Interindividual differences in initial DNA repair capacity when evaluating H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced DNA damage in extended-term cultures of human lymphocytes using the comet assay

It has been suggested that extended-term cultures of human lymphocytes could be used as a complement to cell lines based on transformed cells when testing the genotoxicity of chemicals. To investigate whether the pattern of induced DNA damage and its subsequent repair differs significantly between cultures based on different blood donors, hydrogen peroxide (H₂O₂)-induced DNA damage was measured in cultures from four different subjects using the comet assay. The DNA damage was significantly increased in all cultures after 10 min exposure to 0.25 mmol/L H₂O₂, and there was a significant decrease in the H₂O₂-induced DNA damage in all cultures after 30 min of DNA repair. The level of damage varied between the different donors, especially after the repair. Using PCR and DNA sequencing, exon 5 of the p53 gene was sequenced in the lymphocytes from the donors with the lowest and highest residual damage. No such mutation was found. Mouse lymphoma L5178Y cells carrying the p53 mutation in exon 5 were included as a reference. These cells were found to be less sensitive toward the H₂O₂-induced DNA damage, and they were also found to have a rather low DNA repair capacity. The demonstrated variation in H₂O₂-induced DNA damage and DNA repair capacity between the cultures from the different subjects may be important from a risk assessment perspective, but is obviously not of decisive importance when it comes to the development of a routine assay for genotoxicity.     

Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

Ataxia telangiectasia mutated inhibits oxidative stress-induced apoptosis by regulating heme oxygenase-1 expression

Ataxia telangiectasia (AT) is caused by mutational inactivation of the ataxia telangiectasia mutated (Atm) gene, which is involved in DNA repair. Increased oxidative stress has been shown in human AT cells and neuronal tissues of Atm-deficient mice. Heme oxygenase-1 (HO-1) is an inducible antioxidant enzyme and protects cells against oxidative stress. The purpose of this study is to determine whether ATM induces antioxidant enzyme HO-1 and protects cells from oxidative stress-mediated apoptosis by driving the activation of PKC-δ and NF-κB, by increasing cell viability, and by downregulating DNA fragmentation and apoptotic indicators (apoptosis-inducing factor and cleaved caspase-3). AT fibroblasts stably transfected with human full-length ATM cDNA (YZ5 cells) or the empty vector (MOCK cells) were treated with H₂O₂ as a source of reactive oxygen species (ROS). As a result, transfection with ATM inhibited ROS-induced cell death and DNA fragmentation in MOCK cells. Transfection with ATM induced expression of HO-1 which was mediated by PKC-δ and NF-κB in H₂O₂-treated MOCK cells. ZnPP, an HO-1 inhibitor, and transfection with HO-1 siRNA increased ROS levels and apoptosis, whereas hemin, an HO-1 activator, reduced ROS levels and apoptosis in H₂O₂-treated YZ5 cells. Rottlerin, a PKC-δ inhibitor, inhibited NF-κB activation and HO-1 expression in H₂O₂-treated YZ5 cells. MOCK cells showed increased cell death, DNA fragmentation, and apoptotic indicators compared to YZ5 cells exposed to H₂O₂. In addition, transfection with p65 siRNA increased ROS levels and DNA fragmentation, but decreased HO-1 protein levels in H₂O₂-treated YZ5 cells. In conclusion, ATM induces HO-1 expression via activation of PKC-δ and NF-κB and inhibits oxidative stress-induced apoptosis. A loss of HO-1 induction may explain why AT patients are vulnerable to oxidative stress.     

