A mutual regulatory loop between transcription factor Yin Yang 1 and hepatitis B virus replication influences chronic hepatitis B.

Hepatitis B virus (HBV) infections pose a major threat to human health. HBV can upregulate the expression of the transcription factor Yin Yang 1 (YY1) in in vitro cytological experiments, suggesting an association between YY1 and HBV infection. However, data on YY1 expression in chronic hepatitis B (CHB) patients are lacking. In this study, we aimed to assess the correlation between YY1 expression and HBV infection. We detected serum YY1 levels in 420 patients with chronic HBV infection, 30 patients with chronic hepatitis C virus infection, and 32 healthy controls using an enzyme-linked immunosorbent assay. The correlation between YY1 levels and clinical parameters was analyzed. Meanwhile, the changes of YY1 before and after interferon or entecavir treatment were analyzed. YY1 levels in the liver tissues were detected using immunofluorescence staining. The expression of YY1 in HBV-expressing cells was detected through western blotting. Meanwhile, we explored the effects of YY1 on HBV replication and gene expression. We found that YY1 was highly expressed in the serum and liver tissues of CHB patients. Serum YY1 levels positively correlated with HBV DNA and hepatitis B surface antigen (HBsAg). Additionally, HBV DNA levels increased but HBsAg levels decreased after HBV-expressing cells overexpress YY1. In conclusion, our study demonstrates that YY1 plays an important role in HBV replication and gene expression, providing a potential target for the treatment of CHB.

Inhibition of hepatitis B virus via selective apoptosis modulation by Chinese patent medicine Liuweiwuling Tablet.

BACKGROUND: Liuweiwuling Tablet (LWWL) is a Chinese patent medicine approved for the treatment of chronic inflammation caused by hepatitis B virus (HBV) infection. Previous studies have indicated an anti-HBV effect of LWWL, specifically in terms of antigen inhibition, but the underlying mechanism remains unclear. AIM: To investigate the potential mechanism of action of LWWL against HBV. METHODS: In vitro experiments utilized three HBV-replicating and three non-HBV-replicating cell lines. The in vivo experiment involved a hydrodynamic injection-mediated mouse model with HBV replication. Transcriptomics and metabolomics were used to investigate the underlying mechanisms of action of LWWL. RESULTS: In HepG2.1403F cells, LWWL (0.8 mg/mL) exhibited inhibitory effects on HBV DNA, hepatitis B surface antigen and pregenomic RNA (pgRNA) at rates of 51.36%, 24.74% and 50.74%, respectively. The inhibition rates of LWWL (0.8 mg/mL) on pgRNA/covalently closed circular DNA in HepG2.1403F, HepG2.2.15 and HepG2.A64 cells were 47.78%, 39.51% and 46.74%, respectively. Integration of transcriptomics and metabolomics showed that the anti-HBV effect of LWWL was primarily linked to pathways related to apoptosis (PI3K-AKT, CASP8-CASP3 and P53 pathways). Apoptosis flow analysis revealed that the apoptosis rate in the LWWL-treated group was significantly higher than in the control group (CG) among HBV-replicating cell lines, including HepG2.2.15 (2.92% ± 1.01% vs 6.68% ± 2.04%, P < 0.05), HepG2.A64 (4.89% ± 1.28% vs 8.52% ± 0.50%, P < 0.05) and HepG2.1403F (3.76% ± 1.40% vs 7.57% ± 1.35%, P < 0.05) (CG vs LWWL-treated group). However, there were no significant differences in apoptosis rates between the non-HBV-replicating HepG2 cells (5.04% ± 0.74% vs 5.51% ± 1.57%, P > 0.05), L02 cells (5.49% ± 0.80% vs 5.48% ± 1.01%, P > 0.05) and LX2 cells (6.29% ± 1.54% vs 6.29% ± 0.88%, P > 0.05). TUNEL staining revealed a significantly higher apoptosis rate in the LWWL-treated group than in the CG in the HBV-replicating mouse model, while no noticeable difference in apoptosis rates between the two groups was observed in the non-HBV-replicating mouse model. CONCLUSION: Preliminary results suggest that LWWL exerts a potent inhibitory effect on wild-type and drug-resistant HBV, potentially involving selective regulation of apoptosis. These findings offer novel insights into the anti-HBV activities of LWWL and present a novel mechanism for the development of anti-HBV medications.

