Impact of improving eating habits and rosmarinic acid supplementation on rat vascular and neuronal system in the metabolic syndrome model.

Decreasing high fat and high carbohydrate intake, together with the administration of natural bioactive drugs, is assumed to have a protective effect in the prevention and amelioration of the metabolic syndrome (MetS). The aim of the study was to evaluate effects of diet improvement and/or a phenolic compound (rosmarinic acid; RA) administration (100 mg/kg per d) on metabolic as well as functional changes of vessels and hippocampus caused by the MetS-like conditions. The MetS-like conditions were induced by a high-fat-fructose diet (HFFD) in Prague hereditary hypertriacylglycerolaemic (HTG) rats. The effect of diet improvement and RA administration was studied using biochemical and functional measurements. Consumption of HFFD by HTG rats resulted in the development of conditions like the MetS. The fat and fructose restriction from the diet led to amelioration of basic indicators of metabolic state in rats fed HFFD and to amendment parameters of glucose tolerance test and reduction of the IL-1ß serum levels. Moreover, aortic endothelial function was improved with an impact on blood pressure. The functional measurement of electrophysiology of the hippocampus showed that long-term potentiation of neuronal transmission course deteriorated after HFFD was improved by energy restriction. Oral administration of RA had a supporting effect not only on lipid and glucose metabolism but also on the vascular endothelium. Combination of both types of therapy induced beneficial effect on glucose tolerance and lipid peroxidation. Thus, combined improvement of diet habits and treatment with natural bioactive drugs is assumed to have protective effect in prevention and amelioration of the MetS.

Effect of plant polyphenols on ischemia-reperfusion injury of the isolated rat heart and vessels.

In the present study, we investigated the potential protective effect of selected natural substances in a rat model of heart and mesenteric ischemia-reperfusion (I/R). Experiments were performed on isolated Langendorff-perfused rat hearts, subjected to 30-min global ischemia, followed by 30-min reperfusion. Arbutin, curcumin, rosmarinic acid and extract of Mentha x villosa were applied in the concentration of 1 × 10⁻5 mol/l 10 min before the onset of ischemia and during reperfusion, through the perfusion medium. Mesenteric ischemia was induced by clamping the superior mesenteric artery (SMA) for 60 min, subsequent reperfusion lasted 30 min. Production of reactive oxygen species (ROS) by SMA ex vivo was determined by luminol-enhanced chemiluminiscence (CL). The effect of the substances was tested after their incubation with tissue. Curcumin and extract of Mentha x villosa were found to be the most effective in reducing reperfusion-induced dysrhythmias--ventricular tachycardia and fibrillation. This effect was accompanied by bradycardic effect. The mesenteric I/R induced an increase in CL in vascular tissue which was dampened by substances tested. All substances tested were found to have antioxidant properties, as demonstrated by a reduction in ROS production in mesenteric vessels. This effect was confirmed in curcumin and extract of Mentha x villosa which reduced reperfusion dyshythmias.

Metabolic Syndrome in Hypertriglyceridemic Rats: Effects of Antioxidants.

Long-lasting disturbances in lipid and glucose metabolism present in metabolic syndrome (MetS) lead to serious cardiovascular diseases. The study was aimed to evaluate the effect of natural antioxidant vitamin E (VitE, 100 mg/kg/day, p.o.) on basal biochemical and physiological parameters characterizing MetS and on the changed function of the heart. Furthermore, the possible potentiation of VitE effect by synthetic pyridoindole antioxidant SMe1EC2 (SMe, 15 mg/kg/day, p.o.) was also tested. MetS was induced in hereditary hypertriglyceridemic rats (HTG) by the 5 weeks administration of high-fat fructose diet (HFFD: 1 % cholesterol, 7.5 % pork lard, 10 % fructose). The heart function was tested using Langendorff preparation under constant pressure. The functional parameters of isolated heart, dysrhythmias and evoked fibrillations were evaluated in conditions of ischemia-reperfusion. The HFFD increased body weight gain and serum levels of total cholesterol, low-density lipoproteins and blood glucose. The HFFD significantly increased heart flow and force of contraction, compared to standard diet (SD). During the reperfusion, the HFFD caused the increase of the ventricular premature beats number at the expense of decreasing the duration of serious dysrhythmias (ventricular tachycardias and fibrillations). The addition of VitE, SMe or their combination to the HFFD decreased body weight gain, depressed blood pressure, improved particular biochemical parameters. The combination of VitE and SMe suppressed the occurrence of serious dysrhythmias. Our data indicate that the HFFD-related disturbances led to alterations within pathophysiology in HTG rats. The results showed that a combination of antioxidants might have the potential to amend disorders accompanying MetS.

