H_2O_2诱导Neuro-2a细胞死亡机理的研究

细胞内线粒体呼吸链过程中的电子漏和神经细胞代谢的酶类如单胺氧化酶(MAO)等可产生活性氧物质(ROS)如H_2O_2等。ROS对细胞有毒性作用,导致细胞死亡,在许多疾病特别是神经退行性疾病中具有重要作用。我们用H_2o_2诱导N-2a神经母细胞瘤细胞,利用光镜、荧光显微镜、透射电镜观察了诱导的N-2a细胞的死亡,结果表明其死亡形式不同于典型的细胞凋亡,而类似于Ⅱ型神经细胞编程性死亡,死亡细胞染色质呈团块状凝集,细胞核膜仍保持完整。DNA不降解形成ladder,且不需要caspase-3,1的活性,但是H_2O_2诱导的Neuro-2a细胞死亡可以被Bcl-X_L,抑制。我们的结果可以说明,ROS介导的细胞毒性作用是导致Ⅱ型神经细胞编程性死亡的一个原因。     

ABA诱导蚕豆气孔保卫细胞H2O2的产生

以蚕豆叶片下麦皮为材料,将荧光探针ＨＰＴＳ导入蚕豆忆保卫细胞内,利用荧光光谱和激光共聚焦显微技术,检测了ＡＢＡ诱导蚕豆气孔关闭过程中Ｈ２Ｏ２的产生。结果表明,ＡＢＡ和１００μｍｏｌ／Ｌ的Ｈ２Ｏ２可以迅速猝灭ＨＰＴＳ的荧光,ＣＡＴ几乎可完全阻止９μｌ０．２ｍｍｏｌ／Ｌ的ＡＢＡ引起荧光猝灭。因此,ＡＢＡ可以诱导气孔保卫细胞产生Ｈ２Ｏ２,其产生的强度和速度与ＡＢＡ的浓度直接有关,并且推测：这种Ｈ２Ｏ２产     

UV—B辐射对小麦叶片H2O2代谢的影响

研究了温室种植的小麦在０（ＣＫ）、８．８２ｋＪ／ｍ＾２（Ｔ１）和１２．６ｋＪ／ｍ＾２（Ｔ２）三种剂量的紫外线Ｂ（ＵＶ－Ｂ）辐射下Ｈ２Ｏ２含量的变化及其机理。ＵＶ－Ｂ辐射下Ｈ２Ｏ２、还原型抗坏血酸（ＡｓＡ）和还原型谷胱甘肽（ＧＳＨ）含量增加,抗坏血酸过氧化物酶（ＡＰｘ）和谷胱甘肽不原酶（ＧＲ）活性升高,脂肪酸不饱和度指数（ＩＵＦＡ）降低。ＳＤＳ－ＰＡＧＥ谱图没有质上的差异,但凝胶着色深浅有变化。分析     

植物细胞的氧化猝发和H2O2的信号转导

概述了植物细胞氧化猝发的特性、产生机理、生理作用以及Ｈ２Ｏ２信号转导途径及其对基因表达的调控等的研究进展。     

胞外H2O2及NADPH氧化酶参与了铜胁迫对植物细胞死亡的诱导

以烟草悬浮细胞BY-2(Nicotiana tabacum L.cv.Bright Yellow-2)为材料,探讨了在铜离子胁迫下植物细胞死亡发生过程中胞外H₂O₂及NADPH氧化酶所扮演的角色。实验结果表明,随着外源CuCl₂浓度的上升(从0~700 μmol·L^-1),细胞死亡水平不断上升,且胞外H₂O₂的水平也不断增加。在300 μmol·L^-1的CuCl₂诱导细胞死亡的过程中,加入H₂O₂清除剂N-N-二甲基硫脲(DMTU)降低了胞外CuCl₂胁迫下H₂O₂含量增加的同时也降低了细胞死亡水平的上升,这一观察表明了铜离子胁迫所导致的细胞死亡的发生和胞外H₂O₂的增加有关。进一步的研究表明,300 μmol·L^-1 CuCl₂的胁迫导致了NADPH氧化酶活性的显著性上升,而加入NADPH氧化酶的抑制剂（二亚苯基碘,DPI,)则降低了CuCl₂胁迫所导致的细胞死亡和胞外H₂O₂含量的上升。上述结果表明,胞外H₂O₂和NADPH氧化酶参与了CuCl₂对植物细胞死亡的诱导作用。     

