Chebulinic acid attenuates glutamate-induced HT22 cell death by inhibiting oxidative stress,calcium influx and MAPKs phosphorylation

Glutamate-induced excitotoxicity and oxidative stress is a major causative factor in neuronal cell death in acute brain injuries and chronic neurodegenerative diseases. The prevention of oxidative stress is a potential therapeutic strategy. Therefore, in the present study, we aimed to examine a potential therapeutic agent and its protective mechanism against glutamate-mediated cell death. We first found that chebulinic acid isolated from extracts of the fruit of Terminalia chebula prevented glutamate-induced HT22 cell death. Chebulinic acid significantly reduced intracellular reactive oxygen species (ROS) production and Ca²⁺ influx induced by glutamate. We further demonstrated that chebulinic acid significantly decreased the phosphorylation of mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, and p38, as well as inhibiting pro-apoptotic Bax and increasing anti-apoptotic Bcl-2 protein expression. Moreover, we demonstrated that chebulinic acid significantly reduced the apoptosis induced by glutamate in HT22 cells. In conclusion, our results in this study suggest that chebulinic acid is a potent protectant against glutamate-induced neuronal cell death via inhibiting ROS production, Ca²⁺ influx, and phosphorylation of MAPKs, as well as reducing the ratio of Bax to Bcl-2, which contribute to oxidative stress-mediated neuronal cell death.     

4-O-Methylhonokiol attenuates memory impairment in presenilin 2 mutant mice through reduction of oxidative damage and inactivation of astrocytes and the ERK pathway

Presenilin 2 (PS2) mutation increases Aβ generation and neuronal cell death in the brains of Alzheimer disease (AD) patients. In a previous study, we showed that increased oxidative damage and activation of extracellular signal-regulated kinase (ERK) were associated with Aβ generation and neuronal cell death in neuronal cells expressing mutant PS2. In this study, we show that oral treatment with 4-O-methylhonokiol, a novel compound isolated from Magnolia officinalis, for 3 months (1.0mg/kg) prevented PS2 mutation-induced memory impairment and neuronal cell death accompanied by a reduction in Aβ(1-42) accumulation. We also found that 4-O-methylhonokiol inhibited PS2 mutation-induced activation of ERK and β-secretase, and oxidative protein and lipid damage, but recovered glutathione levels in the cortex and hippocampus of PS2 mutant mice. Additionally, 4-O-methylhonokiol prevented PS2 mutation-induced activation of astrocytes as well as production of TNF-α, IL-1β, reactive oxygen species (ROS), and nitric oxide (NO) in neurons. Generation of TNF-α, IL-1β, ROS, and NO and ERK activation in cultured astrocytes treated with lipopolysaccharide (1μg/ml) were also prevented by 4-O-methylhonokiol in a dose-dependent manner. These results suggest that the improving effects of 4-O-methylhonokiol on memory function may be associated with a suppression of the activation of ERK and astrocytes as well as a reduction in oxidative damage. Thus, 4-O-methylhonokiol may be useful in the prevention and treatment of AD.     

Apigenin-induced apoptosis is mediated by reactive oxygen species and activation of ERK1/2 in rheumatoid fibroblast-like synoviocytes

Fibroblast-like synovial cells play a crucial role in the pathophysiology of rheumatoid arthritis (RA), as these cells are involved in inflammation and joint destruction. Apigenin, a dietary plant-flavonoid, is known to have many functions in animal cells including anti-proliferative and anticancer activities, but its role in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) has not been reported. In this study, we investigated the roles of apigenin in RA-FLSs. The survival rate decreased, and apoptotic cell death was induced by apigenin treatment in RA-FLSs. Apigenin treatment resulted in activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and pretreatment with an ERK inhibitor PD98059 dramatically reduced apigenin-induced apoptosis. We found that apigenin-mediated production of a large amount of intracellular reactive oxygen species (ROS) caused activation of ERK1/2 and apoptosis; treatment with the antioxidant Tiron strongly inhibited the apigenin-induced generation of ROS, phosphorylation of ERK1/2, and apoptotic cell death. Apigenin-induced apoptotic cell death was mediated through activation of the effectors caspase-3 and caspase-7, and was blocked by pretreatment with Z-VAD-FMK (a pan-caspase inhibitor). These results showed that apigenin-induced ROS and oxidative stress-activated ERK1/2 caused apoptotic cell death in apigenin-treated RA-FLSs.     

