The Effect of Garlic and Voluntary Exercise on Cardiac Angiogenesis in Diabetes: The Role of MiR-126 and MiR-210 / O Efeito do Alho e de Exercícios Físicos Voluntários na Angiogênese Cardíaca no Diabetes: O Papel do miRNA-126 e do miRNA-210

Abstract Background: Diabetes mellitus (DM) is one of the major risk factors for cardiovascular disease, leading to endothelial dysfunction and angiogenesis impairment . MiR-126 and miR-210 support angiogenic response in endothelial cells. Objective: The present study sought to explore the effect of garlic and voluntary exercise, alone or together, on miR-126 and miR-210 expressions and cardiac angiogenesis in rats with type 1 diabetes. Methods: Male Wistar rats were divided into five groups (n = 7): Control, Diabetes, Diabetes+Garlic, Diabetes+Exercise, and Diabetes+Garlic+Exercise. Diabetes was induced in the animals by streptozotocin (ip, 50 mg/kg). The rats were then fed raw fresh garlic homogenate (250 mg/kg) or were subjected to voluntary exercise, or to combined garlic and voluntary exercise for 6 weeks. MiR-126 and miR-210 expressions in the myocardium were determined by real time PCR, and the serum lipid profile was measured by enzymatic kits. Angiogenesis was evaluated by immunostaining for PECAM-1/ CD31 in the myocardium. Results: Diabetes reduced both cardiac miR-126 expression and angiogenesis (p < 0.05). On the other hand, there was a miR-210 expression increase in the myocardium of diabetic animals (p < 0.001). However, those effects reversed either with garlic or voluntary exercise (p < 0.01). Moreover, treating diabetic rats with garlic and voluntary exercise combined had an additional effect on the expressions of miR-126 and miR-210 (p < 0.001). Furthermore, both voluntary exercise and garlic significantly improved serum lipid profiles (p < 0.001). Conclusion: The induction of diabetes decreased angiogenesis in the myocardium, whereas our treatment using long-term voluntary exercise and garlic improved myocardial angiogenesis. These changes were possibly owing to the enhancement of myocardial miR-126 and miR-210 expressions.

Resumo Fundamento: O diabetes mellitus (DM) é um dos principais fatores de risco para doenças cardiovasculares, levando à disfunção endotelial e inibição da angiogênese. O miRNA-126 e o miRNA-210 promovem a resposta angiogênica em células endoteliais. Objetivo: O presente estudo buscou explorar o efeito do alho e de exercícios físicos voluntários, isoladamente ou em conjunto, nas expressões do miRNA-126 e do miR-210 e na angiogênese cardíaca em ratos com diabetes tipo 1. Métodos: Ratos Wistar machos foram divididos em cinco grupos (n = 7): Controle, Diabetes, Diabetes+Alho, Diabetes+Exercícios e Diabetes+Alho+Exercícios. Introduziu-se diabetes nos animais por estreptozotocina (ip, 50 mg/kg). Os ratos foram então alimentados com homogenato de alho fresco cru (250 mg/kg), ou foram submetidos a exercícios voluntários, ou a uma combinação de alho e exercícios voluntários, durante 6 semanas. As expressões do miRNA-126 e do miRNA-210 no miocárdio foram determinadas por PCR em tempo real, e o perfil lipídico sérico foi medido por kits enzimáticos. A angiogênese foi avaliada por imunocoloração por PECAM-1/CD31 no miocárdio Resultados: O diabetes reduziu a expressão do miRNA-126 cardíaco e da angiogênese (p < 0,05). Por outro lado, houve um aumento da expressão do miRNA-210 no miocárdio dos animais diabéticos (p < 0,001). No entanto, tais efeitos foram revertidos com alho ou exercícios voluntários (p < 0,01). Além disso, o tratamento de ratos diabéticos conjuntamente com alho e exercícios voluntários teve um efeito adicional sobre as expressões do miRNA-126 e do miRNA-210 (p < 0,001). Além disso, tanto os exercícios voluntários quanto o alho melhoraram significativamente os perfis lipídicos séricos (p < 0,001). Conclusões: A indução de diabetes diminuiu a angiogênese no miocárdio, enquanto nosso tratamento com exercícios voluntários de longa duração e alho melhorou a angiogênese miocárdica. Estas alterações devem-se, possivelmente, ao aumento das expressões do miRNA-126 e do miRNA no miocárdio.

The Effect of Garlic and Voluntary Exercise on Cardiac Angiogenesis in Diabetes: The Role of MiR-126 and MiR-210.

