Protective effect of linoleic acid on liver toxicity induced by methotrexate / Efecto protector del ácido linoleico sobre la toxicidad hepática inducida por metotrexato

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.

Efecto del extracto hidroalcohólico de la planta Lampaya medicinalis Phil. (Verbenaceae) sobre la esteatosis inducida in vitro en hepatocitos humanos / Effect of the hydroalcoholic extract of the plant Lampaya medicinalis Phil. (Verbenaceae) on in vitro-induced steatosis in human hepatocytes

INTRODUCCIÓN: La enfermedad del hígado graso no alcohólico (EHGNA) es la forma más común de enfermedad hepática. A nivel celular se caracteriza por la acumulación de triglicéridos (TG) en forma de gotas lipídicas (GL) dando lugar a esteatosis e inflamación. Entre los factores relevantes para la síntesis de TG se encuentran las enzimas DGAT1/2 que catalizan la etapa final de la síntesis de TG, y la proteína FABP4 que transporta lípidos intracelulares y se expresa en modelos de enfermedad hepática dependiente de obesidad. Por otra parte, TNF-α es una reconocida citoquina involucrada en el proceso inflamatorio en la EHGNA. La medicina popular del norte de Chile ha utilizado la planta Lampaya medicinalis Phil. (Verbenaceae) para el tratamiento de algunas enfermedades inflamatorias. OBJETIVO: Evaluar el efecto de un extracto hidroalcóholico de lampaya (EHL) sobre la esteatosis y expresión de marcadores de inflamación en hepatocitos tratados con ácidos grasos. Diseño experimental: Estudio in vitro en cultivos de la línea celular humana HepG2 tratadas con ácido oleico (AO) y ácido palmítico (AP). MÉTODOS: Se incubó hepatocitos HepG2 con AO/AP por 24 horas en presencia o no de EHL. Se evaluó la presencia de GL y el contenido de TG intracelulares por Oil Red O y Nile Red, respectivamente. La expresión de DGAT1/2, FABP4 y TNF-α fue evaluada por qPCR. RESULTADOS: Los hepatocitos tratados con AO/AP mostraron un aumento en las GL y TG, así como una mayor expresión de DGAT2 en comparación al control. El cotratamiento con EHL revirtió los efectos inducidos por AO/AP. CONCLUSIONES: EHL revierte el incremento en las GL, TG y en la expresión de DGAT2 inducido por AO/AP en células HepG2. Estos hallazgos sugieren un efecto hepatoprotector de la Lampaya contra la esteatosis, y apoyarían su uso complementario en el tratamiento de patologías con componente inflamatorio como la EHGNA.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease. At the cellular level, it is characterized by the accumulation of triglycerides (TG) in the form of lipid droplets (LD), which leads to steatosis and inflammation. Among relevant factors for TG synthesis are the enzymes DGAT1/2 catalyzing the final stage of TG synthesis, and the protein FABP4 which transports intracellular lipids and is expressed in cell models of obesity-dependent liver disease. Additionally, TNF-α is a cytokine involved in the inflammatory process associated to NAFDL. Lampaya medicinalis Phil. (Verbenaceae) is a plant used in folk medicine in northern Chile to treat some inflammatory diseases. OBJECTIVE: To evaluate the effect of the hydroalcoholic extract of lampaya (HEL) on steatosis and the expression of inflammatory markers in hepatocytes treated with fatty acids. Study design: In vitro study in cultures of the human HepG2 cell line treated with oleic acid (OA) and palmitic acid (PA). METHODS: HepG2 hepatocytes were incubated with OA/PA for 24 hours in the presence and absence of HEL. The formation of LD and the accumulation of intracellular TG were assessed by Oil Red O and Nile Red, respectively. The expression of DGAT1/2, FABP4 and TNF-α was assessed by qPCR. RESULTS: The treatment with OA/PA increased the levels of LD and TG as well as the expression of DGAT2 in HepG2 hepatocytes compared to control cells. HEL cotreatment counteracted OA/PA-induced effects. CONCLUSIONS: HEL prevents the increase in LD and TG levels and DGAT2 expression induced by OA/PA in HepG2 cells. These findings suggest that lampaya may have a protective effect against hepatic steatosis, which would support its complementary use in the treatment of pathologies associated with inflammation, such as NAFLD.

Hepatopatia esteroidal em gatas após terapia com prednisolona: aspectos laboratoriais, tomográficos e histopatológicos / [Steroid hepatopathy in cats after prednisolone use: laboratory, tomographic, and histopathologic aspects]

Glicocorticoides são amplamente utilizados na clínica de pequenos animais, entretanto seu uso contínuo pode causar efeitos colaterais. Os gatos são considerados menos susceptíveis a esses efeitos do que outras espécies, mas existem poucos trabalhos abordando os efeitos adversos em felinos. O objetivo deste estudo foi avaliar possíveis alterações laboratoriais, histopatológicas e do grau de atenuação radiográfica do parênquima hepático de gatas submetidas à terapia com prednisolona. Um ensaio clínico foi realizado em quatro gatas hígidas, as quais receberam prednisolona, por via oral, na dose de 3mg/kg, durante 60 dias consecutivos. Nos achados histopatológicos após 60 dias de tratamento, observou-se desorganização dos cordões de hepatócitos e degeneração vacuolar, além de necrose de hepatócitos, porém não foram observados sinais de fibrose no parênquima hepático. Os dados da tomografia computadorizada demonstram aumento do grau de atenuação do parênquima hepático a partir do 30º dia da administração de prednisolona, que persistiu até o final do experimento. No presente estudo, foi possível caracterizar a existência de hepatopatia esteroidal em gatos em estágios precoces da terapia com prednisolona.(AU)

