Adaptation to acrolein through upregulating the protection by glutathione in human bronchial epithelial cells: The materialization of the hormesis concept

Acrolein is a thiol reactive compound present in cigarette smoke and plays a pivotal role in the deleterious effects of smoking. Acrolein causes toxicity in human bronchial epithelial cells in a dose dependent manner. GSH forms the first line of defense against acrolein-induced toxicity. At high doses of acrolein (?10 μM) the capacity of the cellular protection by GSH is overwhelmed and GSH is not able to quench all the acrolein, resulting in cytotoxicity.     

Differential roles of 3H-1,2-dithiole-3-thione-induced glutathione, glutathione S-transferase and aldose reductase in protecting against 4-hydroxy-2-nonenal toxicity in cultured cardiomyocytes

4-hydroxy-2-nonenal (HNE) plays an important role in the pathogenesis of cardiac disorders. While conjugation with glutathione (GSH) catalyzed by GSH S-transferase (GST) has been suggested to be a major detoxification mechanism for HNE in target cells, whether chemically upregulated cellular GSH and GST afford protection against HNE toxicity in cardiac cells has not been investigated. In addition, the differential roles of chemically induced GSH and GST as well as other cellular factors in detoxifying HNE in cardiomyocytes are unclear. In this study, we have characterized the induction of GSH and GST by 3H-1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-elevated cellular defenses on HNE-mediated toxicity in rat H9C2 cardiomyocytes. Treatment of cardiomyocytes with D3T resulted in a significant induction of both GSH and GST as well as the mRNA expression of gamma-glutamylcysteine ligase catalytic subunit and GSTA. Both GSH and GST remained elevated for at least 72 h after removal of D3T from the culture media. Treatment of cells with HNE led to a significant decrease in cell viability and an increased formation of HNE-protein adducts. Pretreatment of cells with D3T dramatically protected against HNE-mediated cytotoxicity and protein-adduct formation. HNE treatment caused a significant decrease in cellular GSH level, which preceded the loss of cell viability. Either depletion of cellular GSH by buthionine sulfoximine (BSO) or inhibition of GST by sulfasalazine markedly sensitized the cells to HNE toxicity. Co-treatment of cardiomyocytes with BSO was found to completely block the D3T-mediated GSH elevation, which however failed to reverse the cytoprotective effects of D3T, suggesting that other cellular factor(s) might be involved in D3T cytotprotection. In this regard, D3T was shown to induce cellular aldose reductase (AR). Surprisingly, inhibition of AR by sorbinil failed to potentiate HNE toxicity in cardiomyocytes. In contrast, sorbinil dramatically augmented HNE cytotoxicity in cells with GSH depletion induced by BSO. Similarly, in BSO-treated cells, D3T cytoprotection was also largely reversed by sorbinil, indicating that AR played a significant role in detoxifying HNE only under the condition of GSH depletion in cardiomyocytes. Taken together, this study demonstrates that D3T can induce GSH, GST, and AR in cardiomyocytes, and that the above cellular factors appear to play differential roles in detoxification of HNE in cardiomyocytes.     

The role of oxidative stress in acrolein-induced DNA damage in HepG2 cells

This study evaluated the role of oxidative stress in acrolein-induced DNA damage, using HepG2 cells. Using the standard single cell gel electrophoresis (SCGE) assay, a significant dose-dependent increment in DNA migration was detected at lower concentrations of acrolein; but at the higher tested concentrations, a reduction in the migration was observed. Post-incubation with proteinase K significantly increased DNA migration in cells exposed to higher concentrations of acrolein. These results indicated that acrolein caused DNA strand breaks and DNA-protein crosslinks (DPC). To elucidate the oxidatively generated DNA damage mechanism, the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) were used to monitor the levels of reactive oxygen species (ROS) and glutathione (GSH), respectively. The present study showed that acrolein induced the increased levels of ROS and depletion of GSH in HepG2 cells. Moreover, acrolein significantly caused 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) formation in HepG2 cells. These results demonstrate that the DNA damage induced by acrolein in HepG2 cells is related to the oxidative stress.     

