PI3K/AKT/eNOS Modulation and Angiotensin II Suppression by <i>Dryopteris crassirhizoma</i> Rhizome Extract: Computational and Experimental Evidence

The rhizome of <i>Dryopteris crassirhizoma</i> (DC) Nakai has long been prescribed in Chinese and Korean medicine to treat parasitic infestations, colds, and hemorrhages. DC is rich in antioxidant phytochemicals that may enhance endothelial nitric oxide bioavailability, thereby promoting vasorelaxation; however, this potential has not been thoroughly investigated. Absorption, distribution, metabolism, excretion, and toxicity-screened DC constituents were retrieved from Traditional Chinese Medicine Systems Pharmacology and incorporated into herb-compound-target-disease and protein-protein interaction networks. Network pharmacology analysis, combined with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment, identified the phosphoinositide 3-kinase/Akt/endothelial nitric oxide synthase (eNOS) signaling cascade and the suppression of angiotensin II signaling as the dominant pathways. Molecular docking, quantitative structure-activity relationship regression, 50 ns molecular dynamics simulations, and Molecular Mechanics Poisson-Boltzmann Surface Area calculations were performed to identify key DC constituents and their potential binding targets. These computational analyses converged on two prominent ligand-target pairs: flavidin-phosphoinositide 3-kinase (PI3K) p110α and deserpidine-angiotensin II type 1 receptor. In isolated thoracic aortic rings from Sprague-Dawley rats, the water extract of DC (DCW; 3, 10, 30, and 100 μg/mL) elicited a concentration-dependent and endothelium-dependent relaxation. This vasorelaxant response was significantly attenuated by the pharmacological inhibitors of PI3K, AKT, eNOS, soluble guanylyl cyclase, and cyclic guanosine monophosphate-dependent protein kinase, but was unaffected by K⁺ or Ca²⁺ channels. Consistent with pathway activation, Western blot analysis showed a significant increase in the p-AKT/AKT ratio at 100 μg/mL DCW<i>.</i> Furthermore, DCW at 100 μg/mL effectively suppressed angiotensin II-induced contractions. Therefore, DCW promotes vasorelaxation through the activation of endothelial PI3K/AKT/eNOS signaling pathway while blocking angiotensin II-mediated vasoconstriction. The integrative computational and ex vivo evidence provides a mechanistic rationale for the potential development of advancing DC as a phytotherapeutic candidate for vascular disorders.