Buyang Huanwu Decoction Group Research Articles

Investigation of the underlying mechanism of Buyang Huanwu decoction in ischemic stroke by integrating systems pharmacology-proteomics and in vivo experiments

Buyang Huanwu Decoction (BHD) has been effective in treating ischemic stroke (IS). However, its mechanism of action remains unclear. The study intended to explore the potential mechanism of BHD against IS using systems pharmacology, proteomics, and animal experiments. The active components of BHD were identified from UPLC-Q-TOF-MS and literature mining. Systems pharmacology and proteomics were employed to investigate the underlying mechanism of BHD against IS. The AutoDock tool was used for molecular docking. A middle cerebral artery occlusion (MCAO) model rat was utilized to explore the therapeutic benefits of BHD. The rats were divided into sham, model, BHD (5, 10, 20 g/kg, ig) groups. The neurological scores, pathological section characteristics, brain infarct volumes, inflammatory cytokines, and signaling pathways were investigated in vivo experiments. The results of systems pharmacology showed that 13 active compounds and 112 common targets were screened in BHD. The docking results suggested that the active compounds in BHD had a high affinity for the key targets. In vivo experiments demonstrated that BHD exhibited neuroprotective benefits by lowering the neurological score, the volume of the cerebral infarct, the release of inflammatory cytokines, and reducing neuroinflammatory damage in MCAO rats. Furthermore, BHD decreased TNF-α and CD38 levels while increasing ATP2B2, PDE1A, CaMK4, p-PI3K, and p-AKT. Combined with systems pharmacology and proteomic studies, we confirmed that PI3K-Akt and calcium signaling pathways are the key mechanisms for BHD against IS. Furthermore, this study demonstrated the feasibility of combining proteomics with systems pharmacology to study the mechanism of herbal medicine.

The Therapeutic Effect of Buyang Huanwu Decoction on Mild Cognitive Impairment (MCI) in Patients with Diabetes

Objective This study aims to comprehensively verify the efficacy of Buyang Huanwu Decoction in improving cognitive function in patients with diabetes. Methods Patients clinically diagnosed with mild cognitive impairment (MCI) assigned to either the placebo group or the Buyang Huanwu Decoction group. After strict screening and exclusions, a total of 156 participants completed the clinical trial, with 76 in the placebo group and 80 in the Buyang Huanwu Decoction group. Results After treatment, Buyang Huanwu Decoction group showed higher Mini-Mental State Examination and Montreal Cognitive Assessment scores compared to placebo (p < 0.05). Memory and Executive Screening, Boston Naming Test, and Animal Fluency Test scores were also higher in the treatment group (p < 0.05). No significant differences were found in DST and CDT scores (p > 0.05). Trail Making Test scores were lower in the treatment group (p < 0.05). No significant difference was observed between the two groups in terms of complications (p > 0.05). Conclusion Patients receiving Buyang Huanwu Decoction treatment demonstrated improvement in cognitive function, showing positive effects and providing preliminary evidence for the role of Buyang Huanwu Decoction in improving cognitive function in patients with diabetes. This suggests its potential for clinical application and further promotion.

[Buyang Huanwu Decoction promotes arteriogenesis after hindlimb ischemia in mice by activating PDGF signaling pathway].

This study aims to investigate the effect of Buyang Huanwu Decoction on blood flow recovery and arteriogenesis after hindlimb ischemia in mice via the platelet-derived growth factor(PDGF) signaling pathway. Forty C57BL/6 mice were randomized into model(clean water, 10 mL·kg~(-1)·d~(-1)), beraprost sodium(positive control, 18 μg·kg~(-1)·d~(-1)), and low-, medium-, and high-dose(10, 20, and 40 g·kg~(-1)·d~(-1), respectively) Buyang Huanwu Decoction groups(n=8). The hindlimb ischemia model was established by femoral artery ligation. The mice were administrated with corresponding agents by gavage daily for 14 days after ligation. For laser Doppler perfusion imaging, the mice were anesthetized and measured under a Periscan PSI imager. The density of capillary and arterio-le in the ischemic gastrocnemius was measured using immunofluorescence staining of the frozen tissue sections. Western blot was employed to determine the expression of PDGF subunit B(PDGFB), phosphorylated mitogen extracellular kinase(p-MEK), MEK, phosphorylated extracellular signal-regulated kinase(p-ERK), and ERK. Real-time PCR was employed to determine the mRNA level of PDGFB. The Buyang Huanwu Decoction-containing serum was used to treat the vascular smooth muscle cells(VSMCs) in hypoxia at doses of 10% and 20%. The proliferation and migration of VSMCs was assessed in vitro. The results showed that compared with the model group, beraprost sodium and Buyang Huanwu Decoction enhanced the blood flow recovery, increased the capillary and arteriole density, and up-regulated the protein levels of PDGFB, p-MEK, p-ERK, and mRNA levels of PDGFB, with the medium-dose Buyang Huanwu Decoction demonstrating the most significant effect. The 10% Buyang Huanwu Decoction-containing serum enhanced the proliferation and migration of VSMCs. Our findings demonstrate that Buyang Huanwu Decoction up-regulates PDGFB transcription and activates PDGF signaling pathway to promote arteriogenesis and blood flow recovery in ischemic gastrocnemius.

