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.     

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.     

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.     

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。     

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.     

Cognitive impairment and increased Aβ levels induced by paraquat exposure are attenuated by enhanced removal of mitochondrial H(2)O(2)

Pesticide exposure is a risk factor of Alzheimer's disease (AD). However, little is known about how pesticide exposure may promote AD pathogenesis. In this study, we investigated the effects of paraquat pesticide exposure on β-amyloid (Aβ) levels and cognition using wild-type (WT) mice and β-amyloid precursor protein (APP) transgenic mice. Our results showed that wild-type mice and APP transgenic mice after paraquat exposure had increased oxidative damage specifically in mitochondria of cerebral cortex and exhibited mitochondrial dysfunction. Moreover, the elevated mitochondrial damage was directly correlated with impaired associative learning and memory and increased Aβ levels in APP transgenic mice exposed to paraquat. Furthermore, overexpression of peroxiredoxin 3, a mitochondrial antioxidant defense enzyme important for H₂O₂ removal, protected against paraquat-induced mitochondrial damage and concomitantly improved cognition and decreased Aβ levels in APP transgenic mice. Therefore, our results demonstrate that mitochondrial damage is a key mechanism underlying cognitive impairment and elevated amyloidogenesis induced by paraquat and that enhanced removal of mitochondrial H₂O₂ could be an effective strategy to ameliorate AD pathogenesis induced by pesticide exposure.     

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.     

Oxidative stress promotes d-GalN/LPS-induced acute hepatotoxicity by increasing glycogen synthase kinase 3β activity

Objective

Our previous studies have demonstrated that glycogen synthase kinase 3β (GSK3β) activity is increased in the progression of acute liver failure (ALF), which aggravates liver injury, while its regulatory mechanism remains elusive. This study is designated to address whether oxidative stress activates GSK3β to promote ALF.

Methods

In a murine model induced by d-galactosamine (d-GalN) (700 mg/kg) and LPS (10 μg/kg), N-acetylcysteine (300 mg/kg) or SB216763 (25 mg/kg) was used to inhibit oxidative stress or GSK3β activity, respectively. Serum alanine aminotransferase and aspartate aminotransferase levels were assessed. The parameters of oxidative stress were evaluated in liver tissue. Whether GSK3β inhibition protects hepatocytes from oxidative stress-induced cell apoptosis was investigated in vitro. Moreover, the activity of GSK3β was measured in the liver of chronic hepatitis B (CHB) patients and ALF patients.

Results

In vivo, N-acetylcysteine ameliorated the d-GalN/LPS-induced hepatotoxicity and reduced GSK3β activity; GSK3β inhibition increased hepatic superoxide dismutase activity and the glutathione content, decreased malondialdehyde production in the liver tissues; while GSK3β inhibition suppressed the JNK activation in the liver and decreased cytochrome c release from mitochondria. In vitro, GSK3β inhibition lessened hepatocytes apoptosis induced by H₂O₂ or Antimycin A, as demonstrated by decreased LDH activity, and reduced cleavage of caspase-3 expression. Furthermore, GSK3β activity in the CHB patients was increased in the phase of ALF.

Conclusions

Results indicate that GSK3β activation contributes to liver injury by participating in oxidative stress response in ALF and is, therefore, a potential therapeutic target for ALF.     

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.     

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.     

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.     

3,4-Dihydroxybenzaldehyde Derived from Prunus mume Seed Inhibits Oxidative Stress and Enhances Estradiol Secretion in Human Ovarian Granulosa Tumor Cells

Granulosa cells form ovarian follicles and play important roles in the growth and maturation of oocytes. The protection of granulosa cells from cellular injury caused by oxidative stress is an effective therapy for female infertility. We here investigated an effective bioactive compound derived from Prunus mume seed extract that protects granulosa cells from hydrogen peroxide (H₂O₂)-induced apoptosis. We detected the bioactive compound, 3,4-dihydroxybenzaldehyde (3,4-DHBA), via bioactivity-guided isolation and found that it inhibited the H₂O₂-induced apoptosis of granulosa cells. We also showed that 3,4-DHBA promoted estradiol secretion in granulosa cells and enhanced the mRNA expression levels of steroidogenic factor 1, a promoter of key steroidogenic enzymes. These results suggest that P. mume seed extract may have clinical potential for the prevention and treatment of female infertility.     