Nucleotide receptor signaling in murine macrophages is linked to reactive oxygen species generation

Macrophage activation is critical in the innate immune response and can be regulated by the nucleotide receptor P2X7. In this regard, P2X7 signaling is not well understood but has been implicated in controlling reactive oxygen species (ROS) generation by various leukocytes. Although ROS can contribute to microbial killing, the role of ROS in nucleotide-mediated cell signaling is unclear. In this study, we report that the P2X7 agonists ATP and 3'-O-(4-benzoyl) benzoic ATP (BzATP) stimulate ROS production by RAW 264.7 murine macrophages. These effects are potentiated in lipopolysaccharide-primed cells, demonstrating an important interaction between extracellular nucleotides and microbial products in ROS generation. In terms of nucleotide receptor specificity, RAW 264.7 macrophages that are deficient in P2X7 are greatly reduced in their capacity to generate ROS in response to BzATP treatment (both with and without LPS priming), thus supporting a role for P2X7 in this process. Because MAP kinase activation is key for nucleotide regulation of macrophage function, we also tested the hypothesis that P2X7-mediated MAP kinase activation is dependent on ROS production. We observed that BzATP stimulates MAP kinase (ERK1/ERK2, p38, and JNK1/JNK2) phosphorylation and that the antioxidants N-acetylcysteine and ascorbic acid strongly attenuate BzATP-mediated JNK1/JNK2 and p38 phosphorylation but only slightly reduce BzATP-induced ERK1/ERK2 phosphorylation. These studies reveal that P2X7 can contribute to macrophage ROS production, that this effect is potentiated upon lipopolysaccharide exposure, and that ROS are important participants in the extracellular nucleotide-mediated activation of several MAP kinase systems.     

Cytoprotection against hydrogen peroxide-induced cell death in cultured mouse mesangial cells by erigeroflavanone, a novel compound from the flowers of Erigeron annuus

Hyperglycemia-induced oxidative stress has been suggested as a mechanism underlying diabetic complications. Oxidative stress triggers cell death in various cell types, including glomerular mesangial cells which play important roles in diabetic nephropathy. In the present study, we investigated the potential cytoprotective effect of erigeroflavanone, a novel flavanone derivative from the flowers of Erigeron annuus, in cultured mouse mesangial cells using hydrogen peroxide (H₂O₂) as an oxidative stress inducer. Our data show that hydrogen peroxide induced a decrease in cell viability that was attenuated by erigeroflavanone. Hydrogen peroxide treatment increased formation of dichlorofluorescein (DCF)-sensitive intracellular reactive oxygen species (ROS). This enhanced ROS formation was significantly reduced by pretreatment with erigeroflavanone in a dose-dependent manner. Hydrogen peroxide treatment also induced phosphorylation of the mitogen-activated protein kinases (MAPKs), c-Jun terminal kinase (JNK), extracellular-regulated kinase (ERK) and p38, and activated caspase-3. Pretreatment with erigeroflavanone inhibited hydrogen peroxide-induced activation of MAPKs and caspase-3. From these data we conclude that erigeroflavanone provides a protective effect against oxidative stress-induced cell death in mesangial cells that is associated with its antioxidant action and inhibition of MAPKs and caspase-3. These results suggest that erigeroflavanone has potential as a therapeutic agent in the treatment of renal diabetic complications.     

