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。     

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.     

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.     

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.     

c-Jun NH₂-terminal kinase contributes to dexmedetomidine-induced contraction in isolated rat aortic smooth muscle

Purpose

Dexmedetomidine, a full agonist of α_2B-adrenoceptors, is used for analgesia and sedation in the intensive care units. Dexmedetomidine produces an initial transient hypertension due to the activation of post-junctional α_2B-adrenoceptors on vascular smooth muscle cells (SMCs). The aims of this in vitro study were to identify mitogen-activated protein kinase (MAPK) isoforms that are primarily involved in full, α_2B-adrenoceptor agonist, dexmedetomidine-induced contraction of isolated rat aortic SMCs.

Materials and Methods

Rat thoracic aortic rings without endothelium were isolated and suspended for isometric tension recording. Cumulative dexmedetomidine (10^-9 to 10^-6 M) dose-response curves were generated in the presence or absence of extracellular signal-regulated kinase (ERK) inhibitor PD 98059, p38 MAPK inhibitor SB 203580, c-Jun NH₂-terminal kinase (JNK) inhibitor SP 600125, L-type calcium channel blocker (verapamil and nifedipine), and α₂-adrenoceptor inhibitor atipamezole. Dexmedetomidine-induced phosphorylation of ERK, JNK, and p38 MAPK in rat aortic SMCs was detected using Western blotting.

Results

SP 600125 (10^-6 to 10^-5 M) attenuated dexmedetomidine-evoked contraction in a concentration-dependent manner, whereas PD 98059 had no effect on dexmedetomidine-induced contraction. SB 203580 (10^-5 M) attenuated dexmedetomidine-induced contraction. Dexmedetomidine-evoked contractions were both abolished by atipamezole and attenuated by verapamil and nifedipine. Dexmedetomidine induced phosphorylation of JNK and p38 MAPK in rat aortic SMCs, but did not induce phosphorylation of ERK.

Conclusion

Dexmedetomidine-induced contraction involves a JNK- and p38 MAPK-mediated pathway downstream of α₂-adrenoceptor stimulation in rat aortic SMCs. In addition, dexmedetomidine-induced contractions are primarily dependent on calcium influx via L-type calcium channels.     

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.     

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.     

Lipid Peroxidation in the Liver of Mice during Development of Granulomatous Inflammation after Combined Infection with <Emphasis Type="Italic">C. albicans</Emphasis> and <Emphasis Type="Italic">M. tuberculosis</Emphasis>

Programmed death of peripheral blood mononuclear cells from healthy donors was studied during culturing with various concentrations of H₂O₂ and selective inhibitors of JNK (SP600125) and p38 MAPK (ML3403). In vitro incubation of mononuclear leukocytes with 1 mM H₂O₂ stimulated apoptotic cell death. Treatment with inhibitors (SP600125 and ML3403) during in vitro oxidative stress prevented the increase in the number of annexin-positive mononuclear cells. Our results indicate that MAP kinases JNK and p38 are involved in the mechanisms of oxidative dysregulation of apoptosis. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 505–508, May, 2008     

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.     

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.     

Differential activation and regulation of mitogen-activated protein kinases through the antigen receptor and CD40 in human B cells.

In human B cells, antigen receptor ligation and CD40 ligation are known to activate the extracellular-regulated kinases (ERK) and c-Jun N-terminal kinase (JNK) pathways, which in turn regulate many important B cell functions. We previously reported that antigen receptor ligation activated the ERK pathway whereas CD40 ligation activated the JNK/stress-activated protein kinase (SAPK) pathway. Here, we demonstrate that another SAPK, p38/Hog1, is activated by both antigen receptor ligation or CD40 ligation in a human B-lymphoblastoid cell line and tonsillar B cells. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, partially inhibited ERK2 and p38 activation triggered through the B cell receptor whereas activation of JNK1 and p38 through CD40 was not affected. PD98059, a specific inhibitor of mitogen-activated extracellular response kinase kinase (MEK), significantly inhibited ERK2 activation and partially inhibited p38 activation triggered by anti-IgM antibody treatment, but did not affect CD40-dependent signaling events. In addition, anti-IgM antibody-induced signaling pathways were shown to be PKC-dependent in contrast to the CD40-induced signaling pathways. Thus, the B cell receptor and CD40 recruit the ERK, JNK and p38 pathways by using different upstream effectors.     

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.     

