过氧化氢预处理对抗氧化应激诱导的PC12细胞凋亡

氧化应激可明显地诱导细胞凋亡。本研究旨在探讨H2O2预处理能否对H2O2诱导的PC12细胞凋亡生产保护作用及ATP敏感性K^ （ATP-sensitive potassinm,KATP）通道在其中的作用。采用PI染色流式细胞仪（flow cytometry, FCM）检测PC12细胞凋亡。结果表明,经10μmol/L H2O2预处理90min的PC12细胞,分别在20、30、50和100μmol/L H2O2作用24h后,其细胞凋亡率明显下降,与未经H2O2的预处理的PC12细胞相比,差异极显著（P＜0.01）,表明H2O2预处理对H2O2诱导PC12细胞凋亡具有保护作用。用10μmol/L的KATP通道激动齐pinacidil(Pin)可显著减少30和50μmol/L H2O2诱导的PC12细胞凋亡,10μmol/L的KATP通道拮抗齐glybenclamide（Gly）则可显著地抑制甚至取消KATP通道激动剂Pin对H2O3诱导PC12细胞凋亡的保护作用,但并不影响H2O2预处理对H2O2诱导PC12细胞凋亡的保护作用;然而,当联合应用H2O2预处理与Pin时,对PC12细胞凋亡的保护作用显大于各自的细胞凋亡作用。提示KATP通道开放不仅对H2O2诱导PC12细胞凋亡具有保护作用,而且与H2O2预处理一起产生抗PC12细胞凋亡的协同作用。但KATP通道开放可能不参与H2O2预处理的适应性保护作用。     

Hydrogen peroxide preconditioning protects PC12 cells against apoptosis induced by dopamine

Dopamine (DA), one of the major sources of reactive oxygen species (ROS), is implicated in neuronal death associated with Parkinson's disease (PD). Preconditioning with oxidative stress has been shown to provide cytoprotection similar to ischemic preconditioning (IPC), against cell apoptosis. In this study, using the model neurosecretory cell line, PC12, we investigated whether hydrogen peroxide (H(2)O(2)) at low concentration (10 microM) can protect PC12 cells against apoptosis induced by DA. PC12 cells were preconditioned with 10 microM H(2)O(2) for 90 min, followed by 24-h recovery and subsequent exposures to different concentrations (20, 50, 100 and 200 microM) of DA for 24-h, respectively. DA induced apoptotic cell death with significant morphological nuclear changes and DNA fragmentation as well as the dysfunction of mitochondria. Preconditioning with H(2)O(2) at 10 microM significantly reduced the percentage of apoptotic cells and partly blocked the decreases in 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction and mitochondrial membrane potential (MMP) induced by DA. These results suggest that preconditioning with low concentration of H(2)O(2) protected PC12 cells against DA-induced apoptosis, the part restoration of the damaged mitochondrial functions might be one of the underlying mechanisms of this cytoprotection.     

Insulin-like growth factor-1 protects H9c2 cardiac myoblasts from oxidative stress-induced apoptosis via phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways

Oxidative stress plays a critical role in cardiac injuries during ischemia/reperfusion. Insulin-like growth factor-1 (IGF-1) promotes cell survival in a number of cell types, but the effect of IGF-1 on the oxidative stress has not been elucidated in cardiac muscle cells. Therefore, we examined the role of IGF-1 signaling pathway in cell survival against H2O2-induced apoptosis in H9c2 cardiac myoblasts. H2O2 treatment induced apoptosis in H9c2 cells, and pretreatment of cells with IGF-1 suppressed apoptotic cell death. The antiapoptotic effect of IGF-1 was blocked by LY294002 (an inhibitor of phosphatidylinositol 3-kinase) and by PD98059 (an inhibitor of extracellular signal-regulated kinase (ERK)). The protective effect of IGF-1 was also blocked by rapamycin (an inhibitor of p70 S6 kinase). Furthermore, H9c2 cells stably transfected with constitutively active PI 3-kinase (H9c2-p110*) and Akt (H9c2-Gag-Akt) constructs were more resistant to H2O2 cytotoxicity than control cells. Although H2O2 activates both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), IGF-1 inhibited only JNK activation. Activated PI 3-kinase (H9c2-p110*) and pretreatment of cells with IGF-1 down-regulated Bax protein levels compared to control cells. Taken together, our results suggest that IGF-1 transmits a survival signal against oxidative stress-induced apoptosis in H9c2 cells via PI 3-kinase and ERK-dependent pathways and the protective effect of IGF-1 is associated with the inhibition of JNK activation and Bax expression.     

