RESUMO
BACKGROUND: Calcitriol has the potential to counteract fibrotic diseases beyond its classical action of maintaining calcium and bone metabolism; however, its functional mechanism remains unknown. Autophagy-related gene 16-like 1 (Atg16l1) is one of the genes related to autophagy and is involved in protecting against fibrotic diseases. The present study aimed to explore the contribution of autophagy to the inhibition of calcitriol-induced hepatic fibrosis, as well as its potential molecular mechanism. METHODS: Carbon tetrachloride (Ccl4)-treated mice were established as hepatic fibrosis models and received calcitriol treatment for 6 weeks. Quantification of Sirius red staining and measurement of key fibrotic markers (collagen-1 and α-SMA) was performed to detect hepatic fibrosis. Chloroquine (CQ) treatment was used to observe autophagic flux, and 3-methyladenine (3-MA) was used to inhibit autophagy. Furthermore, the effects of calcitriol on transforming growth factor ß1 (TGFß1)-stimulated primary hepatic stellate cells (HSCs) were detected. Downregulation of Atg16l1 or vitamin D receptor (VDR) in LX-2 cells was used to explore the mechanism of action of calcitriol in fibrosis and autophagy. Additionally, the electrophoretic mobility shift assay (EMSA) was used to investigate the interactions between VDR and ATG16L1. RESULTS: Calcitriol increased the expression of VDR and ATG16L1, enhanced autophagy and attenuated hepatic fibrosis. 3-MA treatment and VDR silencing abolished the protective effects of calcitriol against fibrosis. Calcitriol-induced anti-fibrosis effects were blocked by ATG16L1 suppression. Furthermore, VDR bound to the ATG16L1 promoter and downregulation of VDR decreased the expression of ATG16L1 in LX-2 cells. CONCLUSION: Calcitriol mitigates hepatic fibrosis partly through ATG16L1-mediated autophagy.
Assuntos
Proteínas Relacionadas à Autofagia , Autofagia , Calcitriol , Células Estreladas do Fígado , Cirrose Hepática , Receptores de Calcitriol , Animais , Humanos , Masculino , Camundongos , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/metabolismo , Calcitriol/farmacologia , Calcitriol/uso terapêutico , Tetracloreto de Carbono/toxicidade , Progressão da Doença , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos Endogâmicos C57BL , Receptores de Calcitriol/efeitos dos fármacos , Receptores de Calcitriol/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor (TGF)-ß superfamily. The rejuvenative effect of GDF11 has been called into question recently, and its role in liver regeneration is unclear. Here, we investigated the pathophysiologic role of GDF11, as well as its plausible signaling mechanisms in a mouse model of partial hepatectomy (PH). We demonstrated that both serum and hepatic GDF11 protein expression increased following PH. Treatment with adeno-associated viruses-GDF11 and recombinant GDF11 protein severely impaired liver regeneration, whereas inhibition of GDF11 activity with neutralizing antibodies significantly improved liver regeneration after PH. In vitro, GDF11 treatment significantly delayed cell proliferation and induced cell-cycle arrest in α mouse liver 12 (AML12) cells. Moreover, GDF11 activated TGF-ß-SMAD2/3 signaling pathway. Inhibition of GDF11-induced SMAD2/3 activity significantly blocked GDF11-mediated reduction in cell proliferation both in vivo and in vitro. In the clinical setting, GDF11 levels were significantly elevated in patients after hepatectomy. Collectively, these results indicate that rather than a 'rejuvenating' agent, GDF11 impairs liver regeneration after PH. Suppression of cell-cycle progression via TGF-ß-SMAD2/3 signaling pathway may be a key mechanism by which GDF11 inhibits liver regeneration.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Fatores de Diferenciação de Crescimento/fisiologia , Regeneração Hepática/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/sangue , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Fatores de Diferenciação de Crescimento/antagonistas & inibidores , Fatores de Diferenciação de Crescimento/sangue , Fatores de Diferenciação de Crescimento/metabolismo , Fatores de Diferenciação de Crescimento/farmacologia , Hepatectomia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Regeneração Hepática/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Período Pós-Operatório , Proteínas Recombinantes/farmacologia , Transdução de Sinais/fisiologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismoRESUMO
Recent studies showing the therapeutic effect of young blood on aging-associated deterioration of organs point to young blood as the solution for clinical problems related to old age. Given that defective autophagy has been implicated in aging and aging-associated organ injuries, this study was designed to determine the effect of young blood on aging-induced alterations in hepatic function and underlying mechanisms, with a focus on autophagy. Aged rats (22 months) were treated with pooled plasma (1 ml, intravenously) collected from young (3 months) or aged rats three times per week for 4 weeks, and 3-methyladenine or wortmannin was used to inhibit young blood-induced autophagy. Aging was associated with elevated levels of alanine transaminase and aspartate aminotransferase, lipofuscin accumulation, steatosis, fibrosis, and defective liver regeneration after partial hepatectomy, which were significantly attenuated by young plasma injections. Young plasma could also restore aging-impaired autophagy activity. Inhibition of the young plasma-restored autophagic activity abrogated the beneficial effect of young plasma against hepatic injury with aging. In vitro, young serum could protect old hepatocytes from senescence, and the antisenescence effect of young serum was abrogated by 3-methyladenine, wortmannin, or small interfering RNA to autophagy-related protein 7. Collectively, our data indicate that young plasma could ameliorate age-dependent alterations in hepatic function partially via the restoration of autophagy.
