RESUMEN
Nitazoxanide is an FDA-approved antiprotozoal drug. Our previous studies find that nitazoxanide and its metabolite tizoxanide affect AMPK, STAT3, and Smad2/3 signals which are involved in the pathogenesis of liver fibrosis, therefore, in the present study, we examined the effect of nitazoxanide on experimental liver fibrosis and elucidated the potential mechanisms. The in vivo experiment results showed that oral nitazoxanide (75, 100 mg·kg-1) significantly improved CCl4- and bile duct ligation-induced liver fibrosis in mice. Oral nitazoxanide activated the inhibited AMPK and inhibited the activated STAT3 in liver tissues from liver fibrosis mice. The in vitro experiment results showed that nitazoxanide and its metabolite tizoxanide activated AMPK and inhibited STAT3 signals in LX-2 cells (human hepatic stellate cells). Nitazoxanide and tizoxanide inhibited cell proliferation and collagen I expression and secretion of LX-2 cells. Nitazoxanide and tizoxanide inhibited transforming growth factor-ß1 (TGF-ß1)- and IL-6-induced increases of cell proliferation, collagen I expression and secretion, inhibited TGF-ß1- and IL-6-induced STAT3 and Smad2/3 activation in LX-2 cells. In mouse primary hepatic stellate cells, nitazoxanide and tizoxanide also activated AMPK, inhibited STAT3 and Smad2/3 activation, inhibited cell proliferation, collagen I expression and secretion. In conclusion, nitazoxanide inhibits liver fibrosis and the underlying mechanisms involve AMPK activation, and STAT3 and Smad2/3 inhibition.
Asunto(s)
Antiprotozoarios , Nitrocompuestos , Tiazoles , Animales , Ratones , Tiazoles/farmacología , Tiazoles/uso terapéutico , Masculino , Humanos , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Línea Celular , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Cirrosis Hepática/inducido químicamente , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Proteína smad3/metabolismo , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/patología , Cirrosis Hepática Experimental/tratamiento farmacológico , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/prevención & control , Ratones Endogámicos C57BL , Proteína Smad2/metabolismoRESUMEN
The effect of liver cirrhosis on vascular remodeling in vivo remains unknown. Therefore, this study investigates the influence of cholestatic liver cirrhosis on carotid arterial remodeling. A total of 79 male Sprague Dawley rats underwent bile duct ligation (cirrhotic group) or sham surgery (control group) and 28 days later left carotid artery balloon dilatation; 3, 7, 14 and 28 days after balloon dilatation, the rats were euthanized and carotid arteries were harvested. Histological sections were planimetrized, cell counts determined, and systemic inflammatory parameters measured. Up to day 14 after balloon dilatation, both groups showed a comparable increase in neointima area and degree of stenosis. By day 28, however, both values were significantly lower in the cirrhotic group (% stenosis: 20 ± 8 vs. 42 ± 10, p = 0.010; neointimal area [mm2]: 0.064 ± 0.025 vs. 0.138 ± 0.025, p = 0.024). Simultaneously, cell density in the neointima (p = 0.034) and inflammatory parameters were significantly higher in cirrhotic rats. This study demonstrates that cholestatic liver cirrhosis in rats substantially increases neointimal cell consolidation between days 14 and 28. Thereby, consolidation proved important for the degree of stenosis. This may suggest that patients with cholestatic cirrhosis are at lower risk for restenosis after coronary intervention.
Asunto(s)
Angioplastia de Balón , Traumatismos de las Arterias Carótidas , Cirrosis Hepática Experimental , Ratas , Masculino , Animales , Ratas Sprague-Dawley , Neointima/patología , Cirrosis Hepática Experimental/patología , Constricción Patológica/patología , Angioplastia de Balón/efectos adversos , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/patología , Hiperplasia/patologíaRESUMEN
Liver cirrhosis is a major underlying factor in the development of hepatocellular carcinoma. Currently, there is an unmet need for midsize experimental vertebrate models that would offer reproducible implantable liver tumors in a cirrhotic liver background. This study establishes a protocol for a syngeneic rabbit model of VX2 liver cancer with underlying liver cirrhosis induced using carbon tetrachloride (CCl4). Male New Zealand white rabbits (n = 3) received CCl4 by intragastric administration once weekly. Concentrations started at 5% v/v CCl4 dissolved in olive oil. CCl4 dosing was progressively increased every week by 2.5% v/v increments for the duration of treatment (16 weeks total). VX2 tumors were then orthotopically implanted into the left hepatic lobe and allowed to grow for 3 weeks. Cross-sectional imaging confirmed the presence of hepatic tumors. Gross and histopathological evaluations showed reproducible tumor growth in the presence of liver cirrhosis in all animals.
