RESUMEN
Nucleic acid polymers (NAPs) are an attractive treatment modality for chronic hepatitis B (CHB), with REP2139 and REP2165 having shown efficacy in CHB patients. A subset of patients achieve functional cure, whereas the others exhibit a moderate response or are non-responders. NAP efficacy has been difficult to recapitulate in animal models, with the duck hepatitis B virus (DHBV) model showing some promise but remaining underexplored for NAP efficacy testing. Here we report on an optimized in vivo DHBV duck model and explore several characteristics of NAP treatment. REP2139 was efficacious in reducing DHBV DNA and DHBsAg levels in approximately half of the treated ducks, whether administered intraperitoneally or subcutaneously. Intrahepatic or serum NAP concentrations did not correlate with efficacy, nor did the appearance of anti-DHBsAg antibodies. Furthermore, NAP efficacy was only observed in experimentally infected ducks, not in endogenously infected ducks (vertical transmission). REP2139 add-on to entecavir treatment induced a deeper and more sustained virological response compared to entecavir monotherapy. Destabilized REP2165 showed a different activity profile with a more homogenous antiviral response followed by a faster rebound. In conclusion, subcutaneous administration of NAPs in the DHBV duck model provides a useful tool for in vivo evaluation of NAPs. It recapitulates many aspects of this class of compound's efficacy in CHB patients, most notably the clear division between responders and non-responders.
Asunto(s)
Infecciones por Hepadnaviridae , Virus de la Hepatitis B del Pato , Hepatitis B Crónica , Hepatitis Viral Animal , Ácidos Nucleicos , Animales , Humanos , Virus de la Hepatitis B del Pato/genética , Hepatitis B Crónica/tratamiento farmacológico , Antivirales/farmacología , Ácidos Nucleicos/uso terapéutico , Polímeros/uso terapéutico , Resultado del Tratamiento , Patos/genética , ADN Viral , Hepatitis Viral Animal/tratamiento farmacológico , Virus de la Hepatitis B , Infecciones por Hepadnaviridae/tratamiento farmacológico , Infecciones por Hepadnaviridae/veterinaria , HígadoRESUMEN
In order to ascertain the mechanism underlying the therapeutic efficacy of Bush sophora root polysaccharides (BSRPS) and phosphorylated Bush sophora root polysaccharides (pBSRPS) in the treatment of in duck viral hepatitis (DVH), an investigation was conducted to assess the protective impact of BSRPS and pBSRPS against duck hepatitis A virus type 1 (DHAV-1) induced mitochondrial dysfunction both in vivo and vitro. The BSRPS underwent modification through the utilization of the sodium trimetaphosphate - sodium tripolyphosphate method, and was subsequently characterized though Fourier infrared spectroscopy and scanning electron microscopy. Following this, the degree of mitochondrial oxidative damage and dysfunction was described through the use of fluorescence probes and various antioxidative enzyme assay kits. Furthermore, the utilization of transmission electron microscopy facilitated the observation of alterations in the mitochondrial ultrastructure within the liver tissue. Our findings demonstrated that both BSRPS and pBSRPS effectively mitigated mitochondrial oxidative stress and conserved mitochondrial functionality, as evidenced by heightened antioxidant enzyme activity, augmented ATP production, and stabilized mitochondrial membrane potential. Meanwhile, the histological and biochemical examinations revealed that the administration of BSRPS and pBSRPS resulted in a reduction of focal necrosis and infiltration of inflammatory cells, thereby mitigating liver injury. Additionally, both BSRPS and pBSRPS exhibited the ability to maintain liver mitochondrial membrane integrity and enhance the survival rate of ducklings infected with DHAV-1. Notably, pBSRPS demonstrated superior performance in all aspects of mitochondrial function compared to BSRPS. The findings indicated that maintaining mitochondrial homeostasis is a crucial factor in DHAV-1 infections, and the administration of BSRPS and pBSRPS may mitigate mitochondrial dysfunction and safeguard liver function.
Asunto(s)
Virus de la Hepatitis del Pato , Hepatitis Viral Animal , Hepatitis Viral Humana , Infecciones por Picornaviridae , Enfermedades de las Aves de Corral , Sophora , Animales , Hepatitis Viral Animal/tratamiento farmacológico , Hepatitis Viral Animal/patología , Patos , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Sophora/química , Mitocondrias , Polisacáridos/química , Hepatitis Viral Humana/tratamiento farmacológico , Infecciones por Picornaviridae/tratamiento farmacológico , Infecciones por Picornaviridae/patología , Enfermedades de las Aves de Corral/tratamiento farmacológicoRESUMEN
Duck hepatitis A virus serotype 1 (DHAV-1) causes acute inflammatory injury with a very high mortality rate in ducklings, leading to severe economic losses worldwide, especially in mainland China. There is an urgent need to find new treatments to prevent and control infection with DHAV-1. Not only is there a shortage of commercial anti-DHAV-1 drugs, but there are also gaps in the use and protection rates of existing commercial vaccines. We previously found that icariin (ICA), an extract of Epimedium, can reduce the mortality rate of ducklings after DHAV-1 infection, and the effect of ICA after phosphorylation modification (pICA) is more evident. In this study, we used duck embryo hepatocytes (DEHs) to investigate the mechanism of the alleviation of DHAV-1-induced inflammation and oxidative stress by ICA and pICA, and to further study their effects on hepatocyte mitochondrial function, apoptosis and cell cycle. It was found that ICA and pICA can inhibit the negative effects of DHAV-1 on apoptosis and cell cycle progression by stabilizing mitochondrial function, thereby reducing inflammation and ultimately protecting liver cells. The effects of pICA are more beneficial than those of ICA. The results of this study may be useful in the development of a new prophylactic and therapeutic strategy against DHAV-1 and other acute inflammatory diseases.
