Study on the Anti-demyelination Mechanism of Bu-Shen-Yi-Sui Capsule in the Central Nervous System Based on Network Pharmacology and Experimental Verification.

Methods: The potential active ingredients and corresponding potential targets of BSYS Capsule were obtained from the TCMSP, BATMAN-TCM, Swiss Target Prediction platform, and literature research. Disease targets of CNSD were explored through the GeneCards and the DisGeNET databases. The matching targets of BSYS in CNSD were identified from a Venn diagram. The protein-protein interaction (PPI) network was constructed using bioinformatics methods. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to predict the mechanisms of BSYS. Furthermore, the neuroprotective effects of BSYS were evaluated using a cell model of hydrogen peroxide- (H2O2-) induced cell death in OLN-93 cells. Results: A total of 59 potential bioactive components of BSYS Capsule and 227 intersection targets were obtained. Topological analysis showed that AKT had the highest connectivity degrees in the PPI network. Enrichment analysis revealed that the targets of BSYS in the treatment of CNSD were the PI3K-Akt and MAPK signaling pathway, among other pathways. GO analysis results showed that the targets were associated with various biological processes, including apoptosis, reactive oxygen species metabolic process, and response to oxidative stress, among others. The experimental results demonstrated that BSYS drug-containing serum alleviated the H2O2-induced increase in LDH, MDA, and ROS levels and reversed the decrease in SOD and mitochondrial membrane potential induced by H2O2. BSYS treatment also decreased the number of TUNEL (+) cells, downregulated Bcl-2 expression, and upregulated Bax and c-caspase-3 expression by promoting Akt phosphorylation. Conclusion: BSYS Capsule alleviated H2O2-induced OLN-93 cell injury by increasing Akt phosphorylation to suppress oxidative stress and cell apoptosis. Therefore, BSYS can be potentially used for CNSD treatment. However, the results of this study are only derived from in vitro experiments, lacking the validation of in vivo animal models, which is a limitation of our study. We will further verify the underlying mechanisms of BSYS in animal experiments in the future.

Effect of catalpol on remyelination through experimental autoimmune encephalomyelitis acting to promote Olig1 and Olig2 expressions in mice.

BACKGROUND: Multiple sclerosis (MS) as an autoimmune disorder is a common disease occurring in central nervous system (CNS) and the remyelination plays a pivotal role in the alleviating neurological impairment in the MS. Catalpol, an effective component extracted from the Chinese herb Radix Rehmanniae, which has been proved protective in cerebral diseases. METHODS: To determine the protective effects and mechanisms of Catalpol on MS, the mice with experimental autoimmune encephalomyelitis (EAE) were induced by myelin oligodendrocyte glycoprotein (MOG) 35-55, as a model for human MS. Th17 cells were counted by flow cytometric (FCM). The expressions of nerve-glial antigen (NG) 2 and myelin basic protein (MBP) were measured by immunohistochemical staining. Olig1+ and Olig2+/BrdU+ cells were counted by immunofluorescence. Olig1 and Olig2 gene expressions were detected by real-time fluorescent quantitative reverse transcription (qRT) -PCR. RESULTS: The results showed that Catalpol improved neurological function, reduced inflammatory cell infiltration and demyelination. It could decrease Th17 cells in the peripheral blood. It increased the protein expressions of NG2 and MBP in mice brains, up-regulated markedly protein and gene expressions of Olig1 and Olig2 in terms of timing, site and targets. CONCLUSIONS: These data demonstrated that Catalpol had a strong neuroprotective effect on EAE mice. Catalpol also plays a role in remyelination by promoting the expressions of Olig1 and Olig2 transcription factors.

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.

Increased plasma levels of epithelial neutrophil-activating peptide 78/CXCL5 during the remission of Neuromyelitis optica.

