[Tetramethylpyrazine regulates angiogenesis of endothelial cells in cerebral ischemic stroke injury via SIRT1/VEGFA signaling pathway].

This study aims to investigate whether tetramethylpyrazine(TMP) can stimulate angiogenesis in cerebral microvascular endothelial cells and alleviate cerebral ischemic stroke(CIS) and to explore the underlying mechanisms. In the animal study, adult Sprague-Dawley rats(n=15) were assigned into sham surgery(sham), middle cerebral artery occlusion/reperfusion(MCAO/R), and MCAO/R+TMP(intraperitoneal injection of 20 mg·kg~(-1)) groups. The neurological function was evaluated by the Z-Longa method. The cerebral infarction volume was detected by TTC staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the expression of vascular endothelial growth factor(VEGF), angiopoietin(Ang), and platelet-derived growth factor(PDGF). Immunofluorescence staining was employed to detect Ki67 and the expression of vascular endothelial growth factor A(VEGFA) and slient information regulator 1(SIRT1). Western blot was employed to determine the expression levels of VEGFA, SIRT1, angiopoietin-2(Ang-2), and platelet-derived growth factor B(PDGFB). In the cell study, mouse brain-derived endothelial cells(Bend.3) were cultured, and the optimal concentration of TMP was determined. Then, VEGF, Ang, and PDGF were detected by ELISA after the addition of cabozantinib. Western blot was employed to measure the expression of VEGFA, Ang-2, and PDGFB. Immunofluorescence staining was used to detect CD31, CD34, and Ki67, and the proliferation, migration, and tube formation ability of Bend.3 cells were observed in vitro. Western blot and immunofluorescence staining were performed to measure the expression of SIRT1 and VEGFA after addition of the SIRT1-specific inhibitor selisistat(EX-527). The results showed that compared with the sham group, the MCAO/R group had severe neurological function damage, increased infarction volume, up-regulated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, and PDGFB, and down-regulated expression of Ki67 and SIRT1(P<0.01). Compared with the MCAO/R group, the MCAO/R+TMP group presented alleviated neurological function damage, reduced infarction volume, and activated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, Ki67, and SIRT1(P<0.01). The cell experiments showed that compared with the normal group, Bend.3 cells were activated by oxygen glucose deprivation/reoxygenation(OGD/R) treatment(P<0.05, P<0.01). Compared with the OGD/R group, the OGD/R+TMP group upregulated the expression levels of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, SIRT1, Ki67, CD31, and CD34, enhanced the angiogenic ability of Bend.3 cells without being inhibited by BMS or EX-527(P<0.05, P<0.01, P<0.001). The results suggest that TMP can activate the SIRT1/VEGFA signaling pathway to stimulate angiogenesis and alleviate CIS injury.

Priming and Combined Strategies for the Application of Mesenchymal Stem Cells in Ischemic Stroke: A Promising Approach.

Ischemic stroke (IS) is a leading cause of death and disability worldwide. Tissue plasminogen activator (tPA) administration and mechanical thrombectomy are the main treatments but have a narrow time window. Mesenchymal stem cells (MSCs), which are easily scalable in vitro and lack ethical concerns, possess the potential to differentiate into various types of cells and secrete a great number of growth factors for neuroprotection and regeneration. Moreover, MSCs have low immunogenicity and tumorigenic properties, showing safety and preliminary efficacy both in preclinical studies and clinical trials of IS. However, it is unlikely that MSC treatment alone will be sufficient to maximize recovery due to the low survival rate of transplanted cells and various mechanisms of ischemic brain damage in the different stages of IS. Preconditioning was used to facilitate the homing, survival, and secretion ability of the grafted MSCs in the ischemic region, while combination therapies are alternatives that can maximize the treatment effects, focusing on multiple therapeutic targets to promote stroke recovery. In this case, the combination therapy can yield a synergistic effect. In this review, we summarize the type of MSCs, preconditioning methods, and combined strategies as well as their therapeutic mechanism in the treatment of IS to accelerate the transformation from basic research to clinical application.

