籽瓜多糖对H_2O_2致PC12细胞氧化损伤的保护作用

本文研究了籽瓜多糖(SWP)对H2O2致PC12细胞氧化应激损伤的影响及其机制。通过建立H2O2诱导PC12细胞氧化损伤模型,CCK-8法测定细胞存活率;硫辛酰胺脱氢酶催化的INT显色反应检测乳酸脱氢酶(LDH)释放量,DCFH-DA检测细胞内ROS;ELISA法检测8-OHd G;JC-1染色检测细胞线粒体膜电位;利用caspase-3可以催化底物Ac-DEVD-p NA的反应检测caspase-3活性;应用caspase-9催化特异性底物Ac-LEHDp NA检测caspase-9活性。结果显示:过氧化氢组与对照组相比,终浓度为500μmol/L H2O2作用细胞24 h后,细胞活力显著下降(P0.01);LDH释放量和细胞内ROS增加(P0.01);8-OHd G含量上升(P0.01);线粒体膜电位下降(P0.01);caspase-3和caspase-9活性增强(P0.01)。与H2O2损伤组相比,不同剂量的SWP预处理后,能显著改善H2O2引起的上述指标的变化(P0.05)。由此得出:SWP对H2O2诱导的PC12细胞的氧化损伤具有一定的保护作用。     

氧化磷酸化中质子的转运机制介绍和讨论

氧化磷酸化过程中电子传递和磷酸化所伴随的质子（H＋）跨线粒体内膜转运,是生物化学教学中的一个重点和难点。该文介绍参与H＋跨膜（线粒体内膜或细菌质膜）转运的复合体Ⅰ（又称为NADH-Q还原酶或NADH脱氢酶）、复合体Ⅲ（又称为细胞色素还原酶或细胞色素bc1复合体）、复合体Ⅳ（又称为细胞色素氧化酶或细胞色素c氧化酶）和复合体Ⅴ（又称为F1F0-ATP合酶）跨膜转运H＋的机制。     

过氧化氢预处理对H9c2心肌细胞氧化应激损伤的保护作用

目的：活性氧介导的氧化损伤是缺血再灌注损伤的重要机制,本研究通过观察H2O2预处理对氧化损伤的H9c2心肌细胞存活率和细胞凋亡的影响,探讨其保护H9c2心肌细胞的作用机制。方法：体外培养H9c2心肌细胞,取对数生长期细胞用于实验研究。建立H2O2预处理抵抗高浓度H：O：诱导的细胞氧化损伤模型,实验分组如下：（1）正常对照组（CTL）;（2）损伤组（INJURY）;（3）预处理组十损伤组（PC）。应用CCK8法检测细胞存活率;试剂盒检测胞内MDA水平和T．sOD活性;Hoechst33258染色观察凋亡形态;Annexin-V／PI双染与流式细胞术检测细胞凋亡率。结果：25vLmol／L的H202预处理90rain能明显地保护H9c2心肌细胞抵抗400μmol／LH2O2诱导的氧化损伤,提高细胞存活率,下调MDA水平,上调SOD活性,抑制细胞凋亡,降低细胞凋亡率。结论：低浓度H2O2预处理能减轻H9c2心肌细胞的氧化损伤,抑制氧化损伤诱导的心肌细胞凋亡,具有很好的抗氧化损伤和抗心肌细胞凋亡的保护作用,其作用机制可能与细胞SOD活性上调有关。H2O2预处理为临床治疗心肌缺血／再灌注损伤提供了一项新策略。     

氧化应激心肌细胞prohibitin表达与分布变化及其生物学意义

目的：探讨prohibitin在氧化应激心肌细胞中的表达与分布变化的特点及其在心肌细胞损伤中的意义。方法：H2O2干预体外培养乳鼠心肌细胞,建立氧化应激心肌细胞损伤模型;采用生物化学法检测细胞培养液中LDH活性及MTT实验观察心肌细胞损伤程度;以Western印迹法检测氧化应激时prohibitin蛋白表达变化与分布变化;线粒体H^＋-ATPase合成活力实验检测线粒体氧化磷酸化功能;流式细胞术检测线粒体跨膜电位。结果：氧化应激组LDH活性显著高于对照组,而细胞存活率低于对照组34．51％～65．5％;线粒体H^＋-ATPase合成活力降低60％;氧化应激组线粒体跨膜电位显著低于对照组;心肌细胞prohibitin表达水平在H2O2,处理3h出现升高然后回落到正常水平,线粒体prohibitin表达水平高于对照组。结论：氧化应激心肌细胞prohibitin表达水平代偿性增加并有向线粒体移位的趋势,氧化应激导致心肌细胞线粒体功能障碍。     

