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Background: Ulcerative colitis (UC), an ongoing inflammatory disorder of the colon, is marked by persistent mucosal surface irritation extending from the rectum to the near-proximal colon. Tiron is a synthetic analogue of vitamin E which is known to have antioxidant and anti-inflammatory effects in various animal models, so the goal of this study was to find out whether Tiron had any preventive impacts on UC inflicted by acetic acid (A.A) exposure in rats., Method: Tiron (235 and 470 mg/kg) was administered intra-peritoneally for 2 weeks, and A.A (2 ml, 3 % v/v) was injected intra-rectally to cause colitis. Colon tissues and blood samples were then collected for measurement of various inflammatory and oxidative stress biomarkers., Results: Tiron administration significantly diminished lactate dehydrogenase (LDH), C-reactive protein (CRP), colon weight, and the weight/length ratio of the colon as compared to A.A-injected rats. Additionally, Tiron attenuated oxidative stress biomarkers. Tiron also enforced the levels of Glucagon-like peptide-1 (GLP-1) and trefoil factor-3 (TFF-3), while it greatly lowered the expression of nuclear factor kappa B (NF-κB), interleukin-6 (IL-6), interferon-γ (IFN-γ), and transforming growth factor-1(TGF-β1), phosphorylated epidermal growth factor receptor (P-EGFR), phosphatidylinositol-3-kinase (PI3K) and protein kinase B (AKT) expression in colonic cellular structures. Furthermore, colonichistopathologic damages, revealed by hematoxylin and eosin (H&E) and Alcian Blue stain, were significantly decreased upon Tiron administration., Conclusion: Tiron prevented A.A-induced colitis in rats via modulating inflammatory pathway TGF-β1/P-EGFR/PI3K/AKT/NF-κB, alongside managing the oxidant/antioxidant equilibrium, and boosting the reliability of the intestinal barrier., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Neurotensin (NTS) receptor 1 regulates the growth non-small cell lung cancer (NSCLC) cells. NTS binds with high affinity to NTSR1, leading to increased tyrosine phosphorylation of the EGFR and HER2. Using Calu3, NCI-H358, or NCI-H441 cells, the effects of NTS on HER3 transactivation were investigated. HER3 tyrosine phosphorylation was increased by NTS or neuregulin (NRG1) addition to NSCLC cells. NCI-H358, NCI-H441, and Calu-3 cells have HER3, NTSR1 and neuregulin (NRG)1 protein. NTSR1 regulation of HER3 transactivation was impaired by SR48692 (NTSR1 antagonist) or monoclonal antibody (mAb)3481 (HER3 blocker). Immunoprecipitation experiments indicated that NTS addition to NCI-H441cells resulted in the formation of EGFR/HER3 and HER2/HER3 heterodimers. The ability of NTS to increase HER3 tyrosine phosphorylation was impaired by GM6001 (MMP inhibitor), PP2 (Src inhibitor), Tiron (superoxide scavenger), or N-acetylcysteine (antioxidant). Adding NTS to NSCLC cells increased phosphorylation of ERK, HER3, and AKT. NTS or NRG1 increased colony formation of NSCLC cells which was strongly inhibited by SR48692 and mAb3481. The results indicate that NTSR1 regulates HER3 transactivation in NSCLC cells leading to increased proliferation., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Published by Elsevier Inc.)

Parkinsonism is a neurodegenerative disease that is common all over the world. This study aimed at exploring the neuroprotective effect of tiron against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism. MPTP (30 mg/kg, intraperitoneally [ip]) was injected in mice daily for 5 consecutive days. Mice were treated with tiron (140 and 280 mg/kg, ip) or levodopa (8.4 mg/kg, orally) for 10 consecutive days starting 5 days before MPTP injection. At the end of the experiment, behavioral tests were conducted to assess the neuroprotective effect of tiron. Moreover, oxidative stress was assessed via measuring antioxidant enzyme, such as catalase, and lipid peroxidation was evaluated as malondialdehyde. Neuronal damage was also detected by histopathological examination and via estimating hippocampal levels of dopamine, γ-aminobutyric acid, and nuclear factor erythroid-derived 2-like 2. In addition, the expression of Kelch-like ECH-associated protein 1 and heme oxygenase-1 was assessed by immunohistochemistry. Compared with the blank control group and the positive control group, the inhibitory effect of tiron on MPTP-induced neurodegenerative injury was statistically significant., (© 2020 Wiley Periodicals LLC.)

Aims: Tobacco smoking is a major health problem associated with lung and liver damage. Lung and liver damage secondary to tobacco smoking is mediated through nicotine-induced oxidative stress. Therefore, we hypothesized that antioxidant treatment with tiron may improve nicotine-induced lung and liver damage., Materials and Methods: Rats were divided into six groups, a control, nicotine (10 mg/kg/day, i.p.; for 8 weeks) and tiron (100 or 200 mg/kg/day, i.p.; for 8 weeks) with or without nicotine administration., Key Findings: Tiron improved survival rate and attenuated lung and liver damage as reflected by decreased total and differential cell counts, lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluid (BALF) and decreased alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in serum; also histopathological examination confirmed the protective effect of tiron in lung and liver tissues of nicotine treated rats. Tiron attenuated dyslipidemia, which is associated with nicotine. These ameliorative effects of tiron may be mainly due to its antioxidant effect as proved by a significant decrease in malondialdehyde (MDA) content, reactive oxygen species (ROS) and total nitrite/nitrate (NOx) levels, and increase in reduced glutathione (GSH) level, catalase (CAT) and superoxide dismutase (SOD) activities. This is likely related to suppression of protein levels of NADPH oxidase enzyme (NOX1), inducible nitric oxide synthase (iNOS), nuclear factor kappa B (NF-κB) and tumor necrosis factor alpha (TNF-α); and up-regulation of protein levels of nuclear factor erythroid-2 (Nrf2)., Significance: This makes tiron (synthetic analogue of vitamin E) good candidate for future use to minimize nicotine's hazards among smokers., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Activation of trypsinogen (formation of trypsin) inside the pancreas is an early pathological event in the development of acute pancreatitis. In our previous studies we identified the activation of trypsinogen within endocytic vacuoles (EVs), cellular organelles that appear in pancreatic acinar cells treated with the inducers of acute pancreatitis. EVs are formed as a result of aberrant compound exocytosis and subsequent internalization of post-exocytic structures. These organelles can be up to 12 μm in diameter and can be actinated (i.e. coated with F-actin). Notably, EVs can undergo intracellular rupture and fusion with the plasma membrane, providing trypsin with access to cytoplasmic and extracellular targets. Unraveling the mechanisms involved in cellular processing of EVs is an interesting cell biological challenge with potential benefits for understanding acute pancreatitis. In this study we have investigated autophagy of EVs and discovered that it involves a non-canonical LC3-conjugation mechanism, reminiscent in its properties to LC3-associated phagocytosis (LAP); in both processes LC3 was recruited to single, outer organellar membranes. Trypsinogen activation peptide was observed in approximately 55% of LC3-coated EVs indicating the relevance of the described process to the early cellular events of acute pancreatitis. We also investigated relationships between actination and non-canonical autophagy of EVs and concluded that these processes represent sequential steps in the evolution of EVs. Our study expands the known roles of LAP and indicates that, in addition to its well-established functions in phagocytosis and macropinocytosis, LAP is also involved in the processing of post-exocytic organelles in exocrine secretory cells., Abbreviations: AP: acute pancreatitis; CCK: cholecystokinin; CLEM: correlative light and electron microscopy; DPI: diphenyleneiodonium; EV: endocytic vacuole; LAP: LC3-associate phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PACs: pancreatic acinar cells; PFA: paraformaldehyde; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; Res: resveratrol; TAP: trypsinogen activation peptide; TEM: transmission electron microscopy; TLC-S: taurolithocholic acid 3-sulfate; TRD: Dextran Texas Red 3000 MW Neutral; ZGs: zymogen granules.

The present study was designed to determine how apelin in paraventricular nucleus (PVN) modulates the renal sympathetic nerve activity (RSNA), arterial blood pressure (ABP), mean arterial pressure (MAP), and heart rate (HR), and whether superoxide anions regulate the performance of PVN apelin in spontaneously hypertensive rats (SHRs). Acute experiment was carried out with 13-week-old male Wistar-Kyoto rats (WKY) and SHRs under anaesthesia. RSNA, ABP, MAP and HR after PVN microinjection were measured. Apelin microinjection into PVN increased RSNA, ABP, MAP and HR in WKY rats and SHRs, more obviously in SHRs. APJ antagonist F13A decreased the RSNA, ABP, MAP and HR in SHRs, and inhibited the effects of apelin. Apelin and APJ mRNA levels were higher in the PVN in SHRs. PVN microinjection of superoxide anion scavengers tempol and tiron, or NAD(P)H oxidase inhibitor apocynin, decreased the RSNA, ABP, MAP and HR in SHRs, and inhibited the effects of apelin, but the superoxide dismutase (SOD) inhibitor diethyldithiocarbamic acid (DETC) potentiated the effects of apelin. NAD(P)H oxidase activity and superoxide anion levels in PVN were increased by apelin, but decreased by APJ antagonist F13A. The apelin-induced increases in NAD(P)H oxidase activity and superoxide anion level were abolished by pre-treatment with F13A. These results indicate that apelin in PVN increases the sympathetic outflow and blood pressure via activating APJ receptor. The enhanced activity of endogenous apelin and APJ receptor in PVN contributes to sympathetic activation in hypertension, and the superoxide anion is involved in these apelin-mediated processes in PVN., (Copyright © 2018. Published by Elsevier Inc.)

