Dichlorofluorescein Fluorescence Research Articles

Detection of H2O2 from the Reduction of Dissolved Oxygen on TCP-Coated AA2024-T3 : Impact on the Transient Formation of Cr(VI)

The detection of H2O2 formed in naturally-aerated electrolyte solution contacting uncoated and TCP-coated AA20243-T3 alloy surfaces is reported on. This molecule is produced from the reduction of dissolved oxygen and it has been implicated in the mechanism of transient formation of Cr(VI) in trivalent chromium process (TCP) conversion coatings. Linear sweep voltammetry and a spectrophotometric assay involving 2’,7’-dichloro-dihydrofluorescein diacetate (DCFH-DA) were used to detect H2O2 produced in 0.5 M Na2SO4. The activation of DCFH-DA to form the redox probe, DCFH, and its oxidation by H2O2 to the fluorescent dichlorofluorescein was used for detection. Immersion tests were performed in naturally-aerated 0.5 M Na2SO4 + 0.1 mM DCFH-DA for periods of time up to 96 h under open circuit conditions. The H2O2 concentration was greater in solution exposed to uncoated than to TCP-coated AA2024-T3 for equivalent times due to the inhibition of the oxygen reduction reaction kinetics by the conversion coating. With this direct evidence of H2O2 production, the following mechanism for transient formation of Cr(VI) in TCP coatings is confirmed: (i) dissolved oxygen is reduced to H2O2 on the alloy surface presumably at cathodic intermetallic sites, (ii) the H2O2 then diffuses to nearby coating sites to oxidize insoluble Cr(OH)3 to soluble Cr(VI) species (e.g., CrO42−) and (iii) the transiently formed Cr(VI) species diffuses to nearby corroding sites on the alloy where the inhibitor gets reduced back to passivating Cr(OH)3.

NOX2 Derived Superoxide Induces Pro-Tumoral p16INK4a Driven Senescence in the AML Bone Marrow Microenvironment

BackgroundAcute myeloid leukaemia (AML) is an age-related disease that is highly dependent on the bone marrow (BM) microenvironment. Senescence is the irreversible arrest of cell proliferation and is associated with the senescence associated secretary phenotype (SASP). The senescent phenotype has been associated with pro-tumoral changes that support proliferation and survival of malignant cells. Because AML is an age-related disease and highly dependent on the BM microenvironment, which naturally becomes senescent with age, we hypothesized, and subsequently tested the idea, that AML not only favors a senescent microenvironment but might actively shape a senescent microenvironment to promote tumor proliferation and survival.MethodsPrimary AML and bone marrow stroma was isolated from bone marrow samples obtained from patients following informed consent and non-malignant CD34+ cells were isolated from cord blood following informed consent. All primary tissue was used under approval from the UK Health Research Authority. NSG, p16-3MR(Demaria et al. 2014 Cell Development) and wildtype C57Bl/6 animals were used and experiments were conducted with approval from the UK Home Office and University of East Anglia Animal Welfare and Ethical Review Board. Primary AML and non-malignant human CD34+ cells were engrafted into NSG mice (AML-NSG and hu-NSG). Bone marrow stromal cells (BMSCs) were isolated by cell sorting for mouse CD45-, Ter119-, CD31-, CD105+, CD140a+. Syngeneic AML cells were generated from lineage negative cells transduced with the oncogene MN1. Senescence was determined by p16, p21 mRNA expression, SASP expression (IL-6 and MIP3a), senescence associated β-Galactosidase activity and using themonomeric red fluorescent protein (mRFP) component of the p16-3MR mouse model. Oxidative stress and superoxide levels were measured using 2‘,7‘-dichlorofluorescein (DCF) fluorescence and the Amplex Red assay. NOX-2 in AML was inhibited by RNAi.ResultsFirst, we isolated and analysed BMSC from AML-NSG and hu-NSG. Using real-time PCR we detected high expression of p16 and p21 in the BMSC isolated from AML-NSG but not hu-NSG. Moreover, we detected high mouse IL-6 and MIP3a cytokine levels from serum isolated from AML-NSG but not hu-NSG. MN1 engrafted into the p16-3MR model induced mRFP expression in the BMSC population. This shows that in both a xenograft and syngeneic model of AML the BMSC population becomes senescent. Using the p16-3MR model we selectively depleted senescent BMSC via the viral thymidine kinase death switch. Treatment of p16-3MR mice engrafted with MN1 with ganciclovir reduced tumour volume and prolonged animal survival. Since hydrogen peroxide has been shown to induce senescence we hypothesised that NOX2 derived superoxide from AML drives the BMSC senescence. BMSCs isolated from the MN1 engrafted p16-3MR model isolated BMSC show increased DCF fluorescence and isolated BM shows elevated superoxide compared to BMSC and BM isolated from control mice. NOX2 knock down resulted in significant reduction in BM derived superoxide and BMSC DCF fluorescence and this resulted in a reduced BMSC senescent phenotype.ConclusionHere we show that in AML, NOX2 derived superoxide induces a p16INK4a drivensenescent phenotype in the BMSC. Moreover, we find that this process forms a fundamental part of the pro-tumoral pathophysiology of the disease. The senescent bone marrow microenvironment may therefore present new treatment targets in AML. DisclosuresNo relevant conflicts of interest to declare.