Asunto(s)
Carcinoma Hepatocelular , Cirrosis Hepática Experimental , Neoplasias Hepáticas Experimentales , Neoplasias Hepáticas , Conejos , Masculino , Animales , Tetracloruro de Carbono/efectos adversos , Hígado/patología , Cirrosis Hepática , Neoplasias Hepáticas/patología , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas Experimentales/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/patologíaRESUMEN
Chronic liver disease such as hepatic fibrosis is a major cause of morbidity and mortality and has been related to high individual risk of hepatocellular carcinoma (HCC). Hepatic stellate cells (HSCs) activation is a central event of hepatic fibrosis progression. In this study, the up-regulation of lncRNA ANXA2P2 (mouse Anxa6) was found in liver fibrosis. Within CCl4-caused liver fibrosis murine model, Anxa6 knockdown partially ameliorated CCl4-induced hepatic fibrosis and blocked the PI3K/Akt signaling activation. In TGF-ß1-stimulated HSCs, Anxa6 knockdown partially inhibited TGF-ß1-induced HSC activation and blocked the PI3K/Akt signaling activation. Mouse Anxa6 downstream mmu-miR-9-5p directly targeted Anxa2; Anxa6 negatively regulated mmu-miR-9-5p, and mmu-miR-9-5p negatively regulated mouse Anxa2. In TGF-ß1-stimulated HSCs, miR-9-5p inhibitor promoted TGF-ß1-induced HSC activation and PI3K/Akt signaling activation, whereas Anxa2 knockdown exerted opposite effects; Anxa2 knockdown significantly attenuated miR-9-5p inhibitor effects upon TGF-ß1-stimulated HSCs. In conclusion, lncRNA ANXA2P2 (mouse Anxa6) expression is up-regulated in hepatic fibrosis and exerts pro-fibrotic effects on CCl4-caused liver fibrosis model mice and TGF-ß1-stimulated HSCs. The mouse Anxa6/miR-9-5p/Anxa2 axis and the PI3K/Akt pathway might participate in the functions of lncRNA ANXA2P2 (mouse Anxa6) on hepatic fibrosis.
Asunto(s)
Anexina A2 , Anexina A6 , Células Estrelladas Hepáticas , Cirrosis Hepática Experimental , MicroARNs , ARN Largo no Codificante , Animales , Anexina A2/metabolismo , Anexina A6/metabolismo , Tetracloruro de Carbono , Proliferación Celular/fisiología , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Ratones , MicroARNs/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Largo no Codificante/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
The effect of ketanserin on inflammation, liver fibrosis, and microviscosity of the plasma and mitochondrial membranes of hepatocytes was studied on young (3 months) and old (9 months) male Wistar rats with experimental liver cirrhosis. Ketanserin reduced inflammation, area of the connective tissue, and liver damage and improved serum biochemical parameters in rats of both age groups; in old rats, the effects were more pronounced than in young animals. In old rats, ketanserin reduced polarity of hepatocyte plasma and mitochondrial membranes in the area of protein-lipid contacts, which determined higher effectiveness of ketanserin during the treatment of liver cirrhosis in aged animals.
Asunto(s)
Cirrosis Hepática Experimental , Hígado , Ratas , Masculino , Animales , Ketanserina/farmacología , Ketanserina/uso terapéutico , Cirrosis Hepática Experimental/patología , Ratas Wistar , Hepatocitos/patología , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/patología , Inflamación/patologíaRESUMEN
The viscosity of plasma and mitochondrial membranes of hepatocytes was studied in young (3-month-old) and old (9-month-old) male Wistar rats. It was shown that viscosity of hepatocyte plasma and mitochondrial membranes in young rats under optimal vital functions in the area of protein-lipid membrane contacts was significantly lower than in old rats. No age-related differences in the viscosity of lipid-lipid membrane contacts and in the polarity of protein-lipid contacts and lipid layers were found. Liver cirrhosis induced by carbon tetrachloride and ethanol administration was associated with increased fluidity of the plasma and mitochondrial membranes of hepatocytes in rats of both age groups. The decrease in membrane viscosity in young rats occurred due to a decrease of the viscosity in the area of protein-lipid and lipid-lipid contacts, while in old rats in the area of protein-lipid contacts. Carbon tetrachloride and ethanol did not affect the polarity of lipid contacts and lipid layers.
