RESUMEN

A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel ß-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4-5 Å, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL-1 and Vmax 690 µmol·min-1.mg-1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.

Asunto(s)

Aspergillus/enzimología , Proteínas Fúngicas/metabolismo , Glicósido Hidrolasas/metabolismo , Catálisis , Estabilidad de Enzimas , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Glicósido Hidrolasas/química , Glicósido Hidrolasas/aislamiento & purificación , Ácidos Hexurónicos/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Pectinas/metabolismo , Conformación Proteica , Estabilidad Proteica , Relación Estructura-Actividad , Especificidad por Sustrato , Temperatura

RESUMEN

Schistosomiasis mansoni is involved in hepatic fibrogenesis and portal hypertension. Previous studies proved that blockade of some components of the renin-angiotensin system (RAS) reduce liver fibrogenesis. However, the effects of inhibition of early stages of RAS pathway in schistosomal fibrosis have not been studied yet. Thus, the aim of this study was to compare the role of different antihypertensive drugs on hepatic fibrosis in murine schistosomiasis. BALB/c mice (n = 50) weighing 20g were subjected to inoculation of 50 cercariae and submitted to different treatments: aliskiren, 50 mg/kg (n = 10); bradykinin, 2 µg/kg (n = 5); losartan, 10 mg/kg (n = 10); lisinopril 10 mg/kg (n = 5) and control, proportional volume vehicle (n = 5); daily for 14 weeks. Six animals were not subjected to cercariae inoculation or any type of treatment. Ultrasound, histological, immunohistochemical and proteomic analyzes were performed to evaluate markers associated with hepatic fibrogenesis. The hepatic areas stained with Sirius red and thenumber of cells marked by α-SMA in animals treated with aliskiren, bradykinin, lisinopril and losartan were diminished when compared to control group, demonstrating reduced hepatic fibrosis after RAS blockade. These results were reinforced by ultrasonography analysis and protein expression of TGFß. These findings demonstrated the effect of RAS inhibition on hepatic fibrosis in murine schistosomiasis, with the most evident results being observed in the losartan and aliskiren treated groups. The main mechanisms underlying this process appear to involve anti-fibrogenic activity through the inhibition of collagen and TGFß synthesis.

Asunto(s)

Cirrosis Hepática/tratamiento farmacológico , Sistema Renina-Angiotensina/efectos de los fármacos , Esquistosomiasis mansoni/complicaciones , Amidas/farmacología , Amidas/uso terapéutico , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Antihipertensivos/farmacología , Antihipertensivos/uso terapéutico , Bradiquinina/farmacología , Bradiquinina/uso terapéutico , Fumaratos/farmacología , Fumaratos/uso terapéutico , Lisinopril/farmacología , Lisinopril/uso terapéutico , Hígado/efectos de los fármacos , Hígado/patología , Cirrosis Hepática/parasitología , Losartán/farmacología , Losartán/uso terapéutico , Masculino , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico Sintasa de Tipo III/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Renina/efectos de los fármacos , Renina/genética , Renina/metabolismo , Esquistosomiasis mansoni/tratamiento farmacológico , Esquistosomiasis mansoni/patología , Inhibidor Tisular de Metaloproteinasa-1/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Factor de Crecimiento Transformador beta1/efectos de los fármacos , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Vasodilatadores/farmacología , Vasodilatadores/uso terapéutico , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, which occurs in the absence of alcohol abuse. NAFLD can evolve into progressive liver injury and fibrosis in the form of nonalcoholic steatohepatitis (NASH). Several animal models have been developed to attempt to represent the morphological, biochemical, and clinical features of human NASH. The actual review presents a critical analysis of the most commonly used experimental models of NAFLD/NASH development. These models can be classified into genetic, nutritional, and a combination of genetic and nutritional factors. The main genetic models are ob/ob and db/db mutant mice and Zucker rats. The principal nutritional models employ methionine- and choline-deficient, high-fat, high-cholesterol and high-cholate, cafeteria, and high-fructose diets. Currently, associations between high-fructose and various compositions of high-fat diets have been widely studied. Previous studies have encountered significant difficulties in developing animal models capable of reproducing human NASH. Some models produce consistent morphological findings, but the induction method differs significantly compared with the pathophysiology of human NASH. Other models precisely represent the clinical and etiological contexts of this disease but fail to provide accurate histopathological representations mainly in the progression from steatosis to liver fibrosis.

