Cell culture and antibodies

HepG2-NTCP and HepAD38 cells were kindly donated by Prof. Ningshao Xia (Xiamen University, China), Primary human hepatocytes (PHHs) were obtained from Liver Biotechnology (China), and Huh-7 cells were obtained from Health Science Research Resource Blank. HepG2-NTCP and Huh-7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. HepAD38 cells were cultured in DMEM medium containing 10% foetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin, with the addition of 400 µg/mL G418 (Merck) to the medium. PHHs were cultured in Williams E medium supplemented with 5 µg/mL transferrin, 10 ng/mL epidermal growth factor (EGF), 3 µg/mL insulin, 2 mM L-glutamine, 18 µg/mL hydrocortisone, 40 ng/mL dexamethasone, 5 ng/mL sodium selenite, 2% dimethyl sulfoxide (DMSO), 100 U/mL penicillin, and 100 µg/mL streptomycin. All cell lines were cultured in a 37ºC cell culture incubator with a 5% CO2 concentration.

Rabbit anti-HBsAg (NB100-62652) was purchased from Novus (USA). Rabbit anti-LC3B (3868 S), Mouse anti-SQSTM1/p62 (88588 S), Rabbit anti-Flag (#14793S) and Rabbit anti-HA (#3724) were purchased from Cell Signaling Technology (USA). Rabbit anti-HBx (RD981038100) was purchased from BioVendor (USA). Rabbit anti-β-actin monoclonal antibody (sc-1616-R) was obtained from Santa Cruz Biotechnology (USA). Rabbit anti-H3K9me3 polyclonal antibody (17–625), rabbit anti-H3K27me3 polyclonal antibody (17–622), mouse anti-H3K4me3 monoclonal antibody (17–678) were obtained from Merck Millpore (Germany).

Nano-Glo® HiBiT lysis assay

To screen for small molecules targeting HBx, we used the Nano-Glo® HiBiT Lytic system (N3030, Promega, Wisconsin, USA) as described in detail previously [19]. In brief, after transfection of HiBiT-HBx plasmid into cells, the cells were treated with different herbal monomer compounds for 2 days. After washing the cells with PBS, 200 µL of Nano-Glo® HiBiT Lytic Buffer was added to each well to lyse the cells. The lysate was transferred to a new 1.5 mL Ep tube. After centrifugation, 10 µL of Nano-Glo® HiBiT Lytic Substrate was added to 50 µL of clarified lysate to measure luciferase activity. The measured luminescence signal was proportional to the amount of HiBiT-tagged HBx in the cell lysate.

MTT assay

(3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to test cell viability. Cells were seeded into 96-well plates, and drug concentrations from 0 µM to 200 µM were added to the plates when the cell density reached 80%. After 72 h of incubation, 5 mg/mL MTT was added to each well, and the plates were incubated for 4 h in the dark. Then, 100 µL of DMSO was added to each well to dissolve the crystals, and a microplate reader was used to measure the half-maximal cytotoxic concentration (CC50).

Alamar blue assay

Various cell lines were seeded in twelve-well culture plates at appropriate densities. The cells were treated with different concentrations of asiatic acid. After the indicated time, 1× Alamar Blue was added to the cells, which were then incubated at 37 °C for 4 h. Absorbance values were detected using an enzyme labeling instrument with the excitation and emission wavelengths set to 560 nm and 590 nm, respectively.

Viruses and infection

HBV virus was collected from the culture supernatant of HepAD38 cells and concentrated with 5% PEG8000. Virus diluted with infection medium was mixed with 5% PEG8000 and added to the well plates. The following day, the medium was discarded and the cells were washed twice with PBS for further experiments.

RNA extraction and realtime PCR

TRNzol Reagent (Tiangen, China) was used to extract RNA. After removing genomic DNA (gDNA), the RNA was reverse transcribed to complementary DNA (cDNA) by using the FastKing RT Kit (KR116-02, Tiangen, China). Then, cDNA was amplified using Fast Start Universal SYBR Green Master (Roche, Switzerland). The fluorescence signals were analyzed by the 2−ΔΔCt method to determine the relative mRNA expression levels. β-actin mRNA was used as an internal reference. The specific primers used are listed in Table S1.

Western blotting

Western blotting was used to analyze proteins expression. First, proteins were extracted from cells by using RIPA with protease inhibitor. Then, the BCA protein assay kit was employed to determine the protein concentration, and the protein content in the lysate was calculated based on a standard curve. Subsequently, 30 µg extracted proteins were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to the polyvinylidene fluoride (PVDF) membranes. Next, the membranes were blocked with 5% non-fat dry milk for 2 h and incubated overnight at 4 °C with diluted primary antibodies. The membranes were then washed and incubated with secondary antibodies at room temperature for 2 h. Finally, the proteins were visualized using enhanced chemiluminescence (ECL) reagents, with β-actin serving as a reference control.

Cycloheximide (CHX) chase assay

HBV-infected HepG2-NTCP cells were treated with 20 µM asiatic acid and then incubated with 10 µg/mL CHX for 1, 2, 3 h. Then, the cells were collected for protein extraction. The level of HBx was measured by Western blotting.

Immunofluorescence staining

Cells were seeded on slides and treated according to the requirements of specific experiments. The cells were fixed with 4% paraformaldehyde for 20 min, permeabilized at room temperature with 0.3% Triton X-100, and blocked with 5% BSA for 1 h. The cells were then incubated with anti-LC3B antibodies for 1 h, followed by staining with fluorochrome conjugated secondary antibodies. The nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI), and images were captured using a confocal laser scanning microscope (Leica DMi8, Germany).