Asunto(s)
Tetracloruro de Carbono/toxicidad , Etanol/toxicidad , Hepatocitos/efectos de los fármacos , Cirrosis Hepática Experimental/metabolismo , Hígado/efectos de los fármacos , Factores de Edad , Animales , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/patología , Masculino , Mitocondrias/química , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/química , Membranas Mitocondriales/efectos de los fármacos , Ratas , Ratas Wistar , Viscosidad/efectos de los fármacosRESUMEN
In patients, advanced cirrhosis only regresses partially once the etiological agent is withdrawn. Animal models for advanced cirrhosis regression are missing. Lifestyle interventions (LIs) have been shown to improve steatosis, inflammation, fibrosis, and portal pressure (PP) in liver disease. We aimed at characterizing cirrhosis regression after etiological agent removal in experimental models of advanced cirrhosis and to study the impact of different LI on it. Advanced cirrhosis was induced in rats either by carbon tetrachloride (CCl4) or by thioacetamide (TAA) administration. Systemic and hepatic hemodynamics, liver fibrosis, hepatic stellate cell (HSC) activation, hepatic macrophage infiltration, and metabolic profile were evaluated after 48 h, 4 wk or 8 wk of etiological agent removal. The impact of LI consisting in caloric restriction (CR) or moderate endurance exercise (MEE) during the 8-wk regression process was analyzed. The effect of MEE was also evaluated in early cirrhotic and in healthy rats. A significant reduction in portal pressure (PP), liver fibrosis, and HSC activation was observed during regression. However, these parameters remained above those in healthy animals. During regression, animals markedly worsened their metabolic profile. CR although preventing those metabolic disturbances did not further reduce PP, hepatic fibrosis, or HSC activation. MEE also prevented metabolic disturbances, without enhancing, but even attenuating the reduction of PP, hepatic fibrosis, and HSC activation achieved by regression. MEE also worsened hepatic fibrosis in early-TAA cirrhosis and in healthy rats.NEW & NOTEWORTHY We have developed two advanced cirrhosis regression experimental models with persistent relevant fibrosis and portal hypertension and an associated deteriorated metabolism that mimic what happens in patients. LI, despite improving metabolism, did not enhance the regression process in our cirrhotic models. CR did not further reduce PP, hepatic fibrosis, or HSC activation. MEE exhibited a profibrogenic effect in the liver blunting cirrhosis regression. One of the potential explanations of this worsening could be ammonia accumulation.
Asunto(s)
Restricción Calórica , Enfermedad Hepática Inducida por Sustancias y Drogas/terapia , Ingestión de Energía , Terapia por Ejercicio , Estilo de Vida Saludable , Cirrosis Hepática Experimental/terapia , Hígado/metabolismo , Animales , Tetracloruro de Carbono , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hipertensión Portal/inducido químicamente , Hipertensión Portal/metabolismo , Hipertensión Portal/fisiopatología , Hipertensión Portal/terapia , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Masculino , Resistencia Física , Ratas Wistar , Conducta de Reducción del Riesgo , Tioacetamida , Factores de TiempoRESUMEN
Sitagliptin is known for its anti-diabetic activity though it has other pleiotropic pharmacological actions. Its effect against concanavalin A (Con A)-induced hepatic fibrosis has not been investigated yet. Our target was to test whether sitagliptin can suppress the development of Con A-induced hepatic fibrosis and if so, what are the mechanisms involved? Con A (6 mg/kg) was injected once weekly to male Swiss albino mice for four weeks. Sitagliptin was daily administered concurrently with Con A. Results have shown the potent hepatoprotective activity of sitagliptin against Con A-induced hepatitis and fibrosis. That was evident through the amelioration of hepatotoxicity serum parameters (ALT, AST, ALP, and LDH) and the increase in the level of serum albumin in sitagliptin treated mice. Simultaneously, there was amendment of the Con A-induced hepatic lesions and repression of fibrosis in sitagliptin-treated animals. Hydroxyproline, collagen content and the immuno-expression of the fibrotic markers, TGF-ß and α-SMA were depressed upon sitagliptin treatment. Sitagliptin suppressed Con A-induced oxidative stress and increased antioxidants. RT-PCR analysis showed enhancement of mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target genes (GCLc, GCLm, NQO-1, HO-1) by sitagliptin. Furthermore, sitagliptin ameliorated the level and immuno-expression of nuclear factor kappa-B (NF-κB) alongside the immuno-expression of the inflammatory cytokine, TNF-α. Taken together, this study demonstrates the hepatoprotective activity of sitagliptin which may be in part related to enhancement of Nrf2 signaling pathway and inhibition of NF-κB which interact inflammatory response in liver. Sitagliptin might be a new candidate to suppress hepatitis-associated fibrosis.
Asunto(s)
Antiinflamatorios/farmacología , Antifibróticos/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Cirrosis Hepática Experimental/prevención & control , Hígado/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Fosfato de Sitagliptina/farmacología , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Concanavalina A , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Masculino , Ratones , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Although macrophages are recognized as important players in the pathogenesis of chronic liver diseases, their roles in cholestatic liver fibrosis remain incompletely understood. We previously reported that long noncoding RNA-H19 (lncRNA-H19) contributes to cholangiocyte proliferation and cholestatic liver fibrosis of biliary atresia (BA). We here show that monocyte/macrophage CD11B mRNA levels are increased significantly in livers of BA patients and positively correlated with the progression of liver inflammation and fibrosis. The macrophages increasingly infiltrate and accumulate in the fibrotic niche and peribiliary areas in livers of BA patients. Selective depletion of macrophages using the transgenic CD11b-diphtheria toxin receptor (CD11b-DTR) mice halts bile duct ligation (BDL)-induced progression of liver damage and fibrosis. Meanwhile, macrophage depletion significantly reduces the BDL-induced hepatic lncRNA-H19. Overexpression of H19 in livers using adeno-associated virus serotype 9 (AAV9) counteracts the effects of macrophage depletion on liver fibrosis and cholangiocyte proliferation. Additionally, both H19 knockout (H19-/-) and conditional deletion of H19 in macrophage (H19ΔCD11B) significantly depress the macrophage polarization and recruitment. lncRNA-H19 overexpressed in THP-1 macrophages enhance expression of Rho-GTPase CDC42 and RhoA. In conclusions, selectively depletion of macrophages suppresses cholestatic liver injuries and fibrosis via the lncRNA-H19 and represents a potential therapeutic strategy for rapid liver fibrosis in BA patients.
