RESUMEN
BACKGROUND: The immunomodulatory oligodeoxynucleotide (ODN) IMT504 might harbor antifibrotic properties within the liver. METHODS: Fibrosis models were induced in mice through thioacetamide (TAA) administration and bile-duct ligation. Cre-loxP mice were utilized to identify GLAST + Wnt1 + bone marrow stromal progenitors (BMSPs) and to examine their contribution with cells in the liver. In vivo and in vitro assays; flow-cytometry, immunohistochemistry, and qPCR were conducted. RESULTS: IMT504 demonstrated significant inhibition of liver fibrogenesis progression and reversal of established fibrosis. Early responses to IMT504 involved the suppression of profibrogenic and proinflammatory markers, coupled with an augmentation of hepatocyte proliferation. Additionally, this ODN stimulated the proliferation and mobilization of GLAST + Wnt1 + BMSPs, likely amplifying their contribution with endothelial- and hepatocytes-like cells. Moreover, IMT504 significantly modulated the expression levels of Wnt ligands and signaling pathway/target genes specifically within GLAST + Wnt1 + BMSPs, with minimal impact on other BMSPs. Intriguingly, both IMT504 and conditioned media from IMT504-pre-treated GLAST + Wnt1 + BMSPs shifted the phenotype of fibrotic macrophages, hepatic stellate cells, and hepatocytes, consistent with the potent antifibrotic effects observed. CONCLUSION: In summary, our findings identify IMT504 as a promising candidate molecule with potent antifibrotic properties, operating through both direct and indirect mechanisms, including the activation of GLAST + Wnt1 + BMSPs.
Asunto(s)
Cirrosis Hepática , Células Madre Mesenquimatosas , Proteína Wnt1 , Animales , Ratones , Cirrosis Hepática/patología , Cirrosis Hepática/tratamiento farmacológico , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Proteína Wnt1/metabolismo , Proteína Wnt1/genética , Hígado/efectos de los fármacos , Hígado/patología , Hígado/metabolismo , Oligodesoxirribonucleótidos/farmacología , Masculino , Ratones Endogámicos C57BL , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , TioacetamidaRESUMEN
Tracking cell death in vivo can enable a better understanding of the biological mechanisms underlying tissue homeostasis and disease. Unfortunately, existing cell death labeling methods lack compatibility with in vivo applications or suffer from low sensitivity, poor tissue penetration, and limited temporal resolution. Here, we fluorescently labeled dead cells in vivo with Trypan Blue (TBlue) to detect single scattered dead cells or to generate whole-mount three-dimensional maps of large areas of necrotic tissue during organ regeneration. TBlue effectively marked different types of cell death, including necrosis induced by CCl4 intoxication in the liver, necrosis caused by ischemia-reperfusion in the skin, and apoptosis triggered by BAX overexpression in hepatocytes. Moreover, due to its short circulating lifespan in blood, TBlue labeling allowed in vivo "pulse and chase" tracking of two temporally spaced populations of dying hepatocytes in regenerating mouse livers. Additionally, upon treatment with cisplatin, TBlue labeled dead cancer cells in livers with cholangiocarcinoma and dead thymocytes due to chemotherapy-induced toxicity, showcasing its utility in assessing anticancer therapies in preclinical models. Thus, TBlue is a sensitive and selective cell death marker for in vivo applications, facilitating the understanding of the fundamental role of cell death in normal biological processes and its implications in disease.
Asunto(s)
Muerte Celular , Azul de Tripano , Animales , Ratones , Muerte Celular/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Hepatocitos/metabolismo , Humanos , Neoplasias/patología , Ratones Endogámicos C57BL , Regeneración Hepática/efectos de los fármacos , Hígado/patología , Hígado/efectos de los fármacos , Rastreo Celular/métodos , Apoptosis/efectos de los fármacos , Imagenología Tridimensional , Regeneración/efectos de los fármacos , Necrosis , MasculinoRESUMEN
Obesity is a complex chronic disease characterized by excess body fat (adipose) that is harmful to health and has been a major global health problem. It may be associated with several diseases, such as nonalcoholic fatty liver disease (NAFLD). Polyunsaturated fatty acids (PUFA) are lipid mediators that have anti-inflammatory characteristics and can be found in animals and plants, with capybara oil (CO) being a promising source. So, we intend to evaluate the hepatic pathophysiological alterations in C57Bl/6 mice with NAFLD, caused by obesity, and the possible beneficial effects of OC in the treatment of this disease. Eighteen 3-month-old male C57Bl/6 mice received a control or high-fat diet for 18 weeks. From the 15th to the 18th week, the animals received treatment-through orogastric gavage-with placebo or free capybara oil (5 g/kg). Parameters inherent to body mass, glucose tolerance, evaluation of liver enzymes, percentage of hepatic steatosis, oxidative stress, the process of cell death with the apoptotic biomarkers (Bax, Bcl2, and Cytochrome C), and the ultrastructure of hepatocytes were analyzed. Even though the treatment with CO was not able to disassemble the effects on the physiological parameters, it proved to be beneficial in reversing the morphological and ultrastructural damage present in the hepatocytes. Thus, demonstrating that CO has beneficial effects in reducing steatosis and the apoptotic pathway, it is a promising treatment for NAFLD.