Hydrogen peroxide induces autophagic cell death in C6 glioma cells via BNIP3-mediated suppression of the mTOR pathway

Oxidative stress by exposure to H₂O₂ induces various types of cell death depending on cell type and conditions. We report herein on a study of the mechanisms underlying H₂O₂-induced cell death in C6 glioma cells. The findings show that H₂O₂ triggers a caspase-independent autophagic cell death in these cells. The findings also show that H₂O₂ induces the dephosphorylation of the mammalian target of rapamycin (mTOR) at Ser 2481 and the p70 ribosomal protein S6 kinase (p70S6K) at Thr389 in a Bcl-2/E1B 19 kDa interacting protein 3 (BNIP3)-dependent manner. BNIP3 has the capacity to inhibit mTOR activity and mTOR inhibition plays a role in autophagic induction. This suggests that BNIP3 may mediate H₂O₂-induced autophagic cell death through the suppression of mTOR. The findings show that the down-regulation of BNIP3 by BNIP3 siRNA prevents C6 cells from undergoing H₂O₂-induced autophagic cell death. Collectively, these results suggest that H₂O₂ induces autophagic cell death in C6 cells via the BNIP3-mediated suppression of the mTOR pathway.     

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.     

Levocarnitine Protects H9c2 Rat Cardiomyocytes from H2O2-induced Mitochondrial Dysfunction and Apoptosis

Background: Although the protective effects of levocarnitine in patients with ischemic heart disease are related to the attenuation of oxidative stress injury, the exact mechanisms involved have yet to be fully understood. Our aim was to investigate the potential protective effects of levocarnitine pretreatment against oxidative stress in rat H9c2 cardiomyocytes.Methods: Cardiomyocytes were exposed to H₂O₂ to create an oxidative stress model. The cells were pretreated with 50, 100, or 200 μM levocarnitine for 1 hour before H₂O₂ exposure.Results: H₂O₂ exposure led to significant activation of oxidative stress in the cells, characterized by reduced viability, increased intracellular reactive oxygen species, lipid peroxidation, and reduced intracellular antioxidant activity. Mitochondrial dysfunction was also observed following H₂O₂ exposure, reflected by the loss of mitochondrial transmembrane potential and intracellular adenosine triphosphate. These pathophysiological processes led to cardiomyocyte apoptosis through activation of the intrinsic apoptotic pathway. More importantly, the levocarnitine pretreatment attenuated the H₂O₂-induced oxidative injury significantly, preserved mitochondrial function, and partially prevented cardiomyocyte apoptosis during the oxidative stress reaction. Western blotting analyses suggested that levocarnitine pretreatment increased plasma protein levels of Bcl-2, reduced Bax, and attenuated cytochrome C leakage from the mitochondria in the cells.Conclusion: Our in vitro study indicated that levocarnitine pretreatment may protect cardiomyocytes from oxidative stress-related damage.     

Manipulating the surface coating of ultra-small Gd2O3 nanoparticles for improved T1-weighted MR imaging

In this report, monodispersed ultra-small Gd₂O₃ nanoparticles capped with hydrophobic oleic acid (OA) were synthesized with average particle size of 2.9 nm. Two methods were introduced to modify the surface coating to hydrophilic for bio-applications. With a hydrophilic coating, the polyvinyl pyrrolidone (PVP) coated Gd₂O₃ nanoparticles (Gd₂O₃-PVP) showed a reduced longitudinal T₁ relaxation time compared with OA and cetyltrimethylammonium bromide (CTAB) co-coated Gd₂O₃ (Gd₂O₃-OA-CTAB) in the relaxation study. The Gd₂O₃-PVP was thus chosen for its further application study in MRI with an improved longitudinal relaxivity r₁ of 12.1 mm⁻¹ s⁻¹ at 7 T, which is around 3 times as that of commercial contrast agent Magnevist^®. In vitro cell viability in HK-2 cell indicated negligible cytotoxicity of Gd₂O₃-PVP within preclinical dosage. In vivo MR imaging study of Gd₂O₃-PVP nanoparticles demonstrated considerable signal enhancement in the liver and kidney with a long blood circulation time. Notably, the OA capping agent was replaced by PVP through ligand exchange on the Gd₂O₃ nanoparticle surface. The hydrophilic PVP grants the Gd₂O₃ nanoparticles with a polar surface for bio-application, and the obtained Gd₂O₃-PVP could be used as an in vivo indicator of reticuloendothelial activity.     

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.