α-tocopherol inhibits oxidative stress induced by cholestanetriol and 25-hydroxycholesterol in porcine ovarian granulosa cells

The cytotoxicity of oxysterols including 7-ketocholesterol, -epoxide, cholestanetriol and 25-hydroxycholesterol and the possible protecting effect of -tocopherol on cholestanetriol and 25-hydroxycholesterol-induced cytotoxicity were investigated in primary cultures of porcine ovarian granulosa cells. Cell viability as determined by % trypan blue staining and mitochondrial function as determined using 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) reduction were decreased significantly after 24 h exposure to 2.5 M -epoxide, cholestanetriol and 25-hydroxycholesterol. 7-ketocholesterol (2.5 M) did not affect cell viability or mitochondrial function under the same culture conditions. The specific activities of catalase and superoxide dismutase, two antioxidant defense enzymes were increased significantly (p < 0.01) following 24 h exposure to 2.5 M concentrations of cholestanetriol while only superoxide dismutase was increased in 25-hydroxycholesterol-treated cells (p < 0.001). Specific activity of glutathione peroxidase was unchanged relative to control cells. Levels of thiobarbituric acid reactive substances remained unchanged after exposure to 7-ketocholesterol, -epoxide, cholestanetriol, 25-hydroxycholesterol and cholesterol. Administration of 1 M -tocopherol to the culture medium significantly improved cell viability and restored both superoxide dismutase and catalase activities to control levels in cholestanetriol -treated cells and only superoxide dismutase in 25-hydroxycholesterol-treated cells. These studies suggest that the cytotoxic nature of physiologically relevant concentrations of cholestanetriol and 25-hydroxycholesterol in granulosa cells is in part due to oxidative stress, but it may be reduced in the presence of a-tocopherol.     

Methyl B12 protects PC12 cells against cytotoxicity induced by Aβ25−35

Alzheimer's disease (AD) is the most common aging-associated dementia. The population of AD patients is increasing as the world age grows. Currently, there is no cure for AD. Given that methyl vitamin B12 (methylcobalamin) deficiency is related to AD and Aβ-induced oxidative damage and that methylcobalamin can scavenge reactive oxygen species (ROS) by direct or indirect ways, we studied the effect of methylcobalamin on the cytotoxicity of Aβ. PC12 cells were chronically exposed (24 hours) to Aβ_25-35 (25 μM) to establish an AD cell model. The cells were pretreated with or without methylcobalamin (1-100 μM) to investigate the role of methylcobalamin. Cell viability and apoptosis were tested, followed by testing of mitochondrial damage, oxidative stress, and mitochondrial calcium concentration. We observed that methylcobalamin improved the cell viability by decreasing the ratio of apoptosis cells in this AD cell model. Further experiments suggested that methylcobalamin functioned as an antioxidant to scavenge ROS, reducing the endoplasmic reticulum-mitochondria calcium flux through IP3R, preventing mitochondria dysfunction, ultimately protecting cells against apoptosis and cell death. Taken together, our results presented, for the first time, that methyl vitamin B12 can protect cells from Aβ-induced cytotoxicity and the mechanism was mainly relevant to the antioxidative function of methyl B12.     

IL-5 blocks apoptosis and tau hyperphosphorylation induced by Aβ<Subscript>25–35</Subscript> peptide in PC12 cells

The primary features of Alzheimer’s disease (AD) are extracellular amyloid plaques consisting mainly of deposits of amyloid β (Aβ) peptides and intracellular neurofibrillary tangles (NFTs). Sets of evidence suggest that interleukin-5 (IL-5) is involved in the pathogenesis of AD. Herein, we investigated the protective role of IL-5 in PC12 cells, to provide new insights into understanding this disease. Western blot was employed to assess the protein levels of Bax and phospho-tau as well as phospho-JAK2; MTT assay was performed to decipher cell viability. Treatment of IL-5 decreased Aβ_25–35-induced tau phosphorylation and apoptosis, effects blunted by JAK2 inhibition. IL-5 prevents Aβ_25–35-evoked tau protein hyperphosphorylation and apoptosis through JAK2 signaling.     