Glucocorticoids are widely used medications in small animal practice; however, its continuous use can have side effects. Cats are considered less susceptible than other species, however, the literature does not usually address adverse effects in felines. The objective of this study was to evaluate possible laboratory and histopathologic changes, as well as changes to the degree of radiographic attenuation of the hepatic parenchyma in cats treated with prednisolone. A clinical trial was done in four healthy cats, who received prednisolone orally at 3mg/kg during 60 consecutive days. In the histopathologic findings at 60 days of treatment, there were disorganized hepatocyte chords and vacuolar degeneration, as well as hepatocyte necrosis, however, there were no signs of fibrosis in the hepatic parenchyma. Data obtained via computed tomography showed increase of the degree of attenuation in the hepatic parenchyma from day 30 of prednisolone therapy, which persisted until the end of the experiment. In the present study, it was possible to characterize the existence of steroidal hepathopathy in cats in the early stages of prednisolone therapy.(AU)

Vitamin C administration attenuated artemether induced hepatic injury in rats / La aAdministración de vitamina C atenúa la lesión hepática inducida por artemeter en ratas

This research was designed to investigate the potential protective effect of vitamin C supplementation against hepatocyte ultrastructural alterations induced by artemether (antimalarial drug) administration. Twenty-four adult male albino rats were used in this study and were divided into four groups (n=6). Group I served as a control and rats in group II administrated artemether (4 mg/kg B.W) orally for three consecutive days. Group III administered artemether plus a low dose of vitamin C (2.86 mg/kg/l water) while group IV received artemether plusa high dose of vitamin C (8.56 mg/kg). At the end of the experimental period (14 days), the harvested liver tissues were examined by transmission electron microscopy (TEM), and blood samples were assayed for biomarkers of liver injury and oxidative stress. Artemether significantly (p<0.05) augmented biomarkers of liver injury such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and oxidative stress such as superoxide dismutase (SOD), Glutathione Peroxidase (GPX), and caused degeneration and damage of the rough endoplasmic reticulum and disrupted mitochondria. The blood sinusoids were also damaged with distortion of their canaliculi. Administration of vitamin C showed improvement of liver biomarkers, and liver parenchyma, especially in a high dose of vitamin C.We concludes that vitamin C is a partial protective agent against artemether-induced liver injury.

Esta investigación fue diseñada para investigar el posible efecto protector de la vitamina C contra las alteraciones ultraestructurales de los hepatocitos, inducidas por la administración de arteméter (medicamento antipalúdico). En el estudio se utilizaron 24 ratas albinas macho adultas y se dividieron en cuatro grupos (n = 6). El grupo I fue designado como control y las ratas en el grupo II se adminstró Arteméter (4 mg / kg de peso corporal) por vía oral durante tres días consecutivos. En el grupo III se administró arteméter, además de una dosis baja de vitamina C (2,86 mg / kg / l de agua) mientras que el grupo IV recibió arteméter más una dosis alta de vitamina C (8,56 mg / kg). Al final del período experimental (14 días), los tejidos hepáticos recolectados se examinaron por microscopía electrónica de transmisión (MET), y las muestras de sangre se analizaron en busca de biomarcadores de daño hepático y estrés oxidativo. El arteméter aumentó significativamente (p <0,05) los biomarcadores de daño hepático como alanina aminotransferasa (ALT), aspartato aminotransferasa (AST) y estrés oxidativo como superóxido dismutasa (SOD), glutatión peroxidasa (GPX) y causó degeneración y daño de la retículo endoplásmico rugoso y mitocondrias alteradas. Los sinusoides sanguíneos también fueron dañados con la distorsión de sus canalículos. La administración de vitamina C mostró una mejoría de los biomarcadores hepáticos y el parénquima hepático, especialmente en una dosis alta de vitamina C. Concluimos que la vitamina C es un agente protector parcial contra la lesión hepática inducida por arteméter.

Possible protective effect of olive leaves extract on paracetamol induced hepatotoxicity in male albino rats / Possível efeito protetor do extrato de folhas de oliveira na hepatotoxicidade induzida por paracetamol em ratos albinos machos

Paracetamol (PCM) overdose can cause hepatotoxicity with oxidative stress; the present study was carried out to establish the possible protective effect of olive leaves extract (OLE) on toxicity induced by paracetamol in adult male rats. Twenty four adult male rats were divided into four equal groups; control, olive leaves extract group, paracetamol group and olive leaves extract plus paracetamol group. Some biochemical parameters and liver histopathology were evaluated. PCM treatment significantly increased serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, gamma-glutamyltransferase (GGT), lactate dehydrogenase (LDH), urea, creatinine and alpha-fetoprotein. Paracetamol was found to significantly increase malonaldehyde (MDA) and decrease glutathione reductase (GR) activity in tissue and significantly decrease total antioxidant capacity (TAC) and superoxide dismutase (SOD) in serum. Administration of OLE caused a significant decrease serum AST, ALT enzyme, total bilirubin, GGT, LDH, creatinine, urea, alpha-fetoprotein. Also, amelioration of oxidant ­ antioxidant status with olive leaves extract was observed in addition to a significant decrease in MDA and a significant increase in TAC in liver tissue with a significant increase in glutathione reductase (GR) and SOD in serum compared to paracetamol treated group The chemical pathological changes were in step with histopathological observation suggesting marked hepatoprotective result of olive leaves extract. It could be concluded that olive leaves extract (OLE) treatment may be effective in decreasing hepatic injury and oxidative stress induced by paracetamol overdose in male albino rats