Angiotensin II-Induced Early and Late Inflammatory Responses Through NOXs and MAPK Pathways

Angiotensin II (Ang II) dysregulation has been determined as cause or an effect of many diseases. The relationship between Ang II and reactive oxygen species (ROS), which are generated by enzymes in the nicotinamide adenine dinucleotide phosphate oxidase (NOX) family, has been the focus of many researchers for years. Inflammation in response to the activities of various NOXs with differing time-dependent characteristics was reported. It is still unclear how these factors interplay over the course of the inflammatory response and how signal transduction through mitogen-activated protein kinase (MAPK) pathways. Our study collected data on the effects of Ang II on human umbilical vascular endothelial cells (HUVECs) over a comprehensive time period. Our results demonstrated that NOXs had two time-dependent reactions in response to Ang II stimulation via MAPK pathways. First, ROS was produced only during the early inflammatory phase. NOX4 promoted more rapid generation of H₂O₂ via the JNK pathway than generation of O₂·^? via ERK1/2 and p38 pathways. During both the early and late phases of the inflammatory response, NOX4 activity was transduced through the JNK pathway, whereas NOX1 and NOX2 signals were transmitted via the ERK1/2 and p38 pathways. Signal transduction via ROS generation was more likely during the early phase of the inflammatory response, and increased cytokine levels were more likely induced by the late phase of the inflammatory response.     

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.     

氧化应激性肠上皮细胞损伤与MAPK信号通路的关系研究

目的观察氧化应激引起肠上皮细胞（IEC-6）损伤后丝裂原活化蛋白激酶（MAPK）信号通路活化的情况,从而探讨氧化应激损伤的机制。方法采用MTT法检测不同浓度（100、200、300、400、500、800、1000、2000μmol/L）H2O2对IEC-6生存率的影响;用WesternBlot法检测H2O2作用不同时间（0.25、0.5、1、2、3、4h）下ERK、JNK以及p38磷酸化的激活情况。结果与正常组相比,随着H2O2刺激浓度的增加,IEC-6的生存率逐渐降低,呈浓度依赖性。当H2O2刺激浓度为200μmol/L时细胞的生存率约为50%。ERK1/2、JNK1/2、p38在H2O2刺激0.25h开始磷酸化,在刺激0.5h达到最高,随后磷酸化水平恢复到基础值。结论氧化应激早期可通过激活ERK、JNK以及p38的磷酸化引起IEC-6的损伤。     

MAPK signaling has stage-dependent osteogenic effects on human adipose-derived stem cells in vitro

Overview: The use of pro-osteogenic growth factors, such as BMP2, in human adipose-derived stem cell (ASC) osteogenesis is well described. Because these growth factors work via signal transduction pathways, such as the mitogen-activated protein kinase (MAPK) cascade, a study of the relationship between MAPK signaling and ASC osteogenesis was conducted. Materials and Methods: ERK, JNK, and p38MAPK activation were measured in ASCs osteo-induced using either dexamethasone or vitamin D3 and correlated with mineralization. Activation and mineralization were also measured without dexamethasone or using the glucocorticoid, cortisone. The expression of the MAPK phosphatase, MKP1, and its relationship to mineralization was also assessed. The effect of decreasing MAPK activation on mineralization through the use of exogenous inhibitors was examined along with siRNA-knockdown and adenoviral overexpression of ERK1/2. Finally, the effect of ERK1/2 overexpression on ASCs induced on PLGA scaffolds was assessed. Results: ASC mineralization in dexamethasone or vitamin D3-induced ASCs correlated with both increased ERK1/2 and JNK1/2 activation. ASCs induced without dexamethasone also mineralized, with JNK1/2 signaling possibly mediating this event. No link between cortisone induction and MAPK signaling could be ascertained. ASCs treated with ERK, JNK, or p38MAPK inhibitors showed decreased osteogenic gene expression and diminished mineralization. Mineralization levels were also affected by viruses designed to inhibit or augment ERK1/2 expression and activity. Finally, ASC mineralization appeared to be a balance between the MAPK kinase activity and MKP1. Conclusions: It is likely that MAPK signaling plays a significant role in ASC osteogenesis, affecting differentiation in kinase- and stage-specific manners.     

Caffeic acid improves cell viability and protects against DNA damage: involvement of reactive oxygen species and extracellular signal-regulated kinase

Hormesis is an adaptive response to a variety of oxidative stresses that renders cells resistant to harmful doses of stressing agents. Caffeic acid (CaA) is an important antioxidant that has protective effects against DNA damage caused by reactive oxygen species (ROS). However, whether CaA-induced protection is a hormetic effect remains unknown, as is the molecular mechanism that is involved. We found that a low concentration (10 μM) of CaA increased human liver L-02 cell viability, attenuated hydrogen peroxide (H₂O₂)-mediated decreases in cell viability, and decreased the extent of H₂O₂-induced DNA double-strand breaks (DSBs). In L-02 cells exposed to H₂O₂, CaA treatment reduced ROS levels, which might have played a protective role. CaA also activated the extracellular signal-regulated kinase (ERK) signal pathway in a time-dependent manner. Inhibition of ERK by its inhibitor U0126 or by its specific small interfering RNA (siRNA) blocked the CaA-induced improvement in cell viability and the protective effects against H₂O₂-mediated DNA damage. This study adds to the understanding of the antioxidant effects of CaA by identifying a novel molecular mechanism of enhanced cell viability and protection against DNA damage.