Antioxidative role of selenoprotein W in oxidant-induced chicken splenic lymphocyte death

To verify the antioxidative role of SelW in oxidant-induced chicken splenic lymphocyte, in this report, the influence of selenite supplementation and SelW gene silence on H₂O₂-mediated cell viability and cell apoptosis in cultured splenic lymphocyte derived from spleen of chicken were examined. The cultured cells were treated with sodium selenite and H₂O₂, or knocked down SelW with small interfering RNAs (siRNAs). The lymphocytes were examined for cell viability, cell apoptosis and mRNA expression levels of SelW and apoptosis-related genes (Bcl-2, Bax, Bak-1, caspase-3 and p53). The results show that the mRNA expression of SelW were effectively increased after treatment with sodium selenite, and H₂O₂-induced cell apoptosis was significantly decreased and cell viability was significantly increased. 20 μM H₂O₂ was found to induce cell apoptosis and decrease cell viability, which was alleviated obviously when cells were pretreated with sodium selenite before exposure to 20 μM H₂O₂. Meanwhile, H₂O₂ induced a significantly up-regulation of the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulation of Bcl-2 (P < 0.05). When lymphocytes were pretreated with Se before treated with H₂O₂, the Bax/Bcl-2 ratio and mRNA expression of those genes were significantly decreased, and Bcl-2 was increased (P < 0.05). SelW siRNA-transfected cells were more sensitive to the oxidative stress induced by treatment of H₂O₂ than control cells. Silencing of the lymphocyte SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H₂O₂. Silencing of SelW significantly up-regulated the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulated Bcl-2 (P < 0.05). The present study demonstrates that SelW plays an important role in protection of splenic lymphocyte of birds from oxidative stress.     

Hydrogen peroxide leads to cell damage and apoptosis in the gill of the freshwater crab Sinopotamon henanense (Crustacea,Decapoda)

Hydrogen peroxide (H₂O₂), a major reactive oxygen species, has been shown to be a critical mediator of apoptosis induced by several toxic metals such as cadmium. In this study, we used the freshwater crab Sinopotamon henanense to study whether H₂O₂ can cause apoptosis in gill cells. The crabs were incubated in H₂O₂ and the DNA fragmentation, ultrastructural changes and caspase-3/8/9 activities were measured. The results showed that in freshwater crab, H₂O₂ was found to induce apoptosis, as confirmed by DNA fragmentation analysis and morphological observation of transmission electron microscopy. This apoptosis occurs in a concentration-dependent pattern. During the apoptotic process, caspase-3, caspase-8 and caspase-9 were activated by H₂O₂. In addition, multiple physiological and pathological changes of gill cells were discovered after 24 h exposure to 5 mM H₂O₂, including aggregation and condensation of nuclear chromatin, appearance of extremely irregular nuclei with finger-like buds, disappearance of the organelles around the nuclei, swollen and dissolved cristae of mitochondria. We propose that H₂O₂-induced stress leads to mitochondria lesions oxidative injury and triggers apoptotic response through the caspase pathway in freshwater crab.     

Hypothermia protects H9c2 cardiomyocytes from H2O2 induced apoptosis

The purpose of our study was to investigate underlying basic mechanisms of hypothermia-induced cardioprotection during oxidative stress in a cardiomyocyte cell culture model. For hypothermic treatment we cooled H9c2 cardiomyocytes to 20 °C, maintained 20 min at 20 °C during which short-term oxidative damage was inflicted with 2 mM H₂O_2, followed by rewarming to 37 °C. Later on, we analyzed lactate dehydrogenase (LDH), caspase-3 cleavage, reactive oxygen species (ROS), mitochondrial activity, intracellular ATP production, cytoprotective signal molecules as well as DNA damage. Hypothermia decreased H₂O₂ damage in cardiomyocytes as demonstrated in a lower LDH release, less caspase-3 cleavage and less M30 CytoDeath staining. After rewarming H₂O₂ damaged cells demonstrated a significantly higher reduction rate of intracellular ROS compared to normothermic H₂O₂ damaged cardiomyocytes_. This was in line with a significantly greater mitochondrial dehydrogenase activity and higher intracellular ATP content in cooled and rewarmed cells. Moreover, hypothermia preserved cell viability by up-regulation of the anti-apoptotic protein Bcl-2 and a reduction of p53 phosphorylation. DNA damage, proven by PARP-1 cleavage and H2AX phosphorylation, was significantly reduced by hypothermia. In conclusion, we could demonstrate that hypothermia protects cardiomyocytes during oxidative stress by preventing apoptosis via inhibiting mitochondrial dysfunction and DNA damage.     

