Efficacy of Heshouwu () on gut mircobiota in mice with autoimmune encephalomyelitis.

OBJECTIVE: To learn the mechanisms between gut microbiome and the autoimmunity benefits on Traditional Chinese Medicine (TCM) in central nervous system (CNS), we investigated the neuro-protection effects and gut mircobiota changes of Heshouwu () on experimental autoimmune encepha-lomyelitis (EAE), an animal model of multiple sclerosis (MS). METHODS: Mice were randomly divided into four groups: EAE mice (control phosphate-buffered saline group), 50 mg·kg·d Heshouwu ()-treated EAE mice, 100 mg·kg·d Heshouwu ()-treated EAE mice, and 200 mg·kg·d Heshouwu ()-treated EAE mice. The spinal cords were stained with hematoxylin and eosin (HE) and luxol fast blue for evaluating inflammatory infiltration and demyelination. The percentages of granulocyte macrophage-colony stimulating factor (GM-CSF)+CD4+, interleukin 17 (IL-17)+CD4+, Foxp3 CD4+, and interferon-γ (IFN-γ)+CD4+ T cells in the inguinal lymph nodes (LNs) and brain were determined by flow cytometry analysis. 16S rRNA gene sequencing was employed to analyze the changes in gut microbiota. RESULTS: We found that Heshouwu () alleviated the disease severity and neuropathology of EAE as evaluated by clinical and histopathologyical scores. Heshouwu () increased the diversity and abundance of the gut microbiota, and decreased / ratio (F/B ratio). Heshouwu () also decreased the concentrations of IL-10, and IL-21 and increase the levels of GM-CSF, IL-17A, IL-17F and IL-22 in serum of EAE mice. Moreover, Heshouwu () modulated the T cell responses by inhibiting Th17 cells and restoring Treg cells in the small intestine lymphoid tissues and inguinal lymph nodes. Microbiota-depleted mice receiving Heshouwu ()-treated fecal microbiota trans-plantation had lower disease severity, neuropathology scores and alleviation of Th17/Treg imbalance compared to ad libitum group. CONCLUSIONS: Our findings suggested that the vital neuro-protection role of Heshouwu () (TCM) in immunomodulation effects partly by regulations of gut microbiome.

Influence of Vitamins A and D on the Expression of MicroRNA27-3p Isoforms and GATA3 in Experimental Autoimmune Encephalomyelitis.

Vitamins A, D, and microRNAs contribute to T cell differentiation into TH2 phenotypes. We investigated the molecular mechanisms and effects of vitamin A and D on the expression of GATA3 and miR-27-3p isoforms in experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis. EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein, mixed with Complete Freund's Adjuvant, together with injection of pertussis toxin. Treatments began one day before immunization with (200 µg and 100 ng of vitamin A and vitamin D per mouse, respectively, and vitamin A+D (100 µg+50 ng) per mouse. Expression levels of GATA3 and miR­27­3p isoforms were measured in the CNS and splenocytes by real-time RT-PCR. The expression level of GATA3 in the mice spinal cords and splenocytes was increased in the vitamin A and A+D-treated EAE mice at 24 h and 48 h after restimulation by 10 µg and 40 µg of myelin oligodendrocyte glycoprotein. Vitamins A and D and their combination upregulated the miR-27-3p isoforms compared with EAE mice with no treatments. We also demonstrated that miR-273p isoform expression was altered in splenocytes of vitamin-treated EAE mice. The results showed a positive correlation between splenocyte GATA3 levels and miR-27-3p isoform expression. The protective impacts of vitamins A and D in EAE mice may be mediated by the upregulation of GATA3. However, it is not specified whether suppression of GATA3-targeting miRNAs of the miR-27-3p family is involved in this effect. These results do not rule out the possibility that miR-27-3p isoforms might have beneficial effects by targeting other transcripts, such as GluA2 and NR2B.

[Therapeutic effect of Shengjiang Powder on experimental autoimmune encephalomyelitis mice through IL-6/JAK2/STAT3 signaling pathway:based on HPLC].

