(Pro)renin receptor (PRR) contributes to regulating many physiological and pathological processes; however, the role of PRR-mediated signaling pathways in myocardial ischemia/reperfusion injury (IRI) remains unclear. In this study, we used an in vitro model of hypoxia/reoxygenation (H/R) to mimic IRI and carried out PRR knockdown by siRNA and PRR overexpression using cDNA in H9c2 cells. Cell proliferation activity was examined by MTT and Cell Counting Kit-8 (CCK-8) assays. Apoptosis-related factors, autophagy markers and β-catenin pathway activity were assessed by real-time PCR and western blotting. After 24 h of hypoxia followed by 2 h of reoxygenation, the expression levels of PRR, LC3B-I/II, Beclin1, cleaved caspase-3, cleaved caspase-9 and Bax were upregulated, suggesting that apoptosis and autophagy were increased in H9c2 cells. Contrary to the effects of PRR downregulation, the overexpression of PRR inhibited proliferation, induced apoptosis, increased the expression of pro-apoptotic factors and autophagy markers, and promoted activation of the β-catenin pathway. Furthermore, all these effects were reversed by treatment with the β-catenin antagonist DKK-1. Thus, we concluded that PRR activation can trigger H/R-induced apoptosis and autophagy in H9c2 cells through the β-catenin signaling pathway, which may provide new therapeutic targets for the prevention and treatment of myocardial IRI.
Adiponectin (APN), an adipose tissue-excreted adipokine, plays protective roles in metabolic and cardiovascular diseases. In this study, the effects and mechanisms of APN on biological functions of rat vascular endothelial progenitor cells (VEPCs) were investigated in vitro . After administrating APN in rat VEPCs, the proliferation was measured by methyl thiazolyl tetrazolium (MTT) method, the apoptotic rate was test by Flow cytometry assay, mRNA expression of B-cell lymphoma-2 (Bcl-2) and vascular endothelial growth factor (VEGF) was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR), and protein expression of mechanistic target of rapamycin (mTOR), signal transducer and activator of transcription 3 (STAT3) and phospho-STAT3 (pSTAT3) was analyzed by Western blot. It was suggested that APN promoted the optical density (OD) value of VEPCs, enhanced mRNA expression of Bcl-2 and VEGF, and inhibited cell apoptotic rate. Furthermore, protein expression of pSTAT3 was also increased in the presence of APN. Moreover, APN changed-proliferation, apoptosis and VEGF expression of VEPCs were partially suppressed after blocking the mTOR-STAT3 signaling pathway by the mTOR inhibitor XL388. It was indicated that APN promoted biological functions of VEPCs through targeting the mTOR-STAT3 signaling pathway., Xiaoying Dong, Xia Yan, Wei Zhang, Shengqiu Tang., and Obsahuje bibliografii
Oxidative stress plays an important role in pressure overloadinduced
cardiac remodeling. The purpose of this study was to determine whether apocynin, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, attenuates pressure overload-induced cardiac remodeling in rats. After abdominal aorta constriction, the surviving rats were randomly divided into four groups: sham group, abdominal aorta constriction group, apocynin group, captopril group. Left ventricular pathological changes were studied using Masson’s trichrome staining. Metalloproteinase-2 (MMP-2) levels in the left ventricle were analyzed by western blot and gelatin zymography. Oxidative stress and apoptotic index were also examined in cardiomyocytes using dihydroethidium and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), respectively. Our results showed that abdominal aorta constriction significantly caused excess collagen deposition and cardiac insult. Treatment with apocynin significantly inhibited deposition of collagen and reduced the level of MMP-2. Furthermore, apocynin also decreased the NADPH oxidase activity, reactive oxygen species production and cardiomyocyte apoptotic index. Interestingly, apocynin only inhibited NADPH oxidase activity without affecting its expression or the level of angiotension II in the left ventricle. In conclusion, apocynin reduced collagen deposition, oxidative stress, and inhibited apoptosis, ultimately ameliorating cardiac remodeling by mechanisms that are independent of the renin-angiotensin system.
Apoptosis was induced by treatment of HL-60 cells with C2-ceramide. Apoptotic damage of DNA was detected according to the sub-Gl peak on a flow cytometer, according to the typical morphology and according to the DNA fragmentation "ladder" after gel electrophoresis. It was shown that the apoptotic cleavage followed after G1 blockade of the cell cycle. A high correlation coefficient (rs=0.957) was found between the percentages of G1 blocked cells and apoptotic cells. This high correlation together with the appearance of the sub-Gl peak suggests that the G1 blocked HL-60 cells were subject to apoptotic death. It is deduced that the mechanisms leading to G1 blockade of the cell cycle and activation of apoptosis in HL-60 cells are interconnected.
