Opposing role of JNK-p38 kinase and ERK1/2 in hydrogen peroxide-induced oxidative damage of human trophoblast-like JEG-3 cells

Trophoblasts play a crucial role in embryo implantation and maintenance of normal pregnancy. Recently, oxidative stress has been considered as one important factor in the pathogenesis of spontaneous abortion and preeclampsia. Many studies have reported that the plasma levels of hydrogen peroxide (H₂O₂) are significantly increased in women with preeclampsia, but the mechanisms involved in H₂O₂-induced cell cytotoxicity in trophoblasts are still not completely explained. Our present study was undertaken to provide a united understanding of the role of oxidative stress generated by H₂O₂ on human trophoblasts and the underlying intracellular signaling pathways. Exposure to H₂O₂ resulted in a concentration-dependent growth decrease and apoptosis in human trophoblast-like JEG-3 cells. H₂O₂ treatment also caused intracellular reactive oxygen species (ROS) production and concomitant dissipation of the mitochondrial membrane potential. The three MAPK subfamilies, ERK1/2, JNK and p38 kinase, were all activated under H₂O₂-induced oxidative stress. Blocking the activation of JNK and p38 kinase increased cell viability and decreased apoptosis induced by H₂O₂ with their respective inhibitors, SP600125 and SB203580. However, preventing ERK1/2 activation further increased H₂O₂-induced cell death with U0126, an inhibitor of ERK upstream kinase MEK1/2. Taken together, these findings suggest that the mitochondria-dependent pathways and JNK-p38 kinase pathways are involved in H₂O₂-induced oxidative damage of human trophoblast-like JEG-3 cells, while ERK1/2 pathway may play an active role in cell survival following oxidant injury.     

H2O2 induced hyperpolarization of pancreatic B-cells

Conventional electrophysiology and the whole-cell patch-clamp technique have been applied to elucidate the effects of H₂O₂ on pancreatic B-cells of the mouse. In these cells, addition of 15 mmol/l glucose leads to depolarization and oscillation of the cell membrane potential. Subsequent addition of H₂O₂ (1 mmol/l) in the presence of glucose was followed by a marked and rapid hyperpolarization of the cell membrane with suppression of the electrical activity. Accordingly, in slow whole-cell patch-clamp experiments (with nystatin in the pipette solution) H₂O₂ induced a marked increase of cell membrane conductance. Tolbutamide, a blocker of K⁺ _ATP channels, only partially blocked the effect of H₂O₂ even at high concentrations. The H₂O₂-induced, tolbutamide-insensitive current component, however, was largely abolished by a high concentration of TEA⁺ (80 mmol/l) or BaCl₂ (10 mmol/l). It is concluded that in B-cells H₂O₂ stimulates a K⁺ current and that this effect leads to marked hyperpolarization and reversal of glucose-induced oscillations of cell membrane potential.     

p62 protects SH-SY5Y neuroblastoma cells against H2O2-induced injury through the PDK1/Akt pathway

The p62 protein has been identified as a major component of the protein aggregations associated with neurodegenerative disease. Oxidative insult has also been identified as a principal cause of neurodegenerative disease. Thus, in the present study, we investigated the potential role of p62 in oxidative stress-induced cell death in SH-SY5Y human neuroblastoma cells. The results indicated that H₂O₂ treatment induced p62 expression in SH-SY5Y cells. In addition, p62 showed neuroprotective effects against H₂O₂-induced cell death in differentiated SH-SY5Y cells. p62 expression prolonged Akt phosphorylation during the later stages of H₂O₂-induced cell death. Furthermore, coexpression of p62 and wild-type PDK1, the upstream kinase of Akt, further increased Akt phosphorylation and cell viability, whereas the expression of kinase-defective PDK1 reversed the cytoprotective effects of p62 under oxidative stress. Overexpression of p62 led to the dissociation of PDK1 from the 14-3-3θ protein, which is thought to be a negative regulator of PDK1 kinase activity. These findings suggest a mechanism that involves the p62-mediated modulation of the interaction between signaling molecules and results in cell survival.     

