Selenoprotein W serves as an antioxidant in chicken myoblasts

Background

Selenoprotein W (SelW) was thought to play an antioxidant role in mammals. Because chicken SelW has no cysteine (Cys) at the residue 37 (Cys37) that is required for the presumed antioxidant function in mammals, this study was conducted to determine whether chicken SelW possessed the same function.

Methods

Small interfering RNAs (siRNAs) technology was applied to suppress the SelW expression in chicken embryonic myoblasts. Thereafter, these myoblasts were treated with different concentrations of H₂O₂ and assayed for cell viability, apoptosis rate, reactive oxygen species (ROS) status, and expression levels of apoptosis-related genes and proteins (Bax, Bcl-2, and caspase-3).

Results

Silencing of the myoblast SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H₂O₂. While the knockout down of SelW up-regulated Bax and caspase-3 and down-regulated Bcl-2, the induced oxidative injuries were alleviated by treatment with a ROS scavenger, N-acetyl-l-cysteine (NAC).

Conclusion

Chicken SelW protected embryonic myoblasts against cell apoptosis mediated by endogenous and exogenous H₂O₂.

General significance

Chicken SelW possesses antioxidant function similar to the mammalian homologues despite the lack of Cys37 in the peptide.     

Effects of N-acetylcystein on bleomycin-induced apoptosis in malignant testicular germ cell tumors

Oxidative stress has been shown to induce apoptosis in cancer cells. Therefore, one might suspect that antioxidants may inhibit reactive oxygen species (ROS) and prevent apoptosis of cancer cells. No study has been carried out so far to elucidate the effects of N-acetylcysteine (NAC) on bleomycin-induced apoptosis in human testicular cancer (NCCIT) cells. We investigated the molecular mechanisms of apoptosis induced by bleomycin and the effect of NAC in NCCIT cells. We compared the effects of bleomycin on apoptosis with H₂O₂ which directly produces ROS. Strong antioxidant NAC was evaluated alone and in combination with bleomycin or H₂O₂ in germ cell tumor-derived NCCIT cell line (embryonal carcinoma, being the nonseminomatous stem cell component). We determined the cytotoxic effect of bleomycin and H₂O₂ on NCCIT cells and measured apoptosis markers such as caspase-3, caspase-8, and caspase-9 activities and Bcl-2, Bax, and cytochrome c (Cyt-c) levels in NCCIT cells incubated with bleomycin, H₂O₂, and/or NAC. We found half of the lethal dose (LD₅₀) of bleomycin on NCCIT cell viability as 120???g/ml after incubation for 72?h. Incubation with bleomycin (LD₅₀) induced increases in caspase-3, caspase-8, and caspase-9 activities and Cyt-c and Bax protein levels and a decrease in Bcl-2 level. Co-incubation of NCCIT cells with bleomycin and 10?mM NAC abolished bleomycin-induced increases in caspase-3 and caspase-9 activities, Bax, and Cyt-c levels and bleomycin-induced decrease in Bcl-2 level. Our results indicate that bleomycin induces apoptosis in NICCT cells and that NAC diminishes bleomycin-induced apoptosis via inhibiting the mitochondrial pathway. We conclude that NAC has negative effects on bleomycin-induced apoptosis in NICCT cells and causes resistance to apoptosis, which is not a desirable effect in the fight against cancer.     

Effects of curcumin on bleomycin-induced apoptosis in human malignant testicular germ cells

Testicular cancer is the most common cancer among young men of reproductive age. Bleomycin is a frequently used drug for the treatment of several malignancies and is part of the chemotherapy protocols in testicular cancer. Bleomycin causes an increase in oxidative stress which has been shown to induce apoptosis in cancer cells. Curcumin (diferuloylmethane), an active component of the spice turmeric, has attracted interest because of its anti-inflammatory and chemopreventive activities. However, no study has been carried out so far to elucidate its interaction with bleomycin in testicular cancer cells. In this study, we investigated the effects of curcumin and bleomycin on apoptosis signalling pathways and compared the effects of bleomycin with H₂O₂ which directly produces reactive oxygen species. We measured apoptosis markers such as caspase-3, caspase-8, and caspase-9 activities and Bcl-2, Bax, and Cyt-c levels in NCCIT cells incubated with curcumin (5 μM), bleomycin (120 μg/ml), bleomycin + curcumin, H₂O₂ (35 μM), and H₂O₂ + curcumin for 72 h. Curcumin, bleomycin, and H₂O₂ caused apoptosis indicated as increases in caspase-3, caspase-8, and caspase-9 activities and Bax and cytoplasmic Cyt-c levels and a decrease in Bcl-2 level. Concurrent use of curcumin with bleomycin decreased caspase activities and Bax and Cyt-c levels compared to their separate effects in NCCIT cells. Our findings suggest that concurrent use of curcumin during chemotherapy in testis cancer should be avoided due to the inhibitory effect of curcumin on bleomycin-induced apoptosis.     