Diquafosol Sodium Inhibits Apoptosis and Inflammation of Corneal Epithelial Cells Via Activation of Erk1/2 and RSK: In Vitro and In Vivo Dry Eye Model.

To evaluate the effect of diquafosol on corneal epithelium in a dry eye model using Transwell culture and a scopolamine-induced dry eye rat model. Desiccation stress induced in an in vitro dry eye model using human corneal epithelial cells was used, and the cells were incubated with or without diquafosol media diluted at 1:100. Reactive oxygen species (ROS) generation was measured using 2',7'-dichlorofluorescein diacetate (DCFH-DA). Apoptosis was analyzed, and levels of phosphorylated Erk1/2, phosphorylated p90RSK, phosphorylated Akt, IκB-α, and NF-κB-p65 were determined. Levels of IL-1β, TNF-α, IL-6, IL-8, and GM-CSF were quantified. To investigate the in vivo effects of diquafosol, we induced dry eye in Wistar rats using scopolamine hydrobromide. The rats were divided into three groups: control, dry eye, and dry eye diquafosol; topical DIQUAS was applied four times daily for 28 days. We used immunohistochemistry to detect the levels of phosphorylated Erk1/2, phosphorylated p90RSK, and IL-1β, and used the TUNEL assay in corneal tissue. The distribution of highly fluorescent dichlorofluorescein and the proportion of annexin V- and PI-positive cells decreased in the diquafosol medium. Diquafosol increased the levels of phospho-Erk1/2, phospho-90RSK, phospho-Akt, and IκB-α, whereas it significantly decreased the levels of NF-κB-p65, IL-1β, and TNF-α. In vivo, apoptosis was enhanced in dry eye group. This response was markedly reduced and the level of phosphorylated p90RSK and phosphorylated ERK1/2 were upregulated and IL-1β was downregulated by DIQUAS. Diquafosol treatment reduced intracellular ROS levels, apoptosis, and inflammation, all of which were increased in the dry eye model through desiccation.

17β-estradiol ameliorates oxidative stress and blue light-emitting diode-induced retinal degeneration by decreasing apoptosis and enhancing autophagy.

PurposeThis study aimed to assess the effects of 17β-estradiol (βE2) on blue light-emitting diode (LED)-induced retinal degeneration (RD) in rats and hydrogen peroxide (H2O2)-induced retinal pigment epithelium cell injury in humans and elucidate the protective mechanism of βE2 underlying these processes.MethodsFemale ovariectomized (OVX) rats were intravitreally injected with βE2 before blue LED exposure (3,000 lux, 2 hours). Retinal function and morphology were assayed via electroretinogram (ERG) and H&E, respectively. Cell viability was assayed using the Cell Counting Kit-8. Cell ROS were measured using dichlorofluorescein fluorescence. Apoptosis was evaluated by TUNEL and Annexin V/propidium iodide staining. Gene expression and protein expression were quantified using quantitative real-time RT-PCR, Western blotting, and immunohistochemistry. Autophagosomes were examined by electron microscopy.ResultsFemale OVX rats were exposed to blue LED, inducing RD. βE2 significantly prevented the reduction in the a- and b-wave ERG amplitudes and the disruption of retinal structure, the loss of photoreceptor cells, and the decrease in the thickness of the outer nuclear layer caused by blue LED exposure. βE2 also decreased cell apoptosis in the retina in blue LED-induced RD. Additionally, βE2 reduced ROS levels and apoptosis in H2O2-treated human retinal pigment epithelial (ARPE-19) cells. Furthermore, βE2 increased the protein expression of p-Akt and Bcl-2 and decreased the protein expression of cleaved caspase-3 and Bax during blue LED-induced retinal damage and in H2O2-treated ARPE-19 cells. βE2 also increased the number of autopha-gosomes and upregulated the expression of LC3-II/LC3-I and Beclin 1 in these processes.ConclusionβE2 protects against blue LED-induced RD and H2O2-induced oxidative stress by acting as an antioxidant, and its protective mechanism might occur by reducing apoptosis and enhancing autophagy; βE2 may be a novel and effective therapy for age-related macular degeneration.

Peroxidase expression is decreased by palmitate in cultured podocytes but increased in podocytes of advanced diabetic nephropathy.