BACKGROUND: In neuromyelitis optica (NMO), one of the underlying pathogenic mechanisms is the formation of antigen-antibody complexes which can trigger an inflammatory response by inducing the infiltration of neutrophils in lesions. Epithelial neutrophil-activating peptide 78 (ENA 78), known as Chemokine (C-X-C motif) ligand 5 (CXCL5), belongs to the ELR-CXCL family. It recruits and activates neutrophils. The aim of this study was to evaluate ENA 78, IL-1ß and TNF-α plasma levels in multiple sclerosis (MS) and neuromyelitis optica (NMO) patients. METHODS: ENA 78, IL-1ß and TNF-α plasma levels were detected in 20 healthy controls (HC), 25 MS and 25 NMO patients using MILLIPLEX® map Human High Sensitivity Cytokine/Chemokine Panels. RESULTS: Plasma levels of ENA 78 were significantly higher in NMO patients than in HC (P < 0.001) and MS patients (P < 0.05). The NMO patients showed higher plasma levels of IL-1ß compared with HC (P < 0.01). Further, increased plasma levels of TNF-α were found in the MS (P < 0.05) and NMO patients (P < 0.001). In addition, NMO patients had higher Expanded Disability Status Scale (EDSS) scores compared with MS patients (P < 0.05). EDSS scores were correlated with plasma levels of ENA 78 in NMO patients (P < 0.05). There were no significant correlations between EDSS scores and plasma levels of ENA 78 in MS patients (P > 0.05). CONCLUSIONS: The overproduction of pro-inflammatory cytokines such as IL-1ß and TNF-α during the remission of NMO activates ENA 78, which in turn leads to neutrophil infiltration in lesions. ENA 78 plasma levels were correlated with EDSS scores in NMO patients. Elevated secretion of ENA 78 may be a critical step in neutrophil recruitment during the remission of NMO.

Effects of Bu Shen Yi Sui Capsule on Th17/Treg cytokines in C57BL/6 mice with experimental autoimmune encephalomyelitis.

BACKGROUND: T helper (Th) 17 and regulatory T (Treg) cells play a critical role in the pathogenesis of multiple sclerosis (MS) disease. Bu Shen Yi Sui Capsule (BSYSC), a traditional Chinese medicine formula, has been used clinically for the treatment of MS patients in China. METHODS: To evaluate the neuroprotective effects and the underlying mechanisms of BSYSC on MS, experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice was induced with myelin oligodendrocyte glycoprotein (MOG) 35-55. Th17 and Treg cells and the related cytokines were detected by flow cytometry, ELISA, real-time quantitative reverse transcription PCR, western blot and immunohistochemistry. RESULTS: We found that BSYSC improved neurological function, reduced inflammatory cell infiltration and damage to the axons and myelin in the brain and spinal cord. BSYSC down-regulated markedly the ratio of CD4 + IL-17+/CD4 + CD25 + FoxP3+ T cells in the spleen, decreased the cytokines of IL-17A, IL-6, IL-23, TGF-beta1 in the brain, and dropped the ratio of IL-17A and FoxP3 mRNA and protein in the brain or spinal cord at different stages. CONCLUSIONS: The study demonstrated that BSYSC had a strong neuroprotective effect on EAE mice. The protective mechanisms of BSYSC might be associated with mediating the regulation of Th17/Treg cells.

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 mL/kg), Tanreqing low dose (2.57 mL/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.

Peroxynitrite reduces Treg cell expansion and function by mediating IL-2R nitration and aggravates multiple sclerosis pathogenesis.