BACKGROUND: Diabetes mellitus (DM) is one of the major risk factors for cardiovascular disease, leading to endothelial dysfunction and angiogenesis impairment . MiR-126 and miR-210 support angiogenic response in endothelial cells. OBJECTIVE: The present study sought to explore the effect of garlic and voluntary exercise, alone or together, on miR-126 and miR-210 expressions and cardiac angiogenesis in rats with type 1 diabetes. METHODS: Male Wistar rats were divided into five groups (n = 7): Control, Diabetes, Diabetes+Garlic, Diabetes+Exercise, and Diabetes+Garlic+Exercise. Diabetes was induced in the animals by streptozotocin (ip, 50 mg/kg). The rats were then fed raw fresh garlic homogenate (250 mg/kg) or were subjected to voluntary exercise, or to combined garlic and voluntary exercise for 6 weeks. MiR-126 and miR-210 expressions in the myocardium were determined by real time PCR, and the serum lipid profile was measured by enzymatic kits. Angiogenesis was evaluated by immunostaining for PECAM-1/ CD31 in the myocardium. RESULTS: Diabetes reduced both cardiac miR-126 expression and angiogenesis (p < 0.05). On the other hand, there was a miR-210 expression increase in the myocardium of diabetic animals (p < 0.001). However, those effects reversed either with garlic or voluntary exercise (p < 0.01). Moreover, treating diabetic rats with garlic and voluntary exercise combined had an additional effect on the expressions of miR-126 and miR-210 (p < 0.001). Furthermore, both voluntary exercise and garlic significantly improved serum lipid profiles (p < 0.001). CONCLUSION: The induction of diabetes decreased angiogenesis in the myocardium, whereas our treatment using long-term voluntary exercise and garlic improved myocardial angiogenesis. These changes were possibly owing to the enhancement of myocardial miR-126 and miR-210 expressions.

Voluntary Exercise Protects Heart from Oxidative Stress in Diabetic Rats.

PURPOSE: Oxidative stress plays a key role in the onset and development of diabetes complications. In this study, we evaluated whether voluntary exercise could alleviate oxidative stress in the heart and blood of streptozotocin - induced diabetic rats. METHODS: 28 male Wistar rats were randomly divided into four groups (n=7): control, exercise, diabetes and exercise + diabetes. Diabetes was induced by injection of streptozotocin in male rats. Rats in the trained groups were subjected to voluntary running wheel exercise for 6 weeks. At the end of six weeks blood and heart tissue samples were collected and used for determination of antioxidant enzymes (including SOD, GPX and CAT activities) and MDA level. RESULTS: Exercise significantly reduced MDA levels both in the heart tissue (p<0.01) and blood samples (p<0.05). In addition, exercise significantly increased SOD (p<0.05), GPX (p<0.001) and CAT (p<0.05) in the heart tissue. Voluntary exercise also significantly increased SOD (p<0.01), GPX (p<0.05) and CAT (p<0.001) in the blood. CONCLUSION: Voluntary exercise diminishes the MDA level in blood and heart tissue of diabetic rats. It also accentuates activities of SOD, GPX and CAT. Therefore, it may be considered a useful tool for the reduction of oxidative stress in diabetes.

Caloric restriction prevents lead-induced oxidative stress and inflammation in rat liver.

The aim of the present study is to investigate the effects of caloric restriction on liver of lead-administered rat. Male Sprague-Dawley rats were randomly divided into two groups: Ad libitum fed group (AL, free access to normal rat chow) and caloric restriction group (CR, fed 65% of AL animals' food intake). After 6 weeks, half of the animals of each group were injected lead acetate and the other half were injected saline. Liver tissue samples were collected at the end of the experiments. Glutathione peroxidase (GPx), superoxide dismutase (SOD), malondialdehyde (MDA), and tumor necrosis factor (TNF-α) were measured in the tissue extracts. Histological studies were also performed. Our results showed that lead administrations (not saline injections) reduced liver SOD and GPx and increased MDA and TNF-α in AL animals, but in the CR animals lead injections did not significantly change the measured parameters. The histological studies supported the biochemical findings. We concluded that 65% CR may prevent lead-induced oxidative stress and inflammation in rat liver.

Voluntary exercise prevents lead-induced elevation of oxidative stress and inflammation markers in male rat blood.

Regular mild exercise enhances antioxidant and anti-inflammatory systems of the body. The present study investigates voluntary exercise effects on lead toxicity as a known oxidative stressor. Male Sprague-Dawley rats were randomly divided into 2 groups. Sedentary control: the animals were housed 7 weeks in the regular cages. Exercise group: the animals were housed 7 weeks in the running wheel equipped cages, that is, the animal model of voluntary exercise. During the 7th week, all animals were administered lead acetate. Blood samples were collected at the end of the 6th week and 7th week (before and after lead administrations). Glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and tumor necrosis factor (TNF-α) were measured in the samples. Our results showed that lead administration reduced blood SOD, GPx and CAT and increased TNF-α; in the controls, but in the exercise group, changes were not statistically significant. MDA in both groups increased after lead injections but it was significantly lower in exercise group compared to the sedentary animals. We concluded that voluntary exercise may be considered as a preventive tool against lead-induced oxidative stress and inflammation.