Modulation of mercury-induced mitochondria-dependent apoptosis by glycine in hepatocytes

Mercury (Hg) is one of the universal environmental pollutants and is responsible for various organ pathophysiology including oxidative stress-induced hepatic disorders. In the present study, we aimed to explore the protective role of glycine in Hg-induced cytotoxicity and cell death in murine hepatocytes. Exposure of mercury (20 μM), in the form HgCl₂ for 1 h, significantly enhanced the ALT and ALP leakage, increased reactive oxygen species production, reduced cell viability and distorted the antioxidant status of hepatocytes. Flow cytometric analyses shows that Hg-induced apoptotic death in hepatocytes. Mechanism of this pathophysiology involves reduced mitochondrial membrane potential, variations in Bcl-2/Bad proteins, activation of caspases and cleavage of PARP protein. In addition, Hg distinctly increased NF-κB phosphorylation in association with IKKα phosphorylation and IκBα degradation. Concurrent treatment with glycine (45 mM), however, reduced Hg-induced oxidative stress, attenuated the changes in NF-κB phosphorylation and protects hepatocytes from Hg-induced apoptotic death. Hg also distinctly increased the phosphorylation of p38, JNK and ERK mitogen-activated protein kinase (MAPKs). Glycine treatment suppressed these apoptotic events, signifying its protective role in Hg-induced hepatocyte apoptosis as referred by reduction of p38, JNK and ERK MAPK signaling pathways. Results suggest that glycine can modulate Hg-induced oxidative stress and apoptosis in hepatocytes probably because of its antioxidant activity and functioning via mitochondria-dependent pathways and could be a beneficial agent in oxidative stress-mediated liver diseases.     

Zn2+-induced ERK activation mediated by reactive oxygen species causes cell death in differentiated PC12 cells

Recent studies have provided evidence that Zn2+ plays a crucial role in ischemia- and seizure-induced neuronal death. However, the intracellular signaling pathways involved in Zn2+-induced cell death are largely unknown. In the present study, we investigated the roles of mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), and of reactive oxygen species (ROS) in Zn2+-induced cell death using differentiated PC12 cells. Intracellular accumulation of Zn2+ induced by the combined application of pyrithione (5 microM), a Zn2+ ionophore, and Zn2+ (10 microM) caused cell death and activated JNK and ERK, but not p38 MAPK. Preventing JNK activation by the expression of dominant negative SEK1 (SEKAL) did not attenuate Zn2+-induced cell death, whereas the inhibition of ERK with PD98059 and the expression of dominant negative Ras mutant (RasN17) significantly prevented cell death. Inhibition of protein kinase C (PKC) and phosphatidylinositol-3 kinase had little effect on Zn2+-induced ERK activation. Intracellular Zn2+ accumulation resulted in the generation of ROS, and antioxidants prevented both the ERK activation and the cell death induced by Zn2+. Therefore, we conclude that although Zn2+ activates JNK and ERK, only ERK contributes to Zn2+-induced cell death, and that ERK activation is mediated by ROS via the Ras/Raf/MEK/ERK signaling pathway.     

Acacetin inhibits neuronal cell death induced by 6-hydroxydopamine in cellular Parkinson’s disease model

Acacetin (5,7-dihydroxy-4′-methoxyflavone), a flavonoid compound isolated from Flos Chrysanthemi Indici, chrysanthemum, safflower, and Calamintha and Linaria species has been shown to have anti-cancer activity, indicating its potential clinical value in cancer treatment. In this study, we sought to study the potentials of acacetin in preventing human dopaminergic neuronal death via inhibition of 6-hydroxydopamine (6-OHDA)-induced neuronal cell death in the SH-SY5Y cells. Our results suggest that acacetin was effective in preventing 6-OHDA-induced neuronal cell death through regulation of mitochondrial-mediated cascade apoptotic cell death. Pretreatment with acacetin significantly inhibited neurotoxicity and neuronal cell death through reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) dysfunction. Acacetin also markedly acted on key molecules in apoptotic cell death pathways and reduced phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinases (PI3K)/Akt, and glycogen synthase kinase-3beta (GSK-3β). These results suggested that acacetin could inhibit 6-OHDA-induced neuronal cell death originating from ROS-mediated cascade apoptosis pathway. Thus, the results of our study suggest that acacetin is a potent therapeutic agent for PD progression.     