3,4-Dihydroxybenzaldehyde purified from the barley seeds (Hordeum vulgare) inhibits oxidative DNA damage and apoptosis via its antioxidant activity

Barley is a major crop worldwide. It has been reported that barley seeds have an effect on scavenging ROS. However, little has been known about the functional role of the barley on the inhibition of DNA damage and apoptosis by ROS. In this study, we purified 3,4-dihydroxybenzaldehyde from the barley with silica gel column chromatography and HPLC and then identified it by GC/MS. And we firstly investigated the inhibitory effects of 3,4-dihydroxybenzaldehyde purified from the barley on oxidative DNA damage and apoptosis induced by H₂O₂, the major mediator of oxidative stress and a potent mutagen. In antioxidant activity assay such as DPPH radical and hydroxyl radical scavenging assay, Fe²⁺ chelating assay, and intracellular ROS scavenging assay by DCF-DA, 3,4-dihydroxybenzaldehyde was found to scavenge DPPH radical, hydroxyl radical and intracellular ROS. Also it chelated Fe²⁺. In in vitro oxidative DNA damage assay and the expression level of phospho-H2A.X, it inhibited oxidative DNA damage and its treatment decreased the expression level of phospho-H2A.X. And in oxidative cell death and apoptosis assay via MTT assay and Hoechst 33342 staining, respectively, the treatment of 3,4-dihydroxybenzaldehyde attenuated H₂O₂-induced cell death and apoptosis. These results suggest that the barley may exert the inhibitory effect on H₂O₂-induced tumor development by blocking H₂O₂-induced oxidative DNA damage, cell death and apoptosis.     

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.     

Vanadate induces apoptosis in epidermal JB6 Pplus cells via hydrogen peroxide-mediated reactions

Apoptosis is a physiological mechanism for the control of DNA integrity in mammalian cells. Vanadium induces both DNA damage and apoptosis. It is suggested that vanadium-induced apoptosis serves to eliminate DNA-damaged cells. This study is designed to clarify a role of reactive oxygen species in the mechanism of apoptosis induced by vanadium. We established apoptosis model with murine epidermal JB6 P⁺ cells in the response to vanadium stimulation. Apoptosis was detected by a cell death ELISA assay and morphological analysis. The result shows that apoptosis induced by vanadate is dose-dependent, reaching its saturation level at a concentration of 100 M vanadate. Vanadyl (IV) can also induce apoptosis albeit with lesser potency. A role of reactive oxygen species was analyzed by multiple reagents including specific scavengers of different reactive oxygen species. The result shows that vanadate-induced apoptosis is enhanced by NADPH, superoxide dismutase and sodium formate, but was inhibited by catalase and deferoxamine. Cells exposed to vanadium consume more molecular oxygen and at the same time, produce more H₂O₂ as measured by the change in fluorescence of scopoletin in the presence of horseradish peroxidase. This change in oxygen consumption and H₂O₂ production is enhanced by NADPH. Taken together, these results show that vanadate induces apoptosis in epidermal cells and H₂O₂ induced by vanadate plays a major role in this process.     

NP7 protects from cell death induced by oxidative stress in neuronal and glial midbrain cultures from parkin null mice

Parkin mutations produce Parkinson’s disease (PD) in humans and nigrostriatal dopamine lesions related to increased free radicals in mice. We examined the effects of NP7, a synthetic, marine derived, free radical scavenger which enters the brain, on H₂O₂ toxicity in cultured neurons and glia from wild-type (WT) and parkin null mice (PK-KO).NP7, 5-10 μM, prevented the H₂O₂ induced apoptosis and necrosis of midbrain neuronal and glial cultures from WT and PK-KO mice. NP7 suppressed microglial activation and the H₂O₂ induced drop-out of dopamine neurons_. Furthermore, NP7 prevented the increased phosphorylation of ERK and AKT induced by H₂O₂. NP7 may be a promising neuroprotector against oxidative stress in PD.     

Activation of AMPK protects against hydrogen peroxide-induced osteoblast apoptosis through autophagy induction and NADPH maintenance: New implications for osteonecrosis treatment?