Asunto(s)
Apoptosis , Hígado , Enfermedad del Hígado Graso no Alcohólico , Aceites , Roedores , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/terapia , Masculino , Animales , Ratones , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Hepatocitos/ultraestructura , Aceites/farmacología , Aceites/uso terapéutico , Obesidad/complicaciones , Apoptosis/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/patología , Hígado/ultraestructura , Oxidorreductasas/metabolismo , Activación Enzimática/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacosRESUMEN
AIMS: Estradiol 17ß-d-glucuronide (E217G) induces cholestasis by triggering endocytosis and further intracellular retention of the canalicular transporters Bsep and Mrp2, in a cPKC- and PI3K-dependent manner, respectively. Pregnancy-induced cholestasis has been associated with E217G cholestatic effect, and is routinely treated with ursodeoxycholic acid (UDCA). Since protective mechanisms of UDCA in E217G-induced cholestasis are still unknown, we ascertained here whether its main metabolite, tauroursodeoxycholate (TUDC), can prevent endocytosis of canalicular transporters by counteracting cPKC and PI3K/Akt activation. MAIN METHODS: Activation of cPKC and PI3K/Akt was evaluated in isolated rat hepatocytes by immunoblotting (assessment of membrane-bound and phosphorylated forms, respectively). Bsep/Mrp2 function was quantified in isolated rat hepatocyte couplets (IRHCs) by assessing the apical accumulation of their fluorescent substrates, CLF and GS-MF, respectively. We also studied, in isolated, perfused rat livers (IPRLs), the status of Bsep and Mrp2 transport function, assessed by the biliary excretion of TC and DNP-SG, respectively, and Bsep/Mrp2 localization by immunofluorescence. KEY FINDINGS: E217G activated both cPKC- and PI3K/Akt-dependent signaling, and pretreatment with TUDC significantly attenuated these activations. In IRHCs, TUDC prevented the E217G-induced decrease in apical accumulation of CLF and GS-MF, and inhibitors of protein phosphatases failed to counteract this protection. In IPRLs, E217G induced an acute decrease in bile flow and in the biliary excretion of TC and DNP-SG, and this was prevented by TUDC. Immunofluorescence studies revealed that TUDC prevented E217G-induced Bsep/Mrp2 endocytosis. SIGNIFICANCE: TUDC restores function and localization of Bsep/Mrp2 impaired by E217G, by preventing both cPKC and PI3K/Akt activation in a protein-phosphatase-independent manner.
Asunto(s)
Colestasis , Endocitosis , Estradiol , Hepatocitos , Fosfatidilinositol 3-Quinasas , Transducción de Señal , Ácido Tauroquenodesoxicólico , Animales , Colestasis/metabolismo , Colestasis/inducido químicamente , Colestasis/prevención & control , Ratas , Transducción de Señal/efectos de los fármacos , Estradiol/metabolismo , Estradiol/farmacología , Estradiol/análogos & derivados , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Endocitosis/efectos de los fármacos , Ácido Tauroquenodesoxicólico/farmacología , Ácido Tauroquenodesoxicólico/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Wistar , Femenino , Masculino , Proteína Quinasa C/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismoRESUMEN
Genetic factors, diet, lifestyle, and other factors lead to various complications in the body, such as obesity and other chronic diseases. The inflammatory state caused by excessive accumulation of body fat affects the pathways related to the control of glycemic homeostasis, leading to a high demand for insulin, to subsequent failure of stressed ß cells, and development of type 2 diabetes mellitus (T2DM). The study of new endocrine signalers, such as bile acids (BAs), becomes necessary as it allows the development of alternatives for T2DM treatment. In this work, a methodology was developed to quantify tauroursodeoxycholic BA (TUDCA) in liver cells of the HepG2 strain treated in hyperlipidic medium. This BA helps to improve insulin clearance by increasing the expression of the insulin-degrading enzyme, restoring sensitivity to this hormone, and making it viable for treating T2DM. Herein, a targeted metabolomic method for TUDCA determination in extracellular medium of hepatocyte matrices by micellar electrokinetic chromatography-UV was optimized, validated, and applied. The optimized background electrolyte was composed of 40 mmol/L sodium cholate and 30 mmol/L sodium tetraborate at pH 9.0. The following figures of merit were evaluated: linearity, limit of quantification, limit of detection, accuracy, and precision. Data obtained with the validated electrophoretic method showed a self-stimulation of TUDCA production in media supplemented only with BA. On the other hand, TUDCA concentration was reduced in the hyperlipidic medium. This suggests that, in these media, the effect of TUDCA is reduced, such as self-stimulated production and consequent regulation of glycemic homeostasis. Therefore, the results reinforce the need for investigating TUDCA as a potential T2DM biomarker as well as its use to treat several comorbidities, such as obesity and diabetes mellitus.