Protective Effect of n-Butanol Extract from Viola yedoensis on Immunological Liver Injury.

Viola yedoensis Makino was used to treat inflammation, viral hepatitis, acute pyogenic infection, and ulcerative carbuncles. However, the protective effect on immunological liver injury (ILI) of V. yedoensis had been rarely reported. This study aimed to explore the protective effect of n-butanol extract (BE) from V. yedoensis on ILI in vitro and in vivo. In vitro, the BE significantly inhibited the secretions of Hepatitis B surface antigen (HBsAg) and Hepatitis B e antigen (HBeAg) in the HepG2.2.15 cells and the replication of HBV DNA. The research data in vivo revealed that the BE reduced the levels of alanine transaminase (ALT), aspartate transaminase (AST), and methane dicarboxylic aldehyde (MDA) in liver tissues of the ConA-induced mice, while increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and the effective contents of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and the BE could ameliorate liver histological lesions. These results motivated a further investigation into the chemical constituents of BE. Four coumarins (esculetin, prionanthoside, cichoriin, and esculin) and one flavonoid (quercetin-3-O-galactoside) were isolated from the BE by silica gel column chromatography and recrystallization, of which structures were eventually confirmed by 1 H-NMR, 13 C-NMR, and MS.

N-Terminal PreS1 Sequence Regulates Efficient Infection of Cell-Culture-Generated Hepatitis B Virus.

BACKGROUND AND AIMS: An efficient cell-culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral reagents. Currently, for the HBV infection assay in cell culture, viruses derived from HBV genome-integrated cell lines of HepG2.2.15 or HepAD-38 are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against antiviral reagents. HBV obtained by the transient transfection of the ordinary HBV molecular clone has limited infection efficiencies in cell culture. APPROACH AND RESULTS: We found that an 11-amino-acid deletion (d11) in the preS1 region enhances the infectivity of cell-culture-generated HBV (HBVcc) to sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. Infection of HBVcc derived from a d11-introduced genotype C strain (GTC-d11) was ~10-fold more efficient than infection of wild-type GTC (GTC-wt), and the number of infected cells was comparable between GTC-d11- and HepG2.2.15-derived viruses when inoculated with the same genome equivalents. A time-dependent increase in pregenomic RNA and efficient synthesis of covalently closed circular DNA were detected after infection with the GTC-d11 virus. The involvement of d11 in the HBV large surface protein in the enhanced infectivity was confirmed by an HBV reporter virus and hepatitis D virus infection system. The binding step of the GTC-d11 virus onto the cell surface was responsible for this efficient infection. CONCLUSIONS: This system provides a powerful tool for studying the infection and propagation of HBV in cell culture and also for developing the antiviral strategy against HBV infection.