Elevated hydrogen peroxide (H₂O₂) causes osteoblast dysfunction and apoptosis, serving as an important contributor to the development of osteonecrosis. Here we aimed to understand the role of AMP-activated protein kinase (AMPK) in the process. We observed a high level of AMPK activation in surgery isolated patients' osteonecrosis tissues. In cultured osteoblastoma MG63 cells, H₂O₂ stimulation induced significant AMPK activation, oxidative stress, cell death and apoptosis. Inhibition of AMPK by its inhibitor (compound C) or by shRNA-mediated knockdown dramatically enhanced H₂O₂-induced MG63 cell apoptosis, while over-expression of AMPK in HEK-293 cells alleviated H₂O₂-induced cell damage. These results confirmed that H₂O₂-activated AMPK is pro-cell survival. We observed that H₂O₂ induced protective autophagy in MG63 cells, and AMPK-dependent Ulk1 activation and mTORC1 (mTOR complex 1) inactivation might involve autophagy activation. Further, AMPK activation inhibited H₂O₂-induced oxidative stress, probably through inhibiting NADPH (nicotinamide adenine dinucleotide phosphate) depletion, since more NADPH depletion and oxidative stress were induced by H₂O₂ in AMPK deficient MG63 cells. Finally, we observed a significant AMPK activation in H₂O₂-treated primary cultured and transformed (MC3T3-E1) osteoblasts, and AMPK inhibitor compound C enhanced death by H₂O₂ in these cells. Based on these results, we concluded that H₂O₂-induced AMPK activation is pro-survival and anti-apoptosis in osteoblasts. Autophagy induction and NADPH maintenance are involved in AMPK-mediated pro-survival effects. AMPK might represent a novel molecular target for osteonecrosis treatment.     

Tumor suppressor Ing1b facilitates DNA repair and prevents oxidative stress induced cell death

Inhibitor of growth (ING) family of proteins are known to coordinate with histone acetyltransferases and regulate the key events of cell cycle and DNA repair. Previous work from our lab showed that Ing1b regulated the nucleotide excision repair by facilitating histone acetylation and subsequent chromatin relaxation. Further, it was also shown that Ing1b protected the cells from genomic instability induced cell death by promoting ubiquitination of proliferating cell nuclear antigen (PCNA). In the present study we explored the role of Ing1b in the repair of oxidized DNA and prevention of oxidative stress induced genotoxic cell death. Using HCT116 cells we show that Ing1b protein expression is induced by treatment with H₂O₂. Ing1b lacking cells showed decreased ability to repair the oxidized DNA. PCNA monoubiquitination, a critical event of DNA repair was blunted in Ing1b knock down cells and augmented in Ing1b over expressing cells. Moreover, oxidative stress induced cell death was higher in cells lacking Ing1b whereas it was lower in Ing1b over expressing cells. Finally we show that inhibition of histone deacetylases, rescued the Ing1b knock down cells from cytotoxic effects of H₂O₂ treatment.     

Effects of pulsed electric fields on DNA of human lymphocytes

The effects of pulsed electric fields of low frequency (50 Hz) on DNA of human lymphocytes were investigated. The influence of additional external factors, such as hydrogen peroxide (H₂O₂) and γ-irradiation, as well as the repair efficiency in these lymphocytes, was also evaluated. The comet assay, a very sensitive and rapid method for detecting DNA damage at the single cells level was the method used. A significant amount of damage was observed after exposure to the electric fields, compared to the controls. After 2 h incubation at 37^°C, a proportion of damage was repaired. H₂O₂ and γ-irradiation increased the damage to lymphocytes exposed to pulsed electric fields according to the dose used, while the amount of the repair was proportional to the damage.     

