RESUMEN
Selenium supplements are beneficial to human health, however, concerns regarding the toxicity of inorganic selenium have stimulated research on safer organic compounds. The main objective of this study was to develop a novel glucosamine-selenium compound (Se-GlcN), clarify its structure, and subsequently investigate its oral toxicity and in vitro anti-hepatitis B virus (HBV) activity. Electron microscopy, infrared, ultraviolet spectroscopy, nuclear magnetic resonance and thermogravimetric analyses revealed a unique binding mode of Se-GlcN, with the introduction of the Se-O bond at the C6 position, resulting in the formation of two carboxyl groups. In acute toxicity studies, the median lethal dose (LD50) of Se-GlcN in ICR mice was 92.31 mg/kg body weight (BW), with a 95 % confidence interval of 81.88-104.07 mg/kg BW. A 30-day subchronic toxicity study showed that 46.16 mg/kg BW Se-GlcN caused livers and kidneys damage in mice, whereas doses of 9.23 mg/kg BW and lower were safe for the livers and kidneys. In vitro studies, Se-GlcN at 1.25 µg/mL exhibited good anti-HBV activity, significantly reducing HBsAg, HBeAg, 3.5 kb HBV RNA and total HBV RNA by 45 %, 54 %, 84 %, 87 %, respectively. In conclusion, the Se-GlcN synthesized in this study provides potential possibilities and theoretical references for its use as an organic selenium supplement.
Asunto(s)
Antivirales , Glucosamina , Virus de la Hepatitis B , Ratones Endogámicos ICR , Animales , Virus de la Hepatitis B/efectos de los fármacos , Glucosamina/química , Glucosamina/farmacología , Ratones , Antivirales/farmacología , Antivirales/síntesis química , Antivirales/química , Antivirales/toxicidad , Administración Oral , Masculino , Selenio/química , Selenio/farmacología , Hígado/efectos de los fármacos , Hígado/patología , Humanos , Femenino , Riñón/efectos de los fármacos , Riñón/patología , Células Hep G2 , Antígenos de Superficie de la Hepatitis B/metabolismoRESUMEN
A substantial body of literature, including our own, points to a connection between hepatitis B virus (HBV) infection and the development of drug resistance in hepatocellular carcinoma (HCC), particularly against sorafenib. However, the influence of HBV on resistance to regorafenib, another therapeutic agent, has been less studied. In this study, we used the GEO database (GSE87630) and clinical samples to demonstrate that C-C motif chemokine receptor 9 (CCR9) was highly expressed in HBV-related HCC and predicted poor overall survival. Its overexpression correlated with HBsAg-positive HCC patients. Both univariate and multivariable Cox regression analysis elucidated CCR9 was an independent risk factor for poor overall survival in HCC patients. Our in vitro findings further revealed that HBV structural proteins, small HBV surface antigen (SHBs), triggered an upregulation of CCR9. Functional assays showed that SHBs enhanced HCC cell proliferation, migration, and invasion, increased ABCB1 and ABCC1 expression, and promoted regorafenib resistance via CCR9. Intriguingly, overexpression of HBV plasmid and an AAV-HBV mouse model both exhibited a significant elevation in global N6-methyladenosine (m6A) levels. Further investigations revealed that SHBs elevated these m6A levels, upregulated CCR9 and stabilized CCR9 mRNA through KIAA1429-mediated m6A modification, with sites 1373 and 1496 on CCR9 mRNA being critical for modification. In conclusion, SHBs promoted HCC progression and regorafenib resistance via KIAA1429-mediated m6A modification of CCR9. Our findings suggested that CCR9 could be a potential prognostic biomarker and a valuable molecular therapeutic target of regorafenib resistance in HBV-related HCC.
Asunto(s)
Carcinoma Hepatocelular , Resistencia a Antineoplásicos , Antígenos de Superficie de la Hepatitis B , Neoplasias Hepáticas , Compuestos de Fenilurea , Piridinas , Carcinoma Hepatocelular/virología , Carcinoma Hepatocelular/tratamiento farmacológico , Humanos , Neoplasias Hepáticas/virología , Neoplasias Hepáticas/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Animales , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , Piridinas/farmacología , Piridinas/uso terapéutico , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Ratones , Masculino , Femenino , Receptores CCR/genética , Receptores CCR/metabolismo , Línea Celular Tumoral , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/efectos de los fármacos , Persona de Mediana Edad , Hepatitis B/virología , Hepatitis B/complicaciones , Hepatitis B/tratamiento farmacológico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Adenosina/análogos & derivadosRESUMEN
Lappanolides A-N (1-14), 14 undescribed sesquiterpenoids, along with 23 known ones (15-37), were isolated from the roots of Saussurea costus, which were primarily categorized into eudesmane, guaiane, and germacrane types. Lappanolide A (1) possessed an unprecedented pseudo-disesquiterpenoids. Their structures and absolute configurations were established using physical data analyses (HRESIMS, IR, 1D and 2D NMR) and ECD calculations. All isolated compounds were tested for anti-hepatitis B virus (anti-HBV) activity. Ten compounds (1, 9, 11, 12, 19, 22, 28, 29, 31, and 36) exhibited activities against HBsAg secretions as determined by ELISA assay, with IC50 values ranging from 5.2 to 45.7 µM. In particular, compounds 28 and 29 showed inhibition of HBsAg secretion with IC50 values of 5.28 and 5.30 µM, and CC50 values of 9.85 and 6.37 µM, respectively, though they all exhibited low selectivity. Several compounds displayed cytotoxicity in the MTT assay. Among them, compound 28 was the most notable and was chosen for further study using flow cytometry. The result showed that it significantly induced HepG2 cell arrest in the S phase and induced apoptosis.