Nuclear morphology and c‐Jun N‐terminal kinase 1 expression differentiate serum‐starved oxidative stress signalling from hydrogen peroxide‐induced apoptosis in retinal neuronal cell line

Oxidative stress induced by serum starvation and H₂O₂ exposure, both triggers apoptosis in retinal neuronal cell line RGC‐5 (retinal ganglion cell‐5). We have examined whether, despite excess generation of ROS (reactive oxygen species) and apoptosis induction, there is any dissimilarity in nuclear morphology and apoptotic signalling pathway in RGC‐5 under these conditions. Sub‐confluent cells were treated either with H₂O₂ or maintained in SFM (serum‐free medium). ROS level was detected along with nuclear morphology and ultrastructural analysis. Generation of excess intracellular ROS, nuclear localization of Bax and caspase 3 activation along with decrease of cellular viability, confirmed apoptosis induction in RGC‐5 by 72 h serum starvation and 500 M H₂O₂ exposure for 1 h. Nuclear swelling as supported by nuclear cytoplasmic ratio and conspicuous black spots with nuclear remodelling were observed only upon SFM, but not with H₂O₂ treatment. Serum starvation did not alter JNK1 (c‐Jun N‐terminal kinase 1) expression, although nuclear translocation and higher level of pJNK (phospho‐JNK) was evident. Conversely, H₂O₂ exposure blocked the expression and activation of JNK1 to phospho‐JNK as a negligible level of pJNK was present in the cytoplasm. Despite similar ROS generation in both the conditions, difference in nuclear morphology and JNK1 expression leads to the hypothesis that RGC‐5 cells may follow different signalling pathways when challenged with serum starvation and H₂O₂.     

Protective effect of sesaminol from Sesamum indicum Linn. against oxidative damage in PC12 cells

Sesaminol is one component of sesame oil and has been widely used as the stabilizer to extend the storage period of food oil in China. In this study, we tried to investigate the antioxidant activity of sesaminol on rat pheochromocytoma (PC12) cells oxidative damaged by H₂O₂. Cell viability, LDH level and apoptosis of the PC12 cells were assayed after treatment with sesaminol for 3 h and exposure to H₂O₂. Furthermore, superoxide (SOD), catalase (CAT), glutathione peroxidase (GSH‐Px) and intracellular ROS were assayed after exposure of the PC12 cells to H₂O₂. The results showed that pre‐treatment with sesaminol prior to H₂O₂ exposure significantly elevated cell survival rate and SOD, CAT and GSH‐Px activity. Meanwhile, sesaminol declined the secreted LDH level, apoptosis rate and ROS level of H₂O₂ exposed cells. Thus, sesaminol may protect PC12 against oxidative injury. Copyright © 2012 John Wiley & Sons, Ltd.     