A high performance liquid chromatography(HPLC) method was established to analyze the components in Shengjiang Powder(SJP) such as emodin and curcumin and explore its therapeutic effect on experimental autoimmune encephalomyelitis(EAE) mice. To be specific, HPLC was performed to determine the content of compounds in SJP such as emodin and curcumin. A total of 72 female SPF C57 BL/6 mice were randomized into control group(equivalent volume of ultrapure water, ig), model group(equivalent volume of ultrapure water, ig), low-, medium-, and high-dose SJP groups(SJP, ig), and positive control group(prednisone acetate, ig), 12 each group. EAE was induced in mice except the control group. Administration began from the first day after immunization. The general conditions, symptom score, and body weight of the mice were recorded. On the 21 st day, mouse brain tissues were separrated. Then hematoxylin-eosin(HE) staining and Luxol Fast Blue(LFB) staining were used to detect the pathological changes of brain tissues. Immunohistochemistry(IHC) was employed to determine the myelin basic protein(MBP) level, and Western blot the expression of occludin and claudin-5, as well as the levels of interleukin-6(IL-6) and proteins in the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) pathway and their phosphorylation levels. The mRNA expression of IL-6, JAK2, and STAT3 was detected by real-time quantitative polymerase chain reaction(qPCR). Finally, molecular docking of six main active components in SJP, including emodin and curcumin, with IL-6, JAK2 and STAT3 was performed, and the binding affinity was evaluated. The results showed that the established HPLC method demonstrated high precision, reproducibility, stability, and high recovery of samples. Compared with the model group, SJP reduced the clinical symptom score and alleviate the inflammatory infiltration of brain white matter and demyelination of EAE mice. At the same time, SJP increased the expression of occludin and claudin-5, down-regulated the mRNA expression of IL-6, JAK2, and STAT3, as well as the levels of IL-6/JAK/STAT3 proteins and the phosphorylation levels, with significant difference. Molecular docking suggested that the six active components in SJP had high binding energy with IL-6, JAK2, and STAT3 proteins. The established HPLC method is simple, accurate, and highly sensitive, which can simultaneously determine the content of emodin and curcumin in SJP. SJP may alleviate the clinical symptoms of EAE by inhibiting IL-6/JAK2/STAT3 signaling pathway, protecting the blood-brain barrier, and relieving the inflammatory response and demyelinization of brain tissue.

DNA/RNA heteroduplex oligonucleotide technology for regulating lymphocytes in vivo.

Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer. Although oligonucleotide therapy has been proven to be successful in treating several conditions, efficient in vivo delivery of oligonucleotide to lymphocyte populations remains a challenge. Here, we demonstrate that intravenous injection of a heteroduplex oligonucleotide (HDO), comprised of an antisense oligonucleotide (ASO) and its complementary RNA conjugated to α-tocopherol, silences lymphocyte endogenous gene expression with higher potency, efficacy, and longer retention time than ASOs. Importantly, reduction of Itga4 by HDO ameliorates symptoms in both adoptive transfer and active experimental autoimmune encephalomyelitis models. Our findings reveal the advantages of HDO with enhanced gene knockdown effect and different delivery mechanisms compared with ASO. Thus, regulation of lymphocyte functions by HDO is a potential therapeutic option for immune-mediated diseases.

Inflammation in an Animal Model of Multiple Sclerosis Leads to MicroRNA-25-3p Dysregulation Associated with Inhibition of Pten and Klf4.

Perturbed expression of microRNAs (miRs) has been reported in different diseases including autoimmune and chronic inflammatory disorders. In this study, we investigated the expression of miR-25-3p and its targets in the central nervous system (CNS) tissue from mice with experimental autoimmune encephalomyelitis (EAE). We also analyzed the expression of miR-25 and its targets in activated macrophages and splenocytes. EAE was induced in 12-week old female C57BL/6 mice; using myelin oligodendrocyte glycoprotein 35-55/complete Freund's adjuvant (MOG35-55/CFA) protocol. The expression of miR-25-3p and its targets, as well as the expression of inflammatory cytokines, were analyzed. We next established primary macrophage cultures as well as splenocyte cultures and evaluated the levels of miR-25-3p and its target genes in these cells following activation with lipopolysaccharide (LPS) and anti-CD3/anti-CD28 antibodies, respectively. MiR-25-3p expression showed a strong positive correlation with the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α, and IL-6 pro-inflammatory cytokines. The expression of phosphatase and tensin homolog (Pten) and Krüppel-like factor 4 (Klf4) was significantly reduced at the peak of the disease. Interestingly, Pten and Klf4 expression showed a significant negative correlation with miR-25-3p. Analysis of miR-25-3p expression in LPS-treated primary macrophages revealed significant upregulation in cells treated with 100ng/ml of LPS. This was associated with suppressed levels of miR-25-3p targets in these cells. However, anti-CD3/anti-CD28-stimulated splenocytes failed to show any alterations in miR-25-3p expression compared with vehicle-treated cells. Our results indicate that miR-25-3p expression is likely induced by inflammatory mediators during autoimmune neuroinflammation. This upregulation is associated with decreased levels of Pten and Klf4, genes with known roles in cell cycle regulation and inflammation.