Asunto(s)
Cromatografía Capilar Electrocinética Micelar , Diabetes Mellitus Tipo 2 , Obesidad , Ácido Tauroquenodesoxicólico , Ácido Tauroquenodesoxicólico/farmacología , Ácido Tauroquenodesoxicólico/análisis , Ácido Tauroquenodesoxicólico/metabolismo , Humanos , Obesidad/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Hep G2 , Cromatografía Capilar Electrocinética Micelar/métodos , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Reproducibilidad de los Resultados , Metabolómica/métodos , Modelos Lineales , Límite de DetecciónRESUMEN
The endogenous metabolite of estradiol, estradiol 17ß-D-glucuronide (E17G), is considered the main responsible of the intrahepatic cholestasis of pregnancy. E17G alters the activity of canalicular transporters through a signaling pathway-dependent cellular internalization, phenomenon that was attributed to oxidative stress in different cholestatic conditions. However, there are no reports involving oxidative stress in E17G-induced cholestasis, representing this the aim of our work. Using polarized hepatocyte cultures, we showed that antioxidant compounds prevented E17G-induced Mrp2 activity alteration, being this alteration equally prevented by the NADPH oxidase (NOX) inhibitor apocynin. The model antioxidant N-acetyl-cysteine prevented, in isolated and perfused rat livers, E17G-induced impairment of bile flow and Mrp2 activity, thus confirming the participation of reactive oxygen species (ROS) in this cholestasis. In primary cultured hepatocytes, pretreatment with specific inhibitors of ERK1/2 and p38MAPK impeded E17G-induced ROS production; contrarily, NOX inhibition did not affect ERK1/2 and p38MAPK phosphorylation. Both, knockdown of p47phox by siRNA and preincubation with apocynin in sandwich-cultured rat hepatocytes significantly prevented E17G-induced internalization of Mrp2, suggesting a crucial role for NOX in this phenomenon. Concluding, E17G-induced cholestasis is partially mediated by NOX-generated ROS through internalization of canalicular transporters like Mrp2, being ERK1/2 and p38MAPK necessary for NOX activation.
Asunto(s)
Estradiol , Hepatocitos , NADPH Oxidasas , Especies Reactivas de Oxígeno , Animales , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ratas , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Estradiol/farmacología , Estradiol/metabolismo , Estradiol/análogos & derivados , Femenino , Colestasis/inducido químicamente , Colestasis/metabolismo , Colestasis/patología , Ratas Wistar , Acetofenonas/farmacología , Estrés Oxidativo/efectos de los fármacos , Acetilcisteína/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células Cultivadas , Antioxidantes/farmacología , Antioxidantes/metabolismo , Colestasis Intrahepática , Complicaciones del Embarazo , Transportadoras de Casetes de Unión a ATPRESUMEN
SUMMARY: We aimed to investigate the protective effect of linoleic acid on liver toxicity induced by methotrexate. The study was carried out in partnership with the Department of Anatomy and Department of Medical Pharmacology of Çukurova University Faculty of Medicine, using the laboratory facilities of the Department of Medical Pharmacology. Human hepatocyte cell line (CRL- 11233) cells obtained from the American Type Culture Collection Organization (ATCC) were used. Expressions of apoptotic pathway markers, apoptosis inducing factor (AIF), BAX, BCL 2, GADD 153, 78-kDa glucose-regulated protein (GRP78), and CASPASE-3 were evaluated. All analyzes were examined in four groups (Group 1; control, Group 2; linoleic acid given, Group 3; methotrexate given and Group 4; linoleic acid and methotrexate given). The mean ± standard error values of the obtained results as nanogram / milliliter (ng / ml) are in Group I, Group II, Group III and Group IV, respectively; AIF values, 0.4150 ± 0.1208, 0.3633 ± 0.2389, 1.792 ± 0.3611 and 1.077 ± 0.1646, BAX values, 0.900 ± 0.1864, 1.002 ± 0.2098, 8.352 ± 1.467 and 4.295 ± 1.522, BCL 2 values, 13.93 ± 1.198, 13.92 ± 1.739, 2.938 ± 1.059 and 9.250 ± 1.492, GADD 153, 0.7333 ± 0.1751, 0.7067 ± 0.2115, 1.650 ± 0.2950 and 1.237 ± 0.1805, GRP78, 0.4767 ± 0.1804, 0.5233 ± 0.1590, 2.183 ± 0.2639 and 1.112 ± 0.2693, CASPASE-3 values , 1.127 ± 0.2033, 0.8317 ± 0.3392, 13.50 ± 1.871 and 8.183 ± 1.030. It was determined that linoleic acid has a protective effect on methotrexate-induced liver toxicity.
