Grape Seed Extract Attenuates Demyelination in Experimental Autoimmune Encephalomyelitis Mice by Inhibiting Inflammatory Response of Immune Cells / 中国结合医学杂志

OBJECTIVE@#To examine the anti-inflammatory effect of grape seed extract (GSE) in animal and cellular models and explore its mechanism of action.@*METHODS@#This study determined the inhibitory effect of GSE on macrophage inflammation and Th1 and Th17 polarization in vitro. Based on the in vitro results, the effects and mechanisms of GSE on multiple sclerosis (MS)-experimental autoimmune encephalomyelitis (EAE) mice model were further explored. The C57BL/6 mice were intragastrically administered with 50 mg/kg of GSE once a day from the 3rd day to the 27th day after immunization. The activation of microglia, the polarization of Th1 and Th17 and the inflammatory factors such as tumor necrosis factor- α (TNF- α), interleukin-1 β (IL-1 β), IL-6, IL-12, IL-17 and interferon-γ (IFN-γ) secreted by them were detected in vitro and in vivo by flow cytometry, enzyme linked immunosorbent assay (ELISA), immunofluorescence staining and Western blot, respectively.@*RESULTS@#GSE reduced the secretion of TNF-α, IL-1 β and IL-6 in bone marrow-derived macrophages stimulated by lipopolysaccharide (P<0.01), inhibited the secretion of TNF-α, IL-1 β, IL-6, IL-12, IL-17 and IFN-γ in spleen cells of EAE mice immunized for 9 days (P<0.05 or P<0.01), and reduced the differentiation of Th1 and Th17 mediated by CD3 and CD28 factors (P<0.01). GSE significantly improved the clinical symptoms of EAE mice, and inhibited spinal cord demyelination and inflammatory cell infiltration. Peripherally, GSE downregulated the expression of toll-like-receptor 4 (TLR4) and Rho-associated kinase (ROCKII, P<0.05 or P<0.01), and inhibited the secretion of inflammatory factors (P<0.01 or P<0.05). In the central nervous system, GSE inhibited the infiltration of CD45+CD11b+ and CD45+CD4+ cells, and weakened the differentiation of Th1 and Th17 (P<0.05). Moreover, it reduced the secretion of inflammatory factors (P<0.01), and prevented the activation of microglia (P<0.05).@*CONCLUSION@#GSE had a beneficial effect on the pathogenesis and progression of EAE by inhibiting inflammatory response as a potential drug and strategy for the treatment of MS.

Progress in Mechanism of Astragalus membranaceus and Its Chemical Constituents on Multiple Sclerosis / 中国结合医学杂志

The primary chemical components of Astragalus membranaceus include polysaccharides, saponins, flavonoids, and amino acids. Recent studies have shown that Astragalus membranaceus has multiple functions, including improving immune function and exerting antioxidative, anti-radiation, anti-tumor, antibacterial, antiviral, and hormone-like effects. Astragalus membranaceus and its extracts are widely used in clinical practice because they have obvious therapeutic effects against various autoimmune diseases and relatively less adverse reaction. Multiple sclerosis (MS) is an autoimmune disease of central nervous system (CNS), which mainly caused by immune disorder that leads to inflammatory demyelination, inflammatory cell infiltration, and axonal degeneration in the CNS. In this review, the authors analyzed the clinical manifestations of MS and experimental autoimmune encephalomyelitis (EAE) and focused on the efficacy of Astragalus membranaceus and its chemical components in the treatment of MS/EAE.

Study on the mechanism of Yishen daluo decoction in alleviating nervous system inflammation in EAE model mice / 中国药房

OBJECTIVE To study the effects of Yishen daluo decoction on inflammatory factors and cyclic adenosine monophosphate（cAMP）/protein kinase A （PKA）/cAMP response element binding protein （CREB） signal pathway in experimental autoimmune encephalomyelitis （EAE） model mice by inhibiting the expressions of β-arrestin1， and to explore the mechanism of Yishen daluo decoction in the treatment of EAE. METHODS Sixty mice were randomly divided into normal group， model group， TCM group （Yishen daluo decoction 20 g/kg）， positive control group （prednisone acetate 3.9 mg/kg）， β-arrestin1 siRNA adeno- associated virus （AAV-β） group， AAV-β+TCM group， with 10 mice in each group. Except for normal group， EAE model was made in other groups. AAV-β group and AAV-β+TCM group were injected with AAV-β via tail vein to interfere with the expression of β -arrestin1 protein. Starting from the 8th day of modeling， they were given corresponding drug solution/normal saline intragastrically， once a day， for consecutive 14 days. The neurological function score of mice was detected； the pathological and morphological changes were observed in the brain and spinal cord tissues of mice； the serum levels of inflammatory factors [interleukin-2 （IL-2）， IL-23， interferon-γ （IFN-γ）] in mice were determined； the expressions of β-arrestin1， cAMP， PKA and CREB in brain and spinal cord were detected. RESULTS Compared with normal group， neurological function scores， serum levels of inflammatory factors， and protein expressions of β-arrestin1 in brain and spinal cord were significantly increased （P＜0.05 or P＜ 0.01）； protein expressions of PKA， CREB and cAMP in brain and spinal cord were decreased significantly（P＜0.05 or P＜0.01）. The deep staining of cellular shrinkage and aggregation of inflammatory cells were observed in most neurons of the brain and spinal cord， with varying degrees of demyelinating. Compared with model group， the neurological function scores， pathological changes in brain and spinal cord tissues， and most indicators （except for CREB and cAMP proteins in the brain tissue of AAV-β group） were significantly reversed （P＜0.05 or P＜0.01）.Compared with AAV- β group， the neurological function scores， the levels of IFN-γ in serum and β-arrestin1 in spinal cord were significantly decreased （P＜0.05 or P＜0.01）， PKA and cAMP in brain and spinal cord tissues were significantly increased in AAV- β +TCM group （P＜0.05 or P＜0.01）. CONCLUSIONS Yishen daluo decoction can inhibit the expression of β-arrestin1 in the central nervous system thus activating the cAMP/PKA/CREB signaling pathway， relieving nervous system inflammation， and ultimately alleviates the symptoms of EAE.