A sobredosagem de paracetamol (PCM) pode causar hepatotoxicidade com estresse oxidativo; o presente estudo foi realizado para estabelecer o possível efeito protetor do extrato de folhas de oliveira (OLE) na toxicidade induzida pelo paracetamol em ratos machos adultos. Vinte e quatro ratos machos adultos foram divididos em quatro grupos iguais: controle, grupo extrato de folhas de oliveira, grupo paracetamol e extrato de folhas de oliveira mais grupo paracetamol. Alguns parâmetros bioquímicos e histopatologia hepática foram avaliados. O tratamento com PCM aumentou significativamente aspartato aminotransferase sérica (AST), alanina aminotransferase (ALT), bilirrubina total, gama-glutamiltransferase (GGT), lactato desidrogenase (LDH), uréia, creatinina e alfa-fetoproteína. Verificou-se que o paracetamol aumenta significativamente o malonaldeído (MDA) e diminui a atividade da glutationa redutase (GR) no tecido e diminui significativamente a capacidade antioxidante total (TAC) e a superóxido dismutase (SOD) no soro. A administração de OLE causou uma diminuição significativa de AST, enzima ALT, bilirrubina total, GGT, LDH, creatinina, uréia, alfa-fetoproteína. Também foi observada melhora do status oxidante - antioxidante com extrato de folhas de oliveira, além de uma diminuição significativa no MDA e um aumento significativo no TAC no tecido hepático, com um aumento significativo na glutationa redutase (GR) e SOD no soro em comparação ao grupo tratado com paracetamol. As alterações patológicas químicas acompanharam a observação histopatológica, sugerindo resultado hepatoprotetor acentuado do extrato de folhas de oliveira. Pode-se concluir que o tratamento com extrato de folhas de oliveira (OLE) pode ser eficaz na diminuição da lesão hepática e do estresse oxidativo induzido pela overdose de paracetamol em ratos albinos machos

Pretreatment of hepatetctomized rats with Coleus forskohlii did not interfere with the course of hepatic hyperplasia

Abstract Purpose Coleus forskohlii Briq., a medicinal plant originally from India, has been indicated against heart disease, expiratory disorders, convulsions, and hepatic changes, among others. In view of the broad pharmacological potential of the plant and the scarce information about its effects, the objective of the present study was to investigate the effect of its use for pretreatment of partially hepatectomized rats. Methods The animals were divided into two experimental groups: Control (CG) receiving physiological saline for 10 days before partial hepatetctomy, and Treated (TG) receiving 40 mg Coleus forskohlii/kg/day for 10 days before partial hepatectomy. The treatments were performed by gastric gavage. After the surgical procedure, treatment was continued according to the following groups: CG 24 h, CG 48 h, TG 24 h, and TG 48 hs, and liver tissue and intracardiac blood samples were obtained for histological and biochemical analysis, respectively. Results No significant differences were observed in mitotic or apoptotic index or in the concentrations of the enzymes AST, ALT and alkaline phosphatase, and no areas of fibrosis were detected. Conclusion Treatment with Coleus forskohlii did not interfere with the course of hepatic hyperplasia.

Vitamin E protects against hepatocyte ultrastructural damage induced by high fat diet in a rat model of pre-diabetes / La vitamina E protege contra el daño ultraestructural de los hepatocitos inducido por la dieta alta en grasas en un modelo de pre-diabetes en ratas

SUMMARY: We sought to investigate the potential protective effect of Vitamin E supplementation against hepatocyte ultrastructural alterations induced by high fat diet (HFD) in a rat model of pre-diabetes. Therefore, rats were either fed with HFD (model group) or a standard laboratory chow (control group) for 12 weeks before being sacrificed. The protective group fed on a HFD and started the treatment with vitamin E (100 mg/kg/day, i.p) from day 1 until being sacrificed at week 12. The harvested liver tissues were examined using transmission electron microscopy (TEM) and blood samples were assayed for biomarkers of liver injury and prediabetes. TEM images showed that HFD induced profound pathological changes to the hepatocyte ultrastructure as demonstrated by degenerated hepatocytes with damaged cytoplasm that have mitochondrial swelling, dilation of endoplasmic reticulum, blebbing of plasma membranes, and cytoplasmic accumulations of lipid droplets and vacuoles, which were substantially but not completely protected with vitamin E. In addition, HFD significantly (p<0.05) augmented biomarkers of liver injury and pre-diabetes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-alpha (TNF-α), malondialdehyde (MDA), total cholesterol (TC), triglycerides (TG), and low density lipoprotein cholesterol (LDL-C), which were significantly (p<0.05) reduced with vitamin E except TNF-α and TC. Furthermore, none of these biomarkers were reduced to the control level by vitamin E. We conclude that vitamin E is a partial protective agent against HFD-induced liver injury and pre-diabetes.