Bu Shen Yi Sui Capsule Alleviates Neuroinflammation and Demyelination by Promoting Microglia toward M2 Polarization, Which Correlates with Changes in miR-124 and miR-155 in Experimental Autoimmune Encephalomyelitis.

BACKGROUND: Bu Shen Yi Sui capsule (BSYS) is a traditional Chinese medicine prescription that has shown antineuroinflammatory and neuroprotective effects in treating multiple sclerosis (MS) and its animal model of experimental autoimmune encephalomyelitis (EAE). Microglia play an important role in neuroinflammation. The M1 phenotype of microglia is involved in the proinflammatory process of the disease, while the M2 phenotype plays an anti-inflammatory role. Promoting the polarization of microglia to M2 in MS/EAE is a promising therapeutic strategy. This study is aimed at exploring the effects of BSYS on microglial polarization in mice with EAE. METHODS: The EAE model was established by the intraperitoneal injection of pertussis toxin and subcutaneous injection of myelin oligodendrocyte glycoprotein (MOG)35-55 in C57BL/6J mice. The mice were treated with BSYS (3.02 g/kg), FTY720 (0.3 mg/kg), or distilled water by intragastric administration. H&E and LFB staining, transmission electron microscopy, qRT-PCR, immunofluorescence, ELISA, fluorescence in situ hybridization, and western blotting were used to detect the histological changes in myelin, microglial M1/M2 polarization markers, and the expression of key genes involved in EAE. Results and Conclusions. BSYS treatment of EAE mice increased the body weight, decreased the clinical score, and reduced demyelination induced by inflammatory infiltration. BSYS also inhibited the mRNA expression of M1 microglial markers while increasing the mRNA level of M2 markers. Additionally, BSYS led to a marked decrease in the ratio of M1 microglia (iNOS+/Iba1+) and an obvious increase in the number of M2 microglia (Arg1+/Iba1+). In the EAE mouse model, miR-124 expression was decreased, and miR-155 expression was increased, while BSYS treatment significantly reversed this effect and modulated the levels of C/EBP α, PU.1, and SOCS1 (target genes of miR-124 and miR-155). Therefore, the neuroprotective effect of BSYS against MS/EAE was related to promoting microglia toward M2 polarization, which may be correlated with changes in miR-124 and miR-155 in vivo.

Microglia Polarization from M1 toward M2 Phenotype Is Promoted by Astragalus Polysaccharides Mediated through Inhibition of miR-155 in Experimental Autoimmune Encephalomyelitis.

Activated microglia is considered to be major mediators of the neuroinflammatory environment in demyelinating diseases of the central nervous system (CNS). Activated microglia are mainly polarized into M1 type, which plays a role in promoting inflammation and demyelinating. However, the proportion of microglia polarized into M2 type is relatively low, which cannot fully play the role of anti-inflammatory and resistance to demyelinating. Our previous study found that Astragalus polysaccharides (APS) has an immunomodulatory effect and can inhibit neuroinflammation and demyelination in experimental autoimmune encephalomyelitis (EAE), which is a classic animal model of CNS demyelinating disease. In this study, we found that APS was effective in treating EAE mice. It restored microglia balance by inhibiting the polarization of microglia to M1-like phenotype and promoting the polarization of microglia to M2-like phenotype in vivo and in vitro. miR-155 is a key factor in regulating microglia polarization. We found that APS could inhibit the expression level of miR-155 in vivo and in vitro. Furthermore, we performed transfection overexpression and blocking experiments. The results showed that miR-155 mediated the polarization of microglia M1/M2 phenotype, while the selective inhibitor of miR-155 attenuated the inhibition of APS on microglia M1 phenotype and eliminated the promotion of APS on microglia M2 phenotype. Microglia can secrete IL-1α, TNF-α, and C1q after polarizing into M1 type and induce the activation of A1 neurotoxic astrocytes, further aggravating neuroinflammation and demyelination. APS reduced the secretion of IL-1α, TNF-α, and C1q by activated microglia, thus inhibited the formation of A1 neurotoxic astrocytes. In summary, our study suggests that APS regulates the polarization of microglia from M1 to M2 phenotype by inhibiting the miR-155, reduces the secretion of inflammatory factors, and inhibits the activation of neurotoxic astrocytes, thus effectively treating EAE.

