Journal: Free radical research
Abstract Myeloperoxidase (MPO), a major constituent of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide (H(2)O(2)) and chloride anion. We have previously reported that MPO-deficient (MPO(-/-)) neutrophils produce greater amount of macrophage inflammatory protein-2 (MIP-2) in vitro than do wild-type when stimulated with zymosan. In this study, we investigated the molecular mechanisms governing the up-regulation of MIP-2 production in the mutant neutrophils. Interestingly, we found that zymosan-induced production of MIP-2 was blocked by pre-treatment with U0126, an inhibitor of mitogen-activated protein kinase/extracellular-signal regulated kinase (ERK), and with BAY11-7082, an inhibitor of nuclear factor (NF)-κB. Western blot analysis indicated that U0126 also inhibited the phosphorylation of p65 subunit of NF-κB (p65), indicating that MIP-2 was produced via the ERK/NF-κB pathway. Intriguingly, we found that ERK1/2, p65, and alpha subunit of inhibitor of κB (IκBα) in the MPO(-/-) neutrophils were phosphorylated more strongly than in the wild-type when stimulated with zymosan. Exogenous H(2)O(2) treatment in addition to zymosan stimulation enhanced the phosphorylation of ERK1/2 without affecting the zymosan-induced MIP-2 production. In contrast, exogenous HOCl inhibited the production of MIP-2 as well as IκBα phosphorylation without affecting ERK activity. The zymosan-induced production of MIP-2 in the wild-type neutrophils was enhanced by pre-treatment of the MPO inhibitor 4-Aminobenzoic acid hydrazide. Collectively, these results strongly suggest that both lack of HOCl and accumulation of H(2)O(2 )due to MPO deficiency contribute to the up-regulation of MIP-2 production in mouse neutrophils stimulated with zymosan.
Production of reactive oxygen species, including hydrogen peroxide (H2O2), is increased in diseased blood vessels. Although H2O2 leads to impairment of the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP signaling pathway, it is not clear whether this reactive molecule affects the redox state of sGC, a key determinant of NO bioavailability. To clarify this issue, mechanical responses of endothelium-denuded rat external iliac arteries to BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator), nitroglycerin (NO donor), acidified NaNO2 (exogenous NO), and 8-Br-cGMP (cGMP analog) were studied under exposure to H2O2. The relaxant response to BAY 41-2272 (pD2: 6.79 ± 0.10 and 6.62 ± 0.17), BAY 60-2770 (pD2: 9.57 ± 0.06 and 9.34 ± 0.15), or 8-Br-cGMP (pD2: 5.19 ± 0.06 and 5.24 ± 0.08) was not apparently affected by exposure to H2O2. In addition, vascular cGMP production stimulated with BAY 41-2272 or BAY 60-2770 in the presence of H2O2 was identical to that in its absence. On the other hand, nitroglycerin-induced relaxation was markedly attenuated by exposing the arteries to H2O2 (pD2: 8.73 ± 0.05 and 8.30 ± 0.05), which was normalized in the presence of catalase (pD2: 8.59 ± 0.05). Likewise, H2O2 exposure impaired the relaxant response to acidified NaNO2 (pD2: 6.52 ± 0.17 and 6.09 ± 0.16). These findings suggest that H2O2 interferes with the NO-mediated action, but the sGC redox equilibrium and the downstream target(s) of cGMP are unlikely to be affected in the vasculature.
Oxidative stress mediated by reactive oxygen species (ROS) contributes to renal tubular atrophy and fibrosis following renal transplantation. Studies have shown that mitochondrial chaperone prohibitin (PHB) has antioxidant effects. Here we used the human renal proximal tubule epithelial cell line, HK-2 cells as an in vitro model to assess the role of PHB in hydrogen peroxide (H2O2)-induced renal tubular oxidative injury. Our results showed that H2O2 treatment inhibited PHB expression in a time-dependent manner in HK-2 cells. PHB overexpression could protect cell from oxidative stress-induced injury by inhibiting H2O2-induced cell apoptosis, intracellular ROS generation and promoting endogenous antioxidant defense components including glutathione peroxidase, catalase, superoxide dismutase, and glutathione. Furthermore, oxidative stress-induced cell injury were suppressed in PHB-overexpressed tubule epithelial cells through mitochondria-mediated pathway, including inhibition of mitochondrial uncoupling protein 2 (UCP2) and Bax expression, mitochondrial ROS production, promotion of ATP production and Bcl-2 expression, inhibition of mitochondrial membrane potential loss and release of cytochrome c from mitochondria to cytoplasma and followed by caspase 3 activation. Meanwhile, inhibition of PHB expression by small interference RNA resulted in less resistance of HK-2 cells to H2O2 toxicity as shown in decreased cell viability, increased apoptosis, ROS production and mitochondrial dysfunction. These data indicated that PHB protected the tubule epithelial cells from oxidative stress-induced damage through the inhibition of oxidative damage, mitochondria dysfunction and ultimately inhibition of cell apoptosis, and that increasing PHB content in mitochondria constituted a new therapeutic target for transient ischemic injury and chronic allograft nephropathy (CAN) following renal transplantation.