Cardiac hypertrophy is commonly involved in cardiac injury. Oxidative stress can induce cardiac hypertrophy with apoptosis. Potassium bromate (KBrO3) has been widely used as a food additive due to its oxidizing properties. In the present study, the rat‑derived heart cell line H9c2 was used to investigate the effect of KBrO3 on cell size. KBrO3 increased cell size at concentrations <250 µM, in a dose‑dependent manner. Additionally, KBrO3 also promoted the gene expression of two biomarkers of cardiac hypertrophy, brain/B‑type natriuretic peptides (BNP) and β‑Myosin Heavy Chain (β‑MHC). However, apoptosis remained unobserved in these cells. Moreover, mediation of free radicals was investigated using a fluorescence assay, and it was observed that superoxide and reactive oxygen species (ROS) levels increased with KBrO3. Effects of KBrO3 were significantly reduced by tiron at concentrations sufficient to produce antioxidant‑like action. Additionally, signals involved in cardiac hypertrophy such as calcineurin and nuclear factor of activated T‑cells (NFAT) were also determined using western blot analysis. KBrO3 increased the protein levels of both these molecules which were decreased by tiron in a dose‑dependent manner. Additionally, cyclosporine A attenuated the cardiac hypertrophy induced by KBrO3 in H9c2 cells at concentrations effective to inhibit calcineurin, in addition to reducing mRNA levels of BNP or β‑MHC. Finally, apoptosis was also identified in H9c2 cells incubated with KBrO3 at concentrations >300 µM. Collectively, these results provided a novel perspective that KBrO3 induces cardiac hypertrophy without apoptosis at a low dose through the generation of ROS, activating the calcineurin/NFAT signaling pathway in H9c2 cells. Therefore, at a dose <250 µM, KBrO3 can be applied as an inducer of cardiac hypertrophy without apoptosis in H9c2 cells. KBrO3 can also be developed as a tool to induce cardiac hypertrophy in animals.

The extensive application of titanium dioxide nanoparticles (TiO 2 NPs) in the food industry arouses a debate regarding the probable risk associated with their use. Several recent studies reported that most nanoparticles (NPs) have adverse actions on the liver. The objective of this study is to examine whether Tiron plays a modulatory role against apoptotic damage induced by TiO 2 NPs in rat livers. Forty rats were randomly divided into 4 groups; a control group received phosphate-buffered saline, an intoxicated group received 100 mg/kg/day of TiO 2 NPs for 60 days, a treated group received 470 mg/kg/day of Tiron for the last 14 days after TiO 2 NPs administration, and a Tiron group received Tiron only as previously mentioned. Oral administration of TiO 2 NPs significantly increased serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). In the liver, TiO 2 NPs increased oxidative stress through increasing lipid peroxidation and decreasing GSH concentration and the levels of the SOD and GPx enzymes. TiO 2 NPs significantly upregulated the proapoptotic Bax gene and downregulated the antiapoptotic Bcl-2 gene. Histopathological examination of hepatic tissue reinforced the previous biochemical results. Apoptotic lesions were also obvious in this group. Treatment with Tiron as an antioxidant significantly decreased serum biochemistry, ameliorated oxidative stress in hepatic tissue, upregulated Bcl-2, decreased Bax expression and attenuated the histopathology of hepatic injury. These findings indicate that Tiron effectively diminishes the hazardous effects of TiO 2 NPs on rat liver., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Purpose: We have demonstrated that RuBPY induces hypotensive effect in hypertensive rats, promotes vasodilation at low concentrations, and presents low cytotoxicity. This study aimed to verify whether the NO donor RuBPY synthesized in our laboratory induces in vitro tolerance and cross-tolerance to acetylcholine (ACh) and sodium nitroprusside (SNP) in rat cava vein., Methods: We compared the maximum relaxing effect (ME) and potency (pD 2 ) of RuBPY and nitroglycerin (GTN) in cava vein rings. Exposure to RuBPY or GTN induced in vitro tolerance. Western Blotting helped to evaluate phosphorylation of endothelial nitric oxide synthase (NOS3/eNOS) at the Ser 1177 activation site and at the Thr 495 inactivation site and to determine the ratio between active eNOS dimers and inactive eNOS monomers. The NO and ROS ratio was assessed by flow citometry., Results: RuBPY did not induce cross-tolerance with ACh, and this NO donor took longer to induce tolerance than GTN. Only GTN elicited phosphorylation of eNOS at Ser 1177 and Thr 495 . In contrast to results obtained with pre-exposure to GTN, pre-exposure to RuBPY did not reduce the formation of NO. The O 2 - ratio increased in cells incubated with GTN., Conclusions: A major contribution of this work has been to evaluate the phenomenon of tolerance induced by GTN and by the new ruthenium complex RuBPY in a venous bed. RuBPY is more advantageous than GTN: RuBPY takes longer to induce tolerance, does not induce endothelial dysfunction or cross-tolerance to ACh, and generates lower amount of ROS., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Although the widespread use of titanium dioxide nanoparticles (TiO 2 NPs), few studies were conducted on its hazard influence on human health. Tiron a synthetic vitamin E analog was proven to be a mitochondrial targeting antioxidant. The current investigation was performed to assess the efficacy of tiron against TiO 2 NPs induced nephrotoxicity. Eighty adult male rats divided into four different groups were used: group I was the control, group II received TiO2 NPs (100mg\Kg BW), group III received TiO2 NPs plus tiron (470mg\kg BW), and group IV received tiron alone. Urea, creatinine and total protein concentrations were measured in serum to assess the renal function. Antioxidant status was estimated by determining the activities of glutathione peroxidase, superoxide dismutase, malondialdehyde (MDA) level and glutathione concentration in renal tissue. As well as Renal fibrosis was evaluated though measuring of transforming growth factor-β1 (TGFβ1) and matrix metalloproteinase 9 (MMP9) expression levels and histopathological examination. TiO 2 NPs treated rats showed marked elevation of renal indices, depletion of renal antioxidant enzymes with marked increase in MDA concentration as well as significant up-regulation in fibrotic biomarkers TGFβ1 and MMP9. Oral administration of tiron to TiO 2 NPs treated rats significantly attenuate the renal dysfunction through decreasing of renal indices, increasing of antioxidant enzymes activities, down-regulate the expression of fibrotic genes and improving the histopathological picture for renal tissue. In conclusion, tiron was proved to attenuate the nephrotoxicity induced by TiO 2 NPs through its radical scavenging and metal chelating potency., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Tiron functions as an effective antioxidant alleviating the intracellular reactive oxygen species (ROS) or the acute toxic metal overload. Previous studies have shown that cardiac myocyte apoptosis can be effectively inhibited by tiron administration in streptozotocin (STZ)-induced diabetic rats, primary neonatal rat cardiomyocytes (NRVMs), and H9c2 embryonic rat cardiomyocytes. However, the underlying signalling mechanism is ill-defined. In the present study, we found that tiron supplementation significantly inhibited apoptosis of high glucose (HG)-treated NRVMs and the left ventricular cardiomyocytes from STZ-diabetic rat, accompanied with a reduction of osteopontin (OPN) levels as well as an inhibition of PKCδ phosphorylation. OPN knockdown protected NRVMs against HG-induced cell apoptosis. In addition, genetic inhibition of PKCδ mitigated HG-stimulated enhancement of intracellular OPN levels in NRVMs. These findings indicate that ROS-mediated activation of PKCδ upregulated OPN expression, leading to cardiac myocyte apoptosis. Interfering with ROS/PKCδ pathway by antioxidants such as tiron provides an optional therapeutic strategy for treatment and prevention of apoptosis-related cardiovascular diseases including diabetic cardiomyopathy., (© 2017 John Wiley & Sons Australia, Ltd.)

Titanium dioxide nanoparticles (TDN) are widely used in paints, plastics, ceramics, cosmetics, printing ink, rubber and paper. Tiron is a water soluble metal chelator and antioxidant. This study was designed to investigate the reproductive toxicity of TDN in male albino rats and the ameliorative role of Tiron to minimize such toxic effects. Eighty adult male albino rats were assigned into 4 equal groups, group 1: control; group 2: received TDN at 100 mg/kg/day orally for 8 weeks; group 3: received Tiron at 470 mg/kg/day intraperitoneally for 2 weeks (the last 2 weeks of the experimental period); group 4: received both TDN and Tiron by the same previously mentioned dose, route and duration. The results revealed that TDN provoked reproductive toxicity which was proved by the deteriorated spermogram picture, high incidence of micronucleated RBCs, elevated oxidative stress parameters and up regulation of Testin gene. Whereas, Tiron co-treatment ameliorated most of these toxic alterations. Our findings highlighted the protective role of tiron against TDN intoxication., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Fusobacterium nucleatum (F. nucleatum) plays a critical role in gastrointestinal inflammation. However, the exact mechanism by which F. nucleatum contributes to inflammation is unclear. In the present study, it was revealed that F. nucleatum could induce the production of proinflammatory cytokines (IL-8, IL-1β and TNF-α) and reactive oxygen species (ROS) in Caco-2 colorectal) adenocarcinoma cells. Furthermore, ROS scavengers (NAC or Tiron) could decrease the production of proinflammatory cytokines during F. nucleatum infection. In addition, we observed that autophagy is impaired in Caco-2 cells after F. nucleatum infection. The production of proinflammatory cytokines and ROS induced by F. nucleatum was enhanced with either autophagy pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (ATG5 or ATG12) in Caco-2 cells. Taken together, these results indicate that F. nucleatum-induced impairment of autophagic flux enhances the expression of proinflammatory cytokines via ROS in Caco-2 Cells., Competing Interests: The authors have declared that no competing interests exist.