瘦素抑制H_2O_2诱导的大鼠心肌细胞凋亡

目的:观察瘦素(leptin)对H2O2诱导的大鼠心肌细胞凋亡的影响并探讨其作用机制。方法:应用脱氧三磷酸尿苷缺口末端标记(TUNEL)法观察瘦素对H2O2诱导的大鼠心肌细胞H9c2凋亡的影响;应用Western blotting法观察瘦素、H2O2对caspase-3、胞外信号调控激酶(ERK)活性的影响。结果:(1)瘦素对H2O2诱导的H9c2细胞凋亡具有显著的抑制作用(与对照组比较P0.01),该作用可被ERK激酶抑制剂PD98059所阻断。(2)H2O2明显抑制ERK活性;而瘦素可激活ERK并部分阻断H2O2诱导的caspase-3激活。结论:瘦素对H2O2诱导的H9c2细胞凋亡具有抑制作用,其机制可能与其激活ERK信号途径有关。     

Diperoxovanadate can substitute for H(2)O(2) at much lower concentration in inducing features of premature cellular senescence in mouse fibroblasts (NIH3T3)

Stress induced premature senescence (SIPS) in mammalian cells is an accelerated ageing response and experimentally obtained on treatment of cells with high concentrations of H₂O₂, albeit at sub-lethal doses, because H₂O₂ gets depleted by abundant cellular catalase. In the present study diperoxovanadate (DPV) was used as it is known to be stable at physiological pH, to be catalase-resistant and to substitute for H₂O₂ in its activities at concentrations order of magnitudes lower. On treating NIH3T3 cells with DPV, SIPS-like morphology was observed along with an immediate response of rounding of the cells by disruption of actin cytoskeleton and transient G2/M arrest. DPV could bring about growth arrest and senescence associated features at 25 μM dose, which were not seen with similar doses of either H₂O₂ or vanadate. A minimal dose of 150 μM of H₂O₂ was required to induce similar affects as 25 μM DPV. Increase in senescent associated markers such as p21, HMGA2 and PAI-1 was more prominent in DPV treated cells compared to similar dose of H₂O₂. DPV-treated cells showed marked relocalization of Cyclin D1 from nucleus to cytoplasm. These results indicate that DPV, stable inorganic peroxide, is more efficient in inducing SIPS at lower concentrations compared to H₂O₂.     

Hydrogen Peroxide Induces G2 Cell Cycle Arrest and Inhibits Cell Proliferation in Osteoblasts

Reactive oxygen species (ROSs) are involved in osteoporosis by inhibiting osteoblastic differentiation and stimulating osteoclastgenesis. Little is known about the role and how ROS controls proliferation of osteoblasts. Mammalian target of rapamycin, mTOR, is a central regulator of cell growth and proliferation. Here, we report for the first time that 5–200 μM hydrogen peroxide (H₂O₂) dose‐ and time‐dependently suppressed cell proliferation without affecting cell viability in mouse osteoblast cell line, MC3T3‐E1, and in human osteoblast‐like cell line, MG63. Further study revealed that protein level of cyclin B1 decreased markedly and the percentage of the cells in G₂/M phase increased about 2‐4 fold by 200 μM H₂O₂ treatment for 24–72 hr. A total of 0.5–5 mM of H₂O₂ but not lower concentrations (5–200 μM) of H₂O₂ inhibited mTOR signaling, as manifested by dephosphorylation of S6K (T389), 4E‐BP1 (T37/46), and S6(S235/236) in MC3T3‐E1 and MG63 cells. Rapamycin, which could inhibit mTOR signaling and cell proliferation, however, did not reduce the protein level of cyclin B1. In a summary, H₂O₂ prevents cell proliferation of osteoblasts by down‐regulating cyclin B1 and inducing G₂ cell cycle arrest. Inhibition of mTOR signaling by H₂O₂ may not be involved in this process. Anat Rec, 292:1107–1113, 2009. © 2009 Wiley‐Liss, Inc.     

