In previous studies, it has been shown that recombinant human neuregulin-1(rhNRG-1) is capable of improving the survival rate in animal models of doxorubicin (DOX)-induced cardiomyopathy; however, the underlying mechanism of this phenomenon remains unknown. In this study, the role of rhNRG-1 in attenuating doxorubicin-induce apoptosis is confirmed. Neonatal rat ventricular myocytes (NRVMs) were subjected to various treatments, in order to both induce apoptosis and determine the effects of rhNRG-1 on the process. Activation of apoptosis was determined by observing increases in the protein levels of classic apoptosis markers (including cleaved caspase-3, cytochrome c, Bcl-2, BAX and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining). The activation of Akt was detected by means of western blot analysis. The study results showed that doxorubicin increased the number of TUNEL positive cells, as well as the protein levels of cleaved caspase-3 and cytochrome c, and reduced the ratio of Bcl-2/Bax. However, all of these effects were markedly antagonized by pretreament with rhNRG-1. It was then further demonstrated that the effects of rhNRG-1 could be blocked by the phosphoinositole-3-kinase inhibitor LY294002, indicating the involvement of the Akt process in mediating the process. RhNRG-1 is a potent inhibitor of doxorubicin-induced apoptosis, which acts through the PI3K-Akt pathway. RhNRG-1 is a novel therapeutic drug which may be effective in preventing further damage from occurring in DOX-induced damaged myocardium., T. An, ... [et al.]., and Obsahuje seznam literatury
The effect of three therapeutically used drugs and five polyphenolic compounds on the mechanism of oxidative burst was compared in whole blood and isolated neutrophils at cellular and molecular level. In 10 μM concentration, the compounds investigated decreased the oxidative burst of whole blood in the rank order of potency: N-feruloylserotonin (N-f-5HT) > curcumin (CUR) > quercetin (QUER) > arbutin (ARB) > resveratrol (RES) > dithiaden (DIT) > carvedilol (CARV) > brompheniramine (BPA). The ratio between the percentage inhibition of extracellular versus intracellular chemiluminescence (CL) followed the rank order QUER > N-f-5HT > RES > CUR > DIT and is indicative of the positive effect of the compounds tested against oxidative burst of neutrophils, demonstrating suppression of reactive oxygen species extracellularly with minimal alteration of intracellular reactive oxygen species (ROS). Activation of protein kinase C was significantly decreased by DIT, CUR, QUER and N-f-5HT. CARV, DIT, QUER and ARB reduced activated neutrophil myeloperoxidase release more significantly compared with the effect on superoxide anion generation. All compounds tested increased the activity of caspase-3 in cell-free system. It is suggested that other regulatory mechanisms than protein kinase C might participate in the inhibition of neutrophil activation with the compounds tested. Different mechanisms are concerned in controlling the assembly of NADPH oxidase and the regulatory role of calcium ions is suggested. Compounds decreasing the amount of extracellular ROS generation, yet affecting but minimally intracellular ROS generation, are promising for further investigation in vivo., R. Nosáľ, K. Drábiková, V. Jančinová, T. Mačičková, J. Pečivová, T. Perečko, J. Harmatha, J. Šmidrkal., and Obsahuje bibliografii
Although statins exert non-lipid cardioprotective effects, their influence on cell death is not fully elucidated. For this purpose, we investigated whether simvastatin treatment (S, 10 mg/kg, 5 days) is capable of mitigating ischemia/reperfusion-induced (IR) apoptosis in the isolated rat hearts, which was examined using immunoblotting analysis. In addition, the content of signal transducer and activator of transcription 3 (STAT3) and its active form, phosphorylated STAT3 (pSTAT3-Thr705), was analyzed. Simvastatin induced neither variations in the plasma lipid levels nor alterations in the baseline content of analysed proteins with the exception of upregulation of cytochrome C. Furthermore, simvastatin significantly increased the baseline levels of pSTAT3 in contrast to the control group. In the IR hearts, simvastatin reduced the expression of Bax and non-cleaved caspase-3. In these hearts, phosphorylation of STAT3 did not differ in comparison to the non-treated IR group, however total STAT3 content was slightly increased. The improved recovery of left ventricular developed pressure co-existed with the increased Bcl- 2/Bax ratio. In conclusion, pleiotropic action of statins may ameliorate viability of cardiomyocytes by favouring the expression of anti-apoptotic Bcl-2 and downregulating the proapoptotic markers; however STAT3 does not seem to be a dominant regulator of this anti-apoptotic action of simvastatin., T. Rajtík, ... [et al.]., and Obsahuje seznam literatury
