Long‐ and short‐term effects of androgens in human umbilical artery smooth muscle

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7‐difluoromethyl‐5,4′‐dimethoxygenistein,a novel agent protecting against vascular endothelial injury caused by oxidative stress

1. Genistein is known to protect the vascular endothelium. However, genistein exhibits poor bioavailability, which limits its use in the treatment of cardiovascular diseases. 7‐Difluoromethyl‐5,4′‐dimethoxygenistein (dFMGEN), prepared by the difluoromethylation and alkylation of genistein, is a new active chemical entity. The protective effects of dFMGEN against vascular endothelial injury caused by oxidative stress were investigated in the present study. 2. Human umbilical vein endothelial cells were treated with either genistein (10 μmol/L) or various concentrations of dFMGEN (0.1, 0.3, 1, 3 and 10 μmol/L) for 30 min before exposure to 1 mmol/L H₂O₂ for 24 h. The generation of reactive oxygen species (ROS) was assessed by fluorescence flow cytometry, the release of lactate dehydrogenase (LDH) was examined by biochemical assay, cell viability was measured by the 3‐(4,5‐dimethyl‐2 thiazoyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide assay, cell apoptosis was detected by flow cytometry and the expression of caspase 3 was examined by western blot analysis. 3. Pretreatment with 0.1, 0.3, 1, 3 and 10 μmol/L dFMGEN decreased the generation of ROS and the release of LDH in H₂O₂‐exposed vascular endothelial cells, enhanced cell viability in a concentration‐dependent manner over the concentration range 0.1–10 μmol/L, suppressed H₂O₂‐induced apoptosis of vascular endothelial cells and downregulated the expression of caspase 3. The protective effect of 10 μmol/L dFMGEN against oxidative stress‐induced endothelial injury was stronger than that of 10 μmol/L genistein. 4. The results of the present study suggest that dFMGEN can protect against vascular endothelial injury caused by oxidative stress.     

2,3,4',5-Tetrahydroxystilbene-2-O-β-d-glucoside inhibits angiotensin II-induced cardiac fibroblast proliferation via suppression of the reactive oxygen species-extracellular signal-regulated kinase 1/2 pathway

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Neuroprotective effects of pramipexole against tunicamycin‐induced cell death in PC12 cells

• 1 Pramipexole (PPX), a dopamine D2 and D3 receptor agonist, exerts neuroprotective effects via both dopamine receptor‐mediated and non‐dopaminergic mechanisms. In the present study, we demonstrate that PPX reduces the toxicity of tunicamycin, a typical endoplasmic reticulum (ER) stressor, in PC12h cells, a subline of PC12 cells.
• 2 The PC12h cells were treated with 300 μmol / L PPX in the presence of 0.5 μmol / L tunicamycin for 24 h. The neuroprotective effects of PPX against tunicamycin‐induced cell death were evaluated using 3‐(4,5‐dimethyl‐2 thiazoyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays, Hoechst 33258 staining and western blot analysis.
• 3 Tunicamycin (0.2, 0.3 and 0.5 μg / mL) dose‐dependently decreased MTT activity and increased LDH release from PC12h cells. Treatment with 300 μmol / L PPX rescued the tunicamycin‐induced decrease in cell viability.
• 4 Spiperone (10 μmol / L), a dopamine D2 and D4 receptor antagonist, had no effect on PPX neuroprotection against tunicamycin in these cells. Marker proteins of ER stress and apoptosis are known to be upregulated by tunicamycin, but we detected no significant effects of PPX on these factors.
• 5 In conclusion, we speculate that a combination of several mechanisms may be involved in PPX‐induced neuroprotection.