Asunto(s)
Antivirales , Virus de la Hepatitis B , Saussurea , Sesquiterpenos , Saussurea/química , Humanos , Virus de la Hepatitis B/efectos de los fármacos , Antivirales/farmacología , Antivirales/química , Antivirales/aislamiento & purificación , Sesquiterpenos/farmacología , Sesquiterpenos/química , Sesquiterpenos/aislamiento & purificación , Células Hep G2 , Estructura Molecular , Antígenos de Superficie de la Hepatitis B/metabolismo , Antígenos de Superficie de la Hepatitis B/efectos de los fármacos , Relación Estructura-Actividad , Raíces de Plantas/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Apoptosis/efectos de los fármacosRESUMEN
BACKGROUND: Interferon-alpha (IFNα) is a common treatment for chronic hepatitis B virus (HBV) infection, but its efficacy varies widely among patients. GTPASE, an interferon-stimulated gene (ISG), has recently been identified as a factor in antiviral immunity, though its role in HBV infection is not fully understood. OBJECTIVE: This study investigates the role of GTPASE in enhancing the antiviral effects of IFNα against HBV and elucidates its mechanism of action. METHODS: We analyzed the impact of GTPASE overexpression and silencing on HBV replication and clearance in HBV-infected cells. Molecular docking studies assessed the interaction between GTPASE and HBV surface antigens (HBs). Clinical samples from HBV patients undergoing Peg-IFNα treatment were also evaluated for GTPASE expression and its correlation with treatment efficacy. RESULTS: Overexpression of GTPASE led to significant inhibition of HBV replication, increased HBeAg seroconversion, and enhanced HBsAg clearance. GTPASE directly bound to HBs proteins, reducing their levels and affecting viral particle formation. Silencing GTPASE reduced these effects, while combined treatment with Peg-IFNα and GTPASE overexpression further improved antiviral outcomes. Mutational analysis revealed that specific sites in GTPASE are crucial for its antiviral activity. CONCLUSIONS: GTPASE acts as a positive regulator in IFNα-induced antiviral immunity against HBV. It enhances the therapeutic efficacy of IFNα by targeting HBs and modulating viral replication. GTPASE levels may serve as a predictive biomarker for response to Peg-IFNα therapy, highlighting its potential for improving individualized treatment strategies for chronic HBV infection.
Asunto(s)
Antivirales , GTP Fosfohidrolasas , Antígenos de Superficie de la Hepatitis B , Virus de la Hepatitis B , Hepatitis B Crónica , Interferón-alfa , Replicación Viral , Humanos , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Replicación Viral/efectos de los fármacos , Antivirales/farmacología , Antivirales/uso terapéutico , Interferón-alfa/farmacología , Interferón-alfa/uso terapéutico , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , Simulación del Acoplamiento Molecular , Adulto , Masculino , Antígenos e de la Hepatitis B/metabolismo , Células Hep G2 , Femenino , Resultado del TratamientoRESUMEN
PURPOSE: This study aimed to assess metabolic changes to monitor the progression from normal liver to hepatitis B virus (HBV)-related hepatitis and liver fibrosis using hyperpolarized 13C magnetic resonance imaging (MRI). PROCEDURES: Hepatitis was induced in mice (n = 16) via hydrodynamic injection of HBV 1.2 plasmid (25 µg). Among them, liver fibrosis was induced in the mice (n = 8) through weight-adapted administration of thioacetamide with ethanol. Normal control mice (n = 8) were injected with a phosphate buffer solution. Subsequently, a hyperpolarized 13C MRI was performed on the mouse liver in vivo. The level of hepatitis B surface antigen (HBsAg) in blood serum was measured. Statistical analysis involved comparing the differential metabolite ratios, blood biochemistry values, and body weight among the three groups using the Kruskal-Wallis one-way analysis of variance. RESULTS: HBsAg was absent in the normal and fibrosis groups, while it was detected in the hepatitis group. The ratios of [1-13C] lactate/pyruvate, [1-13C] alanine/pyruvate, [1-13C] lactate/total carbon, and [1-13C] alanine/total carbon were significantly lower in the normal control group than in the hepatitis and fibrosis groups (p < 0.05). Moreover, these ratios were significantly higher in the fibrosis group than in the hepatitis group (p < 0.05). However, no significant differences were observed in either [1-13C] pyruvate-hydrate/pyruvate or [1-13C] pyruvate-hydrate/total carbon among the three groups. CONCLUSIONS: The levels of [1-13C] lactate and [1-13C] alanine in vivo may serve as valuable indicators for differentiating between HBV-related hepatitis, liver fibrosis, and normal liver.
