Exploring the Mechanism of Action of Isorhynchophylline in Stroke Based on Network Pharmacology and Molecular Docking Technology.

Abstract

Introduction: This study aimed to elucidate the neuroprotective mechanism of Isorhynchophylline (IRN) in stroke.

Methods: Using network pharmacology, the action targets of IRN were predicted using the SwissTargetPrediction, HERB, and TCMIP 2.0 databases. We identified sixty-five targets common to IRN and stroke. We conducted GO and KEGG enrichment analyses using R software and constructed a compound- target- pathway- disease network. Additionally, molecular docking experiments were performed to investigate the binding interactions between IRN and its main targets. Subsequently, an oxygen-glucose deprivation/reoxygenation (OGD/R) model was established using the human neuroblastoma cell line SH-SY5Y under hypoxic conditions (1% O2). The effective working concentration of IRN was determined by measuring cell viability using the CCK-8 assay. Then, an ELISA experiment was conducted to detect the expression of inflammatory factors IL-1β and IL-10. The activity of reactive oxygen species (ROS) was measured using the dual fluorescence detection method. TUNEL staining was employed to assess the apoptosis of cells. Finally, a Western blot was used to examine the effect of IRN on EGFR and AKT expression in cells.

Results: The results of network pharmacology analysis indicated that the main targets of IRN in treating stroke are EGFR, mTOR, JAK2, AKT, and JUN. Enrichment analysis revealed that IRN is associated with neurotransmitter receptor function and participates in the EGFR signaling pathway. Molecular docking results showed that it has a particularly strong binding affinity for AKT1 (binding energy ≈ -8.7 kcal/mol). The in vitro experiments showed that IRN treatment significantly alleviated the damage induced by OGD/R. Specifically, the levels of IL-1β and ROS decreased, the level of IL-10 increased, and the apoptosis rate decreased. Western blot results showed that, compared with the OGD/R group, IRN significantly upregulated the expression of p-EGFR and p-AKT. We demonstrated that IRN employs a multi-target approach to counteract stroke pathology, as revealed by computational predictions and experimental validation.

Discussion: The convergence of network pharmacology, molecular docking, and in vitro assays enabled the identification of key targets, including EGFR, AKT1, and JAK2. These methods also indicate that IRN can simultaneously regulate the inflammatory, oxidative stress, and apoptotic processes. Compared to singletarget agents, IRN exhibits superior multi-target therapeutic potential. These findings, derived from database analyses and cell-based assays, provide a comprehensive overview of IRN's pharmacological effects, which are consistent with its known neuroprotective benefits. Although these results are obtained from cell-based models and predictive databases, they provide a cohesive mechanistic rationale that is consistent with the neuroprotective properties ascribed to IRN. Further studies in animal models and in clinical settings must be conducted to apply these insights for therapeutic purposes.

Conclusion: IRN can exert neuroprotective effects against stroke by regulating key targets, including EGFR. It exhibits anti-inflammatory, anti-oxidative, and anti-apoptotic activities, which provide a reasonable basis for developing IRN as a drug for treating stroke.