Histological investigation of experimentally induced diabetes effects on the distribution of transforming growth factor (TGF beta), nuclear factor kappa b (Nf-KB), heat schock 90 beta (Hsp beta) and E-cadherin proteins in testicular tissue / Investigación histológica de los efectos de la diabetes inducida experimentalmente en la distribución del factor de crecimiento transformante (TGFß), nuclear factor kappa b (Nf-KB), y proteinas heat schock 90ß (Hsp90ß) y E-cadherina en tejido testicular

SUMMARY: Diabetes is a metabolic disorder characterized by high blood sugar levels and it causes complications in many systems, including the reproductive system. As a result of diabetic conditions, one of the mechanisms that can cause repression of reproductive activity is testicular oxidant stress. The identification of diabetes on the cell signaling molecules axis is still under discussion. The aim of this study was to determine the effect of Transforming Growth Factor (TGFβ), Nuclear Factor kappa B (NF-kB), Heat-schock 90β (HSP90β) signal pathways and E-cadherin cell adhesion molecule on infertility in diabetic rat testicular tissue. In our study, includes histological, molecular and biochemical analysis of testicular tissue removed at the end of the 2 weeks experiment period. A total of 14 adult male rats were divided as control and diabetes. No intervention was given to 7 male rats in the control group. For the diabetic group, 7 male rats were injected by intraperitoneal with a single dose of 55 mg/kg streptozotocin (STZ). TGFβ, NF-kB, HSP90β and E-cadherin proteins were immunohistochemically studied to investigate possible tissue damage, inflammatory process, cell stabilization and integrity due to diabetes. In order to determine oxidant stress, lipid peroxidation product malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase (GPx) analyzes were performed. Fibrosis, inflammatory changes and loss of spermatogenetic series are prominent findings in the diabetic group. On analysis of all the samples with immunostaining, in the diabetic group, TGFβ and NF-kB immunoexpression significantly increased, while Hsp90β and E-cadherin immunoexpression significantly decreased compared with control groups. Experimental diabetes was found to cause fibrosis, inflammation, disrupting cell adhesion and stabilization in testicular tissue. These results suggest that cellular therapy studies are needed for possible damage.

RESUMEN: La diabetes es una enfermedad metabólica caracterizada por niveles altos de azúcar en sangre y causa complicaciones en muchos sistemas, incluido el sistema reproductivo. Como resultado de las condiciones diabéticas, uno de los mecanismos que puede causar alteraciones en la actividad reproductiva es el estrés oxidativo testicular. La identificación de la diabetes en el eje de las moléculas de señalización celular aún está en discusión. El objetivo de este estudio fue determinar el efecto del factor de crecimiento transformante (TGFβ), el factor nuclear kappa B (NF-kB), las vías de señalización de Heat-Schock 90b (HSP90β) y la molécula de adhesión celular de E-cadherina sobre la infertilidad en testículo de rata diabética. Al término de dos semanas se realizaron análisis histológico, molecular y bioquímico del tejido testicular extraído. Las 7 ratas macho del grupo control no fueron intervenidas. Para el grupo de diabéticos, 7 ratas macho fueron inyectadas por vía intraperitoneal con una dosis única de 55 mg / kg de estreptozotocina (STZ). Se estudiaron inmunohistoquímicamente las proteínas TGFβ, NF-kB, HSP90β y E-cadherina para investigar el posible daño tisular, el proceso inflamatorio, la estabilización celular y la integridad debido a la diabetes. Para determinar el estrés oxidativo, se realizaron análisis del producto de peroxidación lipídica malondialdehído (MDA), glutatión (GSH) y glutatión peroxidasa (GPx). La fibrosis, los cambios inflamatorios y la pérdida de series espermatogenéticas son hallazgos destacados en el grupo de ratas diabéticas. En el análisis de todas las muestras con inmunotinción, en el grupo diabético, la inmunoexpresión de TGFβ y NF-kB aumentó significativamente, mientras que la inmunoexpresión de Hsp90β y e-cadherina disminuyó significativamente en comparación con los grupos control. Se encontró que la diabetes experimental causa fibrosis, inflamación, alteración de la adhesión celular y estabilización en el tejido testicular. Estos resultados sugieren que son necesarios estudios de terapia celular para verificar posibles daños.

Alterations in L-arginine and inflammatory markers in type 2 diabetic patients with and without microalbuminuria.