Improvement effect and mechanism of matrine on experimental autoimmune encephalomyelitis mice / 中国药房

OBJECTIVE To investigate whether matrine exerts improvement effect on experimental autoimmune encephalomyelitis （EAE） mice by regulating ferroptosis pathway. METHODS Totally 30 female C57BL/6 mice were randomly assigned into normal group， model group and matrine group， with 10 mice in each group. Model group and matrine group were given antigen emulsion containing inactivated Mycobacterium tuberculosis and MOG35-55 to induce EAE model. Matrine group was injected with Matrine injection （50 mg/kg） intraperitoneally since the 7th day after immunization； normal group and model group were given constant volume of normal saline intraperitoneally， once a day， since 18th day after immunization. The neurofunctional score of mice was recorded， and hematoxylin and eosin staining and Luxol fast blue staining were used to observe inflammatory cell infiltration and demyelination in spinal cord tissue. The quantitative reverse transcription PCR and Western blot assay were performed to determine the mRNA expressions of transferrin receptor 1 （TFR1）， nuclear receptor coactivator 4 （NCOA4） and hephaestin （Heph）， and the protein expressions of system Xc－ （xCT） and glutathione peroxidase 4 （GPx4）. RESULTS Compared with normal group， accumulative neurofunctional score was significantly increased in model group （P＜0.01）； inflammatory cell infiltration and demyelination were obvious in spinal cord tissue， and related scores were increased significantly （P＜0.01）. The mRNA expressions of TFR1 and NCOA4 in myelin tissue were up-regulated significantly， while the mRNA expression of Heph and the protein expressions of xCT and GPx4 were down-regulated significantly （P＜0.05 or P＜0.01）. Compared with model group， above indexes of matrine group were all improved significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Matrine can improve EAE mice， the mechanism of which may be associated with regulating iron metabolism pathway and xCT/GPx4 pathway in ferroptosis.

Effect of Yishen Daluo Prescription on Rho/ROCK Signaling Pathway in EAE Mice Based on Silencing of β-arrestin1 / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Yishen Daluo prescription （YSDL） on Ras homolog（Rho）/Rho-associated coiled-coil containing protein kinase（ROCK）signaling pathway in mice with experimental autoimmune encephalomyelitis （EAE） based on the silencing of β-arrestin1 gene. MethodSixty C57BL/6 female mice were randomly divided into a blank group， a model group， a virus group， a YSDL group， a virus + YSDL group， and a prednisone acetate group （hormone group）. The EAE model was induced in mice except for those in the normal group. Adeno-associated virus（AAV）solution （150 μL， 1×1011 vg·mL-1） was injected into the tail vein of each mouse in the virus group and the virus + YSDL group on the 4th day of immunization. Drugs were administered on the 8th day of modeling. Specifically， normal saline was given to the mice in the normal group，the model group，and the virus group at 10 mL∙kg-1， prednisone acetate suspension to those in the hormone group at 3.9 g∙kg-1，and YSDL to those in other groups at 20 g∙kg-1 for 14 consecutive days. The mice were weighed and scored every day. The neurological function scores of mice in each group were recorded every day after immunization. Hematoxylin-eosin （HE） staining was used to determine the inflammatory response and lesion location in the brain tissues and spinal cord tissues of mice. The protein expression of β-arrestin1，Ras homolog gene family member A（RhoA）， and Rho-associated coiled-coil forming protein kinase Ⅰ（ROCK Ⅰ） in spinal cord and brain tissues of EAE mice was determined by Western blot. ResultCompared with the model group， the virus group and the virus + YSDL group showed decreased neurological function scores （P<0.01），and the YSDL group also showed decreased neurological function scores（P<0.05）. HE results showed that there was obvious inflammatory reaction in the central nervous system （CNS） of the model group， which was alleviated to varying degrees in other groups compared with the model group. Western blot results showed that compared with the blank group， the model group showed increased protein expression levels of β-arrestin1， RhoA， and ROCK Ⅰ in the spinal cord tissues （P<0.01）. Compared with the model group， the virus group， the YSDL group， the virus + YSDL group， and the hormone group showed decreased protein expression levels of β-arrestin1， RhoA， and ROCKⅠ in the spinal cord tissues （P<0.01）. Compared with the blank group， the model group showed increased protein expression levels of β-arrestin1， RhoA， and ROCK Ⅰ in the brain tissues （P<0.01）. Compared with the model group， the virus group， the YSDL group， the virus + YSDL group， and the hormone group showed decreased protein expression level of β-arrestin1 in the brain tissues （P<0.01）， and the virus group and the YSDL group showed decreased protein expression levels of RhoA， and ROCKⅠ in the brain tissues （P<0.05）. Additionally， the virus + YSDL group and the hormone group showed decreased protein expression levels of RhoA and ROCKⅠ in the brain tissues （P<0.01）. ConclusionYSDL can improve the clinical symptoms of EAE mice and improve the inflammatory response of CNS. The mechanism is presumably attributed to the fact that YSDL inhibits the expression of β-arrestin1 in CNS，thereby reducing the expression of Rho/ROCK signaling pathway. Furthermore， YSDL may have a synergistic effect with the inhibition of β-arrestin1 gene expression.