UV light suppression of EAE (a mouse model of multiple sclerosis) is independent of vitamin D and its receptor.

Vitamin D and sunlight have each been reported to protect against the development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). To date, the contribution of each has been unclear as ultra violet (UV) exposure also causes the generation of vitamin D in the skin. To examine whether the UV based suppression of EAE results, at least, in part from the production of vitamin D, we studied the effect of UV light on EAE in mice unable to produce 7-dehydroxycholesterol (7-DHC), the required precursor of vitamin D. Furthermore, we examined UV suppression of EAE in mice devoid of the vitamin D receptor (VDR). Our results demonstrate that UV light suppression of EAE occurs in the absence of vitamin D production and in the absence of VDR. Future investigations will focus on identifying the pathway responsible for the protective action of UV in EAE and presumably human MS.

Combination of Cannabinoids, Δ9- Tetrahydrocannabinol and Cannabidiol, Ameliorates Experimental Multiple Sclerosis by Suppressing Neuroinflammation Through Regulation of miRNA-Mediated Signaling Pathways.

Multiple sclerosis (MS) is a chronic and disabling disorder of the central nervous system (CNS) characterized by neuroinflammation leading to demyelination. Recently a combination of Δ9-tetrahydrocannabinol (THC) and Cannabidiol (CBD) extracted from Cannabis has been approved in many parts of the world to treat MS-related spasticity. THC+CBD combination was also shown to suppresses neuroinflammation, although the mechanisms remain to be further elucidated. In the current study, we demonstrate that THC+CBD combination therapy (10 mg/kg each) but not THC or CBD alone, attenuates murine experimental autoimmune encephalomyelitis (EAE) by reducing neuroinflammation and suppression of Th17 and Th1 cells. These effects were mediated through CB1 and CB2 receptors inasmuch as, THC+CBD failed to ameliorate EAE in mice deficient in CB1 and CB2. THC+CBD treatment also caused a decrease in the levels of brain infiltrating CD4+ T cells and pro-inflammatory molecules (IL-17, INF-γ, TNF-α, IL-1ß, IL-6, and TBX21), while increasing anti-inflammatory phenotype such as FoxP3, STAT5b, IL-4, IL-10, and TGF-ß. Also, the brain-derived cells showed increased apoptosis along with decreased percentage in G0/G1 phase with increased percentage in G2/M phase of cell cycle. miRNA microarray analysis of brain-derived CD4+ T cells revealed that THC+CBD treatment significantly down-regulated miR-21a-5p, miR-31-5p, miR-122-5p, miR-146a-5p, miR-150-5p, miR-155-5p, and miR-27b-5p while upregulating miR-706-5p and miR-7116. Pathway analysis showed that majority of the down-regulated miRs targeted molecules involved in cycle arrest and apoptosis such as CDKN2A, BCL2L11, and CCNG1, as well as anti-inflammatory molecules such as SOCS1 and FoxP3. Additionally, transfection studies involving miR-21 and use of Mir21-/- mice suggested that while this miR plays a critical role in EAE, additional miRs may also be involved in THC+CBD-mediated attenuation of EAE. Collectively, this study suggests that combination of THC+CBD suppresses neuroinflammation and attenuates clinical EAE development and that this effect is associated with changes in miRNA profile in brain-infiltrating cells.

Therapeutic impact of orally administered cannabinoid oil extracts in an experimental autoimmune encephalomyelitis animal model of multiple sclerosis.