Mesenchymal stem cells attenuate hydrogen peroxide-induced oxidative stress and enhance neuroprotective effects in retinal ganglion cells

The apoptosis of retinal ganglion cells leads to visual impairment and blindness in ocular neurodegenerative diseases, especially in diabetic retinopathy (DR). Mounting evidence suggests that oxidative stress contributes to the pathogenesis of DR. In the present study, we investigated whether bone mesenchymal stem cells (BMSCs) have protective ability to relieve hydrogen peroxide (H₂O₂)-induced injury on retinal ganglion cells in vitro. An immortalized retinal ganglion cells, RGC-5 cells, were exposed to an indicated concentration of H₂O₂ for 24 h. Cell viability was analyzed by CCK-8 assay to find out a certain concentration to build H₂O₂ oxidative damage model. Morphological changes in RGC-5 cells were observed under optical microscope, and cell apoptosis was detected with Hoechst fluorescence staining. Then, BMSCs were co-cultured with RGC-5 cells in a transwell culture system for 24 h and 48 h. Flow cytometry was performed to qualify the apoptosis rate of RGC-5 cells. Conditioned medium was collected for evaluation the inflammatory cytokines by ELISA. The content of intracellular malondialdehyde (MDA) and superoxide dismutase (SOD) was assayed by thiobarbituric acid and xanthine oxidase method, respectively. qRT-PCR and ELISA were conducted for analysis of the expression changes in brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), respectively. After H₂O₂ exposure, the morphological varieties were observed as cytoplasm shrinking and paramorphia together with nuclear gathering. Meanwhile, the apoptotic cells had hyperfluorescence with Hoechst 33258 staining. Co-culture with BMSCs significantly inhibited retinal cell death. It was found that BMSCs reduced H₂O₂-induced inflammatory factors IL-1β and TNF-α, down-regulated intracellular oxidant factor MDA, up-regulated intracellular antioxidant factor SOD, and increased neurotrophins BDNF and CNTF expression. BMSCs may enhance protective effect of RGC-5 cells in H₂O₂-induced damage through improving antioxidant capacity, inhibiting pro-inflammatory cytokine secretion, and promoting neurotrophin expression.     

Saikosaponin-D Reduces H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced PC12 Cell Apoptosis by Removing ROS and Blocking MAPK-Dependent Oxidative Damage

Neuronal oxidative stress (OS) injury has been proven to be associated with many neurodegenerative diseases, and thus, antioxidation treatment is an effective method for treating these diseases. Saikosaponin-D (SSD) is a sapogenin extracted from Bupleurum falcatum and has been shown to have many pharmacological activities. The main purpose of this study was to investigate whether and how SSD protects PC12 cells from H₂O₂-induced apoptosis. The non-toxic level of SSD significantly mitigated the H₂O₂-induced decrease in cell viability, reduced the apoptosis rate, improved the nuclear morphology, and reduced caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavage. Additionally, exogenous H₂O₂-induced apoptosis by damaging the intracellular antioxidation system. SSD significantly slowed the H₂O₂-induced release of malonic dialdehyde (MDA) and lactate dehydrogenase and increased the activity of superoxide dismutase (SOD) and the total antioxidant capacity, thereby reducing apoptosis. More importantly, SSD effectively blocked H₂O₂-induced phosphorylation of extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38MAPK), and specific inhibitors of ERK, JNK, and p38-reduced OS injury and apoptosis, suggesting that SSD reduces OS injury and apoptosis via MAPK signalling pathways. Finally, we confirmed that SSD significantly reduced H₂O₂-induced reactive oxygen species (ROS) accumulation, and the ROS inhibitor blocked the apoptosis caused by MAPK activation and cellular oxidative damage. In short, our study confirmed that SSD reduces H₂O₂-induced PC12 cell apoptosis by removing ROS and blocking MAPK-dependent oxidative damage.     