Nuestro objetivo fue investigar el efecto protector del ácido linoleico sobre la toxicidad hepática inducida por metotrexato. El estudio se llevó a cabo en colaboración con el Departamento de Anatomía y el Departamento de Farmacología Médica de la Facultad de Medicina de la Universidad de Çukurova, utilizando las instalaciones del laboratorio del Departamento de Farmacología Médica. Se usaron células de la línea celular de hepatocitos humanos (CRL-11233) obtenidas de la American Type Culture Collection Organisation (ATCC). Se evaluaron las expresiones de marcadores de vías apoptóticas, factor inductor de apoptosis (AIF), BAX, BCL 2, GADD 153, proteína regulada por glucosa de 78 kDa (GRP78) y CASPASE-3. Todos los análisis se examinaron en cuatro grupos (Grupo 1; control, Grupo 2; se administró ácido linoleico, Grupo 3; se administró metotrexato y Grupo 4; se administró ácido linoleico y metotrexato). Los valores medios ± error estándar de los resultados obtenidos como nanogramo/mililitro (ng/ml) se encuentran en el Grupo I, Grupo II, Grupo III y Grupo IV, respectivamente; Valores de AIF, 0,4150 ± 0,1208, 0,3633 ± 0,2389, 1,792 ± 0,3611 y 1,077 ± 0,1646, valores de Bax, 0,900 ± 0,1864, 1,002 ± 0,2098, 8,352 ± 1,467 y 4,295 ± 1,522, BCL 2 valores, 13,93 ± 1,199. 2,938 ± 1,059 y 9,250 ± 1,492, GADD 153, 0,7333 ± 0,1751, 0,7067 ± 0,2115, 1,650 ± 0,2950 y 1,237 ± 0,1805, Grp78, 0,4767 ± 0,1804, 0,5233 ± 0,1590, 2,183, ± 1,263. 1,127 ± 0,2033, 0,8317 ± 0,3392, 13,50 ± 1,871 y 8,183 ± 1,030. Se determinó que el ácido linoleico tiene un efecto protector sobre la toxicidad hepática inducida por metotrexato.
Asunto(s)
Humanos , Metotrexato/toxicidad , Ácido Linoleico/administración & dosificación , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Ensayo de Inmunoadsorción Enzimática , Células Cultivadas , Sustancias Protectoras , Hepatocitos/efectos de los fármacos , Factor Inductor de la Apoptosis , Caspasa 3 , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Chaperón BiP del Retículo Endoplásmico , Hígado/citología , Hígado/efectos de los fármacos , Antimetabolitos Antineoplásicos/toxicidadRESUMEN
Doxazosin and carvedilol have been evaluated as an alternative treatment against chronic liver lesions and for their possible role during the regeneration of damage caused by liver fibrosis in a hamster model. However, these drugs have been reported to induce morphological changes in hepatocytes, affecting the recovery of liver parenchyma. The effects of these α/ð½ adrenoblockers on the viability of hepatocytes are unknown. Herein, we demonstrate the protective effect of curcumin against the possible side effects of doxazosin and carvedilol, drugs with proven antifibrotic activity. After pretreatment with 1 µM curcumin for 1 h, HepG2 cells were exposed to 0.1-25 µM doxazosin or carvedilol for 24, 48, and 72 h. Cell viability was assessed using the MTT assay and SYTOX green staining. Morphological changes were detected using the hematoxylin and eosin (H&E) staining and scanning electron microscopy (SEM). An expression of apoptotic and oxidative stress markers was analyzed using reverse transcription-quantitative PCR (RT-qPCR). The results indicate that doxazosin decreases cell viability in a time- and dose-dependent manner, whereas carvedilol increases cell proliferation; however, curcumin increases or maintains cell viability. SEM and H&E staining provided evidence that doxazosin and carvedilol induced morphological changes in HepG2 cells, and curcumin protected against these effects, maintaining the morphology in 90% of treated cells. Furthermore, curcumin positively regulated the expression of Nrf2, HO-1, and SOD1 mRNAs in cells treated with 0.1 and 0.5 µM doxazosin. Moreover, the Bcl-2/Bax ratio was higher in cells that were treated with curcumin before doxazosin or carvedilol. The present study demonstrates that curcumin controls doxazosin- and carvedilol-induced cytotoxicity and morphological changes in HepG2 cells possibly by overexpression of Nrf2.