Tyrosine phosphorylation regulates the proteolytic activation of protein kinase Cdelta in dopaminergic neuronal cells

Oxidative stress is a key apoptotic stimulus in neuronal cell death and has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinson disease (PD). Recently, we demonstrated that protein kinase C-delta (PKCdelta) is an oxidative stress-sensitive kinase that can be activated by caspase-3-dependent proteolytic cleavage to induce apoptotic cell death in cell culture models of Parkinson disease (Kaul, S., Kanthasamy, A., Kitazawa, M., Anantharam, V., and Kanthasamy, A. G. (2003) Eur. J. Neurosci. 18, 1387-1401 and Kanthasamy, A. G., Kitazawa, M., Kanthasamy, A., and Anantharam, V. (2003) Antioxid. Redox. Signal. 5, 609-620). Here we showed that the phosphorylation of a tyrosine residue in PKCdelta can regulate the proteolytic activation of the kinase during oxidative stress, which consequently influences the apoptotic cell death in dopaminergic neuronal cells. Exposure of a mesencephalic dopaminergic neuronal cell line (N27 cells) to H(2)O(2)(0-300 microm) induced a dose-dependent increase in cytotoxicity, caspase-3 activation and PKCdelta cleavage. H(2)O(2)-induced proteolytic activation of PKC was delta mediated by the activation of caspase-3. Most interestingly, both the general Src tyrosine kinase inhibitor genistein (25 microm) and the p60(Src) tyrosine-specific kinase inhibitor (TSKI; 5 microm) dramatically inhibited H(2)O(2) and the Parkinsonian toxin 1-methyl-4-phenylpyridinium-induced PKCdelta cleavage, kinase activation, and apoptotic cell death. H(2)O(2) treatment also increased phosphorylation of PKCdelta at tyrosine site 311, which was effectively blocked by co-treatment with TSKI. Furthermore, N27 cells overexpressing a PKCdelta(Y311F) mutant protein exhibited resistance to H(2)O(2)-induced PKCdelta cleavage, caspase activation, and apoptosis. To our knowledge, these data demonstrate for the first time that phosphorylation of Tyr-311 on PKCdelta can regulate the proteolytic activation and proapoptotic function of the kinase in dopaminergic neuronal cells.     

Syk is required for the activation of Akt survival pathway in B cells exposed to oxidative stress

Syk has been demonstrated to play a crucial role in oxidative stress signaling in B cells. Here we report that Syk is required for the activation of the phosphatidylinositol (PI) 3-kinase-Akt survival pathway in B cells exposed to oxidative stress. Phosphorylation and activation of the serine-threonine kinase Akt were markedly increased in B cells treated with H(2)O(2). In Syk-deficient DT40 cells treated with low doses of H(2)O(2) (10-100 microm), Akt activation was considerably reduced. Pretreatment with wortmannin, a PI 3-kinase-specific inhibitor, completely blocked the Syk-dependent Akt activation. Following stimulation by low doses of H(2)O(2), a significant increase in PI 3-kinase activity was found in wild-type but not in Syk-deficient cells. These findings suggest that PI 3-kinase mediates Syk-dependent Akt activation pathway. Furthermore, viability of Syk-deficient cells, after exposure to H(2)O(2), was dramatically decreased and caspase-9 activity was greatly increased compared with that of the wild-type cells. These results suggest that Syk is essential for the Akt survival pathway in B cells and enhances cellular resistance to oxidative stress-induced apoptosis.     

