Investigating the Protective Mechanism of the Zha-Chong-Shi-San-Pill Ethanol Extract on Cerebral Stroke Based on Network Pharmacology, Molecular Docking, and <i>in vitro</i> Experiments

Objectives To study the antioxidant effects of the traditional Mongolian medicine Zha-Chong-Shi-San-Pill ethanol extract (EEZC) on cerebral stroke for future clinical use. Materials and Methods Active components and mechanisms of EEZC against cerebral stroke were predicted and analyzed via ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS), network pharmacology, and molecular docking. Next, an oxidative damage model was established in bEnd.3 cells using H 2 O 2 to simulate cerebral stroke damage. Multiple cellular tests were performed to verify the mechanisms, including cell counting kit-8 (CCK-8), flow cytometry, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, enzyme-linked immunosorbent assay (ELISA), and Western blotting. Results It was found that EEZC conferred protection against cerebral stroke H 2 O 2 -treated bEnd.3 cells. Moreover, in vitro experiments revealed that EEZC increased cell survival rates and suppressed cell apoptosis and reactive oxygen species (ROS) release. Moreover, EEZC reduced the levels of interleukin-6 (IL-6) and MMP-9, and increased the levels of interleukin-10 (IL-10), superoxide dismutase (SOD), glutathione (GSH), vascular endothelial growth factor (VEGF), phospho-endothelial nitric oxide synthase (p-eNOS), phospho-phosphatidylinositide 3-kinases (p-pi3k), and phospho-protein kinase B (p-AKT). Conclusion These results strongly suggested that EEZC might protect against cerebral stroke via the PI3K/Akt/eNOS signaling pathway.