Buyang Huanwu decoction alleviates oxidative injury of cerebral ischemia-reperfusion through PKCε/Nrf2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ischemic stroke is a significant risk factor for human health, and Buyang Huanwu Decoction is a classical and famous Chinese formula for treating it, but without clear pharmacological mechanism. AIM OF THE STUDY: The aim of this study was to investigate that the molecular mechanism of BYHWD activation of the PKCε/Nrf2 signaling pathway to attenuate cerebral ischemia-reperfusion (I/R) oxidative damage. MATERIALS AND METHODS: The MCAO method was used to establish a brain I/R injury model in SD rats, and neurological deficits were evaluated by neurological function score. Neuronal damage was observed by Nissl staining and immunofluorescence detection of MAP2 expression. Oxidative damage was observed by ROS, SOD, GSH-PX, MDA, and 8-OHdG. Changes in mitochondrial membrane potential were detected by using the fluorescent probe JC-1. The Western blot analysis detected protein expression of PKCε, P-PKCε, total Nrf2, nuclear Nrf2, HO-1, and NQO1. RESULTS: BYHWD significantly enhanced neural function, reduced neuronal damage, inhibited the production of ROS, decreased MDA and 8-OHdG levels, increased SOD and GSH-PX activity to reduce oxidative damage, and restored mitochondrial membrane potential. BYHWD and Nrf2 activator TBHQ increased total Nrf2, nucleus Nrf2 protein expression, and its downstream HO-1 and NQO1 proteins, and the administration of the Nrf2 inhibitor brusatol reduced the enhancing effect of BYHWD. Meanwhile, BYHWD increased the expression of PKCε and P-PKCε and the administration of the PKCε inhibitor εV1-2 reduced the effect of BYHWD in increasing the expression of PKCε, P-PKCε, nuclear Nrf2, and HO-1, as well as promoting the effect of Nrf2 translocation to the nucleus. CONCLUSION: This study marks the first to demonstrate that BYHWD ameliorates oxidative damage and attenuates brain I/R injury by activating the PKCε/Nrf2/HO-1 pathway.

Protective Effect of Buyang Huanwu Decoction on Cerebral Ischemia Reperfusion Injury by Alleviating Autophagy in the Ischemic Penumbra.

OBJECTIVES: To evaluate the protective effect of Buyang Huanwu Decoction (BHD) against cerebral ischemia reperfusion and investigate whether autophagy is involved in its mechanism of action. METHODS: Adult male Sprague Dawley rats were randomly divided into three groups: the sham, cerebral ischemia reperfusion (I/R), and I/R + BHD groups. A rat model of cerebral I/R injury was established via middle cerebral artery occlusion (MCAO) for 2 h, followed by 1, 3, and 7 d of reperfusion. Neurological scores and regional cerebral blood flow were assessed to determine whether the model was successfully established. Brain infarct volume was determined by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. The apoptosis rate was detected using TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, and neuronal damage was evaluated by Nissl staining. The Beclin-1 and LC3 protein levels in the ischemic core, penumbra, and contralateral area were analysed by Western blotting. The occurrence of autophagy in the penumbra was observed by transmission electron microscopy (TEM). RESULTS: BHD treatment alleviated the cerebral infarct volume, neuronal apoptosis rate, and neuronal damage 3 and 7 d after cerebral I/R injury. Furthermore, 3 d after reperfusion, we observed that the Beclin-1 levels were significantly decreased in the core in the I/R group, whereas transformation of LC3 I to LC3 II exhibited no obvious differences between the sham and I/R groups. In the penumbra, the Beclin-1 levels and transformation of LC3 I to LC3 II in the I/R group were significantly increased compared with that in the sham group. However, no significant difference in the contralateral area was noted between the two groups. BHD significantly inhibited the expression of Beclin-1 and the transformation of LC3 I to LC3 II in the penumbra after cerebral I/R injury but yielded no significant changes in the core and contralateral area. CONCLUSIONS: BHD exerts a neuroprotective effect by inhibiting autophagy in neurons in the penumbra after cerebral I/R injury.

[Effect of active component compound of Epimedii Folium,Astragali Radix,and Puerariae Lobatae Radix on expression of ADAM17 in HT22 cells by mediating hepcidin].