T-helper 17 cells and regulatory T cells (Treg) are critical regulators in the pathogenesis of multiple sclerosis (MS) but the factors affecting Treg/Th17 balance remains largely unknown. Redox balance is crucial to maintaining immune homeostasis and reducing the severity of MS but the underlying mechanisms are unclear yet. Herein, we tested the hypothesis that peroxynitrite, a representative molecule of reactive nitrogen species (RNS), could inhibit peripheral Treg cells, disrupt Treg/Th17 balance and aggravate MS pathology by inducing nitration of interleukin-2 receptor (IL-2R) and down-regulating RAS/JNK-AP-1 signalling pathway. Experimental autoimmune encephalomyelitis (EAE) mouse model and serum samples of MS patients were used in the study. We found that the increases of 3-nitrotyrosine and IL-2R nitration in Treg cells were coincided with disease severity in the active EAE mice. Mechanistically, peroxynitrite-induced IL-2R nitration down-regulated RAS/JNK signalling pathway, subsequently impairing peripheral Treg expansion and function, increasing Teff infiltration into the central nerve system (CNS), aggravating demyelination and neurological deficits in the EAE mice. Those changes were abolished by peroxynitrite decomposition catalyst (PDC) treatment. Furthermore, transplantation of the PDC-treated-autologous Treg cells from donor EAE mice significantly decreased Th17 cells in both axillary lymph nodes and lumbar spinal cord, and ameliorated the neuropathology of the recipient EAE mice. Those results suggest that peroxynitrite could disrupt peripheral Treg/Th17 balance, and aggravate neuroinflammation and neurological deficit in active EAE/MS pathogenesis. The underlying mechanisms are related to induce the nitration of IL-2R and inhibit the RAS/JNK-AP-1 signalling pathway in Treg cells. The study highlights that targeting peroxynitrite-mediated peripheral IL-2R nitration in Treg cells could be a novel therapeutic strategy to restore Treg/Th17 balance and ameliorate MS/EAE pathogenesis. The study provides valuable insights into potential role of peripheral redox balance in maintaining CNS immune homeostasis.

[Clinical features of neuromyelitis optica and the distribution of Chinese medical syndrome types: a case report of 63 cases].

OBJECTIVE: To explore the clinical features of neuromyelitis optica (NMO) patients, and to study the distribution of Chinese medical syndrome types and the pathogenesis of NMO. METHODS: The clinical features, figures of tongue and pulse, Chinese medical syndromes were comprehensively analyzed in 63 NMO patients using statistical methods for clinical data. RESULTS: The age ratio of male to female in 63 NMO patients was 1: 6.88. Their average age of first onset was 31.67 +/- 12.44 years old, and 28. 57% of patients had obvious inducing factor. Urgent onset with relieved recurrence were often seen, with the average recurrence times of 4.60. Most patients complained about sensation disorders, vision disorders, and movement disorders as their first attack and visit. The Chinese medical syndrome types included Gan-Shen yin deficiency syndrome and phlegm-heat collateral stagnation syndrome, mainly involved Gan and Shen. Gan-Shen yin deficiency, sputum, blood stasis, and heat were most often seen syndrome elements. CONCLUSIONS: Gan-Shen yin deficiency was dominated in the deficiency in origin of NMO. Phlegm, blood stasis, mingled heat were main dominant evils. Of them, the pathogenesis of Gan-Shen yin deficiency and phlegm-heat collateral stagnation had universality and representativeness, which could be verified from patients' tongue picture and pulse picture.

Association of Pain with Plasma C5a in Patients with Neuromyelitis Optica Spectrum Disorders During Remission.

Objective: To investigate the association of pain with plasma C5a levels and other related inflammatory cytokines in neuromyelitis optica spectrum disorders (NMOSD) patients during remission. Participants and Methods: NMOSD patients (n = 87) and healthy controls (HC; n = 44) were consecutively recruited between January 2017 and April 2018. Plasma complement 5 (C5), C5a, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1ß levels were detected. Visual Analogue Scale (VAS), ID pain scale, 24-item Hamilton Depression Scale (HAMD), Multiple Sclerosis Impact Scale (MSIS-29), and Kurtzke Expanded Disability Status Scale (EDSS) were used to evaluate the degree and types of pain, the existence of depression and anxiety, and the life quality and disability status of patients. Binary logistic regression equation was used to assess the association of pain with plasma C5a levels. Results: Among the 87 NMOSD patients, 40 complained of pain that in 67.5% (27/40) of cases had a neuropathic component (ID pain ≥2). Plasma C5a, IL-6, TNF-α, and IL-1ß levels were significantly elevated in NMOSD patients than in HC. Plasma C5 levels were negatively correlated with the time from sampling to the last relapse or disease onset. NMOSD patients with pain had higher plasma C5a levels, and they suffered from a higher disability, more anxiety, and worse life quality compared to those patients without pain. In NMOSD patients with pain, there were not significant differences between plasma levels of C5, C5a, IL-6, TNF-α, or IL-1ß, regardless of neuropathic pain or not. Binary logistic regression showed that the OR of plasma C5a level was 1.002, with gender and EDSS score were identified as independent factors associated with pain in NMOSD. Conclusion: NMOSD patients during remission had elevated C5a and related inflammatory cytokines levels in peripheral blood. Elevated C5a may have a unique role in the pathogenesis of pain in NMOSD patients.