Low-grade inflammation is closely involved in the pathogenesis of type 2 diabetes and associated micro- and macrovascular complications. The nitric oxide (NO) precursor L-arginine, is relevant to diverse pathological conditions including type 2 diabetes and its complications. High sensitive-CRP (hs-CRP), neopterin and arginine levels were measured in 46 normoalbuminuric, 45 microalbuminuric type 2 diabetics and in 32 healthy controls in order to assess the relationship between markers of inflammation and L: -arginine. Hs-CRP concentrations were higher in microalbuminuric diabetic patients compared to normoalbuminuric patients and controls. Diabetics had higher serum and urine neopterin levels than controls. Urine neopterin and L-arginine levels differed significantly among diabetic patients with and without microalbuminuria. There were significant positive correlations between hs-CRP and BMI in healthy controls and diabetics with and without microalbuminuria. In microalbuminuric diabetics, hs-CRP correlated with microalbuminuria (MAU). Significant predictors for the development of microalbuminuria were higher postprandial glucose levels, lower creatinine clearance and lower serum L-arginine levels. Assessment of early markers of inflammation and endothelial function, such as neopterin and NO precursor L-arginine, may help to predict incipient nephropathy more accurately in type 2 diabetic patients.

Impact of renal function or folate status on altered plasma homocysteine levels in hypothyroidism.

Hyperhomocysteinemia is an independent risk factor for coronary, peripheral and cerebrovascular diseases. Moderately elevated total homocysteine (tHcy) levels have been reported in patients with overt hypothyroidism. Plasma tHcy concentration is affected by several physiological factors and is elevated under conditions of impaired folate and cobalamin status and in renal failure. The aim of this study was to assess plasma tHcy concentrations and to evaluate the role of potential determinants of plasma tHcy levels in hypothyroid patients. Fasting plasma tHcy, serum homocysteine-related vitamins folate and vitamin B(12), serum cystatin C (CysC) and creatinine, were determined in 22 hypothyroid patients and compared with 25 healthy control subjects. Creatinine clearance (CCr) was calculated using the Cockroft-Gault formula. Plasma tHcy levels were determined by HPLC with fluorescence detection and serum CysC by automated particle enhanced immunoturbidimetry. Plasma tHcy, creatinine levels were significantly higher, and serum CysC levels, and creatinine clearance values were lower in hypothyroid patients than in control subjects. Folate levels were lower in hypothyroidic group compared to the control group. There were no differences in vitamin B(12) levels between hypothyroid and control groups. Positive correlation was noted between tHcy and creatinine levels in hypothyroid patients (r = 0.596); however, an inverse correlation was found between tHcy and folate levels (r = -0.705) in hypothyroid patients. In conclusion, tHcy was increased in hypothyroidism, and this increase was more strongly associated with changes in serum folate than in serum creatinine and CysC, suggesting an altered folate status.

Endothelial functions in Behçet's disease.

Behçet's disease (BD) is a systemic vasculitis, capable of involving all types of vessels. Endothelial dysfunction (ED) has been previously documented in BD. The aim of the study was to see whether ED was more prominent in Behçet's patients with vascular involvement (VI) than in those without. The study population consisted of 65 patients with BD, 27 of whom had VI, and 30 healthy controls. High-resolution ultrasound was used to measure endothelium-dependent vasodilatation (EDVD) of brachial artery. Overall, regardless of VI, EDVD was significantly impaired in patients with BD compared with controls (11.4 +/- 6.3 vs 20.4 +/- 9.1%,P = 0.001); however, EDVD was similar in BD patients with and without VI (9.7 +/- 6.3% vs 12.6 +/- 6.1%, P = 0.07). Patients with BD had significantly-higher plasma homocysteine levels than controls (13 +/- 6 micromol/L vs 9 +/- 3 micromol/L, P = 0.001). Plasma homocysteine levels were significantly higher in the subgroup with VI than in those without (15 +/- 7 micromol/L vs 12 +/- 4 micromol/L, P = 0.03); however, we found no positive/significant correlation between plasma homocysteine levels and EDVD. ED is a constant feature of BD, regardless of VI. Etiology of ED in BD is probably multifactorial, including high homocysteine levels. As both ED and elevated homocysteine levels may represent an early atherosclerotic process, a more structured approach to risk factor assessment is needed in BD.

Effects of L-arginine and L-carnitine in hypoxia/reoxygenation-induced intestinal injury.

