Chloromethyl-2',7'-dichlorodihydrofluorescein Diacetate Research Articles

Protective Effects of Safflor Yellow A Against H2O2-induced Oxidative Stress Injury in PC12 Cells via Nrf2/HO-1 Pathway

Background Safflor yellow (SY) is a water-capacitive component of dried flowers, mainly from safflor in the safflower family, which contains a large amount of bruthin A, or Safflor yellow A (SYA) for short. Studies have reported the protective effects of SYA against oxidative stress injury in nerve cells. Materials and Methods In this study, H2O2-treated PC12 cells were used as experimental materials to explore how SYA plays a protective role against nerve cells after oxidative stress and to analyze the molecular mechanism of SYA. PC12 cells were treated with H2O2 and SYA solution. Cell proliferation is then detected with Cell Counting Kit-8 (CCK-8). The detection method is 5-(and 6)-chloromethyl-2′,7′-dichloro-dihydrofluorescein diacetate (CM-H2DCF-DA) staining reactive oxygen species (ROS). Levels of apoptosis-related proteins, including Bcl2-associated X protein (Bax), B-cell lymphoma (Bcl-2), caspase-3, and cleaved caspase-3, and oxidative stress regulators, namely, nuclear factor erythroid-2-related actor 2 (Nrf2) and heme oxygenase 1 (HO-1), were detected by western blotting. Results The CCK-8 assay results showed that the survival rate of H2O2-induced PC12 cells was significantly increased after SYA treatment. Fluorescence microscopy (CM-H2DCF-DA staining) indicated that SYA decreased H2O2-induced apoptosis in PC12 cells by reducing intracellular ROS. Western blotting analysis showed that SYA upregulated the expression of Nrf2, HO-1, and Bcl-2 and downregulated the expression of Bax, caspase 3, and cleaved caspase-3 in H2O2-induced PC12 cells. Conclusion SYA does protect the nerve cells from oxidative stress damage. Its mechanism of action is mainly related to whether the Nrf2/HO-1 pathway is activated.

Concentration Dependence of the Antioxidant and Prooxidant Activity of Trolox in HeLa Cells: Involvement in the Induction of Apoptotic Volume Decrease.

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a hydrophilic analog of vitamin E, is known for its strong antioxidant activity, being a high radical scavenger of peroxyl and alkoxyl radicals. Under particular conditions, Trolox may also exhibit prooxidant properties. The present work aimed at studying the dual antioxidant/prooxidant behavior of Trolox over a wide range of concentrations (from 2.5 to 160 µM) in HeLa cells. In particular, the study addressed the dose-dependent effects of Trolox on the oxidative cell status and vitality of HeLa cells, focusing on the potential role of the vitamin E analog in the induction of one of the first steps of the apoptotic process, Apoptotic Volume Decrease (AVD). In HeLa cells, Trolox showed significant antioxidant activity, expressed as the ability to reduce the endogenous ROS production detected by the ROS-sensitive probe 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), at low concentrations (range: 2.5–15 µM), but exerted a dose-dependent prooxidant effect at higher concentrations after 24 h exposure. The prooxidant effect was paralleled by the reduction in cell viability due to the induction of the apoptotic process. The dual behavior, antioxidant at lower concentrations and prooxidant at higher concentrations, was evident also earlier after 2 h incubation, and it was paralleled by the isotonic shrinkage of the cells, ascribed to AVD. The use of SITS, known Cl− channel blocker, was able to completely inhibit the Trolox-induced isotonic cell shrinkage, demonstrating the involvement of the vitamin E analog in the alteration of cell volume homeostasis and, in turn, in the AVD induction. In conclusion, the study shed light on the concentration dependence of the Trolox antioxidant/prooxidant activity in HeLa cells and revealed its role in the induction of one of the first events of apoptosis, AVD, at high concentrations.