RESUMEN: El objetivo de este estudio fue investigar el posible efecto protector de la administración de suplementos de vitamina E contra las alteraciones ultraestructurales de los hepatocitos inducidas por una dieta rica en grasas (DRG) en un modelo de prediabetes en ratas. Antes de ser sacrificadas las ratas fueron alimentadas con DRG (grupo modelo) o un alimento estándar de laboratorio (grupo control) durante 12 semanas. El grupo protector se alimentó con una DRG y comenzó el tratamiento con vitamina E (100 mg/kg/día, i.p) desde el día 1 hasta sacrificarlo en la semana 12. Los tejidos hepáticos recolectados se examinaron mediante microscopía electrónica de transmisión (MET) y se tomaron muestras de sangre y se analizaron los biomarcadores de daño hepático y prediabetes. Las imágenes de MET mostraron que el DRG indujo cambios patológicos profundos en la ultraestructura de los hepatocitos, como lo demuestran los hepatocitos degenerados con citoplasma dañado e hinchazón mitocondrial, dilatación del retículo endoplasmático, formación de ampollas en las membranas plasmáticas y acumulaciones citoplásmicas de gotas de lípidos y vacuolas, los que fueron sustancialmente protegidas con vitamina E. Además, DRG aumentó significativamente (p <0,05) los biomarcadores de daño hepático y prediabetes como alanina aminotransferasa (ALT), aspartato aminotransferasa (AST), factor de necrosis tumoral alfa (TNF-α), malondialdehído (MDA), colesterol total (CT), triglicéridos (TG) y lipoproteína de colesterol de baja densidad (LDL-C), la cual se redujo significativamente (p <0,05) con vitamina E, excepto TNF-α y CT. Ninguno de estos biomarcadores se redujo al nivel de control por la vitamina E. Concluimos que la vitamina E es un agente protector parcial contra la lesión hepática inducida por DRG y la prediabetes.

Melatonin ameliorates H2O2-induced oxidative stress through modulation of Erk/Akt/NFkB pathway

BACKGROUND: Improper control on reactive oxygen species (ROS) elimination process and formation of free radicals causes tissue dysfunction. Pineal hormone melatonin is considered a potent regulator of such oxidative damage in different vertebrates. Aim of the current communication is to evaluate the levels of oxidative stress and ROS induced damage, and amelioration of oxidative status through melatonin induced activation of signaling pathways. Hepatocytes were isolated from adult Labeo rohita and exposed to H2O2 at three different doses (12.5, 25 and 50 µM) to observe peroxide induced damage in fish hepatocytes. Melatonin (25, 50 and 100 µg/ml) was administered against the highest dose of H2O2. Enzymatic and non-enzymatic antioxidants such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) was measured spectrophotometrically. Expression level of heat shock proteins (HSP70 and HSP90), HSPs-associated signaling molecules (Akt, ERK, cytosolic and nuclear NFkB), and melatonin receptor was also measured by western blotting analysis. RESULTS: H2O2 induced oxidative stress significantly altered (P < 0.05) MDA and GSH level, SOD and CAT activity, and up regulated HSP70 and HSP90 expression in carp hepatocytes. Signaling proteins exhibited differential modulation as revealed from their expression patterns in H2O2-exposed fish hepatocytes, in comparison with control hepatocytes. Melatonin treatment of H2O2-stressed fish hepatocytes restored basal cellular oxidative status in a dose dependent manner. Melatonin was observed to be inducer of signaling process by modulation of signaling molecules and melatonin receptor. CONCLUSIONS: The results suggest that exogenous melatonin at the concentration of 100 µg/ml is required to improve oxidative status of the H2O2-stressed fish hepatocytes. In H2O2 exposed hepatocytes, melatonin modulates expression of HSP70 and HSP90 that enable the hepatocytes to become stress tolerant and survive by altering the actions of ERK, Akt, cytosolic and nuclear NFkB in the signal transduction pathways. Study also confirms that melatonin could act through melatonin receptor coupled to ERK/Akt signaling pathways. This understanding of the mechanism by which melatonin regulates oxidative status in the stressed hepatocytes may initiate the development of novel strategies for hepatic disease therapy in future.

Differential therapeutic effects of Crataegus aronia and simvastatin on the hepatocyte ultrastructure in hepatic steatosis / Efectos terapéuticos diferenciales de Crataegus aronia y simvastatina sobre la estructura del hepatocito en la esteatosis hepática

Complications of fat accumulation in liver, hepatic steatosis such as liver cirrhosis and liver failure are among the common public health problems. We sought to investigate the damage to the hepatocyte ultrastructure induced by high fat diets (HFD) and compared the therapeutic effects at the cellular level of two antioxidant and lipid lowering agents; Crataegus aronia extracts and simvastatin on hepatic steatosis. Rats were either fed with HFD (model group) or low fat diets (LFD) (control group) for 15 weeks before being sacrificed and therapeutic groups started the treatment with these agents after week 11 until the sacrifice day. Harvested liver tissues were examined using transmission electron microscopy (TEM) and liver homogenates were assayed for markers of anti-oxidative stress that are known to be modulated in liver injury. TEM examinations of the model group showed a profound damage to the hepatocytes compared to the control group as demonstrated by steatosis, damaged mitochondria and vaculated cytoplasm, disrupted rough and smooth endoplasmic reticulum and nuclear membrane, dilated intercellular space between hepatocytes, and alterations in lysosomes. In addition, HFD ameliorated the anti-oxidant glutathione (GSH) and augmented the oxidative stress TBARS biomarkers. Both Crataegus aronia and simvastatin significantly reduced lipids and TBARS, and treated damage to hepatic cells, but hepatocyte structures were differentially responded to these agents. However, only Crataegus aronia induced GSH (p=0.001). We conclude that HFD-induced hepatic steatosis caused a substantial damage to the hepatocyte's ultrastructures, and Crataegus aronia and simvastatin treatments differentially reversed hepatic injuries.

