Mechanism of Piper longum L. in the Treatment of Cerebral Ischemic Stroke Based on Network Pharmacology and Molecular Dynamics Simulation, With Verification by Surface Plasmon Resonance

Objective: To investigate the potential mechanisms of Piper longum L. in the treatment of cerebral ischemic stroke (CIS) and conduct experimental verification. Methods: Network pharmacology was used to identify intersecting targets of Piper longum L. and CIS. A drug-active component-target-disease network and the target protein-protein interaction (PPI) network were subsequently constructed, to screen major components and key targets. The intersecting targets were subjected to comprehensive Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Molecular docking and molecular dynamics (MD) simulations were performed to assess the binding energy and stability between the main active components and core targets. Surface plasmon resonance (SPR) was also employed to validate these binding interactions, providing further evidence for the underlying molecular mechanisms. Results: In total, 13 active components were identified in Piper longum L., and 60 intersectional targets were acquired. Among the main active components were piperine, 1,2,5,6-tetrahydrotanshinone, sesamin, N-(2,5-dimethoxyphenyl)-4-methoxybenzamide, and pipernonaline. The key targets predominantly consisted of tumor necrosis factor (TNF), interleukin-6 (IL-6), prostaglandin-endoperoxide synthase 2 (PTGS2), cAMP response element-binding protein (CREB1), and interleukin-1β (IL-1β). These identified targets were enriched in 77 GO terms and involved in multiple signaling pathways, including the TNF and cAMP (cyclic adenosine monophosphate) pathways. The main active components exhibit low binding energies with the key targets. MD simulations further demonstrated the structural stability of the complex formed between the main component, sesamin, and the associated CREB1 target, as well as the significance of key residues ILE240 (isoleucine240), ARG241 (arginine241), and PRO244 (proline244) in the binding interaction. Finally, the SPR method provided experimental evidence of the direct binding between sesamin and CREB1. Conclusions: Piper longum L. may exert pharmacological effects on CIS by acting on targets such as CREB1, PTGS2, and TNF, and by modulating signaling pathways including cAMP and inflammation.