Cornuside ameliorated experimental autoimmune encephalomyelitis by limiting the recruitment of CD4+ T lymphocytes in the spinal cord

We conducted this study to determine whether cornuside could improve the neurological deficit symptoms of experimental autoimmune encephalomyelitis (EAE) rats, as well as determine the potential involvement of CD4+ T lymphocytes, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and tumor necrosis factor-α (TNF-α). Altogether, 32 Lewis rats were randomly divided into control, EAE, EAE/prednisolone, and EAE/cornuside, wherein their neurological function was assessed every day. CD4+ T lymphocyte recruitment into the spinal cord (SC) was evaluated using immunohistochemistry. The VCAM-1, ICAM-1 and TNF-α mRNA expressions in the SC were determined by real-time quantitative PCR, and the VCAM-1 and ICAM-1 proteins were determined by western blotting. Compared to the control group, the EAE group rats with neurological deficits had enhanced CD4+ T lymphocyte infiltration and higher expression levels of VCAM-1, ICAM-1, and TNF-α in the SC. Meanwhile, compared with the EAE group, the EAE/cornuside and EAE/prednisolone groups had lower neurological scores, less CD4+ T lymphocyte infiltrations, and lower expression levels of VCAM-1, ICAM-1, and TNF-α in the SC. Thus, cornuside ameliorated EAE, which could be owed to the inhibition of CD4+ T lymphocyte recruitment and VCAM-1, ICAM-1, and TNF-α expressions in the SC

LncRNA Gm13568 regulates the activation of A1 astrocytes and affects the EAE process in mice / 中华微生物学和免疫学杂志

Objective:To investigate the effects of long non-coding RNA (lncRNA) Gm13568 on the activation of A1 astrocytes and the progress of experimental autoimmune encephalomyelitis (EAE) in mice.Methods:A recombinant lentiviral vector (LV-Inhibit-Gm13568) carrying astrocyte-specific promoter of glial fibrillary acidic protein (GFAP) was established to inhibit the function of endogenous Gm13568. A control vector (LV-ctrl) was established as well. The recombinant vectors were packaged. C57BL/6 mice were injected with 1×10 7 transforming units of viral suspension via the tail vein and 7 d after the injection, myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) was used to establish the mouse model of EAE. Four groups, PBS group, EAE group, LV-ctrl+ EAE group and LV-Inhibit-Gm13568+ EAE group, were included in this study. Clinical signs of the mice were monitored daily in a double-blinded manner. The mice were sacrificed 23 d after the EAE model was established and the spinal cord tissues were collected. The expression of Serping 1, C3, Srgn and H2-T23 at mRNA level was detected by real-time PCR. Changes in the expression of IL-6, TNF-α, macrophage chemotactic protein-1 (MCP-1) and interferon-inducible protein-10 (IP-10) were measured. Western blot was used to investigate the expression of GFAP and Notch1 in spinal cord tissues and the phosphorylation of signal transduction and transcription activator 3 (STAT3). The expression of Notch1 intracellular domain (NICD) and GFAP in spinal cord tissues was detected by immunofluorescence. Furthermore, the infiltration of inflammatory cells and the demyelination of spinal cord were observed using HE and Luxol fast blue (LFB) staining methods. Results:Compared with PBS group, A1 astrocytes were activated and Notch1 expression was significantly up-regulated in EAE group and LV-ctrl+ EAE group. The clinical score of mice in LV-Inhibit-Gm13568+ EAE group was decreased from an average score of 3.5 to less than 1 on 23 d after antigen induction and the clinical symptoms were alleviated as compared with the mice in LV-ctrl+ EAE group. Meanwhile, the activation of A1 astrocytes was down-regulated, and the production of inflammatory cytokines and chemokines was also reduced. The expression of Notch1, GFAP and NICD at protein level and the phosphorylation of STAT3 were significantly reduced. Moreover, the infiltration of inflammatory cells and demyelination of spinal cord tissues were alleviated significantly.Conclusions:LncRNA Gm13568 might regulate the activation of A1 astrocytes via the Notch1/STAT3 pathway, thus affecting the production of inflammatory cytokines and chemokines and participating in the process of EAE.