Asunto(s)
Carvedilol/toxicidad , Curcumina/farmacología , Doxazosina/toxicidad , Estrés Oxidativo/efectos de los fármacos , Apoptosis/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Humanos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
AIM: P-glycoprotein (P-gp) plays a critical role in the excretion of xenobiotics into bile. Previous studies have demonstrated that prolactin (PRL) regulates biotransformation and bile salt transport. Here we investigate whether the capability of the liver to transport xenobiotics into bile is altered in hyperprolactinemic states studying the modulation of hepatic P-gp by PRL. METHODS: We used lactating post-partum rats (PP), as a model of physiological hyperprolactinemia (15 and 21 days after delivery: PP15 and PP21, respectively), and ovariectomized rats treated with PRL (300 µg/day, 7 days, via osmotic minipumps, OVX + PRL). Hepatic P-gp expression and activity were evaluated by western blotting and using rhodamine 123 as substrate in vivo, respectively. Since P-gp is encoded by Mdr1a and Mdr1b in rodents, we quantified their expression by qPCR in primary hepatocyte cultures exposed to 0.1 µg/ml of PRL after 12 h. To further study the mechanism of hepatic P-gp modulation by PRL, hepatocytes were pretreated with actinomycin D and then exposed to PRL (0.1 µg/ml) for 12 h. KEY FINDINGS: We found increased hepatic P-gp protein expression and activity in PP15 and OVX + PRL. Also, a significant increase in Mdr1a and Mdr1b mRNA levels was observed in primary hepatocyte cultures exposed to PRL, pointing out the hormone direct action. Actinomycin D prevented these increases, confirming a transcriptional up-regulation of P-gp by PRL. SIGNIFICANCE: These findings suggest the possibility of an increased biliary excretion of xenobiotics substrates of P-gp, including therapeutic agents, affecting their pharmaco/toxicokinetics in hyperprolactinemic situations.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Hígado/efectos de los fármacos , Hígado/metabolismo , Prolactina/metabolismo , Prolactina/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Lactancia/efectos de los fármacos , Lactancia/metabolismo , Ovariectomía , Ratas , Ratas Wistar , OvinosRESUMEN
Non-alcoholic steatohepatitis (NASH) is a devastating form of non-alcoholic fatty liver disease (NAFLD) with distinguished hallmarks of steatosis and inflammation. Rotundic acid (RA) is a natural pentacyclic triterpene compound extracted from the bank of Ilex rotunda Thunb with a wide range of biological activities. The aim of the study is to evaluate the pharmacological effect and action mechanism of RA on NASH in vitro and in vivo. RA has weak lipid lowering ability in rat primary hepatocytes, significantly decreases serum LDL level, hepatic TG and TC levels and lipid droplets, reduces NAS compared with the NASH group, and alleviates hepatic inflammation. RA also enhances the recovery of intestinal bacterial community and intestinal-derived short-chain fatty acid caused by high food diet (HFD). Further investigation shows that RA protects against HFD-induced NASH via downregulating the expression of SREBP-1c/SCD1 signaling pathway and improving gut microbiota. These findings imply that RA might be helpful for the alleviation of NASH.
Asunto(s)
Microbioma Gastrointestinal/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Sustancias Protectoras/farmacología , Estearoil-CoA Desaturasa/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Triterpenos/farmacología , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Supervivencia Celular/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Ácidos Grasos Volátiles/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Lípidos/sangre , Hígado/efectos de los fármacos , Hígado/lesiones , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Cultivo Primario de Células , Sustancias Protectoras/química , Sustancias Protectoras/uso terapéutico , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Triterpenos/química , Triterpenos/uso terapéuticoRESUMEN
Stearoyl-CoA desaturase (SCD) is a central lipogenic enzyme for the synthesis of monounsaturated fatty acids (MUFA). SCD1 overexpression is associated with a genetic predisposition to hepatocarcinogenesis in mice and rats. This work hypothesized possible roles of SCD1 to genomic stability, lipogenesis, cell proliferation, and survival that contribute to the malignant transformation of non-tumorigenic liver cells. Therefore, HepG2 tumor cells were treated with the SCD1 inhibitor (CAY10566) to ensure a decrease in proliferation/survival, as confirmed by a lipidomic analysis that detected an efficient decrease in the concentration of MUFA. According to that, we switched to a model of normal hepatocytes, the HepaRG cell line, where we: (i) overexpressed SCD1 (HepaRG-SCD1 clones), (ii) inhibited the endogenous SCD1 activity with CAY10566, or (iii) treated with two monounsaturated (oleic OA and/or palmitoleic PA) fatty acids. SCD1 overexpression or MUFA stimulation increased cell proliferation, survival, and the levels of AKT, phospho-AKT(Ser473), and proliferating cell nuclear antigen (PCNA) proteins. By contrast, opposite molecular and cellular responses were observed in HepaRG cells treated with CAY10566. To assess genomic stability, HepaRG-SCD1 clones were treated with ionizing radiation (IR) and presented reduced levels of DNA damage and higher survival at doses of 5 Gy and 10 Gy compared to parental cells. In sum, this work suggests that modulation of SCD1 activity not only plays a role in cell proliferation and survival, but also in maintaining genomic stability, and therefore, contributes to a better understanding of this enzyme in molecular mechanisms of hepatocarcinogenesis projecting SCD1 as a potential translational target.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Ácidos Oléicos/farmacología , Oxadiazoles/farmacología , Piridazinas/farmacología , Estearoil-CoA Desaturasa/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Ciclo Celular , Proliferación Celular , Inestabilidad Genómica , Hepatocitos/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Estearoil-CoA Desaturasa/genética , Células Tumorales CultivadasRESUMEN
In this study, the changes in oncogenic and tumor suppressor signaling pathways in liver and their association with serum and urinary biomarkers of aflatoxin exposure were evaluated in Wistar rats fed diets containing aflatoxin B1 (AFB1) for 90 days. Rats were divided into four groups (n = 15 per group) and assigned to dietary treatments containing 0 (control), 50 (AFB50), 100 (AFB100) and 200 µg AFB1 kg-1 diet (AFB200). Multiple preneoplastic foci of hepatocytes marked with glutathione-S-transferase-placental form (GST-P) were identified in AFB100 and AFB200 groups. Hepatocellular damage induced by AFB1 resulted in overexpression of cyclin D1 and ß-catenin. The liver expression of retinoblastoma (Rb) and p27Kip1 decreased in AFB100 and AFB200 groups, confirming the favorable conditions for neoplastic progression to hepatocellular carcinoma. All samples from rats fed AFB1-contaminated diets had quantifiable AFB1-lysine in serum or urinary AFM1 and AFB1-N7-guanine, with mean levels of 20.42-50.34 ng mL-1, 5.31-37.68 and 39.15-126.37 ng mg-1 creatinine, respectively. Positive correlations were found between AFB1-lysine, AFM1 or AFB1-N7-guanine and GST-P+, ß-catenin+ and cyclin D1+ hepatocytes, while Rb + cells negatively correlated with those AFB1 exposure biomarkers. The pathways evaluated are critical molecular mechanisms of AFB1-induced hepatocarcinogenesis in rats.