Synthetic Pyridoindole and Rutin Affect Upregulation of Endothelial Nitric Oxide Synthase and Heart Function in Rats Fed a High-Fat-Fructose Diet.

Metabolic syndrome (MetS) belongs to the serious health complications expanding in cardiovascular diseases, obesity, insulin resistance, and hyperglycemia. In this study, hypertriacylglycerolemic rats fed a high-fat-fructose diet (HFFD) were used as an experimental model of MetS to explore the effect of tested compounds. Effects of a new prospective pyridoindole derivative coded SMe1EC2 and the natural polyphenol rutin were tested. Endothelial nitric oxide synthase (NOS3) and nuclear factor kappa B (NF-?B) expression were assessed in the left ventricle immunohistochemically and left ventricle activity was monitored in isolated perfused rat hearts. NOS3 activity in the left ventricle decreased markedly as a result of a HFFD. NOS3 expression was upregulated by both substances. NF-?B expression was increased in the MetS group in comparison to control rats and the expression further increased in the SMe1EC2 treatment. This compound significantly improved the coronary flow in comparison to the control group during reperfusion of the heart followed after ischemia. Further, it tended to increase left ventricular systolic pressure, heart product, rate of maximal contraction and relaxation, and coronary flow during baseline assessment. Moreover, the compound SMe1EC2 decreased the sensitivity of hearts to electrically induced ventricular fibrillation. Contrary to this rutin decreased coronary flow in reperfusion. Present results suggest that despite upregulation of NOS3 by both substances tested, pyridoindole SMe1EC2 rather than rutin could be suitable in treatment strategies of cardiovascular disorders in MetS-like conditions.

Carotenoids produced by yeast biomass protect mechanisms regulating endothelial barrier function from lipopolysaccharide-induced damage in the rat heart.

Expression of occludin and junctional adhesion molecule A (JAM-A), transmembrane proteins of tight junctions (TJs), was analysed to characterize endothelial paracellular permeability in the heart of rats subjected to a bolus of bacterial lipopolysaccharide (LPS) at a dose of 1 mg/kg. Potential protective effects of natural carotenoids (10 mg/kg/day) produced by yeast biomass Rhodosporidium kratochvilovae on expression of occludin and JAM-A also examined in LPS-injected rats (n = 6 per group). LPS decreased expression of occludin by 40% (P < 0.01), JAM-A by 36% (P < 0.001) and increased plasma levels of malondialdehyde (MDA) and lysosomal N-acetyl-b-D-glucosaminidase (NAGA) compared to controls. Ten-day diet rich in yeast biomass containing carotenoids (torularhodin, torulene, ß-carotene) attenuated LPS-induced changes in expression of TJ proteins as observed by increased expression of occludin by 30% (P < 0.05) and JAM-A by 61% (P < 0.001) to the control values. Carotenoids also reduced oxidative stress and cellular injury indicated by decreased levels of MDA and NAGA. The results show that diet rich in yeast biomass containing natural carotenoids could protect mechanisms regulating paracellular endothelial barrier function from LPS-induced damage in the heart.

Sulodexide improves endothelial dysfunction in streptozotocin-induced diabetes in rats.