Oxidative stress status in renal transplant recipients.

OBJECTIVES: Despite the demonstration of oxidative stress in patients with end-stage renal disease, the oxidative status during and after a renal transplant are not completely understood. Hepatocyte growth factor is reported to act as an endogenous factor against oxidative stress. The aim of this study was to evaluate the pattern of changes in plasma oxidative status and hepatocyte growth factor levels in living-donor renal transplant recipients during the early phase after transplant. MATERIALS AND METHODS: Nineteen patients who underwent a renal transplant were included. All were on cyclosporine-based immunosuppression. Plasma levels of malondialdehyde, ferric reducing activity, hepatocyte growth factor, vitamin E, erythrocyte glutathione, and superoxide dismutase activities were determined before, and on the second, seventh, and 12th days after the transplant. RESULTS: High malondialdehyde concentration and low superoxide dismutase activity were seen before and 48 hours after transplant compared with healthy subjects. Significant reductions in plasma ferric reducing activity, malondialdehyde, and hepatocyte growth factor were seen on the seventh and twelfth days after transplant, compared with the before-transplant data. Direct correlations were found between hepatocyte growth factor levels and ferric reducing activity of plasma as well as hepatocyte growth factor and creatinine or uric acid. CONCLUSIONS: Renal transplant recipients display persistent oxidative stress during the early phase of transplant. The pattern of oxidative changes should be considered for appropriate time, dosage, type, and the duration of antioxidant therapy in these patients.

Effects of different periods of renal ischemia on liver as a remote organ.

AIM: To assess the hepatic changes after induction of different periods of renal ischemia. METHODS: Rats were subjected to either sham operation or ischemia (30, 45 and 60 min) followed by 60 min reperfusion. Liver and renal functional indices were measured. Hepatic glutathione (GSH) and ferric reducing antioxidant power levels and the concentration of interleukin (IL)-10 and tumor necrosis factor (TNF-alpha) were evaluated. Portions of liver and kidney tissues were fixed for histological evaluation. RESULTS: Forty-five minutes renal ischemia followed by 60 min reperfusion caused significant changes in liver structure and a significant reduction in renal function. These rats showed a significant decrease in liver GSH, as well as a significant increase in TNF-alpha and IL-10 concentrations. These results demonstrated that renal ischemia caused changes in liver histology, function, oxidative stress and inflammatory status, which led to a reduction in hepatic antioxidant capacity. With 30 min ischemia, the magnitude of these changes was less than those with 45 or 60 min ischemia. CONCLUSION: A minimum of 45 min ischemia is needed to study the effects of renal injury on the liver as a remote organ.

Assessment of plasma antioxidant status in hemodialysis patients.

The risk of atherosclerosis and cancer is high in patients on hemodialysis. A breakdown in the natural balance between the activity of the body's antioxidant system and the production of oxidizing agents is suggested to be involved. To investigate the oxidative stress status in Iranian hemodialytic patients, in this study we evaluated plasma vitamin E, malondialdehyde (MDA), reduced glutathione (GSH), and ferric reducing antioxidant power (FRAP) levels in these patients. Twenty-four hemodialytic patients and 24 control subjects (age and sex matched) were included in this study. Each patient was under dialysis, three times per week, four hours in each session. Before and after dialysis, blood was taken for biochemical measurements as well as oxidative stress tests. There was a significant decrease in FRAP and GSH levels after dialysis comparing to before treatment levels. MDA was increased by dialysis and vitamin E levels were less in dialytic patients, both before and after treatment, compared to controls. This study indicates that there is a significant level of oxidative stress in chronic renal patients and this stress is augmented by dialysis. Antioxidant therapy could be considered in these patients.

Effects of nitric oxide on gentamicin toxicity in isolated perfused rat kidneys.