The effect of phagocytosis of living bacteria on apoptotic DNA changes was examined in pig leukocytes in relation to immune system maturation. Blood samples of pigs (aged 6, 12 and 18 weeks) were cultivated with a suspension of bacterial cells Salmonella typhimurium LB 5000 at 37 °C. In the experimental groups, killed bacteria and microspheric particles were used to detect the influence of the phagocytic process. Phagocytic activity and index were determined in each sample by means of microspheric particles. The ability to kill engulfed microbes (bactericidal capacity) was estimated from the decrease in bacterial colony-forming units (CFU). Samples of cultured cells were taken for DNA analysis at given intervals. DNA ladder assay was used for qualitative apoptotic DNA break detection and the TUNEL AP test was employed for quantification of apoptosis. In 18-week-old animals, spontaneous DNA degradation was observed in the control group without phagocytosis after 8 h. In contrast, cells cultivated with microspheric particles or killed bacteria became apoptotic after 4 h. The rate of apoptotic DNA degradation was decreased in the group exposed to living bacteria. This prolonged survival of phagocytes was also detected in 12-week-old animals, but not at 6 weeks of age. These findings were supported by the ability of phagocytes in 6-week-old animals to engulf microbes, but their killing (bactericidal) ability was significantly decreased in comparison with other stages of immune system maturation. These results suggest that the process of phagocytosis itself is accompanied by activation of the apoptotic program in phagocytic cells of the pig immune system, but the presence of phagocyted living bacteria can delay this activation. The prolonged survival of short-lived cells was only observed in later phases of immune system maturation., E. Matalová, A. Španová, F. Kovářů., and Obsahuje bibliografii
Primary graft dysfunction (PGD) is a life-threatening complication among heart transplant recipients and a major cause of early mortality. Although the pathogenesis of PGD is still unclear, ischemia/reperfusion injury has been identified as a predominant factor. Both necrosis and apoptosis contribute to the loss of cardiomyocytes during ischemia/reperfusion injury, and this loss of cells can ultimately lead to PGD. The aim of our prospective study was to find out whether cell death, necrosis and apoptosis markers present in the donor myocardium can predict PGD. The prospective study involved 64 consecutive patients who underwent orthotopic heart transplantation at our institute between September 2010 and January 2013. High-sensitive cardiac troponin T (hs-cTnT) as a marker of minor myocardial necrosis was detected from arterial blood samples before the donor’s pericardium was opened. Apoptosis (caspase-3, active + pro-caspase-3, bcl-2, TUNEL) was assessed from bioptic samples taken from the right ventricle prior graft harvesting. In our study, 14 % of transplant recipients developed PGD classified according to the standardized definition proposed by the ISHLT Working Group. We did not find differences between the groups in regard to hs-cTnT serum levels. The mean hs-cTnT value for the PGD group was 57.4±22.9 ng/l, compared to 68.4±10.8 ng/l in the group without PGD. The presence and severity of apoptosis in grafted hearts did not differ between grafts without PGD and hearts that subsequently developed PGD. In conclusion, our findings did not demonstrate any association between measured myocardial cell death, necrosis or apoptosis markers in donor myocardium and PGD in allograft recipients. More detailed investigations of cell death signaling pathways in transplanted hearts are required., O. Szarszoi, J. Besik, M. Smetana, J. Maly, M. Urban, J. Maluskova, A. Lodererova, L. Hoskova, Z. Tucanova, J. Pirk, I. Netuka., and Obsahuje bibliografii
We aimed to investigate the effects of brain-derived neurotrophic factor (BDNF) on apoptosis of intestinal epithelial cells (IECs) and alterations of intestinal barrier integrity using BDNF knock-out mice model. Colonic tissues from BDNF+/+ mice and BDNF+/- mice were prepared for this study. The integrity of colonic mucosa was evaluated by measuring trans-mucosa electrical resistance and tissue conductance in Ussing chamber. The colonic epithelial structure was analyzed by transmission electron microscopy. Apoptosis involvement was determined with TUNEL staining, active caspase-3 immunostaining and Western blotting for the protein expression of active caspase-3, Bax and Bcl-2. The expression levels and distribution of tight junction proteins were evaluated by immunohistochemistry or Western blots. Compared with BDNF+/+ mice, BDNF+/- mice displayed impaired integrity and ultrastructure alterations in their colonic mucosa, which was characterized by diminished microvilli, mitochondrial swelling and epithelial cells apoptosis. Altered intestinal barrier function was linked to excessive apoptosis of IECs demonstrated by the higher proportion of TUNEL-positive apoptotic cells and enhanced caspase activities in BDNF+/- mice. Increased expression of Bax and claudin-2 proteins and reduced Bcl-2 and tight junction proteins (occludin, ZO-1 and claudin-1) expression were also detected in the colonic mucosa of BDNF+/- mice. BDNF may play a role in the maintenance of intestinal barrier integrity via its anti-apoptotic properties., Dong-Yan Zhao, Wen-Xue Zhang, Qing-Qing Qi, Xin Long, Xia Li, Yan-Bo Yu, Xiu-Li Zuo., and Obsahuje bibliografii