Catalpol Regulates Oligodendrocyte Regeneration and Remyelination by Activating the GEF-Cdc42/Rac1 Signaling Pathway in EAE Mice.

The main obstacle to remyelination in demyelinating diseases, such as multiple sclerosis, is the inability of oligodendrocyte precursor cells (OPCs) to differentiate into mature oligodendrocytes (OLs) in the demyelinating region. Consequently, promoting OL differentiation and myelin remodeling is a key goal in the search for treatments. Rho GTPases play diverse and important roles throughout the development of neuronal axons and the formation of the myelin sheath. The current study aimed to investigate the direct protective effects of catalpol on demyelination damage induced by myelin oligodendrocyte glycoprotein (MOG) immunization and to explore whether the GEF-Cdc42/Rac1 signaling pathway contributes to the regeneration effect induced by catalpol. In the MOG-induced experimental autoimmune encephalomyelitis (EAE) mouse model of demyelination, we observed that catalpol significantly promoted OL development by enhancing the expression of glutathione S-transferase pi (GST-pi) in the affected brain. By Luxol fast blue staining and myelin basic protein (MBP) expression assessment, catalpol was found to increase MBP expression and promote myelin repair. Furthermore, catalpol promoted OL differentiation associated with the upregulation of Cdc42/Rac1 expression and activation in vivo. In addition, PAK1/MRCKα, proteins downstream of Cdc42/Rac1, was positively regulated by catalpol. We also found that catalpol alleviated clinical neurological dysfunction, inhibited inflammatory infiltration, increased the proportion of Treg cells, and suppressed demyelination. Overall, our study is the first to reveal that catalpol can promote OL generation and myelination and contributes to the crucial regulatory process of GEF-Cdc42/Rac1 signaling expression and activation. Therefore, catalpol is a promising drug candidate for the potential treatment of demyelinating diseases.

Identification and Quantification of Bu Shen Yi Sui Capsule by UPLC-LTQ-Orbitrap-MSn and UPLC-QTOF-MS/MS.

Traditional Chinese medicines (TCMs) have been considered as important alternative therapeutics because of their significant medicinal benefits in specific diseases. Chinese herb formula is characterized by a vast molecule that differs in routine medicines. Due to TCMs chemical complexity, proper quality control has been a great challenge. Choosing the appropriate method to identify and qualify these compounds is an important work to ensure its safety, efficacy and quality control. Thus, this study aimed at providing novel information on high-resolution LTQ-Orbitrap mass spectrometer (UPLC-LTQ-Orbitrap-MSn) based identification of Bu Shen Yi Sui capsule (BSYSC), which is used in treating multiple sclerosis as a kind of TCMs. Under the proposed chromatographic conditions, 80 chemical components classified as anthraquinone, phenolic acid and phenylethanoid glycosides were separated and identified from BSYSC. Coupled with the high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) method, eight of them were regarded as marker compounds for the quantitative evaluation of BSYSC. The identification and quantification with precision of UPLC-LTQ-Orbitrap-MSn and UPLC-QTOF-MS/MS could facilitate essential data for further pharmacokinetic studies of BSYSC.

Bu Shen Yi Sui Capsules Promote Remyelination by Regulating MicroRNA-219 and MicroRNA-338 in Exosomes to Promote Oligodendrocyte Precursor Cell Differentiation.

