Hedyotis chrysotricha aqueous extract inhibits hepatitis B surface antigen and viral replication via hepatocyte nuclear factor 4α regulation

Background

Chronic hepatitis B virus (HBV) infection continues to pose a significant global public health challenge, as current antiviral treatments have not yet succeeded in completely eradicating the virus. Traditional Chinese Medicine (TCM) offers unique advantages in treating chronic hepatitis B. Furthermore, the aqueous extract of Hedyotis chrysotricha (Palib.) Merr (HCM), a synonym of Exallage chrysotricha (Palib.) Neupane & N.Wikstr., has shown potential anti-HBV properties. Nevertheless, its pharmacological effects and precise mechanisms of action remain unclear.

Purpose

We aim to evaluate the anti-HBV efficacy of the aqueous extract of HCM and investigate its underlying mechanisms.

Study design

Ultra performance liquid chromatography-triple time-of-flight mass spectrometry (UPLC-Triple-TOF/MS) was used to identify the HCM components, and the anti-HBV efficacy of HCM was evaluated using a transgenic HBV mouse model, as well as the HepG2.2.15 and HepAD38 HBV cell lines.

Methods

We first identified the chemical components of HCM using UPLC-Triple-TOF/MS, combined with relevant reference standards. Quantification was achieved using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), followed by methodology validation. We used a transgenic HBV mouse model along with the HepG2.2.15 and HepAD38 HBV cell lines to assess its anti-HBV efficacy. Besides, network pharmacology, molecular docking and transcriptomics were employed to explore the underlying anti-HBV mechanisms.

Results

UPLC analysis, using authentic reference standards, identified 19 major chemical components in HCM, including isorhamnetin, monotropein, sesamoside, and kaempferol, which were newly identified as components in this matrix. Our study demonstrates that HCM significantly inhibited HBV replication and transcription in both the transgenic HBV mouse model and HBV cell lines, with a notable reduction in HBsAg levels and the potential inhibition of cccDNA, which serves as a stable viral DNA reservoir in the nucleus of infected hepatocytes, driving the replication and persistence of HBV. Further analysis using network pharmacology and transcriptomic approaches suggests that the anti-hepatitis B mechanism may involve the upregulation of the phosphatidylinositol 3-kinase - protein kinase B (PI3K-AKT) and mitogen-activated protein kinase - extracellular signal-regulated kinase 1 and 2 (MAPK-ERK1/2) pathways. Additionally, hepatocyte nuclear factor 4α (HNF4α) was shown to play a critical role in HBV inhibition, with its function negatively regulated by the PI3K-AKT and MAPK-ERK1/2 pathways.

Conclusion

The aqueous extract of HCM not only inhibits HBV replication and transcription but also significantly suppresses HBsAg levels. The underlying mechanism likely involves the concurrent activation of the PI3K-AKT and MAPK-ERK1/2 pathways, resulting in the inhibition of HNF4α activity, which in turn suppresses HBV.