In Type 2 diabetes, it has been proposed that pancreatic beta-cell dysfunction is promoted by oxidative stress caused by NADPH oxidase (NOX) over-activity. Five different NOX enzymes (NOX1-5) have been characterized, among which NOX1 and NOX2 have been proposed to negatively affect beta-cells, but the putative role of NOX4 in type 2 diabetes-associated beta-cell dysfunction and glucose intolerance is largely unknown. Therefore, we presently investigated the importance of NOX4 for high-fat diet (HFD)-induced glucose intolerance using male C57BL/6 mice using the new NOX4 inhibitor GLX351322, which has relative NOX4 selectivity over NOX2. In HFD-treated male C57BL/6 mice a two-week treatment with GLX351322 counteracted non-fasting hyperglycemia and impaired glucose tolerance. This effect occurred without any change in peripheral insulin sensitivity. To ascertain that NOX4 also plays a role for the function of human beta-cells, we observed that glucose- and sodium palmitate-induced insulin release from human islets in vitro was increased in response to NOX4 inhibitors. In long-term experiments (1-3 days), high glucose-induced human islet cell ROS production and death were prevented by GLX351322. We propose that whilst short-term NOX4-generated ROS production is a physiological requirement for beta-cell function, persistent NOX4-activity, e.g. during conditions of high-fat feeding, promotes ROS-mediated beta-cell dysfunction. Thus, selective NOX-inhibition may be a therapeutic strategy in Type 2 diabetes.
Metformin (N,N-dimethylbiguanide), buformin (1-butylbiguanide) and phenformin (1-phenethylbiguanide) are anti-diabetic biguanide drugs, expected to having anti-cancer effect. The mechanism of anti-cancer effect by these drugs is not completely understood. In this study, we demonstrated that these drugs dramatically enhanced oxidative DNA damage under oxidative condition. Metformin, buformin and phenformin enhanced generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in isolated DNA reacted with hydrogen peroxide (H2O2) and Cu(II), although these drugs did not form 8-oxodG in the absence of H2O2 or Cu(II). An electron paramagnetic resonance (EPR) study, utilizing alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide as spin trapping agents, showed that nitrogen-centered radicals were generated from biguanides in the presence of Cu(II) and H2O2, and that these radicals were decreased by the addition of DNA. These results suggest that biguanides enhance Cu(II)/H2O2 -mediated 8-oxodG generation via nitrogen-centered radical formation. The enhancing effect on oxidative DNA damage may play a role on anti-cancer activity.
In vivo decay rates of a nitroxyl contrast agent were estimated by a MR redox imaging (MRRI) technique and compared with the decay rates obtained by the electron paramagnetic resonance spectroscopy (EPRS) and imaging (EPRI). MRRI is a dynamic imaging technique employing T1-weighted pulse sequence, which can visualize a nitroxyl-induced enhancement of signal intensity by T1-weighted contrast. EPR techniques can directly measure the paramagnetic nitroxyl radical. Both the squamous cell carcinoma (SCC) tumor-bearing and normal legs of a female C3H mouse were scanned by T1-weighted SPGR sequence at 4.7 T with the nitroxyl radical, carbamoyl-proxyl (CmP), as the contrast agent. Similarly, the time course of CmP in normal muscle and tumor tissues was obtained using an 700-MHz EPR spectrometer with a surface coil. The time course imaging of CmP was also performed by 300 MHz CW EPR imager. EPRS and EPRI gave slower decay rates of CmP compared to the MRRI. Relatively slow decay rate at peripheral region of the tumor tissues, which was found in the image obtained by MRRI, may contribute to the slower decay rates observed by EPRS and/or the EPRI measurements. To reliably determine the tissue redox status from the reduction rates of nitroxyls such as CmP, heterogenic structure in the tumor tissue must be considered. The high spatial and temporal resolution of T1-weighted MRI and the T1-enhancing capabilities of nitroxyls support the use of this method to map tissue redox status which can be a useful biomarker to guide appropriate treatments based on the tumor microenvironment.
The aim of this study was to investigate the role of miR-138 in osteoporosis and its underlying mechanism.