Genistein attenuates D-galactose-induced oxidative damage through decreased reactive oxygen species and NF-κB binding activity in neuronal PC12 cells

AimsWe investigated the mechanism of D-galactose (DG)-induced oxidative damage and the neuroprotective action of genistein in PC12 cells.Main methodsPC12 cells were treated with 40 mM DG dissolved in medium containing 85% RPMI1640, 10% HBS and 5% FBS with or without genistein. We measured the protein expression of β-amyloid (Aβ), advanced glycation end products (AGEs), IκB-α and manganese-superoxide dismutase (MnSOD) by western blotting, intracellular reactive oxygen species (ROS) by 2, 7-dichlorofluorescin-diacetate, and the binding activity of nuclear factor kappa B (NF-κB) by electrophortic mobility shift assay.Key findingsDG (40 mM) completely retarded cell growth after incubation for 72 h, and this effect was not due to osmotic changes, as 40 mM mannitol had no effect. Mechanistically, we found that DG increased intracellular ROS starting at 4 h and increased Aβ and AGEs at 24 h. DG treatment for 24 h also increased the binding activity of NF-κB but strongly decreased the expression of IκB-α protein. Furthermore, DG treatment for 48 h increased MnSOD protein expression. All these effects of DG were effectively inhibited by genistein (0.5–10 μM).SignificanceThe present study indicates that the protection of genistein against DG-induced oxidative stress in PC12 cells, and the effect is likely mediated by decreased intracellular ROS and binding activity of NF-κB.     

Genistein Inhibits Aβ25–35 –Induced Neurotoxicity in PC12 Cells via PKC Signaling Pathway

Protein kinase C (PKC) signaling pathway is recognized as an important molecular mechanism of Alzheimer??s disease (AD) in the regulation of neuronal plasticity and survival. Genistein, the most active molecule of soy isoflavones, exerts neuroprotective roles in AD. However, the detailed mechanism has not been fully understood yet. The present study aimed to investigate whether the neuroprotective effects of genistein against amyloid ?? (A??)-induced toxicity in cultured rat pheochromocytoma (PC12) cells is involved in PKC signaling pathway. PC12 cells were pretreated with genistein for 2?h following incubation with A??_25?C35 for additional 24?h. Cell viability was assessed by MTT. Hoechst33342/PI staining was applied to determine the apoptotic cells. PKC activity, intracellular calcium level and caspase-3 activity were analyzed by assay kits. The results showed that pretreatment with genistein significantly increased cell viability and PKC activity, decreased the levels of intracellular calcium, attenuated Hoechst/PI staining and blocked caspase-3 activity in A??_25?C35-treated PC12 cells. Pretreatment of Myr, a general PKC inhibitor, significantly attenuated the neuroprotective effect of genistein against A??_25?C35-treated PC12 cells. The present study indicates that PKC signaling pathway is involved in the neuroprotective action of genistein against A??_25?C35-induced toxicity in PC12 cells.     

Vinpocetine attenuates the metabolic dysfunction induced by amyloid β-peptides in PC12 cells

The cytoprotective effect of vinpocetine [14-ethoxycarbonyl-(3α,16α-ethyl)-14,15-eburnamine] was investigated on PC12 cells treated with the amyloid β-peptides (Aβ) for 24 hours. Vinpocetine was shown to protect cells from the inhibition in redox status induced by exposure to Aβ_25–35 and Aβ_1–40, the maximal protection being achieved at a vinpocetine concentration of 40 μM. At this concentration, vinpocetine blocked the inhibition of the mitochondrial respiratory chain complexes II–III and IV and completely abolished the depletion of pyruvate levels induced by toxic concentrations of Aβ peptides. Furthermore, the accumulation of ROS in cells exposed to Aβ_25–35 and Aβ_1–40 evaluated using the fluorescent probe 2′,7′-dichlorofluorescin (DCF), was reduced in the presence of 40 μM vinpocetine. Taken together, the data presented herein demonstrate that vinpocetine protects cells from Aβ toxicity, preventing the generation of oxidative stress due to the excessive accumulation of ROS. This study suggests that vinpocetine can exert neuroprotective properties which might be of importance and contribute to its clinical efficacy in the treatment of Alzheimer's disease or other neurodegenerative disorders in which oxidative stress is involved.     