Protein kinases, transcription factors and other apoptosis- and proliferation-related proteins can regulate reproduction, but their involvement in sexual maturation remains to be elucidated. The general aim of the in vivo and in vitro experiments with porcine ovarian granulosa cells was to identify possible intracellular regulators of female sexual maturation. For this purpose, proliferation (expression of proliferating cell nuclear antigen - PCNA, mitogen-activated protein kinases - ERK 1,2 related MAPK and cyclin B1), apoptosis (expression of the apoptotic protein Bax and apoptosis regulator Bcl-2 protein), expression of some protein kinases (cAMP dependent protein kinase - PKA, cGMPdependent protein kinase - PKG, tyrosine kinase - TK) and cAMP responsive element binding protein 1 (CREB-1) was examined in granulosa cells isolated from ovaries of immature and mature gilts. Expression of PCNA, ERK1,2 related MAPK, cyclin B1, Bcl-2, Bax, PKA, CREB-1, TK and PKG in porcine granulosa cells were detected by immunocytochemistry. Sexual maturation was associated with significant increase in the expression of Bcl-2, Bax, PKA, CREB-1 and TK and with decrease in the expression of ERK1,2 related MAPK, cyclin B1 and PKG in granulosa cells. No significant difference in PCNA expression was noted. The present data obtained from in vitro study indicate that sexual maturation in females is influenced by puberty-related changes in porcine ovarian signaling substances: increase in Bcl-2, Bax, PKA, CREB-1, TK and decrease in ERK1,2 related MAPK, cyclin B1 and PKG. It suggests that these signaling molecules could be potential regulators of porcine sexual maturation., A. Kolesarova, A. V. Sirotkin, M. Mellen, S. Roychoudhury., and Obsahuje bibliografii
Excessive production of oxygen free radicals has been regarded as a causative common denominator of many pathological processes in the animal kingdom. Hydroxyl and nitrosyl radicals represent the major cause of the destruction of biomolecules either by a direct reaction or by triggering a chain reaction of free radicals. Scavenging of free radicals may act preventively or therapeutically. A number of substances that preferentially react with free radicals can serve as scavengers, thus increasing the internal capacity/activity of endogenous antioxidants and protecting cells and tissues against oxidative damage. Molecular hydrogen (H2) reacts with strong oxidants, such as hydroxyl and nitrosyl radicals, in the cells, that enables utilization of its potential for preventive and therapeutic applications. H2 rapidly diffuses into tissues and cells without affecting metabolic redox reactions and signaling reactive species. H2 reduces oxidative stress also by regulating gene expression, and functions as an anti-inflammatory and anti-apoptotic agent. There is a growing body of evidence based on the results of animal experiments and clinical observations that H2 may represent an effective antioxidant for the prevention of oxidative stress-related diseases. Application of molecular hydrogen in situations with excessive production of free radicals, in particular, hydroxyl and nitrosyl radicals is relatively simple and effective, therefore, it deserves special attention., J. Slezák, B. Kura, K. Frimmel, M. Zálešák, T. Ravingerová, C. Viczenczová, Ľ. Okruhlicová, N. Tribulová., and Obsahuje bibliografii