Asunto(s)
Flavonoides , Virus de la Hepatitis del Pato , Hepatitis Viral Animal , Hepatocitos , Infecciones por Picornaviridae , Enfermedades de las Aves de Corral , Animales , Patos , Flavonoides/farmacología , Virus de la Hepatitis del Pato/metabolismo , Hepatitis Viral Animal/tratamiento farmacológico , Hepatocitos/metabolismo , Mitocondrias , Estrés Oxidativo , Infecciones por Picornaviridae/tratamiento farmacológico , Infecciones por Picornaviridae/veterinaria , Enfermedades de las Aves de Corral/tratamiento farmacológico , Enfermedades de las Aves de Corral/prevención & control , SerogrupoRESUMEN
Metformin is not only the first-line medication for the treatment of type 2 diabetes, but it is also effective as an anti-inflammatory, anti-oxidative and anti-tumor agent. However, the effect of metformin during viral hepatitis remains elusive. Using an adenovirus (Ad)-induced viral hepatitis mouse model, we found that metformin treatment significantly attenuated liver injury, with reduced serum aspartate transaminase (AST) and alanine transaminase (ALT) levels and liver histological changes, presumably via decreased effector T cell responses. We then demonstrated that metformin reduced mTORC1 activity in T cells from infected mice, as evidenced by decreased phosphorylation of ribosome protein S6 (p-S6). The inhibitory effects on the mTORC1 signaling by metformin was dependent on the tuberous sclerosis complex 1 (TSC1). Mechanistically, metformin treatment modulated the phosphorylation of dynamin-related protein 1 (Drp-1) and mitochondrial fission 1 protein (FIS1), resulting in increased mass in effector T cells. Moreover, metformin treatment promoted mitochondrial superoxide production, which can inhibit excessive T cell activation in viral hepatitis. Together, our results revealed a protective role and therapeutic potential of metformin against liver injury in acute viral hepatitis via modulating effector T cell activation via regulating the mTORC1 pathway and mitochondrial functions.
Asunto(s)
Infecciones por Adenoviridae/tratamiento farmacológico , Adenoviridae/fisiología , Hepatitis Viral Animal/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Hígado/patología , Metformina/uso terapéutico , Mitocondrias/metabolismo , Infecciones por Adenoviridae/inmunología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Metabolismo Energético , Femenino , Hepatitis Viral Animal/inmunología , Humanos , Hígado/efectos de los fármacos , Activación de Linfocitos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína 1 del Complejo de la Esclerosis Tuberosa/metabolismoRESUMEN
INTRODUCTION: Duck viral hepatitis type I (DVH-I) is a poorly studied contagious disease caused by RNA-containing duck (Anatinae) hepatitis virus type I (Picornaviridae: Avihepatovirus: Avihepatovirus A). This infection is widespread in many countries, including Russia, and causes significant damage to industrial duck breeding. The study of interferonogenic activity of its etiologic agent strains is of great importance in solving the problem of developing effective means to control the disease. MATERIAL AND METHODS: Strain BH-3 of duck hepatitis virus type I isolated from the liver of sick ducklings was used in the study. The strain was adapted to developing 10-12 day old duck embryos, to the cell culture of chicken and duck fibroblasts and deposited in the State Collection of Viruses of the D.I. Ivanovsky Institute of Virology of FSBI «National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya¼ of the Ministry of Health of Russia. Experiments were performed using the standard tissue culture method. RESULTS AND DISCUSSION: Data on the ability of the viral strain BH-3 to induce interferon (IFN) and its sensitivity to the action of exogenous interferon in the culture of duck fibroblasts are presented. It has been shown that the interferonogenic activity of this strain of the hepatitis virus is in direct proportion to the multiplicity of infection. The maximum induction of IFN (1 : 256 CEPD50) was observed at a dose of 1.0 TCD50/cell in 72-96 hrs after inoculation of the cell culture. Exogenous IFN at a dose of 1 : 128 completely suppressed the cytopathic effect and death of duck embryos infected with hepatitis virus at a dose of 100 TCD50/cell. CONCLUSION: The data obtained allow us to state that the vaccine strain BH-3 of duck hepatitis virus type I has a pronounced interferonogenic activity and sensitivity to the action of exogenous IFN. This may have implications for the development of effective therapeutic agents against DVH-I.
