A new perspective on the pleiotropic blood pressure improvement effect of sitagliptin: downregulation of miRNA-155 and miRNA-21.

INTRODUCTION: Deterioration of vascular responses is the crucial event in the initiation of cardiovascular problems in hypertension (HT) and diabetes mellitus (DM). A well-known oral antidiabetic, sitagliptin, has pleiotropic effects besides improving glycemic state in type-2 DM. This study aimed to investigate the therapeutic effect of sitagliptin on blood pressure with previously unassessed parameters of well-known pathophysiological processes and especially at the microRNA (miRNA) level where there are many unknowns. METHODS: N-nitro-L-arginine methyl ester (L-NAME)-induced HT model was performed on nondiabetic male rats. Four groups (including 7 rats in each) were formed: normotensives, sitagliptin-treated, HT and sitagliptin-treated HT. Asymmetric dimethylarginine (ADMA), intercellular adhesion molecule-1 (ICAM-1) and tyrosine hydroxylase (TH), HT related miRNAs were evaluated. In-vitro vessel responses were observed. RESULTS: L-NAME led to a significant increase in blood pressure. Hypertensives exhibited significantly increased contractile responses, consistent with increased ADMA, ICAM-1. Sitagliptin decreased TH levels but not statistically significantly. The new side of the study was the miRNA-21 and miRNA-155 expressions were in line with other parameters in both the HT and sitagliptin-treated HT groups. CONCLUSION: Sitagliptin may control comorbidities, especially HT and introduces new targets to alleviate vascular responses. The new knowledge is; sitagliptin may show these effects through microRNAs (Tab. 2, Fig. 6, Ref. 46).

Effect of melatonin on torsion and reperfusion induced pathogenesis of rat uterus.

We investigated the use of melatonin to improve fertility and reduce uterine damage caused by torsion of the uterus in pregnant rats. We used 35 pregnant rats at gestational age 18 days. The animals were randomized into five groups. Group 1 was anesthetized only. Group 2 was subjected to experimental uterine torsion of 360° and the torsion was corrected after 6 h. Group 3 was subjected to uterine torsion of 360°, the torsion was corrected after 6 h and melatonin was administered at the time of correction. Group 4 rats were subjected to 360º uterine torsion and melatonin was administered 6 h later at the time of correction. Group 5 was administered melatonin followed by uterine torsion of 360 degrees followed by correction of torsion 6 h later. Samples were obtained from the uterine horns on the day 1 postpartum. We used Bax, Bcl-2 and caspase 3 staining to measure apoptosis in the uterine tissues. The mRNA levels of Rho-associated, coiled-coil containing protein kinases 1 (ROCK1), homeobox D10 (Hox4 HoxD10), TLR4, NFκB1, caveolin 1 (Cav1) heat shock protein 90 alpha (cytosolic), class B member 1 (Hsp90ab1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using quantitative real-time polymerase chain reaction analysis (qRT-PCR). Bax, Bcl-2 and caspase 3 were detected using immunohistochemistry. No difference was observed among groups with respect to abortion, neonatal mortality or congenital abnormalities. Compared to the control group, the mRNA levels of Rock1, Hox4, TLR4, NFκB1, Cav1 and Hsp90 genes were decreased significantly in the study groups; the decrease was greater in groups 3 and 4, which were treated with melatonin. The greatest amount of Bax staining was found in group 1 and the least amount of Bcl-2 staining was found in groups 4 and 5; the greatest amount of caspase 3 staining was found in group 2. Our findings indicate that melatonin reduced uterine torsion related tissue damage and that its application during torsion was more effective than application following removal of torsion.

Effects of apocynin, an NADPH oxidase inhibitor, on levels of ADMA, MPO, iNOS and TLR4 induced by myocardial ischemia reperfusion.