Oxidative induction of pro-inflammatory cytokine formation by human monocyte-derived macrophages following exposure to manganese in vitro

Abstract

Manganese (as Mn²⁺), a superoxide dismutase mimetic, catalyzes the formation of the relatively stable membrane-permeable reactive oxygen species (ROS) hydrogen peroxide (H₂O₂), a mediator of intracellular redox signaling in immune and inflammatory cells. The goal of this study was to investigate the potential for Mn²⁺, via its pro-oxidative properties, to activate production of pro-inflammatory cytokines/chemokines IL-1β, IL-6, IL-8, IFNγ, TNFα, and G-CSF by human monocyte-derived macrophages in vitro. For these studies, the cells were isolated from peripheral blood mononuclear leukocytes and matured to generate a population of large CD14/CD16 co-expressing cells. The monocyte-derived macrophages were then exposed to bacterial lipopolysaccharide (LPS, 1?μg/ml) or MnCl₂ (25–100?μM)—alone or in combination—for 24?h at 37?°C, after which cell-free supernatants were analyzed using a multiplex cytokine assay procedure. Exposure of the cells to LPS caused modest statistically insignificant increases in cytokine production; MnCl₂ caused dose-related increases in production of all six cytokines (achieving statistical significance of p?<?0.0171–?<?0.0005 for IL-1β, IL-6, IL-8, IFNγ, and TNFα). In the case of LPS and MnCl₂ combinations, the observed increases in production of IL-1β, IL-6, IL-8, IFNγ, and G-CSF were greater than those seen with cells exposed to the individual agents. The Mn²⁺-mediated induction of cytokine production was associated with increased production of H₂O₂ and completely attenuated by inclusion of the H₂O₂-scavenger dithiothreitol, and partially by inhibitors of NF-κB and p38MAP kinase. The findings from the studies here help to further characterize the pro-inflammatory mechanisms that may underpin clinical disorders associated with excess exposure to Mn²⁺, particularly those disorders seen in the central nervous and respiratory systems.     

Fates of Fe3O4 and Fe3O4@SiO2 nanoparticles in human mesenchymal stem cells assessed by synchrotron radiation-based techniques

Superparamagnetic iron oxide nanoparticles (SPIOs) have been widely used as the magnetic resonance imaging (MRI) contrast agent in biomedical studies and clinical applications, with special interest recently in in vivo stem cell tracking. However, a full understanding of the fate of SPIOs in cells has not been achieved yet, which is particularly important for stem cells since any change of the microenvironment may disturb their propagation and differentiation behaviors. Herein, synchrotron radiation-based X-ray fluorescence (XRF) in combination with X-ray absorption spectroscopy (XAS) were used to in situ reveal the fate of Fe₃O₄ and Fe₃O₄@SiO₂ NPs in human mesenchymal stem cells (hMSCs), in which the dynamic changes of their distribution and chemical speciation were precisely determined. The XAS analysis evidences that Fe₃O₄ NPs cultured with hMSCs are quite stable and almost keep their initial chemical form up to 14 days, which is contradictory to the previous report that Fe₃O₄ NPs were unstable in cell labeling assessed by using a simplified lysosomal model system. Coating with a SiO₂ shell, Fe₃O₄@SiO₂ NPs present higher stability in hMSCs without detectable changes of their chemical form. In addition, XRF analysis demonstrates that Fe₃O₄@SiO₂ NPs can label hMSCs in a high efficiency manner and are solely distributed in cytoplasm during cell proliferation, making it an ideal probe for in vivo stem cell tracking. These findings with the help of synchrotron radiation-based XAS and XRF improve our understanding of the fate of SPIOs administered to hMSCs and will help the future design of SPIOs for safe and efficient stem cells tracking.     

Pigment epithelium-derived factor (PEDF) protects cortical neurons in vitro from oxidant injury by activation of extracellular signal-regulated kinase (ERK) 1/2 and induction of Bcl-2

Mitigating oxidative stress-induced damage is critical to preserve neuronal function in diseased or injured brains. This study explores the mechanisms contributing to the neuroprotective effects of pigment epithelium-derived factor (PEDF) in cortical neurons. Cultured primary neurons are exposed to PEDF and H₂O₂ as well as inhibitors of phosphoinositide-3 kinase (PI3K) or extracellular signal-regulated kinase 1/2 (ERK1/2). Neuronal survival, cell death and levels of caspase 3, PEDF, phosphorylated ERK1/2, and Bcl-2 are measured. The data show cortical cultures release PEDF and that H₂O₂ treatment causes cell death, increases activated caspase 3 levels and decreases release of PEDF. Exogenous PEDF induces a dose-dependent increase in Bcl-2 expression and neuronal survival. Blocking Bcl-2 expression by siRNA reduced PEDF-induced increases in neuronal survival. Treating cortical cultures with PEDF 24 h before H₂O₂ exposure mitigates oxidant-induced decreases in neuronal survival, Bcl-2 expression, and phosphorylation of ERK1/2 and also reduces elevated caspase 3 level and activity. PEDF pretreatment effect on survival is blocked by inhibiting ERK or PI3K. However, only inhibition of ERK reduced the ability of PEDF to protect neurons from H₂O₂-induced Bcl-2 decrease and neuronal death. These data demonstrate PEDF-mediated neuroprotection against oxidant injury is largely mediated via ERK1/2 and Bcl-2 and suggest the utility of PEDF in preserving the viability of oxidatively challenged neurons.     