Pharmacological insights into autophagy modulation in autoimmune diseases

As a cellular bulk degradation and survival mechanism, autophagy is implicated in diverse biological processes. Genome-wide association studies have revealed the link between autophagy gene polymorphisms and susceptibility of autoimmune diseases including systemic lupus erythematosus (SLE) and inflammatory bowel disease (IBD), indicating that autophagy dysregulation may be involved in the development of autoimmune diseases. A series of autophagy modulators have displayed protective effects on autoimmune disease models, highlighting the emerging role of autophagy modulators in treating autoimmune diseases. This review explores the roles of autophagy in the autoimmune diseases, with emphasis on four major autoimmune diseases [SLE, rheumatoid arthritis (RA), IBD, and experimental autoimmune encephalomyelitis (EAE)]. More importantly, the therapeutic potentials of small molecular autophagy modulators (including autophagy inducers and inhibitors) on autoimmune diseases are comprehensively analyzed.

Cornuside alleviates experimental autoimmune encephalomyelitis by inhibiting Th17 cell infiltration into the central nervous system / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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 receptor γ (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 enzyme-linked 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.

Mechanism by which rapamycin protects spinal cord neurons in experimental autoimmune encephalomyelitis mice / 中国组织工程研究

BACKGROUND: There are increasing reports about autophagy, but the relationship between the level of autophagy in neurons and the neuroprotection mechanism is not clear. OBJECTIVE: To investigate whether rapamycin, an mammalian target of rapacmycin (mTOR) autophagy pathway inhibitor, could activate autophagy by mediating the P70s6k and mTOR protein levels to protect spinal cord neurons in experimental autoimmune encephalomyelitis mice. METHODS: Fifty-four healthy female C57BL/6 mice were divided into three groups: control group, model group and treatment group, with 18 mice in each group. Mice in the model group and treatment group were injected with complete Freund’s adjuvant containing MOG35-55 and pertussis diluent for establishing models of experimental autoimmune encephalomyelitis. At the same time, the mice in the treatment group were given rapamycin (1 mg/kg per day), and those in the model and control groups were given the same amount of normal saline. The mice in the model and treatment were sacrificed at the peak of the onset, and the non-morbid mice, including those in the control group, were sacrificed after 4 weeks of feeding. The spinal cord tissue from each animal was taken to isolate the intumescentia lumbalis of the spinal cord. Nissl staining was used for pathological observation of the spinal cord tissue. Immunofluorescence double staining was used to observe the expression and co-localization of autophagy markers LC3 and NeuN in spinal cord tissue. Western blot was used to detect mTOR, P70S6K proteins and their phosphorylation levels in spinal cord tissue. RESULTS AND CONCLUSION: No mice in the control group had an attack, but those in the other groups developed experimental autoimmune encephalomyelitis to different extents. Compared with the model group, the treatment group had prolonged incubation time (P < 0.01), shortened progressive stage (P < 0.01), and decreased neurologic dysfunction score (P < 0.05). Compared with the control group, the model group had the significantly less number of Nissl bodies (P < 0.05), while the number of Nissl bodies in the treatment group was significantly higher than that in the model group, but still lower than that in the control group (P < 0.05). In the model group, LC3 was scattered in the spinal cord neurons and had no obvious dot-like aggregation, whereas in the treatment group, LC3 showed obvious dot-like aggregation, and its distribution was basically consistent with that of NeuN. The phosphorylation levels of mTOR and P70S6K proteins were highest in the model group, followed by the treatment group and control group in turn. To conclude, rapamycin might through inhibiting the phosphorylation levels of mTOR and P70S6K proteins activate the activity of autophagy to protect the spinal cord neurons in experimental autoimmune encephalomyelitis mice.