Whether high oxygen is harmful to the vascular function is unclear. The present study examined if high oxygen modifies vasodilator effect of cysteine via enhanced oxidative stress and thromboxane production. Rat mesenteric arteries with endothelium at 95 or 50 % oxygen were subjected to isometric force recordings, measurement of thromboxane B2 levels, determination of superoxide and peroxynitrite levels and evaluation of NADPH oxidase subunit protein expression, respectively. L-cysteine (0.01-3 mM) constricted or dilated arteries at 95 and 50 % oxygen, respectively. Thromboxane receptor antagonist SQ-29,548 (1 μM) abolished the constriction at 95 % oxygen. L-cysteine (3 mM) increased levels of thromboxane B2 in arteries upon 95 % oxygen application. L-cysteine relaxed arteries treated with superoxide inhibitor tiron (2 mM) or NADPH oxidase inhibitor gp91ds-tat (1 μM) irrespective of the oxygen concentration while ATP-sensitive K(+) channel inhibitor glibenclamide (1 μM) and cystathionine-γ-lyase (CSE) inhibitor DL-propargylglycine (10 mM) similarly abolished the relaxation. L-cysteine (3 mM) with 95 % oxygen augmented levels of superoxide as well as nitrotyrosine within the artery, concomitantly with enhanced membrane protein expression of NADPH oxidase subunit p47phox. The higher concentration of oxygen attenuates L-cysteine-induced vasodilation via superoxide production mediated by NADPH oxidase along with thromboxane A2 production, resulting in vasoconstriction. The increased levels of superoxide, as well as peroxynitrite, coexist with the impaired vasodilation related to ATP-sensitive K(+) channels and CSE. Higher oxygen with plasma cysteine may cause oxidative stress and vasoconstrictor prostanoid production in blood vessels.

Recent research found that Tiron was an effective antioxidant that could act as the intracellular reactive oxygen species (ROS) scavenger or alleviate the acute toxic metal overload in vivo. In this study, we investigated the inhibitory effect of Tiron on matrix metalloproteinase (MMP)-1 and MMP-3 expression in human dermal fibroblast cells. Western blot and ELISA analysis revealed that Tiron inhibited ultraviolet B (UVB)-induced protein expression of MMP-1 and MMP-3. Real-time quantitative PCR confirmed that Tiron could inhibit UVB-induced mRNA expression of MMP-1 and MMP-3. Furthermore, Tiron significantly blocked UVB-induced activation of the MAPK signaling pathway and activator protein (AP)-1 in the downstream of this transduction pathway in fibroblasts. Through the AP-1 binding site mutation, it was found that Tiron could inhibit AP-1-induced upregulation of MMP-1 and MMP-3 expression through blocking AP-1 binding to the AP-1 binding sites in the MMP-1 and MMP-3 promoter region. In conclusion, Tiron may be a novel antioxidant for preventing and treating skin photoaging UV-induced.

Acute pancreatitis (AP) is an acute inflammatory disorder of the pancreas that can be complicated by involvement of other remote organs. Oxidative stress is known to have a crucial role in the development of pancreatic acinar damage and one of the main causes in multisystem organ failure in experimental AP. The aim of the study was to determine the effect of tiron on pancreas and remote organ damage in L-arginine (L-Arg) induced AP rat model. Thirty-two male rats were divided in random into four groups: control, tiron, L-Arg, and tiron with L-Arg. At the end of the experiment, blood samples were withdrawn for biochemical analysis. The pancreas, lung, kidney, and liver were collected for histopathological examination. Estimation of pancreatic water content was done. Analysis of pulmonary, hepatic, renal, and pancreatic lipid peroxide levels (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) were carried out. Finally, nuclear factor kappa B (NF-κB) and transforming growth factor β1 (TGF-β1) expression in pancreatic tissue was determined. Results indicated that treatment with tiron significantly decreased lipid peroxide levels and markedly increased both SOD activity and GSH level. Moreover, histopathological analysis further confirmed that administration of tiron relatively ameliorates pancreatic acinar cells and remote organ damage. Increased immunoreactivity of NF-κB and TGF-β1 were reduced also by tiron treatment. These findings pointed out the protective role of the mitochondrial antioxidant, tiron against AP induced by L-Arg.

The iron-sulfur cluster containing protein mitoNEET is known to modulate the oxidative capacity of cardiac mitochondria but its function during myocardial reperfusion injury after transient ischemia is unknown. The purpose of this study was to analyze the impact of mitoNEET on oxidative stress induced cell death and its relation to the glutathione-redox system in cardiomyocytes in an in vitro model of hypoxia and reoxygenation (H/R). Our results show that siRNA knockdown (KD) of mitoNEET caused an 1.9-fold increase in H/R induced apoptosis compared to H/R control while overexpression of mitoNEET caused a 53% decrease in apoptosis. Necrosis was not affected. Apoptosis of both, mitoNEET-KD and control cells was diminished to comparable levels by using the antioxidants Tiron and glutathione compound glutathione reduced ethyl ester (GSH-MEE), indicating that mitoNEET-dependent apoptosis is mediated by oxidative stress. The interplay between mitoNEET and glutathione redox system was assessed by treating cardiomyocytes with 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthio-carbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), known to effectively inhibit glutathione reductase (GSR) and to decrease the GSH/GSSG ratio. Surprisingly, inhibition of GSR-activity to 20% by 2-AAPA decreased apoptosis of control and mitoNEET-KD cells to 23% and 25% respectively, while at the same time mitoNEET-protein was increased 4-fold. This effect on mitoNEET-protein was not accessible by mitoNEET-KD but was reversed by GSH-MEE. In conclusion we show that mitoNEET protects cardiomyocytes from oxidative stress-induced apoptosis during H/R. Inhibition of GSH-recycling, GSR-activity by 2-AAPA increased mitoNEET-protein, accompanied by reduced apoptosis. Addition of GSH reversed these effects suggesting that mitoNEET can in part compensate for imbalances in the antioxidative glutathione-system and therefore could serve as a potential therapeutic approach for the oxidatively stressed myocardium.

Neutrophils can entrap and kill pathogens by releasing of neutrophil extracellular traps (NETs), in addition to their routine functions such as phagocytosis and degranulation. NETs consist of a DNA backbone supplemented by multiple bactericidal proteins from the nucleus, the cytoplasm and the granules. Neutrophils release NETs after their activation by a number of physiological and pharmacological stimuli. In addition to the antimicrobial function, NETs are involved in the pathogenesis of various autoimmune and inflammatory diseases. Since NET formation predominantly depends on the generation of reactive oxygen species (ROS), all substances that are capable of scavenging ROS or inhibiting the enzymes responsible for their synthesis should prevent ROS-associated NET release. The aim of this study was to test substances with an antioxidant activity, such as Trolox, Tiron, and Tempol, for their capacity to inhibit NET formation by primary human neutrophils in vitro. We revealed for the first time an inhibitory effect of Trolox on ROS-dependent NET release. We also established a suppressive effect of Tempol on NET formation that manifested itself in a wide range of concentrations. In this study, no inhibitory influence of Tiron on NET release was revealed. All tested substances exerted a significant dose-dependent antioxidative effect on ROS generation induced by phorbol 12-myristate 13-acetate (PMA). We suggest that the antioxidants Trolox and Tempol should be recommended for treating autoimmune and inflammatory diseases that implicate ROS-dependent NET release., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Aims: Investigate the effects of chronic ethanol consumption on erectile function and on the corpus cavernosum (CC) reactivity to endothelin-1 (ET-1)., Main Methods: Male Wistar rats were treated with ethanol (20% v/v) for six weeks., Key Findings: Ethanol-treated rats showed impaired erectile function represented by decreased intracavernosal pressure/mean arterial pressure (ICP/MAP) responses. Ethanol consumption increased the contractile response induced by ET-1 in the isolated CC. Tiron increased ET-1-induced contraction in CC from control and ethanol-treated rats. No differences in the maximal contraction to ET-1 were observed after incubation of CC with PEG-catalase. SC560 and SC236 increased ET-1-induced contraction in CC from ethanol-treated rats. Y27632 reduced the contraction induced by ET-1 in CC from control and ethanol-treated rats. Ethanol increased plasma TBARS, superoxide anion (O2(-)) levels and intracellular reactive oxygen species (ROS) generation in the rat CC. Reduced hydrogen peroxide (H2O2) levels in CC and increased catalase (CAT) activity in plasma and CC were detected after treatment with ethanol. Ethanol decreased superoxide dismutase (SOD) activity in the rat CC. Increased expression of COX-1 was observed in CC from ethanol-treated rats. Treatment with ethanol decreased COX-2 expression but did not alter the expression of Nox1, RhoA and p-RhoA (ser(188)) in the rat CC., Significance: The major new findings of our study are that ethanol consumption induces erectile dysfunction (ED) and increases the contraction induced by ET-1 in the rat CC by a mechanism that involves decreased generation of H2O2 and vasodilator prostanoids as well as increased activation of the RhoA/Rho-kinase pathway., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although palmitoleic acid (C16:1) is associated with arrhythmias, and increases in an age-dependent matter, the effects of L-carnitine, which is essential for the transport of long-chain fatty acids into the mitochondria, are unclear. It has been postulated that L-carnitine may attenuate palmitate (C16:0)-induced mitochondrial dysfunction and the apoptosis of cardiomyocytes. The aim of this study was to elucidate the activity of L-carnitine in the prevention of the palmitoleic acid-induced mitochondrial membrane permeability transition and cytochrome c release using isolated cardiac mitochondria from rats. Palmitoleoyl-CoA-induced mitochondrial respiration was not accelerated by L-carnitine treatment, and this respiration was slightly inhibited by oligomycin, which is an inhibitor of ATP synthase. Despite pretreatment with L-carnitine, the mitochondrial membrane potential decreased and mitochondrial swelling was induced by palmitoleoyl-CoA. In the presence of a combination of L-carnitine and tiron, a free radical scavenger, there was attenuated mitochondrial swelling and cytochrome c release following palmitoleoyl-CoA treatment. We concluded that palmitoleic acid, but not palmitate, induces the cardiac mitochondrial membrane permeability transition despite the presence of L-carnitine., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cholecystokinin (CCK) receptors are G-protein coupled receptors (GPCR) which are present on lung cancer cells. CCK-8 stimulates the proliferation of lung cancer cells, whereas the CCK2R receptor antagonist CI-988 inhibits proliferation. GPCR for some gastrointestinal hormones/neurotransmitters mediate lung cancer growth by causing epidermal growth factor receptor (EGFR) transactivation. Here, the role of CCK/gastrin and CI-988 on EGFR transactivation and lung cancer proliferation was investigated. Addition of CCK-8 or gastrin-17 (100 nM) to NCI-H727 human lung cancer cells increased EGFR Tyr(1068) phosphorylation after 2 min. The ability of CCK-8 to cause EGFR tyrosine phosphorylation was blocked by CI-988, gefitinib (EGFR tyrosine kinase inhibitor), PP2 (Src inhibitor), GM6001 (matrix metalloprotease inhibitor), and tiron (superoxide scavenger). CCK-8 nonsulfated and gastrin-17 caused EGFR transactivation and bound with high affinity to NCI-H727 cells, suggesting that the CCK2R is present. CI-988 inhibited the ability of CCK-8 to cause ERK phosphorylation and elevate cytosolic Ca(2+). CI-988 or gefitinib inhibited the basal growth of NCI-H727 cells or that stimulated by CCK-8. The results indicate that CCK/gastrin may increase lung cancer proliferation in an EGFR-dependent manner.

