Astragaloside IV reduces hepatitis B surface antigen level via monocyte/macrophages in chronic HBV infection mice

Background: Astragaloside IV (AS-IV) is one main bioactive component in the herb Astragalus membranaceus that is commonly used to enhance human immune function. However, its extremely low aqueous solubility severely restricts its absorption in the body, thereby limiting its potential for clinical translation. Despite its established immunomodulatory and antiviral properties against several viruses, the therapeutic effect of AS-IV on chronic Hepatitis B Virus (HBV) infection—especially its ability to reduce hepatitis B surface antigen (HBsAg), a key target for functional cure of HBV—remains insufficiently investigated. Purpose: This study aimed to address the poor bioavailability of AS-IV by developing a novel nanoformulation, and investigate its therapeutic efficacy and underlying mechanisms in a chronic recombinant adeno-associated virus (AAV) carrying a replicable HBV genome (AAV-HBV) mouse model. Study Design: AS-IV nanoparticles were prepared using different packaging strategies. Their therapeutic benefit was evaluated on AAV-HBV mouse model by measuring hepatitis B surface antigen (HBsAg) levels. Methods: The nanoparticles were quality controlled by Malvern Laser Particle Size Analyzer, Transmission Electron Microscopy, and Infrared Spectroscopy. Immune mechanisms were dissected by single-cell RNA sequencing (scRNA-seq) and flow cytometry. Results: Among all tested formulations, Pluronic® F-127-formulated AS-IV (P-AS) exhibited the best performance, with significantly improved aqueous solubility, uniform spherical morphology (100–150 nm), and good in vivo safety (no obvious hepatotoxicity or systemic toxicity). P-AS treatment reduced serum HBsAg levels (approximately 50 %, p < 0.0001) without altering HBV RNA expression, suggesting that the reduction of HBsAg is mediated through a post-transcriptional mechanism. scRNA-seq analysis revealed that P-AS markedly increased hepatic infiltration of myeloid cells, especially monocyte/macrophages (over 2-fold). Furthermore, P-AS induced the polarization of these cells toward the classically activated macrophage (M1) phenotype, enhanced their phagocytosis, and activated key pro-inflammatory pathways. Critically, depletion of monocyte/macrophages completely abrogated the HBsAg-lowering effect of P-AS, confirming that the therapeutic efficacy of P-AS depends on the recruitment and functional activation of monocyte/macrophages. Conclusion: This study successfully developed a Pluronic® F-127-based AS-IV nanoformulation (P-AS) with enhanced bioavailability and safety. P-AS effectively reduces serum HBsAg levels in chronic HBV-infected mice through a monocyte/macrophage-dependent mechanism, providing a promising preclinical basis for the development of AS-IV-based nanotherapeutics for chronic hepatitis B treatment.