细胞色素c在后处理抗大鼠肠缺血-再灌注损伤细胞凋亡中的变化

本文旨在研究细胞色素c在后处理抗大鼠肠缺血-再灌注损伤细胞凋亡中的变化。将Sprague-Dawley大鼠32只随机分为4组(n=8):假手术(Sham)组、缺血-再灌注(I/R)组、缺血预处理(IPC)组、缺血后处理(IPOST)组。应用激光共聚焦扫描显微镜检测各组大鼠肠黏膜细胞线粒体跨膜电位的变化。用Western blot方法检测肠黏膜细胞线粒体内细胞色素c及caspase-3表达的变化。末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法(TUNEL)和DNA琼脂糖凝胶电泳方法检测大鼠肠黏膜细胞凋亡发生情况。实验结果显示,与缺血-再灌注组相比,缺血后处理组大鼠肠黏膜细胞线粒体跨膜电位显著升高(P0.05),线粒体内细胞色素c蛋白表达水平显著增加(P0.05),caspase-3蛋白表达降低(P0.05),细胞凋亡率明显降低(P0.05)。缺血后处理组与缺血预处理组相比各项指标差异无统计学意义(P0.05)。上述结果提示缺血后处理可通过阻止线粒体释放细胞色素c抑制凋亡发生,减轻大鼠肠缺血-再灌注损伤。     

外源H2O2对湖北海棠根系线粒体膜透性和细胞核DNA的影响

用0．024％（V／V）H2O2处理湖北海棠（Malus hupehensis Rehd．）实生苗根系,30min后,根系线粒体膜通透,陛明显增大,线粒体膜电位（△ψm）和线粒体内Cyt c／a吸光度比值下降;60min后,根系细胞核DNA发生片段化降解。经荧光染料吖啶橙染色后,可见H2O2处理60min以上的根系压片中出现了清晰致密的黄绿色荧光宽,呈现出细胞程序,陛死亡（PCD）的特征。     

神经酰胺以Caspase依赖和非依赖方式诱导线粒体凋亡蛋白释放

本研究探讨了外源性C2-神经酰胺诱导入结肠癌HT-29细胞凋亡中,线粒体膜间隙凋亡蛋白的释放机制．不同浓度C2-神经酰胺作用HT-29细胞,流式细胞仪检测线粒体膜电位（△ψm）,线粒体／细胞液分离试剂盒分离亚细胞成分,聚丙烯酰胺凝胶电泳检测细胞色素C（Cytc）、高温必需蛋白A2（HtrA2）、线粒体源性半胱天冬氨酸蛋白酶第二活化因子（Smac）、凋亡抑制蛋白（XtAP）和半胱天冬氨酸蛋白酶-3（Caspase-3）蛋白表达水平．实验结果显示25和50μmol／L C2-神经酰胺作用细胞6h,△ψm即开始下降（P〈0．05）,且环孢霉素能通过调节线粒体膜通透性转换孔抑制△ψm的下降．C2-神经酰胺对Cyt c,HtrA2和Smac总蛋白表达没有明显影响,但能诱导Cyt c,HtrA2和Smac从线粒体释放入细胞液中,并下调XIAP蛋白的表达及活化Caspase-3．在Caspase抑制剂存在下,C2-神经酰胺仍能诱导Cyt c和HtrA2从线粒体释放,但不能诱导Smac释放．因此认为C2-神经酰胺能通过线粒体凋亡通路诱导HT-29细胞凋亡,C2-神经酰胺诱导Cytc和HtrA2从线粒体的释放是Caspase非依赖性的,而Smac释放是Caspase依赖性的．     

西达本胺诱导胰腺癌细胞凋亡

目的：观察西达本胺对胰腺癌细胞BxPC-3和PANC-1生长抑制及诱导细胞凋亡作用,探讨西达本胺抗胰腺癌的机制。方法：西达本胺处理BxPC-3和PANC-1细胞后,用流式细胞术检测细胞的凋亡率,用罗丹明123和DCFH—DA染色方法测定细胞线粒体膜跨膜电位变化和活性氧（ROS）的产生,用Western印迹检测Bcl-2家族和γH2AX蛋白表达的变化。结果：西达本胺对胰腺癌细胞BxPC-3和PANC-1具有生长抑制和诱导细胞凋亡的作用,且呈时间和剂量依赖关系;处理72h后,胰腺癌细胞内ROS产生增强导致DNA损伤发生,且线粒体跨膜电位明显下降;促凋亡蛋白Bax的表达,抑制抑凋亡蛋白Bcl-2和Mcl—1的表达。结论：西达本胺具有抑制胰腺癌细胞增殖,诱导细胞凋亡的作用;西达本胺增强胰腺癌细胞内ROS的产生并导致DNA损伤,最终诱导细胞凋亡的发生。     