     

Warifteine,a bisbenzylisoquinoline alkaloid,induces relaxation by activating potassium channels in vascular myocytes

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Adenosine A2A receptor antagonists are broad facilitators of antinicotinic neuromuscular blockade monitored either with 2 Hz train‐of‐four or 50 Hz tetanic stimuli

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Assessment in vitro of the genotoxicity,antigenotoxicity and antioxidant of Ceratonia siliqua L. extracts in murine leukaemia cells L1210 by comet assay

Genotoxicity of Ceratonia siliqua extracts, was investigated by assessing their capacity to induce nucleus DNA degradation of murine leukaemia cells L1210, using the “Comet assay”. The ability of total oligomer flavonoids (TOF) and aqueous extracts to protect cell DNA against oxidative stress induced by H₂O₂, was performed by pre- co or post-treatment of cells with the before mentioned extracts for different periods preceding exposure to H₂O₂ stress. No significant genotoxic effect was detected at different exposure times, except at the lowest concentration of TOF extract (16.25 μg/ml). It appears that extracts decreased DNA damage, induced by H₂O₂.Both of TOF and aqueous extracts exhibited cellular antioxidant capacity, with EC₅₀ values of respectively <16.25 and < 35 μg/ml, as well as, a protective capacity against lipidperoxidation inducing using L1210 cells line as a cellular model. MDA inhibition percentages reached 88.43% and 90.52% with respectively 35.5 μg/ml of TOF extract and 70 μg/ml of aqueous extract.Antioxidant properties of carob leaf extracts revealed by our study make a good antioxidant protection and thus a good candidate as food addition component.     

Cardioprotective and Cardiotoxic Effects of Quercetin and Two of Its In Vivo Metabolites on Differentiated H9c2 Cardiomyocytes

Whilst mitotic rat embryonic cardiomyoblast‐derived H9c2 cells have been widely used as a model system to study the protective mechanisms associated with flavonoids, they are not fully differentiated cardiac cells. Hence, the aim of this study was to investigate the cardioprotective and cardiotoxic actions of quercetin and two of its major in vivo metabolites, quercetin 3‐glucuronide and 3′‐O‐methyl quercetin, using differentiated H9c2 cells. The differentiated cardiomyocyte‐like phenotype was confirmed by monitoring expression of cardiac troponin 1 after 7 days of culture in reduced serum medium containing 10 nM all‐trans retinoic acid. Quercetin‐induced cardiotoxicity was assessed by monitoring MTT reduction, lactate dehydrogenase (LDH) release, caspase 3 activity and reactive oxygen species production after prolonged flavonoid exposure (72 hr). Cardiotoxicity was observed with quercetin and 3′‐O‐methyl quercetin, but not quercetin 3‐glucuronide. Cardioprotection was assessed by pre‐treating differentiated H9c2 cells with quercetin or its metabolites for 24 hr prior to 2‐hr exposure to 600 μM H₂O_2, after which oxidative stress‐induced cell damage was assessed by measuring MTT reduction and LDH release. Cardioprotection was observed with quercetin and 3′‐O‐methyl quercetin, but not with quercetin 3‐glucuronide. Quercetin attenuated H₂O₂‐induced activation of ERK1/2, PKB, p38 MAPK and JNK, but inhibitors of these kinases did not modulate quercetin‐induced protection or H₂O₂‐induced cell death. In summary, quercetin triggers cardioprotection against oxidative stress‐induced cell death and cardiotoxicity after prolonged exposure. Further studies are required to investigate the complex interplay between the numerous signalling pathways that are modulated by quercetin and which may contribute to the cardioprotective and cardiotoxic effects of this important flavonoid.     

Effects of pro-inflammatory cytokines and antioxidants expression in the jejunum of mice induced by hydrogen peroxide

Inflammation-induced jejunal cell death and oxidative stress have been observed in inflammatory bowel disease (IBD), but now, there is still no systematic animal model of jejunal oxidative stress for the evaluation of potential therapies. Thus, the purpose of this study was to evaluate the dynamic changes of pro-inflammatory cytokines and antioxidant expression on the jejunal inflammation. In this study, Hydrogen peroxide(H₂O_2, 10 ml/kg)was administrated intragastrically to mice in a single dose of 1%, 3%, or 5%. The incidence of death, histomorphometry, inflammatory cytokines (including TNF-α,IL-1β,and IL-5), and antioxidant genes were measured via RT-PCR. During the whole period, a massive infiltration of neutrophils was observed in the jejunum. Intragastric administration of H₂O₂ significantly up-regulated the expression of TNF-α,IL-1β, and IL-5 (P < 0.05). Meanwhile, 5% H₂O₂ induced an acute stress in mice which lasted up to 3 days, while 3% H₂O₂ induced a chronic injury in the jejunum that lasted 7 days. In the early stage, H₂O₂ markedly enhanced expression of antioxidant genes and all of the doses used progressively decreased the expression of antioxidant genes in a time-dependent manner. These findings suggested that intragastric administration of 3% H₂O₂ induces relatively stable oxidative stress in the jejunum.     