Asunto(s)
Virus de la Hepatitis del Pato , Hepatitis Viral Animal , Infecciones por Picornaviridae , Enfermedades de las Aves de Corral , Animales , Hidroxitolueno Butilado/análogos & derivados , Patos , Hepatitis Viral Animal/tratamiento farmacológico , Interferones , PicornaviridaeRESUMEN
Various human disorders are cured by the use of licorice, a key ingredient of herbal remedies. Glycyrrhizic acid (GL), a triterpenoid glycoside, is the aqueous extract from licorice root. Glycyrrhetinic acid (GA) has been reported to be a major bioactive hydrolysis product of GL and has been regarded as an anti-inflammatory agent for the treatment of a variety of inflammatory diseases, including hepatitis. However, the mechanism by which GA inhibits viral hepatic inflammatory injury is not completely understood. In this study, we found that, by consecutively treating mice with a traditional herbal recipe, licorice plays an important role in the detoxification of mice. We also employed a murine hepatitis virus (MHV) infection model to illustrate that GA treatment inhibited activation of hepatic inflammatory responses by blocking high-mobility group box 1 (HMGB1) cytokine activity. Furthermore, decreased HMGB1 levels and downstream signaling triggered by injection of a neutralizing HMGB1 antibody or TLR4 gene deficiency, also significantly protected against MHV-induced severe hepatic injury. Thus, our findings characterize GA as a hepatoprotective therapy agent in hepatic infectious disease not only by suppressing HMGB1 release and blocking HMGB1 cytokine activity, but also via an underlying viral-induced HMGB1-TLR4 immunological regulation axis that occurs during the cytokine storm. The present study provides a new therapy strategy for the treatment of acute viral hepatitis in the clinical setting.
Asunto(s)
Antiinflamatorios/uso terapéutico , Medicamentos Herbarios Chinos/uso terapéutico , Ácido Glicirretínico/uso terapéutico , Proteína HMGB1/inmunología , Hepatitis Viral Animal/tratamiento farmacológico , Receptor Toll-Like 4/genética , Animales , Antiinflamatorios/farmacología , Línea Celular , Citocinas/genética , Medicamentos Herbarios Chinos/farmacología , Femenino , Ácido Glicirretínico/farmacología , Glycyrrhiza , Hepatitis Viral Animal/genética , Hepatitis Viral Animal/inmunología , Hígado/efectos de los fármacos , Hígado/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Virus de la Hepatitis Murina , Transducción de Señal/efectos de los fármacosRESUMEN
Duck hepatitis A virus type 1 (DHAV) infection causes duck viral hepatitis and results in enormous loss to poultry farming industry. We reported that phosphorylated Codonopsis pilosula polysaccharide (pCPPS) inhibited DHAV genome replication. Here we further explored its underlying antiviral mechanisms. Autophagosomes formation is essential for the genome replication of picornaviruses. In this study, Western blot, confocal microscopy observation, and ELISA methods were performed to analyze polysaccharides' effects on autophagy by the in vitro and in vivo experiments. Results obtained from in vitro and in vivo experiments showed that Codonopsis pilosula polysaccharide did not play a role in regulating autophagy and had no therapeutic effects on infected ducklings. However, pCPPS treatment downregulated LC3-II expression level activated by DHAV and rapamycin, indicating the inhibition of autophagosomes formation. The interdiction of autophagosomes formation resulted in the inhibition of DHAV genome replication. Further study showed that pCPPS treatment reduced the concentration of phosphatidylinositol-3-phosphate (PI3P), an important component of membrane, in cells and serum, and consequently, autophagosomes formation was downregulated. In vivo experiments also verified the therapeutic effect of pCPPS. Phosphorylated Codonopsis pilosula polysaccharide treatment increased the infected ducklings' survival rate and alleviated hepatic injury. Our studies verified the effects of pCPPS against DHAV infection in duck embryo hepatocytes and ducklings and confirmed that phosphorylated modification enhanced the bioactivities of polysaccharides. The results also stated pCPPS's antiviral mechanisms, provided fundamental basis for the development of new anti-DHAV agents.
Asunto(s)
Antivirales/farmacología , Autofagosomas/efectos de los fármacos , Codonopsis/química , Virus de la Hepatitis del Pato/efectos de los fármacos , Hepatitis Viral Animal/tratamiento farmacológico , Infecciones por Picornaviridae/veterinaria , Polisacáridos/farmacología , Replicación Viral/efectos de los fármacos , Animales , Autofagia/efectos de los fármacos , Virus de la Hepatitis del Pato/fisiología , Fosforilación , Infecciones por Picornaviridae/tratamiento farmacológicoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Formula Le-Cao-Shi (LCS) is a traditional Chinese medicine (TCM), which has long been used as a folk remedy against hepatitis B in China. The present study was conducted to evaluate the anti-hepatitis B effects of aqueous extract of LCS in vivo and in vitro. MATERIALS AND METHOD: we investigated the anti-HBV effects of LCS in vivo and in vitro with duck hepatitis B model and HepG2.2.15â¯cell line model, respectively. The serologic and cellular biomarkers and the histopathological changes were examined. RESULTS: By a duck hepatitis B model, the extract of LCS was found to restrain the expressions of duck hepatitis B surface antigen (DHBsAg), hepatitis B e antigen (DHBeAg), and HBV-DNA (DHBV-DNA). Moreover, LCS could decrease the levels of aspartate and alanine aminotransferases (AST and ALT) and ameliorate duck liver histological lesions. Correspondingly, in a HepG2.2.15 cellular model, LCS could also significantly inhibit the secretions of HBsAg and HBeAg. CONCLUSION: LCS exerted potent anti-hepatitis effects against the infection of HBV. The above results demonstrated the first-hand experimental evidences for the anti-hepatitis B efficiency of LCS. Our study provides a basis for further exploration and development of this promising compound prescription to treat hepatitis B disease.