The role of oxidative stress in acrolein-induced DNA damage in HepG2 cells

This study evaluated the role of oxidative stress in acrolein-induced DNA damage, using HepG2 cells. Using the standard single cell gel electrophoresis (SCGE) assay, a significant dose-dependent increment in DNA migration was detected at lower concentrations of acrolein; but at the higher tested concentrations, a reduction in the migration was observed. Post-incubation with proteinase K significantly increased DNA migration in cells exposed to higher concentrations of acrolein. These results indicated that acrolein caused DNA strand breaks and DNA-protein crosslinks (DPC). To elucidate the oxidatively generated DNA damage mechanism, the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) were used to monitor the levels of reactive oxygen species (ROS) and glutathione (GSH), respectively. The present study showed that acrolein induced the increased levels of ROS and depletion of GSH in HepG2 cells. Moreover, acrolein significantly caused 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) formation in HepG2 cells. These results demonstrate that the DNA damage induced by acrolein in HepG2 cells is related to the oxidative stress.     

Induction of endogenous glutathione by the chemoprotective agent, 3H-1,2-dithiole-3-thione, in human neuroblastoma SH-SY5Y cells affords protection against peroxynitrite-induced cytotoxicity

Substantial evidence suggests that peroxynitrite generated from the bi-radical reaction of nitric oxide and superoxide is critically involved in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Reaction with sulfhydryl (SH)-containing molecules has been proposed to be a major detoxification pathway of peroxynitrite in biological systems. This study was undertaken to determine if chemically elevated intracellular reduced glutathione (GSH), a major SH-containing biomolecule, affords protection against peroxynitrite-mediated toxicity in cultured neuronal cells. Incubation of human neuroblastoma SH-SY5Y cells with the unique chemoprotectant, 3H-1,2-dithiole-3-thione (D3T), led to a significant elevation of cellular GSH in a concentration-dependent fashion. To examine the protective effects of D3T-induced GSH on peroxynitrite-mediated toxicity, SH-SY5Y cells were pretreated with D3T and then exposed to either the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1), or the authentic peroxynitrite. We observed that D3T-pretreated cells showed a markedly increased resistance to SIN-1- or authentic peroxynitrite-induced cytotoxicity, as assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium reduction assay. Conversely, depletion of cellular GSH by buthionine sulfoximine (BSO) caused a marked potentiation of SIN-1- or authentic peroxynitrite-mediated cytotoxicity. To further demonstrate the causal role for GSH induction in D3T-mediated cytoprotection, SH-SY5Y cells were co-treated with BSO to abolish D3T-induced GSH elevation. Co-treatment of the cells with BSO was found to significantly reverse the protective effects of D3T on SIN-1- or authentic peroxynitrite-elicited cytotoxicity. Taken together, this study demonstrates for the first time that D3T can induce GSH in cultured SH-SY5Y cells, and that the D3T-augmented cellular GSH defense affords a marked protection against peroxynitrite-induced toxicity in cultured human neuronal cells.     

Protein alkylation by the α,β-unsaturated aldehyde acrolein. A reversible mechanism of electrophile signaling?

Acrolein, a reactive aldehyde found in cigarette smoke, is thought to induce its biological effects primarily by irreversible adduction to cellular nucleophiles such as cysteine thiols. Here, we demonstrate that acrolein rapidly inactivates the seleno-enzyme thioredoxin reductase (TrxR) in human bronchiolar epithelial HBE1 cells, which recovered over 4–8 h by a mechanism depending on the presence of cellular GSH and thioredoxin 1 (Trx1), and corresponding with reversal of protein–acrolein adduction. Our findings indicate that acrolein-induced protein alkylation is not necessarily a feature of irreversible protein damage, but may reflect a reversible signaling mechanism that is regulated by GSH and Trx1.     

