An assessment on resveratrol and its cellular protective properties

Abstract only availableResveratrol is a polyphenolic compound found abundantly in plants such as knotweed or grapes. Under normal circumstances, it is in plants as a phytoalexin - an antibiotic produced by plants for defense under insults. Based on previous studies, it it also has properties that protect cells from damage caused by free radicals or reactive oxygen species In order to test the effectiveness of resveratrol as a protective agent, an experiment was designed using an immortalized astrocyte cell line, DiTNC. These cells were divided into control, resveratrol and non-resveratrol groups. They were cultured in a standard six well plate, with two wells per group. Menadione (25 and 50 micromolar), a compound that generates reactive oxygen species in cells, was added to four wells, two of which contained pre-incubated resveratrol (50 micromolar). The cells were left to incubate in an incubating oven at 37 Celsius. Photos are taken at intervals of 30 minutes, 60 minutes, and 120 minutes. Menadione caused cells to project processes and then causes them to become shrunken and rounded within 30 min. When the pictures from the resveratrol group was compared to the non-resveratrol group, a significantly less amount of cells from the resveratrol group were found to be either free floating or shrunken, suggesting that these cells survived for a longer period. An MTT test will also be performed in order to quantify the data. The results show that resveratrol has a fair effect on protecting cells from reactive oxygen species and that this chemical warrants further investigations.Alzheimer's disease program project grant 2P01AGO18357 to G. Su

Antioxidants to combat Alzheimer's disease

Abstract only availableAlzheimer's disease (AD) took the life of my grandfather. It is believed that oxidative stress contributes to the progression of this disease. Studies in Dr. Sun's laboratory recognized NADPH oxidase as possible source for production of reactive oxygen species (ROS) which can damage cells in the brain. If this were true then something could be found to stop the activation of NADPH oxidase. This would stop the deterioration of cells, decreasing the amount of Alzheimer's cases. To do my part of this research, we culture immortalized astrocytes (DITNC) and look at how cells react to menadione, a compound that produces ROS, possible through activating NADPH oxidase. We will then see how apocynin stands up against menadione. To test this we had six wells containing the astrocytes. In the first two wells we didn't add anything to the cells, these were our control cells. In the other four wells we added menadione, and then in two of those wells we added apocynin. We then took pictures right after we added the inhibitor, 30 minutes after they were in the incubator, 60 minutes, and then 120 minutes later. Doing this allowed us to see the difference in adding apocynin and not adding it. Looking at our results, we found the wells with only Menadione had more cells that were dead and shriveled up, and there were fewer dying cells in the wells containing Apocynin. This means it is possible for Apocynin to be used to stop the activation of NADPH oxidase; this would keep cells from dying.Missouri Academy at Northwest Missouri State University, Alzheimer disease program project grant 2PO1 AGO18357 to G. Su

Oxidative stress alters cell morphology and cell death indices in immortalized astrocytes

Abstract only availableMenadione, usually known as vitamin K3, also serves as a trigger for oxidative stress, delivering reactive oxygen species (ROS) upon entering the cells. Astrocytes are glial cells that are found in the brain and are extremely important in providing nourishment to cells in the brain, especially neurons. Oxidative stress may cause damage to astrocytes and alter their function. Increase in oxidative stress is the underlying cause for many neurodegenerative diseases like Alzheimer's disease and stroke. In this study, we used menadione as an oxidant compound to study effects of oxidative stress on cell morphology and viability in an immortalized astrocyte cell line (DITNC). Menadione causes cytoskeletal rearrangement and stress fiber protrusions in astrocytes. This event is accomplished by an increase in LDH release and a decrease in MTT release, suggesting loss of cell viability. Resveratrol (enriched in grape) and curcumin (from turmeric), polyphenolic antioxidants, have been shown to inhibit damage caused by ROS. In this study, we also used these botanical compounds to demonstrate their inhibitory properties against menadione-mediated morphological changes and cell damage in DITNC cells.NSF-REU Biology & Biochemistr

Cytosolic phospholipase A 2 plays a crucial role in ROS/NO signaling during microglial activation through the lipoxygenase pathway