重组nkx2.5腺病毒抑制过氧化氢诱导的H9c2细胞凋亡

为研究心脏发育关键基因nkx2.5的功能及应用价值,构建Ad-Nkx2.5重组腺病毒,并检测nkx2.5过表达拮抗氧化应激损伤的效应及机制。采用AdEasy腺病毒表达系统构建Ad-Nkx2.5重组腺病毒,建立H2O2诱导H9c2心肌细胞凋亡模型,分别用Ad-Nkx2.5重组病毒或对照病毒感染细胞,采用Hoechst33342染色观察细胞形态变化、MTT法检测细胞存活率,免疫印迹检测caspase-3活化、细胞色素C的胞浆含量。并通过Real-timePCR检测凋亡相关基因bcl-2和bax表达。结果发现,nkx2.5过表达促进H9c2细胞存活,抑制H2O2诱导的caspase-3活化及线粒体细胞色素C的释放。Nkx2.5过表达上调bcl-2表达,显著下调H2O2诱导的bax表达。并发现H2O2对Nkx2.5核定位无明显影响。结果显示重组腺病毒介导的Nkx2.5过表达可通过调控凋亡相关基因表达,抑制线粒体凋亡途径,保护心肌细胞抗氧化损伤。     

大麦提取物对星形胶质细胞氧化损伤的保护作用

活性氧如H2O2、O-·2以及OH·造成的生物分子及细胞损伤与许多神经退行性疾病如阿尔茨海默病、癫痫和帕金森氏病等有关。本实验对Wistar大鼠脑星形神经胶质细胞进行了分离和原代培养,以此为材料检测了大麦提取物对由H2O2处理引起的细胞生长抑制的保护作用。通过细胞形态学观察和SRB法测定细胞生长率,实验结果表明大麦提取物对H2O2诱导下星形胶质细胞生长率降低具有一定的保护作用,且呈浓度依赖性,当大麦提取物浓度增加到1600μg/m L时,细胞生长率达到100%。     

Dosage effects of EGb761 on hydrogen peroxide-induced cell death in SH-SY5Y cells

Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, is one of the most popular herbal supplements, taken for its multivalent properties. In this study, dosage effects of EGb761 on hydrogen peroxide (H₂O₂)-induced apoptosis of human neuroblastoma SH-SY5Y cells were investigated. It was found that H₂O₂-induced apoptotic cell death in SH-SY5Y cells, which was revealed in DNA fragmentation, mitochondrial membrane potential depolarization, and activation of Akt, c-Jun N-terminal kinases (JNK) and caspase 3. Low doses of EGb761 (50–100 μg/ml) inhibited H₂O₂-induced cell apoptosis via inactivation of Akt, JNK and caspase 3 while high doses of EGb761 (250–500 μg/ml) enhanced H₂O₂ toxicities via inactivation of Akt and enhancement of activation of JNK and caspase 3. Additional experiments revealed that H₂O₂ decreased intracellular GSH content, which was also inhibited by low concentrations of EGb761 but enhanced after high concentrations of EGb761 treatment. This further suggests to us that dosage effects of EGb761 on apoptotic signaling proteins may be correlated with regulation of cell redox state. Therefore, treatment dosage may be one of the vital factors that determine the specific action of EGb761 on oxidative stress-induced cell apoptosis. To understand the mechanisms of dosage effects of EGb761 may have important clinical implications.     

Coenzyme Q10 Protects Astrocytes from ROS-Induced Damage through Inhibition of Mitochondria-Mediated Cell Death Pathway

Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species to protect neuronal cells against oxidative stress in neurodegenerative diseases. The present study was designed to examine whether CoQ10 was capable of protecting astrocytes from reactive oxygen species (ROS) mediated damage. For this purpose, ultraviolet B (UVB) irradiation was used as a tool to induce ROS stress to cultured astrocytes. The cells were treated with 10 and 25 μg/ml of CoQ10 for 3 or 24 h prior to the cells being exposed to UVB irradiation and maintained for 24 h post UVB exposure. Cell viability was assessed by MTT conversion assay. Mitochondrial respiration was assessed by respirometer. While superoxide production and mitochondrial membrane potential were measured using fluorescent probes, levels of cytochrome C (cyto-c), cleaved caspase-9, and caspase-8 were detected using Western blotting and/or immunocytochemistry. The results showed that UVB irradiation decreased cell viability and this damaging effect was associated with superoxide accumulation, mitochondrial membrane potential hyperpolarization, mitochondrial respiration suppression, cyto-c release, and the activation of both caspase-9 and -8. Treatment with CoQ10 at two different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide, normalization of mitochondrial membrane potential, improvement of mitochondrial respiration, inhibition of cyto-c release, suppression of caspase-9. Furthermore, CoQ10 enhanced mitochondrial biogenesis. It is concluded that CoQ10 may protect astrocytes through suppression of oxidative stress, prevention of mitochondrial dysfunction, blockade of mitochondria-mediated cell death pathway, and enhancement of mitochondrial biogenesis.     

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells

Oxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H(2)O(2)) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H(2)O(2)-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H(2)O(2)-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H(2)O(2), and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca(2+)) chelator, overexpression of Homer 1a had no significant effects on H(2)O(2)-induced oxidative stress. These results suggest that Homer 1a has protective effects against H(2)O(2)-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca(2+) homeostasis.     

Anti-apoptotic effect of cGMP in cultured astrocytes: inhibition by cGMP-dependent protein kinase of mitochondrial permeable transition pore.

Reperfusion of cultured astrocytes with normal medium after exposure to H(2)O(2)-containing medium causes apoptosis. We have recently shown that ibudilast, which has been used for bronchial asthma and cerebrovascular disorders, attenuated the H(2)O(2)-induced apoptosis of astrocytes via the cGMP signaling pathway. This study examines the mechanism underlying the protective effect of cGMP. The membrane-permeable cGMP analog dibutyryl-cGMP attenuated the H(2)O(2)-induced decrease in cell viability, DNA ladder formation, nuclear condensation, reduction of the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase-3 activation in cultured astrocytes. These effects of dibutyryl-cGMP were almost completely inhibited by the cGMP-dependent protein kinase (PKG) inhibitor KT5823. In isolated rat brain mitochondria, cGMP in the presence of cytosolic extract from astrocytes inhibited the mitochondrial permeability transition pore (PTP) as determined by monitoring Ca(2+)-induced mitochondrial swelling. This ability of the cytosolic extract was inactivated by heat treatment and was mimicked by exogenous PKG. The effect of cGMP on the mitochondrial swelling was blocked by KT5823. The PTP inhibitors cyclosporin A and bongkrekic acid prevented the H(2)O(2)-induced decrease in cell viability and caspase-3 activation. These findings demonstrate that cGMP inhibits the mitochondrial PTP via the activation of PKG, and the prevention of mitochondrial dysfunction contributes to its anti-apoptotic effect.     

Adrenomedullin protects rat cerebral endothelial cells from oxidant damage in vitro

Increased permeability and reduced cerebral endothelial cell (CEC) viability induced by oxidative stress are the hallmarks of the blood-brain barrier disruption. In our experiments hydrogen peroxide (H2O2, 0.5 mM) induced a continuous decrease of the transendothelial electrical resistance (TEER) and resulted in intercellular gap formations in cultured rat CECs. Adrenomedullin (AM) increased TEER, enhanced peripheral localization of F-actin bands and attenuated the increased permeability induced by H2O2. Furthermore, AM treatment preserved mitochondrial membrane potential, attenuated cytochrome c release, and consequently improved CEC viability in H2O2 treated cultures. These results suggest that AM treatment protects CECs against oxidative injury.     

Caspase-2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis

DNA damage induced by the cancer chemotherapeutic drug etoposide triggers the onset of a series of intracellular events characteristic of apoptosis. Among the early changes observed is the release of cytochrome c from mitochondria, although the mechanism responsible for this effect is unclear. We demonstrate here a role for caspase-2 in etoposide-induced cytochrome c release. In particular, Jurkat T-lymphocytes treated with an irreversible caspase-2 inhibitor, benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethyl ketone (z-VDVAD-fmk), or stably transfected with pro-caspase-2 antisense (Casp-2/AS) are refractory to cytochrome c release stimulated by etoposide. Experiments performed using a reconstituted cell-free system indicate that etoposide-induced cytochrome c release by way of caspase-2 occurs independently of cytosolic factors, suggesting that the nuclear pool of pro-caspase-2 is critical to this process. Apart from inhibiting cytochrome c release, undermining caspase-2 activity results in an attenuation of downstream events, such as pro-caspase-9 and -3 activation, phosphatidylserine exposure on the plasma membrane, and DNA fragmentation. Taken together, our data indicate that caspase-2 provides an important link between etoposide-induced DNA damage and the engagement of the mitochondrial apoptotic pathway.     