Asunto(s)
Cirrosis Hepática Biliar/prevención & control , Cirrosis Hepática Experimental/prevención & control , Hígado/metabolismo , Activación de Macrófagos , Macrófagos/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Estudios de Casos y Controles , Proliferación Celular , Colestasis/complicaciones , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Humanos , Hígado/patología , Cirrosis Hepática Biliar/genética , Cirrosis Hepática Biliar/metabolismo , Cirrosis Hepática Biliar/patología , Cirrosis Hepática Experimental/genética , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Macrófagos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Largo no Codificante/genética , Células THP-1 , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
BACKGROUND AND AIMS: HSCs and portal fibroblasts (PFs) are the major sources of collagen-producing myofibroblasts during liver fibrosis, depending on different etiologies. However, the mechanisms by which their dynamic gene expression directs the transition from the quiescent to the activated state-as well as their contributions to fibrotic myofibroblasts-remain unclear. Here, we analyze the activation of HSCs and PFs in CCL4 -induced and bile duct ligation-induced fibrosis mouse models, using single-cell RNA sequencing and lineage tracing. APPROACH AND RESULTS: We demonstrate that HSCs, rather than PFs, undergo dramatic transcriptomic changes, with the sequential activation of inflammatory, migrative, and extracellular matrix-producing programs. The data also reveal that HSCs are the exclusive source of myofibroblasts in CCL4 -treated liver, while PFs are the major source of myofibroblasts in early cholestatic liver fibrosis. Single-cell and lineage-tracing analysis also uncovers differential gene-expression features between HSCs and PFs; for example, nitric oxide receptor soluble guanylate cyclase is exclusively expressed in HSCs, but not in PFs. The soluble guanylate cyclase stimulator Riociguat potently reduced liver fibrosis in CCL4 -treated livers but showed no therapeutic efficacy in bile duct ligation livers. CONCLUSIONS: This study provides a transcriptional roadmap for the activation of HSCs during liver fibrosis and yields comprehensive evidence that the differential transcriptomic features of HSCs and PFs, along with their relative contributions to liver fibrosis of different etiologies, should be considered in developing effective antifibrotic therapeutic strategies.
Asunto(s)
Células Estrelladas Hepáticas/inmunología , Cirrosis Hepática Experimental/inmunología , Miofibroblastos/inmunología , Animales , Tetracloruro de Carbono/administración & dosificación , Tetracloruro de Carbono/toxicidad , Linaje de la Célula/inmunología , Células Cultivadas , Regulación de la Expresión Génica/inmunología , Técnicas de Sustitución del Gen , Células Estrelladas Hepáticas/metabolismo , Humanos , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/patología , Masculino , Ratones , Ratones Transgénicos , Cultivo Primario de Células , RNA-Seq , Análisis de la Célula IndividualRESUMEN
BACKGROUND AND AIMS: Studies of the identity and pathophysiology of fibrogenic HSCs have been hampered by a lack of genetic tools that permit specific and inducible fate-mapping of these cells in vivo. Here, by single-cell RNA sequencing of nonparenchymal cells from mouse liver, we identified transcription factor 21 (Tcf21) as a unique marker that restricted its expression to quiescent HSCs. APPROACH AND RESULTS: Tracing Tcf21+ cells by Tcf21-CreER (Cre-Estrogen Receptor fusion protein under the control of Tcf21 gene promoter) targeted ~10% of all HSCs, most of which were located at periportal and pericentral zones. These HSCs were quiescent under steady state but became activated on injuries, generating 62%-67% of all myofibroblasts in fibrotic livers and ~85% of all cancer-associated fibroblasts (CAFs) in liver tumors. Conditional deletion of Transforming Growth Factor Beta Receptor 2 (Tgfbr2) by Tcf21-CreER blocked HSC activation, compromised liver fibrosis, and inhibited liver tumor progression. CONCLUSIONS: In conclusion, Tcf21-CreER-targeted perivenous stellate cells are the main source of myofibroblasts and CAFs in chronically injured livers. TGF-ß signaling links HSC activation to liver fibrosis and tumorigenesis.