[Metabolomic study on urine of chronic inflammation rats treated with Buyang Huanwu Decoction based on UPLC-Q-TOF-MS].

The study investigated the effect of Buyang Huanwu Decoction(BYHWD) on endogenous biomarkers in the urine of rats with chronic inflammation induced by lipopolysaccharide(LPS) using ultra-high performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS), aiming to elucidate the molecular mechanism underlying the therapeutic effect of BYHWD on chronic inflammation from a metabolomics perspective. Male SD rats were randomly divided into a normal group, a model group, and low-, medium-, and high-dose BYHWD groups(7.5, 15, and 30 g·kg~(-1)). The model group and BYHWD groups received tail intravenous injection of LPS(200 μg·kg~(-1)) on the first day of each week, followed by oral administration of BYHWD once a day for four consecutive weeks. Urine samples were collected at the end of the administration period, and UPLC-Q-TOF-MS was used to analyze the metabolic profiles of the rat urine in each group. Multivariate statistical analysis methods such as principal component analysis(PCA), partial least squares-discriminant analysis(PLS-DA), and orthogonal partial least squares-discriminant analysis(OPLS-DA) were used to analyze the effect of BYHWD on endogenous metabolites. One-way ANOVA and variable importance for the projection(VIP) were used to screen for potential biomarkers related to chronic inflammation. The identified biomarkers were subjected to pathway and enrichment analysis using MetaboAnalyst 5.0. A total of 25 potential biomarkers were screened and identified in the rat urine in this experiment. Compared with the normal group, the model group showed significant increases in the levels of 14 substances(P&lt;0.05) and significant decreases in the levels of 11 substances(P&lt;0.05). BYHWD was able to effectively reverse the trend of most endogenous biomarkers. Compared with the model group, BYHWD significantly down-regulated 13 biomarkers(P&lt;0.05) and up-regulated 10 biomarkers(P&lt;0.05). The metabolic products were mainly related to the biosynthesis of pantothenic acid and coenzyme A, tryptophan metabolism, retinol metabolism, and propionate metabolism. BYHWD has therapeutic effect on chronic inflammation induced by LPS, which may be related to its ability to improve the levels of endogenous metabolites, enhance the body's anti-inflammatory and antioxidant capabilities, and restore normal metabolic activity.

Buyang Huanwu Decoction alleviates blood stasis, platelet activation, and inflammation and regulates the HMGB1/NF-κB pathway in rats with pulmonary fibrosis

Ethnopharmacological relevanceQi deficiency and blood stasis are identified to be pathological factors of pulmonary fibrosis (PF) in traditional Chinese medicine (TCM) theory. Buyang Huanwu Decoction (BYHWD) is a traditional Chinese prescription ameliorating Qi deficiency and blood stasis. Aim of the studyThe objective of this study was to investigate the anti-fibrosis effect of BYHWD and the potential molecular mechanism in rats. Materials and methodsBleomycin was used to construct PF rat models. 27 PF rats were randomly divided into three groups based on treatments: model group (saline solution, n = 9), low-dose BYHWD group (3.5 g/kg, n = 9), and high-dose BYHWD group (14.0 g/kg, n = 9). Moreover, 9 normal rats were used as the blank group. The blood viscosity, coagulation indexes (APTT, TT, PT, and FIB), platelet-related parameters (PLT, PDW, MPV, PCT, and PLCR), platelet microparticles (PMPs), and inflammatory factors (IL-2, IL-10, IL-1β, IL-6, IL-8, IL-17, IFN-γ, TNF-α, PAC-1, HMGB1, NF-κB, and TF) were determined. The lung tissue samples of rats were observed after hematoxylin-eosin (HE) staining. The full component analysis of the BYHWD extract was performed using the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. The signaling pathway included into the study was selected on the basis of bioinformatics analysis and the results of the phytochemical analysis. The expression levels of genes and proteins involved in the selected signaling pathway were detected. ResultsCompared to the blank group, the whole blood viscosity, PLR, PDW, MPV, PCT, PLCR, PMPs, and the levels of IL-1β, IL-6, IL-8, IL-17, TNF-α, PAC-1, HMGB1, NF-κB, and TF were increased, while the levels of IL-2 and IL-10 were decreased in the model group. Both low-dose BYHWD and high-dose BYHWD reversed these PF-induced effects in spite of the fact that low-dose BYHWD had no significant effect on the level of NF-κB. In addition, BYHWD ameliorated PF-induced inflammation in the rat lung tissue. The phytochemical analysis of the BYHWD extract combined with the bioinformatics analysis suggested that the therapeutical effect of BYHWD on PF was related to the HMGB1/NF-κB pathway, which consisted of NF-κB, IKBKB, ICAM1, VCAM1, HMGB1, and TLR4. Both RT-qPCR and western blot analyses showed that PF induced increases in the expression levels of NF-κB, ICAM1, VCAM1, HMGB1, and TLR4, but a decrease in the expression level of IKBKB. Moreover, both low-dose BYHWD and high-dose BYHWD exerted the opposite effects, and recovered the expression levels of NF-κB, ICAM1, VCAM1, HMGB1, TLR4, and IKBKB, despite the fact that low-dose BYHWD had no effects on the mRNA expression levels of NF-κB or TLR4. ConclusionsIn summary, BYHWD alleviated PF-induced blood stasis, platelet activation, and inflammation in the rats. Our study suggested BYHWD had a therapeutic effect on PF and was a good alternative for the complementary therapy of PF, and the potential molecular mechanism was modulation of HMGB1/NF-κB signaling pathway, and it needs further study.