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.

Bibliometric Analysis of Stem Cells in Ischemic Stroke (2001-2022): Trends, Hotspots and Prospects.

Background: Ischemic stroke is a common cerebrovascular accident with a high risk of neurological deficits. Stem cell therapy has progressively attracted the interest of scientists and clinicians due to the benefits of promoting neural regeneration and regulating the microenvironment surrounding the lesion after ischemic stroke. Our study aimed to evaluate the development trends and research hotspots in the field of stem cells and ischemic stroke. Materials and methods: Publications related to stem cells and ischemic stroke were retrieved from the Web of Science from 2001 to 2022. Data analysis and mapping were performed using VOSviewer, Citespace and ImageGP. Results: In total, 1932 papers were included in the analysis. Publications have steadily increased over the past 22 years. China has contributed the maximum number of publications, whereas the USA ranked first in the total number of citations and was considered the center of the international collaboration network. University of South Florida, Henry Ford Hospital, and Oakland University were the most influential institutions. Stroke, Brain Research, and Neural Regeneration Research were the most productive journals. The research in this field was primarily focused on the effects of stem cells on neurogenesis, inflammation, and angiogenesis following ischemic stroke, as well as their therapeutic potential for the disease. In addition, neural stem cells and mesenchymal stem cells are the most commonly utilized stem cells. The topics related to miRNA, extracellular vesicles, exosomes, mesenchymal stem cells, neuroinflammation, and autophagy are current research hotspots. Conclusion: Our bibliometric study provides a novel perspective on the research trends in the field of stem cells and ischemic stroke. The outcome of this study may benefit scientists to identify research hotspots and development directions, thereby advancing the application of stem cell-based therapy for ischemic stroke.

Investigation on Common Chronic Non-Communicable Diseases and Epidemiological Characteristics of Forsaken Elders Over 60 Years Old in Rural Areas of Datong, China.

Objective: The common chronic non-communicable diseases and epidemiological characteristics of the forsaken elders over 60 in Guangling and Tianzhen were investigated and analyzed to provide reference for health resource allocation, hospital capacity establishing and health management of the forsaken elders in county-level regions. Materials and Methods: The data of 10,331 resident elderly over 60 in Guangling and Tianzhen of Datong Civil Affairs Bureau in the management system for disabled and semi-disabled elderly was collected. The gender, age, main diagnosis and coding of diseases, common chronic non-communicable diseases, and system diseases of the respondents were retrospectively analyzed. Results: The prevalence of the forsaken elders aged over 60 in Guangling and Tianzhen were different. Hypertension, arthritis, type 2 diabetes, hyperlipidemia and cerebral infarction are the top five common chronic non infectious diseases in Guangling, Tianzhen and the two counties. Among the top five common diseases in Guangling, Tianzhen and the two counties, arthritis or rheumatism, hypertension, diabetes or elevated blood sugar were found, which were different in the 60-65, 66-70, 71-75 and 76-80 groups, with the prevalence increasing with age. The top five diseases in Guangling, Tianzhen and the two counties were consistent, while the ranking changed slightly. The proportion of circulatory diseases, musculoskeletal diseases, connective tissue diseases and endocrine/nutritional and metabolic diseases in 60-65, 66-70 and 71-75 groups increased with age, and was much higher than that in other groups. Conclusion: The prevalence and disease spectrum order of common chronic non-communicable diseases and systemic diseases in Guangling and Tianzhen are diverse, also in gender and age groups. As China's county-level local administrative divisions have relatively independent administrative autonomy, medical and health resources can be better configured according to the information mined, accurately maintaining and promoting residents' health. It is suggested to explore the disease management mechanism with county-level administrative divisions as database management units under the background of big data, so as to implement the interconnection and sharing of information among health-related departments in county-level regions.

Exploring Research Trend and Hotspots on Oxidative Stress in Ischemic Stroke (2001-2022): Insights from Bibliometric.

