RESUMEN
High occurrence of obesity currently constitutes the main nutritional disease of the canine species. There is evidence that leptin increases during obesity in dogs. Hyperleptinemia is associated with increased neutrophil oxidative metabolism in obese humans and contributes to oxidative stress. However, in obese dogs, the probable relationship between this condition and the activation of the oxidative metabolism of neutrophils has yet to be established. Thus, we investigated the hypothesis that neutrophil activation and systemic oxidative stress occur in dogs with hyperleptinemia. A control group of 24â¯healthy dogs with a body condition score (BCS) of 4-5, an overweight group of 25 dogs with a BCS of 6-7, and 27 obese dogs with a BCS of 8-9, were composed. Two subgroups were formed composed of dogs with and without hyperleptinemia, grouped according to the 95% confidence interval obtained for plasma leptin values of the control group. Changes in obesity markers (body condition score, adiponectin and plasma leptin) and plasma oxidative stress (lipid peroxidation, total antioxidant and oxidant capacities and oxidative stress index) were measured in all the dogs selected. Neutrophil oxidative metabolism was evaluated in flow cytometry by superoxide production with the probe hydroethidine and by hydrogen peroxide production with the probe 2',7'-dichlorofluorescein diacetate, with or without phorbol myristate acetate (PMA) stimulation. Apoptosis and neutrophil viability were quantified in a capillary flow cytometer using Annexin VPE, with or without camptothecin apoptosis inducing effect. Obese dogs presented higher systemic oxidative stress, hyperleptinemia and preactivated neutrophils with accelerated apoptosis. Dogs with hyperleptinemia and obese dogs presented higher neutrophil superoxide production under PMA stimulation and the presence of systemic oxidative stress compared with control. To our knowledge, this is probably the first evidence that preactivation of the oxidative metabolism of circulating neutrophils occurs in dogs with hyperleptinemia, a condition that can induce systemic oxidative stress in the canine species.
Asunto(s)
Leptina/sangre , Neutrófilos/inmunología , Obesidad/sangre , Estrés Oxidativo , Animales , Antioxidantes/metabolismo , Apoptosis , Perros , Peróxido de Hidrógeno/metabolismo , Neutrófilos/metabolismo , Superóxidos/metabolismoRESUMEN
O sedentarismo é um problema de saúde pública e um dos maiores males da sociedade moderna. Já está bem estabelecido que esforço físico em excesso ou em indivíduos não condicionados acarreta estresse oxidativo e lesões musculares. No presente estudo, foi testada a hipótese de que um único esforço físico é capaz de causar estresse oxidativo e lesão muscular em indivíduos sedentários. Aditivamente foi avaliado efeito antioxidante do polifenol resveratrol (RV) quanto a sua capacidade de atenuar o estresse oxidativo e a lesão muscular causados pelo esforço físico. Para tal, 40 ratos (Rattus norvegicus albinus, Wistar), machos, adultos e sedentários foram aleatoriamente submetidos ou não a 90 minutos de natação, com e sem tratamento com RV (100mg/kg/PV/14dias): N-RV- (n=10) grupo mantido em repouso e não tratado com RV; N-RV+ (n=10) grupo mantido em repouso e tratado com RV; N+RV- (n=10) grupo submetido ao esforço físico de natação e não tratado com RV e N+RV+ (n=10) grupo submetido ao esforço físico de natação e tratado com RV. Em ratos sedentários, o esforço físico da natação promoveu estresse oxidativo (aumento da peroxidação lipídica e diminuição da capacidade antioxidante total do plasma) e aumento significativo da atividade plasmática de creatina quinase (CK) e lactato desidrogenase (LDH). O tratamento com RV diminuiu a peroxidação lipídica e a concentração dos marcadores de lesão muscular (CK e LDH) de ratos sedentários submetidos à natação. Essa é uma das primeiras evidências de que um único esforço físico pode causar estresse oxidativo em indivíduos sedentários e que o RV pode ser uma alternativa para atenuar a lesão muscular causada por esse estresse.(AU)
Physical inactivity is a public health problem when a sedentary population practices physical activity sporadically. Exercise in unconditioned individuals causes oxidative stress and muscle damage. This study tested the hypothesis that a single physical exertion can cause muscle damage and oxidative stress in sedentary individuals, and resveratrol can attenuate it. For this, 40 sedentary adult male rats were equally and randomized into four groups subjected to 90min swimming or rest and administered aqueous resveratrol (100mg/kg/day) or saline for 14 days: N-RV-, rats maintained at rest and administered saline; N-RV+, rats maintained at rest and treated with resveratrol; N+RV-, rats subjected to physical exercise and administered saline; and N+RV+, rats subjected to physical exercise and treated with resveratrol. In sedentary rats, the physical exertion of swimming promoted oxidative stress, i.e. increased lipid peroxidation and decreased plasma total antioxidant capacity, and significant increases in CK and LDH plasma activities. Resveratrol diminished lipid peroxidation and the concentrations of muscle damage markers (CK and LDH) in sedentary rats subjected to swimming. The results provide evidence that a single sudden physical exertion can cause oxidative stress in sedentary rats. Resveratrol showed good results as a treatment for minimizing muscle damage caused by this stress.(AU)