Antiviral activity of a polysaccharide from Radix Isatidis (Isatis indigotica Fortune) against hepatitis B virus (HBV) in vitro via activation of JAK/STAT signal pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatitis B virus (HBV) infection frequently results in both acute and chronic hepatitis and poses serious threats to human health worldwide. Despite the availability of effective HBV vaccine and anti-HBV drugs, apparently inevitable side effects and resistance have limited its efficiency, thus prompt the search for new anti-HBV agents. The traditional Chinese medicine Radix Isatidis has been used for thousands of years, mainly for the treatment of viral and bacterial infection diseases including hepatitis. AIM OF THE STUDY: In this study, antiviral activities of a Radix Isatidis (Isatis indigotica Fortune) polysaccharide (RIP) were evaluated in vitro model using the HepG2.2.15 cell line and the underlying mechanism was elucidated with the aim of developing a novel anti-HBV therapeutic agent. MATERIALS AND METHODS: Structure features of the purified polysaccharide RIP were investigated by a combination of chemical and instrumental analysis. Drug cytotoxicity was assessed using the MTT assay. The contents of HBsAg, HBeAg, intracellular and extracellular IFN-α level were measured using respective commercially available ELISA kit. The HBV DNA expression was evaluated by real-time quantitative polymerase chain reaction (PCR) and the relevant proteins involved in TFN/JAK/STAT signaling pathways were examined by western blot assay. RESULTS: MTT assay showed that RIP had no toxicity on HepG2.2.15 cell line below the concentration 400 µg/ml at Day 3, 6 and 9. Furthermore, RIP at the concentration of 50, 100 and 200 µg/ml significantly reduced extracellular and intracellular level of HBsAg, HBeAg and HBV DNA in HepG2.2.15 cells in a time and dose-dependent manner. Moreover, RIP also enhanced the production of IFN-α in HepG2.2.15 cell via activation of JAK/STAT signal pathway and induction of antiviral proteins, as evidenced by the increased protein expression of p-STAT-1, p-STAT-2, p-JAK1, p-TYK2, OAS1, and Mx in HepG2.2.15 cells. In addition, the over expression of SOCS-1 and SOCS-3 was significantly abolished under same conditions. CONCLUSIONS: These results suggested that the HBV inhibitory effect of RIP was possibly due to the activation of IFN-α-dependent JAK/STAT signal pathway and induction of the anti-HBV protein expression.

Glucosamine promotes hepatitis B virus replication through its dual effects in suppressing autophagic degradation and inhibiting MTORC1 signaling.

Glucosamine (GlcN), a dietary supplement widely utilized to promote joint health and effective in the treatment of osteoarthritis, is an effective macroautophagy/autophagy activator in vitro and in vivo. Previous studies have shown that autophagy is required for hepatitis B virus (HBV) replication and envelopment. The objective of this study was to determine whether and how GlcN affects HBV replication, using in vitro and in vivo experiments. Our data demonstrated that HBsAg production and HBV replication were significantly increased by GlcN treatment. Confocal microscopy and western blot analysis showed that the amount of autophagosomes and the levels of autophagic markers MAP1LC3/LC3-II and SQSTM1 were clearly elevated by GlcN treatment. GlcN strongly blocked autophagic degradation of HBV virions and proteins by inhibiting lysosomal acidification through its amino group. Moreover, GlcN further promoted HBV replication by inducing autophagosome formation via feedback inhibition of mechanistic target of rapamycin kinase complex 1 (MTORC1) signaling in an RRAGA (Ras related GTP binding A) GTPase-dependent manner. In vivo, GlcN application promoted HBV replication and blocked autophagic degradation in an HBV hydrodynamic injection mouse model. In addition, GlcN promoted influenza A virus, enterovirus 71, and vesicular stomatitis virus replication in vitro. In conclusion, GlcN efficiently promotes virus replication by inducing autophagic stress through its dual effects in suppressing autophagic degradation and inhibiting MTORC1 signaling. Thus, there is a potential risk of enhanced viral replication by oral GlcN intake in chronically virally infected patients.Abbreviations: ACTB: actin beta; ATG: autophagy-related; CMIA: chemiluminescence immunoassay; ConA: concanavalin A; CQ: chloroquine; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; EV71: enterovirus 71; GalN: galactosamine; GFP: green fluorescence protein; GlcN: glucosamine; GNPNAT1: glucosamine-phosphate N-acetyltransferase 1; HBP: hexosamine biosynthesis pathway; HBV: hepatitis B virus; HBcAg: hepatitis B core antigen; HBsAg: hepatitis B surface antigen; HBeAg: hepatitis B e antigen; HBV RI: hepatitis B replicative intermediate; IAV: influenza A virus; LAMP1: lysosomal associated membrane protein 1; LAMTOR: late endosomal/lysosomal adaptor, MAPK and MTOR activator; ManN: mannosamine; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PHH: primary human hepatocyte; RAB7: RAB7A, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; RRAGA: Ras related GTP binding A; RT-PCR: reverse transcriptase polymerase chain reaction; SEM: standard error of the mean; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; UAP1: UDP-N-acetylglucosamine pyrophosphorylase 1; VSV: vesicular stomatitis virus.