JAK-STAT pathway modulates the roles of iNOS and COX-2 in the cytoprotection of early phase of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress

Our previous studies have demonstrated that preconditioning with hydrogen peroxide (H₂O₂) activated the JAK-STAT pathway that played an important role in the cytoprotection, and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mediated the late phase of cytoprotection induced by high concentration of H₂O₂ after preconditioning. Here we sought to identify the downstream targets of the JAK-STAT axis that mediated H₂O₂ preconditioning and the expression of iNOS and COX-2 in the early phase of H₂O₂ preconditioning. It was shown that (1) Preconditioning with H₂O₂ at 100 μmol/L for 90 min in PC12 cells induced significant expression of iNOS and COX-2. (2) Pretreatment with the iNOS inhibitor AG (10 μmol/L) or the COX-2 inhibitor NS-398 (10 μmol/L) respectively 20 min before H₂O₂ preconditioning not only inhibits the increased expression of iNOS or COX-2 but also abrogates the protective effects of H₂O₂ preconditioning against apoptosis induced by oxidative stress. (3) Pretreatment with the JAK inhibitor AG-490 (10 μmol/L) 20 min before H₂O₂ preconditioning obviously inhibits the up-regulation of iNOS or COX-2 induced by H₂O₂ preconditioning. These results suggested that JAK-STAT pathway modulates the roles of iNOS and COX-2 in the cytoprotection of early phase of H₂O₂ preconditioning.     

Antioxidative Potential of Duranta Repens (Linn.) Fruits Against H2O2 Induced Cell Death In Vitro

The effects of Duranta repens fruits were investigated on H₂O₂ induced oxidative cell death to evaluate its antioxidative potential in vitro. HEK293T cells were treated with different concentrations [0–1000 µg/ ml] of ethanol extract (E-Ex) and methanol extract (M-Ex) of D. repens for 24h, and then treated with 100 µM H₂O₂ for 24h. Cell viability, antioxidant parameters of cells, and antioxidant constituents of the extracts were determined. Treatment with limited dose of E-Ex or M-Ex increased the survival rate of H₂O₂-treated HEK293T cells, however the extra-high dose showed growth inhibitory effect. Treatment with E-Ex or M-Ex protected cellular lipid per-oxidation. In vitro analyses showed the 2,2-diphenyl-1-picrylhydrazyl and H₂O₂ scavenging activities as well as reducing potential of the extracts. We report here that the limited dose of E-Ex and M-Ex possess antioxidative potential, which can protect H₂O₂-induced oxidative cell damage.     

Guiqi polysaccharide protects the normal human fetal lung fibroblast WI-38 cells from H2O2-induced premature senescence

Objective: This study is to investigate the effects of Guiqi polysaccharide (GQP) on H₂O₂-induced premature senescence in normal human fetal lung fibroblast WI-38 cells. Methods: WI-38 cells were subjected to treatments of GQP, Angelica sinensis polysaccharide (ASP), and Astragalus membranaceus polysaccharide (AMP), and then treated with H₂O₂ to induce premature senescence. Morphological observation, MTT assay, senescence-associated β-galactosidase activity assessment, telomerase activity determination, cell cycle analysis, and Western blot analysis were performed to evaluate cellular senescence. Results: H₂O₂ treatment induced premature senescence in WI-38 cells, as indicated by the decreased fibroblast proliferation activity and changed cellular morphology. When treated with GQP, ASP, or AMP, the morphological changes in WI-38 cells induced by H₂O₂ could be restored. SA-β-gal activity was elevated in H₂O₂-treated WI-38 cells, which could be decreased by GQP treatment. Moreover, compared with the normal control, H₂O₂ treatment significantly inhibited the telomerase activity of WI-38 cells. However, GQP effectively elevated the telomerase activity of these senescent cells. Furthermore, flow cytometry and cell cycle analysis showed that GQP treatment could abrogate the cell cycle arrest in H₂O₂-treated WI-38 cells, which might contribute to the anti-senescent effects. In addition, GQP significantly affected the p53-p21 and p16-pRb pathways in H₂O₂-treated WI-38 cells. The effectiveness of GQP was superior to AMP or ASP treatment alone. Conclusion: GQP has protective effects in oxidative stress-induced senescence. Our findings suggest the promising role of GQP as an attractive and bio-safe agent with the potential to retard senescence and attenuate senescence-related diseases.     