There is a growing surge of investigative research involving the beneficial use of cannabinoids as novel interventional alternatives for multiple sclerosis (MS) and associated neuropathic pain (NPP). Using an experimental autoimmune encephalomyelitis (EAE) animal model of MS, we demonstrate the therapeutic effectiveness of two cannabinoid oil extract formulations (10:10 & 1:20 - tetrahydrocannabinol/cannabidiol) treatment. Our research findings confirm that cannabinoid treatment produces significant improvements in neurological disability scoring and behavioral assessments of NPP that directly result from their ability to reduce tumor necrosis factor alpha (TNF-α) production and enhance brain derived neurotrophic factor (BDNF) production. Henceforth, this research represents a critical step in advancing the literature by scientifically validating the merit for medical cannabinoid use and sets the foundation for future clinical trials.

Regulatory role of oligodendrocyte gap junctions in inflammatory demyelination.

Gap junctions (GJs) coupling oligodendrocytes to astrocytes and to other oligodendrocytes are formed mainly by connexin47 (Cx47) and a smaller portion by connexin32 (Cx32). Mutations in both connexins cause inherited demyelinating disorders, but their expression is also disrupted in multiple sclerosis (MS). To clarify whether the loss of either Cx47 or Cx32 could modify the outcome of inflammation and myelin loss, we induced experimental autoimmune encephalomyelitis (EAE) in fully backcrossed Cx32 knockout (KO) and Cx47KO mice and compared their outcome with wild type (WT, C57BI/6 N) mice. Cx47KO EAE mice developed the most severe phenotype assessed by clinical scores and behavioral testing, followed by Cx32KO and WT mice. Cx47KO more than Cx32KO EAE mice developed more microglial activation, myelin, and axonal loss than did WT mice. Oligodendrocyte apoptosis and precursor proliferation was also higher in Cx47KO than in Cx32KO or WT EAE mice. Similarly, blood-spinal cord barrier (BSCB) disruption and inflammatory infiltrates of macrophages, T- and B-cells were more severe in Cx47KO than either Cx32KO or WT EAE groups. Finally, expression profiling revealed that several proinflammatory cytokines were higher at the peak of inflammation in the Cx47KO mice and persisted at later stages of EAE in contrast to reduction of their levels in WT EAE mice. Thus, loss of oligodendrocyte GJs aggravates BSCB disruption and inflammatory myelin loss, likely due to dysregulation of proinflammatory cytokines. This mechanism may play an important role in MS brain with reduced connexin expression, as well as in patients with inherited mutations in oligodendrocyte connexins and secondary inflammation.

Blockade of sustained tumor necrosis factor in a transgenic model of progressive autoimmune encephalomyelitis limits oligodendrocyte apoptosis and promotes oligodendrocyte maturation.

BACKGROUND: Tumor necrosis factor (TNF) is associated with several neurodegenerative disorders including multiple sclerosis (MS). Although TNF-targeted therapies have been largely unsuccessful in MS, recent preclinical data suggests selective soluble TNF inhibition can promote remyelination. This has renewed interest in regulation of TNF signaling in demyelinating disease, especially given the limited treatment options for progressive MS. Using a mouse model of progressive MS, this study evaluates the effects of sustained TNF on oligodendrocyte (OLG) apoptosis and OLG precursor cell (OPC) differentiation. METHODS: Induction of experimental autoimmune encephalomyelitis (EAE) in transgenic mice expressing a dominant-negative interferon-γ receptor under the human glial fibrillary acidic protein promoter (GFAPγR1Δ) causes severe non-remitting disease associated with sustained TNF. Therapeutic effects in GFAPγR1Δ mice treated with anti-TNF compared to control antibody during acute EAE were evaluated by assessing demyelinating lesion size, remyelination, OLG apoptosis, and OPC differentiation. RESULTS: More severe and enlarged demyelinating lesions in GFAPγR1Δ compared to wild-type (WT) mice were associated with increased OLG apoptosis and reduced differentiated CC1+Olig2+ OLG within lesions, as well as impaired upregulation of TNF receptor-2, suggesting impaired OPC differentiation. TNF blockade during acute EAE in GFAPγR1Δ both limited OLG apoptosis and enhanced OPC differentiation consistent with reduced lesion size and clinical recovery. TNF neutralization further limited increasing endothelin-1 (ET-1) expression in astrocytes and myeloid cells noted in lesions during disease progression in GFAPγR1Δ mice, supporting inhibitory effects of ET-1 on OPC maturation. CONCLUSION: Our data implicate that IFNγ signaling to astrocytes is essential to limit a detrimental positive feedback loop of TNF and ET-1 production, which increases OLG apoptosis and impairs OPC differentiation. Interference of this cycle by TNF blockade promotes repair independent of TNFR2 and supports selective TNF targeting to mitigate progressive forms of MS.

