The mechanisms of Huangqi Guizhi Wuwu decoction in treating ischaemic stroke based on network pharmacology and experiment verification.

CONTEXT: Huangqi Guizhi Wuwu Decoction (HGWD) is effective in treating ischaemic stroke (IS). However, its mechanism of action is still unclear. OBJECTIVE: Network pharmacology integrated with in vivo experiments were used to clarify the underlying mechanisms of HGWD for treating IS. MATERIALS AND METHODS: TCMSP, GeneCards, OMIM and STRING were used to retrieve and construct visual protein interaction networks for the key targets. The AutoDock tool was used for molecular docking between key targets and active compounds. The neuroprotective effect of HGWD were verified in a middle cerebral artery occlusion (MCAO) model rat. The Sprague-Dawley (SD) rats were divided into sham, model, low-dose (5 g/kg, i.g.), high-dose (20 g/kg, i.g.), and nimodipine (20 mg/kg, i.g.) groups once daily for 7 days. The neurological scores, brain infarct volumes, lipid peroxidation, inflammatory cytokines, Nissl bodies, apoptotic neurons, and signalling pathways were all investigated and evaluated in vivo. RESULTS: Network pharmacology identified 117 HGWD targets related to IS and 36 candidate compounds. GO and KEGG analyses showed that HGWD anti-IS effects were mainly associated with PI3K-Akt and HIF-1 signalling pathways. HGWD effectively reduced the cerebral infarct volumes (19.19%), the number of apoptotic neurons (16.78%), and the release of inflammatory cytokines, etc. in MCAO rats. Furthermore, HGWD decreased the levels of HIF-1A, VEGFA, Bax, cleaved caspase-3, p-MAPK1, and p-c-Jun while increasing the expression of p-PI3K, p-AKT1, and Bcl-2. DISCUSSION AND CONCLUSION: This study initially elucidated the mechanism of HGWD anti-IS, which contributed to the further promotion and secondary development of HGWD in clinical practice.

Buyang Huanwu Decoction improves synaptic plasticity of ischemic stroke by regulating the cAMP/PKA/CREB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ischemic stroke is an acute central nervous system disease that poses a threat to human health. It induces a series of severe pathological mechanisms, ultimately leading to neuronal cell death in the brain due to local ischemia and hypoxia. Buyang Huanwu decoction (BYHWD), as a representative formula for treating ischemic stroke, has shown good therapeutic effects in stroke patients. AIM OF THE STUDY: This study aimed to explore the mechanism of BYHWD in promoting neural remodeling after ischemic stroke from the perspective of neuronal synaptic plasticity, based on the cAMP/PKA/CREB signaling pathway. MATERIALS AND METHODS: A modified suture technique was employed to establish a rat model of MCAO. The rats were divided into sham, model, and BYHWD (20 g/kg) groups. After the corresponding intervention, rat brains from each group were collected. TMT quantitative proteomics technology was employed for the research. Following proteomics studies, we investigated the mechanism of BYHWD in the intervention of ischemic stroke through animal experiments and cell experiments. The experimental animals were divided into sham, model, and BYHWD (5 g/kg, 10 g/kg, and 20 g/kg) groups. Infarct volume and severity of brain injury were measured by TTC staining. HE staining was utilized to evaluate alterations in tissue morphology. The Golgi staining was used to observe changes in cell body, dendrites, and dendritic spines. Transmission electron microscopy was used to observe the ultrastructure of synapses in the cortex and hippocampus. TUNEL staining was conducted to identify apoptotic neurons. Meanwhile, a stable and reliable (OGD/R) SH-SY5Y cell model was established. The effect of BYHWD-containing serum on SH-SY5Y cell viability was measured by CCK-8 kit. The apoptosis situation of SH-SY5Y cells was determined by Annexin V-FITC/PI. Immunofluorescence was employed to measure the fluorescence intensity of synaptic-related factors Syt1, Psd95, and Syn1. Synaptic plasticity pathways were assessed by using RT-qPCR and Western blot to determine the expression levels of cAMP, Psd95, Prkacb, CREB1, p-CREB, BDNF, Shank2, Syn1, Syt1, Bcl-2, Bax mRNA and proteins. RESULTS: After treatment with BYHWD, notable alterations were detected in the signaling pathways linked to synaptic plasticity and the cAMP signaling pathway-related targets among the intervention targets. This trend of change was also reflected in other bioinformatics analyses, indicating the important role of synaptic plasticity changes before and after modeling and drug intervention. The results of vivo and vitro experiments showed that BYHWD improved local pathological changes, and reduced cerebral infarct volume, and neurological function scores in MCAO rats. It increased dendritic spine density, improved synaptic structural plasticity, and had a certain neuroprotective effect. BYHWD increased the postsynaptic membrane thickness, synaptic interface curvature, and synaptic quantity. 10% BYHWD-containing serum was determined as the optimal concentration for treatment. 10% BYHWD-containing serum significantly reduced the overall apoptotic rate of (OGD/R) SH-SY5Y cells. Immunofluorescence experiments demonstrated that 10% BYHWD-containing serum could improve synaptic plasticity and increase the relative expression levels of synaptic-related proteins Syt1, Psd95, and Syn1. BYHWD and decoction-containing serum upregulated the mRNA and protein expression levels in (OGD/R) SH-SY5Y cells and MCAO rats, suggesting its ability to improve damaged neuronal synaptic plasticity and enhance transmission efficiency, which might be achieved through the regulation of the cAMP/PKA/CREB pathway. CONCLUSIONS: This study may provide a basis for clinical medication by elucidating the underlying experimental evidence for the promotion of neural plasticity after ischemic stroke by BYHWD.