Hydrogen peroxide induces apoptosis through the mitochondrial pathway in rat Schwann cells

Oxidative stress is one of the several mechanisms that induces apoptosis in cells. It has been shown that hydrogen peroxide (H₂O₂) induces apoptosis in several kinds of cells; however, the role of H₂O₂ in the apoptosis of Schwann cells (SCs) is currently unclear. The objective of this study was to determine whether H₂O₂ is capable of inducing apoptosis in SCs and whether or not such an effect is associated with the activation of mitochondrial pathway. We demonstrated that H₂O₂ induces apoptosis in SCs, and is associated with increased release of cytochrome c from mitochondria and the activation of caspase-3 and -9 by up-regulation of Bax and down-regulation of Bcl-2. These results suggest a potential role for H₂O₂ in SC injury by triggering apoptosis via the mitochondrial pathway under oxidative stress.     

p38β MAPK affords cytoprotection against oxidative stress-induced astrocyte apoptosis via induction of αB-crystallin and its anti-apoptotic function

The activation of p38 mitogen-activated protein kinases (MAPKs) has been implicated in many cellular processes, such as, inflammation, cell death, and survival. In mammals, four distinct genes encode the four known members of p38 MAPKs, p38α, p38β, p38γ, and p38δ. Despite the fact that p38α and p38β MAPKs share over 75% homology sequences, they have distinct, perhaps even opposite roles under stress conditions. In our previous report, we showed that p38β MAPK is induced in activated astrocytes in the penumbra of the postischemic brain, wherein it was co-localized with αB-crystallin and MAPKAPK-2. To investigate the functional significance of p38β MAPK in astrocytes, a C6 astroglioma cell line stably over-expressing p38β MAPK was generated. In these cells, hydrogen peroxide-induced apoptosis was reduced to 44.3% of that obtained from normal C6 cells. Interestingly, we found that expression of a small heat shock protein, αB-crystallin, was significantly increased in these cells, but that the expressions of HSP27 and HSP70 were not. Repression of αB-crystallin expression by αB-crystallin siRNA transfection suppressed the protective effect and recovered caspase 3 activity, indicating that αB-crystallin induction plays a crucial role in the protection against H₂O₂-induced apoptosis observed in p38β-overexpressing C6 astroglioma cells. We found that the binding between αB-crystallin and partially processed caspase-3 (a p24 intermediate) was significantly increased in p38β-overexpressing cells, which might result in suppression of caspase 3 activity in these cells. These results indicate that p38β confers protection against H₂O₂-induced astrocytes apoptosis by inducing a small heat shock protein, αB-crystallin, which inhibits caspase-3 activation.     

Hyperbaric oxygen-stimulated proliferation and growth of osteoblasts may be mediated through the FGF-2/MEK/ERK 1/2/NF-κB and PKC/JNK pathways

We investigated whether the hyperbaric oxygen (O₂) could promote the proliferation of growth-arrested osteoblasts in vitro and the mechanisms involved in this process. Osteoblasts were exposed to different combinations of saturation and pressure of O₂ and evaluated at 3 and 7 days. Control cells were cultured under ambient O₂ and normal pressure [1 atmosphere (ATA)]; high-pressure group cells were treated with high pressure (2.5 ATA) twice daily; high-O₂ group cells were treated with a high concentration O₂ (50% O₂) twice daily; and high pressure plus high-O₂ group cells were treated with high pressure (2.5 ATA) and a high concentration O₂ (50% O₂) twice daily. Hyperbaric O₂ significantly promoted osteoblast proliferation and cell cycle progression after 3 days of treatment. Hyperbaric O₂ treatment stimulated significantly increased mRNA expression of fibroblast growth factor (FGF)-2 as well as protein expression levels of Akt, p70^S6K, phosphorylated ERK, nuclear factor (NF)-κB, protein kinase C (PKC)α, and phosphorylated c-Jun N-terminal kinase (JNK). Our findings indicate that high pressure and high O₂ saturation stimulates growth-arrested osteoblasts to proliferate. These findings suggest that the proliferative effects of hyperbaric O₂ on osteoblasts may contribute to the recruitment of osteoblasts at the fracture site. The FGF-2/MEK/ERK 1/2/Akt/p70^S6K/NF-κB and PKC/JNK pathways may be involved in mediating this process.     

