Investigating the Protective Effects of Rhubarb on Cerebral Ischemic Stroke Rats Using Network Pharmacology and Molecular Docking

Abstract

Objective The objective of this study is to investigate the pharmacological mechanism of rhubarb, a widely used Chinese medicine for the treatment of cerebral ischemic stroke (CIS), which remains poorly understood. Based on network pharmacology and molecular docking techniques, we further to investigate the protective effcts of rhubarb on CIS rats. Methods To evaluate the oral bioavailability and drug similarity, rhubarb compounds were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and subjected to assessment based on absorption, distribution, metabolism, and excretion criteria. A total of 12 compounds were identified through the utilization of network analysis, and their identification was further confirmed by means of mass spectrometry. Additionally, the SwissTargetPrediction tool successfully predicted 296 potential targets associated with rhubarb, while the databases NCBI, GeneCards, Drug Bank, and Online Mendelian Inheritance in Man collectively identified 3027 targets related to CIS. Among these targets, 195 were found to be potentially associated with rhubarb. To analyze the functional annotation of Gene Ontology (GO) pathway enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, a protein-protein interaction network was constructed using Cytoscape 3.7.2. Next, we established the middle cerebral artery occlusion (MCAO) to evalute the protective effect of rhubarb on CIS rats. First, we using neurological function score to observe the neurologic impairment symptoms. TTC staining was used to measure the cerebral infarction volume. We determined the contents of MDA, SOD, and LDH using Biochemical reagent kits and the levels of TNF-α and IL-1β using ELISA kits. Finally, Western blot were employed to examine the protein expressions of PI3K/Akt signaling pathway. Results A comprehensive identification of 20 genes associated with various pathways, including the PI3K/Akt signaling pathway. Additionally, through docking experiments, it was determined that the hub compounds (Eupatin, sitosterol, rhein, and (-)-catechin) may potentially impact the top 5 targets (AKT1, TNF, VEGFA, TP53, and IL1B). After the intervening with rhubarb, rhubarb signalificantly decreased the neurological function score and cerebral infarction volume; It also lowered the MDA and LDH contents and increased SOD content; Similarly, it decreased TNF-α and IL-1β levels; It increased the expreassion of p-PI3K and p-Akt1. Conclusion Our study demonstrated rhubarb exerts a protective effect via regulating PI3K/Akt signal pathway. These findings serve as a valuable reference for the advancement of drug discovery and development in this field.