Estudo da pentoxifilina sobre biomarcadores do estresse glico-oxidativo e da inflamação em camundongos submetidos ao modelo de obesidade e resistência à insulina
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Data
2019-11-28
Autores
Inácio, Maiara Destro [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A obesidade é considerada um problema de saúde pública devido a sua alta prevalência e incidência mundial, e está entre os principais fatores de risco para o desenvolvimento de resistência à insulina e diabetes mellitus tipo 2, ambos levando à hiperglicemia. A manutenção da hiperglicemia por longos períodos é responsável pelo desenvolvimento de complicações microvasculares e macrovasculares, desencadeadas principalmente pelo estresse glico-oxidativo, caracterizado pelo aumento na formação de produtos finais de glicação avançada (AGE). A pentoxifilina, um derivado de metilxantina que atua como um inibidor não-seletivo de fosfodiesterases, tem sido utilizada para o tratamento da claudicação intermitente. No entanto, estudos pré-clínicos indicam que a pentoxifilina é eficaz na promoção de melhorias na resistência à insulina e complicações associadas à obesidade. O objetivo deste estudo foi avaliar os efeitos do tratamento com pentoxifilina em camundongos submetidos ao modelo de obesidade e resistência à insulina, com ênfase nas alterações de parâmetros fisiometabólicos, biomarcadores do estresse glico-oxidativo e da inflamação. Camundongos machos C57BL-6J foram alimentados durante 14 semanas com dieta padrão (P; 3,85 kcal/g, 4% lipídeos) ou com dieta hiperlípidica/hipercalórica (HL; 5,40 kcal/g; 35% de lipídeos). A partir da 7ª semana foram iniciados os tratamentos diários (i.p.) com veículo (salina 0,85%; grupos P e HL) ou com pentoxifilina (50 mg/kg; grupo HPTX), durante as próximas 7 semanas. Peso corporal e ingestão alimentar foram monitorados semanalmente. Nas 12ª e 13ª semanas foram realizados o teste de tolerância à glicose oral (TTGO) e o teste de tolerância à insulina (ITT), respectivamente. Na 14ª semana, os animais foram anestesiados, e em seguida, metade dos animais de cada grupo recebeu insulina (3,8 U/kg, i.p) e a outra metade recebeu salina e, após 10 minutos dos respectivos estímulos, amostras de sangue foram coletadas por punção cardíaca (obtenção de plasma) e foram removidos, pesados e congelados os tecidos adiposos brancos, o tecido adiposo marrom interescapular (iTAM) e o músculo esquelético gastrocnemius para o estudo das alterações na fosforilação de AKT (Ser-473), bem como o fígado e rins para diversas análises: (i) marcadores bioquímicos plasmáticos; (ii) biomarcadores inflamatórios no soro e rim (TNF-α, IL-6 e MCP-1); (iii) hormônios no soro (insulina, leptina e resistina); (iv) atividades das enzimas antioxidantes, superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GSH-Px) em fígado e rim, e paraoxonase 1 (PON 1) no plasma; (v) estimativa dos AGEs fluorescentes e (vi) substâncias reativas ao ácido tiobarbitúrico (TBARS) em plasma, fígado e rim; (vii) conteúdo de triacilgliceróis no fígado e rim; (viii) nos rins foram realizadas análises histológicas e determinados os níveis proteicos do receptor de AGEs (RAGE), marcadores de apoptose (BAX e caspase-3), marcador de inflamação (NF-κB), componentes relacionados à detoxificação de AGEs (GLO 1 e AGE-R1), sensores metabólicos (PGC-1α e AMPK) e do fator de transcrição de enzimas antioxidantes (NRF 2); (ix) por fim, no iTAM foram analisados componentes do programa termogênico (PGC-1α, AMPK, NRF 2, SIRT 1, UCP 1) e do marcador de inflamação (NF-κB). Em comparação aos animais do grupo P, animais HL apresentaram: aumento no ganho de peso corporal e nas massas de tecidos adiposos, fígado e rim; aumento no conteúdo de triacilgliceróis no fígado e rim; aumento nos níveis de marcadores do estresse glico-oxidativo (TBARS e AGEs no plasma, fígado e rim); redução nas defesas antioxidantes endógenas (SOD, CAT, GSH-Px e PON 1); apresentaram intolerância à glicose, redução da