BACKGROUND: This study was designed to show the role of oxidative stress, nitric oxide and glutathione-related antioxidant enzymes in hypoxia/reoxygenation (H/R)-induced intestinal injury model in mice and to evaluate the potential benefits of arginine and carnitine supplementation. METHODS: A total of 28 young Balb/c mice were divided into four groups: Group 1 (untreated) was given physiological saline before the experiment; group 2 H/R mice were supplemented with L-arginine; group 3 H/R mice were given L-carnitine for 7 days; and group 4 mice served as controls. At the end of day 7, H/R injury was induced and intestinal tissue malondialdehyde (MDA), nitrate levels and glutathione peroxidase (GSH-Px), glutathione reductase (GR) and glutathione-S-transferase (GST) activities were measured. RESULTS: MDA levels were higher in the untreated animals than in the other three groups. MDA levels were higher in the L-arginine-treated animals than in the L-carnitine-treated animals. Nitrate levels were found to be increased in the L-arginine-treated group when compared to the controls. GSH-Px and GR activities were increased in the untreated, the L-arginine and the L-carnitine-treated H/R groups when compared to the control group. GST activities were indifferent between the groups. CONCLUSIONS: Oxidative stress contributes to the pathogenesis of H/R-induced intestinal injury. The glutathione redox cycle may have a crucial role in the H/R-induced intestinal injury. L-arginine and L-carnitine supplementations ameliorate the histological evidence of H/R-induced intestinal injury and decrease lipid peroxidation but do not alter the glutathione-related antioxidant enzyme activities.

Amlodipine and glutathione cycle in hypercholesterolaemia.

OBJECTIVE: Effects of amlodipine on lipid peroxidation and alterations in glutathione and related enzymes in blood and aortic tissue were investigated in a cholesterol-induced atherosclerotic rabbit model. METHODS AND RESULTS: New Zealand white male rabbits were fed with regular chow (group I), chow supplemented with I% cholesterol (group II), regular chow plus amlodipine 5 mg/kg/day p.o. (group III) and I% cholesterol diet supplemented with amlodipine (group IV) for 8 weeks. Cholesterol, malondialdehyde (MDA), reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione peroxidase (GSH-PX) and glutathione reductase (GSH-Rd) were determined in blood samples drawn before and after the experimental period. Aortic tissue was examined morphologically for atherosclerotic changes and tissue cholesterol, MDA, GSSG, GSH-PX, GSH-Rd and glutathione-S-transferase (GST) were measured. After 8 weeks, blood cholesterol, MDA, GSSG and GSH-PX were elevated in groups II and IV; GSH was reduced in group IV; MDA levels were higher in group II than in group IV. Aortic tissue investigations revealed higher cholesterol and MDA concentrations in group II than in group IV. Morphological examination of aortic tissues exhibited endothelial disarrangement and lipid deposition in group II. Histopathological alterations related to atherogenesis were less in group IV than in group II. CONCLUSIONS: Amlodipine reduced the increase in oxidative stress by inhibiting excessive MDA production. Accelerated glutathione redox cycle activity of erythrocytes from animals supplemented with amlodipine suggests that this drug may reduce oxidative stress by enhancing the glutathione system. However, this drug does not seem to affect the glutathione redox cycle in the aortic tissue.

Serum and urinary nitric oxide in Type 2 diabetes with or without microalbuminuria: relation to glomerular hyperfiltration.

BACKGROUND: Glomerular hyperfiltration is considered as one of the pathophysiological mechanisms for the development of diabetic nephropathy. Oxidative stress is enhanced in patients with diabetes mellitus. Reportedly, nitric oxide (NO) might be involved in the pathogenesis of hyperfiltration. We investigated the relationship between hyperfiltration and NO system, and malondialdehyde (MDA) levels in Type 2 diabetics with/without microalbuminuria. METHODS: In 39 microalbuminuric, 29 normoalbuminuric Type 2 diabetic patients and 32 healthy controls, serum creatinine, nitrite, nitrate, urinary microalbumin, nitrite, nitrate, plasma MDA and estimated glomerular filtration rate (EGFR) values, calculated according to the Cockcroft and Gault formula, were recorded. RESULTS: Serum and urine NO levels were higher in both microalbuminurics and normoalbuminurics than controls. There were no significant differences in EGFR between groups. However, hyperfiltration was determined in 31% of normoalbuminurics and 20% of microalbuminurics. Serum and urine NO levels were higher in patients with hyperfiltration. Plasma MDA levels were significantly elevated in both microalbuminurics and normoalbuminurics when compared with controls. Serum glucose and microalbuminuria were positively correlated in microalbuminuric diabetics. Serum NO levels were also positively correlated with EGFR in both normoalbuminurics and microalbuminurics. HbA1c levels were positively correlated with both urinary albumin excretion and plasma MDA levels in normoalbuminuric diabetics. CONCLUSIONS: Hyperglycemia is associated with an increased NO biosynthesis and lipid peroxidation. Increased oxidative stress may contribute to the high NO levels in Type 2 diabetes. Furthermore, the high NO levels may lead to hyperfiltration and hyperperfusion, which in turn leads to an increase in urinary albumin excretion and thus causes progression of nephropathy in early Type 2 diabetes.