Abstract Vascular dysfunction is one of the major causes of cardiovascular (CV) mortality and increases with age. Epidemiological studies suggest that Mediterranean diets and high nut consumption reduce CV disease risk and mortality while increasing plasma α-tocopherol. Therefore, we have investigated whether almond supplementation can improve oxidative stress markers and CV risk factors over 4 weeks in young and middle-aged men. Healthy middle-aged men (56+5.8years), healthy young men (22.1+2.9years) and young men with two or more CV risk factors (27.3+5years) consumed 50g almond /day for 4 weeks. A control group maintained habitual diets over the same period. Plasma α-tocopherol/cholesterol ratios were not different between groups at baseline and were significantly elevated by almond intervention with 50g almond/day for four weeks (p<0.05). Plasma protein oxidation and nitrite levels were not different between groups whereas total, HDL and LDL cholesterols and triglycerides were significantly higher in healthy middle-aged and young men with CV risk factors but were not affected by almond intake. In the almond consuming groups, flow mediated dilatation (FMD) was improved and diastolic blood pressure was reduced significantly after 4 weeks, but systolic blood pressure was only reduced in healthy men. In conclusion, a short-term almond-enriched diet can increase plasma α-tocopherol and improve vascular function in asymptomatic healthy mean men aged between 20 and 70 without effect on plasma lipids or markers of oxidative stress.
According to our previous results, resveratrol (RSV, 3, 5, 4-trihydroxystilbene), a naturally polyphenolic phytoalexin, could attenuate myocardial ischemia/reperfusion injury through up-regulation of vascular endothelial growth factor B (VEGF-B) in isolated rat heart or H9c2 cells. However, the molecular mechanism remains unclear. In this study, we investigated the protective effect of RSV on myocardial infarction (MI) in rats and further explored the underlying signal pathway after VEGF-B. Rats received RSV or normal saline by intragastric administration for 7 consecutive days and followed by subcutaneously isoproterenol (ISO) or normal saline injections for another 2 days. We found that RSV pretreatment prevented the unfavorable changes in HW/BW, HW/TL, infarct size and cell apoptosis in ISO treated rats. Moreover, superoxide and malondialdehyde (MDA) production were significantly reduced and superoxide dismutase (SOD) was increased by RSV in ISO treated rats. Furthermore, it showed that RSV pretreatment increased VEGF-B, p-eNOS and p-AMPK expression and NO production in ISO treated rats. Using Neonatal Rat Ventricular Myocytes (NRVM), we found that VEGF-B siRNA could abolish the cardio-protective effect of RSV. The enhanced ratios of eNOS phosphorylation to eNOS expression induced by RSV were markedly reversed by VEGF-B siRNA in NRVM also. Meantime, we found that the effect of VEGF-B knock-down on eNOS activation was rescued by AMPK activator AICAR. L-NAME, a NOS inhibitor, could inhibit RSV enhanced eNOS phosphorylation, but had no effect on VEGF-B expression in NRVM or in rats. Collectively, our results indicate that RSV exerts cardio-protection from ISO-induced myocardial infarction through VEGF-B/AMPK/eNOS/NO signaling pathway.
Oxidative stress is associated with many cardiovascular diseases, such as hypertension and arteriosclerosis. Oxidative stress reportedly activates the L-type voltage-gated calcium channel (VDCCL) and elevates [Ca2+]i in many cells. However, how oxidative stress activates VDCCL under clinical setting and the consequence for arteries are unclear. Here, we examined the hypothesis that hydrogen peroxide (H2O2) regulates membrane potential (Em) by altering Na+ influx through cation channels, which consequently activates VDCCL to induce vasoconstriction in rat mesenteric arteries. To measure the tone of the endothelium-denuded arteries, a conventional isometric organ chamber was used. Membrane currents and Em were recorded by the patch-clamp technique. [Ca2+]i and [Na+]i were measured with microfluorometry using Fura2-AM and SBFI-AM, respectively. We found that H2O2 (10 and 100 µM) increased arterial contraction, and nifedipine blocked the effects of H2O2 on isometric contraction. H2O2 increased [Ca2+]i as well as [Na+]i, and depolarized Em. Gd3+ (1 µM) blocked all these H2O2-induced effects including Em depolarization and increases in [Ca2+]i and [Na+]i. Although both nifedipine (30 nM) and low Na+ bath solution completely prevented the H2O2-induced increase in [Na+], they only partly inhibited the H2O2-induced effects on [Ca2+]i and Em. Taken together, the results suggested that H2O2 constricts rat arteries by causing Em depolarization and VDCCL activation through activating Gd3+-and nifedipine-sensitive, Na+-permeable channels as well as Gd3+-sensitive Ca2+-permeable cation channels. We suggest that unidentified Na+-permeable cation channels as well as Ca2+-permeable cation channels may function as important mediators for oxidative stress-induced vascular dysfunction.