[Mechanism of Buyang Huanwu Decoction in protecting ischemic myocardium by regulating platelet autophagy in rats with acute myocardial infarction].

This study explored the effects of Buyang Huanwu Decoction(BYHWD) on platelet activation and differential gene expression after acute myocardial infarction(AMI). SD rats were randomly divided into a sham-operated group, a model group, a positive drug(aspirin) group, and a BYHWD group. Pre-treatment was conducted for 14 days with a daily oral dose of 1.6 g·kg~(-1) BYHWD and 0.1 g·kg~(-1) aspirin. The AMI model was established using the high ligation of the left anterior descending coronary artery method. The detection indicators included myocardial infarct size, heart function, myocardial tissue pathology, peripheral blood flow perfusion, platelet aggregation rate, platelet membrane glycoprotein CD62p expression, platelet transcriptomics, and differential gene expression. The results showed that compared with the sham-operated group, the model group showed reduced ejection fraction and cardiac output, decreased peripheral blood flow, and increased platelet aggregation rate and CD62p expression, and activated platelets. At the same time, TXB_2 content increased and 6-keto-PGF1α content decreased in serum. Compared with the model group, BYHWD increased ejection fraction and cardiac output, improved blood circulation in the foot and tail regions and cardiomyocytes arrangement, reduced myocardial infarct size and inflammatory infiltration, down-regulated platelet aggregation rate and CD62p expression, reduced serum TXB_2 content, and increased 6-keto-PGF1α content. Platelet transcriptome sequencing results revealed that BYHWD regulated mTOR-autophagy pathway-related genes in platelets. The differential gene expression levels were detected using real-time quantitative PCR. BYHWD up-regulated mTOR, down-regulated autophagy-related FUNDC1 and PINK genes, and up-regulated p62 gene expression. The results demonstrated that BYHWD could regulate platelet activation, improve blood circulation, and protect ischemic myocardium in AMI rats, and its mechanism is related to the regulation of the mTOR-autophagy pathway in platelets.

[Effects of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination on inflammatory responses in atherosclerotic mice].

The study aims to observe the effects and explore the mechanisms of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination in the treatment of the inflammatory response of mice with atherosclerosis(AS) via the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor-κB(NF-κB) signaling pathway. Male ApoE~(-/-) mice were randomly assigned into a model group, a Buyang Huanwu Decoction group, an Astragali Radix-Angelicae Sinensis Radix combination group, and an atorvastatin group, and male C57BL/6J mice of the same weeks old were used as the control group. Other groups except the control group were given high-fat diets for 12 weeks to establish the AS model, and drugs were administrated by gavage. Aortic intimal hyperplasia thickness, blood lipid level, plasma inflammatory cytokine levels, M1/M2 macrophage markers, and expression levels of proteins in TLR4/MyD88/NF-κB pathway in the vessel wall were measured to evaluate the effects of drugs on AS lesions and inflammatory responses. The results showed that the AS model was successfully established with the ApoE~(-/-) mice fed with high-fat diets. Compared with the control group, the model group showed elevated plasma total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c) levels(P&lt;0.05), thickened intima(P&lt;0.01), and increased plasma tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) levels(P&lt;0.01). Moreover, the model group showed increased expression of vascular cell adhesion molecule-1(VCAM-1) and inducible nitric oxide synthase(iNOS)(P&lt;0.01), inhibited expression of endothelial nitric oxide synthase(eNOS) and cluster of differentiation 206(CD206)(P&lt;0.01), and up-regulated mRNA and protein levels of TLR4, MyD88, NF-κB inhibitor alpha(IκBα), and NF-κB in the vessel wall(P&lt;0.05). Compared with the model group, Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination lowered the plasma TC and LDL-c levels(P&lt;0.01), alleviated the intimal hyperplasia(P&lt;0.01), and reduced the plasma TNF-α and IL-6 levels(P&lt;0.05). Moreover, the two interventions promoted the expression of eNOS and CD206(P&lt;0.05), inhibited the expression of VCAM-1 and iNOS(P&lt;0.01), and down-regulated the mRNA and protein levels of TLR4, MyD88, IκBα, and NF-κB(P&lt;0.05) in the vessel wall. This study indicated that Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination could delay the progression of AS, inhibit the polarization of vascular wall macrophages toward M1 type, and attenuate vascular inflammatory response by inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway in the vascular wall. Astragali Radix and Angelicae Sinensis Radix were the main pharmacological substances in Buyang Huanwu Decoction for alleviating the AS vascular inflammatory response.