Protective effects of sulfated chitooligosaccharides against hydrogen peroxide-induced damage in MIN6 cells

Sulfated chitooligosaccharides (COS-S) with different degrees of substitution (DS) were obtained by the chlorosulfuric acid/pyridine method. Protective effects of COS-S against hydrogen peroxide (H₂O₂)-induced damage were investigated in pancreatic β-cells MIN6 cell line. The cell viability, morphology, insulin contents, malondialdehyde (MDA) inhibition, lactate dehydrogenase (LDH) release and the levels of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidise (GPx) were evaluated under oxidative damage by 150 μM H₂O₂ for 6 h. COS-S did not show any harmful or inhibitory effect on cell growth at concentrations ranging from 0.1 to 0.5 mg/ml. While COS-S could enhance the cell viability, decrease the production of ROS, and reduce the MDA level as well as LDH level in oxidative damaged β-cells by being an antioxidant. The underlining mechanisms of protective effects of COS-S are partly due to the enhancement of antioxidant enzyme activity and inhibition of intracellular ROS production, along with suppressing MIN6 cell apoptosis subsequent to the amelioration of ROS. Moreover, increased DS might contribute to the defense mechanisms against H₂O₂-induced oxidative damage in MIN6 cells. These results indicated that the antioxidant properties of COS-S hold great potential for the oxidative diseases treatment, and the sulfate content of polysaccharides made great role in regulating antioxidant activities.     

Neuroprotective Effects of Theaflavins Against Oxidative Stress-Induced Apoptosis in PC12 Cells

Oxidative stress can induce neuronal apoptosis via the production of superoxide and hydroxyl radicals. This process is as a major pathogenic mechanism in neurodegenerative disorders. In this study, we aimed to clarify whether theaflavins protect PC12 cells from oxidative stress damage induced by H₂O₂. A cell model of PC12 cells undergoing oxidative stress was created by exposing cells to 200 μM H₂O₂ in the presence or absence of varying concentrations of theaflavins (5, 10, and 20 μM). Cell viability was monitored using the MTT assay and Hoechst 33258 staining, showing that 10 μM theaflavins enhanced cell survival following 200 μM H₂O₂ induced toxicity and increased cell viability by approximately 40?%. Additionally, we measured levels of intracellular reactive oxygen species (ROS) and antioxidant enzyme activity. This suggested that the neuroprotective effect of theaflavins against oxidative stress in PC12 cells is derived from suppression of oxidant enzyme activity. Furthermore, Western blot analyses indicated that theaflavins downregulated the ratio of pro-apoptosis/anti-apoptosis proteins Bax/Bcl-2. Theaflavins also downregulated the expression of caspase-3 compared with a H₂O₂-treated group that had not been treated with theaflavins. Interestingly, this is the first study to report that the four main components of theaflavins found in black tea can protect neural cells (PC12) from apoptosis induced by H₂O₂. These findings provide the foundations for a new field of using theaflavins or its source, black tea, in the treatment of neurodegenerative diseases caused by oxidative stress.     

EphA2 overexpression reduces H2O2-induced damage of lens epithelial cells

Age-related cataract (ARC) is a progressive lens opacification that occurs from middle to old age. Eph-receptor tyrosinekinase-type A2 (EphA2) has been reported to be associated with ARC. This work aims to investigate the molecular mechanism of EphA2 in ARC. We treated human lens epithelial cells (SRA01/04) with different concentration of H₂O₂ to induce lens epithelial cell damage. Then, we found that H₂O₂ treatment significantly suppressed cell viability and enhanced the expression of EphA2 in the SRA01/04 cells. H₂O₂ treatment repressed cell viability and enhanced the levels of reactive oxygen species (ROS) in SRA01/04 cells, which was partly abolished by EphA2 up-regulation. Moreover, EphA2 overexpression reduced H₂O₂-induced apoptosis of SRA01/04 cells. EphA2 up-regulation caused an up-regulation of Bcl-2, and repressed the expression of Bax and Cleaved-caspase-3 in the SRA01/04 cells following H₂O₂ treatment. In conclusion, our data confirm that EphA2 overexpression enhances cell viability and inhibits apoptosis in the H₂O₂-treated SRA01/04 cells, thereby reducing H₂O₂-induced damage of lens epithelial cells. Thus, this work provides new insights into the mechanism of EphA2 in ARC.     