Las complicaciones de la acumulación de grasa en el hígado, la esteatosis hepática como la cirrosis hepática y la insuficiencia hepática se encuentran entre los problemas comunes de salud pública. Se intentó investigar el daño a la ultraestructura de los hepatocitos inducido por la dieta alta en grasas (DAG) y se compararon los efectos terapéuticos a nivel celular de dos antioxidantes y agentes hipolipemiantes; Extracto de Crataegus aronia y simvastatina sobre esteatosis hepática. Las ratas fueron alimentadas con DAG (grupo modelo) o dieta baja en grasa (DBG) (grupo control) durante 15 semanas antes de sacrificarse y los grupos terapéuticos comenzaron el tratamiento con estos agentes después de la semana 11 hasta el día del sacrificio. Se examinaron los tejidos hepáticos usando microscopía electrónica de transmisión (MET) y se analizaron homogeneizados de hígado para marcadores de estrés anti-oxidativo, que se sabe están modulados en la lesión hepática. Los exámenes MET del grupo DAG mostraron un grave daño de los hepatocitos en comparación con el grupo control, demostrado por esteatosis, daño mitocondrial y citoplasma vacío, retículo endoplásmico rugoso y liso y membrana nuclear, el espacio intercelular dilatado entre hepatocitos y alteraciones en los lisosomas. Además, DAG mejoró el anti-oxidante glutatión (GSH) y aumentó el estrés oxidativo TBARS biomarcadores. Tanto Crataegus aronia como simvastatina redujeron significativamente los lípidos y TBARS, trataron el daño a las células hepáticas, pero las estructuras de hepatocitos respondieron diferencialmente a estos agentes. Sin embargo, sólo Crataegus aronia indujo GSH (p = 0,001). Concluimos que la esteatosis hepática inducida por HFD causó un daño sustancial a la ultraestructura del hepatocito y los tratamientos de Crataegus aronia y simvastatina diferenciaron las lesiones hepáticas.

Hepatic toxicity caused by PLGA-microspheres containing usnic acid from the lichen C ladonia substellata (AHTI) during pregnancy in Wistar rats

ABSTRACT This study aimed to evaluate the teratogenic and hepatotoxic potential of the usnic acid encapsulated into PLGA-microspheres. In total, 12 female Wistar rats in pregnancy were randomly distributed in the control group (n= 6) that received 1.0 mL of physiological solution and treatment group (n= 6) that received 25 mg/kg of encapsulated usnic acid by oral administration. All females were euthanized at day 20 of pregnancy and their fetuses were removed and analyzed. During the pregnancy was observed a reduction in weight gain. There was no difference in serum transaminases levels analyzed as well as any difference in liver weight in both groups. The histomorphometric analysis of the liver from the treatment group revealed an increase in number of hepatocytes and a decrease in nuclear area of these cells. Moreover, no alteration was observed in cell area of hepatocytes or number of Kupffer cells. The fetuses had an increase in total number of hepatocytes and a reduction in the amount of megakaryocytes. These results show the hepatotoxic potential of usnic acid during pregnancy. However, its toxicity can be minimized by encapsulation in microspheres.

Management Strategies for Liver Fibrosis

Abstract: Liver fibrosis resulting from chronic liver injury are major causes of morbidity and mortality worldwide. Among causes of hepatic fibrosis, viral infection is most common (hepatitis B and C). In addition, obesity rates worldwide have accelerated the risk of liver injury due to nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Also liver fibrosis is associated with the consumption of alcohol, or autoimmune hepatitis and chronic cholangiophaties. The response of hepatocytes to inflammation plays a decisive role in the physiopathology of hepatic fibrosis, which involves the recruitment of both pro- and anti-inflammatory cells such as monocytes and macrophages. As well as the production of other cytokines and chemokines, which increase the stimulus of hepatic stellate cells by activating proinflammatory cells. The aim of this review is to identify the therapeutic options available for the treatment of the liver fibrosis, enabling the prevention of progression when is detected in time.

Effect of cigarette smoking on the structure of hepatocytes: TEM study / Efecto del tabaquismo sobre la estructura de los hepatocitos: estudio MET

The liver is one of the major organs that is indirectly affected by cigarette smoke. The aim of this project is to define the histologic and ultrastructural changes in normal liver cells after exposing animals to cigarette smoke. Thirty albino rats were exposed to cigarette smoke for 90 days, followed by morphologic examination of their livers under light microscope and electron microscope. The liver cells of cigarette smoke exposed rats showed mild swelling with increased eosinophilia. Ultrastructural examination of these cells demonstrated cytoplasm with highly proliferated and crowded mitochondria. There were many electron dense mitochondria. These mitochondria were pleomorphic in shape compared to mitochondrias of control rats. Also, loss of mitochondrial cristae and widening of the intermembranous space was noticed. It is concluded that smoking exerts cellular damage and oxidative stress on normal liver cells resulting in ultrastructural changes.

El hígado es uno de los principales órganos indirectamente afectado por el humo del cigarrillo. El objetivo de este trabajo fue definir los cambios histológicos y ultraestructurales de las células normales del hígado después de exponer a los animales al humo del cigarrillo. Treinta ratas albinas fueron expuestas al humo de cigarrillo durante 90 días, seguido de un examen morfológico de los hígados bajo microscopio de luz y microscopio electrónico. Las células hepáticas de las ratas expuestas al humo de cigarrillo mostraron una leve inflamación con un aumento de la eosinofilia. En el examen ultraestructural de estas células se observó el citoplasma mitocondrial altamente proliferado y saturado. Se observó gran cantidad de mitocondrias electrón-densas y éstas presentaban forma pleomórfica en comparación con las mitocondrias del grupo control. Además, se observó pérdida de las crestas mitocondriales y ensanchamiento del espacio intermembranoso. Se concluye que el tabaquismo ejerce daño celular y estrés oxidativo en las células hepáticas normales, lo que resulta en la aparición de cambios ultraestructurales.