The metabolic enhancer piracetam attenuates mitochondrion-specific endonuclease G translocation and oxidative DNA fragmentation

This study was performed to investigate the involvement of mitochondrion-specific endonuclease G in piracetam (P)-induced protective mechanisms. Studies have shown the antiapoptotic effects of piracetam but the mechanism of action of piracetam is still an enigma. To assess the involvement of endonuclease G in piracetam-induced protective effects, astrocyte glial cells were treated with lipopolysaccharide (LPS) and piracetam. LPS treatment caused significantly decreased viability, mitochondrial activity, oxidative stress, chromatin condensation, and DNA fragmentation, which were attenuated by piracetam cotreatment. Cotreatment of astrocytes with piracetam showed its significantly time-dependent absorption as observed with high-performance liquid chromatography. Astrocytes treated with piracetam alone showed enhanced mitochondrial membrane potential (MMP) in comparison to control astrocytes. However, in LPS-treated cells no significant alteration in MMP was observed in comparison to control cells. Protein and mRNA levels of the terminal executor of the caspase-mediated pathway, caspase-3, were not altered significantly in LPS or LPS + piracetam-treated astrocytes, whereas endonuclease G was significantly translocated to the nucleus in LPS-treated astrocytes. Piracetam cotreatment attenuated the LPS-induced endonuclease G translocation. In conclusion this study indicates that LPS treatment of astrocytes caused decreased viability, oxidative stress, mitochondrial dysfunction, chromatin condensation, DNA damage, and translocation of endonuclease G to the nucleus, which was inhibited by piracetam cotreatment, confirming that the mitochondrion-specific endonuclease G is one of the factors involved in piracetam-induced protective mechanisms.     

Erythropoietin protects retinal pigment epithelial cells from oxidative damage

Oxidative damage from reactive oxygen species (ROS) has been implicated in many diseases, including age-related macular degeneration, in which the retinal pigment epithelium (RPE) is considered a primary target. The aim of this study was to determine whether erythropoietin (EPO) protects cultured human RPE cells against oxidative damage and to identify the pathways that may mediate protection. EPO (1 IU/ml) significantly increased the viability of oxidant-treated RPE cells, decreased the release of the inflammatory cytokines tumor necrosis factor-α and interleukin-1β, recovered the RPE cells' barrier integrity disrupted by oxidative stress, prevented oxidant-induced cell DNA fragmentation and membrane phosphatidylserine exposure, and also reduced the levels of oxidant-induced intracellular ROS and restored cellular antioxidant potential, total antioxidant capacity, glutathione peroxidase, and superoxide dismutase and decreased malondialdehyde, the end product of lipid peroxidation. EPO inhibited caspase-3-like activity. Protection by EPO was partly dependent on the activation of Akt1 and the maintenance of the mitochondrial membrane potential. No enhanced or synergistic protection was observed during application of Z-DEVD-FMK (caspase-3 inhibitor) combined with EPO compared with cultures exposed to EPO and H₂O₂ alone. Together, these results suggest that EPO could protect against oxidative injury-induced cell death and mitochondrial dysfunction in RPE cells through modulation of Akt1 phosphorylation, mitochondrial membrane potential, and cysteine protease activity.     

Mitochondria, oxidative stress and aging

In the eighties, Miquel and Fleming suggested that mitochondria play a key role in cellular aging. Mitochondria, and specially mitochondrial DNA (mtDNA), are major targets of free radical attack. At present, it is well established that mitochondrial deficits accumulate upon aging due to oxidative damage. Thus, oxidative lesions to mtDNA accumulate with age in human and rodent tissues. Furthermore, levels of oxidative damage to mtDNA are several times higher than those of nuclear DNA. Mitochondrial size increases whereas mitochondrial membrane potential decreases with age in brain and liver.

Recently, we have shown that treatment with certain antioxidants, such as sulphur-containing antioxidants, vitamins C and E or the Ginkgo biloba extract EGb 761, protects against the age-associated oxidative damage to mtDNA and oxidation of mitochondrial glutathione. Moreover, the extract EGb 761 also prevents changes in mitochondrial morphology and function associated with aging of the brain and liver. Thus, mitochondrial aging may be prevented by antioxidants. Furthermore, late onset administration of certain antioxidants is also able to prevent the impairment in physiological performance, particularly motor co-ordination, that occurs upon aging.