Asunto(s)
Progresión de la Enfermedad , Virus de la Hepatitis B , Cirrosis Hepática , Imagen por Resonancia Magnética , Animales , Cirrosis Hepática/diagnóstico por imagen , Cirrosis Hepática/virología , Cirrosis Hepática/patología , Cirrosis Hepática/metabolismo , Imagen por Resonancia Magnética/métodos , Hepatitis B/complicaciones , Hepatitis B/diagnóstico por imagen , Masculino , Ratones , Hígado/metabolismo , Hígado/diagnóstico por imagen , Hígado/patología , Antígenos de Superficie de la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/metabolismo , Isótopos de CarbonoRESUMEN
Chronic hepatitis B virus (HBV) infection is due to the failure of host immune system to resolve the viral infection. Accordingly, restoration or reconstitution of a functional antiviral immune response to HBV is essential to achieve durable control of HBV replication leading to a functional cure of chronic hepatitis B (CHB). Noninfectious subviral particles (SVPs), comprised of HBV surface antigen (HBsAg), are the predominant viral products secreted by HBV-infected hepatocytes. The high levels of SVPs in the circulation induce immune tolerance and contribute to the establishment of chronic HBV infection. The current standard-of-care medications for CHB efficiently suppress HBV replication but fail to reduce the levels of HBsAg in majority of treated patients. Further understanding the mechanisms underlying SVP morphogenesis, secretion and regulation by viral and host cellular factors are critical for the discovery of therapeutics that can inhibit SVP production and/or induce the degradation of HBV envelope proteins. We describe herein a protocol for intracellular SVP detection by a native agarose gel electrophoresis-based particle gel assy. The method is suitable for quantitative detection of intracellular HBV SVPs and can be applied in dissecting the molecular mechanism of SVP morphogenesis and the discovery of antiviral agents targeting SVP formation in hepatocytes.
Asunto(s)
Virus de la Hepatitis B , Virión , Virus de la Hepatitis B/fisiología , Virus de la Hepatitis B/efectos de los fármacos , Humanos , Hepatocitos/virología , Hepatocitos/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Replicación Viral/efectos de los fármacos , Electroforesis en Gel de Agar/métodos , Células Cultivadas , Hepatitis B Crónica/virología , Hepatitis B Crónica/tratamiento farmacológicoRESUMEN
High levels of hepatitis B virus (HBV) surface antigen (HBsAg) in the blood of chronic HBV carriers are considered to drive the exhaustion of antigen-specific T and B lymphocytes and thus responsible for the persistence of infection. Accordingly, therapeutic elimination of HBsAg may facilitate the activation of adaptive antiviral immune responses against HBV and achieve a functional cure of chronic hepatitis B. We discovered recently that an amphipathic alpha helix spanning W156 to R169 of HBV small envelope (S) protein plays an essential role in the morphogenesis of subviral particles (SVPs) and metabolism of S protein. We thus hypothesized that pharmacological disruption of SVP morphogenesis may induce intracellular degradation of S protein and reduce HBsAg secretion. To identify inhibitors of SVP biogenesis, we screened 4417 bioactive compounds with a HepG2-derived cell line expressing HBV S protein and efficiently secreting small spherical SVPs. The screen identified 24 compounds that reduced intracellular SVPs and secreted HBsAg in a concentration-dependent manner. However, 18 of those compounds inhibited the secretion of HBsAg and HBeAg in HBV replicon transfected HepG2 cells at similar efficiency, suggesting each of those compounds may disrupt a common cellular function required for the synthesis and/or secretion of these viral proteins. Interestingly, lycorine more efficiently inhibited the secretion of HBsAg in HepG2 cells transfected with HBV replicons, HepG2.2.15 cells and HBV infected - HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). The structure activity relationship and antiviral mechanism of lycorine against HBV have been determined.
Asunto(s)
Antivirales , Antígenos de Superficie de la Hepatitis B , Virus de la Hepatitis B , Humanos , Virus de la Hepatitis B/efectos de los fármacos , Antivirales/farmacología , Antígenos de Superficie de la Hepatitis B/metabolismo , Células Hep G2 , Ensamble de Virus/efectos de los fármacos , Virión/efectos de los fármacos , Descubrimiento de Drogas , Replicación Viral/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas del Envoltorio Viral/metabolismo , Antígenos e de la Hepatitis B/metabolismoRESUMEN
Hepatitis B surface antigen (HBsAg) is not only the biomarker of hepatitis B virus (HBV) infection and expression activity in hepatocytes, but it also contributes to viral specific T cell exhaustion and HBV persistent infection. Therefore, anti-HBV therapies targeting HBsAg to achieve HBsAg loss are key approaches for an HBV functional cure. In this study, we found that YZH-106, a rupestonic acid derivative, inhibited HBsAg secretion and viral replication. Further investigation demonstrated that YZH-106 promoted the lysosomal degradation of viral L- and M-HBs proteins. A mechanistic study using Biacore and docking analysis revealed that YZH-106 bound directly to the PreS2 domain of L- and M-HBsAg, thereby blocking their entry into the endoplasmic reticulum (ER) and promoting their degradation in cytoplasm. Our work thereby provides the basis for the design of a novel compound therapy to target HBsAg against HBV infection.