Asunto(s)
Animales , Ratas , Estrés Oxidativo , Ratas/fisiología , Ejercicio Físico , Conducta SedentariaRESUMEN
We aimed to induce and inhibit HO-1, ascertaining its effect on infection rate, parasite load and the levels of superoxide, reactive oxygen species (ROS), nitric oxide (NO), TNF-alpha and IL-10 in cultured macrophages from healthy dogs infected by Leishmania infantum. Macrophages obtained from 15 healthy dogs were cultured alone or infected with L. infantum, with or without association of HO-1 inducer and inhibitor. The infection rate and the parasite load were determined by the number of infected macrophages and number of promastigotes per macrophage, respectively. HO-1 levels and gene expression, as well as IL-10 and TNF-alpha levels were also measured in these cultures. Superoxide, ROS and NO levels in macrophages were measured through flow cytometry. Induction of HO-1 increased the infection rate and parasite load, while its inhibition decreased the infection rate and IL-10 production. There was a positive correlation between HO-1 and infection rate or parasite load. Increased infection rate was associated with decreased superoxide, ROS and NO levels. Induction of HO-1 metabolism in dogs infected by L. infantum is possibly one of the mechanisms responsible for increasing the infection of macrophages, mainly through reduction in the oxidative and nitrosative metabolisms of these cells.
Asunto(s)
Hemo-Oxigenasa 1/metabolismo , Leishmania infantum/fisiología , Leishmaniasis Visceral/veterinaria , Macrófagos/parasitología , Carga de Parásitos , Animales , Células Cultivadas , Enfermedades de los Perros/parasitología , Perros , Expresión Génica , Hemo-Oxigenasa 1/antagonistas & inhibidores , Interleucina-10/genética , Interleucina-10/metabolismo , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We investigated the hypothesis that the increased concentration of plasma methylguanidine (MG) increases oxidative metabolism and accelerates apoptosis of neutrophils from dogs with chronic kidney disease (CKD). To achieve this, the levels of MG were quantified in healthy (n=16) and uremic dogs with CKD stage 4 of according to the guidelines of the International Renal Interest Society (IRIS, 2015) (n=16) using high performance liquid chromatography (HPLC). To evaluate the isolated effect of MG on neutrophil oxidative metabolism and apoptosis, neutrophils isolated from 12 healthy dogs were incubated with the highest concentration of plasma MG (0.005g/L) observed in dogs with CKD. Neutrophil oxidative metabolism was assessed by flow cytometry, using the probes hydroethidine for superoxide production and 2',7'-dichlorofluorescein diacetate for hydrogen peroxide production, with or without phorbol myristate acetate (PMA) stimulus. Neutrophil apoptosis and viability were also evaluated in flow cytometer using the Annexin V-PE system, with or without the apoptosis-inducing effect of camptothecin. Uremic dogs presented higher concentrations of MG (p<0.0001), increased oxidative stress and primed neutrophils with higher apoptosis rate. The neutrophil abnormalities observed in vivo were also reproduced in vitro, using cells isolated from healthy dogs and incubated with MG. We obtained strong evidence that in dogs with CKD, increased MG levels contributed to oxidative stress and potentially compromised the non-specific immune response by altering the oxidative metabolism and viability of canine neutrophils.
Asunto(s)
Apoptosis , Metilguanidina/sangre , Neutrófilos , Insuficiencia Renal Crónica/veterinaria , Animales , Perros , Femenino , Masculino , Estrés Oxidativo , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/inmunología , Uremia/inmunología , Uremia/veterinariaRESUMEN
Canine visceral leishmaniasis (CVL) is caused by the intracellular parasite Leishmania infantum. Increased levels of arginase, nitric oxide (NO2 ) and prostaglandin E2 (PGE2 ) can play a regulatory role regarding the immune response in CVL cases. This study aimed to evaluate the arginase activity in adherent macrophages cultured from the lymph nodes of healthy and naturally infected dogs and to examine the NO2 and PGE2 levels in the supernatant of these cultures. In addition, the regulatory effect of PGE2 on the production of tumour necrosis factor (TNF-α) and interleukin-10 (IL-10) in supernatants from the total lymph node was observed in leucocyte cultures. The arginase activity was lower in the adherent macrophages cultured from the lymph nodes of naturally infected dogs and there were higher concentrations of NO2 and PGE2 in the supernatants of these cultures. Higher TNF-α and IL-10 concentrations were observed in supernatants from total lymph node leucocytes cultures, from infected dogs, and the presence of indomethacin only decreased TNF-α in the supernatant of these cultures. We conclude that the low arginase activity in macrophages suggested that M1 polarization and PGE2 were participating in the immune response and were increasing TNF-α in CVL.
