Icariin ameliorates the cuprizone-induced demyelination associated with antioxidation and anti-inflammation.

The treatment of immunomodulation in multiple sclerosis (MS) can alleviate the severity and relapses. However, it cannot improve the neurological disability of patients due to a lack of myelin protection and regeneration. Therefore, remyelinating therapies may be one of the feasible strategies that can prevent axonal degeneration and restore neurological disability. Natural product icariin (ICA) is a flavonol compound extracted from epimedium flavonoids, which has neuroprotective effects in several models of neurological diseases. Here, we attempt to explore whether ICA has the potential to treat demyelination and its possible mechanisms of action using lipopolysaccharide-treated BV2 microglia, primary microglia, bone marrow-derived macrophages, and cuprizone-induced demyelination model. The indicators of oxidative stress and inflammatory response were evaluated using commercial kits. The results showed that ICA significantly reduced the levels of oxidative intermediates nitric oxide, hydrogen peroxide, malondialdehyde, and inflammatory cytokines TNF-α, IL-1ß, and increased the levels of antioxidants superoxide dismutase, catalase, glutathione peroxidase, and anti-inflammatory cytokines IL-10 and TGF-ß in vitro cell experiments. In vivo demyelination model, ICA significantly alleviated the behavioral abnormalities and enhanced the integrated optical density/mm2 of Black Gold II and myelin basic protein myelin staining, accompanied by the inhibition of oxidative stress/inflammatory response. Immunohistochemical staining showed that ICA significantly induced the expression of nuclear factor erythroid derived 2/heme oxygenase-1 (Nrf2/HO-1) and inhibited the expression of toll-like receptor 4/ nuclear factor kappa B (TLR4/NF-κB), which are two key signaling pathways in antioxidant and anti-inflammatory processes. Our results strongly suggest that ICA may be used as a potential agent to treat demyelination via regulating Nrf2/HO-1-mediated antioxidative stress and TLR4/NF-κB-mediated inflammatory responses.

Efficacy and mechanism of Wuzi Yanzong pill on the prevention and treatment of EAE.

Objective: Studies have shown that Wuzi Yanzong Pill (WYP) can be used to treat neurological diseases, but its mechanisms for multiple sclerosis (MS) remain unclear. This study aims to determine the effect of WYP on MS in an animal model of experimental autoimmune encephalomyelitis (EAE), and explore its mechanism. To provide theoretical basis for the clinical treatment of MS with WYP. Methods: C57BL/6 female mice were randomly divided into Blank control, EAE control, low dose WYP, medium dose WYP, and high dose WYP groups. One week before model generation, the mice were gavaged with saline (50 mL/kg/d) in Blank control and EAE control groups. The treatment groups was gavaged with different doses of WYP solution (4, 8, or 16 g/kg/d respectively) Clinical scores were recorded daily. Sample collection was conducted on the 14th and 28th days, respectively The expressions of IL-10, IL-17, IL-12, TNF-α and IFN-γ in spleen were detected by ELISA. The expressions of ROCKII, P-MYPT1, TLR4, NF-κB/p65, MCP-1, CCR2 in spleen, brain and spinal cord were detected by Western Blot. The types of macrophages and the contents of intracellular IL-10 and IL-12 were detected by Flow Cytometry. The contents of TNF-α and TLR4 mRNA in the spleen were detected by RT-PCR. Results: WYP treatment improved the clinical score of EAE mice in a significant dose-dependent manner, with the WYP high-dose group showed the most significant improvement in clinical score. Compared with the EAE control group, WYP high dose group had significantly lower levels of IL-17, IFN-γ, ROCKII, P-MYPT1, TLR4, NF-κB/p65, MCP-1, and CCR2 as well as TNF-α and TLR4 mRNA, but increased the number of M2 macrophages and IL-10. Conclusion: WYP treatment relieves clinical symptoms in EAE mice, which may be related to regulate inflammatory pathway and inhibiting expressions of inflammatory cytokines.

[Mechanism of bilobalide promoting neuroprotection of macrophages].