Asunto(s)
Antígenos de Superficie de la Hepatitis B , Virus de la Hepatitis B , Lisosomas , Replicación Viral , Humanos , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , Lisosomas/metabolismo , Replicación Viral/efectos de los fármacos , Hepatitis B/virología , Hepatitis B/tratamiento farmacológico , Antivirales/farmacología , Proteolisis , Células Hep G2 , Hepatocitos/virología , Hepatocitos/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Precursores de ProteínasRESUMEN
Interferon-alpha (IFN-α) is a first-line drug for treating chronic hepatitis B (CHB). Guanylate-binding protein 1 (GBP1) is one of the interferon-stimulating factors, which participates in the innate immunity of the host and plays an antiviral and antibacterial role. In this study, we explored how GBP1 is involved in IFN-α antiviral activity against HBV. Before being gathered, HepG2-NTCP and HepG2 2.15 cells were transfected with the wild-type hGBP1 plasmid or si-GBP1, respectively, and followed by stimulation with Peg-IFNα-2b. We systematically explored the role of GBP1 in regulating HBV infection in cell models. Additionally, we also examined GBP1 levels in CHB patients. GBP1 activity increased, and its half-life was prolonged after HBV infection. Overexpression of GBP1 inhibited the production of HBsAg and HBeAg, as well as HBs protein and HBV total RNA levels, whereas silencing of GBP1 inhibited its ability to block viral infections. Interestingly, overexpressing GBP1 co-treatment with Peg-IFNα-2b further increased the antiviral effect of IFN-α, while GBP1 silencing co-treatment with Peg-IFNα-2b partly restored its inhibitory effect on HBV. Mechanistically, GBP1 mediates the anti-HBV response of Peg-IFNα-2b by targeting HBs. Analysis of clinical samples revealed that GBP1 was elevated in CHB patients and increased with Peg-IFNα-2b treatment, while GBP1 showed good stability in the interferon response group. Our study demonstrates that GBP1 inhibits HBV replication and promotes HBsAg clearance. It is possible to achieve antiviral effects through the regulation of IFN-α induced immune responses in response to HBV.
Asunto(s)
Antivirales , Proteínas de Unión al GTP , Virus de la Hepatitis B , Hepatitis B Crónica , Interferón-alfa , Humanos , Interferón-alfa/farmacología , Interferón-alfa/inmunología , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/fisiología , Antivirales/farmacología , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/inmunología , Células Hep G2 , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Hepatitis B Crónica/inmunología , Masculino , Antígenos de Superficie de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/metabolismo , Femenino , Adulto , Replicación Viral/efectos de los fármacos , Hepatitis B/virología , Hepatitis B/inmunología , Hepatitis B/tratamiento farmacológicoRESUMEN
The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions targeting the same organ (liver) as HBV. Until recently, the evolutionary origin of HDV remained largely unknown. The application of bioinformatics on whole sequence databases lead to discoveries of HDV-like agents (DLA) and shed light on HDV's evolution, expanding our understanding of HDV biology. DLA were identified in heterogeneous groups of vertebrates and invertebrates, highlighting that the evolution of HDV, represented by eight distinct genotypes, is broader and more complex than previously foreseen. In this study, we focused on the characterization of three mammalian DLA discovered in woodchuck (Marmota monax), white-tailed deer (Odocoileus virginianus), and lesser dog-like bat (Peropteryx macrotis) in terms of replication, cell-type permissiveness, and spreading pathways. We generated replication-competent constructs expressing 1.1-fold over-length antigenomic RNA of each DLA. Replication was initiated by transfecting the cDNAs into human (HuH7, HeLa, HEK293T, A549) and non-human (Vero E6, CHO, PaKi, LMH) cell lines. Upon transfection and replication establishment, none of the DLA expressed a large delta antigen. A cell division-mediated viral amplification assay demonstrated the capability of non-human DLA to replicate and propagate in hepatic and non-hepatic tissues, without the requirement of envelope proteins from a helper virus. Remarkably L-HDAg but not S-HDAg from HDV can artificially mediate envelopment of WoDV and DeDV ribonucleoproteins (RNPs) by HBsAg to form infectious particles, as demonstrated by co-transfection of HuH7 cells with the respective DLA expression constructs and a plasmid encoding HBV envelope proteins. These chimeric viruses are sensitive to HDV entry inhibitors and allow synchronized infections for comparative replication studies. Our results provide a more detailed understanding of the molecular biology, evolution, and virus-host interaction of this unique group of animal viroid-like agents in relation to HDV.