High levels of serum free fatty acids (FFAs) are associated with lipotoxicity and type 2 diabetes. Palmitic acid (PA) is the predominant circulating saturated FFA. PA induces mitochondrial superoxide and hydrogen peroxide (H2O 2) generation in cultured podocytes. To elucidate the role of PA in antioxidant defense systems in diabetic nephropathy (DN), cultured podocytes were exposed to 250 μM PA for 1–24 hr, and protein expressions of catalase, peroxiredoxins (Prxs), and glutathione peroxidase (GPx) were examined by western blot analysis. PA induced an early transient increase in the Prx1, Prx2, and GPx1 levels in podocytes, but not catalase. Long‐term exposure of PA to podocytes significantly decreased the protein levels of Prx1, Prx2, GPx1, and catalase. Coincubation of PA‐treated cells with oleic acid, however, restored the expression of these proteins. In advanced human diabetic glomeruli, H2O2 generation was elevated as shown by increased fluorescence of dichlorofluorescein. Strong immunostaining for Prx1, Prx2, GPx1, and catalase was observed in the podocytes of advanced human DN, wherein transforming growth factor‐β1 staining was also positive. These results suggest that podocytes are susceptible to PA‐induced oxidative damage with impaired peroxidase activity and that peroxidases have futile antioxidant effects in the podocytes in the late stages of DN. Given this, PA‐induced podocyte injury via inadequate peroxidase response to H2O2 appears to play an important role in the pathogenesis of DN.

Purinergic Vasotoxicity: Role of the Pore/Oxidant/KATP Channel/Ca2+ Pathway in P2X7-Induced Cell Death in Retinal Capillaries.

P2X7 receptor/channels in the retinal microvasculature not only regulate vasomotor activity, but can also trigger cells in the capillaries to die. While it is known that this purinergic vasotoxicity is dependent on the transmembrane pores that form during P2X7 activation, events linking pore formation with cell death remain uncertain. To better understand this pathophysiological process, we used YO-PRO-1 uptake, dichlorofluorescein fluorescence, perforated-patch recordings, fura-2 imaging and trypan blue dye exclusion to assess the effects of the P2X7 agonist, benzoylbenzoyl-ATP (BzATP), on pore formation, oxidant production, ion channel activation, [Ca2+]i and cell viability. Experiments demonstrated that exposure of retinal microvessels to BzATP increases capillary cell oxidants via a mechanism dependent on pore formation and the enzyme, NADPH oxidase. Indicative that oxidation plays a key role in purinergic vasotoxicity, an inhibitor of this enzyme completely prevented BzATP-induced death. We further discovered that vasotoxicity was boosted 4-fold by a pathway involving the oxidation-driven activation of hyperpolarizing KATP channels and the resulting increase in calcium influx. Our findings revealed that the previously unappreciated pore/oxidant/KATP channel/Ca2+ pathway accounts for 75% of the capillary cell death triggered by sustained activation of P2X7 receptor/channels. Elucidation of this pathway is of potential therapeutic importance since purinergic vasotoxicity may play a role in sight-threatening disorders such as diabetic retinopathy.

Ezetimibe Protects Endothelial Cells against Oxidative Stress through Akt/GSK-3β Pathway.

Ezetimibe was reported to pharmacologically defend against oxidative stress. This study was designed to investigate whether ezetimibe can protect against the oxidative stress induced by oxidized low-density lipoprotein (oxLDL) in vitro and the underlying mechanism. Human umbilical vein endothelial cells (HUVECs) were pretreated with ezetimibe and then exposed to oxLDL for 24 h. TUNEL assay and detectionfor the protein levels of cleaved caspase-3, Bcl-xl and Bcl-2 were employed to assess the oxLDL-induced endothelial apoptosis. Intracellular reactive oxygen species (ROS) generation was evaluated by measuring dichlorofluorescein (DCF) fluorescence. The activities of endothelial antioxidant enzymes [superoxide dismutase (SOD) and catalase] were tested via an enzymatic assay. The mitochondrial membrane potential (MMP) was monitored by flow cytometry using JC-1 staining. Phosphorylation levels of glycogen synthase kinase-3p (p-GSK-3P) and Akt (p-Akt), as well as total GSK-3p and Akt were determined by Western blotting. The results showed that ezetimibe treatment inhibited HUVECs apoptosis, intracellular ROS production, and enhanced antioxidant enzyme activities elicited by oxLDL. HUVECs exposed to oxLDL alone had reduced mitochondrial function, while ezetimibe pre-intervention could significantly rescue the MMP. Furthermore, the protein levels of p-GSK-3p and p-Akt in ezetimibe-pretreated HUVECs were markedly increased as compared with those in oxLDL-induced HUVECs. However, no significant effect on total GSK- 3P and Akt was found in ezetimibe-pretreated HUVECs. Taken together, it was concluded that ezetimibe protects against oxLDL-induced oxidative stress through restoring the MMP, which may be mediated by Akt-dependent GSK-3P phosphorylation.

Regional increase in ROS within stretched region exacerbates arrhythmias in rat trabeculae with nonuniform contraction.