Asunto(s)
Antivirales/uso terapéutico , Medicamentos Herbarios Chinos/uso terapéutico , Hepatitis B/tratamiento farmacológico , Hepatitis Viral Animal/tratamiento farmacológico , Animales , Antivirales/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN Viral , Patos , Hepatitis B/inmunología , Hepatitis B/patología , Hepatitis B/virología , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B del Pato/efectos de los fármacos , Virus de la Hepatitis B del Pato/genética , Virus de la Hepatitis B del Pato/inmunología , Antígenos e de la Hepatitis B/inmunología , Hepatitis Viral Animal/inmunología , Hepatitis Viral Animal/patología , Hepatitis Viral Animal/virología , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Medicina Tradicional ChinaRESUMEN
BACKGROUND: Duck viral hepatitis (DVH) is an acute disease of young ducklings with no effective veterinary drugs for treatment. Gynostemma pentaphyllum is a well-known traditional Chinese medicine that plays an important role in the treatment of various diseases. Gypenoside (GP), one of the main ingredients of Gynostemma pentaphyllum, was reported with good hepatoprotective effects. However, its low solubility limits its application in the clinics. To improve its solubility and bioactivity, a phosphorylated derivative of gypenoside (pGP) was prepared by the sodium trimetaphosphate-sodium tripolyphosphate (STMP-STPP) method. An infrared spectroscopy method was applied to analyse the structures of GP and pGP. Then, a methyl thiazolyl tetrazolium (MTT) colorimetric assay was applied to study the hepatocyte protective efficacy of these two drugs against duck hepatitis A virus type 1 (DHAV-1) infection, and qPCR, TUNEL labelling and flow cytometry methods were used to study the relevant hepatocyte protective in vitro. RESULTS: The infrared spectroscopy detection results showed that the phosphorylation modification of GP was successful. The MTT colorimetric assay results showed that both GP and pGP possessed good hepatocyte protective efficacy in vitro, and pGP performed better than GP when the drug was added before or after virus inoculation. Furthermore, the qPCR results revealed that both drugs could effectively inhibit the adsorption (when adding GP and pGP pre-virus inoculation), replication and release of DHAV-1, and the viral inhibition rate of pGP was greater than that of GP. The subsequent TUNEL labelling and flow cytometry assays showed that both GP and pGP could significantly inhibit duck embryo hepatocyte apoptosis induced by DHAV-1, and the inhibition effect of pGP was much stronger than that of GP. CONCLUSIONS: GP exerts good hepatocyte protective efficacy not only by inhibiting the proliferation of DHAV-1 but also by inhibiting duck embryonic hepatocyte apoptosis induced by DHAV-1, and phosphorylation modification significantly improves the antiviral and the anti-apoptotic effects of GP. Therefore, pGP has the potential to be developed into a novel drug against DHAV-1 infection.
Asunto(s)
Virus de la Hepatitis del Pato/efectos de los fármacos , Animales , Antivirales/farmacología , Apoptosis/efectos de los fármacos , Células Cultivadas , Patos , Gynostemma/química , Hepatitis Viral Animal/tratamiento farmacológico , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Fosforilación , Extractos Vegetales/química , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is a malignant disease in ducklings, causing economic losses in the duck industry. However, there is still no antiviral drug against DHAV-1 in the clinic. OBJECTIVE: Our aim is to investigate the anti-DHAV-1 effect of baicalin, which is a flavonoid derived from the Chinese medicinal herb huangqin (Scutellaria baicalensis Georgi). METHODS: Here, we first detected its anti-DHAV-1 ability in vitro and in vivo. At the same time, the inhibition of baicalin on DHAV-1 reproduction was determined. Finally, we tested and verified the anti-oxidative and immuno-enhancing roles of baicalin on its curative effect on DVH. RESULTS: Baicalin possessed anti-DHAV-1 effect. It improved the cytoactive of DEH which was infected by DHAV-1 as well as reduced the DHAV-1 reproduction in DEH. Under baicalin treatment, mortality of ducklings infected by DHAV-1 decreased, additionally the DHAV-1 level and liver injury in such ducklings were significantly reduced or alleviated. The in vitro mechanism study indicated baicalin inhibited DHAV-1 reproduction via interfering the viral replication and release. Furthermore, the in vivo mechanism study manifested both the anti-oxidative and immuno-enhancing abilities of baicalin, which played crucial roles in its curative effect on DVH. CONCLUSION: This study may provide a scientific basis for developing baicalin as one or a part of the anti-DHAV-1 drugs.