Diabetes mellitus is associated with many complications including retinopathy, nephropathy, neuropathy and angiopathy. Increased cardiovascular risk is accompanied with diabetes-induced endothelial dysfunction. Pharmacological agents with endothelium-protective effects may decrease cardiovascular complications. In present study sulodexide (glycosaminoglycans composed from heparin-like and dermatan fractions) was chosen to evaluate its protective properties on endothelial dysfunction in diabetes. Effect of sulodexide treatment (SLX, 100 UI/kg/day, i.p.) in 5 and 10 weeks lasting streptozotocin-induced diabetes (30 mg/kg/day, i.p. administered for three consecutive days) was investigated. Animals were divided into four groups: control (injected with saline solution), control-treated with sulodexide (SLX), diabetic (DM) and diabetic-treated with sulodexide (DM+SLX). The pre-prandial and postprandial plasma glucose levels, number of circulating endothelial cells (EC) and acetylcholine-induced relaxation of isolated aorta and mesenteric artery were evaluated. Streptozotocin elicited hyperglycemia irrespective of SLX treatment. Streptozotocin-induced diabetes enhanced the number of circulating endothelial cells compared to controls. SLX treatment decreased the number of EC in 10-week diabetes. Acetylcholine-induced relaxation of mesenteric arteries was significantly impaired in 5 and 10-week diabetes. SLX administration improved relaxation to acetylcholine in 5 and 10-week diabetes. Diabetes impaired acetylcholine-induced relaxation of rat aorta irrespective of SLX treatment. Our results demonstrate that SLX treatment lowers the number of circulating endothelial cells and improves endothelium-dependent relaxation in small arteries. These findings suggest endothelium-protective effect of sulodexide in streptozotocin-induced diabetes.

Lipopolysaccharide-induced redistribution of myocardial connexin43 is associated with increased macrophage infiltration in both normotensive and spontaneously hypertensive rats.

We investigated whether changes in gap junction alpha-1 protein (Cx43) expression may be associated with macrophage-induced inflammation in the heart of spontaneously hypertensive rats (SHR). To examine mutual interactions of macrophage infiltration with Cx43 expression and redistribution, we applied a bolus of bacterial lipopolysaccharide (LPS) to SHR and age-matched normotensive Wistar rats. The results demonstrated association of Cx43 downregulation with increased infiltration of cardiac CD-68 macrophages and upregulation of nuclear factor-κB (NFκB) and tumor necrosis factor-α (TNF-α) expression in the heart of SHR. LPS application to SHR caused further degradation and redistribution of Cx43 accompanied with extensively increased macrophage infiltration and NFκB and TNF-α expression. LPS administration to Wistar rats resulted in elevation of cardiac CD-68 macrophages but it did not significantly affect total Cx43 expression. Our results are suggestive of regulation of Cx43 expression with macrophages-related inflammation in the heart of SHR. The data also indicate that SHR can be more sensitive to LPS than are normotensive rats.

Mesenteric ischemia/reperfusion-induced intestinal and vascular damage: effect of stobadine.

This study examined the effects of the pyridoindole compound stobadine on intestinal and vascular injury following mesenteric ischemia/reperfusion (I/R) in rats. Ischemia was induced by occlusion of the superior mesenteric artery (SMA) for 60 min, followed by 30 min reperfusion. To characterize gut impairment, some parameters of intestinal damage and biochemical variables, such as GSH content, activity of a lysosomal enzyme N-acetyl-beta-D-glucuronidase and activity of gamma-glutamyl transpeptidase, were determined. Vascular I/R-induced damage was evaluated as changes in acetylcholine evoked relaxation of mesenteric artery rings under isometric conditions. A method of amplified chemiluminescence (CL) was used to detect production of reactive oxygen species (ROS). Following I/R, pronounced intestinal injury of various intensities was observed, with maximal changes occurring in the terminal ileum. The effect of I/R was expressed mainly as increased vascular permeability, with protein leakage and subsequent hemorrhagic injury of the intestine as well as impaired endothelium-dependent SMA relaxation. Vessel dysfunction was manifested by a decrease of the maximal relaxation response to acetylcholine. An increase of CL, indicative of increased ROS production, was observed in both intestinal and vascular tissue. A novel antioxidant, stobadine, was found to reduce the increased vascular permeability and the extent of small intestine injury caused by I/R, to improve biochemical alterations accompanying I/R, to protect endothelial-dependent relaxation of mesenteric arteries, and to attenuate the CL response. The observed beneficial effect of stobadine indicates its possible application in the preventive and/or therapeutic approach to I/R-induced pathologies.