Asunto(s)
Fibroblastos Asociados al Cáncer/citología , Células Estrelladas Hepáticas/citología , Cirrosis Hepática Experimental/patología , Hepatopatías/patología , Neoplasias Hepáticas Experimentales/patología , Miofibroblastos/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Conductos Biliares/cirugía , Tetracloruro de Carbono/toxicidad , Linaje de la Célula , Colestasis , Enfermedad Crónica , Células Estrelladas Hepáticas/metabolismo , Venas Hepáticas/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Cirrosis Hepática Experimental/metabolismo , Hepatopatías/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas Experimentales/metabolismo , Ratones , Miofibroblastos/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Análisis de Secuencia de ARN , Análisis de la Célula IndividualRESUMEN
Liver fibrosis is a compensatory response to the tissue repair process. The activation and proliferation of hepatic stellate cells (HSCs) are thought to be related to the occurrence of hepatic fibrosis. Therefore, inhibiting the activation and proliferation of HSCs is a key step in alleviating liver fibrosis. As a non-specific inhibitor of transient receptor potential melastatin 7 (TRPM7), carvacrol has anti-tumor, anti-inflammatory and anti-hepatic fibrosis activities. This study aimed to explore the protective effect of carvacrol on liver fibrosis and related molecular mechanisms. A CCl4-induced liver fibrosis mouse model and platelet-derived growth factor (PDGF-BB)-activated HSC-T6 cells (a rat hepatic stellate cell line) were employed for in vivo and in vitro experiments. C57BL/6J mice were orally administered different concentrations of carvacrol every day for 6 weeks during the development of CCl4-induced liver fibrosis. The results show that carvacrol could effectively reduce liver damage and the progression of liver fibrosis in mice, which are expressed as fibrotic markers levels were reduced and histopathological characteristics were improved. Moreover, carvacrol inhibited the proliferation and activation of HSC-T6 cells induced by PDGF-BB. In addition, it was found that carvacrol inhibits the expression of TRPM7 and mediated through mitogen-activated protein kinases (MAPK). Collectively, our study shows that carvacrol can reduce liver fibrosis by inhibiting the activation and proliferation of hepatic stellate cells, and the MAPK signaling pathway might be involved in this process.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Cimenos/farmacología , Células Estrelladas Hepáticas/efectos de los fármacos , Cirrosis Hepática Experimental/prevención & control , Hígado/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Canales Catiónicos TRPM/antagonistas & inhibidores , Animales , Becaplermina/farmacología , Tetracloruro de Carbono , Línea Celular , Proliferación Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Colágeno/metabolismo , Células Estrelladas Hepáticas/enzimología , Células Estrelladas Hepáticas/patología , Hígado/enzimología , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/enzimología , Cirrosis Hepática Experimental/patología , Masculino , Ratones Endogámicos C57BL , Ratas , Transducción de Señal , Canales Catiónicos TRPM/metabolismoRESUMEN
Animal models of liver cancer are instrumental in the study of hepatocarcinogenesis and development of novel therapeutic approaches. Here, we describe steps to establish liver cancer in a rat model, via chronic administration of diethylnitrosamine. This causes liver tumors with a sequential progression of hepatitis, cirrhosis, and tumor formation, which closely mimics the development of human liver cancer. This protocol was optimized to significantly increase the incidence of liver tumor formation and reduce the duration of the procedure. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).
Asunto(s)
Carcinogénesis , Dietilnitrosamina/toxicidad , Cirrosis Hepática Experimental , Neoplasias Hepáticas Experimentales , Animales , Carcinogénesis/inducido químicamente , Carcinogénesis/metabolismo , Carcinogénesis/patología , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Liver cirrhosis remains major health problem. Despite the progress in diagnosis of asymptomatic early-stage cirrhosis, prognostic biomarkers are needed to identify cirrhotic patients at high risk developing advanced stage disease. Liver cirrhosis is the result of deregulated wound healing and is featured by aberrant extracellular matrix (ECM) remodeling. However, it is not comprehensively understood how ECM is dynamically remodeled in the progressive development of liver cirrhosis. It is yet unknown whether ECM signature is of predictive value in determining prognosis of early-stage liver cirrhosis. In this study, we systematically analyzed proteomics of decellularized hepatic matrix and identified four unique clusters of ECM proteins at tissue damage/inflammation, transitional ECM remodeling or fibrogenesis stage in carbon tetrachloride-induced liver fibrosis. In particular, basement membrane (BM) was heavily deposited at the fibrogenesis stage. BM component minor type IV collagen α5 chain expression was increased in activated hepatic stellate cells. Knockout of minor type IV collagen α5 chain ameliorated liver fibrosis by hampering hepatic stellate cell activation and promoting hepatocyte proliferation. ECM signatures were differentially enriched in the biopsies of good and poor prognosis early-stage liver cirrhosis patients. Clusters of ECM proteins responsible for homeostatic remodeling and tissue fibrogenesis, as well as basement membrane signature were significantly associated with disease progression and patient survival. In particular, a 14-gene signature consisting of basement membrane proteins is potent in predicting disease progression and patient survival. Thus, the ECM signatures are potential prognostic biomarkers to identify cirrhotic patients at high risk developing advanced stage disease.