Chromatin immunoprecipitation (ChIP)

ChIP assay was performed following the manufacturer’s protocol (17-10086, Merck Millipore, Darmstadt, Germany). Cells cultured in 10-cm dishes were harvested by scraping in cold PBS and centrifuged at 800 × g and 4 °C for 5 min. Cell lysis buffer was used to resuspend the cells. After centrifugation, the nuclei were fixed with 1% formaldehyde at room temperature for 10 min to cross-link DNA with proteins. Then, the pellets were resuspended in nuclear lysis buffer and DNA was fragmented by sonication. The supernatants were diluted with dilution buffer at a 1:10 ratio and 1% of the dilution was used as the input. The remaining diluted chromatin was added with the antibody targeting the protein and 20 µL of protein A/G magnetic beads (17-10086, Merck Millipore, Germany) at 4 °C overnight. On the following day, the supernatant was removed and the beads were washed sequentially with 500 µL each of Low Salt Immune Complex Wash Buffer, High Salt Immune Complex Wash Buffer, LiCl Immune Complex Wash Buffer, and TE Buffer for 5 min each time. The beads were resuspended using 100 µL of ChIP Elution Buffer containing Proteinase K and incubated at 62 °C for 2 h, followed by further incubation at 95 °C for 10 min. Immunoprecipitated chromatin was extracted and purified by using phenolchloroform, isopropanol and ethanol, and analyzed by Taq-man probe PCR. The specific HBV cccDNA primer and probe used are listed in Table S1.

HBV core DNA extraction and quantitation

Cells were treated with the lysate (10 mM Tris-HCl, 1 mM EDTA, 0.5% NP-40, 2% sucrose, pH 8.0) and incubated for 15 min at 37 °C. After centrifugation, 10 mM MgCl2 and 40 U/mL DNase were added to the supernatant of the collected lysate, followed by incubating for 4 h at 37 °C. The HBV core capsids were precipitated with 5% PEG8000. Then, the precipitate was incubated with proteinase K at 45 °C overnight. Finally, HBV core DNA was extracted with phenol-chloroform. Fast Start Universal SYBR Green Master Mix was used for quantitative analysis with the pCH9/3091 plasmid as standard. The specific HBV core DNA primers used are listed in Table S1.

Hirt cccDNA extraction and detection

Cells were lysed in 500 µL cell lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% SDS) at 37 °C for 20 min. Then the lysate solution was transferred to a new tube, 125 µL KCl (2.5 M) was added to the cell lysate, and the solution was incubated overnight at 4 °C. The next day, after centrifugation at 4℃ and 12,000 × g for 30 min, the supernatant was collected. Then 500 µL of phenolchloroform was added with repeated mixing by oscillation. After centrifugation at 16,000 × g for 3 min, the supernatant was collected. An equal volume of isopropanol (approximately 500 µL) and 1 µL of glycogen were added to the supernatant. After centrifugation at 16,000 × g for 30 min at 4 °C, the supernatant was discarded and added 1 mL of 75% ethanol to wash the precipitate. Then, the DNA precipitation was dried at 37 °C in a metal bath, and 20 µL ddH2O was added to dissolve the precipitate overnight. HBV cccDNA expression levels were detected by Taq-man probe PCR. The specific HBVcccDNA primers used are listed in Table S1.

Southern blotting/northern blotting

The DIG-High Prime DNA Labeling and Detection Starter Kit or DIG Northern Starter Kit was used to analyze HBV DNA or total HBV RNA, respectively. DNA and RNA samples were separated on a 1% agarose gel. DNA or RNA in the gel was transferred to a nylon membrane by siphoning. Then DNA or RNA was immobilized on the membrane by using UV cross-linking, and hybridized with labeled HBV DNA or RNA probes overnight at 42 °C (DNA) or 68 °C (RNA). The membrane was washed with washing buffer to remove unbound probes and was then incubated with anti-digoxin antibody. Finally, X-ray film was used to visualize the results.

Enzyme-linked immunosorbent assay (ELISA)

Commercial enzyme linked immunosorbent assay kit (KHB, China) was used to measure the HBsAg in supernatant. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations in mouse serum were measured using a commercially enzyme assay (Wanleibio, China) according to the manufacturer’s protocol.

Mouse model

A precursor cccDNA (prcccDNA)-based model of persistent HBV infection was used to validate the inhibitory effect of asiatic acid on HBV. The 4 µg prcccDNA plasmid and 4 µg Cre recombinase plasmid were injected into mice by hydrodynamic injection. After one week, all the mice were divided into the following four groups: vehicle group, ETV group (0.02 mg/kg), AA (30 mg/kg) and AA + ETV, with six mice per group. The mice were treated with asiatic acid or saline every two days, and serum was collected every four days. After 24 days, the mice were sacrificed to harvest liver tissue. All animal experiments were approved by the Animal Ethics Committee of Chongqing Medical University.

Immunohistochemistry

Liver tissues from the mouse model were used to prepare paraffin sections. After dewaxing, tissue antigens were retrieved by microwaving in sodium citrate buffer (10 mmol/L, pH 6.0). Then, the sections were permeated with 0.5% Triton X-100 (Sigma, USA) and the endogenous peroxidase was sequestered. And goat serum was added for blocking. Next, the specimens were incubated with primary antibody (anti-HBs working solution, ZM-0122, Zhongshan Jinqiao Biological Technology) overnight at 4 °C and were then incubated with a secondary antibody. Finally, the sections were stained with DAB and counterstained with hematoxylin.

Statistical analysis

The data analyses and related statistical graphs in this study were performed using GraphPad Prism 8.0 software. The significance of the results was analyzed using the Mann-Whitney U test. Each experiment was conducted at least three times, and the results were expressed as the mean ± standard deviation. Differences were considered statistically significant when P < 0.05.