Comparative immunohistochemical assessment of the effect of repetitive anesthesia with isoflurane and sevoflurane on rat liver / Avaliação imunohistoquímica comparativa do efeito da anestesia repetitiva com isoflurano e sevoflurano sobre o fígado de rato

Abstract Background and objectives: Inhalation anesthetics are used in human, as well as veterinary medical practice. In the present study we investigated the effect of isoflurane and sevoflurane on rat hepatocytes. Methods: A total of 40 Wistar female rats were used in this study. Animals were divided in groups of 5 rats. Groups IM, SM served as control groups. Groups I1, I2, I3 were used to study isoflurane and S1, S2, S3 for sevoflurane study. They were anesthetized 3 times, for 2 h long, at 2 days interval with a concentration of: 1.5% isoflurane (I1, I2, I3) and 2% sevoflurane (S1, S2, S3). The oxygen supply throughout the anesthesia was 1 L O2/min. Groups IM, IS, I1, S1 were sacrificed immediately after the last anesthesia. Groups I2, S2 were sacrificed 6 h after the last anesthesia, and groups I3, S3, 24 h post-anesthesia. Liver samples were harvested to highlight caspase-3 in apoptotic hepatocytes. Results: Following isoflurane administration, there were less than 1% cells in apoptosis highlighted in rat livers from groups IM, I1 and I2. At 24 h post-anesthesia (group I3), a small number of apoptotic hepatocytes was highlighted (around 3.23% cells in apoptosis), with a strictly periacinar disposition, randomly distributed in a small number of hepatic lobules. After sevoflurane administration, less than 1% apoptotic hepatocytes were identified at all control moments throughout the study. Conclusions: The results suggest that the anesthetics do not present a considerable hepatotoxicity. The comparative assessment of the two anesthetics shows that sevoflurane is superior to isoflurane.

Resumo Justificativa e objetivos: Anestésicos inalatórios são usados em humanos e também na prática médica veterinária. No presente estudo investigamos o efeito de isoflurano e sevoflurano em hepatócitos de rato. Métodos: Foram usados neste estudo 40 ratos Wistar fêmeas. Os animais foram divididos em grupos de cinco. Os grupos IM e SM serviram como controle. Os grupos I1, I2 e I3 foram usados para o estudo de isoflurano e os grupos S1, S2 e S3 para o estudo de sevoflurano. Os ratos foram anestesiados três vezes, durante duas horas em intervalos de dois dias, com uma concentração de 1,5% de isoflurano (I1, I2, I3) e 2% de sevoflurano (S1, S2, S3). O fornecimento de oxigênio durante a anestesia foi de 1 L O2/min. Os grupos IM, IS, I1 e S1 foram sacrificados imediatamente após a última anestesia. Os grupos I2 e S2 foram sacrificados seis horas após a última anestesia e os grupos I3 e S3 foram sacrificados 24 horas após a anestesia. Amostras dos fígados foram colhidas para ressaltar a caspase-3 em hepatócitos apoptóticos. Resultados: Após a administração de isoflurano, havia menos de 1% das células em apoptose em destaque nos fígados dos ratos dos grupos IM, I1 e I2. Às 24 horas após a anestesia (grupo I3), um pequeno número de hepatócitos apoptóticos foi destacado (3,23% de células em apoptose), com uma disposição estritamente periacinar, distribuídos aleatoriamente em um pequeno número de lóbulos hepáticos. Após a administração do sevoflurano, menos de 1% de hepatócitos apoptóticos foi identificado em todos os momentos de controle ao longo do estudo. Conclusões: Os resultados sugerem que os anestésicos não apresentam uma hepatotoxicidade considerável. A avaliação comparativa dos dois anestésicos mostra que sevoflurano é superior ao isoflurano.

The effect of the aqueous extract Peumus boldus on the proliferation of hepatocytes and liver function in rats submitted to expanded hepatectomy

ABSTRACT PURPOSE: To assess the effect of aqueous extract of Peumus Boldus (AEPB) on the liver proliferative response after parcial hepatectomy of 70% (PH) in rodents. METHODS: Twenty Wistar rats were divided in two groups: AEPB100 (whose rats received 100mg/Kg of AEPB, once a day, orally, in 4 days prior to the first surgical procedure) and Vehicle (whose rats were treated similarly with distilled water). Both groups underwent PH. After 24 hours the remaining livers were removed for studying the proliferation of hepatocytes by Ki-67 and 2mL of blood were collected for serological assessment: cholesterol, glucose, triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and total, direct and indirect bilirubin. All data were analyzed by Gaussian distribution. Statistically significant differences between mean values were analyzed using T Student's test. Non-Gaussian data were analyzed using Mann-Whitney's test. RESULTS: The liver of all these rats presented positive staining of Ki-67, indicating liver proliferation. Laboratory results showed no significant difference in serum values between the analyzed groups. The analysis of Ki-67 was significantly more positive in AEPB100 group than in Vehicle group. CONCLUSION: Aqueous extract of Peumus Boldus acute administration exerts significant positive effect on liver regeneration after 24h in rats that underwent parcial hepatectomy, while maintaining unchanged hepatic function.