Nox4 NADPH oxidase-derived reactive oxygen species, via endogenous carbon monoxide, promote survival of brain endothelial cells during TNF-α-induced apoptosis

We investigated the role of reactive oxygen species (ROS) in promoting cell survival during oxidative stress induced by the inflammatory mediator tumor necrosis factor-α (TNF-α) in cerebral microvascular endothelial cells (CMVEC) from newborn piglets. Nox4 is the major isoform of NADPH oxidase responsible for TNF-α-induced oxidative stress and apoptosis in CMVEC. We present novel data that Nox4 NADPH oxidase-derived ROS also initiate a cell survival mechanism by increasing production of a gaseous antioxidant mediator carbon monoxide (CO) by constitutive heme oxygenase-2 (HO-2). TNF-α rapidly enhanced endogenous CO production in a superoxide- and NADPH oxidase-dependent manner in CMVEC with innate, but not with small interfering RNA (siRNA)-downregulated Nox4 activity. CORM-A1, a CO-releasing compound, inhibited Nox4-mediated ROS production and enhanced cell survival in TNF-α-challenged CMVEC. The ROS-induced CO-mediated survival mechanism requires functional interactions between the protein kinase B/Akt and extracellular signal-related kinase (ERK)/p38 MAPK signaling pathways activated by TNF-α. In Akt siRNA-transfected CMVEC and in cells with pharmacologically inhibited Akt, Erk1/2, and p38 mitogen-activated protein kinase (MAPK) activities, CORM-A1 was no longer capable of blocking Nox4 activation and apoptosis caused by TNF-α. Overall, Nox4 NADPH oxidase-derived ROS initiate both death and survival pathways in TNF-α-challenged CMVEC. The ROS-dependent cell survival pathway is mediated by an endogenous antioxidant CO, which inhibits Nox4 activation via a mechanism that includes Akt, ERK1/2, and p38 MAPK signaling pathways. The ability of CO to inhibit TNF-α-induced ERK1/2 and p38 MAPK activities in an Akt-dependent manner appears to be the key element in ROS-dependent survival of endothelial cells during TNF-α-mediated brain inflammatory disease.     

Vibrio vulnificus-induced death of Jurkat T-cells requires activation of p38 mitogen-activated protein kinase by NADPH oxidase-derived reactive oxygen species

Vibrio vulnificus, a pathogenic bacterium causing primary septicemia, exhibited cytotoxicity towards Jurkat cells of T-lymphocytes through intracellular reactive oxygen species (ROS) production. Pretreatment of Jurkat T-cells with diphenyleneiodonium chloride (DPI) abolished V. vulnificus-induced ROS generation and bacterial ability to cause cell death. Jurkat T-cells expressing dominant-negative protein of Rac subunit of NADPH oxidase (NOX) did not show increased ROS production and cell death by V. vulnificus. Vibrio vulnificus also triggered phosphorylation of mitogen-activated protein kinases (MAPKs) including p38 and ERK1/2 in Jurkat T-cells. Experiments using inhibitors or small interfering RNAs for each MAPK showed that both MAPKs are involved in V. vulnificus-induced cell death. DPI only blocked the phosphorylation of p38 MAPK in Jurkat T-cells exposed by V. vulnificus. This study demonstrates that V. vulnificus induces death of Jurkat T-cells via ROS-dependent activation of p38 MAPK, and that NOX plays a major role in ROS generation in V. vulnificus-exposed cells.     

The hierarchical relationship between MAPK signaling and ROS generation in human leukemia cells undergoing apoptosis in response to the proteasome inhibitor Bortezomib

The hierarchy of events accompanying induction of apoptosis by the proteasome inhibitor Bortezomib was investigated in Jurkat lymphoblastic and U937 myelomonocytic leukemia cells. Treatment of Jurkat or U937 cells with Bortezomib resulted in activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK (mitogen-activated protein kinase), inactivation of extracellular signal-regulating kinase 1/2 (ERK1/2), cytochrome c release, caspase-9, -3, and -8 activation, and apoptosis. Bortezomib-mediated cytochrome c release and caspase activation were blocked by the pharmacologic JNK inhibitor SP600125, but lethality was not diminished by the p38 MAPK inhibitor SB203580. Inducible expression of a constitutively active MEK1 construct blocked Bortezomib-mediated ERK1/2 inactivation, significantly attenuated Bortezomib lethality, and unexpectedly prevented JNK activation. Conversely, pharmacologic MEK/ERK1/2 inhibition promoted Bortezomib-mediated JNK activation and apoptosis. Lastly, the antioxidant N-acetyl-l-cysteine (LNAC) attenuated Bortezomib-mediated reactive oxygen species (ROS) generation, ERK inactivation, JNK activation, mitochondrial dysfunction, and apoptosis. In contrast, enforced MEK1 and ERK1/2 activation or JNK inhibition did not modify Bortezomib-induced ROS production. Together, these findings suggest that in human leukemia cells, Bortezomib-induced oxidative injury operates at a proximal point in the cell death cascade to antagonize cytoprotective ERK1/2 signaling, promote activation of the stress-related JNK pathway, and to trigger mitochondrial dysfunction, caspase activation, and apoptosis. They also suggest the presence of a feedback loop wherein Bortezomib-mediated ERK1/2 inactivation contributes to JNK activation, thereby amplifying the cell death process.     