Alzheimer's disease(AD) patients in China have been surging, and the resultant medical burden and care demand have a huge impact on the development of individuals, families, and the society. The active component compound of Epimedii Folium, Astragali Radix, and Puerariae Lobatae Radix(YHG) can regulate the expression of iron metabolism-related proteins to inhibit brain iron overload and relieve hypofunction of central nervous system in AD patients. Hepcidin is an important target regulating iron metabolism. This study investigated the effect of YHG on the expression of a disintegrin and metalloprotease-17(ADAM17), a key enzyme in the hydrolysis of ß amyloid precursor protein(APP) in HT22 cells, by mediating hepcidin. To be specific, HT22 cells were cultured in vitro, followed by liposome-mediated siRNA transfection to silence the expression of hepcidin. Real-time PCR and Western blot were performed to examine the silencing result and the effect of YHG on hepcidin in AD cell model. HT22 cells were randomized into 7 groups: control group, Aß25-35 induction(Aß) group, hepcidin-siRNA(siRNA) group, Aß25-35 + hepcidin-siRNA(Aß + siRNA) group, Aß25-35+YHG(Aß+YHG) group, hepcidin-siRNA+YHG(siRNA+YHG) group, Aß25-35+hepcidin-siRNA+YHG(Aß+siRNA+YHG) group. The expression of ADAM17 mRNA in cells was detected by real-time PCR, and the expression of ADAM17 protein by immunofluorescence and Western blot. Immunofluorescence showed that the ADAM17 protein expression was lower in the Aß group, siRNA group, and Aß+siRNA group than in the control group(P<0.05) and the expression was lower in the Aß+siRNA group(P<0.05) and higher in the Aß+YHG group(P<0.05) than in the Aß group. Moreover, the ADAM17 protein expression was lower in the Aß+siRNA group(P<0.05) and higher in the siRNA+YHG group(P< 0.05) than in the siRNA group. The expression was higher in the Aß+siRNA+YHG group than in the Aß+siRNA group(P<0.05). The results of Western blot and real-time PCR were consistent with those of immunofluorescence. The experiment showed that YHG induced hepcidin to up-regulate the expression of ADAM17 in AD cell model and promote the activation of non-starch metabolic pathways, which might be the internal mechanism of YHG in preventing and treating AD.

Network pharmacology and the experimental findings of Bushenhuoxue formula for improving hippocampal neuron injury in vascular demented rats.

We used network pharmacology to predict the correlation between the pathway of Bushenhuoxue formula in the treatment of vascular dementia and carried out experiments to verify the correlation between drug composition and disease. By screening the active components and key targets through various databases and drawing the topological network diagram, we obtained 502 effective compound targets, 601 disease targets, 95 disease-related compound targets, and 162 pathways. The pathway related to vascular dementia may be neurodegeneration-multiple diseases, PI3K-Akt signaling pathway, Mitogen-activated protein kinase signaling pathway, or HIF-1 signaling pathway. By detecting the learning and memory ability of vascular dementia rats, the morphology of the hippocampus under the electron microscope, the degree of neuronal damage, and autophagy-related proteins, the results showed that the Bushenhuoxue formula could improve the neuronal injury induced by ischemia in the hippocampus, down-regulate the level of autophagy, and thereby improve learning and memory. Therefore, the Bushenhuoxue formula may improve the ischemic injury of neurons by regulating the mechanism of neuronal autophagy.

A Network Pharmacology-Based Identification Study on the Mechanism of Xiao-Xu-Ming Decoction for Cerebral Ischemic Stroke.

OBJECTIVE: We used the network pharmacological analysis method to explore the mechanism of multicomponent, multitarget, and multiway actions of Xiao-Xu-Ming decoction (XXMD) for cerebral ischemic stroke (CIS), which provided a basis on the research of innovative drugs. METHOD: We used the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) to retrieve the active ingredients and targets of 12 herbs of XXMD; we used the Gene Expression Omnibus (GEO) database of the National Center for Biotechnology Information (NCBI) to screen for differentially expressed genes in CIS to obtain the disease targets of CIS and to intersect it with the action targets of XXMD, and then the target drug efficacy is obtained. We used Cytoscape 3.6 software to construct the drug-active ingredient-action target interaction network of XXMD to treat CIS and conduct protein-protein interaction (PPI) network and topology analysis. The action target Gene Ontology (GO) biological processes and metabolic pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG) of XXMD to treat CIS were enrichment analyzed with R software. RESULT: We screened out 226 active ingredients and 3646 action targets for XXMD. Among them, XXMD to treat CIS has 144 active ingredients, 12 targets, and proteins in the core network of PPI having STAT3, HIF1A, etc. Pathway enrichment analysis was based on the GO and KEGG biological processes involved in active oxygen metabolism, smooth muscle cell proliferation, cytokine production, angiogenesis, redox coenzyme metabolism, and oxidative stress. The main action processes are significantly associated with CIS signal pathways involved in microRNAs, ovarian steroid hormones, NF-кB signaling pathway, Th17 cell differentiation pathway, HIF-1 signaling pathway, folic acid synthesis pathway, galactose metabolism, and fructose and mannose metabolism. CONCLUSION: This study initially clarified the main targets and pathways of XXMD in the treatment of CIS, which can lay the foundation for further research on its pharmacological effects.