BACKGROUND: Oxidative stress and inflammation are important factors contributing to the pathophysiology of numerous neurological disorders, including Alzheimer’s disease, Parkinson’s disease, acute stroke, and infections of the brain. There is well-established evidence that proinflammatory cytokines and glutamate, as well as reactive oxygen species (ROS) and nitric oxide (NO), are produced upon microglia activation, and these are important factors contributing to inflammatory responses and cytotoxic damage to surrounding neurons and neighboring cells. Microglial cells express relatively high levels of cytosolic phospholipase A(2) (cPLA(2)), an enzyme known to regulate membrane phospholipid homeostasis and release of arachidonic acid (AA) for synthesis of eicosanoids. The goal for this study is to elucidate the role of cPLA(2)IV in mediating the oxidative and inflammatory responses in microglial cells. METHODS: Experiments involved primary microglia cells isolated from transgenic mice deficient in cPLA(2)α or iPLA(2)β, as well as murine immortalized BV-2 microglial cells. Inhibitors of cPLA(2)/iPLA(2)/cyclooxygenase (COX)/lipoxygenase (LOX) were used in BV-2 microglial cell line. siRNA transfection was employed to knockdown cPLA(2) expression in BV-2 cells. Griess reaction protocol was used to determine NO concentration, and CM-H2DCF-DA was used to detect ROS production in primary microglia and BV-2 cells. WST-1 assay was used to assess cell viability. Western blotting was used to assess protein expression levels. Immunocytochemical staining for phalloidin against F-actin was used to demonstrate cell morphology. RESULTS: In both primary and BV-2 microglial cells, stimulation with lipopolysaccharide (LPS) or interferon gamma (IFNγ) resulted in a time-dependent increase in phosphorylation of cPLA(2) together with ERK1/2. In BV-2 cells, LPS- and IFNγ-induced ROS and NO production was inhibited by arachidonyl trifluoromethyl ketone (AACOCF3) and pyrrophenone as well as RNA interference, but not BEL, suggesting a link between cPLA(2), and not iPLA(2), on LPS/IFNγ-induced nitrosative and oxidative stress in microglial cells. Primary microglial cells isolated from cPLA(2)α-deficient mice generated significantly less NO and ROS as compared with the wild-type mice. Microglia isolated from iPLA(2)β-deficient mice did not show a decrease in LPS-induced NO and ROS production. LPS/IFNγ induced morphological changes in primary microglia, and these changes were mitigated by AACOCF3. Interestingly, despite that LPS and IFNγ induced an increase in phospho-cPLA(2) and prostaglandin E2 (PGE2) release, LPS- and IFNγ-induced NO and ROS production were not altered by the COX-1/2 inhibitor but were suppressed by the LOX-12 and LOX-15 inhibitors instead. CONCLUSIONS: In summary, the results in this study demonstrated the role of cPLA(2) in microglial activation with metabolic links to oxidative and inflammatory responses, and this was in part regulated by the AA metabolic pathways, namely the LOXs. Further studies with targeted inhibition of cPLA(2)/LOX in microglia during neuroinflammatory conditions can be valuable to investigate the therapeutic potential in ameliorating neurological disease pathology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12974-015-0419-0) contains supplementary material, which is available to authorized users

Differential effects of grape seed extract on inflammatory responses of microglial cells [abstract]

Abstract only availableMicroglial activation has been linked to multiple pathologies, including Alzheimer's disease and cerebral ischemia. Nitric oxide (NO) production by inducible nitric oxide synthase (iNOS) and superoxide from NADPH oxidase (NOX) in microglial cells have been regarded as a dual-key mechanism underlying neuro-inflammatory responses leading to neuronal damage. NO reacts with superoxide to produce peroxynitrite, a highly cytotoxic anion. Antioxidants, including resveratrol extracted from grape skin, have previously been shown to attenuate both superoxide and NO production in activated microglia. A mixture of polyphenols found in grape seed extract (GSE) also possess powerful antioxidant properties. Both resveratrol and GSE have been shown to attenuate neuronal death and neuro-behavioral deficits in rodent stroke models. In this study, we tested the hypothesis that GSE is an anti-inflammatory agent capable of suppressing NO production in microglial cells induced by interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). GSE suppressed LPS-induced NO production, but surprisingly promoted IFN-gamma-induced NO production in a dose-dependent manner. At higher concentrations, GSE alone induced NO production. More importantly, in the presence of IFN-gamma, higher concentrations of GSE led to cell death. These results suggest that GSE can either alleviate or exacerbate inflammatory conditions, depending on the types of agonists and inductions.Life Sciences Undergraduate Research Opportunity Progra

Low energy laser light (632.8 nm) suppresses amyloid-β peptide-induced oxidative and inflammatory responses in astrocytes

