Reagents and materials

BA (98% HPLC purity) was purchased from ChromaBio (Chengdu, China). BA was dissolved in DMSO (40 mg/mL) and stored at 4 °C. Before use, the BA stock solution was diluted using cell culture medium and sterilized using a 0.22-μm filter. Dox, compound C, tamoxifen, and metformin were procured from Meilunbio (Dalian, China). Fulvestrant was purchased from Beyotime Biotechnology (Shanghai, China). 17β-estradiol was supplied by Sigma-Aldrich (St. Louis, USA).

Cell culture and transfection

HepG2 cells were cultured in Dulbecco’s Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, Waltham, Australia) at 37 °C in 5% CO2. HepG2 cells were transfected with the pHBV1.2 plasmid using Lipo6000™ Transfection Reagent (Beyotime Biotechnology) according to the manufacturer’s instructions. The pHBV1.2-transfected HepG2 cells were called “pHBV1.2-HepG2” cells [7]. The pHBV1.2 plasmid — which contained a 1.2-fold HBV DNA genome (genotype C, subtype Adr) — was gifted by Prof. Zheng-hong Yuan from Fudan University, China.

The ERα gene was knocked down in HepG2 cells using an shRNA lentiviral vector (LV), which was purchased from GeneChem (Shanghai, China). All protocols were carried out according to the manufacturer’s instructions.

HBV infection and treatment in mice

Male and female BALB/c mice (age: 5–6 weeks, weight: 18–22 g) were purchased from SLACCAS (SPF II Certificate. No. SCXK2012-0002, Shanghai, China). The mice were maintained in plastic cages at 23 ± 2 °C under a relative humidity of 50% ± 10%, with free access to food and water.

Each BALB/c mouse was hydrodynamically injected with 10 μg of the pHBV1.2 plasmid (dissolved in 2 mL of PBS) via the tail vein [15]. The next day, the infected mice were randomly divided into four groups and treated with the relevant treatment agents (blank control, 10 mg/kg BA, 20 mg/kg BA, and 40 mg/kg BA; intragastric administration once a day). Seven days after infection, the mice were sacrificed, and retro-orbital blood samples were collected and centrifuged. The serum was used for ELISA and RT-PCR analysis. Liver tissue was isolated to examine hepatic injury, and a liver tissue homogenate was prepared to extract RNA and proteins for the subsequent analysis of signaling pathway activity.

All animal experiments were performed in accordance with the “Guidelines for the Care and Use of Medical Laboratory Animals” (Ministry of Health of the People’s Republic of China, 1998), and the study was approved by the Ethics Committee of Fudan University (Shanghai, China) (approval No. 2015-O3-HC-SXL-01).

Detection of HBeAg and HBsAg in vivo and in vitro

In vitro: HBsAg and HBeAg were detected using the ELISA method. The kits were purchased from Kehua Bioengineering Co., Ltd., Shanghai, China. pHBV1.2-HepG2 cells were incubated with BA (0–100 μM), and supernatants were collected every 2 days for the ELISA-based quantification of HBeAg and HBsAg.

In vivo: Blood collected from mice was centrifuged to collect serum. The serum was diluted 2000-fold, and the levels of HBeAg and HBsAg were detected using an ELISA kit.

Transcriptomics analysis

The pHBV1.2-HepG2 cells were treated with 100 μM BA for 96 h. The cell samples were used for mRNA isolation, library preparation, and sequencing by the Majorbio Bio-pharm Technology Corporation (Shanghai, China). Differentially expressed genes were subjected to Gene Ontology (GO) and Kyoto Genes and Genomes (KEGG) pathway enrichment analysis via the free online Majorbio Cloud Platform (https://www.majorbio.com). The NCBI accession number is PRJNA 799795.

Molecular docking

Two PDB files of the three-dimensional (3D) structure of ERα (1GWR and 3ERT) were selected for docking analysis and downloaded from the Protein Data Bank (https://www.rcsb.org/). 1GWR was combined with the natural agonist E2, and 3ERT was combined with antagonists. These complexes were preprocessed before Glide Docking using the protein preparation wizard of the Maestro10.2 program by Schrodinger. Meanwhile, the compound was prepared using the Maestro ligand preparation wizard. The Glide Docking module (Glide 5.8) in Maestro 10.2 was used to dock the compound into the binding site of both receptors. The compound was subjected to a Monte Carlo Multiple Minimum conformational search using the OPLS_2005 force field, and the most reasonable conformation was selected. In addition, another molecular docking software, AutoDock 4.2, was used along with the visualization software Pymol 2.4 (Schrodinger) to dock BA with the 3ERT and 1GWR regions of ERα. Based on the results of molecular docking, the free energy of binding between the compound and the protein and the estimated Ki value were predicted. The experimental Ki value was obtained from the PDB database.