Characterization of the in vitro cytotoxic effects of brachydins isolated from Fridericia platyphylla in a prostate cancer cell line

ABSTRACT Brachydins (Br) A, B, and C are flavonoids extracted from Fridericia platyphylla (Cham.) L.G. Lohmann roots (synonym Arrabidaea brachypoda), whose extract previously exhibited cytotoxic and antitumor activity. In vitro cell culture of human prostate tumor cell line (PC-3) was used to determine cell viability as evidenced by MTT, neutral red, and LDH release using nine concentrations (0.24 to 30.72 µM) of each brachydin. A triple-fluorescent staining assay assessed the mechanism resulting in cell death. Genomic instability and protein expression were evaluated using comet assay and western blot analysis, respectively. The pro-oxidant status was analyzed using the5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) probe. The IC50 values for brachydins BrA, BrB, and BrC were 23.41, 4.28, and 4.44 µM, respectively, and all compounds induced apoptosis and necrosis. BrB and BrC increased p21 levels indicating a possible G1 cell cycle arrest. BrA (6 µM) and BrB (3.84 µM) decreased phospho-AKT (AKT serine/threonine kinase) expression, which also influenced cell cycle and proliferation. BrA, BrB, and BrC elevated cleaved PARP (poly (ADP-ribose) polymerase), a protein related to DNA repair and induction of apoptotic processes. Therefore, this study determined the IC50 values of brachydins in the PC-3 cell line as well as the influence on cell proliferation and cell death processes, such as apoptosis and necrosis, indicating the proteins involved in these processes. Abbreviations ANOVA: Analysis of Variance; BrA: Brachydin A; BrB: Brachydin B; BrC: Brachydin C; CGEN: Genetic Heritage Management Council; CID: Compound identification number; CM-H2DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester; CO2: Carbon dioxide; DMSO: Dimethyl sulfoxide; DNA: Deoxyribonucleic acid; DTT: Dithiothreitol; DXR: Doxorubicin; ECL: Chemiluminescence; EDTA: Ethylenediaminetetraacetic acid; FBS: Fetal bovine serum; H2O2: Hydrogen peroxide; HRMS: High-Resolution Mass Spectrometry; IC50: Half maximal inhibitory concentration; LDH: Lactate dehydrogenase; MTT, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Na3VO4: Sodium Orthovanadate; NaOH: Sodium hydroxide; NCBI: National Center for Biotechnology Information; NMR: Nuclear Magnetic Resonance; PBS: Phosphate buffer saline; PCR: Polymerase chain reaction; PSMF: Phenylmethylsulfonyl fluoride; RPMI: Roswell Park Memorial Institute Medium; SDS-PAGE: Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis; STR: Short tandem repeat; TBS-T: Tris-buffered saline and Polysorbate 20; UPHLC: Ultra-Performance Liquid Chromatography

Exendin-4 relieves the inhibitory effects of high glucose on the proliferation and osteoblastic differentiation of periodontal ligament stem cells

BackgroundWith the impaired regenerative potential in patients with diabetes mellitus (DM), Periodontal ligament stem cells (PDLSCs) are regarded as an attractive source of stem cells for periodontal cytotherapy. Recent studies have shown that Exendin-4 (Ex-4) exerts cell-protective effects and bone remodeling ability on many types of cells. The aim of this study was to investigate whether Ex-4 alleviates the inhibition of high glucose on the proliferation and osteogenic differentiation of PDLSCs. MethodsPDLSCs were incubated in medium supplemented with 5.5 mM d-glucose (NG), 30 mM d-glucose (HG), NG plus Ex-4, and HG plus different concentration (1, 10, 20, 100 nM) of Ex-4 respectively. Cell proliferation was detected by CCK-8 assay and cell cycle analysis. Osteogenesis was assessed by Alizarin Red S staining and evaluation of the mRNA expression of Runx2, ALP and Osx at day 7, 14 and 21. Intracellular level of reactive oxygen species (ROS) was detected using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (CMH2DCF-DA). ResultsThe proliferation ability, mineralized nodules forming capacity and the mRNA expression of Runx2, ALP and Osx of PDLSCs in HG group were decreased, the ROS level was increased compared to NG group. With the treatment of Ex-4, the HG-inhibited proliferation ability and osteogenic differentiation ability of PDLSCs were significantly reversed, the HG-increased ROS level could be down-regulated. Moreover, Ex-4 enhanced the osteogenic differentiation of normal PDLSCs. ConclusionsEx-4 alleviates the inhibitory effect of HG on the proliferation and osteoblastic differentiation of PDLSCs, and has a significant enhance in the osteoblastic differentiation of normal PDLSCs, giving new insights into the possible therapeutic method of diabetic periodontitis.

Measurement of ROS in Caenorhabditis elegans Using a Reduced Form of Fluorescein.