This study aims to explore the neuroprotective effect of bilobalide(BB) and the mechanisms such as inhibiting inflammatory response in macrophage/microglia, promoting neurotrophic factor secretion, and interfering with the activation and differentiation of peripheral CD4~+ T cells. BB of different concentration(12.5, 25, 50, 100 µg·mL~(-1)) was used to treat the RAW264.7 and BV2 cells for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and cell counting kit-8(CCK-8) were employed to detect the cytotoxicity of BB and appropriate concentration was selected for further experiment. Lipopolysaccharide(LPS) was applied to elicit inflammation in RAW264.7 and BV2 cells, mouse bone marrow-derived macrophages(BMDMs), and primary microglia, respectively. The effect of BB on cell proliferation and secretion of inflammatory cytokines and neurotrophic factors was detected by enzyme-linked immunosorbent assay(ELISA). Spleen monocytes of C57BL/6 female mice(7-8 weeks old) were isolated, and CD4~+ T cells were separated by magnetic beads under sterile conditions. Th17 cells were induced by CD3/CD28 and the conditioned medium for eliciting the inflammation in BMDMs. The content of IL-17 cytokines in the supernatant was detected by ELISA to determine the effect on the activation and differentiation of CD4~+ T cells. In addition, PC12 cells were incubated with the conditioned medium for eliciting inflammation in BMDMs and primary microglia and the count and morphology of cells were observed. The cytoto-xicity was determined by lactate dehydrogenase(LDH) assay. The result showed that BB with the concentration of 12.5-100 µg·mL~(-1) had no toxicity to RAW264.7 and BV2 cells, and had no significant effect on the activity of cell model with low inflammation. The 50 µg·mL~(-1) BB was selected for further experiment, and the results indicated that BB inhibited LPS-induced secretion of inflammatory cytokines. The experiment on CD4~+ T cells showed that the conditioned medium for LPS-induced inflammation in BMDMs promoted the activation and differentiation of CD4~+ T cells, while the conditioned medium of the experimental group with BB intervention reduced the activation and differentiation of CD4~+ T cells. In addition, BB also enhanced the release of neurotrophic factors from BMDMs and primary microglia. The conditioned medium after BB intervention can significantly reduce the death of PC12 neurons, inhibit neuronal damage, and protect neurons. To sum up, BB plays a neuroprotective role by inhibiting macrophage and microglia-mediated inflammatory response and promoting neurotrophic factors.

Hydroxyfasudil regulates immune balance and suppresses inflammatory responses in the treatment of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a central nervous system (CNS) disease with complicated etiology. Multifocal demyelination and invasion of inflammatory cells are its primary pathological features. Fasudil has been confirmed to improve experimental autoimmune encephalomyelitis (EAE), an animal model of MS. However, Fasudil is accompanied by several shortcomings in the clinical practice. Hydroxyfasudil is a metabolite of Fasudil in the body with better pharmaceutical properties. Therefore, we attempted to study the influence of Hydroxyfasudil upon EAE mice. The results demonstrated that Hydroxyfasudil relieved the symptoms of EAE and the associated pathological damage, reduced the adhesion molecules and chemokines, decreased the invasion of peripheral immune cells. Simultaneously, Hydroxyfasudil modified the rebalance of peripheral T cells. Moreover, Hydroxyfasudil shifted the M1 phenotype to M2 polarization, inhibited inflammatory signaling cascades as well as inflammatory factors, and promoted anti-inflammatory factors in the CNS. In the end, mice in the Hydroxyfasudil group expressed more tight junction proteins, indirectly indicating that the blood-brain barrier (BBB) was protected. Our results indicate that Hydroxyfasudil may be a prospective treatment for MS.

Wuzi Yanzong Pill relieves MPTP-induced motor dysfunction and neuron loss by inhibiting NLRP3 inflammasome-mediated neuroinflammation.

Parkinson disease (PD) is an age-related neurodegenerative disease, which is associated with the loss of dopaminergic neurons (DA neurons) in the substantia nigra pars compacta (SNpc), and neuroinflammation may lead to the occurrence of PD. Wuzi Yanzong Pill (WYP) has demonstrated neuroprotective and anti-inflammatory properties, but its molecular mechanism of action is still unclear. In this study, we used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice and LPS-mediated BV2 microglia to explore WYP intervention, anti-inflammatory effect and molecular mechanism in vivo and in vitro. The results showed that oral administration of WYP in MPTP-induced PD mice for 2 weeks ameliorated abnormal motor dysfunction, attenuated the loss of TH + neurons in SNpc, protected dopaminergic neurons, and inhibited the activation of microglia in MPTP-induced PD mice and LPS-stimulated BV2 cell. Meanwhile, WYP intervention inhibited the expression of IL-6, TNF-α, Pro-IL-1ß, IL-1ß, Pro-IL-18, IL-18 and enhanced the expression of IL-10 in the SNpc of PD mice. Simultaneously, WYP intervention inhibited the expression of NLRP3 inflammasome, accompanied by the decrease of the TLR4/MyD88/NF-κB pathway. However, the exact target and interaction of WYP on NLRP3 inflammasome and TLR4/MyD88/NF-κB pathway still needs to be further investigated.

Grape Seed Extract Attenuates Demyelination in Experimental Autoimmune Encephalomyelitis Mice by Inhibiting Inflammatory Response of Immune Cells.

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 ß (IL-1 ß), 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 ß and IL-6 in bone marrow-derived macrophages stimulated by lipopolysaccharide (P<0.01), inhibited the secretion of TNF-α, IL-1 ß, 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.

Wuzi Yanzong Pill Plays A Neuroprotective Role in Parkinson's Disease Mice via Regulating Unfolded Protein Response Mediated by Endoplasmic Reticulum Stress.