Protection against hydrogen peroxide-induced injury by Z-ligustilide in PC12 cells

Z-ligustilide (Z-LIG) is the primary lipophilic compound of the Chinese medicine Danggui (Radix Angelica sinensis). Previous studies demonstrated that Z-LIG had significant neuroprotective potential in both transient and permanent cerebral ischemia, possibly through antioxidant and anti-apoptotic mechanisms. The present study examined the mechanisms of Z-LIG on hydrogen peroxide (H₂O₂)-induced injury in PC12 cells. Following exposure of the cells to H₂O₂ (500 μM), a significant reduction in cell survival and total antioxidant capacity (TAC), as well as increased intracellular reactive oxygen species (ROS), were observed. In addition, H₂O₂ treatment significantly upregulated Bax expression, cleaved-caspase 3, and cytosolic cytochrome-c, and decreased Bcl-2 protein levels. Pretreatment of the cells with Z-LIG (0.1, 1.0, 2.5, or 5.0 μg/ml) significantly attenuated H₂O₂-induced cell death, attenuated increased intracellular ROS levels, and decreased Bax expression, cleaved-caspase 3, and cytochrome-c. Further, Z-LIG improved cellular TAC and concentration-dependently upregulated Bcl-2 expression. These results demonstrate that Z-LIG has a pronounced protective effect against H₂O₂-induced cytotoxicity, at least partly through improving cellular antioxidant defense and inhibiting the mitochondrial apoptotic pathway. These findings suggest that Z-LIG may be useful in the treatment of neurodegenerative disorders in which oxidative stress and apoptosis are mainly implicated.     

Role of Catalase in H2O2-induced oxidant stress in marsupial erythrocytes

We have examined the catalase activity and H₂O₂-induced oxidant stress on methaemoglobin formation and haemolysis in eight species of marsupials: the black striped wallaby (Macropus dorsalis), bridled nailtail wallaby (Onychogalea fraenata), proserpine rock wallaby (Petrogale persephone), red legged pademelon (Thylogale stigmatica), spectacled hare wallaby (Lagorchestes conspicillatus), whiptail wallaby (Macropus parryi), common brushtail possum (Trichosurus vulpecula), and the koala (Phascolarctos cinereus). The results indicate a significant relationship between the activity of catalase and methaemoglobin formation by H₂O₂.     

Enhanced defense against mitochondrial hydrogen peroxide attenuates age-associated cognition decline

Increased mitochondrial hydrogen peroxide (H₂O₂) is associated with Alzheimer's disease and brain aging. Peroxiredoxin 3 (Prdx3) is the key mitochondrial antioxidant defense enzyme in detoxifying H₂O₂. To investigate the importance of mitochondrial H₂O₂ in age-associated cognitive decline, we compared cognition between aged (17–19 months) APP transgenic mice and APP/Prdx3 double transgenic mice (dTG) and between old (24 months) wild-type mice and Prdx3 transgenic mice (TG). Compared with aged APP mice, aged dTG mice showed improved cognition that was correlated with reduced brain amyloid beta levels and decreased amyloid beta production. Old TG mice also showed significantly increased cognitive ability compared with old wild-type mice. Both aged dTG mice and old TG mice had reduced mitochondrial oxidative stress and increased mitochondrial function. Moreover, CREB signaling, a signaling pathway important for cognition was enhanced in both aged dTG mice and old TG mice. Thus, our results indicate that mitochondrial H₂O₂ is a key culprit of age-associated cognitive impairment, and that a reduction of mitochondrial H₂O₂ could improve cognition by maintaining mitochondrial health and enhancing CREB signaling.