Oxidative stress is widely involved in the pathological process of ischemic stroke and ischemia-reperfusion. Several research have demonstrated that eliminating or reducing oxidative stress can alleviate the pathological changes of ischemic stroke. However, current clinical antioxidant treatment did not always perform as expected. This bibliometric research aims to identify research trends, topics, hotspots, and evolution on oxidative stress in the field of ischemic stroke, and to find potentially antioxidant strategies in future clinical treatment. Relevant publications were searched from the Web of Science (WOS) Core Collection databases (2001-2022). VOSviewer was used to visualize and analyze the development trends and hotspots. In the field of oxidative stress and ischemic stroke, the number of publications increased significantly from 2001 to 2022. China and the USA were the leading countries for publication output. The most prolific institutions were Stanford University. Journal of Cerebral Blood Flow and Metabolism and Stroke were the most cited journals. The research topics in this field include inflammation with oxidative stress, mitochondrial damage with oxidative stress, oxidative stress in reperfusion injury, oxidative stress in cognitive impairment and basic research and clinical translation of oxidative stress. Moreover, "NLRP3 inflammasome," "autophagy," "mitophagy," "miRNA," "ferroptosis," and "signaling pathway" are the emerging research hotspots in recent years. At present, multi-target regulation focusing on multi-mechanism crosstalk has progressed across this period, while challenges come from the transformation of basic research to clinical application. New detection technology and new nanomaterials are expected to integrate oxidative stress into the clinical treatment of ischemic stroke better.

[Eriodictyol improves cognitive function in 5×FAD mice of Alzheimer's disease by inhibiting the microglia NLRP3 inflammasome signaling pathway].

Objective To investigate the therapeutic effects of eriodictyol on transgenic mice with five familial Alzheimer's disease (5×FAD) and the modulation of NOD-like receptor-pyrin domain containing 3 (NLRP3) inflammasome in microglia. Methods The 8-month-old 5×FAD mice were randomly divided into AD model group and eriodictyol-treated AD group. Same-aged wild-type C57BL/6J mice were randomly divided into wild-type (WT) control group and eriodictyol-treated WT group. Morris water maze and Y-maze experiments were performed to assess the cognitive function of each group of mice. Immunofluorescence histochemical staining was performed to detect the expression of NLRP3, caspase-1 and interleukin 18 (IL-18) in mouse brain tissue, and Western blot was performed to detect the protein levels of NLRP3, apoptosis-associated speckle-like protein containing a CARD (ASC), caspase-1, IL-18, IL-1ß and ion calcium-binding adaptor molecule 1 (Iba-1) in mouse brain tissue. Results Compared with the WT group and the eriodictyol-treated WT group, cognitive function was significantly impaired in the AD group mice, and the expression of NLRP3, caspase-1, IL-18, ASC, IL-1ß and Iba1 were increased in microglia of mouse brain tissue. After eriodictyol treatment, learning memory and cognitive function were improved, and the expression of NLRP3, ASC, caspase-1, IL-1ß, IL-18 and Iba1 were all down-regulated in the eriodictyol-treated AD group mice compared with the AD group mice. Conclusion Eriodictyol may improve cognitive function in animal models of AD by inhibiting the activation of the NLRP3 signaling pathway in microglia.

Astrocytic phagocytosis of myelin debris and reactive characteristics in vivo and in vitro.