Study on the action mechanism of Buyang Huanwu Decoction against ischemic stroke based on S1P/S1PR1/PI3K/Akt signaling pathway

Ethnopharmacological relevanceIschemic stroke is a common and frequent clinical disease. Recent studies have demonstrated that sphingolipid plays an important role in the pathological process of ischemic stroke. PI3K-Akt is a classic protective signaling pathway of cerebral ischemic injury. After acting on the S1P receptor, S1P can activate the downstream PI3K/Akt signaling pathway and play an anti-cerebral ischemia role. Buyang Huanwu Decoction (BHD) is a traditional Chinese medicine formula used to treat ischemic stroke. However, the mechanisms of BHD on ischemic stroke remain unclear based on S1P/S1PR1/PI3K/Akt signaling pathway. Aim of the studyThe present study is intended to investigate the action mechanism of BHD on ischemic stroke based on the S1P/S1PR1/PI3K/Akt signaling pathway from multiple perspectives. Materials and methodsThe BHD lyophilized product was prepared by vacuum freeze-drying method, of which the chemical composition was determined by UPLC-Q-TOF/MS. The mouse permanent middle cerebral artery occlusion (pMCAO) model was established by the suture-occluded method. Male KM mice were randomly divided into seven groups: sham group, model group, FTY720 (positive control) group, BHD group, BHD + W146 (selective S1PR1 inhibitor) group, SEW2871 (selective S1PR1 agonist) group, and Calycosin group. Each group was administered continuously for 14 days and evaluated with modified neurological severity score (mNSS) and cerebral infarct volume on the 1st, 4th, 7th, and 14th days. The SphK1, SphK2, S1PR1, PI3K, Akt, and p-Akt protein in the prefrontal lobe, hippocampus, and striatum was quantified by Western blot and immunohistochemical (IHC) experiment respectively. The qRT-PCR method was employed to evaluate SphK1, SphK2, and S1PR1 mRNA expression in the above tissue. ResultsBHD and Calycosin both effectively improved mNSS scores with smaller infarct volumes. The SphK1 level in the prefrontal lobe, hippocampus, and striatum of mice in the BHD group was significantly lower, and SphK2, PI3K, and p-Akt in the hippocampus and striatum were significantly higher than those in the model group.BHD significantly decreased SphK1 mRNA expression in the prefrontal lobe, hippocampus, and striatum, and significantly up-regulated SphK2 mRNA and S1PR1 mRNA expression. Additionally, SphK1 protein expression levels of the prefrontal lobe, hippocampus, and striatum in the BHD group was significantly lower than model group, and SphK2, S1PR1, PI3K, Akt, and p-Akt protein expressions levels were increased obviously.Furthermore, SEW2871 can increase S1PR1 and Akt expression, and up-regulate SphK2 and S1PR1 mRNA expression. The effect of BHD on the expression of S1P/S1PR1/PI3K/Akt signaling pathway-related proteins and mRNA were weakened by BHD + W146. ConclusionBHD and Calycosin significantly improved the symptoms of neurological deficits in pMCAO mice, reduced the cerebral infarction volume, up-regulated SphK2 and S1PR1 mRNA levels, enhanced SphK2, S1PR1, PI3K, Akt, p-Akt protein expression of the prefrontal lobe, hippocampus and striatum, and down-regulated SphK1 mRNA and protein expression, which may be helpful to clarify the mechanism of BHD through S1P/S1PR1/PI3K/Akt signaling pathway to protect against cerebral ischemic injury.