Knockdown of FOXO6 inhibits high glucose–induced oxidative stress and apoptosis in retinal pigment epithelial cells

Oxidative stress and apoptosis in retinal pigment epithelium cells are involved in the pathogenesis of diabetic retinopathy (DR). Forkhead box class O 6 (FOXO6) is a member of the FOXO family that can regulate diabetes-induced oxidative stress. However, the role of FOXO6 in DR has not been clarified. The aim of the present study was to investigate the effects of FOXO6 on high glucose (HG)-induced oxidative stress and apoptosis in ARPE-19 cells. The results showed that FOXO6 was overexpressed in clinical vitreous samples from DR patients and in HG-induced ARPE-19 cells. Knockdown of FOXO6 by small interfeing RNA targeting FOXO6 (si-FOXO6) mitigated the HG-induced the production of reactive oxygen species and malondialdehyde, as well as the inhibition of superoxide dismutase activity. Knockdown of FOXO6 reduced the rate of cell apoptosis in HG-induced ARPE-19 cells. The increase in bax expression and decrease in bcl-2 expression caused by HG stimulation were reversed by si-FOXO6 transfection. Furthermore, knockdown of FOXO6 enhanced the activation of Akt/Nrf2 pathway in HG-stimulated ARPE-19 cells. Taken together, suppression of FOXO6 protects ARPE-19 cells from HG-induced oxidative stress and apoptosis, which is in part mediated by the activation of Akt/Nrf2 pathway.     

Thioredoxin-1 inhibits amyloid-β25–35-induced activation of NLRP1/caspase-1/GSDMD pyroptotic pathway in PC12 cells

Molecular Biology Reports - Alzheimer’s disease (AD), the most common neurodegenerative disease, is charactered by these accepted pathological features, such as β-amyloid (Aβ)...     

5′-Nucleotidase in PC12 cells as revealed by immunocytochemistry

5-Nucleotidase hydrolyzes 5-mononucleotides to their nucleosides but is also thought to have a function in neuronal differentiation and synapse formation. The distribution of the enzyme, a glycosyl-phosphatidylinositol-anchored sialoglycoprotein, was investigated in PC12 cells using immunofluorescence microscopy. 5-Nucleotidase was located both in intracellular compartments and at the cell surface. There was no principal difference in the cellular distribution between undifferentiated cells and after neuritogenic differentiation by nerve growth factor. Intracellularly, 5-nucleotidase often revealed a sickle-shaped perinuclear distribution and a dotted pattern throughout the cytoplasm, including that of neurites and growth cones. The intracellular distribution was clearly different from that of the synaptic vesicle protein synaptophysin. However, the dotted fluorescence resembled that obtained after uptake of the endosomal marker acridine orange. 5-Nucleotidase was present on the entire cell surface including all neurites formed after differentiation. There was no increase in 5-nucleotidase fluorescence at synapse-like contacts between the tips of neurites and other PC12 cells. Surfacelocated 5-nucleotidase could no longer be detected after the application of glycosyl-phosphatidylinositol-specific phospholipase C to cultured cells. This treatment did not affect PC12 cell differentiation. Our results thus reveal 5-nucleotidase both at the surface and within organelles and suggest that PC12 cells may be used as a model system for the study of the physiological function of 5-nucleotidase in neural cells.     