Mechanisms and Therapeutic Effects of Human Olfactory Mucosa-Derived Mesenchymal Stem Cells on Experimental Autoimmune Encephalomyelitis in Mice / 中山大学学报(医学科学版)

@#【Objective】 To study the mechanisms and therapeutic effects of human olfactory mucosa-derived mesenchymal stem cells（OMSC）on experimental autoimmune encephalomyelitis（EAE）in mice.【Methods】Under local anesthesia by using nasal endoscopy，olfactory epithelia of healthy donors were obtained，digested and cultured up to the 5th passage. OMSC were identified，differentiated and stained. EAE models were induced in C57 female mice by myelin oligodendrocyte glycoprotein（MOG35- 55）and pertussis toxin（PT）. Neurological function was documented daily. On day 16 after immunization（peak of incidence），the mice were divided randomly into two groups and treated with OMSC and PBS via tail vein injection respectively. On day 24 after immunization ，blood was collected from angular vein and levels of IL-10，IL-17，IFN-γ and IL-6 were determined by cytometric beads array（CBA）. The size of the spinal cord lesion in mice was observed and measured by using HE and LFB staining. Peripheral blood lymphocytes（PBL）of healthy donors were obtained and then co-cultured with OMSC. The proportions of CD4+ T cells secreting IFN- γ（Th1 cells）in lymphocyte group and co-culture group were compared after 2 days of cultivation. Adding IDO or COX pathway inhibitor to co- culture group and cultivating for 2 days，we observed and compared the proportions of Th1 cells in lymphocyte group，co-culture group and inhibitor treatment group respectively.【Results】OMSC exhibited certain mesenchymal stem cell-like characteristics with respect to expression of stem cell surface markers and multilineage differentiation potentials. After induced by MOG35- 55 and PT，EAE models showed different levels of neurological damage. Compared with those in PBS treatment group，in OMSC treatment group，the severity of neural dysfunction in mice was significantly reduced（P =0.002），the level of IFN-γ in serum was lower（P = 0.032），but no significant differences in the levels of IL-10，IL-17 and IL-6 were found between two groups. HE and LFB staining revealed that the inflammatory infiltration and demyelinating areas in OMSC treatment group were less than those in PBS treatment group. The proportion of Th1 cells was lower in co-culture group than that in lymphocyte group（P = 0.001），higher in IDO inhibitor group than that in co-culture group（P = 0.01），but no significant difference was found between IDO inhibitor group and lymphocyte group or between COX inhibitor group and co-culture group.【Conclusions】OMSC may regulate the proportion of Th1 lymphocytes through IDO pathway so as to inhibit the demyelinating injuries of EAE in mice. This study provides a new idea for the clinical treatment of multiple sclerosis.

Recent advances of long noncoding RNAs involved in the development of multiple sclerosis / 中国天然药物

Given the rapid increase of patients with autoimmune diseases and the lack of satisfactory therapies, the discovery of novel and effective therapeutic targets have been in an urgent demand. Recent studies have revealed that long noncoding RNAs (lncRNAs) play crucial roles in the development of multiple sclerosis (MS), which provides a new opportunity of uncovering novel mechanism associated with the progression of MS. This review highlights the dysregulation of lncRNAs in the development of MS in patients and animal models. Additionally, the potential clinical relevance of lncRNAs severed as therapeutic targets and diagnostic markers are discussed.

Therapeutic Effect of SPK1 Gene Transfected Adipose Derived Mesenchymal Stem Cells on Experimental Autoimmune Encephalomyelitis Mice and Its Effect on T Helper Cell 17/Regulatory T Cells Balance / 中国医学科学院学报

Objective To investigate the therapeutic effect of SPK1 gene transfected adipose derived mesenchymal stem cells(ADMSC)on experimental autoimmune encephalomyelitis mice and the effect on T helper cell 17(Th17)/regulatory T(Treg) cells balance. Methods EAE was induced by myelin oligodendrocyte glycoprotein 35-55 in mice.Totally 44 mice were randomly divided into four groups:normal control group(NC group),model group(EAE group),ADMSC group,and ADMSC-SPK1 group.Forty days after injection,the pathological changes of brain and spinal cord,Th17/Treg-related inflammatory markers in brain tissue,expressions of interleukin-17A(IL-17A)and forkhead box protein p3(Foxp3)in brain and spinal cord tissue,and flow cytometric results of spleen immune cells were detected. Results Forty days after the injection,serious inflammatory cell infiltration and demyelination occurred in the brain and spinal cord of EAE group,whereas demyelination and axonal injury were improved in ADMSC group and ADMSC-SPK1 group.Compared with EAE group,the ADMSC group and ADMSC-SPK1 group had significantly improved levels of IL-17A(

β-Caryophyllene causes remyelination and modifies cytokines expression in C57BL/6 mice with experimental autoimmune encephalomyelitis

The aim of this study is to evaluate the effect of β-Caryophyllene (BCP) on the production of IL-17, transcription factors(T-bet and GATA-3), and remyellination in C57BL/6 mice induced for experimental autoimmune encephalomyelitis(EAE), the model for studying pathogenesis and new therapies for multiple sclerosis. EAE was induced in threegroups of C57BL/6, with administration of BCP in two groups (25 and 50 mg/kg/day) by gavage, after the 10th dayof induction. At 9 days of treatment, mice were euthanized and CNS was removed for the analysis. The profiles ofIL-17, T-bet, GATA-3, and the possible remyelination properties were investigated in the central nervous system(CNS) by immunohistochemistry and Weigert–Pal–Russel’s method, respectively. BCP group (50 mg/kg/day) showeda reduction of IL-17 in brain, cerebellum, and medulla (p < 0.05) and a decrease of T-bet (p < 0.05) in medullaand cerebellum, while GATA-3 was increased (p < 0.05) in cerebellum. In both BCP-treated groups were observedremyelination and better organization of myelin. In conclusion, BCP possesses markedly in vivo anti-inflammatoryand neuroprotective activities and remyelination properties in EAE-mice.