Asunto(s)
Antivirales/farmacología , Flavonoides/farmacología , Virus de la Hepatitis del Pato/efectos de los fármacos , Hepatitis Viral Animal/virología , Animales , Antioxidantes/farmacología , Células Cultivadas , Patos , Virus de la Hepatitis del Pato/genética , Hepatitis Viral Animal/tratamiento farmacológico , Hepatitis Viral Animal/mortalidad , Hepatitis Viral Animal/patología , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Pruebas de Función Hepática , Extractos Vegetales , Scutellaria baicalensis , Liberación del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
CD8 T cells protect the liver against viral infection, but can also cause severe liver damage that may even lead to organ failure. Given the lack of mechanistic insights and specific treatment options in patients with acute fulminant hepatitis, we develop a mouse model reflecting a severe acute virus-induced CD8 T cell-mediated hepatitis. Here we show that antigen-specific CD8 T cells induce liver damage in a perforin-dependent manner, yet liver failure is not caused by effector responses targeting virus-infected hepatocytes alone. Additionally, CD8 T cell mediated elimination of cross-presenting liver sinusoidal endothelial cells causes endothelial damage that leads to a dramatically impaired sinusoidal perfusion and indirectly to hepatocyte death. With the identification of perforin-mediated killing as a critical pathophysiologic mechanism of liver failure and the protective function of a new class of perforin inhibitor, our study opens new potential therapeutic angles for fulminant viral hepatitis.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Hepatitis Viral Animal/tratamiento farmacológico , Hígado/efectos de los fármacos , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inhibidores , Sustancias Protectoras/farmacología , Sulfonamidas/farmacología , Adenoviridae/genética , Adenoviridae/inmunología , Adenoviridae/patogenicidad , Animales , Anticuerpos/administración & dosificación , Antígenos CD40/antagonistas & inhibidores , Antígenos CD40/genética , Antígenos CD40/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Capilares/efectos de los fármacos , Capilares/virología , Modelos Animales de Enfermedad , Células Endoteliales/inmunología , Células Endoteliales/virología , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hepatitis Viral Animal/inmunología , Hepatitis Viral Animal/virología , Hepatocitos/efectos de los fármacos , Hepatocitos/inmunología , Hepatocitos/virología , Humanos , Hígado/irrigación sanguínea , Hígado/patología , Hígado/virología , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/administración & dosificación , Poli I-C/administración & dosificación , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/inmunologíaRESUMEN
Nucleic acid polymer (NAP) REP 2139 treatment was shown to block the release of viral surface antigen in duck HBV (DHBV)-infected ducks and in patients with chronic HBV or HBV/hepatitis D virus infection. In this preclinical study, a combination therapy consisting of REP 2139 with tenofovir disoproxil fumarate (TDF) and entecavir (ETV) was evaluated in vivo in the chronic DHBV infection model. DHBV-infected duck groups were treated as follows: normal saline (control); REP 2139 TDF; REP 2139 + TDF; and REP 2139 + TDF + ETV. After 4 weeks of treatment, all animals were followed for 8 weeks. Serum DHBsAg and anti-DHBsAg antibodies were monitored by enzyme-linked immunosorbent assay and viremia by qPCR. Total viral DNA and covalently closed circular DNA (cccDNA) were quantified in autopsy liver samples by qPCR. Intrahepatic DHBsAg was assessed at the end of follow-up by immunohistochemistry. On-treatment reduction of serum DHBsAg and viremia was more rapid when REP 2139 was combined with TDF or TDF and ETV, and, in contrast to TDF monotherapy, no viral rebound was observed after treatment cessation. Importantly, combination therapy resulted in a significant decrease in intrahepatic viral DNA (>3 log) and cccDNA (>2 log), which were tightly correlated with the clearance of DHBsAg in the liver. CONCLUSION: Synergistic antiviral effects were observed when REP 2139 was combined with TDF or TDF + ETV leading to control of infection in blood and liver, associated with intrahepatic viral surface antigen elimination that persisted after treatment withdrawal. Our findings suggest the potential of developing such combination therapy for treatment of chronically infected patients in the absence of pegylated interferon. (Hepatology 2018;67:2127-2140).