Remyelination is a refractory feature of demyelinating diseases such as multiple sclerosis (MS). Studies have shown that promoting oligodendrocyte precursor cell (OPC) differentiation, which cannot be achieved by currently available therapeutic agents, is the key to enhancing remyelination. Bu Shen Yi Sui capsule (BSYSC) is a traditional Chinese herbal medicine over many years of clinical practice. We have found that BSYSC can effectively treat MS. In this study, the effects of BSYSC in promoting OPCs differentiation and remyelination were assessed using an experimental autoimmune encephalomyelitis (EAE) model in vivo and cultured OPCs in vitro. The results showed that BSYSC reduced clinical function scores and increased neuroprotection. The expression of platelet-derived growth factor receptor α (PDGFR-α) was decreased and the level of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) was increased in the brains and spinal cords of mice as well as in OPCs after treatment with BSYSC. We further found that BSYSC elevated the expression of miR-219 or miR-338 in the serum exosomes of mice with EAE, thereby suppressing the expression of Sox6, Lingo1, and Hes5, which negatively regulate OPCs differentiation. Therefore, serum exosomes of BSYSC-treated mice (exos-BSYSC) were extracted and administered to OPCs in which miR-219 or miR-338 expression was knocked down by adenovirus, and the results showed that Sox6, Lingo1, and Hes5 expression was downregulated, MBP expression was upregulated, OPCs differentiation was increased, and the ability of OPCs to wrap around neuronal axons was improved. In conclusion, BSYSC may exert clinically relevant effects by regulating microRNA (miR) levels in exosomes and thus promoting the differentiation and maturation of OPCs.

Bu Shen Yi Sui Capsule Promotes Myelin Repair by Modulating the Transformation of A1/A2 Reactive Astrocytes In Vivo and In Vitro.

Background/Aims. Multiple sclerosis (MS) is an autoimmune disorder that affects the central nervous system (CNS) primarily hallmarked by neuroinflammation and demyelination. The activation of astrocytes exerts double-edged sword effects, which perform an integral function in demyelination and remyelination. In this research, we examined the therapeutic effects of the Bu Shen Yi Sui capsule (BSYS), a traditional Chinese medicine prescription, in a cuprizone- (CPZ-) triggered demyelination model of MS (CPZ mice). This research intended to evaluate if BSYS might promote remyelination by shifting A1 astrocytes to A2 astrocytes. Methods. The effects of BSYS on astrocyte polarization and the potential mechanisms were explored in vitro and in vivo utilizing real-time quantitative reverse transcription PCR, immunofluorescence, and Western blotting. Histopathology, expression of inflammatory cytokines (IL-10, IL-1ß, and IL-6), growth factors (TGF-ß, BDNF), and motor coordination were assessed to verify the effects of BSYS (3.02 g/kg/d) on CPZ mice. In vitro, A1 astrocytes were induced by TNF-α (30 ng/mL), IL-1α (3 ng/mL), and C1q (400 ng/mL), following which the effect of BSYS-containing serum (concentration of 15%) on the transformation of A1/A2 reactive astrocytes was also evaluated. Results and Conclusions. BSYS treatment improved motor function in CPZ mice as assessed by rotarod tests. Intragastric administration of BSYS considerably lowered the proportion of A1 astrocytes, but the number of A2 astrocytes, MOG+, PLP+, CNPase+, and MBP+ cells was upregulated. Meanwhile, dysregulation of glutathione peroxidase, malondialdehyde, and superoxide dismutase was reversed in CPZ mice after treatment with BSYS. In addition, the lesion area and expression of proinflammatory cytokines were decreased and neuronal protection factors and anti-inflammatory cytokines were increased. In vitro, BSYS-containing serum suppressed the A1 astrocytic markers' expression and elevated the expression levels of A2 markers in primary astrocytes triggered by C1q, TNF-α, and IL-1α. Importantly, the miR-155/SOCS1 signaling pathway was involved in the modulation of the A1/A2 phenotype shift. Overall, this study demonstrated that BSYS has neuroprotective effects in myelin repair by modulating astrocyte polarization via the miR-155/SOCS1 pathway.