Assuntos
Envelhecimento/fisiologia , Autofagia/fisiologia , Hepatócitos/fisiologia , Regeneração Hepática/fisiologia , Animais , Fígado Gorduroso/metabolismo , Hepatectomia/métodos , Fígado/metabolismo , Masculino , Ratos Sprague-DawleyRESUMO
FK866 exhibits a protective effect on D-galactosamine (GaIN)/lipopolysaccharide (LPS) and concanavalin A (ConA)-induced acute liver failure (ALF), but the mechanism by which FK866 affords this benefit has not yet been elucidated. Autophagy has a protective effect on acute liver injury. However, the contribution of autophagy to FK866-conferred hepatoprotection is still unclear. This study aimed to investigate whether FK866 could attenuate GaIN/LPS and ConA-induced ALF through c-jun-N-terminal kinase (JNK)-dependent autophagy. In vivo, Mice were pretreated with FK866 at 24, 12, and 0.5 h before treatment with GaIN/LPS and ConA. 3-methyladenine (3MA) or rapamycin were used to determine the role of autophagy in FK866-conferred hepatoprotection. In primary hepatocytes, autophagy was inhibited by 3MA or autophagy-related protein 7 (Atg7) small interfering RNA (siRNA). JNK was suppressed by SP600125 or Jnk siRNA. FK866 alleviated hepatotoxicity and increased autophagy while decreased JNK activation. Suppression of autophagy abolished the FK866-conferred protection. Inhibition of JNK increased autophagy and exhibited strongly protective effect. Collectively, FK866 could ameliorate GaIN/LPS and ConA-induced ALF through induction of autophagy while suppression of JNK. These findings suggest that FK866 acts as a simple and applicable preconditioning intervention to protect against ALF; autophagy and JNK may also provide therapeutic targets for ALF treatment.
Assuntos
Acrilamidas/farmacologia , Autofagia/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Falência Hepática Aguda/metabolismo , Piperidinas/farmacologia , Animais , Proteína 7 Relacionada à Autofagia/genética , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Hepatócitos/metabolismo , Hepatócitos/patologia , Lipopolissacarídeos/efeitos adversos , Falência Hepática Aguda/tratamento farmacológico , Falência Hepática Aguda/etiologia , Falência Hepática Aguda/patologia , Testes de Função Hepática , Camundongos , Substâncias Protetoras/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: 1,25(OH)2 D3 has been reported to attenuate liver steatosis; however, its exact mechanism of action remains poorly understood. This study aimed to determine whether 1,25(OH)2 D3 can attenuate hepatic steatosis by inducing autophagy. METHODS: Male C57BL/6 mice fed a high-fat diet (HFD) were injected with 1,25(OH)2 D3 for 4 weeks. These mice were given 3-methyladenine (3-MA) to inhibit autophagy. HepG2 cells were preincubated with a free fatty acid (FFA) and then treated with 1,25(OH)2 D3 . Vitamin D receptor (VDR) shRNA and autophagy-related 16-like 1 (ATG16L1) siRNA were used for VDR knockdown or ATG16L1 silencing, respectively. RESULTS: 1,25(OH)2 D3 diminished HFD-induced liver damage and steatosis, changes accompanied by autophagy and ATG16L1 expression upregulation. Inhibition of 1,25(OH)2 D3 -induced autophagy mediated by 3-MA blocked the protective effects of 1,25(OH)2 D3 on hepatic steatosis. Additionally, 1,25(OH)2 D3 -induced autophagy appeared to play a role in anti-inflammation and lipid metabolism modulation in the liver. In HepG2 cells, 1,25(OH)2 D3 reduced lipid accumulation and increased autophagy and ATG16L1 expression; however, this effect was abrogated after VDR knockdown. The protective effects of 1,25(OH)2 D3 -mediated autophagy against lipid accumulation were abolished by 3-MA. Furthermore, siRNA-mediated ATG16L1 knockdown prevented 1,25(OH)2 D3 -induced autophagy, resulting in increased fat accumulation. CONCLUSIONS: The data suggest that 1,25(OH)2 D3 may ameliorate hepatic steatosis by inducing autophagy by upregulating ATG16L1.