Ceramide and reactive oxygen species generated by H2O2 induce caspase-3-independent degradation of Akt/protein kinase B

This study was designed to elucidate the mechanisms leading to down-regulation of the Akt/protein kinase B (PKB) survival pathway during H2O2-induced cell death. H2O2 produced early activation of Akt/PKB and also DNA damage that was followed by stabilization of p53 levels, formation of reactive oxygen species (ROS), and generation of ceramide through activation of a glutathione-sensitive neutral sphingomyelinase. These events correlated with long term dephosphorylation and subsequent degradation of Akt. A membrane-targeted active Akt version attenuated apoptosis but not necrosis induced by H2O2 and was more resistant to dephosphorylation and proteolysis induced by apoptotic concentrations of H2O2. Proteolysis of Akt was prevented by exogenous addition of glutathione, indicating a role of ROS and ceramide in Akt degradation. However, Akt was degraded similarly in cells transfected with wild type and dominant negative p53 mutant, indicating that degradation of Akt under oxidative injury may be p53-independent. Specific inhibitors of caspase groups I and III prevented proteolysis of Akt/PKB and poly(ADP-ribose) polymerase in cells submitted to apoptotic but not necrotic H2O2 concentrations. Surprisingly, in caspase-3-deficient MCF-7 cells Akt was more sensitive to H2O2-induced degradation than the caspase-3 substrate poly(ADP-ribose) polymerase. Moreover, the Akt/PKB double mutant Akt(D108A,D119A), which is not cleaved by caspase-3, and a triple mutant (D453A,D455A,D456A), which lacks the consensus sequence for caspase-3 cleavage, were also degraded in H2O2-treated cells. Our results suggest that strong oxidants generate intracellular ROS and ceramide which in term lead to down-regulation of Akt by dephosphorylation and caspase-3-independent proteolysis.     

Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival

The serine/threonine kinase Akt (also known as protein kinase B) is activated in response to various stimuli by a mechanism involving phosphoinositide 3-kinase (PI3-K). Akt provides a survival signal that protects cells from apoptosis induced by growth factor withdrawal, but its function in other forms of stress is less clear. Here we investigated the role of PI3-K/Akt during the cellular response to oxidant injury. H(2)O(2) treatment elevated Akt activity in multiple cell types in a time- (5-30 min) and dose (400 microM-2 mm)-dependent manner. Expression of a dominant negative mutant of p85 (regulatory component of PI3-K) and treatment with inhibitors of PI3-K (wortmannin and LY294002) prevented H(2)O(2)-induced Akt activation. Akt activation by H(2)O(2) also depended on epidermal growth factor receptor (EGFR) signaling; H(2)O(2) treatment led to EGFR phosphorylation, and inhibition of EGFR activation prevented Akt activation by H(2)O(2). As H(2)O(2) causes apoptosis of HeLa cells, we investigated whether alterations of PI3-K/Akt signaling would affect this response. Wortmannin and LY294002 treatment significantly enhanced H(2)O(2)-induced apoptosis, whereas expression of exogenous myristoylated Akt (an activated form) inhibited cell death. Constitutive expression of v-Akt likewise enhanced survival of H(2)O(2)-treated NIH3T3 cells. These results suggest that H(2)O(2) activates Akt via an EGFR/PI3-K-dependent pathway and that elevated Akt activity confers protection against oxidative stress-induced apoptosis.     

Hydrogen peroxide-mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones: dependence on Ca2+ and PI3-kinase