Nutshell Extracts of Xanthoceras sorbifolia: A New Potential Source of Bioactive Phenolic Compounds as a Natural Antioxidant and Immunomodulator.

The nutshell of Xanthoceras sorbifolia, a waste product in the production of edible oil, is rich in health-promoting phenolic acids. However, the individual constituents, bioactivities, and mechanism of action are largely unknown. In this study, 20 phenolic compounds were characterized in nutshell extract (NE) of X. sorbifolia by gas chromatography-mass spectrometry. Four established in vitro studies showed that NE has significant antioxidant potential. Results in vivo indicated that oral administration of NE effectively ameliorated clinical disease severity of experimental autoimmune encephalomyelitis (EAE) and reduced the neuroinflammation and the central nervous system (CNS) demyelination. The underlying mechanism of NE-induced effects involved decreased penetration of pathogenic immunocyte into the CNS, a reduced production of proinflammatory cytokines and factors, and suppressed differentiation of type 1 T helper and type 17 T helper cells through the Janus kinase/signal transducer and activator of transcription pathway. Taken together, our studies showed that X. sorbifolia nutshell, considered a waste material in the food industry, is a novel source of a natural antioxidant and immunomodulator.

Combination therapy of lovastatin and AMP-activated protein kinase activator improves mitochondrial and peroxisomal functions and clinical disease in experimental autoimmune encephalomyelitis model.

Recent studies report that loss and dysfunction of mitochondria and peroxisomes contribute to the myelin and axonal damage in multiple sclerosis (MS). In this study, we investigated the efficacy of a combination of lovastatin and AMP-activated protein kinase (AMPK) activator (AICAR) on the loss and dysfunction of mitochondria and peroxisomes and myelin and axonal damage in spinal cords, relative to the clinical disease symptoms, using a mouse model of experimental autoimmune encephalomyelitis (EAE, a model for MS). We observed that lovastatin and AICAR treatments individually provided partial protection of mitochondria/peroxisomes and myelin/axons, and therefore partial attenuation of clinical disease in EAE mice. However, treatment of EAE mice with the lovastatin and AICAR combination provided greater protection of mitochondria/peroxisomes and myelin/axons, and greater improvement in clinical disease compared with individual drug treatments. In spinal cords of EAE mice, lovastatin-mediated inhibition of RhoA and AICAR-mediated activation of AMPK cooperatively enhanced the expression of the transcription factors and regulators (e.g. PPARα/ß, SIRT-1, NRF-1, and TFAM) required for biogenesis and the functions of mitochondria (e.g. OXPHOS, MnSOD) and peroxisomes (e.g. PMP70 and catalase). In summary, these studies document that oral medication with a combination of lovastatin and AICAR, which are individually known to have immunomodulatory effects, provides potent protection and repair of inflammation-induced loss and dysfunction of mitochondria and peroxisomes as well as myelin and axonal abnormalities in EAE. As statins are known to provide protection in progressive MS (Phase II study), these studies support that supplementation statin treatment with an AMPK activator may provide greater efficacy against MS.

Effects of Bu Shen Yi sui capsule on NogoA/NgR and its signaling pathways RhoA/ROCK in mice with experimental autoimmune encephalomyelitis.