Oxidative stress is implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease. Oxidative stress is a result of a disruption of the equilibrium between antioxidants and oxidants, in favor of oxidants. Since mitochondria are major sites of production and reduction of reactive oxygen species (ROS), measurement of ROS levels can help us determine if mitochondrial functional integrity has been compromised. In this protocol, we describe a method to measure the level of ROS in the nematode Caenorhabditis elegans, using chloromethyl-2,7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA).

Detection of Reactive Oxygen Species During the Cell Cycle Under Normal Culture Conditions Using a Modified Fixed-Sample Staining Method

We developed an alternative method of simultaneously monitoring the generation of reactive oxygen species (ROS) and cellular oxidative responses using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF) in fixed samples. In this study, we evaluated the ability of this method to detect ROS generation during the cell cycle under normal culture conditions using flow cytometric analyses. Among the fixatives tested, only acetone and paraformaldehyde did not alter the endogenous oxidation of the responsive dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), which is a chloromethyl derivative of H2DCFDA. Only acetone fixation followed by staining with propidium iodide was able to detect ROS generation during the cell cycle without altering DCF oxidation. Further thymidine treatment led to cell cycle arrest at the G1 phase followed by the downregulation of total intracellular ROS. Paraformaldehyde-based fixation enabled the evaluation of ROS generation by immunostaining at a different phase of the cell cycle, whereas MPM2 co-staining enabled identification of the specific mitotic phase. This study demonstrates a modified fixed-sample method that can be used to measure intracellular ROS production during the cell cycle using standard immunostaining techniques.

A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses

The generation of reactive oxygen species (ROS) in a live-cell system is routinely measured using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF). However, it is difficult to simultaneously monitor cellular oxidative responses and ROS generation in cells, and analyses of cellular oxidative responses are typically performed after ROS generation has been evaluated. In this study, we developed a modified fixed staining method that allows the simultaneous analysis of ROS generation and oxidative responses using standard immunostaining techniques. A microplate reader-based assay showed that of the fixatives tested, only methanol did not alter the hydrogen peroxide (H2O2)-mediated oxidation of the responsive dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), a chloromethyl derivative of H2DCFDA, or the fluorescence of oxidized DCF in vitro. Further in vivo assays using flow cytometry showed that both methanol and acetic acid maintained the fluorescence of oxidized DCF in H2O2-, antimycin A-, and serum starvation-treated human lung adenocarcinoma A549 cells and human microvascular endothelial HMEC-1 cells. Following acetic acid-based fixation, the ROS generation in starved HMEC-1 cells could be evaluated by flow cytometric analysis while simultaneously monitoring the phosphorylation status of p38 mitogen-activated protein kinase. Immunostaining also revealed the synchronization of ROS generation and the H2O2-induced phosphorylation of Src homology-2 domain-containing phosphatase2. This study describes a modified method that may be used in future biomedical investigations to simultaneously measure intracellular ROS production and cellular oxidative responses.

Sea urchin spermatozoa generate at least two reactive oxygen species; the type of reactive oxygen species changes under different conditions

Reactive oxygen species (ROS) cause oxidative stress and act as signal transduction molecules in many cells. Spermatozoa from several mammals generate ROS, which are involved in male infertility and signaling during capacitation. In the present study, we investigated ROS generation by sea urchin spermatozoa at the initiation of motility, during dilution with seawater, and following egg jelly treatment. In seawater containing an ROS indicator, 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), fluorescence increased after the addition of spermatozoa. The ROS generation rate was dependent upon the dilution ratio and respiratory rate of the spermatozoa. Spermatozoa in sodium-free seawater did not increase fluorescence, but fluorescence did increase with the addition of NaCl. Sodium chloride also led to the initiation of sperm motility and respiration. Using the indicator MitoSOX Red, ROS generation was detected from spermatozoa exposed to egg jelly dissolved in seawater, but not in normal seawater. Moreover, the respiratory inhibitor antimycin A prevented CM-H(2)DCFDA-detectable ROS and increased MitoSox-detectable ROS at a higher concentration. These findings revealed that the ROS generated were of different species, possibly hydrogen peroxide (H(2)O(2)) and superoxide anion (O(-)(2)), and their detected levels were altered by egg jelly. We concluded that sea urchin spermatozoa generate at least two species of ROS depending on the physiological conditions to which they are exposed. It is possible that the major ROS from sea urchin spermatozoa changes during the course of fertilization.