Chemical constituents from Lonicera japonica flower buds and their anti-hepatoma and anti-HBV activities.

Three new naturally occurring monoterpenoids, japopenoid A (1), japopenoid B (23) japopenoid C (24), and one new caffeoylquinic acid derivative (28), together with thirty-one known compounds (2-22, 25-27, 29-35), were isolated and identified from the flower buds of Lonicera japonica Thunb. Their structures were determined by extensive 1D and 2D NMR spectroscopic methods, high-resolution mass spectrometry, and the absolute configurations of 1, 23, 24 were determined by comparison of their electronic circular dichroism (ECD) spectrum with literature and theoretical calculation. Structurally, compound 1 is a monoterpenoid featured with an unusual tricyclic skeleton. All compounds (1-35) were evaluated for their cytotoxicities against human liver cancer cell lines (HepG 2 and SMMC-7721). Compound 12 exhibited the most potent activity with IC50 values of 26.54 ±â€¯1.95 and 8.72 ±â€¯1.57 µg/ml against HepG 2 and SMMC-7721, and the IC50 values of compound 13 were 26.54 ±â€¯1.95 and 12.35 ±â€¯1.43 µg/ml, respectively. Western blot results further proved that compound 13 induces hepatoma cell apoptosis via the intrinsic apoptosis pathway. In addition, most terpenoids showed inhibitory activity against HBsAg and HBeAg secretion, and HBV DNA replication. In particular, 25 µg/mlof compound 11 inhibits HBsAg and HBeAg secretion, and HBV DNA replication by 39.39 ±â€¯5.25, 15.64 ±â€¯1.25, and 16.13 ±â€¯4.10% compared to the control (p < 0.05). These results indicated that L. japonica flower buds could be served as functional food for anti-hepatoma and anti-HBV activities.

Identification of Retinoic Acid Receptor Agonists as Potent Hepatitis B Virus Inhibitors via a Drug Repurposing Screen.

Currently available therapies for chronic hepatitis B virus (HBV) infection can efficiently reduce viremia but induce hepatitis B surface antigen (HBsAg) loss in very few patients; also, these therapies do not greatly affect the viral covalently closed circular DNA (cccDNA). To discover new agents with complementary anti-HBV effects, we performed a drug repurposing screen of 1,018 Food and Drug Administration (FDA)-approved compounds using HBV-infected primary human hepatocytes (PHH). Several compounds belonging to the family of retinoic acid receptor (RAR) agonists were identified that reduced HBsAg levels in a dose-dependent manner without significant cytotoxicity. Among them, tazarotene exhibited the most potent anti-HBV effect, with a half-maximal inhibitory concentration (IC50) for HBsAg of less than 30 nM in PHH. The inhibitory effect was also observed in HBV-infected differentiated HepaRG (dHepaRG) models, but not in HepG2.215 cells, and HBV genotypes A to D were similarly inhibited. Tazarotene was further demonstrated to repress HBV cccDNA transcription, as determined by the levels of HBV cccDNA and RNAs and the activation of HBV promoters. Moreover, RNA sequence analysis showed that tazarotene did not induce an interferon response but altered the expression of a number of genes associated with RAR and metabolic pathways. Inhibition of RARß, but not RARα, by a specific antagonist significantly attenuated the anti-HBV activity of tazarotene, suggesting that tazarotene inhibits HBV in part through RARß. Finally, a synergistic effect of tazarotene and entecavir on HBV DNA levels was observed. Therefore, RAR agonists as represented by tazarotene were identified as potential novel anti-HBV agents.

Discovery of Oxime Ethers as Hepatitis B Virus (HBV) Inhibitors by Docking, Screening and In Vitro Investigation.