BACKGROUND: Axon growth inhibitory factors NogoA/Nogo receptor (NgR) and its signaling pathways RhoA/Rho kinase (ROCK) play a critical role in the repair of nerve damage in multiple sclerosis (MS). Bu Shen Yi Sui Capsule (BSYSC) is an effective Chinese formula utilized to treat MS in clinical setting and noted for its potent neuroprotective effects. In this study, we focus on the effects of BSYSC on promoting nerve repair and the underlying mechanisms in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. METHODS: The EAE mouse model was induced by injecting subcutaneously with myelin oligodendrocyte glycoprotein (MOG) 35-55 supplemented with pertussis toxin. BSYSC was orally administrated at dose of 3.0 g/kg once a day for 40 days. The levels of protein gene product (PGP) 9.5, p-Tau, growth associated protein (GAP) -43, KI67 and Nestin in the brain or spinal cord on 20 and 40 day post-induction (dpi) were detected via immunofluorescence and Western blot analysis. Furthermore, NogoA/NgR and RhoA/ROCK signaling molecules were studied by qRT-PCR and Western blot analysis. RESULTS: Twenty or 40 days of treatment with BSYSC increased markedly PGP9.5 and GAP-43 levels, reduced p-Tau in the brain or spinal cord of mice with EAE. In addition, BSYSC elevated significantly the expression of KI67 and Nestin in the spinal cord 40 dpi. Further study showed that the activation of NogoA/NgR and RhoA/ROCK were suppressed by the presence of BSYSC. CONCLUSIONS: BSYSC could attenuate axonal injury and promote repair of axonal damage in EAE mice in part through the down-regulation of NogoA/NgR and RhoA/ROCK signaling pathways.

Overexpression of GSN could decrease inflammation and apoptosis in EAE and may enhance vitamin D therapy on EAE/MS.

The decrease of gelsolin (GSN) in the blood has been reported in multiple sclerosis (MS) patients and experimental allergic encephalomyelitis (EAE) animals, but the protective effect of GSN on EAE/MS lacks of evidence. In our study, we increased the GSN level in EAE by injecting GSN-overexpress lentivirus (LV-GSN) into the lateral ventricle and caudal vein and found that GSN administration can delay the onset and decrease the severity of EAE. Vitamin D is proven to have a therapeutic effect on MS/EAE; however, we previously found that vitamin D caused a downregulation of GSN, which might limit vitamin D efficacy. In our current research, we obtained a better symptom and a slowing down progression in EAE after combining vitamin D treatment with a proper increase of GSN. Furthermore, we discovered that the mediation of vitamin D on GSN might occur through the vitamin D receptor (VDR) by using gene interruption and overexpression to regulate the level of VDR in PC12 cells (a rat sympathetic nerve cell line). We also confirmed the anti-apoptotic function of GSN by GSN RNA interference in PC12. Collectively, these results support the therapeutic effect of GSN in EAE, which might enhance Vitamin D therapy in EAE/MS.

miR-219 Cooperates with miR-338 in Myelination and Promotes Myelin Repair in the CNS.

A lack of sufficient oligodendrocyte myelination contributes to remyelination failure in demyelinating disorders. miRNAs have been implicated in oligodendrogenesis; however, their functions in myelin regeneration remained elusive. Through developmentally regulated targeted mutagenesis, we demonstrate that miR-219 alleles are critical for CNS myelination and remyelination after injury. Further deletion of miR-338 exacerbates the miR-219 mutant hypomyelination phenotype. Conversely, miR-219 overexpression promotes precocious oligodendrocyte maturation and regeneration processes in transgenic mice. Integrated transcriptome profiling and biotin-affinity miRNA pull-down approaches reveal stage-specific miR-219 targets in oligodendrocytes and further uncover a novel network for miR-219 targeting of differentiation inhibitors including Lingo1 and Etv5. Inhibition of Lingo1 and Etv5 partially rescues differentiation defects of miR-219-deficient oligodendrocyte precursors. Furthermore, miR-219 mimics enhance myelin restoration following lysolecithin-induced demyelination as well as experimental autoimmune encephalomyelitis, principal animal models of multiple sclerosis. Together, our findings identify context-specific miRNA-regulated checkpoints that control myelinogenesis and a therapeutic role for miR-219 in CNS myelin repair.

Increase in mRNA Level of Orexin1 and 2 Receptors Following Induction of Experimental Autoimmune Encephalomyelitis in Mice.