黄芪皂苷Ⅳ对H2O2诱导的心肌细胞凋亡的保护作用

目的：探讨黄芪皂苷对H2O2诱导的心肌细胞凋亡的保护作用。方法：以H2O2诱导SD大鼠心肌损伤细胞模型为基础,用黄芪皂苷Ⅳ预处理进行干预。MTT法检测不同时段细胞凋亡情况,Western blot和RT-PCR检测24h时段Cyclin D1蛋白和mRNA表达水平。结果：H2O2对SD大鼠心肌细胞的损伤呈时间依赖性。H2O2可显著诱导SD大鼠心肌细胞凋亡,而这一作用可被黄芪皂苷Ⅳ显著抑制。结论：黄芪皂苷Ⅳ对H2O2诱导的SD大鼠心肌细胞损伤有明显保护作用。     

黄芪皂苷IV对H2O2诱导的心肌细胞凋亡的保护作用

目的:探讨黄芪皂苷对H2O2诱导的心肌细胞凋亡的保护作用.方法:以H2O2诱导SD大鼠心肌损伤细胞模型为基础,用黄芪皂苷IV预处理进行干预.MTT法检测不同时段细胞凋亡情况,Western blot和RT-PCR检测24h时段Cyclin D1蛋白和mRNA表达水平.结果:H2O2对SD大鼠心肌细胞的损伤呈时间依赖性.H2O2可显著诱导SD大鼠心肌细胞凋亡.而这一作用可被黄芪皂苷IV显著抑制.结论:黄芪皂苷IV对H2O2诱导的SD大鼠心肌细胞损伤有明显保护作用.     

H2O2诱导Neuro-2a细胞死亡机理的研究

细胞内线粒体呼吸链过程中的电子漏和神经细胞代谢的酶类如单胺氧化酶(MAO)等可产生活性氧物质(ROS)如H2O2等.ROS对细胞有毒性作用,导致细胞死亡,在许多疾病特别是神经退行性疾病中具有重要作用.我们用H2O2诱导N-2a神经母细胞瘤细胞,利用光镜、荧光显微镜、透射电镜观察了诱导的N-2a细胞的死亡,结果表明其死亡形式不同于典型的细胞凋亡,而类似于Ⅱ型神经细胞编程性死亡,死亡细胞染色质呈团块状凝集,细胞核膜仍保持完整.DNA不降解形成ladder,且不需要caspase-3,1的活性,但是H2O2诱导的Neuro-2a细胞死亡可以被Bcl-XL抑制.我们的结果可以说明,ROS介导的细胞毒性作用是导致Ⅱ型神经细胞编程性死亡的一个原因.     

ECS1参与调节CO2诱导的拟南芥气孔关闭和H2O2积累

CO₂浓度升高可以诱导植物叶片气孔关闭, 提高植物对高浓度CO₂的适应性。但植物如何感知CO₂浓度变化并启动气孔关闭反应的分子机制至今仍不十分清楚。利用高通量、非侵入的远红外成像技术, 建立了拟南芥(Arabidopsis thaliana)气孔对CO₂浓度变化反应相关的突变体筛选技术, 筛选出对环境CO₂浓度敏感的拟南芥突变体ecs1。遗传学分析表明, ecs1为单基因隐性突变体, 突变基因ECS1编码一个跨膜钙离子转运蛋白。与野生型拟南芥相比, 360 μL·L^–1CO₂可引起ecs1突变体叶片温度上升和气孔关闭, ecs1突变体对900 μL·L^–1CO₂长时间处理具有较强的适应性。进一步的实验表明, 360μL·L^–1CO₂即可诱导ecs1突变体叶片积累较高浓度的H₂O₂, 而900 μL·L^–1CO₂才能够诱导野生型拟南芥叶片积累H₂O₂。因此, ECS1可能参与调节高浓度CO₂诱导的拟南芥气孔关闭和H₂O₂产生, H₂O₂可能作为第二信号分子介导CO₂诱导拟南芥气孔关闭的反应。     

SB202190 调节蚕豆保卫细胞中SA 诱导H2O2 产生

运用激光共聚焦扫描技术, 在p38 MAP激酶专一抑制剂SB202190处理下, 探索植物促分裂原活化蛋白激酶(mitogenactivated protein kinase, MAP激酶)介导蚕豆(Vicia faba)保卫细胞中H2O2为代表的活性氧(reactive oxygen species, ROS)信号机制, 发现: p38 MAP激酶专一抑制剂SB202190处理没有导致蚕豆保卫细胞中H2O2和Ca2+探针荧光强度增强, 与水杨酸 (salicylic acid, SA) 或脱落酸 (abscisic acid, ABA) 迅速加强2种探针荧光强度形成鲜明对比; 而该抑制剂分别与SA和ABA共同处理, 前者H2O2探针荧光强度没有增加, 而后者荧光强度仍然能够增加; 而进一步使用Ca2+螯合剂BAPTA和SB202190 ＋SA共同处理, H2O2探针荧光强度没有增加。这些结果初步表明: 无论胞质Ca2+浓度高低, SB202190调节蚕豆保卫细胞中SA诱导H2O2产生, 但是不调节植物逆境信使分子ABA 此类的反应。因此推测, 植物细胞中可能有类似动物和酵母细胞中的p38MAP激酶类, 并可能专一调节植物保卫细胞中H2O2信号通路。据我们所知, 这是首次报道SB202190和SA共同调节植物保卫细胞中ROS信号过程。     