OBJECTIVE: To investigate the protective effects and its possible mechanism of Wuzi Yanzong Pill (WYP) on Parkinson's disease (PD) model mice. METHODS: Thirty-six C57BL/6 male mice were randomly assigned to 3 groups including normal, PD, and PD+WYP groups, 12 mice in each group. One week of intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to establish the classical PD model in mice. Meanwhile, mice in the PD+WYP group were administrated with 16 g/kg WYP, twice daily by gavage. After 14 days of administration, gait test, open field test and pole test were measured to evaluate the movement function. Tyrosine hydroxylase (TH) neurons in substantia nigra of midbrain and binding immunoglobulin heavy chain protein (GRP78) in striatum and cortex were observed by immunohistochemistry. The levels of TH, GRP78, p-PERK, p-eIF2α, ATF4, p-IRE1α, XBP1, ATF6, CHOP, ASK1, p-JNK, Caspase-12, -9 and -3 in brain were detected by Western blot. RESULTS: Compared with the PD group, WYP treatment ameliorated gait balance ability in PD mice (P<0.05). Similarly, WYP increased the total distance and average speed (P<0.05 or P<0.01), reduced rest time and pole time (P<0.05). Moreover, WYP significantly increased TH positive cells (P<0.01). Immunofluorescence showed WYP attenuated the levels of GRP78 in striatum and cortex. Meanwhile, WYP treatment significantly decreased the protein expressions of GRP78, p-PERK, p-eIF2α, ATF4, p-IRE1 α, XBP1, CHOP, Caspase-12 and Caspase-9 (P<0.05 or P<0.01). CONCLUSIONS: WYP ameliorated motor symptoms and pathological lesion of PD mice, which may be related to the regulation of unfolded protein response-mediated signaling pathway and inhibiting the endoplasmic reticulum stress-mediated neuronal apoptosis pathway.

The effects and potential of microglial polarization and crosstalk with other cells of the central nervous system in the treatment of Alzheimer's disease.

Microglia are resident immune cells in the central nervous system. During the pathogenesis of Alzheimer's disease, stimulatory factors continuously act on the microglia causing abnormal activation and unbalanced phenotypic changes; these events have become a significant and promising area of research. In this review, we summarize the effects of microglial polarization and crosstalk with other cells in the central nervous system in the treatment of Alzheimer's disease. Our literature search found that phenotypic changes occur continuously in Alzheimer's disease and that microglia exhibit extensive crosstalk with astrocytes, oligodendrocytes, neurons, and penetrated peripheral innate immune cells via specific signaling pathways and cytokines. Collectively, unlike previous efforts to modulate microglial phenotypes at a single level, targeting the phenotypes of microglia and the crosstalk with other cells in the central nervous system may be more effective in reducing inflammation in the central nervous system in Alzheimer's disease. This would establish a theoretical basis for reducing neuronal death from central nervous system inflammation and provide an appropriate environment to promote neuronal regeneration in the treatment of Alzheimer's disease.

Cellular and Molecular Mechanisms Underly the Combined Treatment of Fasudil and Bone Marrow Derived-Neuronal Stem Cells in a Parkinson's Disease Mouse Model.

Bone marrow-derived neural stem cells (BM-NSCs) have shed light on novel therapeutic approaches for PD with the potential to halt or even reverse disease progression. Various strategies have been developed to promote therapeutic efficacy via optimizing implanted cells and the microenvironment of transplantation in the central nervous system (CNS). This current study further proved that the combination of fasudil, a Rho-kinase inhibitor, and BM-NSCs exhibited a synergetic effect on restoring neuron loss in the MPTP-PD mice model. It simultaneously unveiled cellular mechanisms underlying synergistic neuron-protection effects of fasudil and BM-NSCs, which included promoting the proliferation, and migration of endogenous NSCs, and contributing to microglia shift into the M2 phenotype. Corresponding molecular mechanisms were observed, including the inhibition of inflammatory responses, the elevation of neurotrophic factors, and the induction of WNT/ß-catenin and PI3K/Akt/mTOR signaling pathways. Our study provides evidence for the co-intervention of BM-NSCs and fasudil as a promising therapeutic method with enhanced efficacy in treating neurodegenerative diseases.

Neuroprotective effect of hyperoside in MPP+/MPTP -induced dopaminergic neurodegeneration.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the pathological loss of nigrostriatal dopaminergic neurons, which causes an insufficient release of dopamine (DA) and then induces motor and nonmotor symptoms. Hyperoside (HYP) is a lignan component with anti-inflammatory, antioxidant, and neuroprotective effects. In this study, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active neurotoxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) were used to induce dopaminergic neurodegeneration. The results showed that HYP (100 µg/mL) reduced MPTP-mediated cytotoxicity of SH-SY5Y cells in vitro, and HYP [25 mg/(kg d)] alleviated MPTP-induced motor symptoms in vivo. HYP treatment reduced the contents of nitric oxide (NO), H2O2, and malondialdehyde (MDA), as well as the mitochondrial damage of dopaminergic neurons, both in vitro and in vivo. Meanwhile, HYP treatment elevated the levels of neurotrophic factors such as glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, and recombinant cerebral dopamine neurotrophic factor in vivo, but not in vitro. Finally, Akt signaling was activated after the administration of HYP in MPP+/MPTP-induced dopaminergic neurodegeneration. However, the blockage of the Akt pathway with Akt inhibitor did not abolish the neuroprotective effect of HYP on DA neurons. These results showed that HYP protected the dopaminergic neurons from the MPP+- and MPTP-induced injuries, which did not rely on the Akt pathway.