Abnormalcortical thickness in relapsing-remitting multiple sclerosis, correlations with cognition impairment, and effect of modified Bushenyisui decoction on cognitive function of multiple sclerosis.

OBJECTIVE: To investigate the changes of subcortical gray matter volume and cortical thickness, andexplorethe correlations between regional abnormalities of cortical thickness and cognitive impairment and the effect of modified Bushenyisui decoction ( BSYSD) on the cognitive function of multiple sclerosis (MS). METHODS: This prospective study was approved by the institutional review board. 92 subjects were recruited, including 46 relapsing-remitting multiple sclerosis (RRMS) patients and 46 healthy controls (HC). Of the 46 patients, 22 patients experienced the treatment of BSYSD for half a year. A conventional three-dimensional T1-weighted sequence were acquired for all participants on a 3.0 tesla magnetic resonance system. Basic information, detailed cognitive scales Montreal Cognitive Assessment (MoCA), symbol digit modalities test (SDMT), immediate memory, delayed recall, and long-term recognition were evaluated. Subcortical gray matter volume and cortical thickness weremeasured by FreeSurfer. The correlations between cortical thickness which MS patients showed reduced with respect to HC and cognitive scales wereanalyzed by Pearson correlation in RRMS patients. The influence of modified BSYSD on MS patients' cognition was analyzed by paired T Test. RESULTS: MoCA, immediate memory, delayed recall, and long-term delayed recognition in RRMS were significantly decreased than those of HC. Gray matter atrophy measured by FreeSurfer showed mainly in thalamus and hippocampus of RRMS patients. Compared with HC, the cortical thickness of several regions in frontal lobe, parietal lobe, temporal lobe, hippocampal, cingulate gyrus, and fusiform gyrus of RRMS patients were decreased with significant difference. The regions of cortical thickness thinning related to MoCA, immediate memory, delayed recall, and long-term delayed recognition were temporal lobe and fusiform gyrus. Modified BSYSD could improve MoCA, SDMT, immediate memory, delayed recall, and long-term delayed recognition of MS patients, and it could promote the recovery of cognitive function in MS patients. CONCLUSIONS: Gray matter atrophy and cortical thickness thinning were validated in RRMS. Cortical thickness thinning of temporal lobe and fusiform gyrus strongly related to cognitive deficits in RRMS. The modified BSYSD could promote the recovery of cognitive function in MS.

Neuroprotective Effects of Fingolimod Supplement on the Retina and Optic Nerve in the Mouse Model of Experimental Autoimmune Encephalomyelitis.

Favorable effects exerted by long-term administration of fingolimod therapy in multiple sclerosis (MS) patients have been reported, but sporadic side effects, such as reversible macular edema, also have been recorded. The present study aimed to determine whether fingolimod therapy is beneficial to the visual system in experimental autoimmune encephalomyelitis (EAE) mice. A decrease in demyelination and axon loss in the optic nerve as well as cellular infiltration, especially the recruited macrophages, was observed in EAE with fingolimod treatment. Fingolimod administration diminished hypergliosis of macroglia, including astrocytes and Müller cells in the retina and optic nerve in EAE. Microglia were hyperactivated in the retina and optic nerve in the EAE mice compared to controls, which could be alleviated by fingolimod treatment. Moreover, apoptosis of retinal ganglion cells (RGC) and oligodendrocytes in the optic nerve was significantly reduced with fingolimod treatment compared to that in the untreated EAE mice. These results suggested that fingolimod exerts neuroprotective and anti-inflammatory effects on the retina and optic nerve in a mouse model of EAE. Considering the paradox of favorable and side effects of fingolimod on visual system, we speculate that side effects including macular oedema caused by fingolimod during MS treatment is tendency to be vasogenic rather than hypergliosis in optic nerve and retina which warrants further neuroophthalmological investigation.

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.