Metacaspase-8 modulates programmed cell death induced by ultraviolet light and H2O2 in Arabidopsis

Programmed cell death (PCD) is a genetically controlled cell death that is regulated during development and activated in response to environmental stresses or pathogen infection. The degree of conservation of PCD across kingdoms and phylum is not yet clear; however, whereas caspases are proteases that act as key components of animal apoptosis, plants have no orthologous caspase sequences in their genomes. The discovery of plant and fungi metacaspases as proteases most closely related to animal caspases led to the hypothesis that metacaspases are the functional homologues of animal caspases in these organisms. Arabidopsis thaliana has nine metacaspase genes, and so far it is unknown which members of the family if any are involved in the regulation of PCD. We show here that metacaspase-8 (AtMC8) is a member of the gene family strongly up-regulated by oxidative stresses caused by UVC, H(2)O(2), or methyl viologen. This up-regulation was dependent of RCD1, a mediator of the oxidative stress response. Recombinant metacaspase-8 cleaved after arginine, had a pH optimum of 8, and complemented the H(2)O(2) no-death phenotype of a yeast metacaspase knock-out. Overexpressing AtMC8 up-regulated PCD induced by UVC or H(2)O(2), and knocking out AtMC8 reduced cell death triggered by UVC and H(2)O(2) in protoplasts. Knock-out seeds and seedlings had an increased tolerance to the herbicide methyl viologen. We suggest that metacaspase-8 is part of an evolutionary conserved PCD pathway activated by oxidative stress.     

Mitigation of H2O2-induced autophagic cell death by propofol in H9c2 cardiomyocytes

Autophagy, a self-eating process, is responsible for degradation of long-lived proteins and damaged cellular proteins/organelles. Double-membrane autophagosomes, formed during the process, engulf proteins/organelles and fuse with lysosomes to degrade the contents. It is important to maintain cell homeostasis and many physiological processes including cellular responses to oxidative stress. Oxidative stress induced by myocardial infarction is a major factor of heart failures. In this study, we examined how propofol modulates hydrogen peroxide (H₂O₂)-induced autophagic cell death in H9c2 cardiomyocytes. H₂O₂ dramatically induced cell death, which was similarly reduced in the presence of either propofol or autophagy inhibitors (e.g., wortmannin), suggesting that propofol has a protective effect in H₂O₂-induced autophagic cell death. Acidic autophagic vacuoles were elevated in H₂O₂-treated H9c2 cells, but they were largely decreased in the presence of propofol. Furthermore, many autophagy-related proteins such as LC3-II, ATG proteins, p62, AMPK, and JNK were activated in H₂O₂-treated H9c2 cells and were significantly deactivated in the presence of propofol. These results show that propofol regulates oxidative stress-induced autophagic cell death in cardiomyocytes. We further suggest that propofol can act as a cardioprotectant in heart diseases.     

麦冬皂苷D对过氧化氢造模的HUVEC保护作用机制研究

目的:研究麦冬皂苷D对HUVEC凋亡相关分子的影响,探讨其作用机制。方法:用H2O2构建凋亡模型,运用MTT及流式细胞仪检测细胞活性,激光共聚焦方法测定粒体膜电位和钙离子浓度。结果:麦冬皂苷D可以稳定线粒体膜电位,减少钙离子内流,增加细胞的活力。结论:麦冬皂苷D对HUVEC有一定的保护作用。     

The mechanism of dNTP-unbalanced cell death induced by 5-fluorouracil and its derivatives

The mechanism of 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (FdUR)-induced death of mouse mammary tumor FM3A cells was studied. When the cells were exposed to 5-FU or FdUR, an unbalance of intracellular dNTP pool resulted. The unbalance was followed by breakage of mature DNA. DNA double strand breaks were observed in the FdUR (1 microM) treated cells 16 hrs after the administration. We assume that the double strand breaks play an important role in the mechanism of the FdUR-mediated cell death. In addition, the activity that can induce DNA double strand breaks was detected in the lysate of FdUR treated FM3A cells. Since intracellular dNTP pool unbalance seems to be the trigger of these events, this phenomenon may be termed as dNTP-unbalanced cell death.     

