Elucidating the mechanisms of Buyang Huanwu Decoction in treating chronic cerebral ischemia: A combined approach using network pharmacology, molecular docking, and in vivo validation.

OBJECTIVE: This study aimed to explore the potential mechanisms of Buyang Huanwu Decoction (BHD) in regulating the AKT/TP53 pathway and reducing inflammatory responses for the treatment of chronic cerebral ischemia (CCI) using UHPLC-QE-MS combined with network pharmacology, molecular docking techniques, and animal experiment validation. METHODS: Targets of seven herbal components in BHD, such as Astragalus membranaceus, Paeoniae Rubra Radix, and Ligusticum chuanxiong, were identified through TCMSP and HERB databases. CCI-related targets were obtained from DisGeNET and Genecards, with an intersection analysis conducted to determine shared targets between the disease and the herbal components. Functional enrichment analysis of these intersecting targets was performed. Networks of gene ontology and pathway associations with these targets were constructed and visualized. A pharmacological network involving intersecting genes and active components was delineated. A protein-protein interaction network was established for these intersecting targets and visualized using Cytoscape 3.9.1. The top five genes from the PPI network and their corresponding active components underwent molecular docking. Finally, the 2-vessel occlusion (2-VO) induced CCI rat model was treated with BHD, and the network pharmacology findings were validated using Western blot, RT-PCR, behavioral tests, laser speckle imaging, ELISA, HE staining, Nissl staining, LFB staining, and immunohistochemistry and immunofluorescence. RESULTS: After filtration and deduplication, 150 intersecting genes were obtained, with the top five active components by Degree value identified as Quercetin, Beta-Sitosterol, Oleic Acid, Kaempferol, and Succinic Acid. KEGG pathway enrichment analysis linked key target genes significantly with Lipid and atherosclerosis, AGE-RAGE signaling pathway, IL-17 signaling pathway, and TNF signaling pathway. The PPI network highlighted ALB, IL-6, AKT1, TP53, and IL-1ß as key protein targets. Molecular docking results showed the strongest binding affinity between ALB and Beta-Sitosterol. Behavioral tests using the Morris water maze indicated that both medium and high doses of BHD could enhance spatial memory in 2-VO model rats, with high-dose BHD being more effective. Laser speckle results showed that BHD at medium and high doses could facilitate CBF recovery in CCI rats, demonstrating a dose-response relationship. HE staining indicated that all doses of BHD could reduce neuronal damage in the cortex and hippocampal CA1 region to varying extents, with the highest dose being the most efficacious. Nissl staining showed that nimodipine and medium and high doses of BHD could alleviate Nissl body damage. LFB staining indicated that nimodipine and medium and high doses of BHD could reduce the pathological damage to fiber bundles and myelin sheaths in the internal capsule and corpus callosum of CCI rats. ELISA results showed that nimodipine and BHD at medium and high doses could decrease the levels of TNF-α, IL-6, IL-17, and IL-1ß in the serum of CCI rats (p < 0.05). Immunohistochemistry and immunofluorescence demonstrated that BHD could activate the AKT signaling pathway and inhibit TP53 in treating CCI. Western blot and RT-PCR results indicated that nimodipine and all doses of BHD could upregulate Akt1 expression and downregulate Alb, Tp53, Il-1ß, and Il-6 expression in the hippocampus of CCI rats to varying degrees (p < 0.05). CONCLUSION: BHD exerts therapeutic effects in the treatment of CCI by regulating targets, such as AKT1, ALB, TP53, IL-1ß, and IL-6, and reducing inflammatory responses.

Baicalin ameliorates chronic unpredictable mild stress-induced depression through the BDNF/ERK/CREB signaling pathway.

Brain-derived neurotrophic factor (BDNF) can activate the extracellular regulated protein kinase (ERK)/cAMP response element binding protein (CREB) cascade revealing an important role in antidepressant effects. Here, we studied the neuroprotective effect of baicalin (BA) in mice with chronic unpredictable mild stress (CUMS)-induced via a BDNF/ERK/CREB signaling pathway. Depression was induced via six weeks of CUMS in male ICR mice, and drug therapy was given simultaneously for the last three weeks. Cognitive dysfunctions were then evaluated via sucrose preference test (SPT), open field test (OFT), Morris water maze test (MWM), tail suspension test (TST), and novelty suppressed feeding test (NSF). Western blot and real-time PCR were then used to detect the relative expression of ERK, CREB, p-ERK, and p-CREB. Integrated optical density (IOD) tests of p-ERK and p-CREB were then evaluated via immunofluorescence. The behavior results showed that the cognitive dysfunctions increased in the CUMS group versus the control (CON) group (p < 0.01). There were decreases in fluoxetine (FLU) and BA groups (p < 0.05, p < 0.01). The protein ratios of p-ERK/ERK, p-CREB/CREB and ERK mRNA, and CREB mRNA expression decreased in the CUMS group (p < 0.01) and markedly increased in the FLU and BA groups (p < 0.05, p < 0.01). The IOD value of the p-ERK and p-CREB in the CUMS group was decreased versus the CON group (p < 0.01), and these changes were improved via BA and FLU treatment (p < 0.05, p < 0.01). This study indicated that BA can improve cognitive functions and has antidepressant effects in mice, which may be associated with activation of the BDNF/ERK/CREB signaling pathway in the hippocampus.

Baicalin promotes hippocampal neurogenesis via the Wnt/ß-catenin pathway in a chronic unpredictable mild stress-induced mouse model of depression.

Hippocampal neurogenesis is known to be related to depressive symptoms. Increasing evidence indicates that Wnt/ß-catenin signaling regulates multiple aspects of adult hippocampal neurogenesis. Baicalin is a major flavonoid compound with multiple pharmacological effects such as anti-inflammatory, anti-apoptotic, and neuroprotective effects. The current study aimed to explore the antidepressant effects of baicalin and its possible molecular mechanisms affecting hippocampal neurogenesis via the regulation of the Wnt/ß-catenin signaling pathway. A chronic mild unpredictable stress (CUMS) model of depression was used in the study. The CUMS-induced mice were treated with baicalin (50 and 100 mg/kg) for 21 days, orally, and the fluoxetine was used as positive control drug. The results indicated that baicalin alleviated CUMS-induced depression-like behaviour, and improved the nerve cells' survival of the hippocampal dentate gyrus (DG) in CUMS-induced depression of model mice and increased Ki-67- and doublecortin (DCX)-positive cells to restore CUMS-induced suppression of hippocampal neurogenesis. The related proteins in the Wnt/ß-catenin signaling pathway, which declined in the CUMS-induced depression model of mice, were upregulated after baicalin treatment, including Wingless3a (Wnt3a), dishevelled2 (DVL2), and ß-catenin. Further study found that the phosphorylation rate of glycogen synthase kinase-3ß (GSK3ß) and ß-catenin nuclear translocation increased, as the levels of the ß-catenin target genes cyclinD1, c-myc, NeuroD1, and Ngn2 upregulated after baicalin treatment. In conclusion, these findings suggest that baicalin may promote hippocampal neurogenesis, thereby exerting the antidepressant effect via regulation of the Wnt/ß-catenin signaling pathway.