Assuntos
Autofagia/efeitos dos fármacos , Calcitriol/farmacologia , Ácidos Graxos não Esterificados/farmacologia , Fígado Gorduroso/tratamento farmacológico , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Proteínas Relacionadas à Autofagia , Calcitriol/uso terapêutico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Dieta Hiperlipídica , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Inativação Gênica , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Regulação para CimaRESUMO
Carbon monoxide (CO) exerts protective effects on hepatic ischemia/reperfusion injury (IRI), but the underlying molecular mechanisms are not fully understood. High-mobility group box 1 (HMGB1) is an important mediator of injury and inflammation in hepatic IRI. Here, we investigated whether CO could attenuate hepatic IRI via inhibition of HMGB1 release, particularly through sirtuin 1 (SIRT1). CO was released by treatment with carbon monoxide-releasing molecule (CORM)-2. CORM-2-delivered CO ameliorated hepatic IRI, as indicated by lower serum aminotransferase levels, lower hepatic inflammatory responses, and less severe ischemia/reperfusion-associated histopathologic changes. Treatment with CORM-2 significantly inhibited IRI-induced HMGB1 translocation and release. SIRT1 expression was increased by CORM-2 pretreatment. When CORM-2-induced SIRT1 expression was inhibited using EX527, HMGB1 translocation and release were increased and hepatic IRI was worsened, whereas SIRT1 activation by resveratrol reversed this trend. In vitro, CORM-2 reduced hypoxia/reoxygenation-induced HMGB1 translocation and release, these inhibitions were blocked by SIRT1 inhibition using EX527 or SIRT1 small interfering RNA both in alpha mouse liver 12 cells and RAW264.7 macrophages. Moreover, SIRT1 directly interacted with and deacetylated HMGB1. IRI increased HMGB1 acetylation, which was abolished by CORM-2 treatment via SIRT1. In conclusion, these results suggest that CO may increase SIRT1 expression, which may decrease HMGB1 acetylation and subsequently reduce its translocation and release, thereby protecting against hepatic IRI. Liver Transplantation 23 510-526 2017 AASLD.
Assuntos
Monóxido de Carbono/farmacologia , Proteína HMGB1/metabolismo , Inflamação/prevenção & controle , Transplante de Fígado/efeitos adversos , Substâncias Protetoras/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Sirtuína 1/metabolismo , Acetilação , Animais , Carbazóis/farmacologia , Inflamação/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Compostos Organometálicos/uso terapêutico , Células RAW 264.7 , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Resveratrol , Estilbenos/farmacologiaRESUMO
Steatotic livers are more susceptible to ischemia/reperfusion (I/R) injury, which is ameliorated by ischemic preconditioning (IPC). Autophagy possesses protective action on liver I/R injury and declines in steatotic livers. The aim of this study was to test the hypothesis that the increased susceptibility of steatotic livers to I/R injury was associated with defective hepatic autophagy, which could be restored by IPC via heme oxygenase-1 (HO-1) signaling. Obesity and hepatic steatosis was induced using a high fat diet. Obesity impaired hepatic autophagy activity and decreased hepatic HO-1 expression. Induction of HO-1 restored autophagy activity and inhibited calpain 2 activity. Additionally, suppression of calpain 2 activity also restored autophagy activity. Mitochondrial dysfunction and hepatocellular injury were significantly increased in steatotic livers compared to lean livers in response to I/R injury. This increase in sensitivity to I/R injury was associated with defective hepatic autophagy activity in steatotic livers. IPC increased autophagy and reduced mitochondrial dysfunction and hepatocellular damage in steatotic livers following I/R injury. Furthermore, IPC increased HO-1 expression. Inhibition of HO-1 decreased the IPC-induced autophagy, increased calpain 2 activity and diminished the protective effect of IPC against I/R injury. Inhibition of calpain 2 restored autophagic defect and attenuated mitochondrial dysfunction in steatotic livers after I/R. Collectively, IPC might ameliorate steatotic liver damage and restore mitochondrial function via HO-1-mediated autophagy.