Nonsurgical treatment of chronic subdural hematoma with Chinese herbal medicine: A STROBE-compliant retrospective study.

The aim of the study was to observe the efficacy of nonsurgical treatment with Chinese herbal medicine (CHM) for chronic subdural hematoma (CSDH). This study includes clinical results of a STROBE-compliant retrospective study.Forty patients diagnosed with CSDH were recruited from outpatient. Different CHM prescriptions were dispensed for each patient based on syndrome differentiation until the patient had a stable neurologic condition for 2 weeks and/or CSDH completely resolved according to the computed tomography scan. Markwalder grading scale for neurologic symptoms and head computed tomography scan for hematoma volumes were performed before and after CHM treatment to evaluate efficacy.Patients received uninterrupted CHM treatment for 2.81 ±â€Š1.45 months (0.75-6 months). The hematoma volume significantly reduced from 73.49 ±â€Š35.43 mL to 14.72 ±â€Š15.94 mL (P < .001). The Markwalder grading scale scores of patients at the end of CHM treatment decreased significantly, from 1.3 ±â€Š0.69 to 0.15 ±â€Š0.36 (P < .001). Ninety percent of the patients showed >50% decrease in the hematoma volume and complete improvement in neurologic symptoms. The linear regression analysis suggested that change in hematoma was significantly related to the duration of CHM treatment (R = 0.334; P < .001; Y = 25.03 + 11.91X). Leonurus heterophyllus Sweet (Yi-Mu-Cao, 90.5%), Semen persicae (Tao-Ren, 88.8%), and Acorus tatarinowii Schott (Shi-Chang-Pu, 86.2%) were the top 3 single Chinese herbs prescribed in CHM treatment.The CHM treatment for CSDH based on syndrome differentiation with appropriate duration relieved neurologic symptoms quickly and promoted hematoma absorption effectively. It could be an effective nonsurgical therapy for CSDH.

Bu-Shen-Yi-Sui Capsule, an Herbal Medicine Formula, Promotes Remyelination by Modulating the Molecular Signals via Exosomes in Mice with Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a common inflammatory demyelinating disorder of the central nervous system. Bu-shen-yi-sui capsule (BSYSC) could significantly reduce the relapse rate, prevent the progression of MS, and enhance remyelination following neurological injury in experimental autoimmune encephalomyelitis (EAE), an established model of MS; however, the mechanism underlying the effect of BSYSC on remyelination has not been well elucidated. This study showed that exosomes carrying biological information are involved in the pathological process of MS and that modified exosomes can promote remyelination by modulating related proteins and microRNAs (miRs). Here, the mechanism by which BSYSC promoted remyelination via exosome-mediated molecular signals was investigated in EAE mice and oligodendrocyte progenitor cells (OPCs) in vitro. The results showed that BSYSC treatment significantly improved the body weight and clinical scores of EAE mice, alleviated inflammatory infiltration and nerve fiber injury, protected the ultrastructural integrity of the myelin sheath, and significantly increased the expression of myelin basic protein (MBP) in EAE mice. In an in vitro OPC study, BSYSC-containing serum, especially 20% BSYSC, promoted the proliferation and migration of OPCs and induced OPCs to differentiate into mature oligodendrocytes that expressed MBP. Furthermore, BSYSC treatment regulated the expression of neuropilin- (NRP-) 1 and GTX, downregulated the expression of miR-16, let-7, miR-15, miR-98, miR-486, and miR-182, and upregulated the level of miR-146 in serum exosomes of EAE mice. In conclusion, these results suggested that BSYSC has a neuroprotective effect and facilitates remyelination and that the mechanism underlying the effect of BSYSC on remyelination probably involves regulation of the NRP-1 and GTX proteins and miRs in serum exosomes, which drive promyelination.

Protective effects of catalpol and rhein in murine experimental autoimmune encephalomyelitis via regulation of T helper (Th) 1, Th2, Th17, and regulatory T cell responses.

