Multimodal investigation reveals the neuroprotective mechanism of Angong Niuhuang pill for intracerebral hemorrhage: Converging bioinformatics, network pharmacology, and experimental validation

Angong Niuhuang Pill (ANP) is a traditional Chinese medicine formula that has been used clinically for many years in the treatment of cerebral hemorrhage. It is composed of ingredients such as calculus bovis, moschus, and others. Ancient texts have documented that ANP's multiple components possess properties such as heat-clearing, detoxification, and sedation, which can be effective in treating conditions such as coma and stroke. However, the underlying mechanisms of ANP's potential actions are still under investigation.ANP is a Chinese medicine widely utilized for the treatment of intracerebral hemorrhage (ICH). However, the precise mechanism underlying the therapeutic effects remains largely elusive. The present study aims to unravel the effects and pharmacological molecular mechanisms of ANP in combatting ICH, employing a comprehensive network pharmacology approach and experimental validation.The molecular targets of ANP and ICH were obtained from various databases, followed by the construction of protein-protein interaction (PPI) networks using the STRING database. Further, gene ontology (GO) enrichment and Kyoto encyclopedia of genes and genomes (KEGG) analyses were conducted using the Metascape database and Cytoscape, respectively. Finally, molecular docking was performed. We performed a series of behavioral tests, immunohistochemical staining, TUNEL staining, and Western Blot to verify the effects of ANP.IL-6, JUN, MMP9, IL-1β, VEGFA were the main candidate targets and were associated with fluid shear stress and atherosclerosis, TNF signaling pathway, etc. It is suggested that the potential mechanism of ANP against ICH may be mainly related to pyroptosis, inflammation. In vivo validation showed that ANP treatment significantly reduced the number of TUNEL-positive cells and ANP inhibited the activation of Iba-1 positive neurons, and suppressed the expression of inflammatory factors and pyroptosis indicators. In addition, ANP improved the cognitive level and motor ability of ICH mice.The results of the study combined with virtual screening and experimental validation showed that ANP has an important contribution in protecting the brain from neuronal damage by regulating the pathways of inflammation and pyroptosis, laying the foundation and innovative ideas for future studies.