Primary cortical neurones exposed to an oxidative insult in the form of hydrogen peroxide (H(2)O(2)) for 30 min showed a concentration-dependent increase in oxidative stress followed by a delayed NMDA receptor-dependent cell death measured 24 h later. Extracellular signal-regulated protein kinase (ERK1/2), c-jun N-terminal kinase (JNK) and the kinase Akt/PKB may regulate neuronal viability in response to oxidative insults. Using phospho-specific antibodies, a 15-min stimulation of neurones with H(2)O(2) (100 microm - 1 mm) produced a concentration-dependent phosphorylation of ERK1/2 and Akt/PKB that was partly dependent on extracellular Ca(2+) and phosphatidylinositol 3-kinase (PI3-K). Higher concentrations of H(2)O(2) (1 mm) also stimulated a phosphorylation of JNK which was totally dependent on extracellular Ca(2+) but not PI3-K. H(2)O(2)-induced phosphorylation of ERK1/2, Akt/PKB or JNK were unaffected by the NMDA channel blocker MK801. Blocking ERK1/2 activation with the upstream inhibitor U0126 (10 microm) enhanced H(2)O(2)-induced (100-300 microm range) neurotoxicity and inhibited H(2)O(2)-mediated phosphorylation of the cyclic AMP regulatory binding protein (CREB), suggesting that ERK1/2 signals to survival under these conditions. At higher concentrations (mm), H(2)O(2)-stimulated a phosphorylation of c-jun. It is likely, therefore, that subjecting neurones to moderate oxidative-stress recruits pro-survival signals to CREB but during severe oxidative stress pro-death signals through JNK and c-jun are dominant.     

Induction of heat shock proteins (HSPs) by sodium arsenite in cultured astrocytes and reduction of hydrogen peroxide-induced cell death

Induction of heat shock proteins (HSPs) protects cells from oxidative injury. Here Hsp72, Hsp27 and heme oxygenase-1 (HO-1) were induced in cultured rat astrocytes, and protection against oxidative stress was investigated. Astrocytes were treated with sodium arsenite (20-50 micro m) for 1 h, which was non-toxic to cells, 24 h later they were exposed to 400 micro m H2O2 for 1 h, and cell death was evaluated at different time points. Arsenite triggered strong induction of HSPs, which was prevented by 1 micro g/mL cycloheximide (CXH). H2O2 caused cell loss and increased cell death with features of apoptosis, i.e. TdT-mediated dUTP nick-end labelling (TUNEL) reaction and caspase-3 activation. These features were abrogated by pre-treatment with arsenite, which prevented cell loss and significantly reduced the number of dead cells. The protective effect of arsenite was not detected in the presence of CHX. Pre-treatment with arsenite increased protein kinase B (Akt) and extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation after H2O2. However, while Akt phosphorylation was prevented by CHX, Erk1/2 phosphorylation was further enhanced by CHX. The results show that transient arsenite pre-treatment induces Hsp72, HO-1 and, to a lesser extent, Hsp27; it reduces H2O2-induced astrocyte death; and it causes selective activation of Akt following H2O2. It is suggested that HSP expression at the time of H2O2 exposure protects astrocytes from oxidative injury and apoptotic cell death by means of pro-survival Akt.     

Vitamin E protected cultured cortical neurons from oxidative stress-induced cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase

The role of vitamin E in the CNS has not been fully elucidated. In the present study, we found that pre-treatment with vitamin E analogs including alphaT (alpha-tocopherol), alphaT3 (alpha -tocotrienol), gammaT, and gammaT3 for 24 h prevented the cultured cortical neurons from cell death in oxidative stress stimulated by H2O2, while Trolox, a cell-permeable analog of alphaT, did not. The preventive effect of alphaT was dependent on de novo protein synthesis. Furthermore, we found that alphaT exposure induced the activation of both the MAP kinase (MAPK) and PI3 kinase (PI3K) pathways and that the alphaT-dependent survival effect was blocked by the inhibitors, U0126 (an MAPK pathway inhibitor) or LY294002 (a PI3K pathway inhibitor). Interestingly, the up-regulation of Bcl-2 (survival promoting molecule) was induced by alphaT application. The up-regulation of Bcl-2 did not occur in the presence of U0126 or LY294002, suggesting that alphaT-up-regulated Bcl-2 is mediated by these kinase pathways. These observations suggest that vitamin E analogs play an essential role in neuronal maintenance and survival in the CNS.     