Heart failure and angiotensin II modulate atrial Pitx2c promotor methylation

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Vasorelaxation induced by methyl cinnamate,the major constituent of the essential oil of Ocimum micranthum,in rat isolated aorta

The aim of the present study was to investigate the vascular effects of the E‐isomer of methyl cinnamate (E‐MC) in rat isolated aortic rings and the putative mechanisms underlying these effects. At 1–3000 μmol/L, E‐MC concentration‐dependently relaxed endothelium‐intact aortic preparations that had been precontracted with phenylephrine (PHE; 1 μmol/L), with an IC₅₀ value (geometric mean) of 877.6 μmol/L (95% confidence interval (CI) 784.1–982.2 μmol/L). These vasorelaxant effects of E‐MC remained unchanged after removal of the vascular endothelium (IC₅₀ 725.5 μmol/L; 95% CI 546.4–963.6 μmol/L) and pretreatment with 100 μmol/L N^G‐nitro‐l ‐arginine methyl ester (IC₅₀ 749.0 μmol/L; 95% CI 557.8–1005.7 μmol/L) or 10 μmol/L 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (IC₅₀ 837.2 μmol/L; 95% CI 511.4–1370.5 μmol/L). Over the concentration range 1–3000 μmol/L, E‐MC relaxed K⁺‐induced contractions in mesenteric artery preparations (IC₅₀ 314.5 μmol/L; 95% CI 141.9–697.0 μmol/L) with greater potency than in aortic preparations (IC₅₀ 1144.7 μmol/L; 95% CI 823.2–1591.9 μmol/L). In the presence of a saturating contractile concentration of K⁺ (150 mmol/L) in Ca²⁺‐containing medium combined with 3 μmol/L PHE, 1000 μmol/L E‐MC only partially reversed the contractile response. In contrast, under similar conditions, E‐MC nearly fully relaxed PHE‐induced contractions in aortic rings in a Ba²⁺‐containing medium. In preparations that were maintained under Ca²⁺‐free conditions, 600 and 1000 μmol/L E‐MC significantly reduced the contractions induced by exogenous Ca²⁺ or Ba²⁺ in KCl‐precontracted preparations, but not in PHE‐precontracted preparations (in the presence of 1 μmol/L verapamil). In addition, E‐MC (1–3000 μmol/L) concentration‐dependently relaxed the contractions induced by 2 mmol/L sodium orthovanadate. Based on these observations, E‐MC‐induced endothelium‐independent vasorelaxant effects appear to be preferentially mediated by inhibition of plasmalemmal Ca²⁺ influx through voltage‐dependent Ca²⁺ channels. However, the involvement of a myogenic mechanism in the effects of E‐MC is also possible.     

Hydrogen peroxide induces adaptive response and differential gene expression in human embryo lung fibroblast cells