Aucubin modulates Bcl-2 family proteins expression and inhibits caspases cascade in H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced PC12 cells

In this study, the effect of aucubin on H₂O₂-induced apoptosis was studied by using a rat pheochromocytoma (PC12) cell line. We have analyzed the apoptosis of H₂O₂-induced PC12 cells, H₂O₂-induced apoptosis appeared to correlate with lower Bcl-2 expression, higher Bax expression and sequential activation of caspase-3 leading to cleavage of poly-ADP-ribose polymerase (PARP). Aucubin not only inhibited lower Bcl-2 expression, high Bax expression, but also modulated caspase-3 activation, PARP cleavage, and eventually protected against H₂O₂-induced apoptosis. These results indicated that aucubin can obstruct H₂O₂-induced apoptosis by regulating of the expression of Bcl-2 and Bax, as well as suppression of caspases cascade activation.     

Effects of Different Sources and Levels of Zinc on H2O2-Induced Apoptosis in IEC-6 Cells

Zinc has been shown to be an inhibitor of apoptosis for many years. The present study was designed to investigate effects of three zinc chemical forms on H₂O₂-induced cell apoptosis in IEC-6 cells via analysis of cell vitality, LDH activity, apoptosis percentage, caspase-3 activity, and Bcl-2, Bax, and caspase-3, -8, and -9 gene expression. Cells were divided into H₂O₂ and zinc sources+H₂O₂ groups, and there are three different zinc sources [zinc oxide nanoparticle (nano-ZnO), zinc oxide (ZnO), and zinc sulfate (ZnSO₄)] and three concentrations (normal = 25 μM, medium = 50 μM, and high = 100 μM) used in this article. In the present study, we found the striking cytotoxicity of H₂O₂ higher than 200 μM on cell vitality, LDH activity, and apoptosis percentage in the cells using five different concentrations (50, 100, 200, 400, and 800 μM) of H₂O₂ for 4 h. Moreover, we observed that cell vitality was increased, LDH activity and apoptotic percentage were decreased, and gene expression level of Bax and caspase-3 and -9 was markedly reduced, while gene expression level of Bcl-2 and ratio of Bcl-2/Bax were increased in normal concentration groups of nano-ZnO and ZnSO₄ compared with H₂O₂ group, but no significant difference was observed in caspase-8 gene expression. Furthermore, medium or, more intensely, high concentrations of nano-ZnO and ZnSO₄ enhanced H₂O₂-induced cell apoptosis. Compared with nano-ZnO and ZnSO₄, ZnO showed weakest protective effect on H₂O₂-induced apoptosis at normal concentration and was less toxic to cells at high level. Taken together, we proposed that preventive and protective effects of zinc on H₂O₂-induced cell apoptosis varied in IEC-6 cells with its chemical forms and concentrations, and maybe for the first time, we suggested that nano-ZnO have a protective effect on H₂O₂-induced cell apoptosis in IEC-6 cells.     

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.     

Protective effects of a wheat germ peptide (RVF) against H2O2-induced oxidative stress in human neuroblastoma cells

RVF (Arg-Val-Phe), a peptide derived from wheat germ, shows antioxidant properties. Here, the neuroprotective efficacies of RVF were investigated in human neuroblastoma cells (SH-SY5Y) that were pretreated with RVF (150–250 μM, 4 h) and exposed to H₂O₂ (200 μM). RVF increased viable cell numbers by 37 % and reduced the release of lactate dehydrogenase. Pretreatment with RVF also inhibited H₂O₂-induced accumulation of reactive oxygen species and maintained the mitochondrial transmembrane potential as well as preventing intracellular Ca²⁺ dysregulation during H₂O₂ exposure. Furthermore, pretreatment with RVF increased the Bcl-2/Bax ratio and blocked cleavage poly(ADP-ribose) polymerase by inhibiting caspase-3 activation, thus decreasing apoptosis.     