Downregulation of IDH2 exacerbates H2O2-mediated cell death and hypertrophy

Objectives: Reactive oxygen species-mediated cell death contributes to the pathophysiology of cardiovascular disease and myocardial dysfunction. We recently showed that mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) functions as an antioxidant and anti-apoptotic protein by supplying NADPH to antioxidant systems.

Methods: In the present study, we demonstrated that H₂O₂-induced apoptosis and hypertrophy of H9c2 cardiomyoblasts was markedly exacerbated by small interfering RNA (siRNA) specific for IDH2.

Results: Attenuated IDH2 expression resulted in the modulation of cellular and mitochondrial redox status, mitochondrial function, and cellular oxidative damage. MitoTEMPO, a mitochondria-targeted antioxidant, efficiently suppressed increased caspase-3 activity, increased cell size, and depletion of cellular GSH levels in IDH2 siRNA-transfected cells that were treated with H₂O₂.

Discussion: These results indicated that the disruption of cellular redox balance might be responsible for the enhanced H₂O₂-induced apoptosis and hypertrophy of cultured cardiomyocytes by the attenuated IDH2 expression.     

Rapid intracellular acidification and cell death by H2O2 and alloxan in pancreatic beta cells

Pancreatic beta-cell death induced by oxidative stress plays an important role in the pathogenesis of diabetes mellitus. We studied the relation between rapid intracellular acidification and cell death of pancreatic beta-cell line NIT-1 cells exposed to H2O2 or alloxan. Intracellular pH was measured by a pH-sensitive dye, and cell damage by double staining with Annexin-V and propidium iodide using flow cytometry. H2O2 and alloxan caused a rapid fall in intracellular pH and suppressed Na+/H+ exchanger activity in the NH4Cl prepulse method. H2O2 induced necrotic cell death, which shifted to apoptotic cell death when initial acidification was prevented by pH clamping to 7.4 using nigericin (unclamped cells vs clamped cells, necrosis 43.8 +/- 5.8% vs 21.1 +/- 10.6%, P < 0.05; apoptosis 8.0 +/- 1.9% vs 44.5 +/- 5.0%, P < 0.01). pH-clamped cells showed enhanced caspase 3 activity and proapoptotic Bax expression. On the other hand, NIT-1 cells were resistant to alloxan toxicity, but treatment with alloxan and nigericin strikingly enhanced the cytotoxicity. Antioxidants partly prevented cell death, although intracellular pH remained similarly acidic. The rapid intracellular acidification was not the cause of cell death but a significant determinant of the mode of death of H2O2 -treated beta cells, whereas no link between cell death and acidification was demonstrated in alloxan toxicity.     

Natural products inspired synthesis of neuroprotective agents against H2O2-induced cell death

Stroke is a debilitating disease and the third leading cause of death in the USA, where over 2000 new stroke cases are diagnosed every day. Treatment options for stroke-related brain damage are very limited and there is an urgent need for effective neuroprotective agents to treat these conditions. Comparison of the structures of several classes of neuroprotective natural products such as limonoids and cardiac glycosides revealed the presence of a common structural motif which may account for their observed neuroprotective activity. Several natural product mimics that incorporate this shared structural motif were synthesized and were found to possess significant neuroprotective activity. These compounds enhanced cell viability against H₂O₂ induced oxidative stress or cell death in PC12 neuronal cells. The compounds were also found to enhance and modulate Na⁺/K⁺-ATPase activity of PC12 cells, which may suggest that the observed neuroprotective activity is mediated, at least partly, through interaction with Na⁺/K⁺-ATPase.     

Peroxiredoxin I and II inhibit H2O2-induced cell death in MCF-7 cell lines

Apoptosis is known to be induced by direct oxidative damage due to oxygen-free radicals or hydrogen peroxide or by their generation in cells by the actions of injurious agents. Together with glutathione peroxidase and catalase, peroxiredoxin (Prx) enzymes play an important role in eliminating peroxides generated during metabolism. We investigated the role of Prx enzymes during cellular response to oxidative stress. Using Prx isoforms-specific antibodies, we investigated the presence of Prx isoforms by immunoblot analysis in cell lysates of the MCF-7 breast cancer cell line. Treatment of MCF-7 with hydrogen peroxide (H2O2) resulted in the dose-dependent expressions of Prx I and II at the protein and mRNA levels. To investigate the physiologic relevance of the Prx I and II expressions induced by H2O2, we compared the survivals of MCF10A normal breast cell line and MCF-7 breast cancer cell line following exposure to H2O2. The treatment of MCF10A with H2O2 resulted in rapid cell death, whereas MCF-7 was resistant to H2O2. In addition, we found that Prx I and II transfection enabled MCF10A cells to resist H2O2-induced cell death. These findings suggest that Prx I and II have important functions as inhibitors of cell death during cellular response to oxidative stress.