Time-course changes in the expression levels of miR-122, -155, and -21 as markers of liver cell damage, inflammation, and regeneration in acetaminophen-induced liver injury in rats

Drug-induced liver injury (DILI) is a significant threat to patient health and a major concern during drug development. Recently, multiple circulating microRNAs (miRNAs) have been reported to be potential biomarkers for DILI. To adapt and validate miRNAs for clinical use, we investigated the time-course changes in miR-122 expression levels in an acetaminophen-induced liver injury model in rats. In addition, miR-155 and miR-21 were evaluated as makers of inflammation and regeneration, respectively, to characterize liver status. Our results revealed that miR-122 is an early and sensitive biomarker of hepatocellular injury at a stage when alanine transaminase, aspartate transaminase, and total bilirubin were not detectable. However, no significant differences in the expression levels of other miRNAs (miR-155 and -21) were observed between treatment and vehicle groups. Collectively, these time-course changes in the expression levels of miRNAs may be useful as markers for clinical decision-making, in the diagnosis and treatment of DILI.

Ultrastructure of fetal mice hepatocytes exposed in utero to diazepam / Ultraestructura de hepatocitos fetales de ratón expuestos in utero a diazepam

Previous research has shown that fetal mice hepatic cells from females treated with diazepam (Valium) during pregnancy depict cytoplasmic and nuclear modifications when observed with photonic microscope. The purpose of this work is to investigate if diazepam administered subcutaneously (SC) to pregnant mice females induces ultraestructural alterations in the cytoplasmic organelles and nucleus to fetal hepatocytes. Transmission electron microscopy observations of fetal hepatocytes from pregnant females treated with a single daily dose of diazepam 2.7 mg/kg/bw/SC administered from 6th to 15th days of gestation revealed that they frequently presented disorganized and dilated rough endoplasmic reticulum cisterns, membranous elements, abundant Golgi complex and glycogen granules, around large vacuoles. The voluminous nucleus shows atypical distribution of chromatin. These alterations could modify the hepatocyte's physiology and probably persist after birth.

Estudios previos muestran que las células hepáticas de fetos de ratón, de hembras tratadas con diazepam (Valium) durante la gestación, presentan modificaciones citoplásmicas y nucleares que se pueden observar con el microscopio fotónico, por lo que el propósito de este trabajo es determinar si el diazepam administrado por vía subcutánea (SC) a hembras gestantes de ratón, induce alteraciones ultraestructurales de los organelos citoplásmicos y del núcleo de los hepatocitos fetales. En los fetos de ratón del grupo experimental de hembras gestantes, tratadas con dosis únicas diarias de 2,7 mg/kg de peso corporal administradas por vía SC del 6° al 15° día de la gestación, se observó con el microscopio electrónico de transmisión que los hepatocitos fetales presentaban con frecuencia retículo endoplásmico rugoso desorganizado, con cisternas dilatadas; había elementos membranosos y complejo de Golgi abundante, al igual que gránulos de glucógeno que rodeaban a grandes vacuolas. Los núcleos eran voluminosos, con la cromatina distribuida atípicamente. Estas alteraciones podrían modificar la fisiología de los hepatocitos y probablemente persistan después del nacimiento.

Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes

Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H2O2)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H2O2 in a dose-dependent manner. Additionally, H2O2 treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H2O2 significantly attenuated the H2O2-induced decrease of cell viability. H2O2 exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H2O2-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H2O2-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H2O2-induced apoptosis by inhibiting ROS generation.

Efecto del ácido alfa lipoico y la pirfenidona en la modulación antioxidante celular contra el daño oxidativo / Effects of alpha lipoic acid and pirfenidone on liver cells antioxidant modulation against oxidative damage

Background: Liver fibrogenic processes are related to cellular redox state. Glutathione (GSH) is the major cellular antioxidant. GSH induced activation could be related to antifibrogenic effects. Aim: To explore the association between the antifibrogenic effect and pro-antioxidant mechanisms of alpha-lipoic acid (ALA) and pirfenidone (PFD). Material and Methods: HepG2 cells and primary HSC cultures were exposed to menadione 0.1 μM (MEN) as oxidative stress inducer and treated to ALA (5 mM) or PFD (10 μM, 100 μM y 1000 μM). Results: In HSC, PFD decreased cell proliferation and the expression of COL1A1, TGF-β1, TIMP1, IL6, TNFα and MCP1 induced by MEN. Furthermore it was confirmed that ALA and PFD activate diverse antioxidants mediators, however MEN decreases this response. Then, MEN, ALA and PFD induce an antioxidant response, the first one as a response to injury and the latter two as pro-antioxidant inducers. Therefore, when cells are exposed to oxidative stress, endogenous systems activate a battery of mediators that increase the antioxidant potential. When these cells are treated with ALA and PFD, de novo formation of protective genes decreases since previous elicited protection induced in response to injury, enhance ALA and PFD effects. Conclusion: Regardless of the route of action, ALA and PFD induce the biosynthesis of antioxidants mediators which is associated with modulation of fibrogenic processes.

Hemoplasma prevalence and hematological abnormalities associated with infection in three different cat populations from Southern Brazil / Prevalência da infecção por hemoplasmas e alterações hematológicas associadas à infecção em três diferentes populações de gatos do sul do Brasil

Three hemoplasma species are recognized in domestic cats: Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’ and ‘Candidatus Mycoplasma turicensis’. We report the prevalence and hematological abnormalities of hemoplasma infection in 369 domestic cats from three different populations (blood donors, hospitalized cats and shelter cats) from Southern Brazil. Complete blood counts were performed at the time of blood collection, and DNA was extracted and tested by conventional PCR for each hemoplasma species. A total of 79 samples (21.40%) were positive for at least one species. The most prevalent hemoplasma was ‘Candidatus Mycoplasma haemominutum’, with 50/369 (13.55%) positive cats, followed by ‘Candidatus Mycoplasma turicensis’, 10/369 (2.71%), and Mycoplasma haemofelis, 8/369 (2.16%). Mycoplasma haemofelis and ‘Candidatus Mycoplasma haemominutum’ coinfection was observed in 4/369 (1.08%), whereas ‘Candidatus Mycoplasma haemominutum’ and ‘Candidatus Mycoplasma turicensis’ in 5/369 (1.35%). Three cats (0.81%) were infected with all three hemoplasmas. There was no association between infection and the different populations. Anemia was associated with Mycoplasma haemofelis and ‘Candidatus Mycoplasma haemominutum’, but not with ‘Candidatus Mycoplasma turicensis’. Male cats and cats with outdoor access were more likely to be infected. Although ‘Candidatus Mycoplasma haemominutum’ is believed to cause minimal or no hematological alterations, the infected cats studied herein were more likely to be anemic.