A series of oxime ethers with C6-C4 fragment was designed and virtually bioactively screened by docking with a target, then provided by a Friedel-Crafts reaction, esterification (or amidation), and oximation from p-substituted phenyl derivatives (Methylbenzene, Methoxybenzene, Chlorobenzene). Anti-hepatitis B virus (HBV) activities of all synthesized compounds were evaluated with HepG2.2.15 cells in vitro. Results showed that most of compounds exhibited low cytotoxicity on HepG2.2.15 cells and significant inhibition on the secretion of HBsAg and HBeAg. Among them, compound 5c-1 showed the most potent activity on inhibiting HBsAg secretion (IC50 = 39.93 µM, SI = 28.51). Results of the bioactive screening showed that stronger the compounds bound to target human leukocyte antigen A protein in docking, the more active they were in anti-HBV activities in vitro.

Ultrasound assisted extraction of polysaccharides from Lentinus edodes and its anti-hepatitis B activity in vitro.

The aim of this study was to optimize the extraction process of polysaccharides from the fruiting bodies of Lentinus edodes and investigate its anti-hepatitis B virus activity. The extracting parameters including ultrasonic power (240-320W), extraction temperature (40-60°C) and extraction time (15-25min) was optimized by using three-variable-three-level Box-Behnken design based on the single-factor experiments. Data analysis results showed that the optimal conditions for extracting LEPs were an extraction temperature of 45°C, extraction time of 21min and ultrasonic power of 290W. Under these optimal conditions, the experimental yield of LEPs was 9.75%, a 1.62-fold increase compared with conventional heat water extraction (HWE). In addition, crude polysaccharides were purified to obtain two fractions (LEP-1 and LEP-2). Chemical analysis showed that these components were rich in glucose, arabinose and mannose. Furthermore, HepG2.2.15 cells were used as in vitro models to evaluate their anti-hepatitis B virus (HBV) activity. The results suggest that LEPs possesses potent anti-HBV activity in vitro.

Protocatechuic acid inhibits hepatitis B virus replication by activating ERK1/2 pathway and down-regulating HNF4α and HNF1α in vitro.

AIMS: Protocatechuic acid (PCA) is a phenolic compound found in many antiviral Chinese herbal medicines. HNF4α and HNF1α, the members of hepatocyte nuclear factor (HNF) family, play an important regulatory role in the gene transcription of hepatitis B virus (HBV). Previous studies found that PCA inhibited HBV antigen secretion and HBV DNA replication in HepG2.2.15 cells, but its anti-HBV mechanism has not been fully understood. We aim to illustrate the anti-HBV mechanism of PCA. MATERIALS AND METHODS: MTT was used to estimate cytotoxicity. The content of HBsAg or HBeAg was detected using an enzyme-linked immunosorbent assay kit. HBV DNA in cell-free culture media was detected by PCR kit. HNF1α and HNF4α mRNA expression was detected by real-time PCR. HNF1α, HNF4α and ERK1/2 protein expression was detected by western blotting and HBV promoter activity was tested by luciferase reporter assay. KEY FINDINGS: Our results demonstrated that PCA inhibited the gene transcription and protein translation of HNF1α and HNF4α in Huh7 and HepG2.2.15 cells, as well as the promoter activities of HBV X and preS1 in Huh7 cells transfected with the luciferase reporter plasmid of HBV promoter. Further study suggested that PCA induced the phosphorylation of extracellular-signal-related kinase (ERK) 1/2, and thereby inhibited HNF4α and HNF1α expression in HepG2.2.15 cells to exert its antiviral activity. SIGNIFICANCE: To our knowledge, this study is the first to reveal the anti-HBV mechanism of PCA. Our results demonstrate that PCA inhibits HBV replication by activating ERK1/2 pathway and subsequently down-regulating HNF4α and HNF1α in HepG2.2.15 cells.

A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) Adjuvant for Hepatitis B Vaccine.

