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

ABSTRACT

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.