Asunto(s)
Aflatoxina B1/toxicidad , Ciclina D1/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteína de Retinoblastoma/metabolismo , beta Catenina/metabolismo , Aflatoxina B1/análogos & derivados , Aflatoxina B1/sangre , Aflatoxina B1/metabolismo , Aflatoxina B1/orina , Aflatoxina M1/orina , Alanina Transaminasa/metabolismo , Animales , Aspartato Aminotransferasas/metabolismo , Biomarcadores/sangre , Biomarcadores/orina , Expresión Génica/efectos de los fármacos , Guanina/análogos & derivados , Guanina/orina , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/patología , Lisina/sangre , Masculino , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/patología , Ratas WistarRESUMEN
Obesity has a strong relationship to insulin resistance and diabetes mellitus, a chronic metabolic disease that alters many physiological functions. Naturally derived drugs have aroused great interest in treating obesity, and triterpenoids are natural compounds with multiple biological activities and antidiabetic mechanisms. Here, we evaluated the bioactivity of ursolic acid lactone (UAL), a lesser-known triterpenoid, obtained from Eucalyptus tereticornis. We used different cell lines to show for the first time that this molecule exhibits anti-inflammatory properties in a macrophage model, increases glucose uptake in insulin-resistant muscle cells, and reduces triglyceride content in hepatocytes and adipocytes. In 3T3-L1 adipocytes, UAL inhibited the expression of genes involved in adipogenesis and lipogenesis, enhanced the expression of genes involved in fat oxidation, and increased AMP-activated protein kinase phosphorylation. The range of biological activities demonstrated in vitro indicates that UAL is a promising molecule for fighting diabetes.
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Eucalyptus/química , Lactonas/química , Lactonas/farmacología , Triterpenos/química , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Macrófagos , Ratones , Células Musculares/efectos de los fármacos , Fosforilación/efectos de los fármacos , Triglicéridos/metabolismo , Triterpenos/farmacología , Ácido UrsólicoRESUMEN
BACKGROUND: Excess hepatic triglyceride (TG) accumulation (steatosis) commonly observed in obesity, may lead to non-alcoholic fatty liver disease (NAFLD). Altered regulation of intracellular lipid droplets (LD) and TG metabolism, as well as activation of JNK-mediated proinflammatory pathways may trigger liver steatosis-related disorders. Drosophila melanogaster is an animal model used for studying obesity and its associated disorders. In Drosophila, lipids and glycogen are stored in the fat body (FB), which resembles mammalian adipose tissue and liver. Dietary oversupply leads to obesity-related disorders, which are characterized by FB dysfunction. Infusions of Lampaya medicinalis Phil. (Verbenaceae) are used in folk medicine of Chile to counteract inflammatory diseases. Hydroethanolic extract of lampaya (HEL) contains considerable amounts of flavonoids that may explain its anti-inflammatory effect. METHODS: We studied whether HEL affects palmitic acid (PA, C16:0) and oleic acid (OA; C18:1)-induced TG accumulation and proinflammatory marker content in HepG2 hepatocytes as well as impaired lipid storage and proinflammatory molecule expression in Drosophila melanogaster fed a high-fat diet (HFD). RESULTS: In HepG2 hepatocytes, exposure to OA/PA elevated TG content, FABP4, ATGL and DGAT2 expression, and the JNK proinflammatory pathway, as well as TNF-α and IL-6 production, while diminished FAS expression. These effects were prevented by HEL co-treatment. In Drosophila larvae fed a HFD, HEL prevented TG accumulation and downregulated proinflammatory JNK pathway activation. CONCLUSION: HEL effect counteracting OA/PA- and HFD-induced lipid accumulation and proinflammatory marker expression in HepG2 hepatocytes and Drosophila larvae may represent a preventive approach against hepatic steatosis and inflammation, associated to obesity and NAFLD.