Cholesterol inhibits the insertion of the Alzheimer’s peptide Aβ(25–35) in lipid bilayers

The physiological relationship between brain cholesterol content and the action of amyloid β (Aβ) peptide in Alzheimer’s disease (AD) is a highly controversially discussed topic. Evidences for modulations of the Aβ/membrane interaction induced by plasma membrane cholesterol have already been observed. We have recently reported that Aβ(25–35) is capable of inserting in lipid membranes and perturbing their structure. Applying neutron diffraction and selective deuteration, we now demonstrate that cholesterol alters, at the molecular level, the capability of Aβ(25–35) to penetrate into the lipid bilayers; in particular, a molar weight content of 20% of cholesterol hinders the intercalation of monomeric Aβ(25–35) completely. At very low cholesterol content (about 1% molar weight) the location of the C-terminal part of Aβ(25–35) has been unequivocally established in the hydrocarbon region of the membrane, in agreement with our previous results on pure phospholipids membrane. These results link a structural property to a physiological and functional behavior and point to a therapeutical approach to prevent the AD by modulation of membrane properties.     

Sarpogrelate inhibits the expression of ICAM-1 and monocyte–endothelial adhesion induced by high glucose in human endothelial cells

Hyperglycemia is the major cause of diabetic angiopathy. Sarpogrelate hydrochloride is an antiplatelet drug, and expected to be useful in the treatment of chronic arterial occlusive diseases. The aim of our study was to evaluate the possible effects of sarpogrelate hydrochloride on adhesion molecule expression and its underlying mechanism in the prevention and treatment of cardiovascular disorders. Intercellular adhesion molecule-1 (ICAM-1) expression and superoxide dismutase (SOD) activity were determined after endothelial cells were exposed to high glucose in the absence and presence of sarpogrelate hydrochloride. Coincubation of endothelial cells with high glucose for 24 h resulted in a significant increase of monocyte–endothelial cell adhesion and the expression of ICAM-1 (P < 0.01). These effects were abolished by sarpogrelate hydrochloride and sarpogrelate hydrochloride significantly increased SOD activities (40 ± 8 vs. 47 ± 7, n = 8, P < 0.01). The low dose sarpogrelate group (0.1 μM) had significantly higher monocyte–endothelial cell adhesion and the expression of ICAM-1 than medium dose sarpogrelate group (1.0 μM) and high dose sarpogrelate group (10.0 μM) (P < 0.05 for comparison among three groups and P < 0.01 for difference between low and high dose sarpogrelate groups). These findings suggested that sarpogrelate hydrochloride was able to protect vascular endothelium from dysfunction induced by high glucose.     

The lipocalin α1-microglobulin protects erythroid K562 cells against oxidative damage induced by heme and reactive oxygen species

α₁-Microglobulin is a 26 kDa plasma and tissue glycoprotein that belongs to the lipocalin protein superfamily. Recent reports show that it is a reductase and radical scavenger and that it binds heme and has heme-degrading properties. This study has investigated the protective effects of α₁-microglobulin against oxidation by heme and reactive oxygen species in the human erythroid cell line, K562. The results show that α₁-microglobulin prevents intracellular oxidation and up-regulation of heme oxygenase-1 induced by heme, hydrogen peroxide and Fenton reaction-generated hydroxyl radicals in the culture medium. It also reduces the cytosol of non-oxidized cells. Endogeneous expression of α₁-microglobulin was up-regulated by these oxidants and silencing of the α₁-microglobulin expression increased the cytosol oxidation. α₁-microglobulin also inhibited cell death caused by heme and cleared cells from bound heme. Binding of heme to α₁-microglobulin increased the radical reductase activity of the protein as compared to the apo-protein. Finally, α₁-microglobulin was localized mainly at the cell surface both when administered exogeneously and in non-treated cells. The results suggest that α₁-microglobulin is involved in the defence against oxidative cellular injury caused by haemoglobin and heme and that the protein may employ both heme-scavenging and one-electron reduction of radicals to achieve this.     

