Abstract
Introduction: Chronic hepatitis B virus (HBV) infection continues to pose a significant global health burden, and current therapies rarely target the viral covalently closed circular DNA reservoir. Kaempferol (KP), a major flavonoid found in various herbs and plants, exhibits diverse bioactivities, but its potential anti-HBV activity remains unclear. This study aims to investigate the anti-HBV potential of KP and to elucidate its underlying mechanisms. Methods: The HBV-infected Huh7DhNTCP cell, viral stable transfection cell HepG2.2.15, as well as a hydrodynamic injection-based chronic HBV infection mouse model, were established to evaluate the antiviral effects of KP. The levels of HBV RNAs, DNA and proteins were detected using ELISA, western blot, qPCR, immunofluorescence and immunohistochemistry. To investigate the mechanisms, viral promoter activities were assessed via dual-luciferase reporter assays, and relevant transcription factors were validated through qPCR and western blot analysis. Results: KP dose- and time-dependently reduced the levels of viral antigens, RNA, and DNA in vitro, and also significantly lowered viral markers and attenuated HBV-induced hepatic pro-inflammatory cytokines expression in vivo. Furthermore, KP acted in combination with the nucleoside analog entecavir to suppress HBV replication. Mechanistically, KP strongly inhibited the transcriptional activity of the HBV core promoter (Cp), and enhanced the phosphorylation of both extracellular signal-regulated kinase (ERK) and its downstream target forkhead box protein O1 (FOXO1). Importantly, the ERK-specific inhibitor U0126 completely abolished the antiviral effects of KP, confirming that its antiviral activity depended on the ERK/FOXO1 pathway. Discussion: Collectively, our results indicate that KP activates ERK-dependent FOXO1 phosphorylation, leading to transcriptional repression of the HBV Cp and thereby suppression of viral replication. These findings identify KP as a potential candidate for developing novel therapeutics against chronic HBV infection.