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Tejido Adiposo/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Extractos Vegetales/farmacología , Triglicéridos/metabolismo , Verbenaceae/química , Animales , Drosophila melanogaster , Cuerpo Adiposo/efectos de los fármacos , Células Hep G2 , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Inflamación/metabolismoRESUMEN
INTRODUCTION AND OBJECTIVES: It is well-known that signaling mediated by the hepatocyte growth factor (HGF) and its receptor c-Met in the liver is involved in the control of cellular redox status and oxidative stress, particularly through its ability to induce hepatoprotective gene expression by activating survival pathways in hepatocytes. It has been reported that HGF can regulate the expression of some members of the NADPH oxidase family in liver cells, particularly the catalytic subunits and p22phox. In the present work we were focused to characterize the mechanism of regulation of p22phox by HGF and its receptor c-Met in primary mouse hepatocytes as a key determinant for cellular redox regulation. MATERIALS AND METHODS: Primary mouse hepatocytes were treated with HGF (50 ng/mL) at different times. cyba expression (gene encoding p22phox) or protein content were addressed by real time RT-PCR, Western blot or immunofluorescence. Protein interactions were explored by immunoprecipitation and FRET analysis. RESULTS: Our results provided mechanistic information supporting the transcriptional repression of cyba induced by HGF in a mechanism dependent of NF-κB activity. We identified a post-translational regulation mechanism directed by p22phox degradation by proteasome 26S, and a second mechanism mediated by p22phox sequestration by c-Met in plasma membrane. CONCLUSION: Our data clearly show that HGF/c-Met exerts regulation of the NADPH oxidase by a wide-range of molecular mechanisms. NADPH oxidase-derived reactive oxygen species regulated by HGF/c-Met represents one of the main mechanisms of signal transduction elicited by this growth factor.
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Grupo Citocromo b/fisiología , Factor de Crecimiento de Hepatocito/fisiología , Hepatocitos/metabolismo , NADPH Oxidasas/fisiología , Proteínas Proto-Oncogénicas c-met/fisiología , Transducción de Señal/fisiología , Animales , Técnicas de Cultivo de Célula , Hepatocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Biosíntesis de Proteínas , Transcripción GenéticaRESUMEN
The aim of the present study was to elucidate how fructose is able to increase the rate of ethanol metabolism in the liver, an observation previously termed the fructose effect. Previous studies suggest that an increase in ATP consumption driven by glucose synthesis from fructose stimulates the oxidation of NADH in the mitochondrial respiratory chain, allowing faster oxidation of ethanol by alcohol dehydrogenase; however, this idea has been frequently challenged. We tested the effects of fructose, sorbose and tagatose both in vitro and in vivo. Both ethanol and each sugar were either added to isolated hepatocytes or injected intraperitoneally in the rat. In the in vitro experiments, samples were taken from the hepatocyte suspension in a time-dependent manner and deproteinized with perchloric acid. In the in vivo experiments, blood samples were taken every 15 min and the metabolites were determined in the plasma. These metabolites include ethanol, glucose, glycerol, sorbitol, lactate, fructose and sorbose. Ethanol oxidation by rat hepatocytes was increased by more than 50% with the addition of fructose. The stimulation was accompanied by increased glucose, glycerol, lactate and sorbitol production. A similar effect was observed with sorbose, while tagatose had no effect. The same pattern was observed in the in vivo experiments. This effect was abolished by inhibiting alcohol dehydrogenase with 4-methylpyrazole, whereas inhibition of the respiratory chain with cyanide did not affect the fructose effect. In conclusion, present results provide evidence that, by reducing glyceraldehyde and glycerol and fructose to sorbitol, respectively, NADH is consumed, allowing an increase in the elimination of ethanol. Hence, this effect is not linked to a stimulation of mitochondrial re-oxidation of NADH driven by ATP consumption.
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Etanol/metabolismo , Fructosa/administración & dosificación , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Tasa de Depuración Metabólica/efectos de los fármacos , Alcohol Deshidrogenasa/antagonistas & inhibidores , Alcohol Deshidrogenasa/metabolismo , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Inyecciones Intraperitoneales , Masculino , Tasa de Depuración Metabólica/fisiología , RatasRESUMEN
Owing to their antioxidant properties, caffeoylquinic acid (CQA)-derivatives could potentially improve the impaired metabolism in hepatic cells, however, their effect on mitochondrial function has not been demonstrated yet. Here, we evaluated the impact of three CQA-derivatives extracted from purple sweet potato, namely 5-CQA, 3,4- and 4,5-diCQA, on mitochondrial activity in primary hepatocytes using an extracellular flux analyzer. Notably, an increase of maximal respiration and spare respiratory capacity were observed when 5-CQA and 3,4-diCQA were added to the system indicating the improved mitochondrial function. Moreover, 3,4-diCQA was shown to considerably increase glycolytic reserve which is a measure of cell capability to respond to an energy demand through glycolysis. Conversely, 4,5-diCQA did not modify mitochondrial activity but increased glycolysis at low concentration in primary hepatocytes. All compounds tested improved cellular capacity to oxidize fatty acids. Overall, our results demonstrated the potential of test CQA-derivatives to modify mitochondrial function in hepatic cells. It is especially relevant in case of dysfunctional mitochondria in hepatocytes linked to hepatic steatosis during obesity, diabetes, and metabolic syndrome.