Asunto(s)
Antivirales/administración & dosificación , Guanina/análogos & derivados , Infecciones por Hepadnaviridae/tratamiento farmacológico , Virus de la Hepatitis B del Pato/efectos de los fármacos , Hepatitis Viral Animal/tratamiento farmacológico , Ácidos Nucleicos/administración & dosificación , Polímeros/administración & dosificación , Tenofovir/administración & dosificación , Animales , Enfermedad Crónica , Sinergismo Farmacológico , Quimioterapia Combinada , Patos , Guanina/administración & dosificaciónRESUMEN
BACKGROUND: Duck viral hepatitis (DVH), caused by duck hepatitis A virus (DHAV), is a fatal contagious infectious disease which spreads rapidly with high morbidity and high mortality, and there is no effective clinical drug against DVH. MATERIALS AND METHODS: Raw Rehmannia Radix Polysaccharide (RRRP), Lycii Fructus polysaccharides and Astragalus Radix polysaccharides were experimented in vitro and in vivo. Mortality rate, livers change, liver lesion scoring, peroxidative injury evaluation indexes in vitro and in vivo, and hepatic injury evaluation indexes of optimal one were detected and observed in this experiment. RESULTS: RRRP could reduce mortality with the protection rate about 20.0% compared with that of the viral control (VC) group, finding that RRRP was the most effective against DHAV. The average liver scoring of the VC, blank control (BC), RRRP groups were 3.5, 0, 2.1. Significant difference (P<0.05) appeared between any two groups, demonstrating that it can alleviate liver pathological change. RRRP could make the hepatic injury evaluation indexes similar to BC group while the levels of the VC group were higher than other two groups in general. The levels of SOD, GSH-Px, CAT of RRRP group showed significant higher than that of VC group while the levels of NOS and MDA showed the opposite tendency, thus, RRRP could release peroxidative injury. CONCLUSION: RRRP was the most effective against duck hepatitis A virus (DHAV). RRRP could reduce mortality, alleviate liver pathological change, down-regulate liver lesion score, release peroxidative injury and hepatic injury. The antiviral and peroxidative injury releasing activity of RRRP for DHAV provided a platform to test novel drug strategies for hepatitis A virus in human beings.
Asunto(s)
Antivirales/administración & dosificación , Virus de la Hepatitis del Pato/efectos de los fármacos , Hepatitis Viral Animal/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/administración & dosificación , Polisacáridos/administración & dosificación , Enfermedades de las Aves de Corral/tratamiento farmacológico , Rehmannia/química , Animales , Planta del Astrágalo/química , Patos , Virus de la Hepatitis del Pato/fisiología , Hepatitis Viral Animal/diagnóstico por imagen , Hepatitis Viral Animal/metabolismo , Hepatitis Viral Animal/virología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/virología , Raíces de Plantas/química , Enfermedades de las Aves de Corral/metabolismo , Enfermedades de las Aves de Corral/virologíaRESUMEN
The principal target organ of duck hepatitis A virus type 1 (DHAV-1) is duckling liver, which is an energy-intensive organ and plays important roles in body's energy metabolism and conversion. As the "power house" of the hepatocytes, mitochondria provide more than 90% of the energy. However, mitochondria are much vulnerable to the oxidative stress for their rich in polyunsaturated fatty acids. Although previous researches have demonstrated that DHAV-1 could induce the oxidative stress in the serum of the infected ducklings, no related study on the mitochondria during the pathological process of DVH has been reported by far. To address this issue, we examined the HE stained tissue pathological slices, detected the hepatic SOD, CAT and GPX activities and MDA contents and analyzed the ATP content, mitochondrial ultrastructure and the mitochondrial SOD, GPX activities and MDA content in the liver tissues. The results showed that the hepatic redox status was significantly disturbed so that causing the mitochondrial dysfunction, ATP depletion and mitochondrial oxidative stress during the process of the DHAV-1 infection, and a prescription formulated with Hypericum japonicum flavone, Radix Rehmanniae Recens polysaccharide and Salvia plebeia flavone (HRS), which had been demonstrated with good anti-oxidative activity in serum, could effectively alleviate the hepatic injury and the oxidative stress in liver tissue induced by DHAV-1 thus alleviating the mitochondrial injury and oxidative stress. In a word, this research discovers the oxidative stress induced mitochondrial dysfunction and oxidative stress during the DVH pathological process and demonstrates HRS exerts good anti-oxidative activity in liver tissue to protect mitochondria against reactive oxygen species (ROS).
Asunto(s)
Antivirales/farmacología , Medicamentos Herbarios Chinos/farmacología , Virus de la Hepatitis del Pato , Hepatitis Viral Animal/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Infecciones por Picornaviridae/tratamiento farmacológico , Adenosina Trifosfato/metabolismo , Animales , Patos , Flavonas/farmacología , Glutatión Peroxidasa/metabolismo , Hepatitis Viral Animal/metabolismo , Hepatitis Viral Animal/mortalidad , Hepatitis Viral Animal/patología , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Malondialdehído/metabolismo , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Estrés Oxidativo/efectos de los fármacos , Infecciones por Picornaviridae/metabolismo , Infecciones por Picornaviridae/mortalidad , Infecciones por Picornaviridae/patología , Polisacáridos/farmacología , Distribución Aleatoria , Superóxido Dismutasa/metabolismo , Resultado del TratamientoRESUMEN
Although large amounts of vitamin A and its metabolite all-trans retinoic acid (RA) are stored in the liver, how RA regulates liver immune responses during viral infection remains unclear. In this study, we demonstrated that IL-22, mainly produced by hepatic γδ T cells, attenuated liver injury in adenovirus-infected mice. RA can promote γδ T cells to produce mTORC1-dependent IL-22 in the liver, but inhibits IFN-γ and IL-17. RA also affected the aptitude of T cell responses by modulating dendritic cell (DC) migration and costimulatory molecule expression. These results suggested that RA plays an immunomodulatory role in viral infection. Proteomics data revealed that RA downregulated S100 family protein expression in DCs, as well as NF-κB/ERK pathway activation in these cells. Furthermore, adoptive transfer of S100A4-repressed, virus-pulsed DCs into the hind foot of naive mice failed to prime T cell responses in draining lymph nodes. Our study has demonstrated a crucial role for RA in promoting IL-22 production and tempering DC function through downregulating S100 family proteins during viral hepatitis.