In diseased hearts, impaired muscle within the hearts is passively stretched by contractions of the more viable neighboring muscle during the contraction phase. We investigated whether in the myocardium with nonuniform contraction such passive stretch regionally generates ROS within the stretched region and exacerbates arrhythmias. In trabeculae from rat hearts, force, intracellular Ca2+, and membrane potential were measured. To assess regional ROS generation, the slope of the change in the 2',7'-dichlorofluorescein fluorescence (DCFslope) was calculated at the each pixel position along the long axis of trabeculae using DCF fluorescence images. Ca2+ waves and arrhythmias were induced by electrical stimulation. A H2O2 (1mmol/L) jet regionally increased the DCFslope within the jet-exposed region. A blebbistatin (10μmol/L) jet caused passive stretch of the muscle within the jet-exposed region during the contraction phase and increased the DCFslope within the stretched region, the velocity of Ca2+ waves, and the number of beats after electrical stimulation (0.2μmol/L isoproterenol), while 3μmol/L diphenyleneiodonium (DPI), NADPH oxidase inhibitor, decreased them.A jet of a solution containing 0.2 mmol/L H2O2 in addition to 10 µmol/L blebbistatin also increased them. A H2O2 jet within the region where Ca2+ waves propagated increased their velocity. In the myocardium with nonuniform contraction, passive stretch of the muscle by contractions of the neighboring muscle regionally increases ROS within the stretched region, and the regional ROS exacerbates arrhythmias by activating the propagation of Ca2+ waves.

Polysulfide protects midbrain dopaminergic neurons from MPP+-induced degeneration via enhancement of glutathione biosynthesis

Polysulfides are endogenous sulfur-containing molecular species that may regulate various cellular functions. Here we examined the effect of polysulfides exogenously applied to rat midbrain slice cultures, to address their potential neuroprotective actions. Na2S3 at concentrations of 10 μM or higher prevented 1-methyl-4-phenylpyridinium (MPP+)-induced loss of dopaminergic neurons. Na2S4 at 10 μM also protected dopaminergic neurons from MPP+ cytotoxicity, whereas Na2S and Na2S2 at the same concentration had no significant effect. We also found that Na2S3 (10 μM) prevented MPP+-induced increase in intracellular reactive oxygen species as detected by 2′,7′-dichlorofluorescein fluorescence. In addition, the protective effect of Na2S3 was abolished by l-buthionine sulfoximine, an inhibitor of glutathione synthesis. In cellular models of neurons (SH-SY5Y cells) and glial cells (C6 cells), Na2S3 (30 and 100 μM) increased expression of mRNAs encoding the subunits of glutamate cysteine ligase, the rate-limiting enzyme for glutathione biosynthesis. Consistently, the cellular content of total glutathione was increased by Na2S3, and the effect was more prominent in SH-SY5Y cells than in C6 cells. These results suggest that polysulfides are efficient neuroprotectants superior to monosulfur species such as H2S and HS−, and that the neuroprotective effect of polysulfides is mediated by upregulation of glutathione biosynthesis.

N-Acetylcysteine Reduces ROS-Mediated Oxidative DNA Damage and PI3K/Akt Pathway Activation Induced by Helicobacter pylori Infection.

Background H. pylori infection induces reactive oxygen species- (ROS-) related DNA damage and activates the PI3K/Akt pathway in gastric epithelial cells. N-Acetylcysteine (NAC) is known as an inhibitor of ROS; the role of NAC in H. pylori-related diseases is unclear. Aim The aim of this study was to evaluate the role of ROS and the protective role of NAC in the pathogenesis of H. pylori-related diseases. Method An in vitro coculture system and an in vivo Balb/c mouse model of H. pylori-infected gastric epithelial cells were established. The effects of H. pylori infection on DNA damage and ROS were assessed by the comet assay and fluorescent dichlorofluorescein assay. The level of PI3K/Akt pathway-related proteins was evaluated by Western blotting. The protective role of N-acetylcysteine (NAC) was also evaluated with in vitro and in vivo H. pylori infection models. Results The results revealed that, in vitro and in vivo, H. pylori infection increased the ROS level and induced DNA damage in gastric epithelial cells. NAC treatment effectively reduced the ROS level and inhibited DNA damage in GES-1 cells and the gastric mucosa of Balb/c mice. H. pylori infection induced ROS-mediated PI3K/Akt pathway activation, and NAC treatment inhibited this effect. However, the gastric mucosa pathological score of the NAC-treated group was not significantly different from that of the untreated group. Furthermore, chronic H. pylori infection decreased APE-1 expression in the gastric mucosa of Balb/c mice. Conclusions An increased ROS level is a critical mechanism in H. pylori pathogenesis, and NAC may be beneficial for the treatment of H. pylori-related gastric diseases linked to oxidative DNA damage.