Assuntos
Autofagia , Fígado Gorduroso/terapia , Heme Oxigenase (Desciclizante)/metabolismo , Hepatócitos/enzimologia , Precondicionamento Isquêmico/métodos , Fígado/enzimologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Calpaína/metabolismo , Células Cultivadas , Dieta Hiperlipídica , Modelos Animais de Doenças , Fígado Gorduroso/enzimologia , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Heme Oxigenase (Desciclizante)/genética , Hepatócitos/patologia , Fígado/patologia , Masculino , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/patologia , Interferência de RNA , Ratos Sprague-Dawley , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
We previously demonstrated that baicalein could protect against liver ischemia/reperfusion (I/R) injury in mice. The exact mechanism of baicalein remains poorly understood. Autophagy plays an important role in protecting against I/R injury. This study was designed to determine whether baicalein could protect against liver I/R injury via induction of autophagy in rats. Baicalein was intraperitoneally injected 1 h before warm ischemia. Pretreatment with baicalein prior to I/R insult significantly blunted I/R-induced elevations of serum aminotransferase levels and significantly improved the histological status of livers. Electron microscopy and expression of the autophagic marker LC3B-II suggested induction of autophagy after baicalein treatment. Moreover, inhibition of the baicalein-induced autophagy using 3-methyladenine (3-MA) worsened liver injury. Furthermore, baicalein treatment increased heme oxygenase (HO)-1 expression, and pharmacological inhibition of HO-1 with tin protoporphyrin IX (SnPP) abolished the baicalein-mediated autophagy and the hepatocellular protection. In primary rat hepatocytes, baicalein-induced autophagy also protected hepatocytes from hypoxia/reoxygenation injury in vitro and the beneficial effect was abrogated by 3-MA or Atg7 siRNA, respectively. Suppression of HO-1 activity by SnPP or HO-1 siRNA prevented the baicalein-mediated autophagy and resulted in increased hepatocellular injury. Collectively, these results suggest that baicalein prevents hepatocellular injury via induction of HO-1-mediated autophagy.
Assuntos
Autofagia , Flavanonas/metabolismo , Fígado/patologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Modelos Animais de Doenças , Histocitoquímica , Camundongos , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/análise , Ratos , Transaminases/sangueRESUMO
Liver dysfunction has been known to occur frequently in cases of sepsis. Baicalein, the main active ingredient of the Scutellaria root, exerts anti-inflammatory and anti-apoptotic properties in endotoxic shock. However, the role of baicalein in polymicrobial sepsis-induced liver injury and its regulatory mechanisms remain unclear. In this study, we aimed to investigate the protective effects of baicalein on polymicrobial sepsis-induced liver injury and to explore the possible mechanisms. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in C57BL/6 mice. Mice were treated with baicalein (100mg/kg, i.p) at 1h, 6h and 12h following CLP. Baicalein significantly improved the survival of septic mice. Treatment with baicalein ameliorated the CLP-induced liver injury, as indicated by the lower serum aminotransferase levels and the fewer histopathologic abnormalities. Baicalein reduced the neutrophil infiltration and the hepatic inflammatory cytokine expression and release. It also decreased the hepatic and the serum high-mobility group box 1 and macrophage migration inhibitory factor levels in septic mice. Moreover, baicalein significantly inhibited the mitogen-activated protein kinases (MAPKs) activation and suppressed the transcriptional activity of nuclear factor-kappa B (NF-κB). In conclusion, these results suggest that baicalein treatment could protect against the sepsis-induced liver injury, and improve the survival of mice with polymicrobial sepsis. The mechanism of the protective action of baicalein seems to involve its ability to reduce inflammatory response, to inhibit hepatic apoptosis, and to suppress MAPKs and NF-κB activation.