Hydrogen peroxide (H₂O₂), a substance involved in cellular oxidative stress, has been observed to induce an adaptive response, which is characterized by a protection against the toxic effect of H₂O₂ at higher concentrations. However, the molecular mechanism for the adaptive response remains unclear. In particular, the existing reports on H₂O₂‐induced adaptive response are limited to animal cells and human tumor cells, and relatively normal human cells have never been observed for an adaptive response to H₂O₂. In this study, a human embryo lung fibroblast (MRC‐5) cell line was used to model an adaptive response to H₂O₂, and the relevant differential gene expressions by using fluoro mRNA differential display RT‐PCR. The results showed significant suppression of cytotoxicity of H₂O₂ (1100 μM, 1 h) after pretreatment of the cells with H₂O₂ at lower concentrations (0.088–8.8 μM, 24 h), as indicated by cell survival, lactate dehydrogenase release, and the rate of apoptotic cells. Totally 60 mRNA components were differentially expressed compared to untreated cells, and five of them (sizing 400–600 bp) which demonstrated the greatest increase in expression were cloned and sequenced. They showed identity with known genes, such as BCL‐2, eIF3S5, NDUFS4, and RPS10. Real time RT‐PCR analysis of the five genes displayed a pattern of differential expression consistent with that by the last method. These five genes may be involved in the induction of adaptive response by H₂O₂ in human cells, at least in this particular cell type. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 478–485, 2014.     

Protective effects of dehydrocostus lactone against hydrogen peroxide-induced dysfunction and oxidative stress in osteoblastic MC3T3-E1 cells

Oxidative stress regulates cellular functions in multiple pathological conditions, including bone formation by osteoblasic cells. To elucidate the protective effects of dehydrocostus lactone on the response of osteoblast to oxidative stress, osteoblastic MC3T3-E1 cells were incubated with 0.3 mM hydrogen peroxide (H₂O₂) and/or dehydrocostus lactone (0.1–10 μg/ml), and markers of osteoblast function and oxidative damage were examined. Dehydrocostus lactone (0.1–10 μg/ml) significantly increased osteoblast growth compared with control (P < 0.05). H₂O₂-induced reduction of differentiation markers such as alkaline phosphatase (ALP), collagen content, and calcium deposition was recovered in the presence of dehydrocostus lactone (0.4–2 μg/ml). Treatment with dehydrocostus lactone (10 μg/ml) decreased the production of osteoclast differentiation-inducing factors such as interleukin (IL)-6 and receptor activator of nuclear factor-kB ligand (RANKL) in the presence of H₂O₂. Moreover, dehydrocostus lactone (0.4–2 μg/ml) decreased the formation of protein carbonyl (PCO) and malondialdehyde (MDA) induced by H₂O₂ in osteoblasts. Taken together, these results demonstrate that dehydrocostus lactone can protect osteoblasts against H₂O₂-induced cellular dysfunction. These results also suggest that dehydrocostus lactone may be valuable as a protective agent against oxidative damage in osteoblasts.     

Mechanisms of U46619‐induced contraction in mouse intrarenal artery

Thromboxane A₂ (TXA₂) has been implicated in the pathogenesis of vascular complications, but the underlying mechanism remains unclear. The contraction of renal arterial rings in mice was measured by a Multi Myograph System. The intracellular calcium concentration ([Ca²⁺]_i) in vascular smooth muscle cells (VSMCs) was obtained by using a fluo‐4/AM dye and a confocal laser scanning microscopy. The results show that the U46619‐induced vasoconstriction of renal artery was completely blocked by a TXA₂ receptor antagonist GR32191, significantly inhibited by a selective phospholipase C (PI‐PLC) inhibitor U73122 at 10 μmol/L and partially inhibited by a Phosphatidylcholine ‐ specific phospholipase C (PC‐PLC) inhibitor D609 at 50 μmol/L. Moreover, the U46619‐induced vasoconstriction was inhibited by a general protein kinase C (PKC) inhibitor chelerythrine at 10 μmol/L, and a selective PKCδ inhibitor rottlerin at 10 μmol/L. In addition, the PKC‐induced vasoconstriction was partially inhibited by a Rho‐kinase inhibitor Y‐27632 at 10 μmol/L and was further completely inhibited together with a putative IP₃ receptor antagonist and store‐operated Ca²⁺ (SOC) entry inhibitor 2‐APB at 100 μmol/L. On the other hand, U46619‐induced vasoconstriction was partially inhibited by L‐type calcium channel (Cav1.2) inhibitor nifedipine at 1 μmol/L and 2‐APB at 50 and 100 μmol/L. Last, U46619‐induced vasoconstriction was partially inhibited by a cell membrane Ca²⁺ activated C1^? channel blocker 5‐Nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB) at 50 and 100 μmol/L. Our results suggest that the U46619‐induced contraction of mouse intrarenal arteries is mediated by Cav1.2 and SOC channel, through the activation of thromboxane‐prostanoid receptors and its downstream signaling pathway.     