Determination of elemental composition of substance lost following wear of all-ceramic materials.

PURPOSE: The aim of this study was to test the possible elemental release of four different all-ceramic materials in a wear machine to predict results about their long-term behavior in the oral environment. MATERIALS AND METHODS: Four different all-ceramic materials with different chemical compositions were selected for the wear testing. A total of 20 cylindric samples, five for each ceramic group, were prepared according to the manufacturers' instructions. These were subjected to two-body wear testing in an artificial saliva medium under a covered unit with a computer-operated wear machine. The artificial saliva solutions for each material were analyzed for the determination of amounts of sodium, potassium, calcium, magnesium, and lithium elements released from the glass-ceramic materials. The differences between and within groups were statistically analyzed with a one-way ANOVA, followed by Duncan tests. RESULTS: The statistical analyses revealed no significant differences among Na, K, Ca, or Mg levels (P > .05) released from the leucite-reinforced groups, while there was a significant (P < .05) increase in Li release from the lithium disilicate group. CONCLUSION: Considerable element release to the artifical saliva medium was demonstrated in short-term wear testing. The lithia-based ceramic was more prone to Li release when compared with other elements and materials.

Effects of the calcium channel blocker amlodipine on serum and aortic cholesterol, lipid peroxidation, antioxidant status and aortic histology in cholesterol-fed rabbits.

Reactive oxygen metabolites and oxidized fatty acids are proinflammatory and are involved in the pathophysiology of atherosclerosis. Amlodipine, a unique third-generation dihydropyridine-type calcium channel blocker, seems to exert atheroprotective effects through its antioxidant properties related to its chemical structure and independent of its calcium channel-blocking effect. In this study, the interactions of amlodipine with major cellular antioxidants were investigated in order to elucidate the mechanisms underlying its atheroprotective effects. New Zealand white male rabbits were fed regular chow (group 1), chow with 1% cholesterol (group 2), regular chow plus 5 mg/kg/day amlodipine per os (group 3) and 1% cholesterol plus amlodipine (group 4) for 8 weeks. Total cholesterol, malondialdehyde (MDA) and vitamin E concentrations and catalase and superoxide dismutase (SOD) activities were determined in blood drawn before and after the experimental period. Aortic tissue was examined for atherosclerotic changes and aortic total cholesterol, MDA, catalase and SOD were determined. At the end of the 8-week treatment period, serum total cholesterol and plasma MDA were elevated in groups 2 and 4. In group 2, serum vitamin E and plasma SOD diminished (p < 0.05) and catalase increased (p < 0.05). In group 4, SOD activity increased at the end of treatment. MDA levels were lower and plasma SOD activities were higher in group 4 than in group 2. Aortic tissue investigations revealed higher total cholesterol and MDA concentrations and catalase activities in group 2 than in group 4, and the highest tissue SOD activity was recorded in group 4 (p < 0.05 for all comparisons). Morphological examination of aortic tissues exhibited endothelial disarrangement and lipid deposition in group 2. Histopathological alterations related to atherogenesis were less in group 4 than in group 2. Amlodipine seems to exert atheroprotective effects by reducing aortic cholesterol accumulation and blood and aortic lipid peroxidation, enhancing SOD activity both in blood and aortic tissue and suppressing the consumption of vitamin E. On the other hand, the suppression of catalase activity in blood and the aorta interferes with the drug's well-known antioxidant effects.

Comparison of the effects of dietary saturated, mono-, and n-6 polyunsaturated fatty acids on blood lipid profile, oxidant stress, prostanoid synthesis and aortic histology in rabbits.