Saphenous veins used as arterial grafts are exposed to arterial levels of oxygen partial pressure (pO2), which are much greater than what they experience in their native environment. The object of this study is to determine the impact of exposing human saphenous veins to arterial pO2. Saphenous veins and left internal mammary arteries from consenting patients undergoing coronary artery bypass grafting were cultured ex vivo for 2 weeks in the presence of arterial or venous pO2 using an established organ culture model. Saphenous veins cultured with arterial pO2 developed intimal hyperplasia as evidenced by 2.8-fold greater intimal area and 5.8-fold increase in cell proliferation compared to those freshly isolated. Saphenous veins cultured at venous pO2 or internal mammary arteries cultured at arterial pO2 did not develop intimal hyperplasia. Intimal hyperplasia was accompanied by two markers of elevated reactive oxygen species (ROS): increased dihydroethidium associated fluorescence (4-fold, p<0.05) and increased levels of the lipid peroxidation product, 4-hydroxynonenal (10-fold, p<0.05). A functional role of the increased ROS saphenous veins exposed to arterial pO2 is suggested by the observation that chronic exposure to tiron, a ROS scavenger, during the two-week culture period, blocked intimal hyperplasia. Electron paramagnetic resonance based oximetry revealed that the pO2 in the wall of the vessel tracked that of the atmosphere with a ~30 mmHg offset, thus the cells in the vessel wall were directly exposed to variations in pO2. Monolayer cultures of smooth muscle cells isolated from saphenous veins exhibited increased proliferation when exposed to arterial pO2 relative to those cultured at venous pO2. This increased proliferation was blocked by tiron. Taken together, these data suggest that exposure of human SV to arterial pO2 stimulates IH via a ROS-dependent pathway.

Chitosan (CHT) is a non-toxic and inexpensive compound obtained by deacetylation of chitin, the main component of the exoskeleton of arthropods as well as of the cell walls of many fungi. In agriculture CHT is used to control numerous diseases on various horticultural commodities but, although different mechanisms have been proposed, the exact mode of action of CHT is still unknown. In sycamore (Acer pseudoplatanus L.) cultured cells, CHT induces a set of defense/stress responses that includes production of H2O2 and nitric oxide (NO). We investigated the possible signaling role of these reactive molecules in some CHT-induced responses by means of inhibitors of production and/or scavengers. The results show that both reactive nitrogen and oxygen species are not only a mere symptom of stress conditions but are involved in the responses induced by CHT in sycamore cells. In particular, NO appears to be involved in a cell death form induced by CHT that shows apoptotic features like DNA fragmentation, increase in caspase-3-like activity and release of cytochrome c from the mitochondrion. On the contrary, reactive oxygen species (ROS) appear involved in a cell death form induced by CHT that does not show these apoptotic features but presents increase in lipid peroxidation.

Oxygenation with 95%O2 is routinely used in organ bath studies. However, hyperoxia may affect tissue responses, particularly in studies which involve reactive oxygen species (ROS). Here, the effects of the antioxidant, Tiron, were investigated under different gassing conditions in the porcine isolated coronary artery (PCA). Distal PCAs from male and female pigs were mounted in a wire myograph gassed with either 95%O2/5%CO2 or 95% air/5%CO2 and pre-contracted with U46619. Concentration-response curves to bradykinin were constructed in the presence of Tiron (1mM), a cell permeable superoxide scavenger and catalase (1000Uml(-1)) to breakdown H2O2. The H2O2 level in Krebs'-Henseleit solution was detected using Amplex Red. Bradykinin produced concentration-dependent vasorelaxations in male and female PCAs when gassed with either 95%O2 or air, with no differences in the Rmax or EC50. Tiron increased the potency of bradykinin only when gassed with 95%O2 in PCAs from both sexes. At 95%O2, catalase prevented the leftward shift caused by Tiron in both sexes indicating that catalase prevented the formation of H2O2 by Tiron. In female PCAs, addition of catalase to Tiron significantly reduced the Rmax. In the EDH-type response (using L-NAME and indomethacin), Tiron enhanced the potency of the bradykinin-induced vasorelaxation when gassed with 95%O2 in PCAs from both sexes. Biochemical analysis using Amplex Red demonstrated that H2O2 was generated in Krebs'-Henseleit solution when gassed with 95%O2, but not with air. Therefore, hyperoxic gassing conditions could alter the environment generating superoxide within the Krebs'-Henseleit buffer, which may, in turn, influence the in vitro pharmacological responses., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We hypothesized that acute stress would induce endothelial dysfunction. Male Wistar rats were restrained for 2 h within wire mesh. Functional and biochemical analyses were conducted 24 h after the 2-h period of restraint. Stressed rats showed decreased exploration on the open arms of an elevated-plus maze (EPM) and increased plasma corticosterone concentration. Acute restraint stress did not alter systolic blood pressure, whereas it increased the in vitro contractile response to phenylephrine and serotonin in endothelium-intact rat aortas. NG-nitro-l-arginine methyl ester (l-NAME; nitric oxide synthase, NOS, inhibitor) did not alter the contraction induced by phenylephrine in aortic rings from stressed rats. Tiron, indomethacin and SQ29548 reversed the increase in the contractile response to phenylephrine induced by restraint stress. Increased systemic and vascular oxidative stress was evident in stressed rats. Restraint stress decreased plasma and vascular nitrate/nitrite (NOx) concentration and increased aortic expression of inducible (i) NOS, but not endothelial (e) NOS. Reduced expression of cyclooxygenase (COX)-1, but not COX-2, was observed in aortas from stressed rats. Restraint stress increased thromboxane (TX)B(2) (stable TXA(2) metabolite) concentration but did not affect prostaglandin (PG)F2α concentration in the aorta. Restraint reduced superoxide dismutase (SOD) activity, whereas concentrations of hydrogen peroxide (H(2)O(2)) and reduced glutathione (GSH) were not affected. The major new finding of our study is that restraint stress increases vascular contraction by an endothelium-dependent mechanism that involves increased oxidative stress and the generation of COX-derived vasoconstrictor prostanoids. Such stress-induced endothelial dysfunction could predispose to the development of cardiovascular diseases.

Background: It has been demonstrated that photodynamic therapy (PDT) is a promising treatment approach for hyperplastic dermatosis and results in a beneficial outcome. In the present study, PDT involving hematoporphyrin monomethyl ether (HMME) was applied to keloid fibroblasts (KFB), and the effects and the mechanism of action were explored., Methods: Keloid fibroblastic cells were divided into four groups (PDT group, light alone group, HMME alone group, normal cultured group). Cell proliferation and apoptosis were observed. Radical oxygen species (ROS) were detected by means of dihydroethidium (DHE) and dihydrorhodamine (DHR123). ROS in the PDT group were also assessed after addition of tiron., Results: Cell proliferation was inhibited in the PDT group (p < 0.05), while the rate of apoptosis was also clearly increased (p < 0.05). The levels of ROS were significantly higher in the PDT group than was observed in the other three groups (p < 0.05). With the addition of tiron the damaging effects were reduced., Conclusions: Our data indicated that HMME-mediated PDT could inhibit keloid fibroblast proliferation and could also induce apoptosis. This process was associated with the production of ROS.