β-Lapachone induces heart morphogenetic and functional defects by promoting the death of erythrocytes and the endocardium in zebrafish embryos

Backgroundβ-Lapachone has antitumor and wound healing-promoting activities. To address the potential influences of various chemicals on heart development of zebrafish embryos, we previously treated zebrafish embryos with chemicals from a Sigma LOPAC1280™ library and found several chemicals including β-lapachone that affected heart morphogenesis. In this study, we further evaluated the effects of β-lapachone on zebrafish embryonic heart development.MethodsEmbryos were treated with β-lapachone or dimethyl sulfoxide (DMSO) at 24 or 48 hours post fertilization (hpf) for 4 h at 28°C. Heart looping and valve development was analyzed by whole-mount in situ hybridization and histological analysis. For fractional shortening and wall shear stress analyses, AB and Tg (gata1:DsRed) embryos were recorded for their heart pumping and blood cell circulations via time-lapse fluorescence microscopy. Dextran rhodamine dye injection into the tail reticular cells was used to analyze circulation. Reactive oxygen species (ROS) was analyzed by incubating embryos in 5-(and 6-)-chloromethyl-2',7'-dichloro-dihydrofluorescein diacetate (CM-H2DCFDA) and recorded using fluorescence microscopy. o-Dianisidine (ODA) staining and whole mount in situ hybridization were used to analyze erythrocytes. TUNEL assay was used to examine DNA fragmentation.ResultsWe observed a linear arrangement of the ventricle and atrium, bradycardia arrhythmia, reduced fractional shortening, circulation with a few or no erythrocytes, and pericardial edema in β-lapachone-treated 52-hpf embryos. Abnormal expression patterns of cmlc2, nppa, BMP4, versican, and nfatc1, and histological analyses showed defects in heart-looping and valve development of β-lapachone-treated embryos. ROS production was observed in erythrocytes and DNA fragmentation was detected in both erythrocytes and endocardium of β-lapachone-treated embryos. Reduction in wall shear stress was uncovered in β-lapachone-treated embryos. Co-treatment with the NQO1 inhibitor, dicoumarol, or the calcium chelator, BAPTA-AM, rescued the erythrocyte-deficiency in circulation and heart-looping defect phenotypes in β-lapachone-treated embryos. These results suggest that the induction of apoptosis of endocardium and erythrocytes by β-lapachone is mediated through an NQO1- and calcium-dependent pathway.ConclusionsThe novel finding of this study is that β-lapachone affects heart morphogenesis and function through the induction of apoptosis of endocardium and erythrocytes. In addition, this study further demonstrates the importance of endocardium and hemodynamic forces on heart morphogenesis and contractile performance.

108 OXIDATIVE STRESS AND ANTIOXIDANT PARAMETERS IN PATIENTS WITH HYPERLIPIDEMIA

Epidemiological and experimental studies have suggested that high dietary intake of fats is associated with cognitive decline and a significantly increased risk of dementia. Since oxidative stress and inflammation have been speculated to be critical mechanisms underlying neurodegenerative diseases, we hypothesized that a high fat (HF) diet might induce cerebral oxidative stress or neural inflammation and subsequently contribute to the high risk of dementia. To test this hypothesis, male rats were placed on either a HF diet or a low fat (LF) diet starting at 1 month of age and lasting for 5 months. Intracellular reactive oxidative species (ROS) generation in the cerebral cortex was measured by the oxidant-sensitive dye 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). Cortical tissue concentration of prostaglandin E2 (PGE2) was determined using an enzymatic immunoassay. Expression of NADPH oxidase subunits, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), nuclear transcription factor NFkappa-B (NF-κB) p65 subunit, Ikappa B(IκB), and phospho-Ikappa B(phospho-IκB) was evaluated by Western blot analysis. The HF diet significantly increased ROS generation and expression of gp91phox, p22phox, p47phox, and p67phox NADPH oxidase subunits in cerebral cortex. Elevated PGE2 levels and markedly increased COX-2 expression suggested a neural inflammatory response in response to excessive fat intake. These findings were further supported by significantly increased phospho-IκB and nuclear NF-κB expression that suggested a role of IκB phosphorylation in HF diet-induced NF-κB translocation. The present study revealed that HF diet induced neural oxidative stress, inflammation, and NF-κB activation in rat cerebral cortex, and provided novel evidence regarding the link between high dietary fat and increased risk of dementia.