We investigated the detrimental effects of diabetes on myocardium of pregestational streptozo-tocin (STZ)-diabetic mother rats and their neonates via evaluations of oxidative redox, inflammatory and apoptotic pathways, also aiming to characterize whether calcitriol and/or pomegranate peel extract confer myocardial protection in hyperglycaemic dams and their foetuses via modulation of the Raf/MEK/ERK cascade. Sixty Sprague-Dawley female rats were randomized into five groups (N = 12): control, diabetic, diabetic treated with calcitriol and/or pomegranate peel extract (PPE), and mated with non-diabetic healthy males. After confirmation of pregnancy, treatments were kept until gestational day (E-18). Serum and cardiac tissues of mothers and foetuses were collected and processed for bio-chemical, histopathological, and molecular assess-ments., We observed that, compared to the control, diabetic mothers showed dramatically increased hyperglycaemia and hyperlipidaemia associated with decreased myocardial functions and disrupted maternal performance. Also, diabetic mothers and their neonates exhibited elevated levels of myocardial injury (troponin I, endothelin 1, creatine kinase-MB, lactate dehydrogenase), with increased pro-inflammatory cytokines (interleukin 1, interleukin 1β, transforming growth factor β) and oxidative redox. Concurrently, the MAPK pathway was significantly down-regulated with increased myocardial apoptotic activity. Furthermore, mRNA expression of angio-genic and fibrotic markers was significantly increased. Paradoxically, calcitriol and/or pomegranate peel extract alleviated these diabetic myocardial insults and normalized the aforementioned assayed parameters. Our findings hypothesized that calcitriol and/or pomegranate peel extract exerted cardioameliorative impacts due to their unique anti-oxidative and anti-inflammatory properties, and thus may be a promising treatment that directly targets the secondary myocardial complications of diabetes in dams and their offspring., and Corresponding author: Ahmed Elmansi
The effect of ethanol on the structural development of the central nervous system was studied in offspring of Wistar rats, drinking 20 % ethanol during pregnancy and till the 28th day of their postnatal life. The structural changes in the hippocampus and dentate gyrus were analyzed at the age of 18, 35 and 90 days. A lower width of pyramidal and granular cell layers, cell extinction and fragmentation of numerous nuclei were found in all experimental animals compared to control animals. The extent of neural cell loss was similar in all monitored areas and in all age groups. At the age of 18 and 35 days, the degenerating cells were observed in the CA1 and CA3 area of the hippocampus and in the ventral and dorsal blade of the dentate gyrus. Numerous glial cells replaced the neuronal population of this region. Some degenerating cells with fragmented nuclei were observed at the age of 90 days. Our experiments confirmed the vulnerability of the developing central nervous system by ethanol intake during the perinatal period and revealed a long-lasting degeneration process in the hippocampus and dentate gyrus., M. Milotová, V. Riljak, K. Jandová, J. Bortelová, D. Marešová, K. Pokorný, M. Langmeier., and Obsahuje bibliografii a bibliografické odkazy
Mammalian Meckel´s cartilage is a temporary structure
associated with mandible development. Notably, its elimination is
not executed by apoptosis, and autophagy was suggested as the
major mechanism. Simultaneous reports point to pro-apoptotic
caspases as novel participants in autophagic pathways in general.
The aim of this research was to find out whether activation of
pro-apoptotic caspases (-2, -3, -6, -7, -8 and -9) was associated
with autophagy of the Meckel´s cartilage chondrocytes. Active
caspases were examined in serial histological sections of mouse
mandible using immunodetection and were correlated with
incidence of autophagy based on Beclin-1 expression. Caspase-2
and caspase-8 were found in Beclin-1 positive regions, whereas
caspase-3, -6, -7 and -9 were not present. Caspase-8 was further
correlated with Fas/FasL and HIF-1α, potential triggers for its
activation. Some Fas and FasL positivity was observed in the
chondrocytes but caspase-8 activation was found also in FasL
deficient cartilage. HIF-1α was abundantly present in the
hypertrophic chondrocytes. Taken together, caspase-8 activation
in the Meckel´s cartilage was demonstrated for the first time.
Caspase-8 and caspase-2 were the only pro-apoptotic caspases
detected in the Beclin-1 positive segment of the cartilage.
Activation of caspase-8 appears FasL/Fas independent but may
be switched on by HIF-1α.