Background: In this study we investigated the in vitro and in vivo anticancer effect of carnosol, a naturally occurring polyphenol, in triple negative breast cancer., Results: We found that carnosol significantly inhibited the viability and colony growth induced G2 arrest in the triple negative MDA-MB-231. Blockade of the cell cycle was associated with increased p21/WAF1 expression and downregulation of p27. Interestingly, carnosol was found to induce beclin1-independent autophagy and apoptosis in MDA-MB-231 cells. The coexistence of both events, autophagy and apoptosis, was confirmed by electron micrography. Induction of autophagy was found to be an early event, detected within 3 h post-treatment, which subsequently led to apoptosis. Carnosol treatment also caused a dose-dependent increase in the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2). Moreover, we show that carnosol induced DNA damage, reduced the mitochondrial potential and triggered the activation of the intrinsic and extrinsic apoptotic pathway. Furthermore, we found that carnosol induced a dose-dependent generation of reactive oxygen species (ROS) and inhibition of ROS by tiron, a ROS scavenger, blocked the induction of autophagy and apoptosis and attenuated DNA damage. To our knowledge, this is the first report to identify the induction of autophagy by carnosol., Conclusion: In conclusion our findings provide strong evidence that carnosol may be an alternative therapeutic candidate against the aggressive form of breast cancer and hence deserves more exploration.

Propionic acidemia (PA), caused by a deficiency of the mitochondrial biotin dependent enzyme propionyl-CoA carboxylase (PCC) is one of the most frequent organic acidurias in humans. Most PA patients present in the neonatal period with metabolic acidosis and hyperammonemia, developing different neurological symptoms, movement disorders and cardiac complications. There is strong evidence indicating that oxidative damage could be a pathogenic factor in neurodegenerative, mitochondrial and metabolic diseases. Recently, we identified an increase in ROS levels in PA patients-derived fibroblasts. Here, we analyze the capability of seven antioxidants to scavenge ROS production in PA patients' cells. Tiron, trolox, resveratrol and MitoQ significantly reduced ROS content in patients and controls' fibroblasts. In addition, changes in the expression of two antioxidant enzymes, superoxide dismutase and glutathione peroxidase, were observed in PA patients-derived fibroblasts after tiron and resveratrol treatment. Our results in PA cellular models establish the proof of concept of the potential of antioxidants as an adjuvant therapy for PA and pave the way for future assessment of antioxidant strategies in the murine model of PA., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Sympathoexcitation elicited by central command, a parallel activation of the motor and autonomic neural circuits in the brain, has been shown to become exaggerated in chronic heart failure (CHF). The present study tested the hypotheses that oxidative stress in the medulla in CHF plays a role in exaggerating central command-elicited sympathoexcitation, and that exercise training in CHF suppresses central command-elicited sympathoexcitation through its antioxidant effects in the medulla. In decerebrate rats, central command was activated by electrically stimulating the mesencephalic locomotor region (MLR) after neuromuscular blockade. The MLR stimulation at a current intensity greater than locomotion threshold in rats with CHF after myocardial infarction (MI) evoked larger (P < 0.05) increases in renal sympathetic nerve activity and arterial pressure than in sham-operated healthy rats (Sham) and rats with CHF that had completed longterm (8–12 weeks) exercise training (MI + TR). In the Sham and MI + TR rats, bilateral microinjection of a superoxide dismutase (SOD) mimetic Tempol into the rostral ventrolateral medulla (RVLM) had no effects on MLR stimulation-elicited responses. By contrast, in MI rats, Tempol treatment significantly reduced MLR stimulation-elicited responses. In a subset of MI rats, treatment with Tiron, another SOD mimetic, within the RVLM also reduced responses. Superoxide generation in the RVLM, as evaluated by dihydroethidium staining, was enhanced in MI rats compared with that in Sham and MI + TR rats. Collectively, these results support the study hypotheses. We suggest that oxidative stress in the medulla in CHF mediates central command dysfunction, and that exercise training in CHF is capable of normalizing central command dysfunction through its antioxidant effects in the medulla.

Reactive oxygen species (ROS) are metabolism by-products that may act as signaling molecules to sustain tumor growth. Antioxidants have been used to impair cancer cell survival. Our goal was to determine the mechanisms involved in the response to antioxidants of a human cell culture (PT4) containing glioblastoma (GBM) tumorigenic initiating cells (TICs). ROS production in the absence or presence of N-acetyl-L-cysteine (NAC), tiron, and trolox was evaluated by flow cytometry (FCM). The effects of these antioxidants on cell survival and apoptosis were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) and FCM. The biological processes modulated by these drugs were determined by oligonucleotide microarray gene expression profiling. Our results showed that NAC, tiron and trolox impaired PT4 cell survival, had minor effects on ROS levels and caused wide deregulation of cell cycle genes. Furthermore, tiron and trolox caused inhibition of cell survival in two additional cell cultures containing TICs, FO-1 and MM1, established from a melanoma and a mesothelioma patient, respectively. NAC, instead, impaired survival of the MM1 cells but not of the FO-1 cells. However, when used in combination, NAC enhanced the inhibitory effect of PLX4032 (BRAF V600E inhibitor) and Gefitinib (EGFR inhibitor), on FO-1 and PT4 cell survival. Collectively, NAC, tiron and trolox modulated gene expression and impaired the growth of cultures containing TICs primarily by inhibiting cell cycle progression.

Chronic arsenic exposure has been linked to endothelial dysfunction and apoptosis. We investigate the involvement of unfolded protein response (UPR) signaling in the arsenic-mediated cytotoxicity of the SVEC4-10 mouse endothelial cells. The SVEC4-10 cells underwent apoptosis in response to As2O3 dose- and time-dependently, accompanied by increased accumulation of calcium, and activation of caspase-3. These phenomena were completely inhibited by α-lipoic acid (LA), which did not scavenge ROS over-production, but were only partially or not ameliorated by tiron, a potent superoxide scavenger. Moreover, arsenic activated UPR, leading to phosphorylation of eukaryotic translation initiation factor 2 subunit α (eIF2α), induction of ATF4, and processing of ATF6. Treatment with arsenic also triggered the expression of endoplasmic reticulum (ER) stress markers, GRP78 (glucose-regulated protein), and CHOP (C/EBP homologous protein). The activation of eIF2α, ATF4 and ATF6 and expression of GRP78 and CHOP are repressed by both LA and tiron, indicating arsenic-induced UPR is mediated through ROS-dependent and ROS-independent pathways. Arsenic also induced ER stress-inducible genes, BAX, PUMA (p53 upregulated modulator of apoptosis), TRB3 (tribbles-related protein 3), and SNAT2 (sodium-dependent neutral amino acid transporter 2). Consistent with intracellular calcium and cell viability data, ROS may not be important in arsenic-induced death, because tiron did not affect the expression of these pro-apoptotic genes. In addition, pretreatment with salubrinal, a selective inhibitor of eIF2α dephosphorylation, enhanced arsenic-induced GRP78 and CHOP expression and partially prevented arsenic cytotoxicity in SVEC4-10 cells. Taken together, these results suggest that arsenic-induced endothelial cytotoxicity is associated with ER stress, which is mediated by ROS-dependent and ROS-independent signaling.

The blue-green phenazine, Pyocyanin (PYO), is a well-known virulence factor produced by Pseudomonas aeruginosa, notably during cystic fibrosis lung infections. It is toxic to both eukaryotic and bacterial cells and several mechanisms, including the induction of oxidative stress, have been postulated. However, the mechanism of PYO toxicity under the physiological conditions of oxygen limitation that are encountered by P. aeruginosa and by target organisms in vivo remains unclear. In this study, wild-type and mutant strains of the yeast Saccharomyces cerevisiae were used as an effective eukaryotic model to determine the toxicity of PYO (100-500 μmol/L) under key growth conditions. Under respiro-fermentative conditions (with glucose as substrate), WT strains and certain H2 O2 -hypersensitive strains showed a low-toxic response to PYO. Under respiratory conditions (with glycerol as substrate) all the strains tested were significantly more sensitive to PYO. Four antioxidants were tested but only N-acetylcysteine was capable of partially counteracting PYO toxicity. PYO did not appear to affect short-term respiratory O2 uptake, but it did seem to interfere with cyanide-poisoned mitochondria through a complex III-dependent mechanism. Therefore, a combination of oxidative stress and respiration disturbance could partly explain aerobic PYO toxicity. Surprisingly, the toxic effects of PYO were more significant under anaerobic conditions. More pronounced effects were observed in several strains including a 'petite' strain lacking mitochondrial DNA, strains with increased or decreased levels of ABC transporters, and strains deficient in DNA damage repair. Therefore, even though PYO is toxic for actively respiring cells, O2 may indirectly protect the cells from the higher anaerobic-linked toxicity of PYO. The increased sensitivity to PYO under anaerobic conditions is not unique to S. cerevisiae and was also observed in another yeast, Candida albicans., (© 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

There are many evidences indicating a compensatory mechanism in contralateral carotids following balloon injury. Previously it was observed α1-adrenoceptor-mediated hyper-reactivity and impairment of calcium influx in contralateral carotids 4 days after injury. At a later stage, α1-adrenoceptor-mediated contraction is similar to the control and we hypothesized that downstream signaling was normal. In the present study, we aimed to evaluate α1-adrenoceptor-mediated calcium influx in contralateral carotids 15 days after balloon injury. Concentration-response curves for CaCl2 in presence of the α1-adrenoceptor agonist (phenylephrine), measurement of the intracellular calcium transient and the levels of reactive oxygen species using fluorescent dyes were performed in control and contralateral carotids. Phenylephrine-induced intracellular calcium mobilization in contralateral carotids was not altered, while phenylephrine-induced calcium influx was reduced in the contralateral artery. Nitric oxide synthase inhibitors, L-NAME or L-NNA, restored this response, but nitrite and nitrate levels were decreased in contralateral carotids. Additionally, a rise in oxygen free radicals was observed in contralateral carotids. Furthermore, Tiron, a superoxide anion scavenger, restored α1-adrenoceptor-mediated calcium influx in contralateral carotids to the control level. Similar results were observed with the selective potassium channels blockers 4-aminopyridine and charybdotoxin. In conclusion, data showed that balloon catheter injury resulted in increased superoxide anions levels, activation of potassium channels (Kv and BKCa), inhibition of calcium channels (Cav) and preservation of α1-adrenoceptor-mediated contraction at a later stage after injury., (© 2013 Published by Elsevier B.V.)