Although adjuvants are a common component of many vaccines, there are few adjuvants licensed for use in humans due to concerns about their toxic effects. There is a need to develop new and safe adjuvants, because some existing vaccines have low immunogenicity among certain patient groups. In this study, SBP, a hepatitis B surface antigen binding protein that was discovered through screening a human liver cDNA expression library, was introduced into hepatitis B vaccine. A good laboratory practice, non-clinical safety evaluation was performed to identify the side effects of both SBP and SBP-adjuvanted hepatitis B vaccine. The results indicate that SBP could enhance the HBsAg-specific immune response, thus increasing the protection provided by the hepatitis B vaccine. The safety data obtained here warrant further investigation of SBP as a vaccine adjuvant.

Anti-HBV Activities of Three Compounds Extracted and Purified from Herpetospermum Seeds.

The goal of this research was to evaluate the anti-hepatitis B virus (HBV) activities of three compounds extracted and purified from Herpetospermum seeds (HS) on HepG2.2.15 cells. Herpetin (HPT), herpetone (HPO), and herpetfluorenone (HPF) were isolated from HS and identified using HR-ESI-MS and NMR. Different concentrations of the drugs were added to the HepG2.2.15 cells. Cell toxicity was observed with an MTT assay, cell culture supernatants were collected, and HBsAg and HBeAg were detected by ELISA. The content of HBV DNA was determined via quantitative polymerase chain reaction (PCR) with fluorescent probes. The 50% toxicity concentration (TC50) of HPF was 531.48 µg/mL, suggesting that this species is less toxic than HPT and HPO. HPT and HPF showed more potent antiviral activities than HPO. The 50% inhibition concentration (IC50) values of HPF on HBsAg and HBeAg were 176.99 and 134.53 µg/mL, respectively, and the corresponding therapeutic index (TI) values were 2.66 and 3.49, respectively. HPT and HPF were shown to significantly reduce the level of HBV DNA in the HepG2.2.15 culture medium compared to the negative control. This initial investigation of the anti-HBV constituents of HS yielded three compounds that revealed a synergistic effect of multiple components in the ethnopharmacological use of HS.

Mechanisms of Antigen Adsorption Onto an Aluminum-Hydroxide Adjuvant Evaluated by High-Throughput Screening.

The adsorption mechanism of antigen on aluminum adjuvant can affect antigen elution at the injection site and hence the immune response. Our aim was to evaluate adsorption onto aluminum hydroxide (AH) by ligand exchange and electrostatic interactions of model proteins and antigens, bovine serum albumin (BSA), ß-casein, ovalbumin (OVA), hepatitis B surface antigen, and tetanus toxin (TT). A high-throughput screening platform was developed to measure adsorption isotherms in the presence of electrolytes and ligand exchange by a fluorescence-spectroscopy method that detects the catalysis of 6,8-difluoro-4-methylumbelliferyl phosphate by free hydroxyl groups on AH. BSA adsorption depended on predominant electrostatic interactions. Ligand exchange contributes to the adsorption of ß-casein, OVA, hepatitis B surface antigen, and TT onto AH. Based on relative surface phosphophilicity and adsorption isotherms in the presence of phosphate and fluoride, the capacities of the proteins to interact with AH by ligand exchange followed the trend: OVA < ß-casein < BSA < TT. This could be explained by both the content of ligands available in the protein structure for ligand exchange and the antigen's molecular weight. The high-throughput screening platform can be used to better understand the contributions of ligand exchange and electrostatic attractions governing the interactions between an antigen adsorbed onto aluminum-containing adjuvant.

AIC649 Induces a Bi-Phasic Treatment Response in the Woodchuck Model of Chronic Hepatitis B.