Highly sensitive H2O2-scavenging nano-bionic system for precise treatment of atherosclerosis

In atherosclerosis, chronic inflammatory processes in local diseased areas may lead to the accumulation of reactive oxygen species (ROS). In this study, we devised a highly sensitive H₂O₂-scavenging nano-bionic system loaded with probucol (RPP-PU), to treat atherosclerosis more effectively. The RPP material had high sensitivity to H₂O₂, and the response sensitivity could be reduced from 40 to 10 μmol/L which was close to the lowest concentration of H₂O₂ levels of the pathological environment. RPP-PU delayed the release and prolonged the duration of PU in vivo. In Apolipoprotein E deficient (ApoE^?/?) mice, RPP-PU effectively eliminated pathological ROS, reduced the level of lipids and related metabolic enzymes, and significantly decreased the area of vascular plaques and fibers. Our study demonstrated that the H₂O₂-scavenging nano-bionic system could scavenge the abundant ROS in the atherosclerosis lesion, thereby reducing the oxidative stress for treating atherosclerosis and thus achieve the therapeutic goals with atherosclerosis more desirably.     

Chronic mercury exposure at different concentrations produces opposed vascular responses in rat aorta

Mercury chloride exposure for 30 days decreases NO bioavailability and increases oxidative stress. However, the mechanisms underlying the effects of mercury on the cardiovascular system are not completely understood, and it is not known if they are dose‐dependent or if some concentrations have no harmful effects. Thus, we investigated the effects of chronic exposure to doses low (half) and high (2.5‐fold higher) than that needed to obtain 29 nmol/L of HgCl₂ on the vascular function. Three‐month‐old male Wistar rats received intramuscular (i.m.) HgCl₂ for 30 days and were divided in three groups: lower (Low Hg); higher (High Hg); and saline was used as the control. High Hg exposure increased the contractile response to phenylephrine (PHE) in aortic rings, but Low Hg reduced it. The hyporesponsiveness in the Low Hg rats was blunted by endothelial denudation and NOS inhibition with l ‐NAME (100 μmol/L). The phosphorylated‐eNOS/eNOS protein ratio increased in the aortas of Low Hg rats. In the High Hg group, endothelial denudation increased the PHE‐induced contractions, while l ‐NAME had no effects and indomethacin (10 μmol/L), losartan (10 μmol/L) and apocynin (30 μmol/L) reduced this response. In the High Hg group, protein levels of the NADPH oxidase subunit gp91phox and cyclooxygenase‐2 increased. Our results support previous suggestions that High Hg increases oxidative stress that might activate an inflammatory cascade and the renin‐angiotensin system. However, very low Hg concentrations below the level considered safe still reduced vascular reactivity, suggesting the need for special attention to continuous exposure as a putative cause of increased cardiovascular risk.     

The involvement of reactive oxygen species and arachidonic acid in α1-adrenoceptor-induced smooth muscle cell proliferation and migration

1. In a previous study, we demonstrated phenylephrine-stimulated arachidonic acid (AA) release in rabbit cultured aortic smooth muscle cells. Therefore, we have investigated the functional implications of AA which are involved in the cellular response to phenylephrine, particularly proliferation and migration of rabbit cultured aortic smooth muscle cells.
2. First, to determine whether AA directly modifies proliferation and mobility of vascular smooth muscle cells (VSMCs), we exposed the cells to AA. AA induced proliferation and migration of the cells in a dose-dependent fashion. Concomitantly added catalase inhibited the proliferation and chemotaxis induced by AA of VSMCs. Conversely, aminotriazole enhanced the proliferation and migration induced by AA.
3. Secondly, we investigated whether the proliferation and migration of VSMCs by phenylephrine were related to AA and hydrogen peroxide (H₂O₂). The proliferation and chemotaxis of VSMCs by phenylephrine were inhibited by a phospholipase A₂ (PLA₂) inhibitor, or catalase.
4. Lastly, we investigated the effects of AA and phenylephrine on the content of H₂O₂ in VSMCs. AA and phenylephrine treatment led to an increase of H₂O₂ in a dose-dependent manner.
5. These results suggest that the addition of phenylephrine to the cells caused the enhancement of proliferation and migration, probably by mediating AA release and reactive oxygen species (ROS) production.