Assuntos
Apoptose/efeitos dos fármacos , Flavanonas/farmacologia , Fígado/efeitos dos fármacos , Fígado/lesões , Substâncias Protetoras/farmacologia , Sepse/complicações , Sepse/microbiologia , Animais , Ceco/cirurgia , Flavanonas/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína HMGB1/metabolismo , Inflamação/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Oxirredutases Intramoleculares/metabolismo , Ligadura/efeitos adversos , Fígado/imunologia , Fígado/patologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Masculino , Camundongos , Infiltração de Neutrófilos/efeitos dos fármacos , Substâncias Protetoras/uso terapêutico , Punções/efeitos adversos , Sepse/etiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: To explore the effect of the cytoplasmic DNA sensor DAI on replication of hepatitis B virus (HBV) and its possible mechanism. METHODS: The hepatocyte-derived cell line HepG2 was co-transfected with DAI siRNA and the HBV1.3 replicative plasmid PHY106, and the cells were divided into two experimental groups. Six hours later, total RNA was extracted from the first group of cells and expression of IFIT1 and IL-6 were detected by real-time RT-PCR. The second group of cells was incubated for 4 days, after which the cell supernatant was collected and the HBV surface antigen (HBsAg) and envelope antigen (HBeAg) were detected by ELISA. In addition, HBV core particles were extracted and applied to southern blot assay to detect the intracellular HBV replication intermediates (rcDNA, dlDNA and ssDNA). Next, the HepG2 cells were triple transfected with siRNA targeting the type I interferon pathway molecule TBK1 and DAI simultaneously and HBV1.3, after which HBV viral proteins were detected. Two-group comparisons were made using the independent sample t-test, and more-than-2-group comparisons were made using ANOVA. RESULTS: DAI gene expression was down-regulated in response to DAI siRNA transfection. Cells with down-regulated DAI showed inhibited HBV replication (in a dose-dependent manner), accompanied by reduced levels of HBsAg (0.0195+/-0.0050 vs. CONTROL: 0.3150+/-0.0200, P less than 0.05, t = 14.77) and HBeAg (0.0140+/-0.0040 vs. CONTROL: 0.01235+/-0.0135, P less than 0.05, t = 7.777). No effect of down-regulated DAI was observed for the expression of IFIT1 of IL-6. siRNA-mediated down-regulation of TBK1 and DAI simultaneously led to reduced expression of HBsAg and HBeAg. CONCLUSION: Down-regulation of DAI gene expression inhibited HBV replication and HBV protein expression, but the underlying mechanism was not related to the type I interferon or NF-kB signaling pathway.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Vírus da Hepatite B/fisiologia , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Regulação da Expressão Gênica , Células Hep G2 , Antígenos de Superfície da Hepatite B/isolamento & purificação , Antígenos E da Hepatite B/isolamento & purificação , Humanos , Interleucina-6/metabolismo , NF-kappa B/metabolismo , Plasmídeos , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA , Transdução de Sinais , TransfecçãoRESUMO
Ischemia/reperfusion (I/R) is a pathophysiologic process that occurs during hemorrhagic shock, liver resection and liver transplantation. Baicalein, the main active ingredient of the Scutellaria root, exerts anti-inflammatory and anti-apoptotic properties in the setting of I/R injury in the heart and brain. However, the role of baicalein in liver I/R injury and its regulatory mechanisms remain poorly understood. This study was designed to evaluate the effects of baicalein in a model of liver I/R in mice and to explore the possible mechanisms. Baicalein (100mg/kg) was intraperitoneally injected 1h before warm ischemia. Pretreatment with baicalein protected against liver I/R injury, as indicated by the decreased serum aminotransferase levels and the reduced histopathologic abnormalities. Baicalein also significantly reduced cellular hepatic apoptosis in response to I/R injury. Moreover, pretreatment with baicalein significantly inhibited nuclear factor-kappa B (NF-κB) activation and the subsequent proinflammatory cytokine production, and decreased leukocyte infiltration. In vitro studies, baicalein treatment inhibited the proinflammatory cytokine production via the modulation of NF-κB signaling pathway in lipopolysaccharide-stimulated macrophages. Taken together, these results suggest that baicalein could protect against liver I/R injury via inhibition of inflammation by down-regulating NF-κB activity, and suppression of cellular hepatic apoptosis.