Asunto(s)
Adenoviridae/inmunología , Células Dendríticas/efectos de los fármacos , Hepatitis Viral Animal/tratamiento farmacológico , Factores Inmunológicos/uso terapéutico , Interleucinas/metabolismo , Hígado/inmunología , Proteína de Unión al Calcio S100A4/metabolismo , Tretinoina/uso terapéutico , Animales , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/virología , Femenino , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/genética , Interleucinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Linfocitos T/inmunología , Linfocitos T/virología , Interleucina-22RESUMEN
An estimated 350 million people are chronically infected with hepatitis B virus (HBV), and over one million people die each year due to HBV-associated liver diseases, such as cirrhosis and liver cancer. Current therapeutics for chronic HBV infection are limited to nucleos(t)ide analogs and interferon. These anti-HBV drugs in general reduce viral load and improve the long-term outcome of infection but very rarely lead to a cure. Thus, new therapies for chronic HBV infection need to be developed by utilizing liver cell lines and primary cultures and small laboratory animals capable of replicating HBV or surrogate hepadnaviruses for antiviral testing. Natural infection with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to HBV, occurs in woodchucks. Chronic WHV infection has been established over decades as a suitable model for evaluating direct-acting antivirals as well as vaccines, vaccine adjuvants, and immunotherapeutics because animals are fully immunocompetent. Before HBV-specific compounds are applied to woodchucks, they are usually tested in primary woodchuck hepatocytes (PWHs) replicating WHV at high levels for confirming drug specificity against viral or host targets. Here we describe a protocol for the isolation of PWHs from liver of WHV-infected woodchucks, maintenance in culture, and use in assays for determining antiviral efficacy, safety, and associated host innate immune response of new experimental drugs. Exemplary assays were performed with the nucleoside analog, lamivudine, and the immunomodulator, interferon-alpha.
Asunto(s)
Antivirales/farmacología , Virus de la Hepatitis B de la Marmota/inmunología , Hepatitis Viral Animal/inmunología , Hepatitis Viral Animal/virología , Hepatocitos/efectos de los fármacos , Hepatocitos/inmunología , Inmunidad Innata , Animales , Separación Celular , Supervivencia Celular , Regulación de la Expresión Génica/efectos de los fármacos , Hepatitis Viral Animal/tratamiento farmacológico , Hepatocitos/virología , Inmunidad Innata/genética , Factores Inmunológicos/farmacología , Interferón-alfa/farmacología , ARN Viral , Replicación ViralRESUMEN
Duck hepatitis A virus (DHAV) (Picornaviridae) causes an infectious disease in ducks which results in severe losses in duck industry. However, the proper antiviral supportive drugs for this disease have not been discovered. Polysaccharide is the main ingredient of Astragalus that has been demonstrated to directly and indirectly inhibit RNA of viruses replication. In this study, the antiviral activities of Astragalus polysaccharide (APS) and its derivatives against DHAV were evaluated and compared. APS was modified via the sodium trimetaphosphate and sodium tripolyphosphate (STMP-STPP) method and chlorosulfonic acid-pyridine method to obtain its phosphate (pAPS) and sulfate (sAPS), respectively. The infrared structures of APS, pAPS, and sAPS were analyzed with the potassium bromide disc method. Additionally, the antiviral activities were evaluated with the MTT ((4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) method in vitro and the artificial inoculation method in vivo. The clinical therapy effects were evaluated by mortality rate, liver function-related biochemical indicators, and visual changes in pathological anatomy. The anti-DHAV proliferation effects of APS, pAPS, and sAPS on the viral multiplication process in cell and blood were observed with the reverse transcription-polymerase chain reaction method. The results revealed that pAPS inhibited DHAV proliferation more efficiently in the entire process of viral multiplication than APS and sAPS. Moreover, only pAPS significantly improved the survival rate to 33.5% and reduced the DHAV particle titer in the blood as well as liver lesions in clinical trials. The results indicated that pAPS exhibited greater anti-DHAV activity than APS and sAPS both in vitro and in vivo.