PURPOSE: In this study, the effects of apocynin, an NADPH oxidase inhibitor, on the levels of inducible nitric oxide synthase (iNOS) and the toll-like receptor 4 (TLR4), which are inflammatory mediators in myocardial ischemia-reperfusion (MIR) injury, and myeloperoxidase (MPO), which is the indicator of neutrophil infiltration and the endogenous nitric oxide synthase inhibitor asymmetric dimethyl arginine (ADMA) increasing with oxidative stress were investigated. METHODS: MIR injury was accomplished by the application of occlusion for 30 minutes and reperfusion for 120 minutes in the left anterior descending artery (LAD). In the study, 21 Sprague-Dawley male rats were divided into three groups: a sham group (n = 7); a MIR group (n = 7); and a MIR + apocynin treatment group (n = 7, before the procedure, an intraperitoneal administration of 10 mg/kg of apocynin for 15 days). After reperfusion, iNOS, TLR4, MPO and ADMA levels in myocardial tissue were measured by ELISA. RESULTS: While myocardial TLR4, MPO and ADMA levels increased in the MIR group, these parameters were found to be decreased significantly in the group treated with apocynin. Although iNOS levels showed an increase in the MIR group compared to the sham group and a reduction in the MIR+apocynin group, there was no statistically significant difference between the groups. DISCUSSION: In our study, the effect of the treatment of apocynin in MIR on ADMA, MPO, iNOS and TLR4 levels in myocardial tissue was shown for the first time. It is thought that apocynin treatment may show a protective effect in MIR injury by affecting oxidative stress (ADMA) and inflammatory parameters (iNOS, MPO).

Effects of caffeic acid phenethyl ester on cerebral cortex: structural changes resulting from middle cerebral artery ischemia reperfusion.

Overproduction of free radicals is important in the pathogenesis of the cerebral damage induced by ischemia reperfusion. Caffeic acid phenethyl ester, an active component of propolis extract, exhibits antioxidant properties. The study was carried out in 16 male Wistar albino rats, divided into two groups: ischemia reperfusion and ischemia reperfusion with caffeic acid phenethyl ester. The middle cerebral artery was occluded for 60 min with an intraluminal suture, followed by 24-h reperfusion. In this study, widespread infarcted areas, red neurons (eosinophilic degeneration), pyknotic cells, vacuolization and neuroglial cell infiltration were observed in the cerebral cortex in the ischemia reperfusion group. In the caffeic acid phenethyl ester group, slightly infarcted areas were observed and neuroglial cell infiltration was not determined. Congestion of choroid plexus and pia mater was found more severe in the ischemia reperfusion group than in the caffeic acid phenethyl ester group. In the caffeic acid group, neuroglial cell activation was rare. Vacuolization, an indication of brain edema, was prevented by caffeic acid phenethyl ester. In the present study, we showed that pre-treatment with a single i.p. injection of caffeic acid phenethyl ester at 50 microM/kg dose reduced the structural changes.

Protective effect of caffeic acid phenethyl ester (CAPE) on myocardial ischemia-reperfusion-induced apoptotic cell death.

Occlusion of coronary artery causes cardiomyocyte dysfunction. Reperfusion relieves ischemia by providing cells with metabolites and oxygen, thereby preventing extensive tissue damage. Although reperfusion salvages the myocardium, it also initiates a series of events including myocardial apoptosis and necrosis. The common inducers of apoptosis include reactive oxygen species (ROS). Caffeic acid phenethyl ester (CAPE) is known as an antioxidative, anti-inflammatory effects, may protect myocardial ischemia-reperfusion (MI/R)-induced apoptosis. We have previously reported that CAPE reduced MI/R-induced necrosis. Therefore, this study was focused to investigate protective effect of CAPE on the distinct form of cell death; apoptosis in an in vivo rat model. To produce MI/R, a branch of the descending left coronary artery was occluded for 30 min followed by 2 h reperfusion. ECG changes, blood pressure (BP), and heart rate (HR) were measured before occlusion and continued both occlusion and reperfusion. CAPE (50 micromol/kg) was given 10 min before ischemia via jugular vein. Extensive formation of DNA strand breaks, the typical biochemical feature of apoptosis, was detected with the use of the terminal deoxynucleotidyl transferase (TdT)-mediated d UTP-biotin nick and labeling (TUNEL) method. Also, cysteine aspartate specific proteinase (caspase)-3 and caspase-9 activities a universal effector of apoptosis, were determined. Trunk blood was extracted to determine the serum contents related to oxidant-antioxidant status. In hemodynamic parameters, there was no significant difference in HR or BP values among any group. CAPE administration had no a significant effect on hemodynamic parameters during ischemia or reperfusion. Control group revealed extensive TUNEL-positive cardiomyocytes especially in free wall of left ventricule, interventiculare septum and nearly apex zone. Intensity of TUNEL-positive cardiomyocytes reduced as a result of CAPE treatment compared to control group in the same sections. Result of the caspase activities was found to correlate with TUNEL evaluation. CAPE also, ameliorated antioxidant status. We propose that CAPE acts in the heart as a potent scavenger of free radicals to prevent the apoptotic effect of I/R. Further studies are needed to elucidate the mechanisms of apoptotic death machinery.