Assuntos
Antioxidantes/administração & dosagem , Flavanonas/administração & dosagem , Fígado/irrigação sanguínea , Fígado/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Traumatismo por Reperfusão/prevenção & controle , Alanina Transaminase/sangue , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Creatinina/sangue , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Lipopolissacarídeos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacosRESUMO
Liver dysfunction has been known to occur frequently in cases of sepsis. Excessive inflammation and apoptosis are pathological features of acute liver failure. Recent studies suggest that activation of glycogen synthase kinase- (GSK-) 3ß is involved in inflammation and apoptosis. We aimed to investigate the protective effects of GSK-3ß inhibition on polymicrobial sepsis-induced liver injury and to explore the possible mechanisms. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP), and SB216763 was used to inhibit GSK-3ß in C57BL/6 mice. GSK-3ß was activated following CLP. Administration of SB216763 decreased mortality, ameliorated liver injury, and reduced hepatic apoptosis. The inhibition of GSK-3ß also reduced leukocyte infiltration and hepatic inflammatory cytokine expression and release. Moreover, GSK-3ß inhibition suppressed the transcriptional activity of nuclear factor-kappa B (NF-κB) but enhanced the transcriptional activity of cAMP response element binding protein (CREB) in the liver. In in vitro studies, GSK-3ß inhibition reduced inflammatory cytokine production via modulation of NF-κB and CREB signaling pathways in lipopolysaccharide-stimulated macrophages. In conclusion, these findings suggest that GSK-3ß blockade protects against CLP-induced liver via inhibition of inflammation by modulating NF-κB and CREB activity and suppression of hepatic apoptosis.
Assuntos
Apoptose , Quinase 3 da Glicogênio Sintase/metabolismo , Inflamação/metabolismo , Fígado/patologia , Animais , Linhagem Celular , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Glicogênio Sintase Quinase 3 beta , Imuno-Histoquímica , Indóis/química , Interleucina-6/metabolismo , Leucócitos/citologia , Fígado/lesões , Fígado/metabolismo , Masculino , Maleimidas/química , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Peroxidase/metabolismo , Transdução de SinaisRESUMO
The inflammatory response after liver ischemia/reperfusion (I/R) contributes to increased risk of liver failure after liver surgery. Strategies aimed to preventing inflammation could be beneficial in reducing liver I/R injury. Recent studies have demonstrated that peptide Bß15-42 is able to decrease the injury of I/R in heart and kidney by inhibition of leukocyte migration and preserving endothelial barrier function. Prompted by these results, we hypothesized that Bß15-42 could also possess anti-inflammatory abilities to protect from or reduce hepatic I/R injury. Therefore, in this study, we aimed to evaluate the effects of Bß15-42 in a model of liver I/R injury in rats. Rats were treated with Bß15-42 at initiation of reperfusion and 2 h thereafter. Rats were killed at 0.5, 6, 24, and 48 h after reperfusion. Hepatic mRNA levels of fibrinogen-α (Fgα), Fgß, Fgγ were significantly increased after I/R. Treatment with Fg-derived Bß15-42 ameliorated liver I/R injury, as indicated by lower serum aminotransferase levels and fewer I/R-associated histopathologic changes. Bß15-42 treatment decreased leukocyte infiltration and expression of hepatic inflammatory cytokines. Moreover, Bß15-42 significantly reduced high-mobility group box 1 release and altered mitogen-activated protein kinase activation. In conclusion, Bß15-42 treatment protected against liver warm I/R injury. The mechanism of protective action of Bß15-42 seemed to involve its ability to reduce hepatic inflammatory response through preventing high-mobility group box 1 release and altering mitogen-activated protein kinase activation.