Unprecedented chemosensing behavior of novel tetra-substituted benzimidazole zinc(II) phthalocynine for selective detection of Bi3 + ion: Synthesis, characterization and ROS generation

In this work, synthesis of novel symmetrical 4-(2-bromo-4-(5-bromo-1H-benzo[d] imidazol-2-yl) phenoxy) tetra substituted zinc phthalocyanine has been reported. The novel benzimidazole zinc phthlocynine compound (3) has been characterized by MALDI-TOF MS, FT-IR, UV–vis, and 1H NMR spectroscopy. This new compound 3 displayed excellent selectivity towards Bi3+ ion in the presence of other competitive ions including Ca2+, Cd2+, Co2+ Cu2+, Fe3+, Hg2+, Sn2+, Mg2+, Na+, Ni2+ and Pb2+ respectively. Upon addition of Bi3+ into the solution of compound 3 in DMSO, dramatic change was observed in the Q- and the B-bands in UV–visible spectra as a result of donor acceptor interactions. Reactive oxygen species (ROS) were also studied using 2,7-dichlorofluorescin diacetate (DCFH-DA) a fluorescent probe which is converted to highly fluorescent dichlorofluorescein (DCF) in the presence of ROS. This property of non-aggregating zinc phthalocyanine is promising as a photosensitizer in photodynamic therapy of cancer.

Down-regulation of NOX4 by betulinic acid protects against cerebral ischemia-reperfusion in mice.

Ischemic stroke leads to high potentiality of mortality and disability. The current treatment for ischemic stroke is mainly focused on intravenous thrombolytic therapy. However, ischemia/ reperfusion induces neuronal damage, which significantly influences the outcome of patients with ischemic stroke, and the exact mechanism implicated in ischemia/reperfusion injury remains unclear, although evidence shows that oxidative stress is likely to be involved. Betulinic acid is mainly known for its anti-tumor and anti-inflammatory activities. Our previous study showed that betulinic acid could decrease the reactive oxygen species (ROS) production by regulating the expression of NADPH oxidase. Thus, we hypothesized that betulinic acid may protect against brain ischemic injury in the animal model of stroke. Focal cerebral ischemia was achieved by using the standard intraluminal occlusion method and reperfusion enabled after 2 h ischemia. Neurological deficits were scored. Infarct size was determined with 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining and the mRNA expression of NADPH oxidase 4 (NOX4) was determined by RT-PCR in infarct tissue. ROS generation and apoptosis in ischemic tissue were analyzed by measuring the oxidative conversion of cell permeable 2',7'-dichloro-fluorescein diacetate (DCF-DA) to fluorescent dichlorofluorescein (DCF) in fluorescence microplate reader and TUNEL assay, respectively. In Kunming mice, 2 h of middle cerebral artery (MCA) occlusion followed by 24 or 72 h of reperfusion led to an enhanced NOX4 expression in the ischemic hemisphere. This was associated with elevated levels of ROS generation and neuronal apoptosis. Pre-treatment with betulinic acid (50 mg/kg/day for 7 days via gavage) prior to MCA occlusion prevented the ischemia/reperfusion-induced up-regulation of NOX4 and ROS production. In addition, treatment with betulinic acid could markedly blunt the ischemia/reperfusion-induced neuronal apoptosis. Finally, betulinic acid reduced infarct volume and ameliorated the neurological deficit in this stroke mouse model. Our results suggest that betulinic acid protects against cerebral ischemia/reperfusion injury in mice and the down-regulation of NOX4 may represent a mechanism contributing to this effect.

Aloe-emodin triggers ROS and Ca 2+ production and decreases the levels of mitochondrial membrane potential of human brain capillary endothelial cells

<p>The aim of this work was to investigate the mechanisms of cytotoxicity of phyto-hydroxyanthraquinone aloe-emodin (AE) on human brain microvascular endothelial cell line hCMEC/D3 and to assess the cellular response in the early stage of treatment in order to extend the knowledge of AE’s anti-angiogenic properties. The immortalized human brain capillary endothelial cells hCMEC/D3 were treated with a series of AE concentrations (5 - 200 μM) for a period of 24 hours. The cell viability was determined by MTS assay. The cellular adenosine triphosphate (ATP) levels were evaluated by CellTiter-Glo® luminescent assay. The intracellular reactive oxygen species (ROS) were determined by 2’,7’-dichlorofluorescein (CM- H2DCFDA) fluorescence assay. The mitochondrial membrane potential (MMP) was assessed using tetramethylrhodamine methyl ester (TMRM) staining, while Fluo-4 was used to measure 2 the intracellular free Ca 2+ concentrations inside living cells analysed by High Content Analysis using the Arrayscan VTI 740. Twenty-four- hour treatment of hCMEC/D3 cells with AE, in concentrations between 50 and 200 µM, decreased the cell viability as well as the intracellular ATP levels in a dose- dependent manner. Increased ROS production and disruption of the mitochondrial membrane potential have also been detected. Notably, AE at a concentration greater than 5 µM dramatically increased intracellular calcium levels. Our results collectively indicate that AE inhibits proliferation of human brain microvascular cells via a mechanism involving ROS generation, disruption of Ca 2+ homeostasis and mitochondrial damage.</p>

Glycation of high-density lipoprotein triggers oxidative stress and promotes the proliferation and migration of vascular smooth muscle cells.