Zinc Inhibits H2O2-Induced MC3T3-E1 Cells Apoptosis via MAPK and PI3K/AKT Pathways

Zinc has been shown to increase bone mass and promote bone cell proliferation and differentiation. We, therefore, hypothesized that zinc might be cytoprotective for bone cells during oxidative stress. The cells were divided into H(2)O(2), zinc and zinc+H(2)O(2) groups. In the present study, zinc was found to inhibit H(2)O(2)-induced apoptosis in MC3T3-E1 cells, as shown by analysis of Annexin V/PI double staining. Western blot data showed that in zinc+H(2)O(2)-treated cells, zinc decreased the levels of AIF, Bax and active caspase-9 and -3, which are pro-apoptotic factors. And zinc inhibited release of cytochrome c from mitochondria to cytosol in zinc+H(2)O(2)-treated cells. Further investigation shows that protection is via activation of PI3K/Akt/mTor and MAPK /ERK pathways and inhibition of MAPK/P38 and MAPK/JNK pathways. Protecting osteoblast cells from oxidative damage presents a potential application in the treatment of osteoporosis.     

Diminished Akt phosphorylation in neurons lacking glutathione peroxidase-1 (Gpx1) leads to increased susceptibility to oxidative stress-induced cell death

We have previously identified an increased susceptibility of glutathione peroxidase-1 (Gpx1)-/- mice to neuronal apoptosis following mid-cerebral artery (MCA) occlusion. This study was designed to elucidate the mechanisms involved in elevated neuronal cell death arising from an altered endogenous oxidant state. This was addressed in both an in vitro and in vivo model of oxidative stress in the form of exogenous H2O2 and cerebral ischaemia, respectively. Increased levels of cell death were detected in primary neurons lacking Gpx1 following the addition of exogenous H2O2. This increased apoptosis correlated with a down-regulation in the activation of the phospho-inositide 3-kinase [PI3K]-Akt survival pathway. The importance of this pathway in protecting against H2O2-induced cell death was highlighted by the increased susceptibility of wildtype neurons to apoptosis when treated with the PI3K inhibitor, LY294002. The Gpx1-/- mice also demonstrated elevated neuronal cell death following MCA occlusion. Although Akt phosphorylation was detected in the Gpx1-/- brains, activation was not seen in later reperfusion events, as demonstrated in wildtype brains. Previous studies have highlighted the importance of Akt phosphorylation in protecting against neuronal cell death following cerebral ischaemia-reperfusion. Our results suggest that the increased susceptibility of Gpx1-/- neurons to H2O2-induced apoptosis and neuronal cell death in vivo following cerebral ischaemia-reperfusion injury can be attributed in part to diminished activation of Akt. Perturbations in key anti-apoptotic mechanisms as a result of an altered redox state may have implications in the study of oxidative stress-mediated neuropathologies.     

Correlation of mitogen-activated protein kinase activities with cell survival and apoptosis in porcine granulosa cells

The regulation of granulosa cell survival and death is critical for determining the fate of ovarian follicles. Mitogen-activated protein kinases (MAPKs) play central roles in various cellular responses, but the relationship between MAPK activities and granulosa cell survival as well as death is poorly understood. The present study examines the roles of the extracellular signal-regulated kinase (ERK) and p38 MAPK activities in porcine granulosa cells in response to survival factors and oxidative stress. Cell survival and apoptosis were evaluated by Trypan blue staining, DNA fragmentation, and chromatin staining with Hoechst 33342. Cell survival induced by serum or by follicle-stimulating hormone (FSH) was inhibited when ERK activity was attenuated with PD98059, which led to the induction of apoptosis. The p38 inhibitor SB203580 significantly decreased the cell survival evoked by FSH, but not by serum. Even in the presence of 10% serum, H(2)O(2) caused apoptosis, indicating that H(2)O (2) may be an atretogenic factor or its mediator. Interestingly, this induction of apoptosis was also prevented by SB203580, suggesting that p38 is involved in an apoptotic pathway induced by H(2)O (2) as well as in a survival pathway evoked by FSH in granulosa cells. These results indicate that whereas ERK activity is critical to the survival of granulosa cells, p38 activity contributes to their survival or apoptosis depending on the stimulus.     