Cytoprotective effect of dieckol on human endothelial progenitor cells (hEPCs) from oxidative stress-induced apoptosis

Abstract

Although endothelial progenitor cells (EPCs) have been used to promote revascularization after peripheral or myocardial ischemia, excess amounts of reactive oxygen species (ROS) are often involved in senescence and apoptosis of EPCs, thereby causing defective neovascularization and reduced or failed recovery. Here, we examined the cytoprotective effect of Ecklonia cava-derived antioxidant dieckol (DK) on oxidative stress-induced apoptosis in EPCs to improve EPC bioactivity for vessel repair. Although H₂O₂ (10 ^{? 3} M) increased the intracellular ROS level in EPCs, DK (10ug/ml) pretreatment suppressed the H₂O₂-induced ROS increase and drastically reduced the ratios of apoptotic cells. H₂O₂-induced ROS increased the phosphorylation of p38 MAPK and JNK; this was inhibited by DK pretreatment. H₂O₂ treatment increased the phosphorylation of NF-κB, which was blocked by pretreatment with SB 203580, a p38 MAPK inhibitor, or SP 600125, a JNK inhibitor. H₂O₂ decreased the cellular levels of Bcl-2 and c-IAPs, cellular inhibitors of apoptosis proteins, but increased caspase-3 activation. However, all these effects were inhibited by pretreatment with DK. Injection of DK-mixed EPCs (DK + EPCs) into myocardial ischemic sites in vivo induced cellular proliferation and survival of cells at the ischemic sites and, thereby, enhanced the secretion of angiogenic cytokines at the ischemic sites. These results show that DK + EPC exhibit markedly enhanced anti-apoptotic and antioxidative capabilities, unlike that shown by EPCs alone; thus, they contribute to improved repair of ischemic myocardial injury through cell survival and angiogenic cytokine production.     

Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom

L-amino acid oxidase (LAAO) from the Malayan pit viper induces both necrosis and apoptosis in Jurkat cells. Cell death by necrosis is attributed to H₂O₂ produced by oxidation of α-amino acids. In the presence of catalase that effectively scavenges H₂O_2, a switch to apoptosis is observed. The major factors contributing to apoptosis are proposed to be: (i) generation of toxic intermediates from fetal calf serum (ii) binding and internalization of LAAO. The latter process appears to be mediated by the glycan moiety of the enzyme as desialylation reduces cytotoxicity. D-amino acid oxidase (DAAO), which catalyzes the same reaction as LAAO but lacks glycosylation, triggers necrosis as a consequence of H₂O₂ production but not apoptosis in the presence of catalase. Thus induction of cell death by LAAO appears to involve both the generation of H₂O₂ and the molecular interaction of the glycan moiety of the enzyme with structures at the cell surface. S. R. Ande, P. R. Kommoju contributed equally to this work.     

Neuroprotective potential of epigallo catechin-3-gallate in PC-12 cells

Oxidative stress is a major player in aging and neurodegenerative disorders. Macromolecular damage occurs as a result of oxidative stress that affects the mitochondria. Mitochondrial damage leads to cell death by apoptosis or necrosis. EGCG is a tea polyphenol that protects the cells against oxidative stress. Neuroprotective potential of EGCG was tested against H₂O₂ induced oxidative stress in PC-12 cells. PC-12 cells were grown in tissue culture flasks. Oxidative stress was induced by adding H₂O₂ to the cells. EGCG was also added and the cell death was assessed using MTT assay. Oxidative stress was assessed by protein carbonyl and thiol status. Mitochondrial membrane potential was studied using JC-1 staining. TNF-α levels were assessed using ELISA. H₂O₂ increased the protein carbonyl content and reduced the thiol status in the PC-12 cells. Cell death was increased in H₂O₂ treated cells as shown by MTT assay. Mitochondrial membrane potential was also decreased along with increase in TNF-α level in H₂O₂ treated cells. EGCG brought about an increase in the cellular thiol status and decreased the protein carbonyl content in the PC-12 cells. Cell death was attenuated by EGCG treatment along with an increase in mitochondrial membrane potential and decrease in TNF-α level. EGCG conferred its antioxidant potential to PC-12 cells as evident by decreased protein damage. Mitochondrial membrane potential was improved along with a decrement in the cell death in PC-12 cells. EGCG acts as a good neutraceutical antioxidant to render neuroprotectivity to PC-12 cells.     