Changes in the function and ultrastructure of vessels in the rat model of multiple low dose streptozotocin-induced diabetes.

In this study we investigated functional changes in the femoral artery and ultrastructural alterations in mesenteric vessels and capillaries in the rat model of multiple low dose streptozotocin (STZ)-induced diabetes. Participation of oxidative stress in this model of diabetes was established by studying the effect of the pyridoindole antioxidant stobadine (STB) on diabetes-induced impairment. Experimental diabetes was induced by i.v. bolus of STZ (20 mg/kg) given for three consecutive days to male rats. At the 12(th) week following STZ administration, the animals revealed typical signs of diabetes, such as polyphagia, polydypsia and polyuria. There was no weight gain in the diabetic groups throughout the experiment. No exitus occurred in any group. Diabetes was characterised with high levels of plasma glucose, no significant changes in lipid metabolism, decreased serum levels of glutathione, increased serum levels of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (NAGA), injured endothelial relaxant capacity of the femoral artery and alterations in ultrastructure of mesenteric arteries and capillaries. Antioxidant STB in the dose of 25 mg/kg body weight i.p. (5 times per week) did not influence glucose levels, however, it mitigated biochemical, functional and ultrastructural changes induced by diabetes, suggesting a role of reactive oxygen species in diabetes-induced tissue damage.

In vitro inhibition of lens aldose reductase by (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid in enzyme preparations isolated from diabetic rats.

(2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (compound 1), a novel aldose reductase inhibitor, was assayed for efficacy and selectivity to inhibit rat lens aldose reductase under in vitro conditions by using enzyme preparations obtained from diabetic animals. The inhibitory efficiency was characterized by IC(50) in micromolar region. Enzyme kinetics analysis revealed uncompetitive type of inhibition, both in relation to the D,L-glyceraldehyde substrate and to the NADPH cofactor. In testing for selectivity, comparisons to rat kidney aldehyde reductase, an enzyme with the highest homology to aldose reductase, was used. The inhibition selectivity of the compound tested was characterized by selectivity factor around 20 and was even slightly improved under conditions of prolonged experimental diabetes. These findings were identical with those in the control rats. To conclude, the inhibitory mode, efficacy and selectivity of compound 1, a novel aldose reductase inhibitor, was preserved even under the conditions of prolonged STZ-induced experimental diabetes of rats.

Hypertriglyceridemic rats fed high fat diet as a model of metabolic syndrome.

People with metabolic syndrome have higher risk of cardiovascular diseases then those without. The aim of the work was to investigate whether high fat diet administered to Prague hereditary hypertriglyceridemic (HTG) rats can induce signs of metabolic syndrome (MetS). Our results showed that HTG rats fed high fat diet (HTGch) had disturbed glucose metabolism and also lipid metabolism - increased serum triacylglycerols (TAG), total cholesterol (Ch), low-density lipoprotein-Ch (LDL-Ch), and decreased high-density lipoprotein-Ch (HDL-Ch). Their livers proved markers of developing steatosis. Moreover, HTGch had increased blood pressure, yet the vascular endothelium was not significantly damaged. All these changes were accompanied with oxidative stress and tissue damage identified as increased liver concentrations of thiobarbituric acid reactive substances (TBARS) and activity of the lysosomal enzyme N-acetyl-D-glucosaminidase (NAGA). We assume that the model used may be suitable for the study of MetS with no evidence of obesity. Prolongation of the high fat diet duration might have a major impact on all parameters tested, especially on vascular endothelial function.

Omega-3 fatty acids reduce lipopolysaccharide-induced abnormalities in expression of connexin-40 in aorta of hereditary hypertriglyceridemic rats.