Cardiac-specific overexpression of metallothionein rescues nicotine-induced cardiac contractile dysfunction and interstitial fibrosis

Cigarette smoking is a devastating risk factor for cardiovascular diseases and nicotine is believed the main toxin component responsible for the toxic myocardial effects of smoking. Nonetheless, neither the precise mechanism of nicotine-induced cardiac dysfunction nor effective treatment is elucidated. The aim of this study was to evaluate the impact of cardiac-specific overexpression of heavy metal scavenger metallothionein on myocardial geometry and mechanical function following nicotine exposure. Adult male friend virus B (FVB) wild-type and metallothionein mice were injected with nicotine (2mg/kg/d) intraperitoneally for 10 days. Mechanical and intracellular Ca2+ properties were examined. Myocardial histology (cross-sectional area and fibrosis) was evaluated by hematoxylin and eosin (H&E) and Masson trichrome staining, respectively. Oxidative stress and apoptosis were measured by fluoroprobe 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) fluorescence and caspase-3 activity, respectively. Nicotine exposure failed to affect the protein abundance of metallothionein. Our data revealed reduced echocardiographic contractile capacity (fractional shortening), altered cardiomyocyte contractile and intracellular Ca2+ properties including depressed peak shortening amplitude, maximal velocity of shortening/relengthening, resting and electrically-stimulated rise in intracellular Ca2+, as well as prolonged duration of relengthening and intracellular Ca2+ clearance in hearts from nicotine-treated FVB mice, the effect of which was ameliorated by metallothionein. Biochemical and histological findings depicted overt accumulation of reactive oxygen species (ROS), apoptosis and myocardial fibrosis without any change in myocardial cross-sectional area following nicotine treatment, which was mitigated by metallothionein. Taken together, our findings suggest the antioxidant metallothionein may reconcile short-term nicotine exposure-induced myocardial contractile dysfunction and fibrosis possibly through inhibition of ROS accumulation and apoptosis.

Apocynin Reversibly Inhibits L-type Ca2+ Channel Current Involvement of Reactive Oxygen Species

L-type Ca2+ channel current (ICa,L) is a major Ca2+ entry mechanism and contraction of arterial smooth muscle (ASM) is regulated by reactive oxygen species (ROS). We utilized apocynin (APO) as a tool to clarify the contribution of ROS in the regulation of ICa,L. APO, a natural organic compound contained in a variety of plants, is widely used as inhibitor of NADPH oxidase (NOX) that reduces oxygen to superoxide in the presence of NADPH to generate ROS including H2O2. We recorded whole cell ICa,L with Ba2+ as charge carrier from isolated bovine coronary ASM (BCASM) and HEK293 cells transiently expressing human cardiac CaV1.2. APO was introduced and washed out during continuous application of depolarization step to 0 mV from HP −80 mV at 1/min. We found that APO inhibited ICa,L in a dose-dependent manner between 0.1 and 10 mM decreasing its amplitude to ∼20% of control. APO also accelerated ICa,L decay during depolarization. Surprisingly, washout of the high concentration of APO caused rapid recovery of ICa,L. It could even produce a rebound increase of ICa,L with its peak at ∼3 min in BCASM. We performed fluorometric analysis of APO-induced change of cellular ROS by loading cells with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). Oxidation of CM-H2DCF to CM-DCF by ROS induces an increase of fluorescence. APO markedly inhibited the fluorescence increase, while washout of APO caused more intensive increase of fluorescence than control. The results are explained by the inhibition of NOX by APO which results in decrease of ROS and overproduction of ROS during washout utilizing accumulated NADPH produced by prior NOX inhibition. ROS which changes dynamically in situ, e.g. hypoxia and reoxygenation, seems to be vital to sustain ICa,L.