Effect and mechanism of Buyang Huanwu decoction on retinal inflammation in streptozotocin-induced diabetic rats

ObjectiveTo observe the protective effect of Buyang Huanwu decoction (BYHWD) against retinal inflammation in diabetic rats. MethodsHealthy male Wistar rats were randomly divided into normal and diabetic model groups. Diabetic rats were given a one-time intraperitoneal injection of streptozotocin (STZ) to establish a diabetic phenotype and were randomly assigned to the negative control (saline), positive control (calcium dobesilate), and BYHWD treatment groups. After 8 weeks of treatment, fluorescein-labeled dextran and Evans blue dye were injected into the tail vein to observe vascular permeability and vascular leakage in the retina, respectively. Perfusion of fluorescein-labeled concanavalin was used to detect adhesion in rat retinal leukocytes. Western blotting was used to quantify the expression levels of the inflammatory factors TNF-α, NF-κBp65, and p-NF-κBp65 in retinal tissue. ResultsCompared with nondiabetic rats, saline-treated diabetic rats showed significantly increased retinal vascular leakage, leukocyte adhesion, and protein levels of TNF-α and p-NF-κBp65 (P<0.05). These effects were significantly reduced in the positive control and BYHWD groups (P<0.05). ConclusionBuyang Huanwu decoction can ameliorate retinal inflammation in STZ-induced diabetic rats. Its mechanism of action may be related to the inhibition of TNF-α expression and NF-κB pathway activation.

Effects of Buyang Huanwu Decoction on Intestinal Barrier, Intestinal Flora, and Trimethylamine Oxide in Rats with Heart Failure.

To explore the mechanisms of Buyang Huanwu Decoction (BYHWD) modulating the gut microbiome and trimethylamine oxide (TAMO) to exert cardioprotective effects. Ligation of the left anterior descending coronary artery was performed in rats to induce heart failure (HF). Except for the sham-operation group (n=10), 36 operation-induced models were randomized into 3 groups using a random number table (n=12 in each group): the model group, the BYHWD group (15.02 g/kg BYHWD), and the positive group (4.99 g/kg metoprolol succinate). After 4-week treatment (once daily by gavage), echocardiography was applied to evaluate the cardiac function and the Tei index (the ratio of ventricular isovolumic contraction time (IVCT) and isovolumic diastolic time (IVRT) to ejection time (ET)) was calculated; hematoxylin-eosin (HE) staining was observed to characterize the pathology of the myocardium and small intestinal villi. D-lactic acid was detected by an enzyme-linked immunosorbent assay (ELISA). Expressions of occludin, claudin-1, and zonula occludens (ZO-1) were detected by Western blot. 16S ribosomal ribonucleic acid (16S rRNA) sequencing was used to explore the changes in the intestinal flora. TMAO was detected via liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the echocardiography, the Tei index was considerably lower in the positive and BYHWD groups compared with the model group (P<0.05). Besides, BYHWD improved the pathology of myocardium and small intestine of HF rats and lowered the D-lactic acid content in the serum, when compared with the model group (P<0.05). BYHWD also improved the expression of occludin and claudin-1 (P<0.05); in the gut microbiota analysis, BYHWD slowed down modifications in the structure distribution of gut microbiota and regulated the diversity of intestinal flora in HF rats. The content of TMAO in the serum was significantly lowered by BYWHT compared with the model group (P<0.05). BYHWD may delay progression of HF by enhancing the intestinal barrier structure, and regulating intestinal flora and TAMO.

Buyang Huanwu Decoction: A Traditional Chinese Medicine, Promotes Lactate-Induced Angiogenesis in Experimental Intracerebral Hemorrhage.

Identifying the underlying mechanisms and exploring effective therapies for intracerebral hemorrhage (ICH) are urgently needed. Here, we aim to elucidate the potential roles and underlying mechanisms of Buyang Huanwu decoction (BYHWD) in ICH. In the first set of experiments, rats were randomly divided into five groups: Sham, ICH, ICH + sodium oxamate (OXA), ICH + BYHWD, and ICH + BYHWD + OXA. The lactate level around the hematoma was evaluated. PCNA+/vWF+ nuclei were observed. Additionally, an online bioinformatics analysis tool was used to predict the BYHWD druggable targets related to angiogenesis. Then, we validated these predictions. In the second set, exogenous sodium L-lactate (Lac) was infused into the intact brains of rats. Rats were randomly divided into three groups: Sham, Lac, and Lac + YC-1. The numbers of PCNA+/vWF+ nuclei and the expression of HIF-1α and VEGF were evaluated. In the first set of experiments, compared with the ICH group, the BYHWD group exhibited significantly increased numbers of PCNA+/vWF+ nuclei, and neurological dysfunction was markedly improved. Bioinformatics analysis revealed that the improvements caused by BYHWD indicated a role for the HIF-1α pathway. The HIF-1α and VEGF protein levels were upregulated after BYHWD administration. Moreover, we verified that lactate was involved in the predicted mechanisms. In the second set, lactate facilitated angiogenesis and HIF-1α and VEGF expression. Co-infusion with a HIF-1α inhibitor, YC-1, significantly inhibited these effects. Our data suggest that the pharmacological effects of BYHWD involve lactate-induced angiogenesis, these data may provide new evidence for its use in ICH.