Asunto(s)
Virus de la Hepatitis B , Virus de la Hepatitis Delta , Marmota , Replicación Viral , Animales , Virus de la Hepatitis Delta/genética , Virus de la Hepatitis Delta/fisiología , Humanos , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Marmota/virología , División Celular , Quirópteros/virología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Línea Celular , Hepatitis B/virología , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , Genotipo , Células HEK293 , Hepatitis D/virología , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
Occult hepatitis B virus infection (OBI) is characterized by the presence of HBV DNA in the absence of detectable HBsAg. OBI is an important risk factor for cirrhosis and hepatocellular carcinoma, but its pathogenesis has not been fully elucidated. Mutations in the HBV preS/S genes can lead to impaired secretion of either HBsAg or S-protein resulting in the accumulation of defective viruses or S protein in cells. In our previous work, the M133S mutation was present in the HBV S gene of maintenance hemodialysis (MHD) patients with OBI. In this study, we investigated the potential role of amino acid substitutions in S proteins in S protein production and secretion through the construction of mutant S gene plasmids, structural prediction, transcriptome sequencing analysis, and in vitro functional studies. Protein structure prediction showed that the S protein M133S mutant exhibited hydrophilic modifications, with greater aggregation and accumulation of the entire structure within the membrane phospholipid bilayer. Differential gene enrichment analysis of transcriptome sequencing data showed that differentially expressed genes were mainly concentrated in protein processing in the endoplasmic reticulum (ER). The expression of heat shock family proteins and ER chaperone molecules was significantly increased in the wild-type and mutant groups, whereas the expression of mitochondria-associated proteins was decreased. Immunofluorescence staining and protein blotting showed that the endoplasmic reticulum-associated protein PDI, the autophagy marker LC3, and the lysosome-associated protein LAMP2 co-localized with the S proteins in the wild-type and mutant strains, and their expression was increased. The mitochondria-associated TOMM20 protein was also co-expressed with the S protein, but expression was significantly reduced in the mutant. The M133S mutation in the S gene is expressed as a defective and misfolded protein that accumulates in the endoplasmic reticulum causing secretion-impaired endoplasmic reticulum stress, which in turn triggers mitochondrial autophagy and recruits lysosomes to fuse with the autophagosome, leading to mitochondrial clearance. This study preliminarily demonstrated that the mutation of M133S in the S gene can cause OBI and is associated with disease progression, providing a theoretical basis for the diagnosis and treatment of OBI.
Asunto(s)
Estrés del Retículo Endoplásmico , Antígenos de Superficie de la Hepatitis B , Virus de la Hepatitis B , Hepatitis B , Mitofagia , Diálisis Renal , Humanos , Mitofagia/genética , Hepatitis B/virología , Hepatitis B/genética , Hepatitis B/metabolismo , Hepatitis B/complicaciones , Virus de la Hepatitis B/genética , Estrés del Retículo Endoplásmico/genética , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , Masculino , Mutación , Femenino , Persona de Mediana Edad , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Mitocondrias/metabolismo , Mitocondrias/genética , Sustitución de Aminoácidos , AdultoRESUMEN
BACKGROUND: In recent years, a gene-editing technology known as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been developed and is progressively advancing into clinical trials. While current antiviral therapies are unable to eliminate the Hepatitis B virus (HBV), it stands as a prime target for the CRISPR/Cas9 technology. The objective of this study was to enhance the efficacy of CRISPR/Cas9 in suppressing HBV replication, lowering HBsAg and HBeAg levels, and eliminating covalently closed circular DNA (cccDNA). METHODS: To enhance the anti-HBV effectiveness of CRISPR/Cas9, our study delved into a dual-guide RNA (gRNA) strategy. After evaluating the antiviral activities of multiple gRNAs that effectively impeded HBV replication, we identified three specific gRNAs-namely 10, 4, and 21. These gRNAs were selected for their targeting of distinct yet conserved regions within the HBV genome. RESULTS: In HBV-stable cell lines, namely HepAD38, and HBV infection models of HepG2-NTCP cells, our investigation revealed that the co-application of gRNA-10 with either gRNA-4 or gRNA-21 within the CRISPR/Cas9 system demonstrated heightened efficacy in impeding HBV replication, reducing the levels of HBsAg, HBeAg, and cccDNA levels, along with a more pronounced promotion of HBsAg clearance when compared to the use of a single gRNA. CONCLUSIONS: The CRISPR/Cas9 system employing dual gRNAs has proven highly effective in both suppressing HBV replication and facilitating HBsAg clearance. This promising outcome suggests that it holds potential to emerge as a novel approach for achieving the functional cure of patients with HBV infection.