Asunto(s)

Modelos Animales de Enfermedad , Cirrosis Hepática/fisiopatología , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Animales , Progresión de la Enfermedad , Humanos , Cirrosis Hepática/genética , Ratones , Enfermedad del Hígado Graso no Alcohólico/genética , Ratas

RESUMEN

Rosmarinic acid (RosmA) demonstrates antioxidant and anti-inflammatory properties. We investigated the effect of RosmA on liver ischemia/reperfusion injury. Rats were submitted to 60 min of ischemia plus saline or RosmA treatment (150 mg/kg BW intraperitoneally) followed by 6 h of reperfusion. Hepatocellular injury was evaluated according to aminotransferase activity and histological damage. Hepatic neutrophil accumulation was also evaluated. Oxidative/nitrosative stress was estimated by measuring the reduced glutathione, lipid hydroperoxide and nitrotyrosine levels. Endothelial and inducible nitric oxide synthase (eNOS/iNOS) and nitric oxide (NO) were assessed with immunoblotting and chemiluminescence assays. Hepatic tumor necrosis factor-alpha (TNF-α) and interleukin-1beta mRNA were assessed using real-time PCR, and nuclear factor-kappaB (NF-κB) activation was estimated by immunostaining. RosmA treatment reduced hepatocellular damage, neutrophil infiltration and all oxidative/nitrosative stress parameters. RosmA decreased the liver content of eNOS/iNOS and NO, attenuated NF-κB activation, and down-regulated TNF-α and interleukin-1beta gene expression. These data indicate that RosmA exerts anti-inflammatory and antioxidant effects in the ischemic liver, thereby protecting hepatocytes against ischemia/reperfusion injury. The mechanisms underlying these effects may be related to the inhibitory potential of RosmA on the NF-κB signaling pathway and the reduction of iNOS and eNOS expressions and NO levels, in addition to its natural antioxidant capability.

Asunto(s)

Antiinflamatorios/uso terapéutico , Antioxidantes/uso terapéutico , Cinamatos/uso terapéutico , Depsidos/uso terapéutico , Hepatopatías/tratamiento farmacológico , Daño por Reperfusión/tratamiento farmacológico , Animales , Glutatión/metabolismo , Interleucina-1beta/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hepatopatías/patología , Pruebas de Función Hepática , Masculino , Infiltración Neutrófila , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/análisis , Peroxidasa/metabolismo , Ratas , Ratas Wistar , Daño por Reperfusión/patología , Factor de Necrosis Tumoral alfa/metabolismo , Ácido Rosmarínico

RESUMEN

Riboflavin has been shown to exhibit anti-inflammatory and antioxidant properties in the settings of experimental sepsis and ischaemia/reperfusion (I/R) injury. We investigated the effect of riboflavin on normothermic liver I/R injury. Mice were submitted to 60 min of ischaemia plus saline or riboflavin treatment (30 µmoles/kg BW) followed by 6 h of reperfusion. Hepatocellular injury was evaluated by aminotransferase levels, reduced glutathione (GSH) content and the histological damage score. Hepatic neutrophil accumulation was assessed using the naphthol method and by measuring myeloperoxidase activity. Hepatic oxidative/nitrosative stress was estimated by immunohistochemistry. Liver endothelial and inducible nitric oxide synthase (eNOS/iNOS) and nitric oxide (NO) amounts were assessed by immunoblotting and a chemiluminescence assay. Riboflavin significantly reduced serum and histological parameters of hepatocellular damage, neutrophil infiltration and oxidative/nitrosative stress. Furthermore, riboflavin infusion partially recovered hepatic GSH reserves and decreased the liver contents of eNOS/iNOS and NO. These data indicate that riboflavin exerts antioxidant and anti-inflammatory effects in the ischaemic liver, protecting hepatocytes against I/R injury. The mechanism of these effects appears to be related to the intrinsic antioxidant potential of riboflavin/dihydroriboflavin and to reduced hepatic expression of eNOS/iNOS and reduced NO levels, culminating in attenuation of oxidative/nitrosative stress and the acute inflammatory response.

Asunto(s)

Isquemia/patología , Hepatopatías/complicaciones , Reperfusión , Riboflavina/farmacología , Animales , Hepatopatías/patología , Ratones