The role of mitochondrial glutathione and cellular protein sulfhydryls in formaldehyde toxicity in glutathione-depleted rat hepatocytes

Depletion of cellular GSH by diethyl maleate (DEM) potentiates CH2O toxicity in isolated rat hepatocytes and it was postulated that this increase in toxicity is due to the further decrease in GSH caused by CH2O in DEM-pretreated hepatocytes (1). The present investigation was conducted to investigate further the effects of CH2O, DEM, and acrolein (a compound which is structurally related to CH2O and DEM) on subcellular GSH pools and on protein sulfhydryl groups (PSH). CH2O caused a decrease in cytosolic GSH but had no effect on mitochondrial GSH either in previously untreated hepatocytes or in DEM-pretreated hepatocytes in which GSH was approximately 25% of control. DEM decreased both cytosolic and mitochondrial GSH but it did not produce toxicity. Neither CH2O (up to 7.5 mM) nor DEM (20 mM) decreased PSH. However, in cells pretreated with 1 mM DEM, CH2O (7.5 mM) decreased PSH and this effect preceded cell death. Acrolein decreased both cytosolic and mitochondrial GSH and it also decreased PSH significantly prior to causing cell death. CH2O and acrolein stimulated phosphorylase alpha activity, indicative of an increase in cytosolic free Ca2+, by a PSH-independent and PSH-dependent mechanism, respectively. These results suggest that the further depletion of cellular GSH by CH2O in DEM-pretreated cells is not due to the depletion of mitochondrial GSH. CH2O toxicity in DEM-pretreated cells is, however, correlated with depletion of PSH. The critical sulfhydryl protein(s) responsible for cell death remain to be more clearly defined.     

Implications of altered glutathione metabolism in aspirin-induced oxidative stress and mitochondrial dysfunction in HepG2 cells

We have previously reported that acetylsalicylic acid (aspirin, ASA) induces cell cycle arrest, oxidative stress and mitochondrial dysfunction in HepG2 cells. In the present study, we have further elucidated that altered glutathione (GSH)-redox metabolism in HepG2 cells play a critical role in ASA-induced cytotoxicity. Using selected doses and time point for ASA toxicity, we have demonstrated that when GSH synthesis is inhibited in HepG2 cells by buthionine sulfoximine (BSO), prior to ASA treatment, cytotoxicity of the drug is augmented. On the other hand, when GSH-depleted cells were treated with N-acetyl cysteine (NAC), cytotoxicity/apoptosis caused by ASA was attenuated with a significant recovery in oxidative stress, GSH homeostasis, DNA fragmentation and some of the mitochondrial functions. NAC treatment, however, had no significant effects on the drug-induced inhibition of mitochondrial aconitase activity and ATP synthesis in GSH-depleted cells. Our results have confirmed that aspirin increases apoptosis by increased reactive oxygen species production, loss of mitochondrial membrane potential and inhibition of mitochondrial respiratory functions. These effects were further amplified when GSH-depleted cells were treated with ASA. We have also shown that some of the effects of aspirin might be associated with reduced GSH homeostasis, as treatment of cells with NAC attenuated the effects of BSO and aspirin. Our results strongly suggest that GSH dependent redox homeostasis in HepG2 cells is critical in preserving mitochondrial functions and preventing oxidative stress associated complications caused by aspirin treatment.     

Upregulation of endogenous glutathione system by 3H-1,2-dithiole-3-thione in pancreatic RINm5F beta-cells as a novel strategy for protecting against oxidative beta-cell injury

This study was undertaken to investigate the inducibility of glutathione (GSH), glutathione reductase (GR) and glutathione peroxidase (GPx) by 3H-1,2-dithiole-3-thione (D3T) in beta-cells, and the resultant cytoprotection against oxidant injury. Incubation of the insulin-secreting RINm5F cells with D3T led to significant induction of GSH, GR and GPx. D3T-mediated induction of GSH was abolished by buthionine sulfoximine (BSO), suggesting a critical involvement of γ-glutamylcysteine ligase (γGCL). Consistently, incubation of RINm5F cells with D3T resulted in increased expression of γGCL protein and mRNA. Pretreatment of RINm5F cells with D3T provided remarkable protection against oxidant-elicited cytotoxicity. On the other hand, depletion of cellular GSH by BSO sensitized RINm5F cells to oxidant injury. Furthermore, cotreatment of RINm5F cells with BSO to reverse D3T-mediated GSH induction abolished the cytoprotective effects of D3T on oxidant injury. Taken together, this study demonstrates that upregulation of glutathione system by D3T is effective for protecting against oxidative beta-cell injury.     