BACKGROUND/AIMS: To compare the effects of saturated, monounsaturated and polyunsaturated n-6 fatty acid-enriched diets on the development of atherosclerosis and thrombosis in New Zealand white male rabbits, 3- to 6-month-old animals were supplemented daily (10 g/100 g diet) with butter (n = 8), olive oil (n = 8) or corn oil (n = 8) by oral administration for 7 weeks. METHODS: Total cholesterol (TC), HDL- (HDL-C) and LDL-cholesterol (LDL-C), triglycerides (TG), apolipoprotein A-1 (ApoA-1), apolipoprotein B (ApoB), lipid peroxides as thiobarbituric acid-reactive substances (TBARS), thromboxane B2 (TXB2) and 6-ketoprostaglandin F(1alpha) (6-ketoPGF(1alpha)) concentrations were determined in blood samples drawn before and after each group was fed the different dietary regimens. Histological examination was performed on the aortic tissues. RESULTS: After 7 weeks, TC, ApoB and TXB2 increased significantly (p < 0.05) in the butter-fed animals compared to pre-experimental concentrations. Olive oil administration lead to a significant (p < 0.05) decrease in TC and ApoB levels. The corn oil-enriched diet decreased TC, LDL-C concentrations, TC/HDL-C ratios and 6-ketoPGF(1alpha) (stable metabolite of prostacyclin-PGI2; p < 0.05 for all) but increased TBARS levels and TXB2/6-ketoPGF(1alpha) ratios. Light microscopic findings were in accordance with these biochemical alterations. CONCLUSION: Although effective in lipid lowering, corn oil increased oxidant stress as evidenced by increased TBARS and induced endothelial damage which lead to a reduction in PGI2 synthesis and consequently to an increase in the TXB2/6-ketoPGF(1alpha) ratio. Olive oil administration did not induce oxidant stress and it had no affect on PGI2 and TXB2 levels which are implicated in platelet aggregation. These findings suggest that oleic acid is more effective than linoleic acid in the protection of endothelial integrity.

Association between homocysteinemia and renal function in patients with type 2 diabetes mellitus.

Homocysteinemia is an independent risk factor for cardiovascular disease, but information on its association with type 2 diabetes and mild renal dysfunction is limited. Plasma total homocysteine (tHcy) concentration is partly determined by renal plasma clearance. Serum cystatin C (Cys C) concentration has been introduced as a marker of renal function, specifically as an indicator of glomerular filtration rate (GFR). The aim of this study was to explore the relationships among tHcy, creatinine clearance (Ccr), serum Cys C, and microalbuminuria in a population with type 2 diabetes. Fasting plasma tHcy, serum homocysteine-related vitamins (folate and vitamin B12), serum Cys C, serum creatinine, urine microalbumin, and creatinine clearance were determined in 75 type 2 diabetic patients and 40 healthy control subjects. The patients were assigned to two groups based on urinary albumin excretion (UAE): normoalbuminuric (NAU, UAE < 30 mg/24 hr, n = 35) and microalbuminuric (MAU, UAE 30-300 mg/24 hr, n = 40). Ccr was calculated using the Cockroft-Gault formula. Plasma Hcy levels were determined by HPLC with fluorescence detection and serum Cys C by automated particle enhanced immunoturbidimetry. Plasma tHcy levels were significantly higher in normoalbuminuric and microalbuminuric patients than in controls (10.64 +/- 0.53, 13.29 +/- 0.78, 6.91 +/- 0.37 mmol/L, respectively). Serum Cys C levels in microalbuminuric diabetics were higher than in normoalbuminurics and controls (1.36 +/- 0.06, 1.12 +/- 0.04, 1.10 +/- 0.06 mg/ L, respectively). Positive correlations were noted between tHcy and Cys C levels in normoalbuminuric and microalbuminuric diabetics (r = 0.72, r = 0.64, respectively). Homocysteine and creatinine concentrations were correlated in both diabetic groups (r = 0.89, r = 0.93, NAU and MAU, respectively). Elevated plasma total homocysteine concentrations in type 2 diabetics suggest an association between homocysteinemia and deterioration of renal function, evidenced by increased serum creatinine and Cys C, Ccr, and microalbuminuria. These findings implicate homocysteinemia in the relationship between diabetic nephropathy and cardiovascular complications of diabetes.