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Hepatocitos/efectos de los fármacos , Ipomoea batatas/química , Mitocondrias/efectos de los fármacos , Extractos Vegetales/farmacología , Ácido Quínico/análogos & derivados , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Hepatocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Ácido Quínico/química , Ácido Quínico/aislamiento & purificación , Ácido Quínico/farmacologíaRESUMEN
Because the liver plays a major role in metabolic homeostasis and secretion of clotting factors and inflammatory innate immune proteins, there is interest in understanding the mechanisms of hepatic cell activation under hyperglycaemia and whether this can be attenuated pharmacologically. We have previously shown that hyperglycaemia stimulates major changes in chromatin organization and metabolism in hepatocytes, and that the histone deacetylase inhibitor valproic acid (VPA) is able to reverse some of these metabolic changes. In this study, we have used RNA-sequencing (RNA-seq) to investigate how VPA influences gene expression in hepatocytes. Interesting, we observed that VPA attenuates hyperglycaemia-induced activation of complement and coagulation cascade genes. We also observe that many of the gene activation events coincide with changes to histone acetylation at the promoter of these genes indicating that epigenetic regulation is involved in VPA action.
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Coagulación Sanguínea/genética , Proteínas del Sistema Complemento/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hiperglucemia/sangre , Hiperglucemia/genética , Ácido Valproico/farmacología , Coagulación Sanguínea/efectos de los fármacos , Proteínas del Sistema Complemento/efectos de los fármacos , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Histonas/metabolismo , Humanos , Regiones Promotoras Genéticas/genética , Reproducibilidad de los ResultadosRESUMEN
3D Cell culture is an alternative to animal use in many drug development and toxicity studies. The 3D cell culture can mimic and reproduce the original tissue microenvironment, morphology, and mechanical and physiological characteristics, to provide a more realistic and reliable response as compared to two-dimensional cultures. 3D cell culture encapsulated in alginate beads is a very simple and relatively inexpensive tool that is easy to handle and to maintain. The alginate beads function as a scaffold that imprisons cells and allows 3D cell growth, to generate spheroids that can have greater genic expression and cell-cell communication as a nano or microtissue. The HepG2 cell line is a human hepatocellular carcinoma cell derivative. HepG2 cells preserve several of the characteristics of hepatocytes and are therefore often used in toxicity studies. Here, we describe HepG2 cell encapsulation in alginate beads and analyze the resulting spheroids formed within the alginate beads by immunocytochemistry, by staining a certain structure with a specific antibody coupled with a fluorophore. This method preserves the beads and enables cell analysis by confocal microscopy.
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Alginatos/química , Técnicas de Cultivo de Célula , Técnica del Anticuerpo Fluorescente , Hepatocitos/efectos de los fármacos , Microscopía Confocal , Pruebas de Toxicidad , Biomarcadores/metabolismo , Células Hep G2 , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Esferoides CelularesRESUMEN
BACKGROUND: Brazilian flora is rich in plants with medicinal properties, which though popular, has contributed to the development of a range of phytotherapic products that use plants to treat and cure diseases. However, studies that use Brazilian plants in the treatment of metabolic disorders are still scarce in the literature. OBJECTIVE: The aim of this study was to analyze the effect of hepatotoxicity Lafoensia pacari on the metabolism of mice with obesity induced by a high-fat diet and to verify the phytochemical difference between the Lafoensia pacari bark of the trunk, leaves, and branches. METHODS: The plant material was collected from April to May in the municipality of Bonito de Minas, MG, Brazil. Qualitative tests for the presence of secondary metabolite classes were performed for leaves, branches and bark of the trunk. Through histological analysis, we evaluated hepatocytes and cell lesions in the liver. RESULTS: The comparative phytochemical analysis of the plant did not reveal alterations between the different plant parts. The phytochemical test showed that is preferable to use the leaves to make the extract to be applied, aiming to reduce the plant aggression. After treatment, greater changes were observed in the animals that received the high-fat diet and the hydroethanolic extract; the levels of AST, ALT, albumin and creatinine that were increased, thus demonstrating a possible toxicity. There were no significant differences in body weight. In the histological analysis, the animals without plant treatment displayed decreased liver weight and reduction in the inflammatory infiltrate. CONCLUSION: We conclude that Lafoensia pacari should be better evaluated for oral consumption and may cause liver damage.