OBJECTIVE: To examine the effects of catalpol and rhein on pro- and anti-inflammatory responses in C57BL/6 mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. METHODS: Female C57BL/6 mice were randomly divided into four groups (n = 30): (a) normal saline control, (b) EAE control, (c) EAE + prednisone acetate (PA, 6 mg/kg), and (d) EAE + catalpol (40 mg/kg) and rhein (5 mg/kg). EAE was induced by injection of myelin oligodendrocyte glycoprotein 35-55 plus pertussis toxin. Treatments were orally administered daily for 40 d. Disease progression and neurological function were assessed using a semi-quantitative scale of tail and limb paralysis. Brains and spinal cords were collected on Days 6, 20, and 40 and assessed for histopathological changes by hematoxylin and eosin staining. Production of interleukin (IL)-2, IL-4, IL-10, and IL-17A protein was measured by enzyme-linked immunosorbent assay. Expression of the T helper (Th)1-, Th2-, Th17-, and regulatory T cell (Treg)-specific transcription factors T-bet, GATA3, ROR-γt, and Foxp3, respectively, were analyzed by quantitative reverse-transcription polymerase chain reaction and western blot analysis. RESULTS: Combination treatment with catalpol and rhein significantly alleviated the clinical disability and neurological dysfunction of mice with EAE. Catalpol and rhein treatment also reduced the infiltration of pro-inflammatory T cells into pathological lesions; significantly increased the expression of the anti-inflammatory factors GATA3, Foxp3, IL-4, and IL-10; and significantly decreased the expression of the pro-inflammatory factors T-bet, ROR-γt, IL-2, and IL-17A. CONCLUSION: Catalpol and rhein reduced the neurological disabilities of mice with EAE, at least in part by rebalancing the pro- and anti-inflammatory environment in the brains and spinal cords.

The Bu Shen Yi Sui Formula Promotes Axonal Regeneration via Regulating the Neurotrophic Factor BDNF/TrkB and the Downstream PI3K/Akt Signaling Pathway.

Axonal damage is recognized as an important pathological feature in the chronic progressive neurological disorder multiple sclerosis (MS). Promoting axonal regeneration is a critical strategy for the treatment of MS. Our clinical and experimental studies have shown that the Bu Shen Yi Sui formula (BSYS) promotes axonal regeneration in MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS, but the exact mechanism has not been thoroughly elucidated to date. In this study, we investigated the effects of BSYS and its two decomposed formulas-the Bu Shen formula (BS) and the Hua Tan Huo Xue formula (HTHX)-on brain-derived neurotrophic factor (BDNF)/TrkB and related signaling pathways to explore the mechanism by which axonal regeneration is promoted in vitro and in vivo. Damaged SH-SY5Y cells incubated with low serum were treated with BSYS-, BS-, and HTHX-containing serum, and EAE mice induced by the myelin oligodendrocyte glycoprotein (MOG)35-55 peptide were treated with BSYS. The results showed that the BSYS-containing serum markedly increased cell viability and increased the levels of growth associated protein (GAP)-43, phosphorylated (p)-cAMP-response element binding protein (CREB), BDNF, TrkB, and p-PI3K. The BS and HTHX treatments also induced the protein expression of GAP-43 and p-extracellular signal-regulated kinase (ERK) in the cells. Furthermore, the effects of BSYS on cell viability, GAP-43, p-CREB, and neurite outgrowth were clearly inhibited by LY294002, a specific antagonist of the PI3K signaling pathways. The addition of U0126 and U73122, antagonists of the ERK and PLCγ pathway, respectively, significantly inhibited cell viability and GAP-43 protein expression. Moreover, BSYS treatment significantly increased the expression of the 68-, 160-, and 200-kDa neurofilaments (NFs) of proteins and the BDNF, TrkB, PI3K, and Akt mRNA and proteins in the brain or spinal cord of mice at different stages. These results indicated that BSYS promotes nerve regeneration, and its mechanism is mainly related to the upregulation of the BDNF/TrkB and PI3K/Akt signaling pathways. BS and HTHX also promoted nerve regeneration, and this effect involved the ERK pathway.