Curcumin attenuates glutamate-induced HT22 cell death by suppressing MAP kinase signaling

Glutamate induces cell death by upsetting the cellular redox homeostasis, termed oxidative glutamate toxicity, in a mouse hippocampal cell line, HT22. Extracellular signal-regulated kinases (ERK) 1/2 are known key players in this process. Here we characterized the roles of both MAP kinases and cell cycle regulators in mediating oxidative glutamate toxicity and the neuroprotective mechanisms of curcumin in HT22 cells. c-Jun N-terminal kinase (JNK) and p38 kinase were activated during the glutamate-induced HT22 cell death, but at a later stage than ERK activation. Treatment with a JNK inhibitor, SP600125, or a p38 kinase inhibitor, SB203580, partly attenuated this cell death. Curcumin, a natural inhibitor of JNK signaling, protected the HT22 cells from glutamate-induced death at nanomolar concentrations more efficiently than SP600125. These doses of curcumin affected neither the level of intracellular glutathione nor the level of reactive oxygen species, but inactivated JNK and p38 significantly. Moreover, curcumin markedly upregulated a cell-cycle inhibitory protein, p21cip1, and downregulated cyclin D1 levels, which might help the cell death prevention. Our results suggest that curcumin has a neuroprotective effect against oxidative glutamate toxicity by inhibiting MAP kinase signaling and influencing cell-cycle regulation.     

Crosstalk from survival to necrotic death coexists in DU-145 cells by curcumin treatment

Curcumin as an anticancer agent was investigated in regards to its ability to regulate the switching of cancer cells from survival to necrotic cell death. At higher concentrations, curcumin induced ROS production leading to JNK and p38 phosphorylation in DU-145 prostate cancer cells. Of the MAP kinases, ERK or p38/JNK were phosphorylated earlier during curcumin treatment, and were responsible for curcumin-induced cell survival at early time of treatment with the help of phosphorylated Akt, while significant amounts of ROS production in later periods stimulated cell death with caspase degradation. In addition to autophagic signaling, necrosis was dominant with little apoptotic cell death. Caspase activation was completely prohibited by procaspase degradation, which contributed to curcumin-induced early necrosis. At the later incubation period (24 h), cytotoxicity caused by curcumin peaked, at which time survival or proliferation signals, such as phosphorylated Akt and phosphorylated ERK, was almost completely diminished. Curcumin-induced ROS were shown to function, biphasically depending on the incubation period; facilitating survival, in the earlier incubation period, and necrotic death in the later. Based on all of these results, we concluded that curcumin contributes to a complex signaling network, affecting cell survival and necrotic cell death, which in turn could inhibit apoptotic cell death.     

Arrestin regulates MAPK activation and prevents NADPH oxidase-dependent death of cells expressing CXCR2