Orexin A and B are hypothalamic peptides with a wide variety of effects such as anti-inflammation and neuroprotection. Impaired function of orexin system has been reported in some neurodegenerative diseases like Parkinson, Huntington and Alzheimer. In this study, the mRNA expression levels of some hypothalamic peptides were investigated in C57BL/6 female mice with experimental autoimmune encephalomyelitis (EAE). Animals were randomly divided into two control and EAE groups. EAE was induced by administration of myelin oligodendrocyte glycoprotein (MOG) with complete Ferund's adjuvant and pertussis toxin. Twenty-first days following immunization, mice were decapitated to remove the brains. Then, the expression profiles of prepro-orexin, orexin 1 receptors (OX1R) and orexin 2 receptors (OX2R) in hypothalamic region were assessed using real-time PCR method. In this study, we found a considerable increase in the mRNA expression of OX1R and OX2R following EAE induction in C57BL/6 mice. Elevation levels of OX1R and OX2R following EAE induction suggest that alteration in orexinergic system may involve in pathogenesis of multiple sclerosis.

Effect of Bushen Yisui Capsule () on oligodendrocyte lineage genes 1 and 2 in mice with experimental autoimmune encephalomyelitis.

OBJECTIVE: To study the effects of Bushen Yisui Capsule (, BSYSC) on the oligodendrocyte lineage genes (Olig) 1 and Olig2 in C57BL/6 mice with experimental autoimmune encephalomyelitis (EAE) in order to explore the remyelination effect of BSYSC. METHODS: The mice were randomly divided into normal control (NC), EAE model (EAE-M), prednisone acetate (PA, 6 mg/kg), BSYSC high-dose (3.02 g/kg) and BSYSC low-dose (1.51 g/kg) groups. The mice were induced by immunization with myelin oligodendrocyte glycoprotein (MOG) 35-55. The neurological function scores were assessed once daily. The pathological changes in mice brains were observed with hematoxylin-eosin (HE) staining and transmission electron microscope (TEM). The protein expressions of myelin basic protein (MBP), Olig1 and Olig2 in brains were measured by immunohistochemistry. The mRNA expressions of Olig1 and Olig 2 was also determined by quantitative real-time polymerase chain reaction. RESULTS: Compared with the EAE-M mice, (1) the neurological function scores were significantly decreased in BSYSC-treated mice on days 22 to 40 (P<0.01); (2) the inflammatory cells and demyelination in brains were reduced in BSYSC-treated EAE mice; (3) the protein expression of MBP was markedly increased in BSYSC-treated groups on day 18 and 40 respectively (P<0.05 or P<0.01); (4) the protein expression of Olig1 was increased in BSYSC (3.02 g/kg)-treated EAE mice on day 40 (P<0.01). Protein and mRNA expression of Olig2 was increased in BSYSC-treated EAE mice on day 18 and 40 (P<0.01). CONCLUSION: The effects of BSYSC on reducing demyelination and promoting remyelination might be associated with the increase of Olig1 and Olig2.

Enhanced stability of tristetraprolin mRNA protects mice against immune-mediated inflammatory pathologies.

Tristetraprolin (TTP) is an inducible, tandem zinc-finger mRNA binding protein that binds to adenylate-uridylate-rich elements (AREs) in the 3'-untranslated regions (3'UTRs) of specific mRNAs, such as that encoding TNF, and increases their rates of deadenylation and turnover. Stabilization of Tnf mRNA and other cytokine transcripts in TTP-deficient mice results in the development of a profound, chronic inflammatory syndrome characterized by polyarticular arthritis, dermatitis, myeloid hyperplasia, and autoimmunity. To address the hypothesis that increasing endogenous levels of TTP in an intact animal might be beneficial in the treatment of inflammatory diseases, we generated a mouse model (TTPΔARE) in which a 136-base instability motif in the 3'UTR of TTP mRNA was deleted in the endogenous genetic locus. These mice appeared normal, but cultured fibroblasts and macrophages derived from them exhibited increased stability of the otherwise highly labile TTP mRNA. This resulted in increased TTP protein expression in LPS-stimulated macrophages and increased levels of TTP protein in mouse tissues. TTPΔARE mice were protected from collagen antibody-induced arthritis, exhibited significantly reduced inflammation in imiquimod-induced dermatitis, and were resistant to induction of experimental autoimmune encephalomyelitis, presumably by dampening the excessive production of proinflammatory mediators in all cases. These data suggest that increased systemic levels of TTP, secondary to increased stability of its mRNA throughout the body, can be protective against inflammatory disease in certain models and might be viewed as an attractive therapeutic target for the treatment of human inflammatory diseases.