BACKGROUND INFORMATION: Persistent myelin debris can inhibit axonal regeneration, thereby hindering remyelination. Effective removal of myelin debris is essential to eliminate the interference of myelin debris in oligodendrocyte progenitor cell (OPC) activation, recruitment to demyelinating sites and/or differentiation into mature oligodendrocytes (OLs). In addition to microglia, it has been reported that astrocytic phagocytosis of myelin debris is a feature of early demyelination. RESULTS: In the present study, astrocytes effectively phagocytized myelin debris in vitro and in vivo. On the 5th day after injecting myelin debris into the brain, astrocytes were enriched in the area injected with myelin debris compared with microglia, and their ability to engulf myelin debris was stronger than that of microglia. When exposed to myelin debris, astrocytes phagocytizing myelin debris triggered self-apoptosis, accompanied by the activation of NF-κB, down-regulation of Nrf2, and the increase of ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (bFGF). However, the activation of astrocytic NF-κB did not influence the inflammatory cytokines IL-1ß, IL-6, and TNF-α, and the anti-inflammatory factor IL-10. The proliferation of astrocytes and mobilization of OPCs in the subventricular zone were elevated on the 5th day after intracerebral injection of myelin debris. CONCLUSIONS: The results suggested that myelin phagocytosis of astrocytes should help improve the microenvironment and promote myelin regeneration by increasing CNTF and bFGF within the central nervous system. SIGNIFICANCE: However, the molecular interaction of astrocytes acting as phagocytes remains to be further explored. Therefore, an improvement of astrocytes to phagocytize myelin debris may be a promising treatment measure to prevent demyelination and promote remyelination in MS and other diseases with prominent myelin injury.

[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.

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.

Astragaloside IV inhibits experimental autoimmune encephalomyelitis by modulating the polarization of both microglia/macrophages and astrocytes.

Astragaloside IV (AST IV), a major saponin component and active ingredient isolated from Astragalus membranaceus, has been well known to exhibit neuroprotective effects on diverse models of neurological diseases. Accumulating evidence suggests that dynamic balance of microglia/macrophages and astrocytes plays a vital role in neuroprotection and remyelination. However, dysregulation of microglia/macrophages and astrocytes orchestrate the pathogenesis of nervous system disorders. Therefore, we hypothesized that switching the transformation of microglia/macrophages and astrocytes into the neuroprotective M2 and A2 phenotypes, respectively, could be a potential target for therapeutic intervention. In the present study, we evaluate the efficacy of AST IV intervention on the effects of microglia/macrophages and astrocytes in an experimental autoimmune encephalomyelitis (EAE) model. AST IV improved paralysis and pathology of EAE by inhibiting the neurotoxic M1 microglia/macrophage phenotype, promoting M2 phenotype, shifting astrocytes towards a neuroprotective A2 phenotype, and protecting neurons from apoptosis through inhibition of TLR4/Myd88/NF-kB signalling pathway. Our study showed that AST IV could be a potential and promising drug for multiple sclerosis treatment.

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.

[Knock-down of ROCK2 gene improves cognitive function and reduces neuronal apoptosis in AD mice by promoting mitochondrial fusion and inhibiting its division].

Objective To explore the effect of knocking down Rho-associated coiled-coil kinase (ROCK2) gene on the cognitive function of amyloid precursor protein/presenilin-1 (APP/PS1) double transgenic mice and its mechanism. Methods APP/PS1 double transgenic mice were randomly divided into AD model group (AD group), ROCK2 gene knock-down group (shROCK2 group), ROCK2 gene knock-down control group (shNCgroup), and wild-type C57BL/6 mice of the same age served as the wild-type control (WT group). Morris water maze and Y maze were employed to test the cognitive function of mice. Neuron morphology was detected by Nissl staining. Immunofluorescence histochemical staining was used to detect the expression of phosphorylated dynamin-related protein 1 (p-Drp1) and mitochondrial fusion 1 (Mfn1). Western blot analysis was used to detect the expression ROCK2, cleaved-caspase-3 (c-caspase-3), B-cell lymphoma 2 (Bcl2), Bcl2-related protein X (BAX), p-Drp1, mitochondrial fission 1 (Fis1), optic atrophy 1 (OPA1), Mfn1 and Mfn2. Results Compared with AD group mice, the expression of ROCK2 in shROCK2 group mice was significantly reduced; the cognitive function was significantly improved with the number of neurons in the hippocampal CA3 and DG areas increasing, and nissl bodies were deeply stained; the expression of c-caspase-3 and BAX was decreased, while the expression of Bcl2 was increased; the expression of mitochondrial division related proteins p-Drp1 and Fis1 were decreased, while the expression of mitochondrial fusion-related proteins OPA1, Mfn1 and Mfn2 were increased. Conclusion Knock-down of ROCK2 gene can significantly improve the cognitive function and inhibit the apoptosis of nerve cells of APP/PS1 mice. The mechanism may be related to promoting mitochondrial fusion and inhibiting its division.

