Background/objectivesLiver fibrosis is the consequence of the wound-healing response of the liver to frequent injury. Calycosin is a flavonoid, which has been documented for its hepatoprotective properties. The aim of this study was to evaluate the hepatoprotective action of calycosin in a model of ethanol-induced liver injury (EILI) in rats. For mechanistic insights, we aimed to measure tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB), and hypoxia-inducible factor-1 alpha (HIF-1α) protein levels in liver samples and evaluate the ability of calycosin to bind these pathologic molecules.MethodsNetwork pharmacology and molecular docking were performed for discovering the interacting target proteins. Forty male rats were assigned into five groups: 1) the vehicle, 2) the calycosin per se group, 3) the EILI control group, and 4) and 5) the EILI + calycosin (5 or 10 mg/kg) groups. The liver homogenates were used for ELISA measurement of TNF-α, NF-κB, and HIF-1α. Furthermore, liver specimens were used for histopathological and ultrastructural investigations.ResultsThe network pharmacology study confirmed the role of TNF-α/NF-κB/HIF-1α signaling in EILI, and molecular docking explored the possible interaction between calycosin and these three molecules (docking score = −7.6, −7, and −7.6 kcal/mol). The rat study showed that calycosin was able to attenuate histopathological and ultrastructural changes in rat livers, reduce collagen accumulation, and prevent the increases shown in liver enzyme activities (2.17-fold for ALT and 2.26-fold for AST).ConclusionThus, these integrated in silico and in vivo studies confirmed the hepatoprotective effect of calycosin against the EILI rat model and provided a mechanistic insight through TNF-α/NF-κB/HIF-1α signaling. Further studies are warranted to fully explore the protective mechanism of calycosin in EILI.