Hydrogen peroxide induces oxidative stress and the mitochondrial pathway of apoptosis in RAT intestinal epithelial cells (IEC-6)

In order to investigate the mechanism of apoptosis in rat intestinal epithelial cells (IEC-6) induced by hydrogen peroxide (H₂O₂), IEC-6 cells were subjected to 20 μmol/L H₂O₂ and cell proliferation activity was determined using 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide. Cell morphology was observed by microscopy and cell apoptosis was detected by acridine orange and ethidium bromide staining and the portion of apoptotic cells was measured by flow cytometry. Genes and proteins related to cell apoptosis were detected by RT-PCR and Western blotting, and the mitochondrial membrane potential was evaluated by fluorescence probes. Results: Significant morphology damage was caused by exposure to H₂O₂, and results showed that ROS generation significantly increased (P < 0.01). The activity of superoxide dismutase decreased significantly (P < 0.05), malondialdehyde content increased (P < 0.05), and expression of both catalase and glutathione peroxidase decreased significantly (P < 0.05) in the H₂O₂ treatment group. Mitochondrion membrane potential was reduced, cytochrome released into the cytoplasm and caspase-9 and caspase-3 were significantly increased (P < 0.01) after treatment with H₂O₂. Moreover, the ratio of Bax/Bcl-2 and apoptosis were significantly increased (P < 0.01) in the H₂O₂ group. In conclusion, the present study indicated that the mitochondrial pathway plays a vital role in H₂O₂ induced IEC-6 cell apoptosis.     

Endogenous Expression of ODN-Related Peptides in Astrocytes Contributes to Cell Protection Against Oxidative Stress: Astrocyte-Neuron Crosstalk Relevance for Neuronal Survival

Astroglial cells are important actors in the defense of brain against oxidative stress injuries. Glial cells synthesize and release the octadecaneuropeptide ODN, a diazepam-binding inhibitor (DBI)-related peptide, which acts through its metabotropic receptor to protect neurons and astrocytes from oxidative stress-induced apoptosis. The purpose of the present study is to examine the contribution of the endogenous ODN in the protection of astrocytes and neurons from moderate oxidative stress. The administration of H₂O₂ (50 μM, 6 h) induced a moderate oxidative stress in cultured astrocytes, i.e., an increase in reactive oxygen species, malondialdehyde, and carbonyl group levels, but it had no effect on astrocyte death. Mass spectrometry and QPCR analysis revealed that 50 μM H₂O₂ increased ODN release and DBI mRNA levels. The inhibition of ODN release or pharmacological blockage of the effects of ODN revealed that in these conditions, 50 μM H₂O₂ induced the death of astrocytes. The transfection of astrocytes with DBI siRNA increased the vulnerability of cells to moderate stress. Finally, the addition of 1 nM ODN to culture media reversed cell death observed in DBI-deficient astrocytes. The treatment of neurons with media from 50 μM H₂O₂-stressed astrocytes significantly reduced the neuronal death induced by H₂O₂; this effect is greatly attenuated by the administration of an ODN metabotropic receptor antagonist. Overall, these results indicate that astrocytes produce authentic ODN, notably in a moderate oxidative stress situation, and this glio- and neuro-protective agent may form part of the brain defense mechanisms against oxidative stress injury.     