BackgroundIn type 2 diabetes mellitus (T2DM), high-density lipoprotein (HDL) impairs its anti-atherogenic properties and even develops to a pro-inflammatory and pro-atherogenic phenotype because of abnormal compositions and modifications. In this study, we examined the effects and the related mechanisms of glycation of HDL on the proliferation and migration of vascular smooth muscle cells (VSMCs).Methods & ResultsGlycated HDL (G-HDL) was modified with D-glucose (25 mmol/L) in vitro. Diabetic HDL (D-HDL) was isolated from T2DM patients. Rat VSMCs were isolated from the thoracic aortas. Human VSMCs were obtained from ScienCell Research Laboratories. Alpha-actin was detected through immunofluorescence. VSMC proliferation was assayed by Cell Count. VSMC migration was determined by transwell chamber and scratch-wound assay. Intracellular reactive oxygen species (ROS) was detected based on ROS-mediated 2′,7′-dichlorofluorescein (DCFH-DA) fluorescence. Compared to native HDL (N-HDL), G-HDL remarkably promoted VSMC proliferation and migration in the dose and time-dependent manners. In addition, G-HDL enhanced ROS generation in VSMCs. However, the ROS scavenger, N-acetylcysteine, efficiently decreased ROS production and subsequently inhibited the proliferation of VSMCs induced by G-HDL. Similarly, D-HDL from T2DM patients also promoted ROS release and VSMC proliferation and migration.ConclusionsHDL either glycated in vitro or isolated from T2DM patients triggered VSMC proliferation, migration, and oxidative stress. These results might partly interpret the higher morbidity of cardiovascular disease in T2DM patients.

CD47 overexpression is associated with decreased neutrophil apoptosis/phagocytosis and poor prognosis in non-small-cell lung cancer patients

Background:Non-small-cell lung cancer (NSCLC) patients often exhibit neutrophilia, which has been associated with poor clinical outcomes. However, the mechanisms that lead to neutrophilia have not been fully established. CD47 is an antiphagocytic molecule that promotes neutrophil recruitment.Methods:Blood was collected from 50 treatment-naive patients with advanced NSCLC and from 25 healthy subjects. The frequency of CD66b+ cells and the expression of CD47 were determined by flow cytometry. Neutrophil apoptosis was determined by 7-amino-actinomycin D/Annexin V-APC staining. Phagocytosis was assessed by flow cytometry. Reactive oxygen species production after phorbol 12-myristate 13-acetate treatment was quantified by 2′,7′-dichlorofluorescein fluorescence. Pro-inflammatory plasma cytokines were quantified using a cytometric bead array assay.Results:The percentage of circulating neutrophils was significantly higher in patients than in controls (P<0.001). Patient-derived neutrophils had a higher oxidative potential than those of controls (P=0.0286). The number of neutrophils in late apoptosis/necrosis was lower in patients than in controls (P=0.0317). Caspase 3/7 activation was also lower in patients than in controls (P=0.0079). CD47 expression in whole-blood samples and in the neutrophil fraction was higher in NSCLC patients than in controls (P=0.0408 and P<0.001). Patient-derived neutrophils were phagocytosed at a lower rate than those of controls (P=0.0445). CD47 expression in neutrophils negatively correlated with their ingestion by macrophages (P=0.0039). High CD47 expression was associated with a lower overall survival.Conclusions:Increased CD47 expression on the surface of neutrophils was associated with a delay in neutrophil apoptosis and with an impairment in their phagocytic clearance by macrophages, suggesting that CD47 overexpression may be one of the underlying mechanisms leading to neutrophilia in NSCLC patients.

Palmitate induces mitochondrial superoxide generation and activates AMPK in podocytes.