Differential Cytotoxicity of Acetaminophen in Mouse Macrophage J774.2 and Human Hepatoma HepG2 Cells: Protection by Diallyl Sulfide

Non-steroidal anti-inflammatory drugs (NSAIDs), including acetaminophen (APAP), have been reported to induce cytotoxicity in cancer and non-cancerous cells. Overdose of acetaminophen (APAP) causes liver injury in humans and animals. Hepatic glutathione (GSH) depletion followed by oxidative stress and mitochondrial dysfunction are believed to be the main causes of APAP toxicity. The precise molecular mechanism of APAP toxicity in different cellular systems is, however, not clearly understood. Our previous studies on mouse macrophage J774.2 cells treated with APAP strongly suggest induction of apoptosis associated with mitochondrial dysfunction and oxidative stress. In the present study, using human hepatoma HepG2 cells, we have further demonstrated that macrophages are a more sensitive target for APAP—induced toxicity than HepG2 cells. Using similar dose- and time-point studies, a marked increase in apoptosis and DNA fragmentation were seen in macrophages compared to HepG2 cells. Differential effects of APAP on mitochondrial respiratory functions and oxidative stress were observed in the two cell lines which are presumably dependent on the varying degree of drug metabolism by the different cytochrome P450s and detoxification by glutathione S-transferase enzyme systems. Our results demonstrate a marked increase in the activity and expression of glutathione transferase (GST) and multidrug resistance (MDR1) proteins in APAP-treated HepG2 cells compared to macrophages. This may explain the apparent resistance of HepG2 cells to APAP toxicity. However, treatment of these cells with diallyl sulfide (DAS, 200 μM), a known chemopreventive agent from garlic extract, 24 h prior to APAP (10 μmol/ml for 18h) exhibited comparable cytoprotective effects in the two cell lines. These results may help in better understanding the mechanism of cytotoxicity caused by APAP and cytoprotection by chemopreventive agents in cancer and non-cancerous cellular systems.     

Upregulation of endogenous glutathione system by 3H-1,2-dithiole-3-thione in pancreatic RINm5F beta-cells as a novel strategy for protecting against oxidative beta-cell injury

This study was undertaken to investigate the inducibility of glutathione (GSH), glutathione reductase (GR) and glutathione peroxidase (GPx) by 3H-1,2-dithiole-3-thione (D3T) in beta-cells, and the resultant cytoprotection against oxidant injury. Incubation of the insulin-secreting RINm5F cells with D3T led to significant induction of GSH, GR and GPx. D3T-mediated induction of GSH was abolished by buthionine sulfoximine (BSO), suggesting a critical involvement of γ-glutamylcysteine ligase (γGCL). Consistently, incubation of RINm5F cells with D3T resulted in increased expression of γGCL protein and mRNA. Pretreatment of RINm5F cells with D3T provided remarkable protection against oxidant-elicited cytotoxicity. On the other hand, depletion of cellular GSH by BSO sensitized RINm5F cells to oxidant injury. Furthermore, cotreatment of RINm5F cells with BSO to reverse D3T-mediated GSH induction abolished the cytoprotective effects of D3T on oxidant injury. Taken together, this study demonstrates that upregulation of glutathione system by D3T is effective for protecting against oxidative beta-cell injury.     