Preventing development of post-stroke hyperexcitability by optogenetic or pharmacological stimulation of cortical excitatory activity.

Stroke is the most common cause of acquired epilepsy, but treatment for preventing the development of post-stroke epilepsy is still unavailable. Since stroke results in neuronal damage and death as well as initial loss of activity in the affected brain region, homeostatic plasticity may be trigged and contribute to an increase in network hyperexcitability that underlies epileptogenesis. Correspondingly, enhancing brain activity may inhibit hyperexcitability from enhanced homeostatic plasticity and prevent post-stroke epileptogenesis. To test these hypotheses, we first used in vivo two-photon and mesoscopic imaging of activity of cortical pyramidal neurons in Thy1-GCaMP6 transgenic mice to determine longitudinal changes in excitatory activity after a photothrombotic ischemic stroke. At 3-days post-stroke, there was a significant loss of neuronal activity in the peri-injury area as indicated by reductions in the frequency of calcium spikes and percentage of active neurons, which recovered to baseline level at day 7, supporting a homeostatic activity regulation of the surviving neurons in the peri-injury area. We further used optogenetic stimulation to specifically stimulate activity of pyramidal neurons in the peri-injury area of Thy-1 channelrhodopsin transgenic mice from day 5 to day 15 after stroke. Using pentylenetetrazole test to evaluate seizure susceptibility, we showed that stroke mice are more susceptible to Racine stage V seizures (time latency 54.3 ± 12.9 min) compared to sham mice (107.1 ± 13.6 min), but optogenetic stimulation reversed the increase in seizure susceptibility (114.0 ± 9.2 min) in mice with stroke. Similarly, administration of D-cycloserine, a partial N-methyl-d-aspartate (NMDA) receptor agonist that can mildly enhance neuronal activity without causing post-stroke seizure, from day 5 to day 15 after a stroke significantly reversed the increase in seizure susceptibility. The treatment also resulted in an increased survival of glutamic acid decarboxylase 67 (GAD67) positive interneurons and a reduced activation of glial fibrillary acidic protein (GFAP) positive reactive astrocytes. Thus, this study supports the involvement of homeostatic activity regulation in the development of post-stroke hyperexcitability and potential application of activity enhancement as a novel strategy to prevent post-stroke late-onset seizure and epilepsy through regulating cortical homeostatic plasticity.

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.

Wuzi Yanzong Pill protects neural tube defects by activating PI3K/Akt signaling pathway.

Neural tube defects (NTDs) are severe congenital malformations that can lead to lifelong disability. Wuzi Yanzong Pill (WYP) is an herbal formula of traditional Chinese medicine (TCM) that has been shown to have a protective effect against NTDs in a rodent model induced by all-trans retinoic acid (atRA), but the mechanism remains unclear. In this study, the neuroprotective effect and mechanism of WYP on NTDs were investigated in vivo using an atRA-induced mouse model and in vitro using cell injury model induced by atRA in Chinese hamster ovary (CHO) cells and Chinese hamster dihydrofolate reductase-deficient (CHO/dhFr) cells. Our findings suggest that WYP has an excellent preventive effect on atRA-induced NTDs in mouse embryos, which may be related to the activation of the PI3K/Akt signaling pathway, improved embryonic antioxidant capacity, and anti-apoptotic effects, and this effect is not dependent on folic acid (FA). Our results demonstrated that WYP significantly reduced the incidence of NTDs induced by atRA; increased the activity of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and content of glutathione (GSH); decreased the apoptosis of neural tube cells; up-regulated the expression of phosphatidylinositol 3 kinase (PI3K), phospho protein kinase B (p-Akt), nuclear factor erythroid-2 related factor (Nrf2), and b-cell lymphoma-2 (Bcl-2); and down-regulated the expression of bcl-2-associated X protein (Bax). Our in vitro studies suggested that the preventive effect of WYP on atRA-treated NTDs was independent of FA, which might be attributed to the herbal ingredients of WYP. The results suggest that WYP had an excellent prevention effect on atRA-induced NTDs mouse embryos, which may be independent of FA but related to the activation of the PI3K/Akt signaling pathway and improvement of embryonic antioxidant capacity and anti-apoptosis.