Buyang Huanwu decoction promotes angiogenesis of rat brain microvascular endothelial cells after oxygen-glucose deprivation reperfusion injury via activation of PI3K-AKT signaling pathway.

To investigate the effect and mechanism of Buyang Huanwu decoction (BYHWD) on angiogenesis of rat brain microvascular endothelial cells (RBMECs) after oxygen-glucose deprivation reperfusion (OGD/R) injury. RBMECs were pretreated with BYHWD containing serum 24 h before OGD/R injury was induced. Cells were randomly divided into blank control group, model control group, BYHWD group (provided BYHWD containing serum) and LY294002 group [treated with phosphoinositide 3-kinase (PI3K) inhibitor LY294002 for 1 h before provided BYHWD containing serum]. The cell viability, migration and tube formation abilities of RBMECs were detected by CCK-8, scratch wound healing, Transwell migration and tube formation assays, respectively. The protein expression levels of PI3K, p-PI3K, protein kinase B (AKT), p-AKT, hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were determined by Western blotting. Compared with model control group, cell viability, migration and tube formation abilities of RBMECs were significantly improved in BYHWD group (all P<0.01), the protein expression levels of p-PI3K, p-AKT, HIF-1α and VEGF were up-regulated (all P<0.05); while above effects were blocked by LY294002. BYHWD can promote angiogenesis of RBMECs after OGD/R injury, which may be related to the increased protein expression of HIF-1α and VEGF through activation of PI3K-AKT signaling pathway.

Construction of a circRNA-miRNA-mRNA network revealed the potential mechanism of Buyang Huanwu Decoction in the treatment of cerebral ischemia

Buyang Huanwu Decoction (BHD) is a traditional Chinese herbal medicine that is effective for treating cerebral ischemia (CI). However, the molecular mechanisms of BHD in CI have not been fully elucidated. In this study, we integrated the circular RNA (circRNA)-microRNA (miRNA)-messenger RNA (mRNA) network of middle cerebral artery occlusion (MACO) rats treated with BHD. SD rats were randomly divided into a control group, model group, model+BHD group (2.5, 5, 10g/kg) and model+butylphthalide (NBP) group (54mg/kg). The neurological functions of the rats were evaluated by a modified neurological severity scoring (mNSS) system. Pathological lesions were assessed by Nissl staining, and the effects of BHD on neurovascular unit (NVU) associated protein microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP) and von Willebrand factor (VWF) were assessed by immunohistochemistry. CeRNA and miRNA microarrays were used to establish the circRNA, miRNA, and mRNA profiles. Finally, a circRNA-miRNA-mRNA ternary transcription network was constructed. BHD improved the neurobehavioral test scores (P<0.01) and the histopathological changes in ischemic brain tissue in MCAO rats. The expression of MAP2 and VWF decreased and the expression of GFAP increased in the ischemic side brain tissue of MCAO rats (P<0.01), and treatment with BHD reversed the above changes (P<0.01 or 0.05). We identified seven, three, and 86 significantly dysregulated circRNAs, miRNAs, and mRNAs, respectively, that were associated with the neuroprotective effects of BHD. Furthermore, bioinformatics analysis showed that these targets may exert therapeutic effects through multiple pathways, such as the VEGF and Hippo signaling pathways. Finally, we constructed a circRNA-miRNA-mRNA network. In brief, our study provides novel insights into ceRNA-mediated gene regulation in the progression of NVU after CI and the mechanism of action for BHD.

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.

BYHWD Alleviates Inflammatory Response by NIK-Mediated Repression of the Noncanonical NF-κB Pathway During ICH Recovery.

Intracerebral hemorrhage (ICH) is a life-threatening type of stroke that lacks effective treatments. The inflammatory response following ICH is a vital response that affects brain repair and organism recovery. The nuclear factor κB (NF-κB) signaling pathway is considered one of the most important inflammatory response pathways and one of its response pathways, the noncanonical NF-κB signaling pathway, is known to be associated with persistent effect and chronic inflammation. NF-κB–inducing kinase (NIK) via the noncanonical NF-κB signaling plays a key role in controlling inflammation. Here, we investigated potential effects of the traditional Chinese medicine formula Buyang Huanwu Decoction (BYHWD) on inflammatory response in a rat model of ICH recovery by inhibiting the NIK-mediated the noncanonical NF-κB signaling pathway. In the first part, rats were randomly divided into three groups: the sham group, the ICH group, and the BYHWD group. ICH was induced in rats by injecting collagenase (type VII) into the right globus pallidus of rats' brain. For the BYHWD group, rats were administered BYHWD (4.36 g/kg) once a day by intragastric administration until they were sacrificed. Neurological function was evaluated in rats by a modified neurological severity score (mNSS), the corner turn test, and the foot-fault test. The cerebral edema showed the degree of inflammatory response by sacrificed brain water content. Western blot and real-time quantitative reverse transcription PCR tested the activity of inflammatory response and noncanonical NF-κB signaling. In the second part, siRNA treatment and assessment of inflammation level as well as alterations in the noncanonical NF-κB signaling were performed to determine whether the effect of BYHWD on inflammatory response was mediated by suppression of NIK via the noncanonical NF-κB signaling pathway. We show that BYHWD treated rats exhibited: (i) better health conditions and better neural functional recovery; (ii) decreased inflammatory cytokine and the edema; (iii) reduced expression of NIK, a key protein in unregulated the noncanonical NF-κB signaling pathways; (iv) when compared with pretreated rats with NIK targeting (NIK siRNAs), showed the same effect of inhibiting the pathway and decreased inflammatory cytokine. BYHWD can attenuate the inflammatory response during ICH recovery in rats by inhibiting the NIK-mediated noncanonical NF-κB signaling pathway.

