Regulación dual de la NADPH oxidasa por el factor de crecimiento de hepatocitos (HGF) en cultivo primario de hepatocitos de ratón

El factor de crecimiento de hepatocitos (HGF) y su receptor c-Met protegen contra el daño celular provocado por el estrés oxidante mediante la inducción de proteínas antiapoptóticas y antioxidantes. Nrf2 es un factor de transcripción que regula positivamente la expresión de genes antioxidantes en el hígado, sin embargo, ahora se sabe que las especies reactivas de oxigeno (ERO) a niveles basales son necesarias para la activación de vías de señalización. Datos previos de nuestro grupo de investigación revelan que los hepatocitos de ratón con eliminación condicional de c-Met muestran un incremento en la activación de la NADPH oxidasa y la sobreexpresión de algunos de sus subunidades como Nox4, Nox2, p22 y p67. El objetivo de este trabajo fue identificar el mecanismo de regulación de la NADPH oxidasa ejercida por el HGF/c-Met y la inducción de la activación de Nrf2 por un mecanismo dependiente de este complejo enzimático. Los cultivos primarios de hepatocitos se aislaron usando el método de perfusión en dos pasos con colagenasa, se usaron ratones macho de la cepa CD1, las células se pre-trataron o no con 50 ng/ml de HGF. Los datos obtenidos muestran que HGF indujo un incremento en la actividad de la NADPH oxidasa y en la producción de ERO en tiempos tempranos (1-30 min) pero una disminución en tiempos más largos incluso por debajo de los niveles normales con respecto a las células no tratadas. El incremento temprano en el contenido de las ERO se relacionó con la activación de Nrf2 la cual fue analizada por medio de un ensayo de retardo de la movilidad electroforética (EMSA) y por microscopia confocal. Para verificar la activación de Nrf2 llevada a cabo por el HGF, se analizó la expresión de sus principales proteínas blanco SOD, -GCS y NQO1, donde se encontró que estas enzimas se incrementaron 2, 2.8 y 3 veces respectivamente con respecto a las células no tratadas. Para confirmar que el HGF tenía un efecto protector en los hepatocitos, se evaluó la viabilidad celular por medio de un estudio de tinción celular con cristal violeta. Debido a que HGF induce la disminución en la actividad de la NADPH oxidasa en tiempos más largos, decidimos caracterizar la expresión de ARNm y de la proteína de sus subunidades por medio de qRT-PCR e Inmunoblot respectivamente, los datos muestran que HGF induce una desregulación en Nox2, Nox4, p47 y p22. Finalmente, decidimos explorar la posibilidad de que c-Met estuviera interactuando con la NADPH oxidasa por medio de una de sus subunidades ancladas a la membrana esto se analizó con una inmunoprecipitación, los datos muestran una interacción entre c-Met y p22 la cual disminuye después de 30 min de tratamiento con HGF, este dato fue comprobado por un ensayo de entrecruzamiento. En conclusión nuestros datos muestran que HGF ejerce una regulación dual sobre la NADPH oxidasa, activándola en tiempos cortos como respuesta de sobrevivencia mediada por Nrf2 y disminuye su expresión en tiempos más largos quizá para evitar el uso de ésta oxidasa por citocinas citotóxicas

Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress

Cholesterol overload in the liver has shown toxic effects by inducing the aggravation of nonalcoholic fatty liver disease to steatohepatitis and sensitizing to damage. Although the mechanism of damage is complex, it has been demonstrated that oxidative stress plays a prominent role in the process. In addition, we have proved that hepatocyte growth factor induces an antioxidant response in hepatic cells; in the present work we aimed to figure out the protective effect of this growth factor in hepatocytes overloaded with free cholesterol. Hepatocytes from mice fed with a high-cholesterol diet were treated or not with HGF, reactive oxygen species present in cholesterol overloaded hepatocytes significantly decreased, and this effect was particularly associated with the increase in glutathione and related enzymes, such as γ-gamma glutamyl cysteine synthetase, GSH peroxidase, and GSH-S-transferase. Our data clearly indicate that HGF displays an antioxidant response by inducing the glutathione-related protection system