Omega-3 fatty acids (omega3FA) are known to reduce hypertriglyceridemia- and inflammation-induced vascular wall diseases. However, mechanisms of their effects are not completely clear. We examined, whether 10-day omega3FA diet can reduce bacterial lipopolysaccharide-induced changes in expression of gap junction protein connexin40 (Cx40) in the aorta of hereditary hypertriglyceridemic (hHTG) rats. After administration of a single dose of lipopolysaccharide (LPS, 1 mg/kg, i.p.) to adult hHTG rats, animals were fed with omega3FA diet (30 mg/kg/day) for 10 days. LPS decreased Cx40 expression that was associated with reduced acetylcholine-induced relaxation of aorta. Omega3FA administration to LPS rats had partial anti-inflammatory effects, associated with increased Cx40 expression and improved endothelium dependent relaxation of the aorta. Our results suggest that 10-day omega3FA diet could protect endothelium-dependent relaxation of the aorta of hHTG rats against LPS-induced damage through the modulation of endothelial Cx40 expression.

Effect of stobadine on cardiac injury induced by ischemia and reperfusion.

The aim of this work was to evaluate the effect of the antioxidant stobadine on ischemia/reperfusion-induced injury of the isolated rat heart. Experiments were performed according to Langendorff. Ischemia was induced by stop-flow lasting 30 minutes and the duration of repefusion was 30 minutes. Reperfusion of the ischemic heart induced dysrhythmias, with the most severe ones occurring in the first minutes of reperfusion. A significant increase in coronary perfusion pressure was observed starting after 15 min of reperfusion. Stobadine (10(-6) M applied 3 minutes before onset of ischemia and during reperfusion) prevented the deleterious effects to develop fully. The protective effect of stobadine observed in our experiments seems to be a consequence of its antioxidant properties.

Reactive oxygen species induced smooth muscle responses in the intestine, vessels and airways and the effect of antioxidants.

Numerous experimental data confirm the importance of reactive oxygen species (ROS) in physiological activities of smooth muscles and in the pathogenesis of various diseases with altered function of smooth muscles. The present study shows that smooth muscles of the intestine, airways and vessels, as well as their epithelium, endothelium and innervations, might be important targets of the ROS action. We demonstrated differences among the actions of various ROS (endogenous, exogenous, produced enzymatically, non-enzymatically) as well as among their actions in different smooth muscle tissues. Our results indicate that ROS are involved in changes in muscle tone, membrane conductance, calcium homeostasis, calcium-dependent processes, as well as in eicosanoid and nitric oxide metabolism. The effects of antioxidative enzymes (superoxide dismutase, catalase), of several drugs of natural origin (e.g. Kampo Medicines) and synthetic agents (e.g. stobadine, nitrosopine, ACE inhibitors) suggest that smooth muscle tissues are useful models to study ROS action and drug intervention in ROS induced injuries.

Effect of dietary supplementation with the pyridoindole antioxidant stobadine on antioxidant state and ultrastructure of diabetic rat myocardium.

Consistent with the postulated role of oxidative stress in the etiology of late diabetic complications, pharmacological interventions based on biological antioxidants have been suggested. The aim of the present study was to investigate the effect of dietary supplementation with the pyridoindole antioxidant stobadine on the myocardial antioxidant status and ultrastructure of streptozotocin-diabetic rats. Diabetic male Wistar rats were fed for 32 weeks a standard diet or a diet supplemented with stobadine (0.05% w/w). Control rats received a standard diet or stobadine-supplemented diet (0.16% w/w). Plasma levels of glucose, cholesterol and triglycerides were increased significantly by diabetes. Activities of superoxide dismutase and catalase were markedly elevated in the diabetic myocardium. Myocardial levels of conjugated dienes increased after eight months of diabetes, in spite of significantly increased myocardial alpha-tocopherol and coenzyme Q9 content. The long-term treatment of diabetic animals with stobadine (i) reduced plasma cholesterol and triglyceride levels yet left the severe hyperglycemia unaffected, (ii) reduced oxidative damage of myocardial tissue as measured by conjugated dienes, (iii) reversed myocardial levels of alpha-tocopherol and coenzyme Q9 to near control values, (iv) reduced elevated activity of superoxide dismutase in the diabetic myocardium, and (v) attenuated angiopathic and atherogenic processes in the myocardium as assessed by electron microscopy examination. These results are in accordance with the postulated prooxidant role of chronic hyperglycemia and provide further evidence that development of pathological changes in diabetic myocardium is amenable to pharmacological intervention by biological antioxidants.