     

ROS activates JNK-mediated autophagy to counteract apoptosis in mouse mesenchymal stem cells in vitro

Aim:

Transplantation of mesenchymal stem cells (MSCs) for the treatment of diabetic erectile dysfunction (ED) is hampered by apoptosis of the transplanted cells. In diabetic ED, there is increased oxidative stress and decreased NO in the corpora cavernosa, and reactive oxygen species (ROS) induce apoptosis of the transplanted cells. In this study we examined whether and how autophagy was involved in ROS-induced apoptosis of MSCs.

Methods:

Mouse C3H10 MSCs were treated with H₂O₂ to simulate the high oxidative condition in diabetic ED. Cell viability was measured using MTT assay. Apoptosis was analyzed by flow cytometry. Apoptosis- and autophagy-related proteins were detected with Western blot assays. Intracellular autophagosome accumulation was studied using transmission electron microscopy.

Results:

Treatment of MSCs with H₂O₂ (50–400 μmol/L) inhibited the cell viability in concentration- and time-dependent manners. Furthermore, H₂O₂ (300 μmol/L) induced apoptosis, as well as activated autophagy in MSCs. Pretreatment with lysosome inhibitor chloroquine (10 μmol/L) or PI3K inhibitor 3-methyladenine (5 mmol/L) significantly enhanced H₂O₂-induced cell death. Pretreatment with JNK inhibitor SP600125 (10 μmol/L) abrogated H₂O₂-induced accumulation of LC3-II, and attenuated H₂O₂-induced reduction of Bcl-2 levels in MSCs.

Conclusion:

ROS induce autophagy to counteract apoptosis in MSCs by activation of JNK. Thus, augmentation of autophagy may reduce apoptosis, prolonging MSC survival and improving MSC-based therapeutic efficacy for diabetic ED.     

Hydrogen sulphide protects H9c2 cells against chemical hypoxia‐induced injury

1. The aim of the present study was to investigate the effect of hydrogen sulphide (H₂S) on cobalt chloride (CoCl₂)‐induced injury in H9c2 embryonic rat cardiac cells. 2. After 36 h incubation in the presence of 600 μmol/L CoCl₂, reduced cell viability of H9c2 cells was observed, as well as the induction of apoptosis. In addition, CoCl₂ (600 μmol/L) enhanced the production of reactive oxygen species (ROS) and the expression of cleaved caspase 3, induced a loss of mitochondrial membrane potential (MMP) and decreased reduced glutathione (GSH) production. These results suggest that CoCl₂ induces similar responses to hypoxia/ischaemia. 3. Pretreatment of cells with 400 μmol/L NaHS (a H₂S donor) for 30 min prior to exposure to CoCl₂ (600 μmol/L) significantly protected H9c2 cells against CoCl₂‐induced injury. Specifically, increased cell viability and decreased apoptosis were observed. In addition, NaHS pretreatment blocked the CoCl₂‐induced increases in ROS production and cleaved caspase 3 expression, as well as the decreases in GSH production and loss of MMP. 4. Pretreatment of cells with 2000 μmol/L N‐acetylcysteine (NAC), a ROS scavenger, for 1 h prior to CoCl₂ exposure significantly protected H9c2 cells against CoCl₂‐induced injury, specifically enhancing cell viability, decreasing ROS production and preventing loss of MMP. 5. The findings of the present study suggest that H₂S protects H9c2 cells against CoCl₂‐induced injury by suppressing oxidative stress and caspase 3 activation.