Asunto(s)
Sistemas CRISPR-Cas , Virus de la Hepatitis B , ARN Guía de Sistemas CRISPR-Cas , Replicación Viral , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Humanos , Replicación Viral/genética , Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/genética , Células Hep G2 , Edición Génica/métodos , ADN Circular/genética , ADN Circular/metabolismo , ADN Viral/genética , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , Antígenos e de la Hepatitis B/genética , Antígenos e de la Hepatitis B/metabolismo , Antivirales/farmacología , Hepatitis B/virología , Hepatitis B/genética , Hepatitis B/terapiaRESUMEN
Chronic hepatitis B virus (HBV) infection remains a significant global health challenge due to its link to severe conditions like HBV-related cirrhosis and hepatocellular carcinoma (HCC). Although current treatments effectively reduce viral levels, they have limited impact on certain HBV elements, namely hepatitis B surface antigen (HBsAg) and covalently closed circular DNA (cccDNA). This highlights the urgent need for innovative pharmaceutical and biological interventions that can disrupt HBsAg production originating from cccDNA. In this study, we identified a natural furanocoumarin compound, Imperatorin, which markedly inhibited the expression of HBsAg from cccDNA, by screening a library of natural compounds derived from Chinese herbal medicines using ELISA assay and qRT-PCR. The pharmacodynamics study of Imperatorin was explored on HBV infected HepG2-NTCP/PHHs and HBV-infected humanized mouse model. Proteome analysis was performed on HBV infected HepG2-NTCP cells following Imperatorin treatment. Molecular docking and bio-layer interferometry (BLI) were used for finding the target of Imperatorin. Our findings demonstrated Imperatorin remarkably reduced the level of HBsAg, HBV RNAs, HBV DNA and transcriptional activity of cccDNA both in vitro and in vivo. Additionally, Imperatorin effectively restrained the actions of HBV promoters responsible for cccDNA transcription. Mechanistic study revealed that Imperatorin directly binds to ERK and subsequently interfering with the activation of CAMP response element-binding protein (CREB), a crucial transcriptional factor for HBV and has been demonstrated to bind to the PreS2/S and X promoter regions of HBV. Importantly, the absence of ERK could nullify the antiviral impact triggered by Imperatorin. Collectively, the natural compound Imperatorin may be an effective candidate agent for inhibiting HBsAg production and cccDNA transcription by impeding the activities of HBV promoters through ERK-CREB axis.
Asunto(s)
ADN Circular , Furocumarinas , Antígenos de Superficie de la Hepatitis B , Virus de la Hepatitis B , Transcripción Genética , Furocumarinas/farmacología , Humanos , Animales , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , Antígenos de Superficie de la Hepatitis B/genética , Células Hep G2 , Ratones , ADN Circular/genética , ADN Circular/metabolismo , Transcripción Genética/efectos de los fármacos , Antivirales/farmacología , ADN Viral , Simulación del Acoplamiento Molecular , Replicación Viral/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Modelos Animales de Enfermedad , Regiones Promotoras GenéticasRESUMEN
This study explore the molecular mechanism of the synergistic effect of Chinese Yam polysaccharides and nucleoside analogues(NAs) on hepatitis B virus(HBV) resistance. Different concentrations of Chinese Yam polysaccharide and entecavir were ad-ded to HepG2.2.15 cells. After the cytotoxicity was detected by cell counting kit-8(CCK-8), the optimal concentration and time of the two drugs to inhibit HepG2.2.15 cells were screened out. They were divided into control group, Chinese Yam polysaccharide group, entecavir group and combination drug group(Chinese Yam polysaccharide + entecavir). The drugs were added to HepG2.2.15 cells, ELISA was used to detect the effects of each group of drugs on the secretion of hepatitis B virus surface antigen(HBsAg) and hepatitis B virus e antigen(HBeAg) in cell supernatant, probe quantitative real-time PCR(probe qRT-PCR) was used to detect the effects of drugs on HBV-DNA in HepG2.2.15 cells, and Western blot was used to detect the effects of each group of drugs on the expression of p38 MAPK, p-p38 MAPK, NTCP proteins in HepG2.2.15 cells. The qRT-PCR was used to detect the effect of drugs on the expression of p38 MAPK and NTCP mRNA in HepG2.2.15 cells. The results showed that compared with control group, the concentrations of HBeAg and HBsAg in Chinese Yam polysaccharide group, entecavir group and combination group decreased(P<0.01 or P<0.001), and both of them inhibited HBV-DNA in HepG2.2.15 cells(P<0.01), and the HBV-DNA inhibition of HepG2.2.15 cells in the combination group was more obvious(P<0.001), and the protein expression levels of p-p38 MAPK and NTCP were significantly decreased(P<0.05 or P<0.01), the mRNA expression level of p38 MAPK increased, and the mRNA expression level of NTCP decreased(P<0.05 or P<0.01). To sum up, Chinese Yam polysaccharide can reduce the expression of NTCP protein and mRNA through p38 MAPK signaling pathway and cooperate with entecavir in anti-HBV.