Asunto(s)
Planta del Astrágalo/química , Virus de la Hepatitis del Pato/efectos de los fármacos , Hepatitis Viral Animal/tratamiento farmacológico , Infecciones por Picornaviridae/tratamiento farmacológico , Polisacáridos/farmacología , Enfermedades de las Aves de Corral/tratamiento farmacológico , Replicación Viral/efectos de los fármacos , Animales , Patos/virología , Medicina Tradicional China , Fosforilación , Preparaciones de Plantas/farmacología , Polisacáridos/química , Enfermedades de las Aves de Corral/virología , Sulfatos/química , Sulfatos/farmacologíaRESUMEN
To screen effective anti-duck hepatitis A virus (DHAV) drugs, we applied STMP-STPP method to prepare phosphorylated Codonopsis pilosula polysaccharide (pCPPS), the phosphorylation-modified product of Codonopsis pilosula polysaccharide (CPPS). The IR spectrum and field emission scanning electron microscope (FE-SEM) were subsequently used to analyze the structure of pCPPS. Several tests were conducted to compare the anti-DHAV activities of CPPS and pCPPS. The MTT method was used to compare the effect of the drugs on DHAV-infected duck embryonic hepatocytes (DEHs), and the Reed-Muench assay was employed to observe changes in the virulence of DHAV. We also applied real-time PCR to examine the relationship between virus replication and the expression of IFN-ß. The results indicated that CPPS could not inhibit the replication of DHAV. In contrast, pCPPS increased the virus TCID50, inhibited viral replication and, accordingly, increased the survival rate of DEHs infected with DHAV. Because DHAV induced the expression of IFN-ß, and the IFN-ß expression level was positively associated with the number of DHAV, the reduction of IFN-ß expression levels after pCPPS treatment demonstrated a decrease in the number of virus particles. These results indicated that pCPPS, which reduces the number of DHAV, was more effective than CPPS in anti-DHAV activity.
Asunto(s)
Antivirales/farmacología , Virus de la Hepatitis del Pato/efectos de los fármacos , Hepatitis Viral Animal/tratamiento farmacológico , Extractos Vegetales/farmacología , Polisacáridos/farmacología , Enfermedades de las Aves de Corral/tratamiento farmacológico , Animales , Proteínas Aviares/metabolismo , Células Cultivadas , Codonopsis/química , Evaluación Preclínica de Medicamentos , Patos , Virus de la Hepatitis del Pato/fisiología , Hepatitis Viral Animal/virología , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Interferón beta/metabolismo , Enfermedades de las Aves de Corral/virología , Virulencia , Replicación Viral/efectos de los fármacosRESUMEN
The sphingosine kinase (SphK)1/sphingosine-1-phosphate (S1P) pathway is involved in multiple biological processes, including liver diseases. This study investigate whether modulation of the SphK1/S1P system associates to the beneficial effects of melatonin in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10(4) hemagglutination units of a RHDV isolate and received 20 mg/kg of melatonin at 0, 12, and 24 hr postinfection. Liver mRNA levels, protein concentration, and immunohistochemical labeling for SphK1 increased in RHDV-infected rabbits. S1P production and protein expression of the S1PR1 receptor were significantly elevated following RHDV infection. These effects were significantly reduced by melatonin. Rabbits also exhibited increased expression of toll-like receptor (TLR)4, tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, nuclear factor-kappa B (NF-κB) p50 and p65 subunits, and phosphorylated inhibitor of kappa B (IκB)α. Melatonin administration significantly inhibited those changes and induced a decreased immunoreactivity for RHDV viral VP60 antigen in the liver. Results obtained indicate that the SphK1/S1P system activates in parallel to viral replication and the inflammatory process induced by the virus. Inhibition of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in this animal model of ALF, and supports the potential of melatonin as an antiviral agent.
Asunto(s)
Infecciones por Caliciviridae/metabolismo , Virus de la Enfermedad Hemorrágica del Conejo , Hepatitis Viral Animal/metabolismo , Fallo Hepático Agudo/metabolismo , Lisofosfolípidos/metabolismo , Melatonina/farmacocinética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal/efectos de los fármacos , Esfingosina/análogos & derivados , Animales , Infecciones por Caliciviridae/tratamiento farmacológico , Hepatitis Viral Animal/tratamiento farmacológico , Fallo Hepático Agudo/tratamiento farmacológico , Masculino , Conejos , Esfingosina/metabolismoRESUMEN
Because polysaccharide and flavone ingredients display good antiviral activity, we developed a flavone/polysaccharide-containing prescription that would be effective against duck viral hepatitis (DVH) and investigated its hepatoprotective effects. Flavones were derived from Hypericum japonicum (HJF) (entire herb of Hypericum japonicum Thunb) and Salvia plebeia (SPF) (entire herb of Salvia plebeia R. Br.), and polysaccharides were derived from Radix Rehmanniae Recens (RRRP) (dried root of Rehmannia glutinosa Libosch). This prescription combination was based on the theory of syndrome differentiation and treatment in traditional Chinese veterinary medicine. In vitro and in vivo experiments were conducted using the three single ingredients compared to the combined HRS prescription to determine their anti-duck hepatitis A viral (anti-DHAV) activity. The results showed that all experimental conditions displayed anti-DHAV activity, but the HRS prescription presented the best effect. To further investigate the hepatoprotective effect of the HRS prescription on DHAV-induced hepatic injury, we tested the mortality rate, the hepatic pathological severity score, plasma biochemical indexes of hepatic function, blood DHAV gene expression levels and peroxidation damage evaluation indexes and then analyzed correlations among these indexes. The results demonstrated that the HRS prescription significantly decreased the mortality rate, reduced the severity of hepatic injury, decreased the hepatic pathological severity score, depressed blood DHAV gene expression levels, and returned the indexes of hepatic function and peroxidation almost to a normal level. These results indicate that the HRS prescription confers an outstanding hepatoprotective effect, and we expect that it will be developed into a new candidate anti-DHAV drug.