Inhibitory effects of short-term administration of dl-α-lipoic acid on oxidative vulnerability induced by Aβ amyloid fibrils (25–35) in mice

Aβ amyloid peptide is believed to induce oxidative stress leading to inflammation, which is postulated to play a significant role in the toxicity of Alzheimer’s disease (AD). This study was designed to investigate the inhibitory effects of dl-α lipoic acid (LA), a potential free radical scavenger, on oxidative vulnerability induced by intraperitoneal injection of Aβ_25–35 amyloid fibrils in mice. Mice were divided into three groups: control, Aβ amyloid toxicity induced (AT), and LA treated (ATL). Blood Plasma was separated, liver, spleen and brain were dissected and analysis of oxidants, antioxidants, ATPases, glial fibrillary acidic protein (GFAP) and nuclear factor kappa-B (NFκB) were carried out. Results show biochemical parameters such as reactive oxygen species (ROS) and lipid peroxidation (LPO) were significantly lowered (P < 0.05) and levels of antioxidants and ATPase (P < 0.05) were significantly increased (P < 0.05) in hepatocytes, splenocytes and astrocytes of the ATL group. Moreover, our histological results revealed a decreased GFAP immunoreactivity in the neocortical region and NFκB immunoreactivity in neocortex, liver and spleen. This study reiterates LA as a potent free radical scavenger to combat oxidative vulnerability in the treatment for Aβ amyloid toxicity.     

Analysis of DNA damage induced by aflatoxin B1 in Dunkin–Hartley guinea pigs

Aflatoxin B1 (AFB1) is among the most potent naturally occurring carcinogens and classified as a group I carcinogen. Since the ingestion of aflatoxin-contaminated food is associated with several liver diseases, the aim of the present study was to evaluate the effect of 2, 20, and 200 ppb of AFB1 on DNA damage in peripheral blood lymphocytes and liver cells in Dunkin-Hartley guinea pigs. The animals were divided into four groups according to the given diet. After the treatment the lymphocytes and liver cells were isolated and DNA damage determined by Comet assay. The levels of DNA damage in lymphocytes were higher animals treated with 200 ppb of AFB1-enriched diet (P = 0.02). In the liver cells there were a relationship between the levels of DNA damage and the consumption of AFB1 in all studied groups. These results suggest that Comet assay performed on lymphocytes is a valuable genotoxic marker for high levels of exposure to AFB1 in guinea pig. Additionally our results indicate that the exposure to this toxin increases significantly and increases the level of DNA damage in liver cells, which is a key step on liver cancer development. We also suggest that the Comet assay is an useful tool for monitoring the genotoxicity of AFB1 in liver.     

FISH-based analysis of 10- and 25-kV soft X-ray–induced DNA damage in 184A1 human mammary epithelial cells

Over the past years, several in vitro studies have been performed on DNA damage induced by soft X-rays, especially in the energy range below 50 keV. Radiation effects originating from such low-energy photons are relevant in the context of medical diagnostics, for example, mammography, or of accidental exposure to scattered radiation. The present study was initiated to investigate the X-ray energy-dependent induction of stable and unstable chromosomal aberrations in the human mammary epithelial cell line 184A1. Three colour fluorescence in situ hybridisation was applied to identify chromosomal damage in chromosomes 1, 8 and 17, induced by 10-kV or 25-kV soft X-rays as well as by 200-kV X-rays as a reference quality. The overall results confirm the X-ray energy dependencies published for human lymphocytes showing increasing chromosomal aberration frequencies and higher aberration complexity with decreasing X-ray energy and increasing dose. Comparing the obtained dose dependencies, ratios of 0.84 ± 0.09 and 1.22 ± 0.18 were revealed for stable translocations induced by 25- and 10-kV X-rays, respectively, using 200-kV X-rays as reference. Moreover, the analysis of the minimum number of breaks required to form the visible chromosomal damage resulted in similar ratios of 0.93 ± 0.07 for 25-kV X-rays and 1.25 ± 0.10 for 10-kV X-rays relative to 200-kV X-rays. In addition, non-DNA-proportional contributions of chromosomes 8 and 17 to the whole DNA damage and deviations from the expected 1:1 ratio of translocations and dicentrics were observed for cell line 184A1.