Três espécies de hemoplasmas são reconhecidas em gatos domésticos: Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’ e ‘Candidatus Mycoplasma turicensis’. A prevalência e alterações hematológicas associadas à infecção por hemoplasmas foi estudada, em 369 gatos domésticos de três populações distintas (doadores de sangue, hospitais e gatos de abrigo) do Sul do Brasil. Foram realizados hemogramas completos no momento da coleta de sangue e as amostras tiveram seu DNA extraído e testado por PCR convencional para cada espécie de hemoplasmas. Setenta e nove amostras (21,40%) foram positivas para pelo menos uma espécie. O mais prevalente foi ‘Candidatus Mycoplasma haemominutum’ com 50/369 (13,55%) gatos positivos, seguidos por ‘Candidatus Mycoplasma turicensis’ com 10/369 (2,71%) e Mycoplasma haemofelis com 8/369 (2,16%). Coinfecção por Mycoplasma haemofelis e ‘Candidatus Mycoplasma haemominutum’ foi observada em 4/369 (1,08%), enquanto ‘Candidatus Mycoplasma haemominutum’ e ‘Candidatus Mycoplasma turicensis’ coinfectaram 5/369 (1,35%) gatos. Três (0,81%) gatos apresentaram infecção pelos três hemoplasmas. Não houve associação entre a infecção e as diferentes populações. Anemia foi associada com a infecção por Mycoplasma haemofelis e ‘Candidatus Mycoplasma haemominutum’, mas não com ‘Candidatus Mycoplasma turicensis’. Gatos machos e com acesso à rua apresentaram maior probabilidade de serem infectados. Embora se acredite que ‘Candidatus Mycoplasma haemominutum’ possa causar alterações hematológicas mínimas ou ausentes, gatos infectados encontrados neste estudo foram mais propensos à anemia.

Expression of Glucose Transporters 1 and 3 in Metastatic and Non-Metastatic Lower Lip Squamous Cell Carcinoma

This study aimed to evaluate the immunoexpression of glucose transporters 1 (GLUT-1) and 3 (GLUT-3) in metastatic and non-metastatic lower lip squamous cell carcinoma (LLSCC). Twenty LLSCCs with regional nodal metastasis and 20 LLSCCs without metastasis were selected. The distribution of staining and the percentage of GLUT-1 and GLUT-3 staining in each tumor core and at the deep invasive front were assessed. Most tumors (70%) exhibited peripheral staining for GLUT-1 in nests, sheets and islands of neoplastic cells, whereas predominantly central staining was observed for GLUT-3 (72.5%). A high percentage of GLUT-1-positive cells was observed at the deep invasive front and in the tumor core of metastatic and non-metastatic tumors (p>0.05). The percentage of GLUT-1-positive cells was much higher than that of GLUT-3-positive cells both in the deep invasive front (p<0.001) and in the tumor core (p<0.001) of LLSCCs. No significant differences in the percentage of GLUT-1- and GLUT-3-positive cells were observed according to nodal metastasis, clinical stage or histological grade of malignancy (p>0.05). In conclusion, the results of the present study suggest an important role of GLUT-1 in glucose uptake in LLSCCs, although this protein does not seem to be involved in the progression of these tumors. On the other hand, GLUT-3 expression may represent a secondary glucose uptake mechanism in LLSCCs.

Este estudo objetivou avaliar a imunoexpressão dos transportadores de glicose 1 (GLUT-1) e 3 (GLUT-3) em carcinomas de células escamosas de lábio inferior (CCELI) metastáticos e não-metastáticos. Vinte CCELIs com metástase nodal regional e 20 CCELI sem metástase foram selecionados. Foram analisados a distribuição da imunomarcação e o percentual de imunorreatividade para GLUT-1 e GLUT-3 no centro tumoral e no front de invasão tumoral. A maioria dos tumores (70%) revelou marcação para GLUT-1 em áreas periféricas dos ninhos, lençóis e ilhas de células neoplásicas, ao passo que GLUT-3 revelou predomínio de marcação em áreas centrais (72.5%). Um alto percentual de células positivas para GLUT-1 foi observado no front de invasão e no centro tumoral das lesões metastáticas e não-metastáticas (p>0,05). O percentual de células positivas para GLUT-1 foi superior ao percentual de células positivas para GLUT-3, tanto no front de invasão (p<0,001) quanto no centro tumoral (p<0,001) dos CCELIs. Não foram observadas diferenças significativas no percentual de células positivas para GLUT-1 e GLUT-3 em relação à mestástase nodal, ao estádio clínico ou ao grau histológico de malignidade (p>0,05). Em conclusão, os resultados do presente estudo sugerem um importante papel para GLUT-1 na absorção de glicose nos CCELIs, embora esta proteína não pareça estar envolvida na progressão destes tumores. Por outro lado, a expressão de GLUT-3 pode representar um mecanismo secundário para a absorção de glicose nos CCELIs.