Asunto(s)
Dinoprostona/metabolismo , Enfermedades de los Perros/inmunología , Perros/inmunología , Leishmania infantum/fisiología , Leishmaniasis Visceral/veterinaria , Ganglios Linfáticos/inmunología , Macrófagos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Arginasa/análisis , Arginasa/metabolismo , Dinoprostona/análisis , Enfermedades de los Perros/patología , Leishmaniasis Visceral/patología , Ganglios Linfáticos/citología , Ganglios Linfáticos/parasitología , Ganglios Linfáticos/patología , Macrófagos/química , Óxido Nítrico/análisisRESUMEN
Leishmania (L.) chagasi is the etiologic agent of visceral leishmaniasis (VL) that can be transmitted to humans and dogs. VL in Brazil represents a serious public health problem; therefore, it is important to study new alternatives to treat infected dogs. In dogs, the therapeutic arsenal against canine VL is limited. The immunomodulator protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA) improves immunocompetence when the immune system is impaired, but its dependence on Toll-like receptors (TLRs) and the mechanisms involved in immune response remain unclear. The in vitro action of P-MAPA on the expression of TLR2 and TLR4, reactive oxygen species (ROS), nitric oxide (NO) and p38 mitogen-activated protein kinase (p38 MAPK) and IKK phosphorylation was studied in mononuclear cells from peripheral blood and macrophages from healthy and Leishmania-infected dogs. The PBMC or macrophages were isolated and cultured with different concentrations of P-MAPA (20,100 and 200 µg/ml) in a humid environment at 37°C with 5% CO(2). Observation revealed that Leishmania-infected dogs showed a decrease in TLR2 in macrophages compared with healthy dogs and in induction with P-MAPA. ROS were increased in PBMCs from Leishmania spp.-infected dogs compared with healthy dogs and P-MAPA improved ROS production. NO production was increased in culture supernatant from macrophages stimulated by P-MAPA in both healthy and Leishmania spp. infected dogs. Treatment of macrophages from healthy dogs with immunomodulatory P-MAPA induced p38 MAPK and IKK phosphorylation, suggesting signal transduction by this pathway. These findings suggest that P-MAPA has potential as a therapeutic drug in the treatment of canine visceral leishmaniasis.
Asunto(s)
Enfermedades de los Perros/inmunología , Factores Inmunológicos/farmacología , Leishmaniasis Visceral/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Macrófagos/efectos de los fármacos , Animales , Perros , Femenino , Factores de Transcripción de Tipo Kruppel/inmunología , Leishmaniasis Visceral/veterinaria , Leucocitos Mononucleares/inmunología , Macrófagos/inmunología , Masculino , Óxido Nítrico/inmunología , Especies Reactivas de Oxígeno/inmunología , Receptor Toll-Like 2/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunologíaRESUMEN
The aim of the present study was to test the hypothesis that oxidative stress and alteration of oxidative metabolism and apoptosis of neutrophils in dogs vary with the stage of leishmaniasis and to determine the contribution of uremia to such alterations. Dogs with leishmaniasis were classified into two stages: moderate (Leish II, n=20) or very severe (i.e. with concurrent uremia; Leish IV, n=20) according to the LeishVet Consensus. The two leishmaniasis groups were compared with uremic dogs without leishmaniasis (Uremic, n=10) and to healthy dogs (Control, n=30). To determine oxidative stress, total antioxidant/oxidant capacity, lipid peroxidation, total glutathione and the plasma antioxidants albumin, uric acid and bilirubin were quantified. Superoxide production was determined using the hydroethidine probe and viability and apoptosis were measured using annexin V-PE by capillary flow cytometry. Oxidative stress was present in both uremia and leishmaniasis with reduced total antioxidant capacity and was associated with increased induced production of superoxide and apoptosis. The greatest amount of oxidants was observed in animals with moderate disease only. Neutrophils from uremic dogs with and without leishmaniasis had decreased viability and an increased apoptosis rate in addition to increased lipid peroxidation. In conclusion, oxidative stress occurs in both stages of leishmaniasis with differences in intensity and levels of plasma markers; however, uremia does contribute to the decreased spontaneous viability of neutrophils in dogs in the final stage of the disease.