Assuntos
Fármacos Cardiovasculares/farmacologia , Produtos de Degradação da Fibrina e do Fibrinogênio/farmacologia , Fígado/irrigação sanguínea , Fragmentos de Peptídeos/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Biomarcadores/metabolismo , Citocinas/metabolismo , Proteína HMGB1/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Infiltração de Neutrófilos/efeitos dos fármacos , Ratos , Ratos Endogâmicos Lew , Transaminases/metabolismoRESUMO
OBJECTIVE: To evaluate the effect of heat shock protein 90 (HSP90) on hepatitis B virus (HBV) replication in hepatocytes and to investigate the related molecular mechanism. METHODS: A eukaryotic plasmid expressing human HSP90 was constructed (designated as HA-HSP90). HepG2 cells were co-transfected with HA-HSP90 and the HBV replicative plasmid HBV1.3. Expression of the exogenous HSP90 was assessed by Western blotting. Expression of the HBV surface antigen (HBsAg) was determined by enzyme-linked immunosorbent assay, and HBV replicative intermediates were detected by Southern blotting. Small interfering (si)RNAs were designed against HSP90 and TBK1 and transfected into the HepG2 cells to further assess the effects of HSP90 and its underlying mechanism. HSP90-mediated effects on the expression of interleukins IL-1b and IL-6 and the interferon response gene IFIT1 were assessed by quantitating mRNA levels with real time RT-PCR. RESULTS: The HA-HSP90 plasmid successfully expressed exogenous HSP90 protein in HepG2 cells. The exogenous HSP90 was able to inhibit HBV replication and HBsAg expression. IFIT1 expression was up-regulated after HA-HSP90 transfection, but neither IL-1b nor IL-6 were affected. The siRNA-mediated TBK1 down-regulation had no effect on the HSP90-inhibited HBV replication. CONCLUSION: HSP90 can inhibit HBV replication and TBK1 is not involved in this process.
Assuntos
Proteínas de Choque Térmico HSP90/genética , Vírus da Hepatite B/fisiologia , Replicação Viral , Células Hep G2 , Antígenos E da Hepatite B/metabolismo , Humanos , Proteínas Serina-Treonina Quinases/genética , TransfecçãoRESUMO
microRNAs (miRNAs) have regulated the expression and function of genes implicated in many pathological settings, but their impact on the pathoetiological characteristics of overactive bladder (OAB) largely remains unknown. We have generated a mouse model in which adult mice can be induced for detrusor deletion of Dicer, an enzyme essential for miRNA processing. Targeted deletion of Dicer did not lead to a significant change for detrusor functionality under physiological conditions; however, loss of Dicer exacerbated cyclophosphamide-induced OAB, manifested by the higher severity of altered detrusor contractile force and sensitivity, abnormal urodynamics, and enhanced macrophage infiltration. Mechanistic studies revealed that loss of Dicer may impair the expression of miRNAs that are capable of targeting P2x mRNAs. As a result, mice deficient in Dicer manifest enhanced P2X expression in the detrusor on cyclophosphamide treatment, predisposing to the increased risk for OAB development. More important, studies using bladder biopsy samples of patients with OAB also demonstrated similar results as those found in animals. Taken together, our results suggest that miRNAs modulate OAB susceptibility by regulating purinergic signaling, in which the pathogenic insult induces the expression of miRNAs capable of targeting P2X mRNAs to suppress OAB symptoms.
Assuntos
RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/metabolismo , Receptores Purinérgicos/metabolismo , Ribonuclease III/deficiência , Ribonuclease III/metabolismo , Transdução de Sinais , Bexiga Urinária Hiperativa/metabolismo , Animais , Sequência de Bases , Western Blotting , Biologia Computacional , Ciclofosfamida , Deleção de Genes , Regulação da Expressão Gênica , Marcação de Genes , Humanos , Imuno-Histoquímica , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Contração Muscular/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Bexiga Urinária/patologia , Bexiga Urinária/fisiopatologia , Bexiga Urinária Hiperativa/induzido quimicamente , Bexiga Urinária Hiperativa/patologia , Bexiga Urinária Hiperativa/fisiopatologiaRESUMO
OBJECTIVE: This report aims to investigate the Toll-like receptor (TLR) signaling pathways and induced antiviral activity in hepatocytes. METHODS: We isolated primary hepatocytes from wild-type C57BL/6 mice and examined the expression of TLR by realtime RT-PCR. Hepatocytes were stimulated with TLR 1-9 agonists and the supernatants were harvested. The secretion of cytokines were tested by ELISA. The antiviral effectors in supernatants were assayed via virus protection assay (in EMCV system) and the control of HBV replication were assessed via Southern blotting (in HBV system). RESULTS: We demonstrated that hepatocytes expressed TLR1-9. In accordance with these TLR expression profiles, hepatocytes responded to all TLR ligands by producing inflammatory cytokines (TNF-α or IL-6), to TLR -1,-3,-7 and -9 ligands by producing type I IFN (IFN-α or IFN-ß). Only TLR 3 and TLR 7 agonists could stimulate the production of high amounts of antiviral mediators by hepatocytes in virus protection assay. By contrast, supernatants from TLR1, -3 and -4 directly stimulated hepatocytes and TLR 3, -7 and -9 transfected hepatocytes were able to potently suppress HBV replication. CONCLUSION: Primary hepatocytes display a unique TLR signaling pathway and can control HBV replication after stimulation by TLR agonists in mice. It may be helpful for the development of TLR-based therapeutic approaches against hepatotropic virus.