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Colágeno Tipo IV/metabolismo , Matriz Extracelular/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática Experimental/metabolismo , Hígado/metabolismo , Animales , Tetracloruro de Carbono , Línea Celular , Proliferación Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Colágeno Tipo IV/genética , Bases de Datos Genéticas , Progresión de la Enfermedad , Matriz Extracelular/patología , Células Estrelladas Hepáticas/patología , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/genética , Cirrosis Hepática Experimental/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Pronóstico , Proteoma , Factores de Tiempo , TranscriptomaRESUMEN
Nutritional ketosis as a therapeutic tool has been extended to the treatment of metabolic diseases, including obesity, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD). The purpose of this study was to determine whether dietary administration of the ketone ester (KE) R,S-1,3-butanediol diacetoacetate (BD-AcAc2) attenuates markers of hepatic stellate cell (HSC) activation and hepatic fibrosis in the context of high-fat diet (HFD)-induced obesity. Six-week-old male C57BL/6J mice were placed on a 10-wk ad libitum HFD (45% fat, 32% carbohydrates, 23% proteins). Mice were then randomized to one of three groups (n = 10 per group) for an additional 12 wk: 1) control (CON), continuous HFD; 2) pair-fed (PF) to KE, and 3) KE (HFD + 30% energy from BD-AcAc2, KE). KE feeding significantly reduced histological steatosis, inflammation, and total NAFLD activity score versus CON, beyond improvements observed for calorie restriction alone (PF). Dietary KE supplementation also reduced the protein content and gene expression of profibrotic markers (α-SMA, COL1A1, PDGF-ß, MMP9) versus CON (P < 0.05), beyond reductions observed for PF versus CON. Furthermore, KE feeding increased hepatic markers of anti-inflammatory M2 macrophages (CD163) and also reduced proinflammatory markers [tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and cellular communication network factor 1 (CCN1)] versus CON and PF (P ≤ 0.05), in the absence of changes in markers of total hepatic macrophage content (F4/80 and CD68; P > 0.05). These data highlight that the dietary ketone ester BD-AcAc2 ameliorates histological NAFLD and inflammation and reduces profibrotic and proinflammatory markers. Future studies to further explore potential mechanisms are warranted.NEW & NOTEWORTHY To our knowledge, this is the first study focusing on hepatic outcomes in response to dietary ketone ester feeding in male mice with HFD-induced NAFLD. Novel findings include that dietary ketone ester feeding ameliorates NAFLD outcomes via reductions in histological steatosis and inflammation. These improvements were beyond those observed for caloric restriction alone. Furthermore, dietary ketone ester feeding was associated with greater reductions in markers of hepatic fibrogenesis and inflammation compared with control and calorie-restricted mice.
Asunto(s)
Acetoacetatos/farmacología , Butileno Glicoles/farmacología , Dieta Alta en Grasa , Cirrosis Hepática Experimental/prevención & control , Hígado/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Animales , Biomarcadores/metabolismo , Restricción Calórica , Regulación de la Expresión Génica , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Mediadores de Inflamación/metabolismo , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/genética , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Activación de Macrófagos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , FenotipoRESUMEN
Normally, there are multiple microRNAs involved in the pathogenesis of liver fibrosis. In our work, we aimed at identifying the role of miR-34c in the hepatic stellate cell (HSC) activation and liver fibrosis and its potential mechanism. Our results have shown that during natural activation of HSC, the level of miR-34c was increased significantly whereas acyl-CoA synthetase long-chain family member-1(ACSL1), which is a key enzyme can affect fatty acid(FA) synthesis, was decreased. A double fluorescence reporter assay further confirmed that ACSL1 is a direct target gene of miR-34c. Moreover, the inhibition of miR-34C can attenuate the synthesis of collagen in HSC-T6. In our rescue assay, ACSL1 expression was 1.49-fold higher compared to normal control cells which were transfected with the miR-34c inhibitor in a stable low expression ACSL1 cell line. While at the same time, α-SMA and Col1α expression decreased by 18.22% and 2.58%, respectively. Moreover, we performed an in vivo model using dimethylnitrosamine (DMN) in conjunction with the miR-34c agomir, combined with the treatment of DMN and the miR-34c agomir can increase liver fibrosis. Meanwhile, the degree of hepatic fibrosis was increased and lipid droplets reduced dramatically in rats and HSC-T6 cell treated with miR-34c mimics alone compared to untreated groups. Our results indicate that miR-34c plays an essential role in liver fibrosis by targeting ACSL1 closely associated with lipid droplets, and it might be used as a potential therapeutic target.