Ginsenoside-Rd potentiates apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells through the mitochondrial pathway

Our previous studies showed that ginsenoside-Rd, a purified component from Panax notoginseng, inhibited cell proliferation and reversed basilar artery remodeling. The aim of this study was to investigate whether ginsenoside- Rd influences H₂O₂-induced apoptosis in basilar artery smooth muscle cells (BASMCs). The results showed that ginsenoside-Rd significantly potentiated H₂O₂-induced cell death and cell apoptosis. This resulted in a concentration-dependent reduction of the cell viability. Ginsenoside-Rd further increased cytochrome C release and caspase-9/caspase-3 activations, and reduced the stability of mitochondrial membrane potential (MMP) and the ratio of Bcl-2/Bax. Cyclosporine A, an inhibitor of mitochondrial-permeability transition, inhibited alteration of mitochondrial permeability induced by H₂O₂ and reversed the effect of ginsenoside-Rd on MMP. Our data strongly suggest that ginsenoside-Rd potentiated H₂O₂-induced apoptosis of BASMCs through the mitochondria-dependent pathway.     

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.     

Mitochondria dependent pathway is involved in the protective effect of bestrophin-3 on hydrogen peroxide-induced apoptosis in basilar artery smooth muscle cells

Bestrophin 3 (Best-3) is expressed in a variety of tissues, such as cardiac, smooth muscle and renal tissues, and it is highly expressed in rat basilar arterial smooth muscle cells (BASMCs). Lee et al. (Biochim Biophys Acta 1823:1864–1876, 2012) reported that Best-3 prevented apoptotic cell death induced by endoplasmic reticulum stress. In the present study, we used small interference RNA (siRNA) and bestrophin 3 cDNA transfection strategy to investigate whether Best-3 can provide a protective effect on apoptosis induced by hydrogen peroxide (H₂O₂) in BASMCs and studied the underlying mechanisms. We found that silencing of Best-3 with siRNA resulted in an increased H₂O₂-induced apoptosis and a decreased cell viability, whereas overexpression of Best-3 significantly prevented the apoptotic cell death and increased the cell viability. Overexpression of Best-3 could stabilize the mitochondrial membrane potential, increase the ratio of Bcl-2/Bax, and decrease cytochrome c release and caspase-3 activation. In contrast, silencing of Best-3 produced the opposite effects. Our present data strongly suggest that Best-3 inhibits apoptosis induced by H₂O₂ in BASMCs through mitochondria dependent pathway.     

Hydrogen peroxide-induced Akt phosphorylation regulates Bax activation

Reactive oxygen species such as hydrogen peroxide (H₂O₂) are involved in many cellular processes that positively and negatively regulate cell fate. H₂O₂, acting as an intracellular messenger, activates phosphatidylinositol-3 kinase (PI3K) and its downstream target Akt, and promotes cell survival. The aim of the current study was to understand the mechanism by which PI3K/Akt signaling promotes survival in SH-SY5Y neuroblastoma cells. We demonstrate that PI3K/Akt mediates phosphorylation of the pro-apoptotic Bcl-2 family member Bax. This phosphorylation suppresses apoptosis and promotes cell survival. Increased survival in the presence of H₂O₂ was blocked by LY294002, an inhibitor of PI3K activation. LY294002 prevented Bax phosphorylation and resulted in Bax translocation to the mitochondria, cytochrome c release, caspase-3 activation, and cell death. Collectively, these findings reveal a mechanism by which H₂O₂-induced activation of PI3K/Akt influences post-translational modification of Bax and inactivates a key component of the cell death machinery.     