Protective effects of melatonin on myocardial ischemia/reperfusion induced infarct size and oxidative changes.

Free radicals, calcium overloading and loss of membrane phospholipids play an important role in the development of ischemia/reperfusion (I/R) injury. Melatonin is a well-known antioxidant and free radical scavenger. Melatonin may also reduce the intracellular calcium overloading and inhibit lipid peroxidation. This study was designed to investigate the effects of melatonin on the I/R-induced cardiac infarct size in an in vivo rat model. We also investigated glutathione (GSH) levels, an antioxidant the levels of which are influenced by oxidative stress, and malondialdehyde (MDA) levels, which is an index of lipid peroxidation. To produce cardiac damage, the left main coronary artery was occluded for 30 min, followed by 120 min reperfusion, in anesthetized rats. Melatonin (10 mg/kg) or vehicle was given 10 min before ischemia via the jugular vein. Infarct size, expressed as the percentage of the risk zone, was found significantly greater in I/R group than in the melatonin-treated I/R group. MDA levels were significantly higher, but GSH levels were lower in the I/R group than in the control group. Melatonin significantly reduced the MDA values and increased the GSH levels. These results suggest that oxidative stress contributes to myocardial I/R injury and melatonin administration exerts a mitigating effect on infarct size. Furthermore, the results indicated that melatonin improves the antioxidant capacity of the heart and attenuates the degree of lipid peroxidation after I/R.

The attenuation of vasospasm by using a sod mimetic after experimental subarachnoidal haemorrhage in rats.

BACKGROUND: Delayed cerebral vasoconstriction and brain ischemia, are critical problems in the management of a patient affected by rupture of an intracranial aneurysm. Overexpression of Cu-Zn superoxide dismutase (Cu-Zn SOD) can reduce the extent of cerebral vasospasm. We, therefore investigated if vasospasm, can be prevented by a novel, stable, and cell permeable SOD mimetic, MnTBAP [Mn(III) tetrakis (4-benzoic acid) porphyrin] which permeates the biological membranes and scavenges superoxide anions and peroxynitrite. METHODS: 28 rats (225-250 g) were divided equally into four groups: group 1: control; group 2: SAH only; group 3: SAH plus placebo; and group 4: SAH plus MnTBAP. We used a double haemorrhage method to produce SAH. Starting six hours after SAH, 5 mg/kg MnTBAP (Calbiochem, Darmstadt-Germany; Cat. No 475870)) or an equal volume of 0.9% saline (37 degrees C) was administered by intraperitoneal injection twice daily for 5 days to groups 4 and 3 respectively. MnTBAP or 0.9% saline injections were continued up to fifth day after SAH and rats were sacrificed on the fifth day. Brain sections at the level of the pons were examined by light microscopy. Planimetric measurements were made for the cross-sectional areas of the lumen and the vessel wall (intima plus media) of the basilar artery by a micrometer. FINDING: Administration of MnTBAP significantly attenuated the vasoconstriction of the basilar artery in group 4 compared with the groups 2 and 3 (p<0.001). INTERPRETATION: These results suggest that this SOD mimetic (MnTBAP) attenuates delayed cerebral vasoconstriction following experimental SAH and that superoxide anions have a role in the pathogenesis of vasospasm after SAH.

The beneficial effect of melatonin on chronic cyclosporin A nephrotoxicity in rats.

Cyclosporin A (CsA)-induced nephrotoxicity may be the consequence of oxidative stress. Anti-oxidant agents could be useful in reducing CsA toxicity. In this light microscopy study, tubular dilatation, atrophy, vacuolization and tubulointerstitial fibrosis were observed in rats given CsA, whereas in rats given CsA plus melatonin, no histological changes occurred. It is concluded that melatonin could be useful for reducing the nephrotoxic effects of CsA.

Effects of captopril and losartan on myocardial ischemia-reperfusion induced arrhythmias and necrosis in rats.

Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II type 1 (AT (1)) receptor blockers improve ischemia-reperfusion induced arrhythmias and infarct size in several animal models. However, the effects of pretreatment with ACEIs or AT (1) receptor blockers on acute myocardial infarct size and arrhythmias are controversial. Thus, we sought to assess the comparative effects of pretreatment with ACEI captopril and AT (1)-receptor blocker losartan on myocardial infarct size and arrhythmias in a rat model of ischemia-reperfusion. We randomly assigned 92 male Wistar rats for arrhythmias ( n= 60) and necrosis ( n= 32) experiments. To produce arrhythmia, the left main coronary artery was occluded for 7 min, followed by 7 min of reperfusion and to produce necrosis, the the left main coronary artery was occluded for 30 min, followed by 120 min of reperfusion. Captopril (3 mg kg (-1)) and losartan (0.2 and 2 mg kg (-1)) were given intravenously 10 min before occlusion. Captopril reduced the incidences of ventricular fibrillation (VF) and mortality associated with irreversible VF, whereas the studied doses of losartan did not. Captopril also decreased the number of ventricular beats on reperfusion. Losartan 2 mg kg (-1) reduced both the number of ventricular premature beats and the incidence of ventricular tachycardia (VT) on reperfusion, while losartan at dose of 0.2 mg kg (-1) had no effect on these arrhythmias. Compared to the control group, both captopril and losartan reduced myocardial infarct size in the rat model of ischemia-reperfusion, but this was statistically significant for captopril only. In this experimental model, although captopril did not reduce the incidence of reperfusion-induced VT, it was more effective than the AT (1)-receptor blocker losartan at preventing mortality associated with irreversible VF and to reduce myocardial infarct size in rat model of ischemia-reperfusion.

Effects of chronic ethanol consumption on alpha-adrenergic-induced contractions and endothelium-dependent relaxations in rat thoracic aorta.

The effects of chronic oral administration of ethanol (7.2% daily during 24 weeks) on the contractions induced by phenylephrine (Phe) and the endothelium-dependent relaxation responses to acetylcholine (ACh) were studied in rat thoracic aorta. Ethanol pretreatment significantly attenuated the contractile responses to Phe, resulting in parallel shift of the concentration-response curve to the right. EC(50)values of Phe were 64.6+/-11.2 and 95.5+/-8.5 nmol l(-1)in control and ethanol-fed rats, respectively. On the other hand, either calcium-induced contractions or relaxation responses to ACh and sodium nitroprusside were similar in the vessels of the control and ethanol-treated rats. These results suggest that chronic ethanol ingestion significantly attenuates the alpha(1)-adrenergic-induced contractions but does not affect the relaxation responses mediated by nitric oxide in rat aortic rings.

Melatonin administration prevents the nephrotoxicity induced by gentamicin.

OBJECTIVE: To investigate the effect of melatonin on the antioxidant enzyme activity and renal tubular necrosis induced by gentamicin. MATERIALS AND METHODS: Twenty-four adult male Sprague-Dawley rats were divided into three equal groups. In group 1, the rats were injected with vehicle (controls), in group 2 they were injected with gentamicin for 5 days and in group 3 injected with gentamicin plus melatonin for 5 days. At 24 h after the last injection, rats were killed and the renal cortex separated from the medulla. Most of the cortex was homogenized but a small sample was fixed in formaldehyde solution for histological examination by light microscopy. Blood samples were also taken to assess the serum levels of urea, creatinine, Na+, K+ and gamma-glutamyl transpeptidase (gamma-GT); before death, urine samples were analysed for protein content. Crude extracts of the cortex were used to determine lipoperoxides, reduced glutathione (GSH-Px), catalase and superoxide dismutase (SOD). The results were compared using the Mann-Whitney U-test. RESULTS: Compared with the controls rats, gentamicin caused hyperproteinuria, an increase in the level of gamma-GT in serum, a marked increase in lipoperoxides and a significant decrease of GSH-Px, catalase and SOD activity in the kidney. In the rats in group 3 there was a marked restoration in lipid peroxidation, GSH-Px, catalase, SOD activity and proteinuria, and in gamma-GT in serum. In rats in group 2 there was widespread tubular necrosis (grade 2-4) but in rats in group 3 there was a marked reduction in the extent of tubular damage. There was no significant difference in serum levels of Na+, K+, blood urea nitrogen and creatinine. CONCLUSION: These results indicate that melatonin prevents the tubular necrosis induced by gentamicin in rats, presumably because it is a potent antioxidant and restores antioxidant enzyme activity in the rat kidney.