Asunto(s)
Coenzima A Ligasas/genética , Células Estrelladas Hepáticas/patología , Cirrosis Hepática Experimental/genética , Hígado/patología , MicroARNs/metabolismo , Animales , Coenzima A Ligasas/metabolismo , Colágeno/biosíntesis , Dimetilnitrosamina/administración & dosificación , Dimetilnitrosamina/toxicidad , Células Estrelladas Hepáticas/efectos de los fármacos , Humanos , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos/genética , Hígado/citología , Hígado/efectos de los fármacos , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/patología , MicroARNs/agonistas , MicroARNs/antagonistas & inhibidores , RatasRESUMEN
Nonalcoholic steatohepatitis (NASH) could progress to hepatic fibrosis in the absence of effective control. The purpose of our experiment was to investigate the protective effect of drinking water with a high concentration of hydrogen, namely, hydrogen-rich water (HRW), on mice with nonalcoholic fatty liver disease to elucidate the mechanism underlying the therapeutic action of molecular hydrogen. The choline-supplemented, l-amino acid-defined (CSAA) or the choline-deficient, l-amino acid-defined (CDAA) diet for 20 wk was used to induce NASH and fibrosis in the mice model and simultaneously treated with the high-concentration 7-ppm HRW for different periods (4 wk, 8 wk, and 20 wk). Primary hepatocytes were stimulated by palmitate to mimic liver lipid metabolism during fatty liver formation. Primary hepatocytes were cultured in a closed vessel filled with 21% O2 + 5% CO2 + 3.8% H2 and N2 as the base gas to verify the response of primary hepatocytes in a high concentration of hydrogen gas in vitro. Mice in the CSAA + HRW group had lower serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and milder histological damage. The inflammatory cytokines were expressed at lower levels in the HRW group than in the CSAA group. Importantly, HRW reversed hepatocyte fatty acid oxidation and lipogenesis as well as hepatic inflammation and fibrosis in preexisting hepatic fibrosis specimens. Molecular hydrogen inhibits the lipopolysaccharide-induced production of inflammation cytokines through increasing heme oxygenase-1 (HO-1) expression. Furthermore, HRW improved hepatic steatosis in the CSAA + HRW group. Sirtuin 1 (Sirt1) induction by molecular hydrogen via the HO-1/adenosine monophosphate activated protein kinase (AMPK)/peroxisome proliferator-activated receptor α (PPARα)/peroxisome proliferator-activated receptor γ (PPAR-γ) pathway suppresses palmitate-mediated abnormal fat metabolism. Orally administered HRW suppressed steatosis induced by CSAA and attenuated fibrosis induced by CDAA, possibly by reducing oxidative stress and the inflammation response.NEW & NOTEWORTHY The mRNA expression of inflammatory cytokines in the HRW group was lower than in the CSAA group. HRW reversed hepatocyte apoptosis as well as hepatic inflammation and fibrosis in NASH specimens. Molecular hydrogen inhibits LPS-induced inflammation via an HO-1/interleukin 10 (IL-10)-independent pathway. HRW improved hepatic steatosis in the CSAA + HRW group. Sirt1 induction by molecular hydrogen via the HO-1/AMPK/PPARα/PPARγ pathway suppresses palmitate-mediated abnormal fat metabolism.
Asunto(s)
Hemo-Oxigenasa 1/metabolismo , Hepatocitos/efectos de los fármacos , Hidrógeno/farmacología , Interleucina-10/metabolismo , Cirrosis Hepática Experimental/prevención & control , Hígado/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Sirtuina 1/metabolismo , Agua/farmacología , Animales , Hepatocitos/enzimología , Hepatocitos/patología , Hidrógeno/química , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/metabolismo , Lipólisis/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Cirrosis Hepática Experimental/enzimología , Cirrosis Hepática Experimental/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/patología , Células RAW 264.7 , Transducción de SeñalRESUMEN
Polycystic liver disease (PLD) is a hereditary liver disease in which the number of cysts increases over time, causing various abdominal symptoms and poor quality of life. Although effective treatment for PLD has not been established, we recently reported that long-term exercise ameliorated liver cyst formation and fibrosis with the activation of AMP-activated protein kinase (AMPK) in polycystic kidney (PCK) rats, a PLD model. Therefore, the aim of this study was to investigate whether metformin, an indirect AMPK activator, was effective in PCK rats. PCK rats were randomly divided into a control (Con) group and a metformin-treated (Met) group. The Met group was treated orally with metformin in drinking water. After 12 wk, liver function, histology, and signaling cascades of PLD were examined in the groups. Metformin did not affect the body weight or liver weight, but it reduced liver cyst formation, cholangiocyte proliferation, and fibrosis around the cyst. Metformin increased the phosphorylation of AMPK and tuberous sclerosis complex 2 and decreased the phosphorylation of mammalian target of rapamycin, S6, and extracellular signal-regulated kinase and the expression of cystic fibrosis transmembrane conductance regulator, aquaporin I, transforming growth factor-ß, and type 1 collagen without changes in apoptosis or collagen degradation factors in the liver. Metformin slows the development of cyst formation and fibrosis with the activation of AMPK and inhibition of signaling cascades responsible for cellular proliferation and fibrosis in the liver of PCK rats.NEW & NOTEWORTHY This study indicates that metformin, an indirect AMPK activator slows liver cyst formation and fibrosis in PLD rat model. Metformin attenuates excessive cell proliferation in the liver with the inactivation of mTOR and ERK pathways. Metformin also reduces the expression of proteins responsible for cystic fluid secretion and liver fibrosis. Metformin and AMPK activators may be potent drugs for polycystic liver disease.