BACKGROUND: There have been many studies in recent years concerning the role of nitric oxide (NO) in acute renal failure (ARF). In this study, the effects of the inhibition or the induction of NO synthase (NOS) on gentamicin-induced ARF was investigated in isolated perfused rat kidneys. METHODS: Kidneys from male Sprague-Dawley rats were perfused in situ for 90 min. Perfusion was conducted in the presence of inulin (60 mg/dL in perfusion buffer) as a glomerular filtration rate (GFR) marker. Six groups (total: 42 rats) were studied: group 1, controls with no treatment; group 2, L-arginine (2 mM in perfusate); group 3, L-nitro-arginine-methyl ester (L-NAME, 0.1 mM in perfusate); group 4, gentamicin (GM, 0.5 mg/mL in perfusate); group 5, GM + L-arginine (same dose as groups 2 and 4) and; group 6, GM + L-NAME (same dose as groups 3 and 4). Cell injury was assessed by measuring N-acetyl-beta-D-glucosaminidase (NAG), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity in urine. RESULTS: L-arginine prevented, whereas L-NAME enhanced, GM-induced enzyme release and GFR reduction. Histological studies showed that GM-treated kidneys had clear signs of tubular damage and this damage was increased by simultaneous L-NAME and GM administration. CONCLUSION: This study suggests that NO formation could prevent the GM-induced nephrotoxicity in this ARF model.

Beneficial effects of MnTBAP, a broad-spectrum reactive species scavenger, in rat renal ischemia/reperfusion injury.

BACKGROUND: In recent years, several lines of evidence have implicated reactive species as contributors to renal ischemia/reperfusion injury (I/R). This study was designed to investigate the effect of Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a broad-spectrum reactive species scavenger, in the prevention of renal I/R injury. METHODS: Experiments were performed on rats anesthetized with pentobarbital. After tracheotomy, the right femoral artery was cannulated and the mean arterial pressure and heart rate were recorded. A midline laparatomy was performed, and the renal arteries were carefully separated from surrounding tissues. After surgery and a stabilization period (60 min), the animals were randomly assigned to four groups: sham-operated; sham+MnTBAP; I/R; I/R+MnTBAP. In I/R groups, the rats were subjected to bilateral renal artery occlusion for 40 min followed by 6 h reperfusion. Other groups underwent the surgery protocol but did not undergo renal artery occlusion, and were maintained under anesthesia for the duration of the experiment. Rats were administered either MnTBAP (10 mg kg(-1), i.v. bolus, 15 min prior to I/R) or saline. Renal function was assessed by plasma creatinine (Cr), blood urea nitrogen (BUN), and aspartate aminotransferase (AST) measurements. The fractional excretion of Na(+) (FE(Na+)) and urinary N-acetyl-beta-D-glucosaminidase (NAG) activities were also measured. Renal section damage was evaluated by light microscopy, and oxidative stress status was evaluated by measurements of plasma and renal vitamin E levels. RESULTS: We found that MnTBAP significantly reduced the I/R-mediated increases in plasma Cr, BUN, AST, FE(Na+), and NAG and improved the renal tissue histology. CONCLUSION: Our results showed that MnTBAP was effective in preventing the development of I/R-induced renal injury.

Effects of co-supplementation of vitamins E and C on gentamicin-induced nephrotoxicity in rat.

Gentamicin (GM) is an effective antibiotic against severe gram-negative infections. However it can produce nephrotoxicity in human. Reactive oxygen species (ROS) have been proposed as the causative factors of the renal side effects the drug. This study was performed to investigate the protective role of antioxidant vitamins against GM-mediated nephropathy in an in situ model of isolated rat kidney. Male Sprague-Dawley rats were randomly assigned to one of the following groups of seven rats: group 1 (Control) was perfused with Tyrode solution; group 2 (GM), 200 microg ml(-1) GM was added to the perfusate; group 3 (GM + Vit C), as group 2 with vitamin C added to the drinking water for 3 days (200 mg l(-1)) and to the perfusate (100 mg l(-1)); group 4 (GM + Vit E), as group 2 with vitamin E (100 mg (100 g body weight)(-1), i.m.) injected 12 h before the start of the experiment; group 5 (GM + Vit C + Vit E) as group 2 with vitamin E and C co-administered (concentrations and conditions as in groups 3 and 4). To compare the groups, urinary lactate dehydrogenase (LDH), N-acetyle-beta-D-glucosaminidase (NAG) and alkaline phosphatase (ALP) activities, inulin clearance (glomerular filtration rate, GFR) and renal tissue glutathione (GSH) content were measured. GM caused a significant nephrotoxicity demonstrated by an increase in urinary LDH, NAG and ALP activities. Reduction in GSH content and a marked decrease in GFR were observed compared to controls. Vitamin C inhibited the GM-induced increase in urinary enzyme activities but did not show a significant effect on the GSH content or GFR. Vitamin E prevented the GM-induced reduction in GSH level without a significant improvement in GFR. Co-administration of vitamins C and E significantly prevented the GM-induced nephrotoxicity demonstrating by preservation of GFR and GSH levels and prevention of increase in urinary enzyme activities. We conclude that co-administration of moderate doses of vitamins C and E has beneficial effects on renal preservation in GM-induced nephrotoxicity.