Role of Nrf2 signaling in regulation of antioxidants and phase 2 enzymes in cardiac fibroblasts: protection against reactive oxygen and nitrogen species-induced cell injury

Understanding the molecular pathway(s) of antioxidant gene regulation is of crucial importance for developing antioxidant-inducing agents for the intervention of oxidative cardiac disorders. Accordingly, this study was undertaken to determine the role of Nrf2 signaling in the basal expression as well as the chemical inducibility of endogenous antioxidants and phase 2 enzymes in cardiac fibroblasts. The basal expression of a scope of key cellular antioxidants and phase 2 enzymes was significantly lower in cardiac fibroblasts derived from Nrf2-/- mice than those from wild type control. These include catalase, reduced glutathione (GSH), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase-1 (NQO1). Incubation of Nrf2+/+ cardiac fibroblasts with 3H-1,2-dithiole-3-thione (D3T) led to a significant induction of superoxide dismutase (SOD), catalase, GSH, GR, glutathione peroxidase (GPx), GST, and NQO1. The inducibility of SOD, catalase, GSH, GR, GST, and NQO1, but not GPx by D3T was completely abolished in Nrf2-/- cells. The Nrf2-/- cardiac fibroblasts were much more sensitive to reactive oxygen and nitrogen species-mediated cytotoxicity. Upregulation of antioxidants and phase 2 enzymes by D3T in Nrf2+/+ cardiac fibroblasts resulted in a dramatically increased resistance to the above species-induced cytotoxicity. In contrast, D3T-treatment of the Nrf2-/- cells only provided a slight cytoprotection. Taken together, this study demonstrates for the first time that Nrf2 is critically involved in the regulation of the basal expression and chemical induction of a number of antioxidants and phase 2 enzymes in cardiac fibroblasts, and is an important factor in controlling cardiac cellular susceptibility to reactive oxygen and nitrogen species-induced cytotoxicity.     

Involvement of Oxidative Stress in Paraquat-Induced Metallothionein Synthesis Under Glutathione Depletion

The inhibition of glutathione (GSH) synthesis by -buthionine-SR-sulfoximine (BSO) causes aggravation of hepatotoxicity of paraquat (PQ), an oxidative-stress inducing substance, in mice. On the other hand, synthesis of metallothionein (MT), a cysteine-rich protein having radical scavenging activity, is induced by PQ, and the induction by PQ is significantly enhanced by pretreatment of mice with BSO. The purpose of present study is to examine whether generation of reactive oxygens is involved in the induction of MT synthesis by PQ under inhibition of GSH synthesis. Administration of PQ to BSO-pretreated mice increased hepatic lipid peroxidation and frequency of DNA single strand breakage followed by manifestation of the liver injury and induction of MT synthesis. Both vitamin E and deferoxamine prevented MT induction as well as lipid peroxidation in the liver of mice caused by administration of BSO and PQ. In cultured colon 26 cells, both cytotoxicity and the increase in MT mRNA level caused by PQ were significantly enhanced by pretreatment with BSO. Facilitation of PQ-induced reactive oxygen generation was also observed by BSO treatment. These results suggest that reactive oxygens generated by PQ under inhibition of GSH synthesis may stimulate MT synthesis. GSH depletion markedly increased reactive oxygen generation induced by PQ, probably due to the reduced cellular capability to remove the radical species produced.     

Water and methanolic extracts of Salvia officinalis protect HepG2 cells from t-BHP induced oxidative damage

Common sage (Salvia officinalis L., Lamiaceae) is an aromatic and medicinal plant well known for its antioxidant properties. Some in vivo studies have shown the biological antioxidant effects of sage. However, the intracellular antioxidant mechanisms of action are still poorly understood. In this study, we evaluated the cytoprotective effects of two sage extracts (a water and a methanolic extract) against tert-butyl hydroperoxide (t-BHP)-induced toxicity in HepG2 cells. The most abundant phenolic compounds present in the extracts were rosmarinic acid and luteolin-7-glucoside. Both extracts, when co-incubated with the toxicant, protected significantly HepG2 cells against cell death. The methanolic extract, with a higher content of phenolic compounds than the water extract, conferred better protection in this in vitro model of oxidative stress with liver cells. Both extracts, tested in a concentration that protects 80% against cell death (IC(80)), significantly prevented t-BHP-induced lipid peroxidation and GSH depletion, but not DNA damage assessed by the comet assay. The ability of sage extracts to reduce t-BHP-induced GSH depletion by 62% was probably the most relevant contributor to the observed cytoprotection. A good correlation between the above cellular effects of sage and the effects of their main phenolic compounds was found. When incubated alone for 5h, sage extracts induced an increase in basal GSH levels of HepG2 cells, which indicates an improvement of the antioxidant potential of the cells. Compounds present in sage extracts other than phenolics may also contribute to this latter effect. Based in these results, it would be of interest to investigate whether sage has protective effects in suitable in vivo models of liver diseases, where it is known that oxidative stress is involved.     

Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells

Aldose reductase (AR), a glucose-metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (K_m aldehydes 5–30 µM) relative to glucose. Acrolein, a major endogenous lipid peroxidation product as well as a component of environmental pollutants and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders, but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells (SAECs). Exposure of SAECs to varying concentrations of acrolein caused cell death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low-dose (5–10 µM) but not the high-dose (>10 µM) acrolein-induced SAEC death. AR inhibition protected SAECs from low-dose (5 µM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail moment, and annexin V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of the proapoptotic proteins Bax and Bad from the cytosol to the mitochondria and that of Bcl2 and Bcl_XL from the mitochondria to the cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases 1 and 2, stress-activated protein kinase/c-Jun NH₂-terminal kinase, and p38MAPK, and c-Jun were transiently activated in airway epithelial cells by acrolein in a concentration- and time-dependent fashion, which was significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells.     

Induction of COX-2 by acrolein in rat lung epithelial cells

Acrolein is a highly reactive alpha, beta-unsaturated aldehyde, and a product of lipid peroxidation reactions. Acrolein is also an environmental pollutant and a key component of cigarette smoke, and has been implicated in multiple respiratory diseases. Lung tissue is a primary target for acrolein toxicity in smokers and may lead to chronic lung inflammation and lung cancer. Chronic inflammation, associated with expression of cyclooxygenase-2 (COX-2) and prostaglandins, are predisposing factors for malignancy. In this study, we investigated the induction of COX-2 by acrolein in rat lung epithelial cells and its related signaling cascade. Induction of COX-2 by acrolein was significant at 6 h post-treatment and was dependent upon NFκB activation. The activation of NFκB by acrolein was induced as a result of degradation of IκBα over the time of treatment. In addition, the upstream signaling cascade involved Raf-1/ERK activation by acrolein in the COX-2 induction and was inhibited by GW5074 (a Ras/Raf-1/ERK inhibitor), thereby providing evidence for the role of this cascade in this process. The results of these studies offer an explanation for the mechanism of COX-2 induction by acrolein in rat lung epithelial cells.     

Acrolein-induced cell death in PC12 cells: role of mitochondria-mediated oxidative stress

Oxidative stress has been implicated in acrolein cytotoxicity in various cell types, including mammalian spinal cord tissue. In this study we report that acrolein also decreases PC12 cell viability in a reactive oxygen species (ROS)-dependent manner. Specifically, acrolein-induced cell death, mainly necrosis, is accompanied by the accumulation of cellular ROS. Elevating ROS scavengers can alleviate acrolein-induced cell death. Furthermore, we show that exposure to acrolein leads to mitochondrial dysfunction, denoted by the loss of mitochondrial transmembrane potential, reduction of cellular oxygen consumption, and decrease of ATP level. This raises the possibility that the cellular accumulation of ROS could result from the increased production of ROS in the mitochondria of PC12 cells as a result of exposure to acrolein. The acrolein-induced significant decrease of ATP production in mitochondria may also explain why necrosis, not apoptosis, is the dominant type of cell death. In conclusion, our data suggest that one possible mechanism of acrolein-induced cell death could be through mitochondria as its initial target. The subsequent increase of ROS then inflicts cell death and further worsens mitochondria function. Such mechanism may play an important role in CNS trauma and neurodegenerative diseases.