Gene Expression Profile of Olfactory Transduction Signaling in an Animal Model of Human Multiple Sclerosis

Olfactory dysfunction occurs in multiple sclerosis in humans, as well as in an animal model of experimental autoimmune encephalomyelitis (EAE). The aim of this study was to analyze differentially expressed genes (DEGs) in olfactory bulb of EAE-affected mice by next generation sequencing, with a particular focus on changes in olfaction-related signals. EAE was induced in C57BL/6 mice following immunization with myelin oligodendrocyte glycoprotein and adjuvant. Inflammatory lesions were identified in the olfactory bulbs as well as in the spinal cord of immunized mice. Analysis of DEGs in the olfactory bulb of EAE-affected mice revealed that 44 genes were upregulated (and which were primarily related to inflammatory mediators), while 519 genes were downregulated; among the latter, olfactory marker protein and stomatin-like 3, which have been linked to olfactory signal transduction, were significantly downregulated (log2 [fold change] >1 and p-value < 0.05). These findings suggest that inflammation in the olfactory bulb of EAE-affected mice is associated with the downregulation of some olfactory signal transduction genes, particularly olfactory marker protein and stomatin-like 3, which may lead to olfactory dysfunction in an animal model of human multiple sclerosis.

Efeitos da exposição à fumaça do cigarro e suas implicações na neuroinflamação / Effects of exposure to cigarette smoke and its implications on neuroinflammation

O Sistema Nervoso Central (SNC) humano é formado por cerca de 86,1 bilhões de neurônios entre o encéfalo e a medula espinhal. O desenvolvimento pré-natal humano (tempo da concepção ao nascimento) possui cerca de 38 semanas, e é dividido na fase embrionária que corresponde ao período das 8 semanas iniciais da gestação, seguido pela fase fetal. A fase embrionária é o período mais vulnerável à ocorrência de anormalidades congênitas. Por ser um órgão com grande período de desenvolvimento, o SNC está sujeito às alterações genéticas, epigenéticas e ambientais. Durante a fase de implantação do embrião, o DNA é mais vulnerável às influências externas, como à fumaça do cigarro, aumentando o risco de retardo do desenvolvimento fetal, o risco de morte súbita pós-natal e de anormalidades do sistema imune. Neste contexto, o objetivo deste trabalho é avaliar os efeitos da exposição à fumaça do cigarro sobre o processo de neuroinflamação da prole de camundongos C57BL/6 expostos à fumaça do cigarro durante a gestação e desafiados ou não com LPS. Para tanto, camundongos C57BL/6 fêmeas prenhes foram expostas à fumaça do cigarro desde o plug vaginal até o nascimento da prole. No 3º dia de vida, os filhotes foram separados para três linhas de trabalho: 1) in vivo: os animais foram desafiados com LPS pelo período de 4h, seguidos de eutanasia e análises de PCR Array do SNC. 2) in vitro: os encéfalos dissecados foram utilizados para a preparação de cultura mista de glia e da cultura enriquecida com neurônio. Após a maturação celular, as células foram estimuladas com LPS 100 ng/mL e, após 24h, foram realizados ensaios de CBA, citometria de fluxo, PCR, dosagem de NO, avaliação de morte celular e metilação global. 3) Encefalomielite Autoimune Experimental (EAE): após o desmame, os animais foram mantidos em suas caixas moradia por 8 semanas sem nenhum estímulo externo, e então foram imunizados com MOG35-55 para o desenvolvimento da EAE. Nos experimentos in vivo observamos o aumento da transcrição de genes relacionados ao processo inflamatório, como interleucinas e quimiocinas. Em relação aos experimentos in vitro observamos maior crescimento de células astrocitárias (astrogliose), e células da microglia com aumento de moléculas co-estimuladoras (CD80 e CD86) bem como da transcrição e concentração de citocinas pró-inflamatórias e produção de NO. Em cultura enriquecida de neurônio, foi observado aumento na porcentagem de células em apoptose no grupo exposto à fumaça do cigarro desafiados ou não com LPS. O bloqueio da atividade da microglia pela minociclina reverteu a apoptose e diminuiu a produção de NO minimizando a morte celular. Em relação aos experimentos de EAE, os animais expostos à fumaça do cigarro no período gestacional, quando imunizados na vida adulta apresentam aumento no grau da doença bem como maior persistência da mesma quando observado escore clínico, além de acompanhados de um grau maior de infiltrado celular e desmielinização. Desta forma podemos concluir que a exposição à fumaça do cigarro durante o período gestacional leva a uma programação fetal com aumento da resposta neuroinflamatória frente a um estimulo sistêmico, trazendo consequências na vida adulta