Endothelial protective effect of stobadine on ischaemia/reperfusion-induced injury.

The aim of the present study was to evaluate the influence of the antioxidant stobadine on changes in the reactivity of the rat abdominal aorta induced by ischaemia and reperfusion (I/R). In anaesthetized male rats, in vivo ischaemia was elicited by occlusion of the abdominal aorta for 18 hours; reperfusion lasted 30 minutes. The aortal rings were taken from the reperfused portion. Decreased relaxant response to acetylcholine, as a consequence of endothelial injury, was seen after I/R. We also demonstrated I/R-induced reversible ultrastructural changes both in endothelial and smooth muscle cells, predominantly in the mitochondria. Lipid peroxidation was increased in homogenates of I/R aortae; the concentration of thiobarbituric acid reactive substances (TBARS) increased from a control value of 0.97 +/- 0.03 to 2.57 +/- 0.06 nmol/l/mg protein. Stobadine (2 mg/kg i.v., 5 minutes before starting reperfusion) protected the abdominal aorta against I/R-induced decrease of acetylcholine relaxation, and prevented changes in mitochondria and an increase of TBARS concentration. The protective effect of stobadine seems to be due to its antioxidant properties.

Streptozotocin diabetes-induced changes in aorta, peripheral nerves and stomach of Wistar rats.

In rats with diabetes induced by streptozotocin (STZ), we studied the reactivity of the aorta in response to vasoconstrictor and vasorelaxant agents, changes in conduction velocity in the sciatic nerve, and glutathion (GSH) content in the gastric mucosa as well as the occurrence of spontaneous gastric lesions. STZ-induced diabetes was found to be accompanied by endothelial injury, exhibited by diminished endothelium-dependent relaxation and by increased noradrenaline- and H2O2-induced contraction. Conduction velocity in the nerves from STZ-treated animals was significantly lower compared to that in nerves from control animals. Moreover, gastric hyperaemia, occasional gastric lesions, and a significant depletion of GSH in the gastric mucosa were observed in STZ-treated rats. Our experiments confirmed the suitability of Wistar rats for the model of STZ-induced diabetes.

Effect of a new antiarrhythmic BK 129 on canine vascular smooth muscle in vitro.

The effect of a new carbanilate derivative BK 129, 1-methoxymethyl-2(1-perhydroazepinyl)ethyl ester 2-(n)-pentyloxycarbanilic acid hydrochloride, was tested on preparations of canine dorsal pedal and coronary artery in vitro. The drug produced relaxation of arterial rings both under resting conditions and under precontraction with KCl. BK 129, at a concentration of 10(-5) mol/l, produced a shift to the right of the concentration-response curve of phenylephrine, with a decrease in the slope and a depression of the maximal response. BK 129 inhibited the response to norepinephrine and blocked histamine- and norepinephrine-induced contractions in Ca(2+)-free physiological salt solution. The electrical stimulation-induced contractions of arterial preparation were also inhibited. BK 129 shifted the concentration-response curve of CaCl2 in Ca(2+)-free depolarizing solution to the right in a non-competitive manner. It may be concluded that BK 129 is a local anesthetic which possesses relaxant properties. It appears to inhibit Ca2+ entry into the smooth muscle cell and Ca2+ release from sarcoplasmic reticulum.

Differences in the responses of the canine basilar and dorsal pedal artery to phenylephrine, clonidine and serotonin.

The effects of phenylephrine, clonidine and serotonin were studied on isolated preparations of the canine basilar and dorsal pedal artery. Differences were mainly observed in the responses of the two types of arterial preparations to clonidine and phenylephrine. While in the dorsal pedal artery, alpha 1- and alpha 2-receptors were involved in the response to alpha-agonists, in the basilar artery contractions induced by alpha 2-receptor stimulation prevailed. Moreover, in the latter artery the contractile responses induced by each of the agonists used were more dependent on the presence of extracellular calcium than those in the dorsal pedal artery.