Diosgenin in Dioscorea spongiosa Suppresses Glycolysis-Driven Angiogenesis as a ROCK1 Inhibitor

The rhizome of Dioscorea spongiosa is used in Chinese herbal medicine as Bixie (BX), a drug with antirheumatic effects. To explore the mechanisms underlying its antiangiogenic actions, adjuvant-induced arthritis (AIA) rats were administered with BX extract for 28 days. The therapeutic efficacy was evaluated by serological, histological, and immunohistochemical tests. Metabolites were quantified by kits and UPLC–MS. The BX extract was separated into 30 fractions by silica gel chromatography. The antiangiogenic effects of reagents were assessed based on wound-healing, migration, and tube-formation experiments using human umbilical vein endothelial cells (HUVECs). Protein targets were pulled down by a biotin-conjugated compound. These proteins and constituents in BX-related samples were identified by LC–MS. The target was validated by molecular simulation, immunoprecipitation, thermal shift assays, and enzymatic digestion. BX mitigated the metabolic abnormalities, inflammation, and synovial invasion of AIA rats. Angiogenesis in white adipose tissues and joints was suppressed, shown by the decrease of vessels, angiogenic cytokines, and CD31. The BX extract inhibited glycolysis and angiogenic capabilities of HUVECs. The expression of some genes/proteins related to VEGF and glycolytic signals was reduced. Diosgenin was the only component with high concentrations in the two most effective fractions and a BX-treated rat’s serum. It curbed the glycolysis-fueled activation of HUVECs. Diosgenin competed with biotin–diosgenin when bound to ROCK1. It stabilized ROCK1 and inhibited ROCK1 phosphorylation. Its effects on VEGF-treated cells were similar to GSK429286 (a ROCK1 inhibitor) and weakened by arachidonate (a ROCK1 activator). It hardly affected ROCK1-silenced HUVECs. In short, diosgenin inhibits ROCK1 and suppresses angiogenesis via glycolysis downregulation.