Bushenhuoxue improves cognitive function and activates brain-derived neurotrophic factor-mediated signaling in a rat model of vascular dementia.

OBJECTIVE: To explore the protective mechanisms of the Traditional Chinese Medicine Bushenhuoxue (BSHX) in a rat model of vascular dementia (VD). METHODS: A rat model of VD was developed using bilateral common carotid artery occlusion (BCCAO). Rats were administered BSHX (10.14 or 5.07 g/kg), nimodipine (11.06 mg/kg; positive control), or saline (control) by gavage daily for 30 d post-surgery. Learning and memory abilities were assessed using the Morris water maze. Morphological changes in the hippocampus were observed using light microscopy (hematoxylin and eosin staining) and transmission electron microscopy (TEM). The mRNA and protein expression levels of brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase B (TrkB), phosphatidyl inositol 3-kinase (PI3K), serine/threonine kinase (AKT), and cAMP response element binding protein (CREB) were measured by real-time polymerase chain reaction (RT-PCR) and Western blot, respectively. RESULTS: Compared with the sham group, rats with BCCAO exhibited impaired learning and memory abilities (Morris water maze) and showed abnormalities in neuronal morphology (light microscopy) and ultrastructure (TEM) in the hippocampus. They also had decreased mRNA and protein expressions of BDNF, TrkB, PI3K, AKT, and CREB in hippocampal tissue (all P < 0.05). In rats with BCCAO, administration of BSHX attenuated deficits in learning and memory, improved the morphology and ultrastructure of hippocampal neurons, and enhanced mRNA and protein expression levels of BDNF, TrkB, PI3K, AKT, and CREB (all P < 0.05). CONCLUSION: BSHX may protect hippocampal neurons and improve learning and memory abilities, at least in part via the activation of BDNF/TrkB/PI3K/AKT/CREB signaling.

Application frequency and therapeutic effect of Astragalus in treating ischemic cerebrovascular disease.

In recent years, Traditional Chinese Medicine (TCM) therapy has achieved substantial progress and development, playing an important role in clinical practice. In this paper, to explore the therapeutic effect of Astragalus in treating ischemic cerebrovascular disease, 2687 patients with ischemic cerebrovascular disease who had been treated with Astragalus therapy from Jan 2014-Oct 2015 were selected as the research objects. Through retrospective analysis of their medical records, we analyzed the application frequency of Astragalus using quantitative method of information, and conducted specific analysis on the therapeutic effect of Astragalus after fully surveyed patients' physical conditions after treatment. Results show that in the treatment of ischemic cerebrovascular disease by TCM, Astragalus is of high application frequency and sound therapeutic effect, which is deserved to be applied and promoted in clinical practices.

Neuroprotective effect of the active components of three Chinese herbs on brain iron load in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative brain disorder and the most common cause of dementia. New treatments for AD are required due to its increasing prevalence in aging populations. The present study evaluated the effects of the active components of Epimedium, Astragalus and Radix Puerariae on learning and memory impairment, ß-amyloid (Aß) reduction and brain iron load in an APPswe/PS1ΔE9 transgenic mouse model of AD. Increasing evidence indicates that a disturbance of normal iron homeostasis may contribute to the pathology of AD. However, the underlying mechanisms resulting in abnormal iron load in the AD brain remain unclear. It has been hypothesized that the brain iron load is influenced by the deregulation of certain proteins associated with brain iron metabolism, including divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1). The present study investigated the effects of the active components of Epimedium, Astragalus and Radix Puerariae on the expression levels of DMT1 and FPN1. The treatment with the active components reduced cognitive deficits, inhibited Aß plaque accumulation, reversed Aß burden and reduced the brain iron load in AD model mice. A significant increase was observed in the levels of DMT1-iron-responsive element (IRE) and DMT1-nonIRE in the hippocampus of the AD mouse brain, which was reduced by treatment with the active components. In addition, the levels of FPN1 were significantly reduced in the hippocampus of the AD mouse brain compared with those of control mice, and these levels were increased following treatment with the active components. Thus, the present study indicated that the active components of Epimedium, Astragalus and Radix Puerariae may exert a neuroprotective effect against AD by reducing iron overload in the AD brain and may provide a novel approach for the development of drugs for the treatment of AD.