The human central nervous system (CNS) is made up of about 86.1 billion neurons between the brain and the spinal cord. The human prenatal development (time from conception to birth) is about 38 weeks, and is divided into the embryonic phase that corresponds to the period of the initial 8 weeks of gestation, followed by the fetal phase. The embryonic stage is the period most vulnerable to the occurrence of congenital abnormalities. Because it is an organ with a long period of development, the CNS is subject to genetic, epigenetic and environmental changes. During the embryo implantation phase, DNA is more vulnerable to external influences such as cigarette smoke, increasing the risk of delay on fetal development, risk of sudden postnatal death, and abnormalities of the immune system. In this context, the aim of this work is to evaluate the effects of exposure to cigarette smoke on the neuroinflammation process of offspring of C57BL/6 mice exposed to cigarette smoke during gestation and challenged or not with LPS. For this, pregnant female C57BL/6 mice were exposed to cigarette smoke from vaginal plug to offspring birth. On the 3rd day of life the offspring were separated into three lines of work: 1) in vivo: the animals were challenged with 1mg/Kg LPS and after 4h they followed to euthanasia; PCR analysis of the CNS was made in this period. 2) in vitro: dissected encephalons were used for the preparation of mixed culture of glia and the culture enriched with neuron. After cell maturation, the cells were stimulated with 100 ng/mL LPS and, after 24 hours, CBA, flow cytometry, PCR, NO assay, cell death and global methylation assays were performed. 3) Experimental Autoimmune Encephalomyelitis (EAE): After weaning, the animals were kept in their housing for 8 weeks without any external stimulus, and then were immunized with MOG35-55 for the development of EAE. In the in vivo experiments we observed increased transcription of genes related to the inflammatory process, such as interleukins and chemokines. In vitro experiments showed higher growth of astrocytes (astrogliosis) and microglia cells with increased stimulatory molecules (CD80 and CD86) as well as the transcription and concentration of proinflammatory cytokines and NO production. In the enriched neuron culture, an increase in the percentage of cells in apoptosis was observed in the group exposed to cigarette smoke challenged or not with LPS. Blocking microglial activity by minocycline reversed apoptosis and decreased NO production by minimizing cell death. The EAE experiments shows that the animals exposed to cigarette smoke in the gestational period, when immunized in adulthood, present an increase in the degree of the disease as well as a greater persistence of the disease; The higher as the clinical score higher is the degree of cellular infiltration and demyelination. In this way we can conclude that the exposure to cigarette smoke during the gestational period leads to a fetal programming with increased neuroinflammatory response to a systemic stimulus and that this is able to last until the adult stage

Effect and mechanism of natural compounds in treatment of EAE / 中草药

Experimental autoimmune encephalomyelitis (EAE) is a classic animal model for the study of human multiple sclerosis (MS). A number of studies have found that natural compounds possessed effects on the treatment of EAE. In this paper, we consulted related literatures, classified natural compounds such as alkaloids, flavonoids, terpenoids, and polyphenols, and explained the treatment mechanisms against EAE of Chinese materia medica, and to provide references for clinical research and treatment of neurological autoimmune diseases such as MS.

Autophagy regulation and anti-inflammatory effect of curcumin on the mouse with experimental autoimmune encephalomyelitis / 解放军医学杂志