Hydrogen peroxide is critical for UV-induced apoptosis inhibition

Abstract

Apoptosis is an important cell death system that deletes damaged and mutated cells, preventing the induction of cancer. We previously have reported that UV irradiation inhibited the apoptosis induced by serum starvation and cell detachment. This phenomenon is suitable for clarifying the relationship between cancer and the dysregulation of apoptosis by UV irradiation. Here, we have studied the factors responsible for this inhibition of apoptosis, focusing on reactive oxygen species (ROS) and DNA damage. Treatment with xanthine oxidase in the presence of hypoxanthine, which is known to produce superoxide anion (O₂^??) and hydrogen peroxide (H₂O₂), inhibited the induction of apoptosis. The xanthine oxidase-induced anti-apoptotic effect was suppressed in the presence of an H₂O₂-eliminating enzyme, catalase, but not in the presence of an O₂^??-eliminating enzyme, superoxide dismutase. Treatment with H₂O₂ itself significantly inhibited the induction of apoptosis. Furthermore, the effect of the inhibition of cell death by UVB irradiation and by H₂O₂ treatment decreased in H₂O₂-resistant cells. Although both UVB and H₂O₂ are known to induce DNA damage, other DNA damaging agents, like γ-irradiation and treatment with cisplatin and bleomycin, showed no inhibition of apoptosis. These findings suggested that H₂O₂ was essential to the inhibition of apoptosis, in which DNA damage had no role.     

Human polynucleotide phosphorylase reduces oxidative RNA damage and protects HeLa cell against oxidative stress

We examined HeLa cell viability and RNA oxidative damage in response to hydrogen peroxide (H₂O₂) treatment. The level of damaged RNA, measured by the content of 8-hydroxyguanosine (7,8-dihydro-8-oxoguanosine, 8-oxoG), increases depending on H₂O₂ dosage and is inversely correlated with cell viability. The elevated level of 8-oxoG in RNA decreases after removal of oxidative challenge, suggesting the existence of surveillance mechanism(s) for cleaning up oxidized RNA. Human polynucleotide phosphorylase (hPNPase), an exoribonuclease primarily located in mitochondria, has been previously shown to bind 8-oxoG-RNA with high affinity. The role of hPNPase in HeLa cell under oxidative stress conditions is examined here. Overexpression of hPNPase reduces RNA oxidation and increases cell viability against H₂O₂ insult. Conversely, hPNPase knockdown decreases viability and increases 8-oxoG level in HeLa cell exposed to H₂O₂. Our results suggest that hPNPase plays an important role in protecting cells and limiting damaged RNA under oxidative stress.     

Olfactory Ensheathing Cell-Conditioned Medium Reverts Aβ<Subscript>25–35</Subscript>-Induced Oxidative Damage in SH-SY5Y Cells by Modulating the Mitochondria-Mediated Apoptotic Pathway

Olfactory ensheathing cells (OECs) are a type of glia from the mammalian olfactory system, with neuroprotective and regenerative properties. β-Amyloid peptides are a major component of the senile plaques characteristic of the Alzheimer brain. The amyloid beta (Aβ) precursor protein is cleaved to amyloid peptides, and Aβ_25–35 is regarded to be the functional domain of Aβ, responsible for its neurotoxic properties. It has been reported that Aβ_25–35 triggers reactive oxygen species (ROS)-mediated oxidative damage, altering the structure and function of mitochondria, leading to the activation of the mitochondrial intrinsic apoptotic pathway. Our goal is to investigate the effects of OECs on the toxicity of aggregated Aβ_25–35, in human neuroblastoma SH-SY5Y cells. For such purpose, SH-SY5Y cells were incubated with Aβ_25–35 and OEC-conditioned medium (OECCM). OECCM promoted the cell viability and reduced the apoptosis, and decreased the intracellular ROS and the lipid peroxidation. In the presence of OECCM, mRNA and protein levels of antioxidant enzymes (SOD1 and SOD2) were upregulated. Concomitantly, OECCM decreased mRNA and the protein expression levels of cytochrome c, caspase-9, caspase-3, and Bax in SH-SY5Y cells, and increased mRNA and the protein expression level of Bcl-2. However, OECCM did not alter intracellular Ca²⁺ concentration in SH-SY5Y cells. Taken together, our data suggest that OECCM ameliorates Aβ_25–35-induced oxidative damage in neuroblastoma SH-SY5Y cells by inhibiting the mitochondrial intrinsic pathway. These data provide new insights into the functional actions of OECCM on oxidative stress-induced cell damage.     

