Novel mechanism of Guizhi Jia Huangqi decoction disrupting HBV lifecycle via cAMP/PKC/PKB axis beyond NTCP blockade.

BACKGROUND AND PURPOSE: The eradication of covalently closed circular DNA (cccDNA) remains the primary obstacle to a functional cure for chronic hepatitis B virus (HBV) infection. While the entry inhibitor Myrcludex B, which targets the sodium taurocholate co-transporting polypeptide (NTCP), represents a significant advance. It does not address the pre-existing cccDNA pool. In contrast, Guizhi Jia Huangqi Decoction (GZHQ), a classic traditional Chinese medicine (TCM) formulation, exhibits multi-targeted therapeutic potential. Although both GZHQ and Myrcludex B modulate NTCP to impede HBV entry, their distinct mechanisms and comparative efficacy, particularly concerning cccDNA suppression, have not been systematically elucidated. This study was designed to conduct a rigorous comparative analysis of these two therapeutic strategies.

STUDY DESIGN AND

METHODS

Using a HepG2.2.15-hNTCP cell model of HBV infection, we treated cells with either GZHQ-containing serum or Myrcludex B. Key virological markers, including hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and HBV DNA, were quantified by enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (qPCR). The expression of NTCP and its associated endocytic machinery was assessed by Western blot. Furthermore, network pharmacology integrated with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed to identify the bioactive constituents of GZHQ and predict their molecular targets.

RESULTS

Both GZHQ-containing serum and Myrcludex B significantly reduced the levels of HBsAg, HBeAg, and extracellular HBV DNA. Mechanistically, Myrcludex B inhibited HBV entry by downregulating epidermal growth factor receptor (EGFR), epsin-15 (EPS15), and Ras-related protein Rab-5 (Rab5), thereby blocking clathrin-mediated endocytosis. However, it exerted no inhibitory effect on cccDNA levels or its transcriptional activity. In stark contrast, GZHQ suppressed the cyclic adenosine monophosphate (cAMP)/protein kinase C (PKC)/protein kinase B (AKT) signaling axis, leading to reduced NTCP expression and, critically, the direct inhibition of cccDNA transcription. GZHQ did not interfere with the clathrin-mediated endocytic axis or phosphodiesterase function. Network pharmacology identified 39 bioactive components in GZHQ, which synergistically targeted the phosphatidylinositol 3-kinase (PI3K)/AKT and cAMP axes to exert their antiviral effects.

CONCLUSION

GZHQ effectively inhibited HBV invasion by modulating the cAMP/PKC/AKT/NTCP signaling axis and uniquely suppressed cccDNA transcriptional activity. By targeting both viral entry and the cccDNA reservoir, GZHQ demonstrated superior antiviral efficacy in vitro compared to Myrcludex B. These findings warrant further in vivo validation to explore the full pharmacological potential and safety profile of GZHQ as a promising multi-targeted therapy for chronic HBV infection.