sensibilidade à insulina e nos níveis de fosforilação de AKT estimulada por insulina nos tecidos adiposos e no músculo esquelético gastrocnemius; aumento nos níveis séricos de insulina, leptina, resistina, MCP-1 e TNF-α. Nos rins de animais HL, houve aumento na área glomerular; aumento nos níveis renais de MCP-1, TNF-α e IL-6; aumento nos níveis proteicos de RAGE, NF-κB, BAX e caspase-3 clivada; redução nos níveis proteicos de NRF 2, AGE-R1 e nos níveis proteicos/fosforilação de AMPK (Thr 172), mas não observou-se alteração em GLO 1. No iTAM de animais HL, houve diminuição nos níveis de SIRT 1 e nos níveis de fosforilação de AMPK, e aumento nos níveis de PGC-1α, UCP 1 e NF-κB, quando comparados aos animais do grupo P. Animais HPTX apresentaram menor ganho de peso corporal e menores massas dos tecidos adiposos, fígado e rim; redução no conteúdo de triacilgliceróis no fígado de rim; atenuação nas alterações morfológicas renais promovidas pela ingestão de dieta hiperlipídica; redução nos níveis dos biomarcadores do estresse glico-oxidativo (TBARS e AGEs no plasma, fígado e rim), bem como aumento nas atividades de enzimas antioxidantes (SOD, CAT, GSH-Px e PON 1). O tratamento com pentoxifilina melhorou a tolerância à glicose, aumentou a sensibilidade à insulina e os níveis de fosforilação de AKT estimulada por insulina nos tecidos adiposos e no músculo esquelético gastrocnemius, além de reduzir os níveis séricos de insulina e leptina. Avanços foram conquistados na compreensão das ações protetoras da pentoxifilina nos rins de animais alimentados com dieta hiperlipídica, incluindo a redução de biomarcadores inflamatórios (TNF-α, MCP-1 e IL-6) e nos níveis proteicos de RAGE, BAX, caspase-3 e NF-κB, e aumento nos níveis de fosforilação de AMPK (Thr 172); e em relação aos componentes de detoxificação de AGEs, animais HPTX apresentaram aumento nos níveis proteicos de GLO 1 e redução de AGE-R1. No iTAM de animais HPTX, houve o aumento nos níveis proteicos de componentes do programa termogênico (NRF 2, SIRT 1, UCP 1) e redução do NF-κB. Em conjunto, os achados deste estudo apresentam evidências pré-clínicas que podem futuramente contribuir para o reposicionamento da pentoxifilina como uma opção para o tratamento da obesidade, diabetes mellitus e complicações associadas, com ênfase nas complicações renais.
Obesity is considered a public health problem due to its increased prevalence and incidence worldwide, and is one main risk factor for the development of insulin resistance and type 2 diabetes mellitus, both leading to hyperglycemia. The maintenance of hyperglycemia for long periods is responsible for the onset of microvascular and macrovascular complications, mainly triggered by glycoxidative stress, characterized by the increased generation of advanced glycation end products (AGEs). Pentoxifylline, a methylxanthine derivative that acts as a non-selective inhibitor of phosphodiesterases, has been used for the treatment of intermittent claudication. However, preclinical studies indicate that pentoxifylline is effective in promoting improvements in insulin resistance and complications associated with obesity. The objective of this study was to investigate with emphasis in the changes in physiometabolic parameters, biomarkers of glyco-oxidative stress and inflammation of mice under an experimental model of high-fat diet-induced obesity and treated with pentoxifylline. C57BL-6J male mice were fed a standard diet (P; 3.85 kcal/g, 4% lipids) or a high-fat/hypercaloric diet (HFD, 5.40 kcal/g, 35% lipid) for 14 weeks. Daily treatments (i.p.) were performed with vehicle (0.85% saline, P and HL groups) or with pentoxifylline (50 mg/kg, HPTX group) from the 7th week, during the next 7 weeks. Body weight and food intake were monitored weekly. At 12th and 13th weeks, the oral glucose tolerance test (OGTT) and the insulin tolerance test (ITT) were performed, respectively. At the 14th week, the animals were anesthetized, and then, half of the animals from each group received