Protective Effect of Selenoprotein X Against Oxidative Stress-Induced Cell Apoptosis in Human Hepatocyte (LO2) Cells via the p38 Pathway

Oxidative stress, as mediated by ROS (reactive oxygen species), is a significant factor in initiating the cells damaged by affecting cellular macromolecules and impairing their biological functions; SelX, a selenoprotein also known as MsrB1 belonging to the methionine sulfoxide reductase (Msr) family, is the redox repairing enzyme and involved in redox-related functions. In order to more precisely analyze the relationship between oxidative stress, cell oxidative damage, and SelX, we stably overexpressed porcine Selx full-length cDNA in human normal hepatocyte (LO2) cells. Cell viability, cell apoptosis rate, intracellular ROS, and the expression levels of mRNA or protein of apoptosis-related genes under H₂O₂-induced oxidative stress were detected. We found that overexpression of SelX can prevent the oxidative damage caused by H₂O₂ and propose that the main mechanism underlying the protective effects of SelX is the inhibition of LO2 cell apoptosis. The results revealed that overexpressed SelX reduced the H₂O₂-induced intracellular ROS generation, inhibited the H₂O₂-induced upregulation of Bax and downregulation of Bcl-2, and increased the mRNA and protein ratio of Bcl-2/Bax. Furthermore, it inhibited H₂O₂-induced p38 MAPK phosphorylation. Taken together, our findings suggested that SelX played important roles in protecting LO2 cells against oxidative damage and that its protective effect is partly via the p38 pathway by acting as a ROS scavenger.     

Fibroblast growth factor-8 inhibits oxidative stress-induced apoptosis in H9c2 cells

Fibroblast growth factors (FGFs) comprise a large family of signaling molecules that involve cell patterning, mobilization, differentiation, and proliferation. Various FGFs, including FGF-1, FGF-2, and FGF-5, have been shown to play a role in cytoprotection during adverse cardiac events; however, whether FGF-8 is a cytoprotective remains unclear. The current study was designed to evaluate the effect of FGF-8 treatment on oxidative stress-induced apoptosis in H9c2 cells. Cells were divided into three groups: control, H₂O₂ (400 µm H₂O₂), and H₂O₂ + FGF-8 (4 ng/ml FGF-8). Our results suggest apoptosis was significantly (p < 0.05) enhanced in the H₂O₂ group relative to control. Moreover, a significant (p < 0.05) decline in apoptosis was observed in the H₂O₂ + FGF-8 group compared to H₂O₂-treated cells as evidenced by TUNEL staining, a cell death detection ELISA, and cell viability. Levels of downstream apoptotic mediators, caspase-3 and caspase-9, were significantly (p < 0.05) upregulated following H₂O₂ treatment but were abrogated following FGF-8 application. Expression levels of Forkhead box protein O1 (FoxO-1), MnSOD, catalase, pAKT, and p-mTOR were significantly (p < 0.05) reduced in the H₂O₂ group (p < 0.05). Notably, these levels were significantly (p < 0.05) reversed following FGF-8 treatment. Our data, for the first time, suggest FGF-8 is an anti-apoptotic mediator in oxidative-stressed H9c2 cells. Furthermore, our data demonstrate that apoptotic inhibition by FGF-8 is consequent to FoxO-1 oxidative detoxification as well as augmentation to the PI3K/AKT cell survival pathway.     