Asunto(s)
Antivirales , Dioscorea , Virus de la Hepatitis B , Polisacáridos , Proteínas Quinasas p38 Activadas por Mitógenos , Humanos , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Polisacáridos/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Células Hep G2 , Antivirales/farmacología , Dioscorea/química , Sinergismo Farmacológico , Nucleósidos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Antígenos de Superficie de la Hepatitis B/metabolismo , Antígenos de Superficie de la Hepatitis B/genética , Antígenos e de la Hepatitis B/metabolismo , Hepatitis B/tratamiento farmacológico , Hepatitis B/virología , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/química , Guanina/análogos & derivados , Guanina/farmacologíaRESUMEN
Objective: To explore the antiviral activity of the small-molecule compound AM679 in hepatitis B virus (HBV) replication and infection cell models. Methods: The positive regulatory effect of AM679 on EFTUD2 expression was validated by qPCR and Western blotting. HepAD38 and HepG2-NTCP cells were treated with AM679 (0.5, 1, and 2 nmol/L). Negative control, positive control, and AM679 combined with the entecavir group were set up. HBV DNA intra-and extracellularly, as well as the expression levels of intracellular HBV total RNAs and 3.5kb-RNA changes, were detected with qPCR. Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels were measured in the cell supernatant by an enzyme-linked immunosorbent assay (ELISA). The t-test method was used for the statistical analysis of the mean difference between groups. Results: EFTUD2 mRNA and protein expression levels were significantly increased in HepAD38 and HepG2-NTCP cells following AM679 treatment, with a statistically significant difference (Pâ <â 0.001). Intra-and extracellular indicators such as HBV DNA, HBV RNAs, HBV 3.5kb-RNA, HBsAg, and HBeAg were decreased to varying degrees in both cell models, and the decrease in these indicators was more pronounced with the increase in AM679 concentration and prolonged treatment duration, while the combined use of AM679 and entecavir had a more significant antiviral effect. The HBV DNA inhibition rates in the supernatant of HepAD38 cells with the use of 2 nmol/L AM679 were 21% and 48% on days three and nine, respectively. The AM679 combined with the ETV treatment group had the most significant inhibitory effect (62%), with a Pâ <â 0.01. More active HBV replication was observed after silencing EFTUD2, while the antiviral activity of AM679 was significantly weakened. Conclusion: AM679 exerts anti-HBV activity in vitro by targeting the regulation of EFTUD2 expression.
Asunto(s)
Antivirales , Virus de la Hepatitis B , Replicación Viral , Humanos , Antivirales/farmacología , ADN Viral , Guanina/análogos & derivados , Células Hep G2 , Antígenos e de la Hepatitis B/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Indoles/química , Indoles/farmacología , Ácidos Pentanoicos/química , Ácidos Pentanoicos/farmacología , Factores de Elongación de Péptidos/antagonistas & inhibidores , Factores de Elongación de Péptidos/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/antagonistas & inhibidores , Ribonucleoproteína Nuclear Pequeña U5/metabolismoRESUMEN
Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.
Asunto(s)
Virus de la Hepatitis B , Hepatitis B Crónica , Hepatocitos , Humanos , Hepatocitos/virología , Hepatocitos/efectos de los fármacos , Animales , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/fisiología , Ratones , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Proteínas del Núcleo Viral/metabolismo , Antivirales/farmacología , Antivirales/uso terapéutico , Antígenos del Núcleo de la Hepatitis B/metabolismo , Cápside/metabolismo , Cápside/efectos de los fármacos , Hígado/virología , Hígado/efectos de los fármacos , Hígado/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Ensamble de Virus/efectos de los fármacos , Apoptosis/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND & AIMS: The immune tolerance induced by hepatitis B virus (HBV) is a major challenge for achieving effective viral clearance, and the mechanisms involved are not well-understood. One potential factor involved in modulating immune responses is mesencephalic astrocyte-derived neurotrophic factor (MANF), which has been reported to be increased in patients with chronic hepatitis B. In this study, our objective is to examine the role of MANF in regulating immune responses to HBV. METHODS: We utilized a commonly used HBV-harboring mouse model, where mice were hydrodynamically injected with the pAAV/HBV1.2 plasmid. We assessed the HBV load by measuring the levels of various markers including hepatitis B surface antigen, hepatitis B envelope antigen, hepatitis B core antigen, HBV DNA, and HBV RNA. RESULTS: Our study revealed that following HBV infection, both myeloid cells and hepatocytes exhibited increased expression of MANF. Moreover, we observed that mice with myeloid-specific MANF knockout (ManfMye-/-) displayed reduced HBV load and improved HBV-specific T cell responses. The decreased HBV-induced tolerance in ManfMye-/- mice was associated with reduced accumulation of myeloid-derived suppressor cells (MDSCs) in the liver. Restoring MDSC levels in ManfMye-/- mice through MDSC adoptive transfer reinstated HBV-induced tolerance. Mechanistically, we found that MANF promoted MDSC expansion by activating the IL-6/STAT3 pathway. Importantly, our study demonstrated the effectiveness of a combination therapy involving an hepatitis B surface antigen vaccine and nanoparticle-encapsulated MANF siRNA in effectively clearing HBV in HBV-carrier mice. CONCLUSION: The current study reveals that MANF plays a previously unrecognized regulatory role in liver tolerance by expanding MDSCs in the liver through IL-6/STAT3 signaling, leading to MDSC-mediated CD8+ T cell exhaustion.