Fibrosis is the final disorder of end-stage renal disease. Activation of fibroblast growth factor (FGF) 23-klotho axis could suppress renal fibrosis in mice. Also, a marked decrease of klotho expression was observed in the kidney of streptozotocin-induced diabetic rats (STZ rats). However, relation of FGF in renal fibrosis remained unclear. This study was aimed to screen the effect of hyperglycemia on FGF receptor (FGFR) and fibrosis in kidney of rats with diabetic nephropathy and investigate this potential mechanism in cultured Madin-Darby Canine Kidney (MDCK) epithelial cells. STZ rats were used to treat with insulin or phloridzin at the dose sufficient to correct hyperglycemia for understanding the changes of renal dysfunction. The cultured MDCK cells were also used to treat with high glucose, hydrogen peroxide, or tiron in addition to transfection of siRNA to silence the klotho. Both insulin and phloridzin reversed fibrosis and FGFR expressions in kidney of STZ rats. It was confirmed in high glucose-exposed MDCK cells. However, klotho failed to modify the level of FGFR in MDCK cells. Meanwhile, FGFR was restored by tiron in MDCK cells and in diabetic rats without changing blood glucose. In conclusion, interstitial fibrosis and decreased FGFR expression are observed in the kidney of diabetic rats. This change is reversed by tiron without the correction of blood glucose. Also, klotho has no effect on expression of FGFR. Thus, decrease of oxidative stress is useful for the recovery of FGFR expression and improvement of renal fibrosis in type-1 like diabetic rats., (© Georg Thieme Verlag KG Stuttgart · New York.)

Skin cancer and aging are linked to increased cellular reactive oxygen species (ROS), particularly following exposure to ultraviolet A (UVA) in sunlight. As mitochondria are the main source of cellular ROS, this study compared the protective effects of mitochondria-targeted and -localized antioxidants (MitoQ and tiron, respectively) with cellular antioxidants against oxidative stress-induced [UVA and hydrogen peroxide (H2O2)] mitochondrial DNA (mtDNA) damage in human dermal fibroblasts. With the use of a long quantitative PCR assay, tiron (EC50 10 mM) was found to confer complete (100%) protection (P<0.001) against both UVA- and H2O2-induced mtDNA damage, whereas MitoQ (EC50 750 nM) provided less protection (17 and 32%, respectively; P<0.05). This particular protective effect of tiron was greater than a range of cellular antioxidants investigated. The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway provides cellular protection against oxidative stress. An ELISA assay for the Nrf2 target gene heme oxygenase-1 (HO-1) and studies using Nrf2 small interfering RNA both indicated that tiron's mode of action was Nrf2 independent. The comet assay showed that tiron's protective effect against H2O2-induced nuclear DNA damage was greater than the cellular antioxidants and MitoQ (P<0.001). This study provides a platform to investigate molecules with similar structure to tiron as potent and clinically relevant antioxidants.

Protein damage mediated by oxidation has been associated with aging and age-related diseases, in particular neurodegenerative diseases. The protein that is known to be one of the major targets of oxidative stress is glyceraldehyde- 3-phosphate dehydrogenase. GAPDH is believed to play a key role in certain neurodegenerative disorders, such as Alzheimer's, Parkinson's, and Huntington's diseases. Several recent studies have suggested that a wide range of variety of polyphenols including resveratrol may have neuroprotective effects. Here, we present data that clearly indicate the prooxidative properties of resveratrol and tiron in the inactivation of GAPDH induced by the superoxide anion generated via xanthine oxidase mediated oxidation of xanthine. Generated in the studied system tiron and resveratrol radicals are much more efficient in the inactivation of GAPDH than the superoxide anion alone. The analysis of CD spectra of protein exposed to the tiron and resveratrol radicals revealed little effect on the secondary structure of GAPDH. In both cases reduction of &#945;-helical structure was followed by the increase in β-sheet conformation. Thus, the most probable mechanism of inactivation of GAPDH in the studied system is oxidation of cysteine residues in the catalytic center of the enzyme. Finally, molecular modeling of the resveratrol - GAPDH and tiron - GAPDH complexes showed potential binding sites for those antioxidants with binding affinity -45 kcal/mol and -48 kcal/mol respectively.

A close relationship between cell death and pathological calcification has recently been reported, such as vascular calcification in atherosclerosis. However, the roles of cell death in calcification by osteoblast lineage have not been elucidated in detail. In this study, we investigated whether cell death is involved in the calcification on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hMSC) under osteogenic culture in vitro. Apoptosis and necrosis occurred in an osteogenic culture of hMSC, and cell death preceded calcification. The generation of intracellular reactive oxygen species, chromatin condensation and fragmentation, and caspase-3 activation increased in this culture. A pan-caspase inhibitor (Z-VAD-FMK) and anti-oxidants (Tiron and n-acetylcysteine) inhibited osteogenic culture-induced cell death and calcification. Furthermore, calcification was significantly promoted by the addition of necrotic dead cells or its membrane fraction. Spontaneously dead cells by osteogenic culture and exogenously added necrotic cells were surrounded by calcium deposits. Induction of localized cell death by photodynamic treatment in the osteogenic culture resulted in co-localized calcification. These findings show that necrotic and apoptotic cell deaths were induced in an osteogenic culture of hMSC and indicated that both necrotic and apoptotic cells of osteoblast lineage served as nuclei for calcification on osteoblastic differentiation of hMSC in vitro., (Copyright © 2013 John Wiley & Sons, Ltd.)

Myofibroblast transformation is a key process in the pathogenesis of lung fibrosis. We have previously reported that hyperoxia induces RhoA activation in HFL-1 lung fibroblasts and RhoA mediates collagen synthesis in hyperoxic lung fibrosis. In this study, we investigated the role of RhoA and actin cytoskeleton in hyperoxia-induced myofibroblast transformation. Exposure of HFL-1 lung fibroblasts to hyperoxia stimulated actin filament formation, shift of G-actin to F-actin, nuclear colocalization of myocardin-related transcription factor-A (MRTF-A), recruitment of MRTF-A to the α-smooth muscle actin (α-SMA) gene promoter, myofibroblast transformation, and collagen-I synthesis. Inhibition of RhoA by C3 transferase CT-04 or dominant-negative RhoA mutant T19N, and inhibition of ROCK by Y27632, prevented myofibroblast transformation and collagen-I synthesis. Moreover, inhibition of RhoA by CT-04 prevented hyperoxia-induced actin filament formation, shift of G-actin to F-actin, and nuclear colocalization of MRTF-A. In addition, disrupting actin filaments with cytochalasin D or scavenging reactive oxygen species (ROS) with tiron attenuated actin filament formation, nuclear colocalization of MRTF-A, myofibroblast transformation, and collagen-I synthesis. Furthermore, overexpression of constitutively active RhoA mutant Q63L or stabilization of actin filaments recapitulated the effects of hyperoxia on the actin cytoskeleton and nuclear colocalization of MRTF-A, myofibroblast transformation, and collagen-I synthesis. Interestingly, knocking down MRTF-A prevented hyperoxia-induced increase in the recruitment of MRTF-A to the serum response factor transcriptional complex on the α-SMA gene promoter, myofibroblast transformation, and collagen-I synthesis. Finally, Y27632 and tiron attenuated hyperoxia-induced increases in α-SMA and collagen-I in mouse lungs. Together, these results indicate that the actin cytoskeletal reorganization due to the ROS/RhoA-ROCK pathway mediates myofibroblast transformation and collagen synthesis in lung fibrosis of oxygen toxicity. MRTF-A contributes to the regulatory effect of the actin cytoskeleton on myofibroblast transformation during hyperoxia., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Reactive oxygen species and norepinephrine are known as physiological active substances which cause cell damage and cardiac dysfunction in myocardial ischemia/reperfusion injury. We investigated the role of reactive oxygen species, especially superoxide (O2(-)), in ischemia-induced norepinephrine overflow and cardiac dysfunction using superoxide scavengers tempol and tiron. According to the Langendorff technique, isolated rat hearts were subjected to 40-min global ischemia followed by 30-min reperfusion. Tempol (10 and 100 µM) and tiron (100 and 500 µM) were perfused 15 min before ischemia and during reperfusion. Cardiac levels of oxidative stress markers such as O2(-) and malondialdehyde were notably increased during ischemia and following reperfusion, which were suppressed by the administration of tempol or tiron. These agents significantly improved ischemia/reperfusion-induced cardiac dysfunction such as decreased left ventricular developed pressure and the maximum and minimum value of the first derivative of left ventricular pressure and increased left ventricular end-diastolic pressure. Furthermore, norepinephrine overflow in the coronary effluent after ischemia/reperfusion was significantly suppressed by the administration of each agent. These results suggest that endogenously increased O2(-) is involved in norepinephrine overflow and cardiac dysfunction after myocardial ischemia/reperfusion., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Mechanical ventilation with high tidal volumes has been associated with pulmonary alveolar flooding. Understanding the mechanisms underlying cyclic stretch-induced increases in alveolar epithelial permeability may be important in designing preventive measures for acute lung injury. In this work, we assessed whether cyclic stretch leads to the generation of reactive oxygen species in type I-like alveolar epithelial cells, which increase monolayer permeability via activation of NF-κB and extracellular signal-regulated kinase (ERK). We cyclically stretched type I-like rat primary alveolar epithelial cells at magnitudes of 12, 25, and 37% change in surface area (ΔSA) for 10 to 120 minutes. High levels of reactive oxygen species and of superoxide and NO specifically were detected in cells stretched at 37% ΔSA for 10 to 120 minutes. Exogenous superoxide and NO stimulation increased epithelial permeability in unstretched cells, which was preventable by the NF-κB inhibitor MG132. The cyclic stretch-induced increase in permeability was decreased by the superoxide scavenger tiron and by MG132. Furthermore, tiron had a dramatic protective effect on in vivo lung permeability under mechanical ventilation conditions. Cyclic stretch increased the activation of the NF-κB signaling pathway, which was significantly decreased with the ERK inhibitor U0126. Altogether, our in vitro and in vivo data demonstrate the sensitivity of permeability to stretch- and ventilation-induced superoxide production, suggesting that using antioxidants may be helpful in the prevention and treatment of ventilator-induced lung injury.