Surface plasmon resonance (SPR) analysis of the kinetic constants of BA against ERα

The SPR technique was applied to characterize the kinetic constants of BA against human ERα (ab82606, Abcam, London, UK) using a Biacore T200 instrument (GE Healthcare, Chicago, USA). Experiments were performed at 25 °C. ERα was immobilized onto the CM5 sensor chip surface using the Amine Coupling kit. BA was serially diluted two-fold from 58.9 μM to 0.12 μM using the running buffer (HBS-EP+). Then, the mixtures were injected at a flow rate of 30 μL/min, with an association time of 45 s and a dissociation time of 60 s. The surface was then regenerated with HBS-EP+ buffer for 30 s at a flow rate of 30 μL/min.

The kinetic data were processed with Evaluation Software version 3.0. The data were fitted globally to a 1:1 binding model. The association Ka (M−1·s−1) and dissociation Kd (s−1) kinetic rate constants were obtained. The equilibrium dissociation constant KD (M) was determined based on the equation KD = Kd/Ka.

All the reagents and kits were purchased from GE Healthcare.

ERE luciferase reporter gene experiment

The pHBV1.2 plasmid was transfected into HepG2 cells using the Lipo6000™ transfection reagent. The cells were then transfected with the ERE luciferase reporter plasmid (11528ES03; Yeasen Biotechnology, Shanghai, China) and ESR1-expressing plasmid (constructed by Fenghui Biotechnology, Beijing, China) using the Lipo6000™ Transfection Reagent for 12 h. This was followed by treatment with BA alone or BA + tamoxifen (100 nM) for 12 h. E2 (10 nM) was used as the positive control. The cells were pretreated with reagents from a luciferase reporter gene assay kit (Yeasen Biotechnology) according to the manufacturer’s instructions. The relative light unit (RLU) values were detected with an automatic fluorescence chemiluminescence analyzer (Fluoroskan Ascent FL, Thermo Scientific, Waltham, USA).

Gene transcription analysis

Total RNA was isolated from HepG2 cells using the TRIzol reagent and reverse transcribed to cDNA using the PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa Bio, Beijing, China), as previously reported [16]. qRT-PCR was performed to analyze gene transcription using the SYBR Premix Ex Taq™ (TaKaRa Biomedical Technology, Beijing, China) and StepOne Plus Real-Time PCR System (Thermo Scientific). The following thermocycling parameters were applied: 95 °C for 15 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The RQ values were calculated using the ΔΔCT method. GAPDH was used as the internal reference (Table 1).

Table 1 Primers used for RT-PCR.Western blot

The culture medium was discarded from the culture flasks and plates, and the cells were washed with PBS. Western and immunoprecipitation lysis buffer (Beyotime Biotechnology) was added to the cells, and the supernatant was collected after centrifugation (12,000 × g for 15 min). Subsequently, a BCA detection kit (Beyotime Biotechnology) was used to measure protein concentrations. Protein samples were separated using SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Scientific). PVDF membranes were then blocked with Western blocking solution (Beyotime Biotechnology) for 1 h. Subsequently, the membranes were incubated overnight with the indicated primary antibodies (1:1000 dilution) on a shaker at 4 °C. The membranes were then incubated with the secondary antibody (1:1000) on a shaker at room temperature (25 °C) for 1.5 h. The obtained bands were visualized via chemiluminescence reactions (ECL, Merck Millipore, Birica, USA), and gray values were analyzed using Image J software (NIH, Bethesda, Rockville, USA).

Antibodies against ERα, p-ERα, p-p38MAPK, C/EBPα, AMPKα, p-AMPKα, LKB1, p-LKB1, β-actin, and GAPDH were obtained from Cell Signaling Technology (Danvers, USA). The antibody against ERβ was purchased from Wanlei Biotechnology (Shenyang, China).

Cellular ATP and AMP measurement

The treated HepG2 cells were washed with PBS, lysed on ice using a lysis buffer for 10 min, and centrifuged at 4 °C and 12,000 × g for 5 min to obtain the supernatant. The cellular ATP and AMP levels in the supernatant were detected using an ATP detection kit (Beyotime Biotechnology) and AMP detection kit (Fantaibio, Shanghai, China) according to the manufacturer’s instructions. For ATP detection, chemiluminescence was determined using an automatic fluorescence chemiluminescence analyzer. Meanwhile, AMP content was measured using a microplate reader at A450 nm. The ATP and AMP concentrations of the samples were calculated according to standard curves.

Statistical analysis

Data were presented as the mean ± standard deviation. All experiments were repeated at least thrice. The obtained data were analyzed using Student’s t-tests or one-way ANOVA followed by Bonferroni’s test. The associations between HBeAg and HBsAg levels were analyzed using Pearson’s correlation coefficient. P < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 8 (La Jolla, USA).