[Chinese medicine Buyang Huanwu decoction promotes neurogenesis and angiogenesis in ischemic stroke rats by upregulating miR-199a-5p expression].

To investigate the mechanism of Chinese medicine Buyang Huanwu decoction (BYHWD) promoting neurogenesis and angiogenesis in ischemic stroke rats. Male SD rats were randomly divided into sham operation group, model group, BYHWD group, antagonist group and antagonist control group with 14 rats in each. Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery for 90 min with intraluminal filament and reperfusion for 14 d in all groups except sham operation group. BYHWD (13 g/kg) was administrated by gastrogavage in BYHWD group, antagonist group and antagonist control group at 24 h after modeling respectively, and BrdU (50 mg/kg) was injected intraperitoneally in all groups once a day for 14 consecutive days. miR-199a-5p antagomir or NC (10 nmol) was injected into the lateral ventricle at d5 after ischemia in antagonist and antagonist control groups, respectively. The neurological deficits were evaluated by the modified neurological severity score (mNSS) and the corner test, and the infract volume was measured by toluidine blue staining. Neurogenesis and angiogenesis were detected by immunofluorescence double labeling method. The expression level of miR-199a-5p was tested by real-time RT-PCR, and the protein expressions of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) were determined by Western blotting. BYHWD treatment significantly promoted the recovery of neurological function (P<0.05 or P<0.01), reduced the infarct volume (P<0.05), increased the number of BrdU+/DCX+, BrdU+/NeuN+ and BrdU+/vWF+ cells (all P<0.01), upregulated the expression of miR-199a-5p (P<0.01), and increased the protein expression of VEGF and BDNF at d14 after cerebral ischemia (all P<0.05). The above effects were reversed by intracerebroventricular injection of miR-199a-5p antagomir. Buyang Huanwu decoction promotes neurogenesis and angiogenesis in rats with cerebral ischemia, which may be related to increased protein expression of VEGF and BDNF through upregulating miR-199a-5p.

Buyang Huanwu Decoction Targets SIRT1/VEGF Pathway to Promote Angiogenesis After Cerebral Ischemia/Reperfusion Injury

Background: Ischemia stroke is known as the major cause of morbidity and mortality. Buyang Huanwu Decoction (BHD), a classical traditional Chinese medicine (TCM) formula, has been used to prevent and treat stoke for hundreds of years. The purpose of present study is to investigate the effects of BHD on angiogenesis in rats after cerebral ischemia/reperfusion (I/R) injury targeting Silent information regulator 1 (SIRT1) / Vascular endothelial growth factor (VEGF) pathway.Methods: The cerebral I/R injury model was induced by middle cerebral artery occlusion (MCAO). Adult Sprag-Dawley (SD) rats were randomly divided into five groups: sham group, normal saline (NS) group, BHD group, BHD+EX527 (SIRT1 specific inhibitor) group, and NS+EX527 group. Each group was divided into the subgroups according to 1, 3, 7, or 14 days time-point after cerebral ischemia/reperfusion, respectively. Neurological function score (NFS) was evaluated by the Rogers scale; microvascular density (MVD) in brain tissue around infarction area was observed by immunofluorescence; and the expression of SIRT1 and VEGF was assessed by Western Blot and Quantitative Real-time-PCR.Results: BHD can significantly improve NFS (P < 0.05), increase the MVD in the boundary ischemic area (P < 0.01) and elevate the expression of protein and mRNA of SIRT1 and VEGF following I/R injury (P < 0.01). In contrast, treatment with EX527 reversed the BHD-induced improvements in NFS (P < 0.01) and decreased the MVD (P < 0.01) and the expression of SIRT1 and VEGF (P < 0.05).Conclusion: BHD exerts neuroprotection targeting angiogenesis through the up-regulation of SIRT1/VEGF pathway against cerebral ischemic injury in rats.