This study describes the mechanism of trolox and tiron induced potentiation of cytotoxicity caused by Ery5, an analog of magnolol, in human myeloid leukemia HL-60 cells. Ery5 induced cytotoxicity in HL-60 cells by involving activation of bax and cleavage of caspase 3, which contributed towards activation of both apoptotic and autophagic pathways. Trolox and tiron, even at non-toxic concentrations, contributed to the cytotoxicity of Ery5 by activation of autophagic proteins like ATG7, ATG12 and LC3-II. Z-VAD-fmk mediated reduction in the cytotoxicity and expression of autophagic proteins, further suggested that autophagy induced by Ery5 is largely dependent upon caspases. Interestingly, Ery5 induced autophagy was accompanied by the downregulation of PI3K/AKT pathway whereas, trolox and tiron strongly enhanced this effect. In addition to that treatment of cells with Ery5, trolox and tiron individually, displayed a marked upregulation of Bax. The involvement of Bax in trolox and tiron induced enhancement of the cytotoxicity of Ery5 was confirmed, when siRNA induced silencing of Bax led to increased viability of the cells and exerted a strong inhibitory effect on LC3-II accumulation and p62 degradation in case of cells treated by the combination of Ery5 with trolox or tiron. Additionally, an important role of PARP in Ery5 mediated cell death has been suggested by PARP silencing experiments, however, potentiation of autophagic cytotoxicity by trolox and tiron did not seem to be dependent on PARP-1. Therefore, Bax seems to play a vital role in trolox and tiron mediated potentiation of autophagic cell death by Ery5 in HL-60 cells.

Endothelial dysfunction is associated with KCa3.1 dysfunction and contributes to the development of hypertension in preeclampsia. However, evidence of endothelial KCa3.1 dysfunction in the vascular system from women with preeclampsia is still lacking. Therefore, we examined whether endothelial KCa3.1 dysfunction occurs in vessels from women with preeclampsia. We compared KCa3.1 and NADPH oxidase (NOX) expression in umbilical vessels and primary cultured human umbilical vein endothelial cells (HUVECs) from normal (NP; n=17) and preeclamptic pregnancy (PE; n=19) and examined the effects of plasma from NP or PE on KCa3.1 and NOX2 expression in primary cultured HUVECs from NP or human uterine microvascular endothelial cells. The endothelial KCa3.1 was downregulated, and NOX2 was upregulated, in umbilical vessels and HUVECs from PE, compared with those from NP. In addition, HUVECs from PE showed a significant decrease in KCa3.1 current. Plasma from PE induced KCa3.1 down regulation, NOX2 upregulation, phosphorylated-p38 mitogen-activated protein kinase downregulation, and superoxide generation, and these effects were prevented by antioxidants (tempol or tiron), NOX2 inhibition, or anti-lectin-like oxidized low-density lipoprotein (LDL) receptor 1 (LOX1) antibody. Oxidized LDL and the superoxide donor xanthine/xanthine oxidase mixture induced KCa3.1 downregulation. In contrast, plasma from PE did not generate hydrogen peroxide, and the hydrogen peroxide donor tert-butylhydroperoxide induced KCa3.1 upregulation. These results provide the first evidence that plasma from PE generates superoxide via a LOX1-NOX2-mediated pathway and downregulates endothelial KCa3.1, which may contribute to endothelial dysfunction and vasculopathy in preeclampsia. This suggests KCa3.1as a novel target for patients with preeclampsia., (Copyright © 2012 Elsevier Inc. All rights reserved.)

"Ecstasy" (3,4-methylenedioxymethamphetamine or MDMA) is a widely abused recreational drug, reported to produce neurotoxic effects, both in laboratory animals and in humans. MDMA metabolites can be major contributors for MDMA neurotoxicity. This work studied the neurotoxicity of MDMA and its catechol metabolites, α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA) in human dopaminergic SH-SY5Y cells differentiated with retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate. Differentiation led to SH-SY5Y neurons with higher ability to accumulate dopamine and higher resistance towards dopamine neurotoxicity. MDMA catechol metabolites were neurotoxic to SH-SY5Y neurons, leading to caspase 3-independent cell death in a concentration- and time-dependent manner. MDMA did not show a concentration- and time-dependent death. Pre-treatment with the antioxidant and glutathione precursor, N-acetylcysteine (NAC), resulted in strong protection against the MDMA metabolites' neurotoxicity. Neither the superoxide radical scavenger, tiron, nor the inhibitor of the dopamine (DA) transporter, GBR 12909, prevented the metabolites' toxicity. Cells exposed to α-MeDA showed an increase in intracellular glutathione (GSH) levels, which, at the 48 h time-point, was not dependent in the activity increase of γ-glutamylcysteine synthetase (γ-GCS), revealing a possible transient effect. Importantly, pre-treatment with buthionine sulfoximine (BSO), an inhibitor of γ-GCS, prevented α-MeDA induced increase in GSH levels, but did not augment this metabolite cytotoxicity. Even so, BSO pre-treatment abolished NAC protective effects against α-MeDA neurotoxicity, which were, at least partially, due to GSH de novo synthesis. Inversely, pre-treatment of cells with BSO augmented N-Me-α-MeDA-induced neurotoxicity, but only slightly affected NAC neuroprotection. In conclusion, MDMA catechol metabolites promote differential toxic effects to differentiated dopaminergic human SH-SY5Y cells., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Menopause is associated with endothelial dysfunction and oxidative stress. In this condition, reduced n-3 polyunsaturated fatty acids (n-3 PUFAs) contribute to cardiovascular disease. We investigated whether treatment with n-3 PUFA reverses endothelial dysfunction and oxidative stress in experimental menopause. Thirty female rats underwent either sham-surgery or bilateral ovariectomy or bilateral ovariectomy+oral n-3 PUFA (0.8 g kg(-1) day(-1) for 2 months). Ovariectomy caused endothelial dysfunction to acetylcholine, which was reversed by superoxide scavenger Tiron. Erythrocyte membrane lipid composition was characterized by reduced n-3 PUFA total content and omega-3 index, and by concomitant increase in n-6:n-3 PUFA ratio. Ovariectomy-related oxidative stress, demonstrated by both enhanced superoxide production and 3-nitrotyrosine expression in aorta, was associated with increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit NOX-4 protein expression. Endothelial nitric oxide synthase (eNOS) functional inhibition by l-NG-nitroarginine methyl ester, protein expression and activity did not change. In ovariectomized rats, treatment with n-3 PUFA increased n-3 PUFA total content and omega-3 index and decreased n-6:n-3 PUFA ratio in erythrocyte membrane, reversed vascular oxidative stress, endothelial dysfunction, aortic 3-nitrotyrosine and markedly lowered NOX-4 protein expression; eNOS protein expression also increased, paralleled by reversal of inhibitory binding to Caveolin-1, while ex-vivo functional inhibition and NOS synthesis were unchanged. These findings demonstrate in vivo a therapeutic benefit of n-3 PUFA on menopause-associated endothelial dysfunction by reversal of alterations in membrane lipid composition induced by ovariectomy and by reduction of vascular oxidative stress. In this setting they also identify NOX-4 as a potential target to reduce oxidative stress-mediated vascular complications., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aims: Endothelin-1 (ET-1) has been shown to increase endothelial superoxide (O(2)(-)) production in experimental animal models. It is unclear whether ET-1 increases O(2)(-) production in humans. We sought to elucidate whether ET-1 increases O(2)(-) production in human vessels and to identify the mechanism behind this effect., Main Methods: Segments of internal mammary artery (IMA) and human saphenous vein (HSV) were harvested from 90 patients undergoing elective coronary artery bypass graft surgery. Paired vessel rings were incubated in the presence and absence of ET-1 (10(-10)M), the ET(A) receptor antagonist BQ123 alone, or in combination with the ET(B) receptor antagonist BQ788 (dual BQ) and known inhibitors of sources of O(2)(-) and further analysed for O(2)(-) production using lucigenin-enhanced chemiluminescence and DHE fluorescence., Key Findings: ET-1 increased O(2)(-) production in both IMA (2.6 ± 1.5 vs. 1.4 ± 0.8 relative light units/s/mg tissue (RLU); n=33; p < 0.0001) and HSV (1.4 ± 0.8 vs. 1.1 ± 0.6 RLU; n=24; p<0.05). The increase in O(2)(-)production induced by ET-1 in IMA was inhibited by co-incubation with dual BQ (p < 0.05; n=15) and BQ123 (p<0.05; n = 17). Of known O(2)(-) inhibitors, only incubation with Tiron and diphenyleneiodonium resulted in a significant reduction in ET-mediated O(2)(-) production., Significance: ET-1 increases O(2)(-) production especially in human arteries and less so in veins from patients with coronary artery disease via a receptor-dependent pathway involving a flavin dependent enzyme which is likely to be NADPH oxidase. Production of O(2)(-) may be an important factor underlying the negative effects of ET-1 on vascular function such as impairment of endothelium-dependent vasodilatation and pro-inflammatory effects., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background/objectives:   Free radicals and reactive oxygen species (ROS), which are generated by UV irradiation, may induce an irreversible growth arrest similar to senescence. Tiron, 4,5-dihydroxy-1,3-benzene disulfonic acid, is a widely used antioxidant to rescue ROS-evoked cell death. The aim of the article was to explore the effects of tiron on skin photoaging and associated mechanisms., Methods:   The effects of tiron on cell proliferation were determined using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide. Senescent cells were determined by morphology and senescence-associated β-galactosidase activity analysis. Intracellular hydrogen peroxide, superoxide anion and glutathione concentration were analysed by a fluorescent probe. The concomitant changes of protein expression were analysed with Western blot., Results:   Human dermal fibroblasts were induced to premature senescence by sub-cytotoxic doses of irradiated UVB. Strong senescence-associated β-galactosidase activity and increased intracellular superoxide anion were observed in human dermal fibroblasts irradiated by UVB. Tiron blocks UVB-induced glutathione depletion and increase of superoxide anion and protects against UVB-induced senescence-like characteristics in human dermal fibroblasts. Compared with normal fibroblasts, UVB-irradiated human dermal fibroblasts showed a higher ratio of active (hypophosphorylated) to inactive (phosphorylated) forms of Rb and p38, upregulation of p53 or p16 and c-Myc and insulin-like growth factor 1 (IGF-1) downregulation. After treatment with tiron, p53, p16 c-Myc and IGF-1 as well as phosphorylation Rb and p38 could partially recover., Conclusion:   These results indicate that tiron protects against UVB-induced senescence-like characteristics in human dermal fibroblasts via the inhibition of production of superoxide anion and glutathione depletion, and modulation of related senescence proteins., (© 2012 The Authors. Australasian Journal of Dermatology © 2012 The Australasian College of Dermatologists.)