AIC649 has been shown to directly address the antigen presenting cell arm of the host immune defense leading to a regulated cytokine release and activation of T cell responses. In the present study we analyzed the antiviral efficacy of AIC649 as well as its potential to induce functional cure in animal models for chronic hepatitis B. Hepatitis B virus transgenic mice and chronically woodchuck hepatitis virus (WHV) infected woodchucks were treated with AIC649, respectively. In the mouse system AIC649 decreased the hepatitis B virus titer as effective as the "gold standard", Tenofovir. Interestingly, AIC649-treated chronically WHV infected woodchucks displayed a bi-phasic pattern of response: The marker for functional cure--hepatitis surface antigen--first increased but subsequently decreased even after cessation of treatment to significantly reduced levels. We hypothesize that the observed bi-phasic response pattern to AIC649 treatment reflects a physiologically "concerted", reconstituted immune response against WHV and therefore may indicate a potential for inducing functional cure in HBV-infected patients.

Substitution at rt269 in Hepatitis B Virus Polymerase Is a Compensatory Mutation Associated with Multi-Drug Resistance.

The emergence of compensatory mutations in the polymerase gene of drug resistant hepatitis B virus (HBV) is associated with treatment failure. We previously identified a multi-drug resistant HBV mutant, which displayed resistance towards lamivudine (LMV), clevudine (CLV), and entecavir (ETV), along with a strong replication capacity. The aim of this study was to identify the previously unknown compensatory mutations, and to determine the clinical relevance of this mutation during antiviral therapy. In vitro mutagenesis, drug susceptibility assay, and molecular modeling studies were performed. The rtL269I substitution conferred 2- to 7-fold higher replication capacity in the wild-type (WT) or YMDD mutation backbone, regardless of drug treatment. The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF). However, upon combination with YMDD mutation, the replication capacity under LMV or ETV treatment was enhanced by several folds. Molecular modeling studies suggested that the rtL269I substitution affects template binding, which may eventually lead to the enhanced activity of rtI269-HBV polymerase in both WT virus and YMDD mutant. The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV. Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.

Effect of Radix Trichosanthis and Trichosanthin on Hepatitis B Virus in HepG2.2.15 Cells.

Radix Trichosanthis is a Chinese herbal medicine that has great medical value and pharmacological actions. There is already a long history of using the plant Radix Trichosanthis as treatment for hepatitis B virus in China. This research mainly focused on investigating the therapeutic effect of different extracts from Radix Trichosanthis on hepatitis B virus, on a cellular level (ex vivo). Cell survival rate of HepG2.2.15 cells was detected by MTT assay. HBsAg and HBeAg in HepG 2.2.15 cell supernatant were evaluated by enzyme linked immunosorbent assay (ELISA). Results showed that water extract from Radix Trichosanthis had a stronger inhibitive effect on expression of HBsAg and HBeAg in HepG2.2.15 cells than the alcohol extract from the same plant. Considering that the most active component of Radix Trichosanthis was in its aqueous extract and this might be related to the active component Trichosanthin. Trichosanthin was further used for related experiments to confirm this hypothesis. The results showed that Trichosanthin, in the aqueous extract from Radix Trichosanthis, is likely the main component responsible for the anti-hepatitis B viral effect.

Anti-hepatitis B virus activities of friedelolactones from Viola diffusa Ging.

BACKGROUND: Hepatitis B virus (HBV) infection is the major factor of causing hepatitis B, cirrhosis and liver cancer. Interferon and nucleoside drugs, the main drugs to treat HBV infection, have disadvantages of scavenge difficulty and drug resistance respectively. Viola diffusa Ging is used as a traditional Chinese herbal medicine for the treatment of hepatitis. PURPOSE: The aim of the study is to investigate the chemical constituents of Viola diffusa Ging and their anti-HBV activity. METHODS: Chemical constituents were extracted and purified by ethanol reflux extraction and chromatographic separation technology including D-101 Macroporous resin, silica gel, Sephadex LH-20 and preparative thin-layer chromatography. Their structures were elucidated on the basis of extensive NMR and MS data. Cytotoxicity and inhibiting effects on HBsAg and HBeAg secretion of HepG2.2.15 of all compounds except 10 were studied by MTT method and ELISA method. RESULTS: Three friedelolactones with naturally occurring seco-ring-A friedelane triterpenoids, 2ß-hydroxy-3, 4-seco-friedelolactone-27-oic acid (1), 2ß, 28ß-dihydroxy-3,4-seco-friedelolactone-27-oic acid (2) and 2ß, 30ß-dihydroxy-3,4-seco-friedelolactone-27-lactone (3), and a stigmastane, stigmast-25-ene-3ß,5α,6ß-triol (11) together with nine known compounds were isolated from the whole plant of Viola diffusa G. (Violaceae). Compounds 1-3, 9, 11, 12 exhibited significant activities of blocking both HBsAg and HBeAg secretion, and compound 4, 6, 7, 8 selectively inhibited HBeAg secretion while compound 13 selectively inhibited HBsAg secretion. IC50 values of compounds 1 and 2, 26.2 µM and 33.7 µM for HBsAg, 8.0 µM and 15.2 µM for HBeAg, was significantly lower than that of positive control lamivudine. CONCLUSION: Compounds 1-3, 11 are new compounds never reported before and the promising results demonstrate the potential of compound 1-3, 9, 11, 12 for the treatment of HBV infection.