Establishment and Evaluation of a Rat Model of Spinal Cord Injury with the Pathopattern of Qi-Deficiency and Blood-Stasis in Traditional Chinese Medicine

ABSTRACT Objective To establish a rat model of Spinal Cord Injury (SCI) with the pathopattern of Qi-deficiency and Blood-stasis (QDBS) in Traditional Chinese Medicine (TCM), and then assess its feasibility. Methods Swimming exhaustion method and Rubrospinal Tract (RST) transection were combined to establish arat model of SCI with pathopattern of QDBS in TCM. Macroscopic representation, including the body weight, food intake and tongue scores, was recorded. Behavioristics was scored with spontaneous vertical exploration test. Hemorrheology was analyzed using hemorheological analyzer and morphology of spinal cord was observed. The feasibility of the model was confirmed on the basis of the reversion caused by administration of Bu Yang Huan Wu Decoction (BYHWD). Results The findings in the QDBS group were consistent with the symptoms of QDBS syndrome, including reduced body weight, food intake, and forelimb utilization rate and increased tongue scores. However, the corresponding findings in the BYHWD group were superior to those in the QDBS group after 35 d (P Conclusion Swimming exhaustion method combined with RST transection operation can successfully establish a rat model of SCI with pathopattern of QDBS.

Buyang Huanwu Decoction Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Rats via Downregulation of Related Protein and Gene Expression.

Little is known about the effects of Buyang Huanwu decoction on pulmonary fibrosis. Herein, 144 healthy SD rats were randomly divided into six groups: blank control group (B), model control group (M), positive medicine control group (Mp), and high-, moderate-, and low-dose Buyang Huanwu decoction groups (Hd, Md, and Ld). A pulmonary fibrosis model was established by endotracheal injection of bleomycin. On the second day of modeling, the corresponding saline, methylprednisolone suspension, and the three doses of Buyang Huanwu decoction were used to treat the 6 groups of rats by intragastric administration for 7, 14, and 28 consecutive days. After 7, 14, and 28 days of treatment, the mRNA expression of CTGF and AKT, the protein level of CTGF, p-AKT, and collagen types I and III were tested. Finally, we found that the serum collagen type I and III level in Hd, Md, and Ld rats on the 14th and 28th day and the collagen type I and III level in Hd rats on 7th day were significantly lower than in M rats (P < 0.01). The protein level of p-AKT and CTGF in Hd and Md rats on the 7th and 14th days and the protein level of p-AKT in Hd rats on the 28th day were lower than in M rats (P < 0.01, P < 0.05). The level of CTGF mRNA in Hd, Md, and Ld rats and the level of AKT mRNA in Hd and Md rats on the 7th, 14th, and 28th days and the expression level of AKT mRNA in Ld rats on the 14th and 28th days were significantly lower than in M rats (P < 0.01). The study suggests that Buyang Huanwu decoction alleviated pulmonary fibrosis of rats by improvement of lung tissue morphology, low level of serum collagen types I and III, and the reduced expression of CTGF and p-AKT protein, which might be a result of its downregulated expression of CTGF and AKT mRNA levels.

Differential gene expression profile of Buyanghuanwu decoction in rats with ventricular remodeling post-myocardial infarction

ObjectiveTo investigate the effect of Buyanghuanwu decoction (BYHWD) on gene expression in ventricular remodeling post-myocardial infarction in rats. MethodsAnimal models of myocardial infarction were established by permanent ligation of the left anterior descending coronary artery. Echocardiography measurements were performed after the treatment of BYHWD (18 g·kg−1·dΔ1) for 90 days. Myocardial collagen was observed by mallory trichrome staining. Capillary density was quantified by using Factor VIII immunohistochemical staining. Differentially expressed genes were explored by a short-read sequencing technology combined with a tag-based digital gene expression profiling (DGE) system. Real-time quantitative polymerase chain reaction detecting system (qPCR) was used to validate the sequencing results. After assembling the gene information from Sham, model and BYHWD groups, we constructed three DGE libraries based on each group. The sequencing of three libraries generated 66 000-73 000 unique tags, which were mapped to reference sequences for annotation of expressed genes. ResultsAmong them, 511 and 352 differentially expressed genes were found in comparison with sham/model and model/BYHWD, respectively. Fifty-five genes exhibited reversed direction of gene expression differences between Sham/Model and Model/BYHWD groups. We found that transforming growth factor beta receptor-1, junctophilin-2, monocyte chemotactic protein 1, neuropeptide Y, arachidonate 5-Lipoxygenase, arachidonate 15-Lipoxygenase were significantly modulated, which suggested the involvement of these genes in BYHWD treatment. ConclusionThe DGE profiling data provide comprehensive gene expression information at the transcriptional level that could facilitate our understanding of the pharmacological mechanisms of BYHWD in ventricular remodeling post-myocardial infarction.