SIRT3 protects cardiomyocytes from oxidative stress-mediated cell death by activating NF-κB

Oxidative stress-mediated cell death in cardiomyocytes reportedly plays an important role in many cardiac pathologies. Our previous report demonstrated that mitochondrial SIRT3 plays an essential role in mediating cell survival in cardiac myocytes, and that resveratrol protects cardiomyocytes from oxidative stress-induced apoptosis by activating SIRT3. However, the exact mechanism by which SIRT3 prevents oxidative stress remains unknown. Here, we show that exposure of H9c2 cells to 50 μM H₂O₂ for 6 h caused a significant increase in cell death and the down-regulation of SIRT3. Reactive oxygen species (ROS)-mediated NF-κB activation was involved in this SIRT3 down-regulation. The SIRT3 activator, resveratrol, which is considered an important antioxidant, protected against H₂O₂-induced cell death, whereas the SIRT inhibitor, nicotinamide, enhanced cell death. Moreover, resveratrol negatively regulated H₂O₂-induced NF-κB activation, whereas nicotinamide enhanced H₂O₂-induced NF-κB activation. We also found that SOD2, Bcl-2 and Bax, the downstream genes of NF-κB, were involved in this pathological process. These results suggest that SIRT3 protects cardiomyocytes exposed to oxidative stress from apoptosis via a mechanism that may involve the NF-κB pathway.     

Regulation of apoptosis/necrosis execution in cadmium-treated human promonocytic cells under different forms of oxidative stress

Pulse-treatment of U-937 human promonocytic cells with cadmium chloride followed by recovery caused caspase-9/caspase-3-dependent, caspase-8-independent apoptosis. However, pre-incubation with the glutathione (GSH)-suppressing agent DL-buthionine-(S,R)-sulfoximine (cadmium/BSO), or co-treatment with H₂O₂ (cadmium/H₂O₂), switched the mode of death to caspase-independent necrosis. The switch from apoptosis to necrosis did not involve gross alterations in Apaf-1 and pro-caspase-9 expression, nor inhibition of cytochrome c release from mitochondria. However, cadmium/H₂O₂-induced necrosis involved ATP depletion and was prevented by 3-aminobenzamide, while cadmium/BSO-induced necrosis was ATP independent. Pre-incubation with BSO increased the intracellular cadmium accumulation, while co-treatment with H₂O₂ did not. Both treatments caused intracellular peroxide over-accumulation and disruption of mitochondrial transmembrane potential (ΔΨm). However, while post-treatment with N-acetyl-L-cysteine or butylated hydroxyanisole reduced the cadmium/BSO-mediated necrosis and ΔΨm disruption, it did not reduce the effects of cadmium/H₂O₂. Bcl-2 over-expression, which reduced peroxide accumulation without affecting the intracellular GSH content, attenuated necrosis generation by cadmium/H₂O₂ but not by cadmium/BSO. By contrast, AIF suppression, which reduced peroxide accumulation and increased the GSH content, attenuated the toxicity of both treatments. These results unravel the existence of two different oxidation-mediated necrotic pathways in cadmium-treated cells, one of them resulting from ATP-dependent apoptosis blockade, and the other involving the concurrence of multiple regulatory factors.     

Bradykinin Inhibits Oxidative Stress-Induced Cardiomyocytes Senescence via Regulating Redox State

Background

Cell senescence is central to a large body of age related pathology, and accordingly, cardiomyocytes senescence is involved in many age related cardiovascular diseases. In consideration of that, delaying cardiomyocytes senescence is of great importance to control clinical cardiovascular diseases. Previous study indicated that bradykinin (BK) protected endothelial cells from senescence induced by oxidative stress. However, the effects of bradykinin on cardiomyocytes senescence remain to be elucidated. In this study, we investigated the effect of bradykinin on H₂O₂-induced H9C2 cells senescence.