Inhibition of hydrogen sulfide synthesis provides protection for severe acute pancreatitis rats via apoptosis pathway

We aimed to investigate the relationship between the synthesis of hydrogen sulfide (H₂S) and the pancreatic acinar cell apoptosis in severe acute pancreatitis (SAP) rats, as well as analyse the potential apoptotic pathway involved in this process. Sixty rats had been equally divided into four groups: sham, SAP, SAP + sodium hydrosulfide (NaHS) and SAP + DL-propargylglycine (PAG). 24 h after SAP induction, all surviving animals of each group were sacrificed to collect blood and tissue samples for the following measurements: the level of serum H₂S as well as the levels of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), H₂S synthesizing activity, CSE mRNA and protein expression, maleic dialdehyde (MDA) and myeloperoxidase (MPO) activity, the expression of Bax, Bcl-2, caspase-3, -8 and -9, the release of cytochrome c and the activation of nuclear factor-kappa B (NF-κB), ERK1/2, JNK1/2 and p38 in pancreas. Furthermore, in situ detection of cell apoptosis was examined and the severity of pancreatic damage was analyzed by pathological grading and scoring. Results Significant differences in every index except IL-10 had been found between the SAP, NaHS and PAG groups (P < 0.05). Treatment with PAG obviously induced the pancreatic acinar cell apoptosis as well as improved all the pathological changes and inflammatory parameters. In contrast, administration of NaHS significantly attenuated apoptosis in the pancreas and aggravated the severity of pancreatic damage. Moreover, the expressions of caspase-3, -8, -9 and the release of cytochrome c were all increased in the apoptotic cells, and the activity of NF-κB as well as the phosphorylation of ERK1/2, JNK1/2 and p38 decreased accompanying with the reduction of the serum H₂S level. H₂S plays a pivotal role in the regulation of pancreatic acinar cell apoptosis in SAP rats. The present results showed that inhibition of H₂S synthesis provided protection for SAP rats via inducing acinar cell apoptosis. This process acted through both extrinsic and intrinsic apoptotic pathways, and may be regulated by reducing the activity of NF-κB.     

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.     

Neuroprotective Effect of <Emphasis Type="Italic">Salvia sahendica</Emphasis> is Mediated by Restoration of Mitochondrial Function and Inhibition of Endoplasmic Reticulum Stress

Herein, we investigated the protective effect of Salvia sahendica against H₂O₂-induced cell death in rat pheochromocytoma (PC12) cells. Our data show that S. sahendica blocks apoptosis pathway by inhibition of cytochrome c release from mitochondria and leakage of calcium from endoplasmic reticulum. It also activates/inactivates two members of Bcl-2 family, Bax and Bcl-2. Bax inhibition and Bcl-2 activation suppress release of cytochrome c from mitochondria that prevents cleavage of caspase-3. Besides S. sahendica suppresses ER stress via attenuation of intracellular levels of calcium. Suppression of ER stress decreased calpain activation and subsequently cleavage of caspase-12. Altogether, these results indicate that S. sahendica protects PC12 cells treated with H₂O₂ via suppression of upstream factors of apoptosis pathway. While oxidative stress is an early event in Alzheimer disease, it seems that S. sahendica prevents deleterious effects of reactive oxygen species by stabilizing mitochondrial membranes and inhibiting ER 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.     

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.     

Increase of autophagy and attenuation of apoptosis by Salvigenin promote survival of SH-SY5Y cells following treatment with H2O2

Oxidative stress is a major component of harmful cascades activated in neurodegenerative disorders. Here, we tried to elucidate the possible neuroprotective effect of Salvigenin, a natural polyphenolic compound, on oxidative stress-induced apoptosis and autophagy in human neuroblastoma SH-SY5Y cells. We measured cell viability by MTT test and found that 25?μM is the best protective concentration of Salvigenin. GSH and SOD assays suggested that Salvigenin activates antioxidant factors. At the same time, measurement of ER stress-associated proteins including calpain and caspase-12 showed the ability of Salvigenin to decrease ER stress. We found that Salvigenin could decrease the apoptotic factors. Salvigenin inhibited H₂O₂-induced caspase-3 which is a hallmark of apoptosis in addition to reducing Bax\Bcl-2 ratio by 1.45 fold. Additionally, Salvigenin increased the levels of autophagic factors. Our results showed an increase in LC3-II/LC3-I ratio, Atg7, and Atg12 in the presence of 25?μM of Salvigenin by about 1.28, 1.25, and 1.54 folds, respectively, compared to H₂O₂-treated cells. So it seems that H₂O₂ cytotoxicity mainly results from apoptosis. Besides, Salvigenin helps cells to survive by inhibiting apoptosis and enhancing autophagy that opens a new horizon for the future experiments.     