Identification of Active Ingredients of Huangqi Guizhi Wuwu Decoction for Promoting Nerve Function Recovery After Ischemic Stroke Using HT22 Live-Cell-Based Affinity Chromatography Combined with HPLC-MS/MS.

OBJECTIVE: The Chinese medicine Huangqi Guizhi Wuwu Decoction (HGWD) has been reported to improve the clinical symptoms and restore nerve function after ischemic stroke; however, its active ingredients are not well-determined. Therefore, this study aimed to investigate the bioactive compounds of HGWD and explore the possible mechanism of action. METHODS: The methods, including live HT22 cells, solid-phase extraction, and HPLC-MS/MS were utilized. The potential ingredients were identified through comparisons with literature and monomer compounds. Then, oxygen-glucose deprivation reperfusion (OGD/R)-treated HT22 cells were utilized to investigate the effect of HGWD components with specific binding affinities. Reactive oxygen species (ROS), superoxide dismutase (SOD), lactate dehydrogenase (LDH), and Tunel staining were used as testing indexes to analyze the protective effects of potential active ingredients on OGD/R-induced damage. RESULTS: Eleven compounds with specific binding affinities were identified as calycosin-7-O-glucoside, calycosin, formononetin, cinnamic alcohol, cinnamic acid, betaine, dl-2-phenylpropionic acid, 4-hydroxycinnamic acid, 6-methylcoumarin, wogonin, and paeoniflorin. Among them, six compounds had a protective effect on OGD/R-treated HT22 cells. Furthermore, calycosin-7-O-glucoside, calycosin, paeoniflorin, 4-hydroxycinnamic acid, wogonin, and formononetin could regulate oxidative stress and apoptosis to attenuate the cell damage caused by OGD/R. CONCLUSION: The mechanism of action of HGWD to promote neurological recovery after ischemic stroke was related to the regulation of oxidative stress and apoptosis. This study suggested that cell membrane affinity chromatography combined with HPLC-MS/MS could be applied to screen potential active components in traditional Chinese medicines (TCM).

Piggery wastewater treatment by Acinetobacter sp. TX5 immobilized with spent mushroom substrate in a fixed-bed reactor.

Acinetobacter sp. TX5 immobilized with spent Hypsizygus marmoreus substrate (SHMS) was used to treat the raw piggery wastewater (RPW). In batch experiments, NH4+-N in the diluted RPW decreased from initial 34.95 mg/L to 3.83 mg/L at 8 h with the removal efficiency (RE) being 89%, and the beads immobilized with SHMS were comparable to those immobilized with activated carbon. In continuous experiments, the RE ranged from 74% to 95% for NH4+-N, from 73% to 93% for TN and from 54% to 82% for COD when the RPW was treated in a fixed-bed reactor packed with SHMS-immobilized TX5. The isotope analysis and enzyme purification indicated simultaneous nitrification and denitrification existing in TX5. This is the first time that spent mushroom substrates have been used to immobilize Acinetobacter species to treat the real RPW and a denitrifying nitrite reductase (dNiR) has been purified to make the nitrogen removal pathway in this species clearer.