Response of Lens Epithelial Cells to Hydrogen Peroxide Stress and the Protective Effect of Caloric Restriction

Hydrogen peroxide (H₂O₂) has been reported to be present at significant levels in the lens and aqueous humor in some cataract patients and suggested as a possible source of chronically inflicted damage to lens epithelial (LE) cells. We measured H₂O₂effects on bovine and mouse LE cells and determined whether LE cells from old calorically restricted mice were more resistant to H₂O₂-induced cellular damage than those of same age ad libitum fed (AL) mice. Bovine lens epithelial cells were exposed to H₂O₂at 40 or 400 μM for 2 h and then allowed to recover from the stress. The cells were assayed for DNA damage, DNA synthesis, cell viability, cell morphology, response to growth stimuli, and proliferation potential. Hydrogen peroxide-treated cells showed an increased DNA unwinding 50% greater than that for untreated controls. These DNA strand breaks appeared to be almost completely rejoined by 30 min following removal of the cells from a 2-h exposure. The 40 μM exposure did not produce a significantly lower DNA synthesis rate than the control, it responded to growth factor stimuli, and it replicated as did the control cells after removal of H₂O₂. The 400 μM H₂O₂severely affected DNA synthesis and replication, as shown by increased cell size and by markedly reduced clonal cell growth. The cells did not respond to growth stimulation by serum or growth factors and lost irreversibly the capacity to proliferate. The responses of LE cells from old adlib diet (AL) and calorically restricted (CR) mice to H₂O₂were significantly different. Exposure of LE cells to 20, 40, or 100 μM H₂O₂for 1 h induces a significant loss of cellular proliferation in cells from old AL mice. LE cells from long-term CR mice of the same strain and age were more resistant to oxidative damage at all three concentrations of H₂O₂than those of both old and young AL mice and showed a significantly higher proliferation potential following treatment. It is concluded that CR results in superior resistance to reactive oxygen radicals in the lens epithelium.     

Hydrogen Peroxide Induces Lysosomal Protease Alterations in PC12 Cells

Alterations in lysosomal proteases have been implicated in many neurodegenerative diseases. The current study demonstrates a concentration-dependent decrease in PC12 cell viability and transient changes in cystatin C (CYSC), cathepsin B (CATB), cathepsin D (CATD) and caspase-3 following exposure to H₂O₂. Furthermore, activation of CATD occurred following exposure to H₂O₂ and cysteine protease suppression, while inhibition of CATD with pepstatin A significantly improved cell viability. Additionally, significant PARP cleavage, suggestive of caspase-3-like activity, was observed following H₂O₂ exposure, while inhibition of caspase-3 significantly increased cell viability compared to H₂O₂ administration alone. Collectively, our data suggest that H₂O₂ induced cell death is regulated at least in part by caspase-3 and CATD. Furthermore, cysteine protease suppression increases CATD expression and activity. These studies provide insight for alternate pathways and potential therapeutic targets of cell death associated with oxidative stress and lysosomal protease alterations.     

Sonic Hedgehog Protects Cortical Neurons Against Oxidative Stress

Oxidative stress is one of the most important pathological mechanisms in neurodegenerative diseases and ischemia. Recent studies have indicated that the sonic hedgehog (SHH) signaling pathway is involved in these diseases, but the underlying mechanisms remains elusive. Here we report that the SHH pathway was activated in primary cultured cortical neurons after exposure to hydrogen peroxide (H₂O₂). H₂O₂ treatment decreased the cell viability of neurons, and inhibition of endogenous SHH signaling exacerbated its neurotoxicity. Activation of SHH signaling protected neurons from H₂O₂-induced apoptosis and increased the cell viability while those effects were partially reversed by blocking SHH signals. Exogenous SHH increased the activities of Superoxide dismutase (SOD) and Glutathione peroxidase (GSH-PX) in H₂O₂-treated neurons and decreased production of Malondialdehyde (MDA). It also promoted expression of the anti-apoptotic gene Bcl-2 and inhibited expression of pro-apoptotic gene Bax. Activation of SHH signals upregulated both Neurotrophic factors vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). Pretreatment with SHH inhibited the activation of ERK (extracellular signal-regulated kinases) signals induced by H₂O₂. Our findings demonstrate that activation of SHH signaling protects cortical neurons against oxidative stress and suggest a potential role of SHH for the clinic treatments of brain ischemia and neurodegenerative disorders.