OP0319 DIAGNOSTIC ACCURACY OF ULTRASOUND IN CALCIUM PYROPHOSPHATE DEPOSITION DISEASE: PRELIMINARY RESULTS OF THE OMERACT US IN CPPD SUB-GROUP

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The Overexpression of NALP3 Inflammasome in Knee Osteoarthritis Is Associated with Synovial Membrane Prolidase and NADPH Oxidase 2

Osteoarthritis is characterized by the presence of proinflammatory cytokines and reactive oxygen species. We aimed to clarify the role of prooxidant enzyme content at the synovial membrane level and how it correlates with the inflammatory process in patients with knee osteoarthritis (KOA). In synovial membranes from KOA patients and control group, we analyzed the protein content of prooxidant enzymes such as Nox2, xanthine oxidase (XO), and prolidase as well as the proinflammatory NALP3. Results show that protein content of prolidase and Nox2 increased 4.8- and 8.4-fold, respectively, and XO showed an increasing trend, while the NALP3 inflammasome increased 5.4-fold with respect to control group. Levels of prolidase and XO had a positive correlation between the levels of NALP3 and Nox2. By principal component analysis the protein expression pattern by study groups was evaluated. Three clusters were identified; protein expression patterns were higher for clusters two (prolidase) and three (XO and Nox2) between KOA patients and controls. Data suggest that prooxidant enzymes increase in synovial membrane of KOA patients and may contribute to the inflammatory state and degradation of the articular cartilage

Fast Morphological Gallbladder Changes Triggered by a Hypercholesterolemic Diet

Introduction and aim. Obesity is a worldwide epidemic problem, described as a risk factor for hepatic diseases, such as non-alcoholic fatty liver disease and other pathologies related to development of cholesterol crystals and cholesterol gallbladder stones. It has been reported that cholesterol overload may cause hepatic damage; however, little is known about the effects of an acute hypercholesterolemic diet on the gallbladder. The aim of this manuscript was to evaluate the impact of a cholesterol-rich diet on the gallbladder.Material and methods. The study included ten eight-week-old C57BL6 male mice, which were divided into two study groups and fed different diets for 48 h: a hypercholesterolemic diet and a balanced Chow diet. After 48 h, the mice were analyzed by US with a Siemens Acuson Antares equipment. Mice were subsequently sacrificed to carry out a cholesterol analysis with a Refloton System (Roche), a crystal analysis with a Carl Zeiss microscope with polarized light, and a histological analysis with Hematoxylin-eosin staining.Results. The hypercholesterolemic diet induced an increase in gallbladder size and total cholesterol content in the bile, along with important histological changes.Conclusion. Cholesterol overloads not only trigger hepatic damage, but also affect the gallbladder significantly

Hyperlipidemic microenvironment conditionates damage mechanisms in human chondrocytes by oxidative stress

Abstract Background Currently, two pathogenic pathways describe the role of obesity in osteoarthritis (OA); one through biomechanical stress, and the other by the contribution of systemic inflammation. The aim of this study was to evaluate the effect of free fatty acids (FFA) in human chondrocytes (HC) expression of proinflammatory factors and reactive oxygen species (ROS). Methods HC were exposed to two different concentrations of FFA in order to evaluate the secretion of adipokines through cytokines immunoassays panel, quantify the protein secretion of FFA-treated chondrocytes, and fluorescent cytometry assays were performed to evaluate the reactive oxygen species (ROS) production. Results HC injury was observed at 48 h of treatment with FFA. In the FFA-treated HC the production of reactive oxygen species such as superoxide radical, hydrogen peroxide, and the reactive nitrogen species increased significantly in a at the two-dose tested (250 and 500 μM). In addition, we found an increase in the cytokine secretion of IL-6 and chemokine IL-8 in FFA-treated HC in comparison to the untreated HC. Conclusion In our in vitro model of HC, a hyperlipidemia microenvironment induces an oxidative stress state that enhances the inflammatory process mediated by adipokines secretion in HC