Oxidative stress and inflammation are important processes in the progression of Alzheimer's disease (AD). Recent studies have implicated the role of amyloid β-peptides (Aβ) in mediating these processes. In astrocytes, oligomeric Aβ induces the assembly of NADPH oxidase complexes resulting in its activation to produce anionic superoxide. Aβ also promotes production of pro-inflammatory factors in astrocytes. Since low energy laser has previously been reported to attenuate oxidative stress and inflammation in biological systems, the objective of this study was to examine whether this type of laser light was able to abrogate the oxidative and inflammatory responses induced by Aβ. Primary rat astrocytes were exposed to Helium-Neon laser (λ=632.8 nm), followed by the treatment with oligomeric Aβ. Primary rat astrocytes were used to measure Aβ-induced production of superoxide anions using fluorescence microscopy of dihydroethidium (DHE), assembly of NADPH oxidase subunits by the colocalization between the cytosolic p47phox subunit and the membrane gp91phox subunit using fluorescent confocal microscopy, phosphorylation of cytosolic phospholipase A2 (cPLA2), and expressions of pro-inflammatory factors including interleukin-1β (IL-1β) and inducible nitric-oxide synthase (iNOS) using Western blot Analysis. Our data showed that laser light at 632.8 nm suppressed Aβ-induced superoxide production, colocalization between NADPH oxidase gp91phox and p47phox subunits, phosphorylation of cPLA2, and the expressions of IL-1β and iNOS in primary astrocytes. We demonstrated for the first time that 632.8 nm laser was capable of suppressing cellular pathways of oxidative stress and inflammatory responses critical in the pathogenesis in AD. This study should prove to provide the groundwork for further investigations for the potential use of laser therapy as a treatment for AD

ROS from menadione induces astrocytic damage: protective effects of apocynin

Abstract only availableOxidative stress is a core cause of neurodegenerative diseases such as Alzheimer's disease. When cells are under oxidative stress, they will produce a high amount of reactive oxygen species (ROS). ROS are small and highly reactive and include compounds such as oxygen ions, free radicals, and peroxides. Understanding what triggers oxidative stress and how to ameliorate its damaging effects is a crucial step in discovering a cure for Alzheimer's disease. Menadione, a vitamin precursor of K2, is an oxidative compound that is capable of delivering ROS to the cells. Apocynin, a natural organic compound that has been isolated from Picrorhiza curroa grown in the Himalayan Mountains, is an inhibitor of NADPH oxidase, an enzyme for ROS production in cells. In this experiment, we studied whether apocynin may neutralize the effects of menadione using an immortalized astrocyte cell line DITNC. Astrocytes are glial cells that play a crucial role in the brain by providing necessary nutrient to surrounding neurons. We had three sample groups and treated each group with different drugs. The first group was the control, the second group was treated with menadione, and the third group was treated with both menadione and apocynin. After treating the cells, we recorded morphological changes of the cells by taking pictures of each sample group at three different time intervals (30 min, 1 and 2 hours). In addition to the morphological evidence, we also did a MTT assay to assess cell viability and later a data analysis based on the result from the MTT test. MTT assay measures mitochondrial activity and thus indirectly measures cell viability. Both morphological data and MTT analysis showed menadione caused DITNC cell damage with decreased mitochondrial activity. When cells are treated with menadione, they formed processes, shrink, and then round up. We also found apocynin protects against the oxidative damage caused by menadione to a certain extent. Since apocynin is an inhibitor of NADPH oxidase, this also indicates oxidative stress is generated by NADPH oxidase, suggesting apocynin may be a potential means to treat Alzheimer's disease.Alzheimer disease program project grant 2P01 AG018357 to G. Su

Low energy laser light (632.8 nm) suppresses amyloid-β peptide-induced oxidative and inflammatory responses in astrocytes

Oxidative stress and inflammation are important processes in the progression of Alzheimer's disease (AD). Recent studies have implicated the role of amyloid β-peptides (Aβ) in mediating these processes. In astrocytes, oligomeric Aβ induces the assembly of NADPH oxidase complexes resulting in its activation to produce anionic superoxide. Aβ also promotes production of pro-inflammatory factors in astrocytes. Since low energy laser has previously been reported to attenuate oxidative stress and inflammation in biological systems, the objective of this study was to examine whether this type of laser light was able to abrogate the oxidative and inflammatory responses induced by Aβ. Primary rat astrocytes were exposed to Helium-Neon laser (λ=632.8 nm), followed by the treatment with oligomeric Aβ. Primary rat astrocytes were used to measure Aβ-induced production of superoxide anions using fluorescence microscopy of dihydroethidium (DHE), assembly of NADPH oxidase subunits by the colocalization between the cytosolic p47phox subunit and the membrane gp91phox subunit using fluorescent confocal microscopy, phosphorylation of cytosolic phospholipase A2 (cPLA2), and expressions of pro-inflammatory factors including interleukin-1β (IL-1β) and inducible nitric-oxide synthase (iNOS) using Western blot Analysis. Our data showed that laser light at 632.8 nm suppressed Aβ-induced superoxide production, colocalization between NADPH oxidase gp91phox and p47phox subunits, phosphorylation of cPLA2, and the expressions of IL-1β and iNOS in primary astrocytes. We demonstrated for the first time that 632.8 nm laser was capable of suppressing cellular pathways of oxidative stress and inflammatory responses critical in the pathogenesis in AD. This study should prove to provide the groundwork for further investigations for the potential use of laser therapy as a treatment for AD