Objective To investigate the anti-inflammatory effect of curcumin on the mouse with experimental autoimmune encephalomyelitis (EAE), and explore its mechanism of action. Methods Forty female C57BL/6 mice were randomly divided into EAE+DMSO group, EAE+curcumin group, DMSO group and curcumin group with10 mice in each group. Mice in EAE+DMSO group and EAE+curcumin group were used to establish the EAE model. DMSO group was the solvent control of EAE+DMSO group, and 0.2 ml 20% DMSO solution daily were intraperitoneally injected in the both groups a week after immunization; and mice in curcumin group and EAE+curcumin group were daily intraperitoneally injected with 10 mg/kg curcumin solution a week after immunization. From the date of immunization, the mice in each group were evaluated for clinical symptoms every day. Mice were anesthetized and sacrificed at the peak period of EAE (21 d), their spinal cord tissues were taken for HE staining, serum concentrations of INF-γ and IL-17 were measured by ELISA, spleen lymphocytes were sorted for CD4+ T. The expression levels of Atg-5 mRNA and LC3-Ⅱ mRNA in spinal cord tissue were determined by RT-PCR, and of LC3-Ⅱ was estimated by immunohistochemical staining. Results Based on the successful establishment of mouse EAE model, there were no obvious neurological injury symptoms in DMSO group and curcumin group, and no inflammatory reaction by HE staining in spinal cord tissue. The clinical symptom score in EAE+DMSO group (3.312±0.347) was significantly higher than that in DMSO group (P0.05). The serum concentrations of INF-γ and IL-17 were significantly higher in EAE+DMSO group (210.56±47.74 and 218.09±35.33) than in DMSO group (P0.05); Compared with the DMSO group, the Atg-5 level (0.70±0.06), LC3-Ⅱ gene expression level (0.67±0.10) and LC3-Ⅱimmunohistochemical positive cell count [(8.33±2.05)/HP] were down-regulated significantly in EAE+DMSO group (P<0.05); the Atg-5 level (1.56±0.08), LC3-Ⅱ gene expression level (1.99±0.11) and LC3-Ⅱimmunohistochemical positive cell count [(19.67±1.70)/HP] raised significantly in EAE+curcumin group when compared with that in EAE+DMSO group (P<0.05). Conclusions Curcumin may relieve the clinical nerve function injury in the course of EAE, downgrade the peripheral and central inflammatory response, meanwhile up-regulate the expression levels of Atg-5 and LC3-Ⅱ and upgrade the autophagy level of central nerve cells. Thus the inflammatory injury process in EAE course may be limited.

Neuroprotective Mechanism of Buyang Huanwu Tang on Experimental Autoimmune Encephalomyelitis Mice / 中国实验方剂学杂志

Objective:To explore the neuroprotective effect and mechanism of Buyang Huanwu Tang (BYHWT) on experimental autoimmune encephalomyelitis (EAE) at different stages. Method:The 36 female C57BL/6 mice were immunized subcutaneously with myelin oligodendrocyte glycoprotein peptides (MOG35-55),then randomly divided into 9, 17, 28 d EAE control group. Each BYHWT group was orally given drugs on the 3rd day after immunization (50 g·kg-1·d-1), and EAE control group was given the same volume of normal saline in the same way once a day for 9, 17 and 28 d after immunization. The effect of BYHWT on EAE mice was observed with internationally accepted clinical score. Brain and spinal cord specimens were collected at 9, 17 and 28 d after immunization. The neuroprotective effect of BYHWT was observed by hematoxylin-eosin(HE)staining and solid blue staining (LFB). The expressions of BDNF and GAP-43 in spinal cord and brain were detected by Western blot. Result:After treatment, BYHWT can significantly inhibit myelitis cell infiltration and alleviate myelin loss. Compared with EAE group, the expression of Nogo-A in the spinal cord of each BYHWT group was significantly down-regulated (PPPPConclusion:BYHWT can improve the local nerve growth microenvironment and promote the expression of NTFs, reduce the expressions of neuroinhibitory factors, and play a role in neuroprotection.

Therapeutic effect of regulatory T cells on mice with experimental autoimmune encephalomyelitis / 中华微生物学和免疫学杂志

Objective@#To study the role of Treg cells in the development of mouse experimental autoimmune encephalomyelitis (EAE) through depleting or transplanting Treg cells.@*Methods@#C57BL/6 mice were injected with anti-CD25 monoclonal antibody to deplete natural CD25-expressing Treg cells in vivo, and then treated with MOG35-55/CFA to induce EAE. Their EAE scores were compared with those of the mice without Treg cell deletion (control group). The numbers and percentages of CD4+ Foxp3+ cells in mouse blood samples on 6 d, 10 d, 20 d and 35 d were quantified using flow cytometry. To evaluate the therapeutic effect of Treg cells transplantation on EAE, magnetic activated cell sorting (MACS) combined with fluorescence-activated cell sorting (FACS) was used to isolate Treg cells from spleen and lymph nodes of Foxp3GFP+ transgenic mice on 6 d after EAE induction. Then the cells were injected through tail vein into wild-type mice on 6 d after EAE induction. The EAE scores of both recipient and control mice were recorded and compared.@*Results@#The efficiency of natural Treg cells depletion with anti-CD25 antibody was above 95%. The mice with Treg cell depletion developed significantly more severe EAE than the control mice after MOG35-55/CFA induction. FACS analysis of Treg cells during the development of EAE demonstrated that the lowest Treg cell percentage was detected on 6 d after EAE induction, hence it was the time point for the transplantation of Treg cells. CD4+ GFP+ Treg cells were isolated from Foxp3GFP+ transgenic mice on 6 d after EAE induction and immediately transplanted into wild-type mice on 6 d after EAE induction. The transplantation of isolated Treg cells significantly alleviated the EAE in mice as compared with the control group.@*Conclusions@#Mice with Treg cell depletion developed severer EAE than the control mice after induction, but the EAE score could be significantly reduced with the transplantation of Treg cells. This study showed that the transplanted Treg cells had protective effect on mice during the course of EAE development. Thus, Treg cell transplantation could be used as an effective therapeutic approach for the treatment of multiple sclerosis (MS).