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Proliferación Celular/efectos de los fármacos , Quistes/tratamiento farmacológico , Activadores de Enzimas/farmacología , Cirrosis Hepática Experimental/tratamiento farmacológico , Hepatopatías/tratamiento farmacológico , Hígado/efectos de los fármacos , Metformina/farmacología , Animales , Quistes/enzimología , Quistes/patología , Progresión de la Enfermedad , Activación Enzimática , Hígado/enzimología , Hígado/patología , Cirrosis Hepática Experimental/enzimología , Cirrosis Hepática Experimental/patología , Hepatopatías/enzimología , Hepatopatías/patología , Masculino , Fosforilación , Ratas , Transducción de Señal , Factores de TiempoRESUMEN
Branched-chain amino acids (BCAA) reverse malnutrition and l-carnitine leads to the reduction of hyperammonemia and muscle cramps in cirrhotic patients. BCAA and l-carnitine are involved in glucose and fatty acid metabolism, however their mechanistic activity in cirrhotic liver is not fully understood. We aim to define the molecular mechanism(s) and combined effects of BCAA and l-carnitine using a cirrhotic rat model. Rats were administered carbon tetrachloride for 10 weeks to induce cirrhosis. During the last 6 weeks of administration, cirrhotic rats received BCAA, l-carnitine or a combination of BCAA and l-carnitine daily via gavage. We found that BCAA and l-carnitine treatments significantly improved hepatocellular function associated with reduced triglyceride level, lipid deposition and adipophilin expression, in cirrhotic liver. Lipidomic analysis revealed dynamic changes in hepatic lipid composition by BCAA and l-carnitine administrations. BCAA and l-carnitine globally increased molecular species of phosphatidylcholine. Liver triacylglycerol and phosphatidylcholine hydroperoxides were significantly decreased by BCAA and l-carnitine. Furthermore, serum and liver ATP levels were significantly increased in all treatments, which were attributed to the elevation of mature cardiolipins and mitochondrial component gene expressions. Finally, BCAA and l-carnitine dramatically reduced hepatocellular death. In conclusion, BCAA and l-carnitine treatments attenuate hepatocellular damage through the reduction of lipid peroxides and the overall maintenance of mitochondrial integrity within the cirrhotic liver. These effectiveness of BCAA and l-carnitine support the therapeutic strategies in human chronic liver diseases.
Asunto(s)
Aminoácidos de Cadena Ramificada/farmacología , Carnitina/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Hígado Graso/prevención & control , Hepatocitos/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Cirrosis Hepática Experimental/prevención & control , Hígado/efectos de los fármacos , Animales , Tetracloruro de Carbono , Muerte Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hígado Graso/inducido químicamente , Hígado Graso/metabolismo , Hígado Graso/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/patología , Ratas WistarRESUMEN
Progression of nonalcoholic steatohepatitis (NASH) is attributed to several factors, including inflammation and oxidative stress. In recent years, renalase has been reported to suppress oxidative stress, apoptosis and inflammation. A number of studies have suggested that renalase may be associated with protecting the liver from injury. The present study aimed to clarify the effects of renalase knockout (KO) in mice with NASH that were induced with a cholinedeficient highfat diet (CDAHFD) supplemented with 0.1% methionine. Wild type (WT) and KO mice (6weekold) were fed a normal diet (ND) or CDAHFD for 6 weeks, followed by analysis of the blood liver function markers and liver tissues. CDAHFD intake was revealed to increase blood hepatic function markers, lipid accumulation and oxidative stress compared with ND, but no significant differences were observed between the WT and KO mice. However, in the KOCDAHFD group, the Adgre1 and Tgfb1 mRNA levels were significantly higher, and αSMA expression was significantly lower compared with the WTCDAHFD group. Furthermore, the Gclc mRNA and phosphorylated protein kinase B (Akt) levels were significantly lower in the KOND group compared with the WTND group. The results of the current study indicated that as NASH progressed in the absence of renalase, oxidative stress, macrophage infiltration and TGFß expression were enhanced, while αSMA expression in NASH may be partly suppressed due to the decreased phosphorylation of Akt level.