Although epidemiological data associate hypertension with a strong predisposition to develop Alzheimer disease, no mechanistic explanation exists so far. We developed a model of hypertension, obtained by transverse aortic constriction, leading to alterations typical of Alzheimer disease, such as amyloid plaques, neuroinflammation, blood-brain barrier dysfunction, and cognitive impairment, shown here for the first time. The aim of this work was to investigate the mechanisms involved in Alzheimer disease of hypertensive mice. We focused on receptor for advanced glycation end products (RAGE) that critically regulates Aβ transport at the blood-brain barrier and could be influenced by vascular factors. The hypertensive challenge had an early and sustained effect on RAGE upregulation in brain vessels of the cortex and hippocampus. Interestingly, RAGE inhibition protected from hypertension-induced Alzheimer pathology, as showed by rescue from cognitive impairment and parenchymal Aβ deposition. The increased RAGE expression in transverse aortic coarctation mice was induced by increased circulating advanced glycation end products and sustained by their later deposition in brain vessels. Interestingly, a daily treatment with an advanced glycation end product inhibitor or antioxidant prevented the development of Alzheimer traits. So far, Alzheimer pathology in experimental animal models has been recognized using only transgenic mice overexpressing amyloid precursor. This is the first study demonstrating that a chronic vascular insult can activate brain vascular RAGE, favoring parenchymal Aβ deposition and the onset of cognitive deterioration. Overall we demonstrate that RAGE activation in brain vessels is a crucial pathogenetic event in hypertension-induced Alzheimer disease, suggesting that inhibiting this target can limit the onset of vascular-related Alzheimer disease.

In pathological conditions, such as hypertension, there is impairment in the autonomic control of blood pressure resulting in changes in baroreflex sensitivity. In the present study we tested the hypothesis that acute superoxide scavenging would restore the depressed baroreflex sensitivity (BRS) in spontaneously hypertensive rats (SHR). Male 10-week-old SHR (n = 14) and their controls (Wistar-Kyoto (WKY) rats; n = 14) underwent femoral artery and vein catheterization for conscious blood pressure recording and drug administration. The BRS was obtained by the drug-induced method using phenylephrine (8 μg/kg, i.v.) and sodium nitroprusside (25 μg/kg, i.v.) before and after the administration of tiron (30 mg/kg, i.v.), a superoxide dismutase mimetic, or apocynin (30 μg/kg), an NADPH oxidase inhibitor. Spontaneously hypertensive rats was significantly hypertensive compared with WKY rats (160 ± 7 vs 105 ± 2 mmHg, respectively). However, there was no significant difference in heart rate between the two groups (388 ± 10 vs 370 ± 20 b.p.m.). In addition, SHR exhibited a diminished BRS compared with WKY rats (-1.34 ± 0.11 vs -2.91 ± 0.20 b.p.m./mmHg, respectively). Administration of tiron improved BRS in SHR (from -1.34 ± 0.11 to 2.26 ± 0.21 b.p.m./mmHg), as did apocynin (to -2.14 ± 0.23 b.p.m./mmHg). Serum samples from SHR (n = 20) and WKY rats (n = 20) were collected for thiobarbituric acid-reactive substances assays before and after tiron or apocynin to confirm the reduction in oxidative stress. There was considerably greater oxidative stress in SHR compared with WKY rats (36.2 ± 3.0 vs 13.3 ± 2.6 nmol/L, respectively). Both apocynin and tiron treatment reduced the oxidative stress in SHR (from 36.2 ± 3.0 to 21.5 ± 3.0 nmol/L and from 37.2 ± 3.9 to 21.9 ± 1.6 nmol/L, respectively). The data suggest that acute scavenging of NADPH oxidase-derived superoxide improves baroreflex sensitivity in SHR., (© 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Blackwell Publishing Asia Pty Ltd.)

Transient receptor potential vanilloid 1 (TRPV1) is a Ca(2+)-permeable cation channel activated by a variety of physicochemical stimuli. The effect of hypoxia (P(O(2)), 3%) on rat TRPV1 overexpressed in HEK293T has been studied. The basal TRPV1 current (I (TRPV1)) was partly activated by hypoxia, whereas capsaicin-induced TRPV1 (I (TRPV1,Cap)) was attenuated. Such changes were also suggested from hypoxia- and capsaicin-induced Ca(2+) signals in TRPV1-expressing cells. Regarding plausible changes of reactive oxygen species (ROS) under hypoxia, the effects of antioxidants, vitamin C and tiron, as membrane-impermeable and -permeable, respectively, were tested. Both I (TRPV1) and I (TRPV1,Cap) were increased by vitamin C, while only I (TRPV1) was slightly increased by tiron. The hypoxic inhibition of I (TRPV1,Cap) was still persistent under hypoxia/vitamin C. Interestingly, hypoxia/tiron strongly inhibited both I (TRPV1) and I (TRPV1,Cap). Also, with vitamin C applied through a pipette solution, hypoxia inhibited I (TRPV1) and I (TRPV1,Cap). In contrast, hypoxia and hypoxia/tiron had no effect on the I (TRPV1) induced by acid (pH 6.2, I (TRPV1,Acid)). Taken together, hypoxia partly activated TRPV1 while it decreased their sensitivity to capsaicin. Putative changes of ROS under hypoxia might underlie the side-specific effects of ROS on TRPV1: inhibitory at the extracellular and stimulatory at the intracellular side, respectively. The differential effects of hypoxia on I (TRPV1,Cap) and I (TRPV1,Acid) suggested that the intracellular ROS increase might attenuate the pharmacological potency of capsaicin.

Background: An impairment of cardiovascular function in streptozotocin (STZ)-diabetic rats has been mentioned within 5 days-to-3 months of induction. ATP-sensitive potassium (K(ATP)) channels are expressed on cardiac sarcolemmal membranes. It is highly responsive to metabolic fluctuations and can have effects on cardiac contractility. The present study attempted to clarify the changes of cardiac K(ATP) channels in diabetic disorders., Methods: Streptozotocin-induced diabetic rats and neonatal rat cardiomyocytes treated with a high concentration of glucose (a D-glucose concentration of 30 mM was used and cells were cultured for 24 hr) were used to examine the effect of hyperglycemia on cardiac function and the expression of K(ATP) channels. K(ATP) channels expression was found to be linked to cardiac tonic dysfunction, and we evaluated the expression levels of K(ATP) channels by Western blot and Northern blot analysis., Results: The result shows diazoxide produced a marked reduction of heart rate in control group. Furthermore, the methods of Northern blotting and Western blotting were employed to identify the gene expression of K(ATP) channel. Two subunits of cardiac K(ATP) channel (SUR2A and kir 6.2) were purchased as indicators and showed significantly decreased in both diabetic rats and high glucose treated rat cardiac myocytes. Correction of hyperglycemia by insulin or phlorizin restored the gene expression of cardiac K(ATP) in these diabetic rats., Conclusions: Both mRNA and protein expression of cardiac K(ATP) channels are decreased in diabetic rats induced by STZ for 8 weeks. This phenomenon leads to result in desensitization of some K(ATP) channel drugs.