Antioxidant potential of Minocycline in Japanese Encephalitis Virus infection in murine neuroblastoma cells: Correlation with membrane fluidity and cell death

Minocycline is neuroprotective in animal models of a number of acute CNS injuries, neurodegenerative diseases and CNS infection. While anti-inflammatory and anti-apoptotic effects of Minocycline have been characterized, the molecular basis for the neuroprotective effects of Minocycline remains unclear. We report here that Minocycline and two classical antioxidant compounds inhibit the Japanese Encephalitis Virus (JEV)-induced free radical generation in mouse neuroblastoma. In cultures of Neuro2a (N2a) cells infected with JEV for up to 24 h, the number of cells undergoing cell death was also reduced by Minocycline (20 μM). JEV infection resulted in increased oxidative stress, as revealed by an increase in the fluorescence intensity for 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), a reactive oxygen species (ROS) indicator. Minocycline at 20 μM inhibited this ROS production. Cells were moderately protected from JEV-induced death by diphenyleneiodonium (DPI), an inhibitor of flavon-containing enzyme inhibitor, whereas common antioxidants such as N-acetyl-cysteine (NAC) turned out to be ineffective. Direct antioxidant property of Minocycline and reference antioxidant compounds is evaluated by LDH assay, ROS measurement and mitochondrial membrane potential measurement. Our findings suggest that Minocycline reduces the neuronal damage seen in JEV infection in neuronal cell culture models at least in part through inhibition of oxidative stress.

Dynein Light Chain LC8 Negatively Regulates NF-κB through the Redox-dependent Interaction with IκBα

Redox regulation of nuclear factor kappaB (NF-kappaB) has been described, but the molecular mechanism underlying such regulation has remained unclear. We recently showed that a novel disulfide reductase, TRP14, inhibits tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation, and we identified the dynein light chain LC8, which interacts with the NF-kappaB inhibitor IkappaBalpha, as a potential substrate of TRP14. We now show the molecular mechanism by which NF-kappaB activation is redox-dependently regulated through LC8. LC8 inhibited TNFalpha-induced NF-kappaB activation in HeLa cells by interacting with IkappaBalpha and thereby preventing its phosphorylation by IkappaB kinase (IKK), without affecting the activity of IKK itself. TNFalpha induced the production of reactive oxygen species, which oxidized LC8 to a homodimer linked by the reversible formation of a disulfide bond between the Cys(2) residues of each subunit and thereby resulted in its dissociation from IkappaBalpha. Butylated hydroxyanisol, an antioxidant, and diphenyleneiodonium, an inhibitor of NADPH oxidase, attenuated the phosphorylation and degradation of IkappaBalpha by TNFalpha stimulation. In addition LC8 inhibited NF-kappaB activation by other stimuli including interleukin-1beta and lipopolysaccharide, both of which generated reactive oxygen species. Furthermore, TRP14 catalyzed reduction of oxidized LC8. Together, our results indicate that LC8 binds IkappaBalpha in a redox-dependent manner and thereby prevents its phosphorylation by IKK. TRP14 contributes to this inhibitory activity by maintaining LC8 in a reduced state.

Hyperglycemia-induced reactive oxygen species toxicity to endothelial cells is dependent on paracrine mediators.

OBJECTIVE—This study determined the effects of high glucose exposure and cytokine treatment on generation of reactive oxygen species (ROS) and activation of inflammatory and apoptotic pathways in human retinal endothelial cells (HRECs).RESEARCH DESIGN AND METHODS—Glucose consumption of HRECs, human retinal pigment epithelial cells (HRPEs), and human Müller cells (HMCs) under elevated glucose conditions was measured and compared with cytokine treatment. Production of ROS in HRECs was examined using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), spin-trap electron paramagnetic resonance, and MitoTracker Red staining after high glucose and cytokine treatment. The activation of different signaling cascades, including the mitogen-activated protein kinase pathways, tyrosine phosphorylation pathways, and apoptosis by high glucose and cytokines in HRECs, was determined.RESULTS—HRECs, in contrast to HRPEs and HMCs, did not increase glucose consumption in response to increasing glucose concentrations. Exposure of HRECs to 25 mmol/l glucose did not stimulate endogenous ROS production, activation of nuclear factor-κB (NF-κB), extracellular signal–related kinase (ERK), p38 and Jun NH2-terminal kinase (JNK), tyrosine phosphorylation, interleukin (IL)-1β, or tumor necrosis factor-α (TNF-α) production and only slightly affected apoptotic cell death pathways compared with normal glucose (5 mmol/l). In marked contrast, exposure of HRECs to proinflammatory cytokines IL-1β or TNF-α increased glucose consumption, mitochondrial superoxide production, ERK and JNK phosphorylation, tyrosine phosphorylation, NF-κB activation, and caspase activation.CONCLUSIONS—Our in vitro results indicate that HRECs respond to cytokines rather than high glucose, suggesting that in vivo diabetes–related endothelial injury in the retina may be due to glucose-induced cytokine release by other retinal cells and not a direct effect of high glucose.