过氧化氢预处理对H9c2心肌细胞氧化应激损伤的保护作用

目的：活性氧介导的氧化损伤是缺血再灌注损伤的重要机制,本研究通过观察H2O2预处理对氧化损伤的H9c2心肌细胞存活率和细胞凋亡的影响,探讨其保护H9c2心肌细胞的作用机制。方法：体外培养H9c2心肌细胞,取对数生长期细胞用于实验研究。建立H2O2预处理抵抗高浓度H：O：诱导的细胞氧化损伤模型,实验分组如下：（1）正常对照组（CTL）;（2）损伤组（INJURY）;（3）预处理组十损伤组（PC）。应用CCK8法检测细胞存活率;试剂盒检测胞内MDA水平和T．sOD活性;Hoechst33258染色观察凋亡形态;Annexin-V／PI双染与流式细胞术检测细胞凋亡率。结果：25vLmol／L的H202预处理90rain能明显地保护H9c2心肌细胞抵抗400μmol／LH2O2诱导的氧化损伤,提高细胞存活率,下调MDA水平,上调SOD活性,抑制细胞凋亡,降低细胞凋亡率。结论：低浓度H2O2预处理能减轻H9c2心肌细胞的氧化损伤,抑制氧化损伤诱导的心肌细胞凋亡,具有很好的抗氧化损伤和抗心肌细胞凋亡的保护作用,其作用机制可能与细胞SOD活性上调有关。H2O2预处理为临床治疗心肌缺血／再灌注损伤提供了一项新策略。     

Angiopoietin-1 attenuates H2O2-induced SEK1/JNK phosphorylation through the phosphatidylinositol 3-kinase/Akt pathway in vascular endothelial cells

Oxidative stress activates various signal transduction pathways, including Jun N-terminal kinase (JNK) and its substrates, that induce apoptosis. We reported here the role of angiopoietin-1 (Ang1), which is a prosurvival factor in endothelial cells, during endothelial cell damage induced by oxidative stress. Hydrogen peroxide (H2O2) increased apoptosis of endothelial cells through JNK activation, whereas Ang1 inhibited H2O2-induced apoptosis and concomitant JNK phosphorylation. The inhibition of H2O2-induced JNK phosphorylation was reversed by inhibitors of phosphatidylinositol (PI) 3-kinase and dominant-negative Akt, and constitutively active-Akt attenuated JNK phosphorylation without Ang1. These data suggested that Ang1-dependent Akt phosphorylation through PI 3-kinase leads to the inhibition of JNK phosphorylation. H2O2-induced phosphorylation of SAPK/Erk kinase (SEK1) at Thr261, which is an upstream regulator of JNK, was also attenuated by Ang1-dependent activation of the PI 3-kinase/Akt pathway. In addition, Ang1 induced SEK1 phosphorylation at Ser80, suggesting the existence of an additional signal transduction pathway through which Ang1 attenuates JNK phosphorylation. These results demonstrated that Ang1 attenuates H2O2-induced SEK1/JNK phosphorylation through the PI 3-kinase/Akt pathway and inhibits the apoptosis of endothelial cells to oxidative stress.     

Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells

Vascular endothelial growth factor (VEGF) utilizes a phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling pathway to protect endothelial cells from apoptotic death. Here we show that PI 3-kinase/Akt signaling promotes endothelial cell survival by inhibiting p38 mitogen-activated protein kinase (MAPK)-dependent apoptosis. Blockade of the PI 3-kinase or Akt pathways in conjunction with serum withdrawal stimulates p38-dependent apoptosis. Blockade of PI 3-kinase/Akt also led to enhanced VEGF activation of p38 and apoptosis. In this context, the pro-apoptotic effect of VEGF is attenuated by the p38 MAPK inhibitor SB203580. VEGF stimulation of endothelial cells or infection with an adenovirus expressing constitutively active Akt causes MEKK3 phosphorylation, which is associated with decreased MEKK3 kinase activity and down-regulation of MKK3/6 and p38 MAPK activation. Conversely, activation-deficient Akt decreases VEGF-stimulated MEKK3 phosphorylation and increases MKK/p38 activation. Activation of MKK3/6 is not dependent on Rac activation since dominant negative Rac does not decrease p38 activation triggered by inhibition of PI 3-kinase. Thus, cross-talk between the Akt and p38 MAPK pathways may regulate the level of cytoprotection versus apoptosis and is a new mechanism to explain the cytoprotective actions of Akt.