Torulene and torularhodin,protects human prostate stromal cells from hydrogen peroxide-induced oxidative stress damage through the regulation of Bcl-2/Bax mediated apoptosis

The current study was designed to elucidate the cytoprotective effects and possible mechanisms of torulene and torularhodin on hydrogen peroxide (H₂O₂)-induced oxidative stress damage in human prostate stromal cells (WPMY-1). After treated with H₂O₂, a notable decrease was appeared in cell viability, yet the decrease was attenuated when cells were pretreated with torulene and torularhodin (0.5–10?μM) as evaluated by WST-1 assay. Pretreatment with these two carotenoids significantly attenuated H₂O₂-induced apoptosis in WPMY-1 cells through the inhibition of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) overproduction, as well as the activation of the activities in catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Finally, pretreatment of cells with carotenoids resulted in the regulation of the mRNA and protein expression of Bcl-2 and Bax in H₂O₂-exposed prostate stromal cells. The present results indicate that both torulene and torularhodin can protect human prostate stromal cells from oxidative stress damage via Bcl-2/Bax mediated apoptosis.     

αB-crystallin regulates oxidative stress-induced apoptosis in cardiac H9c2 cells via the PI3K/AKT pathway

The present study was carried out to observe the protective effects of αB-crystallin protein on hydrogen peroxide (H₂O₂)-induced injury in rat myocardial cells (H9c2) and to investigate the mechanisms of these protective effects at the cellular level, which could provide the experimental basis for future applications of αB-crystallin in the treatment of cardiovascular disease. Western blotting was used to measure the expression of αB-crystallin in cultured H9c2 cells in vitro. A αB-crystallin recombinant expression vector, pcDNA3.1-Cryab, was constructed to transfect H9c2 cells for the establishment of cells that stably expressed αB-crystallin. A tetrazolium-based colorimetric assay (MTT test) was used to measure changes in the viability of the H9c2 cells at 1, 2, 3 and 4 h after induced by 150 μM H₂O₂ to establish a model of H₂O₂ injury to cells. H₂O₂ was applied to H9c2 cells that were stably transfected with αB-crystallin, and the effect of αB-crystallin overexpression on the viability of myocardial cells subjected to H₂O₂-induced injury was measured by the MTT assay. The effect of αB-crystallin overexpression on the H₂O₂-induced injury of H9c2 cells was also analyzed by flow cytometry. The mitochondrial components and cytoplasmic components of H9c2 cells were separated, and western blotting was used to measure the effect of αB-crystallin overexpression on the release of cytochrome c from the mitochondria. Western blotting was also used to measure the effect of αB-crystallin overexpression on the expression of the anti-apoptosis protein Bcl-2 and components of the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathway. The αB-crystallin recombinant expression vector pcDNA3.1-Cryab successfully transfected H9c2 cells, and H9c2 cells that were stably transfected with αB-crystallin were established after G418 selection. The measurements carried out by western blotting showed that αB-crystallin proteins are expressed in normal H9c2 cells, but the proteins’ expression was much higher in pcDNA3.1-Cryab transfected cells (P < 0.01). The MTT assays showed that 4 h of H₂O₂ treatment induced significant injury in H9c2 cells (P < 0.01), but αB-crystallin overexpression can effectively antagonize the H₂O₂-induced injury to H9c2 cells (P < 0.05). The results of flow cytometry analysis showed that αB-crystallin overexpression can significantly reduce apoptosis in H₂O₂-injured H9c2 cells (P < 0.05). The results of western blotting showed that αB-crystallin overexpression in myocardial cells can reduce the H₂O₂-induced release of cytochrome c from the mitochondria (P < 0.05), antagonize the H₂O₂-induced downregulation of Bcl-2 (P < 0.05) and magnify the decrease in phosphorylated AKT levels induced by H₂O₂ injury (P < 0.05). The overexpression of αB-crystallin has a protective effect on H₂O₂-injured H9c2 cells, and αB-crystallin can play a protective role by reducing apoptosis, reducing the release of cytochrome c from the mitochondria and antagonizing the downregulation of Bcl-2 expression. The protective effects of αB-crystallin may be related to the PI3K/AKT pathway.