insulin (3.8 U/kg, i.p.) and the other half received saline and, after 10 minutes of the respective stimulus , blood samples were collected by cardiac puncture (to obtain plasma), white adipose tissues, interscapular brown adipose tissues (iBAT) and skeletal muscles gastrocnemius were removed, weighed and frozen for the study of the changes in the phosphorylation of AKT (Ser-473) as well as liver and kidneys for several analyzes: (i) plasma biochemical markers; (ii) serum and renal inflammatory biomarkers (TNF-α, IL-6 and MCP-1); (iii) serum hormonal (insulin, leptin and resistin); (iv) activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) in liver and kidney, and paraoxonase 1 (PON 1) in plasma; (v) estimation of fluorescent AGEs and (vi) thiobarbituric acid reactive substances (TBARS) in plasma, liver and kidney; (vii) triacylglycerol content in liver and kidney; (viii) in the kidneys were performed histological analysis and determined the protein levels of the AGE receptor (RAGE), markers of apoptosis (BAX and caspase-3), inflammation marker (NF-𝜅B), components related to the detoxification of AGEs (GLO 1 and AGE-R1), metabolic sensors (PGC-1α and AMPK) and transcription factor for antioxidant enzymes and GLO 1 (NRF 2); (ix) finally, in the iBAT were analyzed components of the thermogenic program (PGC-1α, AMPK, NRF 2, SIRT 1, UCP 1) and the inflammation marker (NF-κB). In comparison to P animals, HL animals showed: increase in body weight gain and in the weights of adipose tissues, liver and kidney; increased levels of glycoxidative stress markers (TBARS and AGEs in plasma, liver and kidney); increased content of triacylglycerols in liver and kidney; reduction in endogenous antioxidant defenses (SOD, CAT, GSH-Px, and PON 1); HL animals showed glucose intolerance, reduced insulin sensitivity and phosphorylation levels of insulin-stimulated AKT in adipose tissues and skeletal muscle gastrocnemius; increased serum insulin, leptin, resistin levels; and increased renal TNF-α and MCP-1 levels. In the kidneys of HL animals, there was an increase in the glomerular area; increased renal levels of MCP-1, TNF-α and IL-6; increase in the protein levels of RAGE, NF-𝜅B, BAX and cleaved caspase-3, and reduction in the protein levels of NRF 2, AMPK protein levels/phosphorylation (Thr 172) and AGE-R1, however there was no change in GLO 1. In iBAT of HL animals, there was a reduction in the levels of SIRT 1 and in the AMPK phosphorylation, and an increase in the levels of PGC-1α, UCP 1 and NF-κB, when compared to P animals. HPTX animals had lower body weight gain, minor weights of adipose tissues, liver and kidney, attenuation in renal morphological alterations caused by the ingestion of high-fat diet, as well as reduction in biomarkers of glycoxidative stress (TBARS and AGEs in plasma, liver and kidney), increases in the activities of antioxidant enzymes (SOD, CAT, GSH-Px and PON 1). The treatment with pentoxifylline improved the insulin sensitivity, increased glucose tolerande and the phosphorylation levels of insulin-stimulated AKT in adipose tissues and skeletal muscle gastrocnemius, as well as reducing serum insulin and leptin levels. Advances were achieved in the understanding of the protective actions of pentoxifylline in kidneys of animals fed a high-fat diet, including reduction of inflammatory biomarkers (TNF-α, MCP-1 and IL-6) and protein levels of the receptor RAGE, BAX, caspase-3 and NF-κB, and increase in AMPK phosphorylation levels (Thr 172); about AGE detoxification-related components, HPTX animals showed increased GLO 1 protein levels and reduced AGE-R1. There was an increase in the protein levels of NRF 2, SIRT 1 and UCP 1, and reduction of NF-κB in iBAT of HPTX animals. Taken together, the findings of this study indicate preclinical evidence that may contribute, in the future, to repositioning pentoxifylline as an option for the treatment of obesity, diabetes mellitus and associated complications, with emphasis on renal complications.