Studies have shown that high levels of serum free fatty acids (FFAs) are associated with lipotoxicity and type 2 diabetes. Palmitic acid (PA) is the predominant circulating saturated FFA, yet its role in the pathogenesis of diabetic nephropathy (DN) is not clear. Recently, one study suggested that mitochondrial superoxide production is related to AMP-activated protein kinase (AMPK) activity in diabetic mice kidneys. To elucidate the link between PA and oxidative stress and AMPK activity in DN, we compared the cultured murine podocytes exposed to PA and oleic acid (OA). Incubation of cells with 250 μM PA or OA induced a translocation of CD36, a fatty acid transport protein, with intracellular lipid accumulation. PA, but not OA, induced mitochondrial superoxide and hydrogen peroxide (H2 O2 ) generation in podocytes, as shown by enhanced fluorescence of MitoSOX Red and dichlorofluorescein (DCF), respectively. Costimulation of PA-treated cells with the H2 O2 scavenger catalase abolished the PA-induced DCF fluorescence. Only PA induced mitochondrial damage as shown by electron microscopy. The AMPK activity was determined by immunoblotting, measuring the ratio of phosphorylated AMPK (p-AMPK) to total AMPK. Only PA significantly increased the p-AMPK levels compared with controls. Addition of catalase to PA-treated cells did not affect the PA-stimulated p-AMPK levels. Collectively, our results indicate that PA induces mitochondrial superoxide and H2 O2 generation in cultured podocytes, which may not be directly linked to AMPK activation. Given that, PA seems to play an important role in the pathogenesis of DN through lipotoxicity initiated by mitochondrial superoxide overproduction.

Bioaccumulation, stress, and swimming impairment in Daphnia magna exposed to multiwalled carbon nanotubes, graphene, and graphene oxide.

The use of carbon-based nanomaterials (CNMs) such as multiwalled carbon nanotubes (MWCNTs), graphene, and graphene oxide (GO) is increasing across many applications because of their unique and versatile properties. These CNMs may enter the aquatic environment through many pathways, creating the potential for organism exposure. The present study addresses the bioaccumulation and toxicity seen in Daphnia magna exposed to CNMs dispersed in sodium dodecyl benzene sulfonate (SDBS). In study I, D. magna were exposed to varying outer diameters of MWCNTs for 24 h in moderately hard or hard freshwater. Bioaccumulation of MWCNT was found in all treatments, with the highest concentrations (0.53 ± 0.27 μg/g) in D. magna exposed in hard freshwater (p < 0.005). The median lethal concentration (LC50) was determined for D. magna exposed to CNMs in moderately hard and hard freshwater. In study II, D. magna were exposed to CNMs for 72 h in moderately hard freshwater to assess swimming velocity and generation of reactive oxygen species (ROS) detected by dichlorofluorescein fluorescence. An overall decrease was seen in D. magna swimming velocity after exposure to CNMs. The generation of ROS was significantly higher (1.54 ± 0.38 dichlorofluorescein mM/mg dry wt) in D. magna exposed to MWCNTs of smaller outer diameters than in controls after 72 h (p < 0.05). These results suggest that further investigation of CNM toxicity and behavior in the aquatic environment is needed. Environ Toxicol Chem 2017;36:2199-2204. © 2017 SETAC.

Protective effects of hydrogen sulfide against angiotensinII-induced endoplasmic reticulum stress in HUVECs.

The inhibitory effects of hydrogen sulfide (H2S) on angiotensinII (AngII)-stimulated human umbilical vein endothelial cell (HUVEC) dysfunction remain to be elucidated. Endoplasmic reticulum (ER) stress has been detected in endothelial dysfunction (ED). The present study aimed to determine whether H2S may exert an inhibitory effect on AngII‑induced ERstress. Using HUVECs as a model system, the present study used western blotting to detect protein expression, intracellular reactive oxygen species (ROS) levels were determined by oxidative conversion of cell permeable DCFH‑DA to fluorescent dichlorofluorescein, CCK‑8 assay was used to investigate the cell viability, methylene blue was used to investigate the CSE activity, TUNEL was used to investigate the cells apoptosis. The present study demonstrated that AngII not only upregulated the expression levels of inducible nitric oxide synthase (iNOS), stimulated ROS production and increased cell apoptosis, but also downregulated the expression levels of phosphorylated‑endothelial nitric oxide synthase, decreased the expression and activity of cystathionine‑c‑lyase (CSE) and decreased cell viability. Furthermore, hydrogen peroxide (H2O2; an exogenous ROS) downregulated the expression and activity of CSE, and had similar effects as AngII, whereas the inhibitory effects of AngII were completely suppressed by N-acetyl-L-cysteine (a ROS scavenger). In addition, AngII induced the expression of glucose‑regulated protein78 (GRPP78) and C/EBP homologous protein (CHOP), which are markers of ER stress. Conversely, the stimulatory effects of AngII were completely inhibited by sodium hydrosulfide (NaHS; a H2S donor). Treatment with NaHS attenuated ROS production, inhibited CHOP and GRP78 expression, and decreased cell apoptosis. The present study indicated that AngII induced ED via the activation of ERstress in HUVECs. In addition, the effects of AngII on ER stress could be suppressed by H2S.