Flavonoids, polyphenol derivatives of plant origin, possess a broad range of pharmacological properties. A number of studies have found both pro/anti-apoptotic effects for many of these compounds. For these reasons we investigated whether ProvinolsTM, flavonoids obtained from red wine, have anti-apoptotic properties. The investigations have been carried out in rats treated with Cyclosporine A (CsA). In particular, four groups of rats have been treated for 21 days with either olive oil (control group), with CsA, with ProvinolsTM, or with CsA and ProvinolsTM simultaneously. Oxidative stress, systolic blood pressure, body weight, biochemical parameters and different markers of pro/anti-apoptotic pathway were measured. CsA produced an increase of systolic blood pressure, a decrease in body weight, serum creatinine levels, urinary total protein concentration and creatinine clearance. Moreover, CsA induced renal alterations and the translocation of Bax and cytochrome c from cytoplasm to mitochondria and vice versa. These changes activated the caspase cascade pathway, that leads to morphological and biochemical features of apoptosis. ProvinolsTM restored morphological and biochemical alterations and prevented nephrotoxicity. In conclusion, this study may augment our current understanding of the controversial pro-/anti-apoptotic properties of flavonoids and their molecular mechanisms., R. Rezzani ... [et al.]., and Obsahuje seznam literatury
Remodeled pulmonary arteries return to normal structural conditions after the increase in pulmonary artery flow resistance is reversed. We studied whether proteolysis of extracellular matrix proteins and apoptosis occur during reversal of remodeling produced by chronic hypoxia in the rat. Main pulmonary arteries were removed at different times during a 10-day period of exposure to 10% O2 and 14 days after return to air. Content and rates of degradation of collagen and elastin as well as immunoreactive collagenase in tissue and isolated mast cells were measured. Immunoblots for collagenase and tissue inhibitor of metalloproteinases (TIMP) were performed. Apoptosis was assessed by cleavage of DNA and TUNEL assay. Excess collagen and elastin present at 10 days of hypoxia decreased to near normal levels after 3-5 days of air. Transient increases in collagenolytic and elastolytic enzyme activities accompanied the rapid decrease in matrix proteins. Mast cells containing collagenase accumulated in remodeled pulmonary arteries, and the active form of collagenase appeared at the time of peak proteolytic activity. TIMP increased during remodeling. Apoptosis was maximal 3 days after return to air. Our results suggest that activation of enzymes, which degrade matrix proteins, and apoptosis play a role in resolution of vascular remodeling., D. J. Riley, S. Thakker-Varia, F. J. Wilson, G. J. Poiani, C. A. Tozzi., and Obsahuje bibliografii
In vitro models serve as a tool for studies of steatosis. Palmitic and oleic acids can induce steatosis in cultured hepatocytes. The aim of our study was to verify steatogenic and cytotoxic effects of palmitic acid (PA), oleic acid (OA) and their combinations as well as their impact on functional capacity of rat primary hepatocytes. Hepatocytes were exposed to OA or PA (0.125-2 mmol/l) or their combination at ratios of 3:1, 2:1 or 1:1 at the final concentrations of 0.5-1 mmol/l. Both OA and PA caused a dose-dependent increase in triacylglycerol content in hepatocytes. PA was more steatogenic at 0.25 and 0.5 mmol/l while OA at 0.75 and 1 mmol/l. PA exhibited a dose-dependent cytotoxic effect associated with ROS production, present markers of apoptosis and necrosis and a decrease in albumin production. OA induced a damage of the cytoplasmic membrane from 1 mM concentration. Mixture of OA and PA induced lower cytotoxicity with less weakened functional capacity than did PA alone. Extent of steatosis was comparable to that after exposure to OA alone. In conclusion, OA or combination of OA with PA is more suitable for simulation of simple steatosis than PA alone., A. Moravcová, Z. Červinková, O. Kučera, V. Mezera, D. Rychtrmoc, H. Lotková., and Obsahuje bibliografii
