encephalomyelitis(EAE),which was induced by myelin oligodendrocyte glycoprotein 35–55 peptides.EAE mice showed decreased mean body weight but increased mean clinical scores compared with vehicle or control mice.And inflammatory infiltration was found in the lumbosacral spinal cord of EAE mice.In addition,VDR expression was significantly lower while the expression of miR-125a-5p was markedly higher in the spinal ventral horn of EAE mice than in vehicle or control mice.Importantly,activation of VDRs by paricalcitol or inhibition of miR-125a-5p by its antagomir markedly decreased the mean clinical scores in EAE mice.Interestingly,VDR and miR-125a-5p were co-localized in the same neurons of the ventral horn.More importantly,inhibition of miR-125a-5p remarkably blocked the decrease of VDRs in EAE mice.These results support a critical role for miR-125a-5p in modulating VDR activity in EAE and suggest potential novel therapeutic interventions.

MiR-30a Positively Regulates the Inflammatory Response of Microglia in Experimental Autoimmune Encephalomyelitis

Multiple sclerosis（MS） is a classical inflammatory demyelinating disease of the central nervous system（CNS）. Microglia are the main resident immune cells in the CNS and are closely associated with the pathogenesis of MS.In the present study, we found that mi R-30 a was highly expressed in jellyfish-like microglia in chronic active lesions of MS patients, as well as in the microglia of mice with experimental autoimmune encephalomyelitis（EAE） at the chronic phase. In vitro, the conditioned supernatant of mouse microglia overexpressing miR-30 a promoted the apoptosis of oligodendrocyte precursor cells（OPCs）, and inhibited OPC differentiation. In vivo, overexpressing miR-30 a in transplanted microglia exacerbated the progression of EAE.Overexpression and knock-down experiments in primary cultured mouse microglia showed that mi R-30 a increased the expression of IL-1 b and i NOS, which are pro-inflammatory, while inhibiting the expression of Ym-1 and CD206.Mechanistically, mi R-30 a inhibited the expression of Ppargc1 b, which is the co-activator of peroxisome proliferator-activated receptor gamma, resulting in pro-inflammatory effects. Our work shows that mi R-30 a is an important regulator of the inflammatory response in microglia, and may be a promising therapeutic target for inflammatory diseases like MS in the CNS.

Protective effect of Tanreqing injection on axon myelin damage in the brain of mouse model for experimental autoimmune encephalomyelitis

OBJECTIVE: To evaluate the effect of Tanreqing injection on axon myelin in the mouse brain of experimental autoimmune encephalomyelitis(EAE).METHODS: An EAE model was established by myelin oligodendrocyte glycoprotein(MOG)35-55 immunization in C57BL/6 mice. Mice were randomly divided into the following groups: normal, model,prednisone acetate(PA)(6 mg/kg), Tanreqing high dose(5.14 m L/kg), Tanreqing low dose(2.57 m L/kg). On the day of immunization, both Tanreqing groups were treated by intraperitoneal injection,with the PA group treated by intragastrical perfusion after T cell response, and the other groups treated with saline. Changes in body weight, neurological deficit score, incidence rate, mortality rate,and course of disease were observed for all mice.Brain tissue was isolated and stained with hematoxylin-eosin, and pathological investigations performed to evaluate axon myelin damage by transmission electron microscopy(TEM). Myelin basic protein and microtubule associated protein-2 were analyzed by immunohistochemistry.RESULTS: Tanreqing injection significantly prolonged EAE latency and decreased the neurological deficit score, alleviated infiltration of inflammatory cells in the focus area, up-regulated hippocampal MBP expression at the acute stage and the remission stage, and increased microtubule associated protein-2 expression in the EAE brain to varying degrees in the acute stage. TEM analysis indicated that Tanreqing injection alleviates myelin damage in the EAE mouse and maintains the integrity of circular layer structures and alleviates axon mitochondrial swelling.CONCLUSION: Tanreqing injection alleviates EAE symptoms.

Experimental Autoimmune Encephalomyelitis Inhibited by Huangqi Guizhi Wuwu Decoction via Th2 Cytokine Enhancement

Background:Huangqi Guizhi Wuwu decoction(HQGZWW)exhibits good effects when administered to treat multiple sclerosis(MS)and its animal model,experimental autoimmune encephalomyelitis(EAE).Understanding the precise mechanism of this decoction is thus important.Based on the findings of our previous study,the aim of the present study was to understand the role of antigen-specific CD8^(+)T-cells on the pathogene sis of MS/EAE when HQGZWW is administered as treatment.Methods:Myelin oligodendrocyte glycoprotein(MOG);-induced mice were administered distilled water,prednisone,and high dose or low dose HQGZWW.After purified CD4^(+)and CD8^(+)T-cells were stimulated with the MOG;peptide,proliferation and cytokine secretion assays were performed.To establish the adoptive transfer EAE model,naive mice were injected with MOG;-CD8^(+)or CD4^(+)T-cells.Results:Significant improvements in EAE score and pathology were observed in the high dose HQGZWW and prednisone groups.Compared to the low dose HQGZWW and distilled water groups,lower antigen-specific re sponses,lower levels of interferon-gamma,and higher levels of interleukin(IL)-4 and IL-10 from CD8^(+)and CD4^(+)T cells were observed in the high dose HQGZWW and prednisone groups.Finally,the EAE score was observed to be similar between the high dose HQGZWW group and prednisone group;however,this finding was not observed in the low dose HQGZWW group.Conclusion:Our findings suggest that high dose HQGZWW has similar effects on cell proliferation,cytokine secretion,and EAE score to prednisone,while low dose HQGZWW does not have such effect.The protective role of HQGZWW against EAE might thus depend on the Th2 cytokine secretion profile induced by either MOG;specific CD8^(+)or CD4^(+)T-cells.