Fasudil alleviates cerebral ischemia­reperfusion injury by inhibiting inflammation and improving neurotrophic factor expression in rats.

The Rho kinase inhibitor fasudil exerts neuroprotective effects. We previously showed that fasudil can regulate M1/M2 microglia polarization and inhibit neuroinflammation. Here, the therapeutic effect of fasudil on cerebral ischemia­reperfusion (I/R) injury was investigated using the middle cerebral artery occlusion and reperfusion (MCAO/R) model in Sprague­Dawley rats. The effect of fasudil on the phenotype of microglia and neurotrophic factors in the I/R brain and its potential molecular mechanism was also explored. It was found that fasudil ameliorated neurological deficits, neuronal apoptosis, and inflammatory response in rats with cerebral I/R injury. Fasudil also promoted the polarization of microglia into the M2 phenotype, in turn promoting the secretion of neurotrophic factors. Furthermore, fasudil significantly inhibited the expression of TLR4 and NF­κB. These findings suggest that fasudil could inhibit the neuroinflammatory response and reduce brain injury after I/R injury by regulating the shift of microglia from an inflammatory M1 phenotype to an anti­inflammatory M2 phenotype, which may be related to the regulation of the TLR4/ NF­κB signal pathway.

An optimal combination of five main monomer components in Wuzi Yanzong Pill that prevents neural tube defects and reduces apoptosis and oxidative stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Wuzi Yanzong Pill (WYP) is a classic traditional Chinese medicine (TCM) formula that is used for reproductive system diseases. Previous studies showed that WYP had a preventive effect on the development of neural tube defects (NTDs) induced by all-trans retinoic acid (atRA) in mice. AIM OF THE STUDY: This study aimed to determine the optimal combination of main monomer components in WYP on preventing NTDs and to understand the underlying mechanism. MATERIALS AND METHODS: An optimal combination was made from five representative components in WYP including hyperoside, acteoside, schizandrol A, kaempferide and ellagic acid by orthogonal design method. In a mouse model of NTDs induced by intraperitoneal injection of atRA, pathological changes of neural tube tissues were observed by Hematoxylin & Eosin (HE) staining, neural tube epithelial cells apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), protein changes related to apoptosis, anti-apoptosis, and antioxidant factors were detected with Western blot. Potential targets and mechanisms of monomer compatibility group (MCG) acting on NTDs were analyzed by bioinformatics. RESULTS: Treatment with different combinations of WYP bioactive ingredients resulted in varying decreases in the incidence of NTDs in mice embryos. The combination of MCG15 (200 mg/kg of hyperoside, 100 mg/kg of acteoside, 10 mg/kg of schizandrol A, 100 mg/kg of kaempferide and 1 mg/kg of ellagic acid) showed the most significant reduction in NTD incidence. Mechanistically, MCG15 inhibited apoptosis and oxidative stress, as evidenced by reduced TUNEL-positive cells, downregulation of caspase-9, cleaved caspase-3, Bad, and Bax, and upregulation of Bcl-2, as well as decreased MDA and increased SOD, CAT, GSH, HO-1, and GPX1 levels. Bioinformatics analysis showed that MCG15 acted on the PI3K/Akt signaling pathway, which was confirmed by Western blot analysis showing increased expression of p-PI3K, p-Akt/Akt, and Nrf2 related indicators. CONCLUSION: We have identified an optimal combination of five bioactive components in WYP (MCG15) that prevented NTDs in mice embryos induced by atRA by activating the PI3K/Akt signaling pathway and inhibiting apoptosis and oxidative stress.

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.