Assuntos
Vírus da Hepatite B/fisiologia , Hepatócitos/imunologia , Imunidade Inata , Receptores Toll-Like/imunologia , Replicação Viral , Animais , Células Cultivadas , Vírus da Hepatite B/imunologia , Hepatócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Receptores Toll-Like/metabolismoRESUMO
To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR H1b) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH1a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector pIRES2EGFP, pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRH1a and H2c in 4-1-6 were confirmed by RT-PCR, Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H1b/pCDNA3.1 (neo) was transfected into cell line 4-1-6, H1b did not down-regulate the ligand binding ability of ASGPR. The eukaryotic expression plasmid H1b/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single H1b nor H1b and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both H1a and H2c stably was established. The new split variant H1b has no effect on ASGPR binding to ASOR. ASGPRH1b alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.
Assuntos
Receptor de Asialoglicoproteína/biossíntese , Hepatócitos/metabolismo , Transfecção , Receptor de Asialoglicoproteína/genética , Sítios de Ligação , Linhagem Celular , Vetores Genéticos/genética , Células HeLa , Humanos , Ligantes , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
BACKGROUND: The human asialoglycoprotein receptor (ASGPR) is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR genomic sequence. Thus, ASGPR variant H1b transcript encodes a protein lacking the transmembrane domain. Using an H1b-specific antibody, H1b protein and a functional soluble ASGPR (sASGPR) composed of H1b and H2 in human sera and in hepatoma cell culture supernatant were identified. The expression of ASGPR H1a and H1b in Hela cells demonstrated the different cellular loctions of H1a and H1b proteins at cellular membranes and in intracellular compartments, respectively. In vitro binding assays using fluorescence-labeled sASGPR or the substract ASOR revealed that the presence of sASGPR reduced the binding of ASOR to cells. However, ASOR itself was able to enhance the binding of sASGPR to cells expressing membrane-bound ASGPR. Further, H1b expression is reduced in liver tissues from patients with viral hepatitis. CONCLUSIONS: We conclude that two naturally occurring ASGPR H1 splice variants are produced in human hepatocytes. A hetero-oligomeric complex sASGPR consists of the secreted form of H1 and H2 and may bind to free substrates in circulation and carry them to liver tissue for uptake by ASGPR-expressing hepatocytes.
Assuntos
Receptor de Asialoglicoproteína/genética , Receptor de Asialoglicoproteína/metabolismo , Espaço Extracelular/metabolismo , Hepatócitos/metabolismo , Splicing de RNA , Receptor de Asialoglicoproteína/química , Sequência de Bases , Linhagem Celular , Células Cultivadas , Espaço Extracelular/genética , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
Hepatitis B virus surface antigen (HBsAg), a specific antigen on the membrane of Hepatitis B virus (HBV)-infected cells, provides a perfect target for therapeutic drugs. The development of reagents with high affinity and specificity to the HBsAg is of great significance to the early-stage diagnosis and treatment of HBV infection. Herein, we report the selection of RNA aptamers that can specifically bind to HBsAg protein and HBsAg-positive hepatocytes. One high affinity aptamer, HBs-A22, was isolated from an initial 115 mer library of ~1.1 x 10¹5 random-sequence RNA molecules using the SELEX procedure. The selected aptamer HBs-A22 bound specifically to hepatoma cell line HepG2.2.15 that expresses HBsAg but did not bind to HBsAg-devoid HepG2 cells. This is the first reported RNA aptamer which could bind to a HBV specific antigen. This newly isolated aptamer could be modified to deliver imaging, diagnostic, and therapeutic agents targeted at HBV-infected cells.