stilbenes and diacetyldichlorofluorescein

Vascular endothelial cells play a vital role in the health and maintenance of vascular homeostasis, but hyperglycemia disrupts their function by increasing cellular oxidative stress. Resveratrol, a plant polyphenol, possesses antioxidant properties that can mitigate oxidative stress. Addressing the challenges of its limited solubility and stability, gold nanoparticles (GNps) were utilized as carriers. A microfluidic chip (MFC) with dynamic flow conditions was designed to simulate body vessels and to investigate the antioxidant properties of resveratrol gold nanoparticles (RGNps), citrate gold nanoparticles (CGNps), and free Resveratrol on human umbilical vein endothelial cells (HUVEC). The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay was employed to measure the extracellular antioxidant potential, and cell viability was determined using the Alamar Blue test. For assessing intracellular oxidative stress, the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay was conducted, and results from both the cell culture plate and MFC were compared. Free Resveratrol demonstrated peak DPPH scavenging activity but had a cell viability of about 24-35%. RGNPs, both 3.0 ± 0.5 nm and 20.2 ± 4.7 nm, consistently showed high cell viability (more than about 90%) across tested concentrations. Notably, RGNPs (20 nm) exhibited antioxidative properties through DPPH scavenging activity (%) in the range of approximately 38-86% which was greater than that of CGNps at about 21-32%. In the MFC,the DCFH-DA analysis indicated that RGNPs (20 nm) reduced cellular oxidative stress by 57-82%, surpassing both CGNps and free Resveratrol. Morphologically, cells in the MFC presented superior structure compared to those in traditional cell culture plates, and the induction of hyperglycemia successfully led to the formation of multinucleated variant endothelial cells (MVECs). The MFC provides a distinct advantage in observing cell morphology and inducing endothelial cell dysfunction. RGNps have demonstrated significant potential in alleviating oxidative stress and preventing endothelial cell disorders.

Topics: Antioxidants; Endothelium; Gold; Human Umbilical Vein Endothelial Cells; Humans; Hyperglycemia; Lab-On-A-Chip Devices; Metal Nanoparticles; Oxidative Stress; Resveratrol; Stilbenes

Ammonia is a metabolite that, at high concentrations, is implicated in neurological disorders, such as hepatic encephalopathy (HE), which is associated with acute or chronic liver failure. Astrocytes are considered the primary target of ammonia toxicity in the central nervous system (CNS) because glutamine synthetase (GS), responsible for ammonia metabolism in CNS, is an astrocytic enzyme. Thus, neuronal dysfunction has been associated as secondary to astrocytic impairment. However, we demonstrated that ammonia can induce direct effects on neuronal cells. The cell viability was decreased by ammonia in SH-SY5Y cells and cerebellar granule neurons. In addition, ammonia induced increased reactive oxygen species (ROS) production and decreased GSH intracellular content, the main antioxidant in CNS. As ammonia neurotoxicity is strongly associated with oxidative stress, we also investigated the potential neuroprotective roles of the antioxidants, resveratrol (RSV) and lipoic acid (LA), against ammonia toxicity in cerebellar granule neurons. RSV and LA were able to prevent the oxidative damage induced by ammonia, maintaining the levels of ROS production and GSH close to basal values. Both antioxidants also decreased ROS production and increased GSH content under basal conditions (in the absence of ammonia). Moreover, we showed that heme oxygenase 1 (HO1), a protein associated with protection against stress conditions, is involved in the beneficial effects of RSV and LA in cerebellar granule neurons. Thus, this study reinforces the neuroprotective effects of RSV and LA. Although more studies in vivo are required, RSV and LA could represent interesting therapeutic strategies for the management of HE.

Topics: Ammonia; Animals; Animals, Newborn; Anti-Inflammatory Agents, Non-Steroidal; Cells, Cultured; Cerebellum; Dose-Response Relationship, Drug; Fluoresceins; Glutathione; Heme Oxygenase-1; Humans; Neurons; Oxidative Stress; Rats; Rats, Wistar; Resveratrol; Stilbenes

Resveratrol has been shown to be a potential chemopreventive and anticancer agent, inducing apoptosis in a variety of cancer cells. The present study was performed to evaluate the effect of resveratrol on A549 human lung adenocarcinoma epithelial cells. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide evaluation demonstrated that the exposure of cells to increasing concentrations of resveratrol (0-175 µM) for 24 h resulted in a decrease in cell viability (IC50 85.5 µM). Annexin V/propidium iodide double stain verified apoptosis in A549 cells, while negligible cell cytotoxity (≥ 0.5 %) was observed in all untreated incubations. Using colorimetric assay kits, induction of caspase-3, but not of caspase-8, activity was detected in response to resveratrol (> 130 µM). Confirmatory evidence of this finding was provided by Western blotting, indicating expression of cleaved caspase-3 levels in a concentration-dependent manner with a minimum resveratrol concentration of 65 µM required for activation of this protease, while that of caspase-8 remained unaffected. The apoptotic process was associated with reactive oxygen species production in a concentration-dependent manner, evidenced by microscopic examination and fluorescence-activated cell sorting analysis using the 2',7'-dichlorofluorescein diacetate assay. In the presence of the mitochondrial electron transport chain inhibitor rotenone, reactive oxygen species production and the concomitant apoptotic cell population were significantly reduced. This finding suggested that the resveratrol-induced apoptosis was mediated via a mitochondrial pathway alignment in human A549 cells. Although effective levels were observed at high concentrations, the outcome may well differ under in vivo conditions. Finally, experiments reaffirmed the chemical instability of trans-resveratrol, suggesting the need for protection of the solutions from extended exposure to light.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Caspase 3; Caspase 8; Cell Line, Tumor; Cell Survival; Epithelial Cells; Fluoresceins; Humans; Lung Neoplasms; Mitochondria; Reactive Oxygen Species; Resveratrol; Stilbenes; Tetrazolium Salts; Thiazoles

Resveratrol (3,5,4'-trihydroxystilbene) is a natural occurring molecule, synthesized by plants in response to different stresses. Recent literature data seem to converge in indicating Resveratrol as an agent possessing protective effects against oxidative stresses through different mechanisms. Furthermore conflicting data are present in relation to its activity of free radical scavenger. Here we studied the antioxidant activity actually exerted by the agent against reactive oxygen species induced by H(2)O(2) treatments in CHO cells. Our attention has been focused on two major potential mechanisms: scavenging activity and interference with oxidative metabolism, by the analysis of three important targets: intracellular oxidation (Dichlorofluorescein Test), primary DNA damage (Comet Assay) and fixed DNA damage (chromosomal aberrations). Cells were treated with a single H(2)O(2) dose (2x10(-4) M) in order to induce Reactive Oxygen Species and than challenged with Resveratrol to test its ability in modulating damage. Two experimental protocols have been applied: (i) simultaneous treatment and (ii) a 3 h Resveratrol pre-treatment. In our experimental conditions Resveratrol does not appear able, 'per se', to induce primary DNA damage whereas a slight increase in endogenous oxidation and chromosomal aberrations at the highest dose have to be noticed. In combined treatments the molecule appears to differently affect primary and fixed DNA damage.

Topics: Animals; Antioxidants; CHO Cells; Chromosome Aberrations; Comet Assay; Cricetinae; DNA Damage; Drug Interactions; Flow Cytometry; Fluoresceins; Free Radical Scavengers; Hydrogen Peroxide; Reactive Oxygen Species; Resveratrol; Stilbenes