Asunto(s)
Enfermedades de los Perros/metabolismo , Leishmaniasis Visceral/veterinaria , Neutrófilos/metabolismo , Estrés Oxidativo , Uremia/veterinaria , Animales , Antioxidantes/metabolismo , Apoptosis , Enfermedades de los Perros/parasitología , Perros , Femenino , Leishmaniasis Visceral/metabolismo , Leishmaniasis Visceral/parasitología , Peroxidación de Lípido , Masculino , Oxidantes/metabolismo , Uremia/metabolismo , Uremia/parasitologíaRESUMEN
Canine visceral leishmaniosis (CVL) causes a dependent-stage alteration in neutrophil oxidative metabolism. When production of reactive oxygen species (ROS) exceeds the antioxidant capacity of neutrophils, apoptosis is triggered, impairing the viability and function of these cells, which can predispose dogs to infection. However, the uremic condition observed in late-stage CVL can also alter the viability and function of human neutrophils. To more clearly understand this relationship, the apoptosis rate and oxidative metabolism of neutrophils from control dogs (n=20) were compared to dogs in moderate (n=15) and very severe (n=15) stage CVL, classified according to LeishVet Consensus. To assess neutrophil oxidative metabolism, superoxide production was measured using the nitroblue tetrazolium reduction test (NBT) in isolated neutrophils. The apoptosis rate of neutrophils was estimated using the morphological method. Moderate-stage dogs presented increased superoxide production, while dogs with very severe stage CVL presented decreased superoxide production and an increase neutrophil apoptosis rate. Leishmaniosis causes differential neutrophil dysfunction according to disease stage. In moderate stage CVL, increased superoxide production is observed with no change in neutrophil viability. However, in very severe stage CVL, decreased superoxide production and increased apoptosis occur associated with uremia.
Asunto(s)
Enfermedades de los Perros/patología , Leishmaniasis Visceral/veterinaria , Neutrófilos/metabolismo , Animales , Apoptosis , Enfermedades de los Perros/metabolismo , Perros , Femenino , Leishmaniasis Visceral/metabolismo , Leishmaniasis Visceral/patología , Masculino , Oxidación-Reducción , Estrés OxidativoRESUMEN
O presente trabalho tem como objetivo testar a hipótese de que, à semelhança do que ocorre na uremia, cães com azotemia pré-renal sofrem estresse oxidativo, o qual está relacionado com alterações do metabolismo oxidativo e apoptose dos neutrófilos. Para tal, foi determinada a peroxidação lipídica pela quantificação do malondialdeído (MDA) e o status antioxidante total do plasma de 15 cães normais e 10 com azotemia pré-renal, correlacionando-os com a produção de superóxido e o índice apoptótico dos neutrófilos. As determinações do MDA e do status antioxidante total foram estabelecidas empregando-se um conjunto de reagentes comerciais. Por meio de citometria de fluxo capilar, a produção de superóxido e a apoptose de neutrófilos isolados de sangue periférico foram determinadas utilizando-se a sonda hidroetidina e o sistema anexina V-PE, respectivamente. Cães azotêmicos (26,29±5,32g/L) apresentaram menor concentração (p=0,0264) do antioxidante albumina em relação ao grupo-controle (30,36±3,29g/L) e também uma menor (p=0,0027) capacidade antioxidante total (2,36±0,32 versus 2,73±0,24mmol/L), enquanto não houve alteração da peroxidação lipídica plasmática e da produção de superóxido neutrofílica. Concluiu-se que, à semelhança do que ocorre na uremia, condições azotêmicas pré-renais no cão causam estresse oxidativo e aceleração da apoptose dos neutrófilos.
This study aims to test the hypothesis that, similarly to what occurs in uremia, dogs with prerenal azotemia suffer oxidative stress associated with changes in oxidative metabolism and apoptosis in neutrophils. For this purpose, fifteen normal dogs and ten with prerenal azotemia had lipid peroxidation determined by quantifying the malondialdehyde (MDA) and had plasma total antioxidant status evaluated, correlating them with the superoxide production and apoptotic index of neutrophils. MDA and plasma total antioxidant status were determined using commercial reagents. Using capillary flow cytometry, superoxide production and apoptosis were determined from isolated neutrophils of peripheral blood using the hydrithidine and Annexin V-PE probe system, respectively. Azotemic dogs (26.29±5.32g/L) had a lower concentration (p=0.0264) of the plasma antioxidant albumin than the control group (30.36±3.29g/L) and also had lower (p=0.0027) total antioxidant status (2.36±0.32 versus 2.73±0.24mmol/L), while no alterations were observed in plasma lipid peroxidation and superoxide production. It was concluded that, similarly to what occurs in uremia, prerenal azotemia causes oxidative stress and acceleration of neutrophil apoptosis in dogs.