Activation of CXCR2 IL-8 receptor leads to activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and rapid receptor endocytosis. Co-immunoprecipitation and co-localization experiments showed that arrestin and CXCR2 form complexes with components of the ERK1/2 cascade following ligand stimulation. However, in contrast to the activation of the beta2-adrenergic receptor, arrestin was not necessary for ERK1/2 phosphorylation or receptor endocytosis. In contrast, beta-arrestin 1/2 double knockout cells showed greatly enhanced phosphorylation of ERK1/2, as well as phosphorylation of the stress kinases p38 and c-Jun N-terminal protein kinase. The stimulation of stress kinases in arrestin double knockout cells could be attenuated in the presence of diphenylene iodonium (DPI), an inhibitor of the NADPH oxidase, suggesting that reactive oxidant species (ROS) participated in mitogen-activated protein kinase (MAPK) activation. ROS could indeed be detected in IL-8-stimulated beta-arrestin 1/2 knockout cells, and cytoplasmic Rac was translocated to the membrane fraction, which is a prerequisite for oxidant formation. The oxidative burst induced cell death within 6 h of IL-8 stimulation of these cells, which could be prevented in the presence of DPI. These results indicate a novel function for arrestin, which is protection from an excessive oxidative burst, resulting from the sustained stimulation of G-protein-coupled receptors that cause Rac translocation.     

Paclitaxel induces prolonged activation of the Ras/MEK/ERK pathway independently of activating the programmed cell death machinery

Paclitaxel is a widely used chemotherapeutic agent and is known to induce programmed cell death (apoptosis) in a variety of cell types, but the precise underlying mechanisms are poorly understood. To elucidate these mechanisms, we challenged human esophageal squamous cancer cell lines with paclitaxel and investigated its effects upon signal transduction pathways. Physiologically relevant concentrations of paclitaxel (1-1,000 nm) induced apoptosis. All three mitogen-activated protein kinase (MAPK) family members, c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK) were activated upon paclitaxel treatment. Interestingly, JNK activation and p38 MAPK activation were delayed and peaked at 48 h, whereas ERK activity was sustained over 72 h. In addition, Ras activation and MAPK/ERK kinase (MEK) phosphorylation were observed in concordance with ERK activation. While ERK activation was completely ablated by MEK inhibitors, immunoprecipitation and Western blot analysis revealed that neither MEK-1 nor MEK-2 was involved, but instead another member of the MEK family may potentially participate. Although pretreatment with a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone rescued the cell death, it did not prevent Ras or ERK activation. Furthermore, inhibition of JNK, p38 MAPK, or MEK did not alter PARP cleavage and the cell death induced by paclitaxel. These results in aggregate suggest that the delayed activation of JNK, p38 MAPK, and ERK was not linked to activation of the cell death machinery.     

Involvement of heme oxygenase-1 induction via Nrf2/ARE activation in protection against H2O2-induced PC12 cell death by a metabolite of sesamin contained in sesame seeds

Induction of phase II antioxidant enzymes by activation of Nrf2/ARE (antioxidant response element) signaling has been considered as a promising strategy to combat with oxidative stress-related diseases. In the present study, we tested for potential effects of sesamin, a major lignan contained in sesame seeds, its stereoisomer episesamin, and their metabolites on Nrf2/ARE activation in rat pheochromocytoma PC12 cells. Luciferase reporter assays showed that primary metabolites of sesamin and episesamin, SC-1 and EC-1 were the most potent ARE activators among all tested compounds. SC-1 {(1R,2S,5R,6S)-6-(3,4-dihydroxyphenyl)-2-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo-[3,3,0]octane} enhanced nuclear translocation of Nrf2 and up-regulated expression of phase II antioxidant enzymes including heme oxygenase-1 (HO-1). Treatment with SC-1 resulted in increased phosphorylation of p38 MAP kinase and transient increase in intracellular ROS levels. N-acetylcysteine (NAC) treatment abolished p38 phosphorylation as well as HO-1 induction caused by SC-1, indicating that ROS are upstream signals of p38 in Nrf2/ARE activation by SC-1. Furthermore, preconditioning with SC-1 attenuated H(2)O(2)-induced cell death in a dose-dependent manner. Finally, treatment with a HO-1 inhibitor, Zn-protoporphyrin (ZnPP), and overexpression of a dominant-negative mutant of Nrf2 diminished SC-1-mediated neuroprotection. Our results demonstrate that SC-1 is capable of protecting against oxidative stress-induced neuronal cell death in part through induction of HO-1 via Nrf2/ARE activation, suggesting its potential to reduce oxidative stress and ameliorate oxidative stress-related neurodegenerative diseases.