Anti-oxidative and photo-protective effects of coumarins isolated fromFraxinus chinensis

Free radicals and reactive oxygen species (ROS), which are generated by UV irradiation, may cause serious injury to skin cell membranes, DNA and functional proteins. In addition, these agents stimulate the expressions of matrix metalloproteinases (MMPs), which can degrade most components of the extracellular matrix (ECM), including collagen. In order to develop new anti-photoaging agents, five major components from the extract of Fraxinus chinensis extract (FCE) were identified. Two of the major components of FCE were found to be esculin (11.2%) and esculetin (1.9%). FCE (IC50: 50.0 microg/mL 1, 1-diphenyl-2-picrylhydrazyl (DPPH); 19.8 microg/mL, superoxide anion radical) and esculetin (IC50: 2.1 microg/mL DPPH; 0.6 microg/mL, superoxide anion radical) showed strong antioxidative activities. Of the compounds tested, esculetin showed the strongest scavenging activity against DPPH radicals, followed by superoxide anions from the xanthine/xanthine oxidase system. The intracellular ROS scavenging activity showed that oxidation of 5-(6-)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) was effectively inhibited by esculetin, with potent free radical scavenging activity was also shown in UVB-irradiated human dermal fibroblasts (HDFs). Moreover, treatment of UVA-irradiated HDFs with esculetin resulted in dose-dependent decreases in the expression levels of MMP-1 mRNA and protein. From these results, FCE and one of its components, esculetin, were predicted to be potentially useful as ingredients in cosmetics for protecting against photoaging.

Antioxidative Activity of the Hydrolytic Enzyme Treated Sorbus commixta Hedl. and its Inhibitory Effect on Matrix Metallopro- teinase- 1 in UV Irradiated Human Dermal Fibroblasts

Superoxide radical scavenging activity and DPPH radical scavenging activity were assessed in order to evaluate the antioxidant effect of the Sorbus commixta Hedl. extract (SCoE). SCoE was also treated with several carbohydrate-hydrolytic enzymes that significantly increased the total phenol and flavonoid composition of SCoE. The enzymatically treated SCoE was then assessed for antioxidative activity. The most efficient radical scavenging activity was observed when SCoE was treated with -glucanase. The radical scavenging activity of beta-glucanase-treated SCoE (beta-GSCoE) enhanced the viability of human dermal fibroblasts (HDFs) exposed to ultraviolet (UV) light. The intracellular reactive oxygen species (ROS) scavenging activity of beta-GSCoE was assessed using UVB (20 mJ/cm2)-irradiated HDFs. UVB irradiation increased dichlorofluorescein (DCF) fluorescence, which was measured by a 5-(6-)chloromethyl-2',7'- dichlorodihydrofluorescein diacetate (CM-H2DCFDA). DCF-fluorescence was significantly decreased in the beta-GSCoE-containing culture medium, suggesting that beta-GSCoE scavenges free radicals. The protective effect was further verified by assessing the expression of matrix metalloproteinase-1 (MMP-1) in UVA-irradiated HDFs. The treatment of UVA-irradiated HDFs with beta-GSCoE resulted in a dose-dependent decrease in the expression level of MMP-1 protein and mRNA. These results suggest that beta-GSCoE may mitigate the effects of photoaging in skin by reducing UV-induced adverse skin reactions.