a1_We investigated the potential neuroprotective effect of transient hypertension on neuronal cell death induced by ischemia-reperfusion. Recovery of neurons, terminally differentiated cells, is almost entirely dependent upon active transcription and repair of DNA damage. We focused on the histochemical detection of distribution of NOR (argyrophylic nucleolar proteins) reflecting nucleolar integrity, immunohistochemical detection of PARP-1 (poly(ADP-ribose) polymerase-1), MADD (mitogen-activated death domain), a protein accumulated in nucleoli upon stimulation by ischemia, the active form of caspase-3, a universal proteolytic enzyme of apoptosis. The terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick-end-labeling method (TUNEL) proved the presence of in situ DNA fragmentation. We used the model of transient focal cerebral ischemia in rats with occlusion of middle cerebral artery. In experimental group of rats, the transient hypertension was induced by constriction of the abdominal aorta. The period of ischemia lasted 15, 30, 60 and 120 min followed by 48 h of reperfusion. We examined the frontal lobe of the ipsilateral hemisphere for apoptosis of neurons and compared it with the intact brain tissue. In normotensive rats with transient focal cerebral ischemia, we found disintegrated nucleoli of cortical as well as subcortical neurons at all investigated periods of ischemia, whereas the neurons of intact animals showed compact nucleoli with a few satellites. Nuclear positivity for MADD and PARP-1 was apparent in the neocortex after 15 min and peaked after 30 min of ischemia. On the other hand, the subcortical neurons showed nuclear positivity after 60 and 120 min. The immunohistochemical reaction for active caspase 3 was apparent after 30 min onwards predominantly in the cortex. The TUNEL staining was distinct after 60 and 120 min., a2_In hypertensive rats, we found nucleolar disintegration, positivity for MADD, PARP-1 and caspase 3 after 30 min cortically and subcortically, followed by TUNEL positive staining of cortical neurons after 60 and 120 min. In summary, we detected delayed activation of neuronal apoptosis in transiently hypertensive rats with focal cerebral ischemia compared to normotensive animals. The apoptotic phenotype was confirmed by a panel of complementary methods showing rapid proteolysis-nucleolar segregation, MADD, PARP-1 and caspase-3 positivity as well as ultimate DNA fragmentation proved by the TUNEL assay., M. Smrčka, M. Horký, F. Otevřel, Š. Kuchtíčková, V. Kotala, J. Mužík., and Obsahuje bibliografii
Genes encoding enzymes involved in fatty acids (FA) and glucose oxidation are transcriptionally regulated by peroxisome proliferator-activated receptors (PPAR), members of the nuclear receptor superfamily. Under conditions associated with O 2 deficiency, PPAR-α modulates substrate switch (between FA and glucose) aimed at the adequate energy production to maintain basic cardiac function. Both, positive and negative effects of PPAR-α activation on myoc ardial ischemia/reperfusion (I/R) injury have been reported. Moreover, the role of PPAR-mediated metabolic shifts in cardioprotective mechanisms of preconditioning (PC) is relatively less investigated. We explored the effects of PPAR-α upregulation mimicking a delayed “second window” of PC on I/R injury in the rat heart and potential downstream mechanisms involved. Pretreatment of rats with PPAR-α agonist WY-14643 (WY, 1 mg/kg, i.p.) 24 h prior to I/R reduced post-ischemic stunning, arrhythmias and the extent of lethal injury (infarct size) and ap optosis (caspase-3 expression) in isolated hearts exposed to 30-min global ischemia and 2-h reperfusion. Protection was associated with remarkably increased expression of PPAR- α target genes promoting FA utilization (medium-chain acyl-CoA de hydrogenase, pyruvate dehydrogenase kinase-4 and carnitine palmitoyltransferase I) and reduced expression of glucose transporter GLUT-4 responsible for glucose transport and metabolism. In addition, enhanced Akt phosphorylation and protein levels of eNOS, in conjunction with blunting of cardioprotection by NOS inhibitor L-NAME, were observed in the WY-treated hearts. Conclusions: upregulation of PPAR-α target metabolic genes involved in FA oxidation may underlie a delayed phase PC-like protection in the rat heart. Potential non-genomic effects of PPAR-α-mediated cardioprotection may involve activation of prosurvival PI3K/Akt pathway and its downstream targets such as eNOS and subsequently reduced apoptosis., T. Ravingerová ... [et al.]., and Obsahuje bibliografii a bibliografické odkazy