Asunto(s)
Virus de la Hepatitis B , Tolerancia Inmunológica , Ratones Noqueados , Factores de Crecimiento Nervioso , Factor de Transcripción STAT3 , Animales , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/genética , Virus de la Hepatitis B/inmunología , Ratones , Factor de Transcripción STAT3/metabolismo , Modelos Animales de Enfermedad , Humanos , Hepatocitos/metabolismo , Hepatocitos/virología , Hepatocitos/inmunología , Hepatitis B/inmunología , Hepatitis B/virología , Carga Viral , Antígenos de Superficie de la Hepatitis B/metabolismo , Antígenos de Superficie de la Hepatitis B/inmunología , Hígado/inmunología , Hígado/metabolismo , Hígado/virología , Ratones Endogámicos C57BL , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virologíaRESUMEN
In subunit vaccines, aluminum salts (Alum) are commonly used as adjuvants, but with limited cellular immune responses. To overcome this limitation, CpG oligodeoxynucleotides (ODNs) have been used in combination with Alum. However, current combined usage of Alum and CpG is limited to linear mixtures, and the underlying interaction mechanism between CpG and Alum is not well understood. Thus, we propose to chemically conjugate Alum nanoparticles and CpG (with 5' or 3' end exposed) to design combination adjuvants. Our study demonstrates that compared to the 3'-end exposure, the 5'-end exposure of CpG in combination adjuvants (Al-CpG-5') enhances the activation of bone-marrow derived dendritic cells (BMDCs) and promotes Th1 and Th2 cytokine secretion. We used the SARS-CoV-2 receptor binding domain (RBD) and hepatitis B surface antigen (HBsAg) as model antigens to demonstrate that Al-CpG-5' enhanced antigen-specific antibody production and upregulated cytotoxic T lymphocyte markers. Additionally, Al-CpG-5' allows for coordinated adaptive immune responses even at lower doses of both CpG ODNs and HBsAg antigens, and enhances lymph node transport of antigens and activation of dendritic cells, promoting Tfh cell differentiation and B cell activation. Our novel Alum-CPG strategy points the way towards broadening the use of nanoadjuvants for both prophylactic and therapeutic vaccines.
Asunto(s)
Adyuvantes Inmunológicos , Hidróxido de Aluminio , Óxido de Aluminio , Células Dendríticas , Antígenos de Superficie de la Hepatitis B , Nanopartículas , Oligodesoxirribonucleótidos , Adyuvantes Inmunológicos/farmacología , Animales , Nanopartículas/química , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacología , Antígenos de Superficie de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/metabolismo , Hidróxido de Aluminio/química , Hidróxido de Aluminio/farmacología , Ratones , Ratones Endogámicos C57BL , Femenino , Citocinas/metabolismo , Compuestos de Alumbre/química , Compuestos de Alumbre/farmacologíaRESUMEN
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.
Asunto(s)
ADN Viral , Antígenos de Superficie de la Hepatitis B , Virus de la Hepatitis B , Hepatitis B Crónica , Hígado , Replicación Viral , Factor de Transcripción YY1 , Humanos , Factor de Transcripción YY1/metabolismo , Factor de Transcripción YY1/genética , Hepatitis B Crónica/virología , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/metabolismo , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Masculino , Femenino , Adulto , Persona de Mediana Edad , ADN Viral/genética , ADN Viral/sangre , Antígenos de Superficie de la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/metabolismo , Hígado/virología , Hígado/metabolismo , Guanina/análogos & derivados , Antivirales/uso terapéutico , Antivirales/farmacología , Interferones/metabolismo , Células Hep G2RESUMEN
Chronic liver diseases caused by hepatitis B virus (HBV) are the accepted main cause leading to liver cirrhosis, hepatic fibrosis, and hepatic carcinoma. Sodium taurocholate cotransporting polypeptide (NTCP), a specific membrane receptor of hepatocytes for triggering HBV infection, is a promising target against HBV entry. In this study, pentacyclic triterpenoids (PTs) including glycyrrhetinic acid (GA), oleanolic acid (OA), ursolic acid (UA) and betulinic acid (BA) were modified via molecular hybridization with podophyllotoxin respectively, and resulted in thirty-two novel conjugates. The anti-HBV activities of conjugates were evaluated in HepG2.2.15 cells. The results showed that 66% of the conjugates exhibited lower toxicity to the host cells and had significant inhibitory effects on the two HBV antigens, especially HBsAg. Notably, the compounds BA-PPT1, BA-PPT3, BA-PPT4, and UA-PPT3 not only inhibited the secretion of HBsAg but also suppressed HBV DNA replication. A significant difference in the binding of active conjugates to NTCP compared to the HBV PreS1 antigen was observed by SPR assays. The mechanism of action was found to be the competitive binding of these compounds to the NTCP 157-165 epitopes, blocking HBV entry into host cells. Molecular docking results indicated that BA-PPT3 interacted with the amino acid residues of the target protein mainly through π-cation, hydrogen bond and hydrophobic interaction, suggesting its potential as a promising HBV entry inhibitor targeting the NTCP receptor.