Thrombin induces oxidative mitochondrial (mt) DNA damage through a membrane NADPH oxidase in pulmonary microvascular endothelial cells

Recent evidence suggests that mtDNA mutations may play a role in the pathogenesis of atherosclerosis (Mutat. Res., 570: 80, 2005). Though oxidative damage to mtDNA is a precursor to mtDNA mutations, the source of oxidants leading to mtDNA damage in ECs is unclear. Thrombin and other inflammatory mediators incriminated in development of atherosclerosis are known to utilize reactive oxygen species (ROS) generated from a membrane-bound NADPH oxidase as second messengers. In this study, we tested the hypothesis that thrombin-induced ROS production causes mtDNA damage in pulmonary microvascular endothelial cells. Fluorescence microscopy with 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) showed that thrombin caused ROS production that was sensitive to diphenyliodonium (DPI), thus supporting involvement of NADPH oxidase. In addition, quantitative Southern blot analyses indicated that thrombin increased the equilibrium lesion density of oxidative lesions within the mitochondrial genome. Like the thrombin-induced ROS production, the increase in mtDNA lesion density also was suppressed by DPI. These findings raise the possibility that ROS used as second messengers in inflammatory signaling damage mtDNA in ECs thus leading to formation of mtDNA mutations that contribute to atherogenesis. Supported by NIH

Attenuation of acetaldehyde-induced cell injury by overexpression of aldehyde dehydrogenase-2 (ALDH2) transgene in human cardiac myocytes: role of MAP kinase signaling

Acetaldehyde, the major metabolite of ethanol, which is far more toxic and reactive than ethanol, may be responsible for alcohol-induced cardiac damage. This study was designed to examine the impact of facilitated acetaldehyde metabolism using transfection of human aldehyde dehydrogenase-2 (ALDH2) transgene on acetaldehyde- and ethanol-induced cell injury. Fetal human cardiac myocytes were transfected with ALDH2, the efficacy of which was verified by flow cytometry, Western blot and ALDH2 activity assays. Generation of reactive oxygen species (ROS) was detected using 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). Apoptosis was evaluated by 4′,6′-diamidino-2′-phenylindoladihydrochloride (DAPI) fluorescence microscopy, quantitative DNA fragmentation ELISA and caspase 3 activity. Acetaldehyde and ethanol elicited overt ROS generation and apoptosis in human cardiac myocytes following 24–48 h of incubation. Immunostaining revealed activation of the MAP kinase cascades ERK1/2, SAPK/JNK and p38 MAP kinase in acetaldehyde-treated myocytes. Interestingly, ALDH2 transgene significantly attenuated acetaldehyde-induced ROS generation, apoptosis and phosphorylation of ERK1/2 and SAPK/JNK. Time-dependent response (0–12 h) revealed ROS accumulation and activation of MAP kinases prior to acetaldehyde-induced apoptosis. In addition, acetaldehyde-induced ROS generation and apoptosis were antagonized by non-enzymatic antioxidants. Our results suggested that ALDH2 transgene overexpression may effectively alleviate acetaldehyde-elicited cell injury through an ERK1/2 and SPAK/JNK-dependent mechanism. Our data are consistent with the notion of acetaldehyde as a contributor to alcoholic cardiomyopathy and implicate the therapeutic potential of ALDH2 enzyme in alcoholic complications.

The isolation and antioxidative effects of vitexin fromAcer palmatum

Free radicals and reactive oxygen species (ROS) caused by UV exposure or other environmental factors are critical players in cellular damage and aging. In order to develop a new anti-photoaging agent, this work focused on the antioxidant effects of the extract of tinged autumnal leaves of Acer palmatum. One compound was isolated from an ethyl acetate soluble fraction of the A. palmatum extract using silica gel column chromatography. The chemical structure was identified as apigenin-8-C-beta-D-glucopyranoside, more commonly known as vitexin, by spectral analysis including LC-MS, FT-IR, UV, 1H-, and 13C-NMR. The biological activities of vitexin were investigated for the potential application of its anti-aging effects in the cosmetic field. Vitexin inhibited superoxide radicals by about 70% at a concentration of 100 microg/mL and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals by about 60% at a concentration of 100 microg/mL. Intracellular ROS scavenging activity was indicated by increases in dichlorofluorescein (DCF) fluorescence upon exposure to UVB 20 mJ/cm2 in cultured human dermal fibroblasts (HDFs) after the treatment of vitexin. The results show that oxidation of 5-(6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) is inhibited by vitexin effectively and that vitexin has a potent free radical scavenging activity in UVB-irradiated HDFs. In ROS imaging using a confocal microscope we visualized DCF fluorescence in HDFs directly. In conclusion, our findings suggest that vitexin can be effectively used for the prevention of UV-induced adverse skin reactions such as free radical production and skin cell damage.