Preventive effects of a major component of green tea, epigallocathechin-3-gallate, on hepatitis-B virus DNA replication.

BACKGROUND: Hepatitis B virus infection is one of the major world health problems. Epigallocatechin-3 gallate is the major component of the polyphenolic fraction of green tea and it has an anti-viral, anti-mutagenic, anti- tumorigenic, anti-angiogenic, anti-proliferative, and/or pro-apoptotic effects on mammalian cells. In this study, our aim was to investigate the inhibition of HBV replication by epigallocatechin-3 gallate in the Hep3B2.1-7 hepatocellular carcinoma cell line. MATERIALS AND METHODS: HBV-replicating Hep3B2.1-7 cells were used to investigate the preventive effects of epigallocatechin-3 gallate on HBV DNA replication. The expression levels of HBsAg and HBeAg were determined using ELISA. Quantitative real-time-PCR was applied for the determination of the expression level of HBV DNA. RESULTS: Cytotoxicity of epigallocathechin-3-gallate was not observed in the hepatic carcinoma cell line when the dose was lower than 100 µM. The ELISA method demonstrated that epigallocatechin-3 gallate have strong effects on HBsAg and HBeAg levels. Also it was detected by real-time PCR that epigallocatechin-3 gallate could prevent HBV DNA replication. CONCLUSIONS: The obtained data pointed out that although the exact mechanism of HBV DNA replication and related diseases remains unclear, epigallocatechin-3 gallate has a potential as an effective anti-HBV agent with low toxicity.

Hepatitis B Virus Pre-S2 Mutant Induces Aerobic Glycolysis through Mammalian Target of Rapamycin Signal Cascade.

Hepatitis B virus (HBV) pre-S2 mutant can induce hepatocellular carcinoma (HCC) via the induction of endoplasmic reticulum stress to activate mammalian target of rapamycin (MTOR) signaling. The association of metabolic syndrome with HBV-related HCC raises the possibility that pre-S2 mutant-induced MTOR activation may drive the development of metabolic disorders to promote tumorigenesis in chronic HBV infection. To address this issue, glucose metabolism and gene expression profiles were analyzed in transgenic mice livers harboring pre-S2 mutant and in an in vitro culture system. The pre-S2 mutant transgenic HCCs showed glycogen depletion. The pre-S2 mutant initiated an MTOR-dependent glycolytic pathway, involving the eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), Yin Yang 1 (YY1), and myelocytomatosis oncogene (MYC) to activate the solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1), contributing to aberrant glucose uptake and lactate production at the advanced stage of pre-S2 mutant transgenic tumorigenesis. Such a glycolysis-associated MTOR signal cascade was validated in human HBV-related HCC tissues and shown to mediate the inhibitory effect of a model of combined resveratrol and silymarin product on tumor growth. Our results provide the mechanism of pre-S2 mutant-induced MTOR activation in the metabolic switch in HBV tumorigenesis. Chemoprevention can be designed along this line to prevent HCC development in high-risk HBV carriers.