Methods and Results

Bradykinin pretreatment decreased the senescence induced by H₂O₂ in cultured H9C2 cells in a dose dependent manner. Interestingly, 1 nmol/L of BK almost completely inhibited the increase in senescent cell number and p21 expression induced by H₂O₂. Since H₂O₂ induces senescence through superoxide-induced DNA damage, we also observed the DNA damage by comet assay, and BK markedly reduced DNA damage induced by H₂O₂, and moreover, BK treatment significantly prevented reactive oxygen species (ROS) production in H9C2 cells treated with H₂O₂. Importantly, when co-incubated with bradykinin B2 receptor antagonist HOE-140 or eNOS inhibitor N-methyl-L-arginine acetate salt (L-NAME), the protective effects of bradykinin on H9C2 senescence were totally blocked. Furthermore, BK administration significantly prevented the increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity characterized by increased ROS generation and gp91 expression and increased translocation of p47 and p67 to the membrane and the decrease in superoxide dismutase (SOD) activity and expression induced by H₂O₂ in H9C2 cells, which was dependent on BK B2 receptor mediated nitric oxide (NO) release.

Conclusions

Bradykinin, acting through BK B2 receptor induced NO release, upregulated antioxidant Cu/Zn-SOD and Mn-SOD activity and expression while downregulating NADPH oxidase activity and subsequently inhibited ROS production, and finally protected against cardiomyocytes senescence induced by oxidative stress.     

H2O2-Induced Cell Death in Human Glioma Cells: Role of Lipid Peroxidation and PARP Activation

underlying mechanism of ROS-induced cell injury remains to be defined. This study was undertaken to examine the role of lipid peroxidation and poly (ADP-ribose) polymerase (PARP) activation in H₂O₂-induced cell death in A172 cells, a human glioma cell line. H₂O₂ induced a dose- and time-dependent cell death. The cell death was prevented by thiols (dithiothreitol and glutathione), iron chelators (deferoxamine and phenanthroline), H₂O₂ scavengers (catalase and pyruvate), and a hydroxyl radical scavenger (dimethylthiourea). Antioxidants N,N-diphenyl-p-phenylenediamine (DPPD) and Trolox had no effect on the H₂O₂-induced cell death. Lipid peroxidation did not increase in human glioma cells exposed to H₂O₂. The PARP inhibitor 3-aminobenzamide prevented the cell death induced by H₂O₂. The PARP activity was increased by H₂O₂ and the H₂O₂ effect was prevented by 3-aminobenzamide, dithiothreitol, and phenanthroline. The ATP depletion induced by H₂O₂ was prevented by catalase, dithiothreitol, phenanthroline, and 3-aminobenzamide, but not by DPPD. These results indicate that the H₂O₂-induced cell death is mediated by PARP activation but not by lipid peroxidation in human glioma cells.     

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.     

MicroRNA‐328 is involved in the effect of selenium on hydrogen peroxide‐induced injury in H9c2 cells

Oxidative stress induces apoptosis in cardiac cells, and antioxidants attenuate the injury. MicroRNAs (miRNAs) are also involved in cell death; therefore, this study aimed to investigate the role of miRNAs in the effect of selenium on oxidative stress‐induced apoptosis. The effects of sodium selenite were analyzed via cell viability, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration. Flow cytometry was used to evaluate cell apoptosis. Fura‐2AM was used to calculate intracellular Ca²⁺ concentration. Sodium selenite could ameliorate hydrogen peroxide (H₂O₂)‐induced cell apoptosis and improve expression levels of glutathione peroxidase and thioredoxin reductase. Pretreatment with sodium selenite improved SOD activity and reduced MDA concentration. Treatments with H₂O₂ or sodium selenite decreased miR‐328 levels. MiR‐328 overexpression enhanced cell apoptosis, reduced ATP2A2 levels, and increased intracellular Ca²⁺ concentration, while inhibition produced opposite effects. MiR‐328 might be involved in the effect of sodium selenite on H₂O₂‐induced cell death in H9c2 cells.