Palmitic Acid Curcumin Ester Facilitates Protection of Neuroblastoma against Oligomeric Aβ40 Insult

Background/Aims: The generation of reactive oxygen species (ROS) caused by amyloid-β (Aβ) is considered to be one of mechanisms underlying the development of Alzheimer’s disease. Curcumin can attenuate Aβ-induced neurotoxicity through ROS scavenging, but the protective effect of intracellular curcumin on neurocyte membranes against extracellular Aβ may be compromised. To address this issue, we synthesized a palmitic acid curcumin ester (P-curcumin) which can be cultivated on the cell membrane and investigated the neuroprotective effect of P-curcumin and its interaction with Aβ. Methods: P-curcumin was prepared through chemical synthesis. Its structure was determined via nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). An MTT assay was used to assess Aβ cytotoxicity and the protective effect of P-curcumin on SH-SY5Y cells. The effect of P-curcumin on Aβ-induced ROS production in vitro and in vivo were assessed based on changes in dichlorofluorescein (DCF) fluorescence. A spectrophotometric method was employed to detect lipid peroxidation. To mimic the interaction of P-curcumin on cell membranes with Aβ, liposomes were prepared by thin film method. Finally, the interactions between free P-curcumin and P-curcumin cultivated on liposomes and Aβ were determined via spectrophotometry. Results: A novel derivative, palmitic acid curcumin ester was prepared and characterized. This curcumin, cultivated on the membranes of neurocytes, may prevent Aβ-mediated ROS production and may inhibit the direct interaction between Aβ and the cellular membrane. Furthermore, P-curcumin could scavenge Aβ-mediated ROS as curcumin in vitro and in vivo, and had the potential to prevent lipid peroxidation. Morphological analyses showed that P-curcumin was better than curcumin at protecting cell shape. To examine P-curcumin’s ability to attenuate direct interaction between Aβ and cell membranes, the binding affinity of Aβ to curcumin and P-curcumin was determined. The association constants for free P-curcumin and curcumin were 7.66 × 10⁴ M^-1 and 7.61 × 10⁵ M^-1, respectively. In the liposome-trapped state, the association constants were 3.71 × 10⁵ M^-1 for P-curcumin and 1.44× 10⁶ M^-1 for curcumin. With this data, the thermodynamic constants of P-curcumin association with soluble Aβ (ΔH, ΔS, and ΔG) were also determined. Conclusion: Cultivated curcumin weakened the direct interaction between Aβ and cell membranes and showed greater neuroprotective effects against Aβ insult than free curcumin.

Induction of Apoptosis and Cell Cycle Arrest by Flavokawain C on HT-29 Human Colon Adenocarcinoma via Enhancement of Reactive Oxygen Species Generation, Upregulation of p21, p27, and GADD153, and Inactivation of Inhibitor of Apoptosis Proteins.

Chalcones have been shown to exhibit anti-cancer properties by targeting multiple molecular pathways. It was, therefore, of interest to investigate flavokawain C (FKC), a naturally occurring chalcone, which can be isolated from Kava (Piper methysticum Forst) root extract. The aim of this study was to investigate the inhibitory effect of FKC on the growth of HT-29 cells and its underlying mechanism of action. Cell viability of HT-29 cells was assessed by Sulforhodamine B assay after FKC treatment. Induction of apoptosis was examined by established morphological and biochemical assays. ROS generation was determined by dichlorofluorescein fluorescence staining, and superoxide dismutase activity was measured using the spectrophotometric method. Western blotting was used to examine the changes in the protein levels. FKC markedly decreased the cell viability of HT-29 cells and the cells showed dramatic changes in cellular and nuclear morphologies with typical apoptotic features. The induction of apoptosis correlated well with the externalization of phosphatidylserine, DNA fragmentation, decreased mitochondrial membrane potential, activation of caspases, and PARP cleavage. This was associated with an increase in reactive oxygen species and a decrease in SOD activity. The protein levels of XIAP, c-IAP1, and c-IAP2 were downregulated, whereas the GADD153 was upregulated after FKC treatment. FKC induced cell cycle arrest at the G1 and G2/M phases via upregulation of p21 and p27 in a p53-independent manner. Our results provide evidence that FKC has the potential to be developed into chemotherapeutic drug for the treatment of colon adenocarcinoma.SUMMARY: Flavokawain C inhibited the growth of HT-29 human colon adenocarcinoma cellsFlavokawain C induced apoptosis in HT-29 cells, associated with an increase in reactive oxygen species and a decrease in SOD activityFlavokawain C induced cell cycle arrest at the G1 and G2/M phases via upregulation of p21 and p27 in HT-29 cellsHT-29 cells treated with flavokawain C caused downregulation of XIAP, c-IAP1, and c-IAP2, and upregulation of GADD153. Abbreviations used: FKC: Flavokawain C; SRB: Sulforhodamine B; ROS: Reactive oxygen species; SOD: Superoxide dismutase; PARP: Poly(ADP-ribose) polymerase; ER: Endoplasmic reticulum; IAPs: Inhibitor of apoptosis proteins; TUNEL: Transferase dUTP nick end labeling; Annexin V-FITC: Annexin V conjugated with fluorescein isothicyanate