Obesity is considered a public health problem due to its increased prevalence and incidence worldwide, and is one main risk factor for the development of insulin resistance and type 2 diabetes mellitus, both leading to hyperglycemia. The maintenance of hyperglycemia for long periods is responsible for the onset of microvascular and macrovascular complications, mainly triggered by glycoxidative stress, characterized by the increased generation of advanced glycation end products (AGEs). Pentoxifylline, a methylxanthine derivative that acts as a non-selective inhibitor of phosphodiesterases, has been used for the treatment of intermittent claudication. However, preclinical studies indicate that pentoxifylline is effective in promoting improvements in insulin resistance and complications associated with obesity. The objective of this study was to investigate with emphasis in the changes in physiometabolic parameters, biomarkers of glyco-oxidative stress and inflammation of mice under an experimental model of high-fat diet-induced obesity and treated with pentoxifylline. C57BL-6J male mice were fed a standard diet (P; 3.85 kcal/g, 4% lipids) or a high-fat/hypercaloric diet (HFD, 5.40 kcal/g, 35% lipid) for 14 weeks. Daily treatments (i.p.) were performed with vehicle (0.85% saline, P and HL groups) or with pentoxifylline (50 mg/kg, HPTX group) from the 7th week, during the next 7 weeks. Body weight and food intake were monitored weekly. At 12th and 13th weeks, the oral glucose tolerance test (OGTT) and the insulin tolerance test (ITT) were performed, respectively. At the 14th week, the animals were anesthetized, and then, half of the animals from each group received insulin (3.8 U/kg, i.p.) and the other half received saline and, after 10 minutes of the respective stimulus , blood samples were collected by cardiac puncture (to obtain plasma), white adipose tissues, interscapular brown adipose tissues (iBAT) and skeletal muscles gastrocnemius were removed, weighed and frozen for the study of the changes in the phosphorylation of AKT (Ser-473) as well as liver and kidneys for several analyzes: (i) plasma biochemical markers; (ii) serum and renal inflammatory biomarkers (TNF-α, IL-6 and MCP-1); (iii) serum hormonal (insulin, leptin and resistin); (iv) activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) in liver and kidney, and paraoxonase 1 (PON 1) in plasma; (v) estimation of fluorescent AGEs and (vi) thiobarbituric acid reactive substances (TBARS) in plasma, liver and kidney; (vii) triacylglycerol content in liver and kidney; (viii) in the kidneys were performed histological analysis and determined the protein levels of the AGE receptor (RAGE), markers of apoptosis (BAX and caspase-3), inflammation marker (NF-𝜅B), components related to the detoxification of AGEs (GLO 1 and AGE-R1), metabolic sensors (PGC-1α and AMPK) and transcription factor for antioxidant enzymes and GLO 1 (NRF 2); (ix) finally, in the iBAT were analyzed components of the thermogenic program (PGC-1α, AMPK, NRF 2, SIRT 1, UCP 1) and the inflammation marker (NF-κB). In comparison to P animals, HL animals showed: increase in body weight gain and in the weights of adipose tissues, liver and kidney; increased levels of glycoxidative stress markers (TBARS and AGEs in plasma, liver and kidney); increased content of triacylglycerols in liver and kidney; reduction in endogenous antioxidant defenses (SOD, CAT, GSH-Px, and PON 1); HL animals showed glucose intolerance, reduced insulin sensitivity and phosphorylation levels of insulin-stimulated AKT in adipose tissues and skeletal muscle gastrocnemius; increased serum insulin, leptin, resistin levels; and increased renal TNF-α and MCP-1 levels. In the kidneys of HL animals, there was an increase in the glomerular area; increased renal levels of MCP-1, TNF-α and IL-6; increase in the protein levels of RAGE, NF-𝜅B, BAX and cleaved caspase-3, and reduction in the protein levels of NRF 2, AMPK protein levels/phosphorylation (Thr 172) and AGE-R1, however there was no change in GLO 1. In iBAT of HL animals, there was a reduction in the levels of SIRT 1 and in the AMPK phosphorylation, and an increase in the levels of PGC-1α, UCP 1 and NF-κB, when compared to P animals. HPTX animals had lower body weight gain, minor weights of adipose tissues, liver and kidney, attenuation in renal morphological alterations caused by the ingestion of high-fat diet, as well as reduction in biomarkers of glycoxidative stress (TBARS and AGEs in plasma, liver and kidney), increases in the activities of antioxidant enzymes (SOD, CAT, GSH-Px and PON 1). The treatment with pentoxifylline improved the insulin sensitivity, increased glucose tolerande and the phosphorylation levels of insulin-stimulated AKT in adipose tissues and skeletal muscle gastrocnemius, as well as reducing serum insulin and leptin levels. Advances were achieved in the understanding of the protective actions of pentoxifylline in kidneys of animals fed a high-fat diet, including reduction of inflammatory biomarkers (TNF-α, MCP-1 and IL-6) and protein levels of the receptor RAGE, BAX, caspase-3 and NF-κB, and increase in AMPK phosphorylation levels (Thr 172); about AGE detoxification-related components, HPTX animals showed increased GLO 1 protein levels and reduced AGE-R1. There was an increase in the protein levels of NRF 2, SIRT 1 and UCP 1, and reduction of NF-κB in iBAT of HPTX animals. Taken together, the findings of this study indicate preclinical evidence that may contribute, in the future, to repositioning pentoxifylline as an option for the treatment of obesity, diabetes mellitus and associated complications, with emphasis on renal complications.
Descrição
Palavras-chave
obesidade, resistência à insulina, diabetes mellitus, estresse glico-oxidativo, pentoxifilina, glycoxidative stress, pentoxifylline, insulin resistance, Obesity