Sodium pyruvate protects against H(2)O(2) mediated apoptosis in human neuroblastoma cell line-SK-N-MC

Free radicals are involved in neuronal damage. The present study was aimed to investigate the protective effect of sodium pyruvate-a free radical scavenger against hydrogen peroxide (H(2)O(2)) induced apoptosis in human neuroblastoma cell line-SK-N-MC. On exposure to H(2)O(2) (0.025 mM) cells exhibited apoptosis within 24 h, demonstrating a high caspase 3 activity by 3 h followed by cleavage of PARP that was maximum at 24 h. A break down in the mitochondrial membrane potential was observed 3 h onwards. Sodium pyruvate protected cells significantly (P<0.05) against apoptosis in a dose dependent manner as assessed for cell viability by dye exclusion method and apoptosis by TUNEL. Sodium pyruvate significantly inhibited caspase 3 activity, cleavage of PARP and breakdown of mitochondrial membrane potential. These data suggest that sodium pyruvate protects neuronal damage caused by H(2)O(2).     

神经节苷脂调节PKC信号通路及其对PC12细胞去血清损伤的保护作用

目的 研究去血清损伤中PKC信号通路的改变,同时阐明神经节苷脂对大鼠嗜铬细胞瘤细胞株 (PC12)细胞去血清损伤的保护作用及其作用机制.方法 采用去血清损伤24h作为损伤模型,MTT测定细胞存活率,Hoechst 33258/PI观察细胞损伤后的死亡类型及坏死、凋亡的细胞数;Western blotting检测PC12细胞在损伤后细胞浆和细胞膜PKC:蛋白的变化.结果 去血清损伤时间依赖性对PC12细胞有损伤,多数细胞呈凋亡征象,而神经节苷脂对去血清损伤有明显的保护作用;去血清损伤时,PKC信号通路被活化,主要表现为PC12细胞胞浆中的PKC:蛋白减少,胞膜上的PKC蛋白逐渐增加,实现 PKC 蛋白转位;而神经节苷脂可以抑制这种转位,从而起到保护作用.结论 神经节苷脂对去血清损伤有保护作用,其保护作用部分是由于神经节苷脂抑制PKC信号通路的活化.     

Protection by puerarin against MPP+-induced neurotoxicity in PC12 cells mediated by inhibiting mitochondrial dysfunction and caspase-3-like activation

Puerarin, a main isoflavone glycoside distributed in Pueraria lobata (Willd.) Ohwi, showed inhibitory activity on H2O2-induced PC12 cells damage in our previous work. However, there is insufficient evidence in protective mechanism of puerarin, especially that relating to the mitochondrial function. In this study, when cells were pretreated with puerarin prior to 0.4 mM MPP+, protective roles were accompanied by a reduction of cell viability loss, morphological changes of apoptosis and apoptotic rate. To explore the protective mechanism of puerarin in MPP+-induced PC12 cells, mitochondrial function and caspase-3-like activity were measured. The results indicated that puerarin inhibited the release of mitochondrial cytochrome c to cytosol and the loss of mitochondrial membrane potentials. In addition, puerarin also reduced MPP+-induced caspase-3-like activation. Taken together, the above results suggest that pretreatment of PC12 cells with puerarin could block MPP+-mediated apoptosis by mitochondria-dependent caspase cascade.     

Comparative study of survival signal withdrawal- and 4-hydroxynonenal-induced cell death in cerebellar granule cells

The lipid peroxidation product, 4-hydroxynonenal (HNE), has been shown to induce apoptosis in PC12 cells and hippocampal neurons. We compared the degree of cell death induced by survival signal withdrawal (K+ and serum deprivation) with that induced by HNE, and investigated whether agents that block survival signal withdrawal-induced apoptosis could also prevent HNE-induced cell death in cultured cerebellar granule cells. Cell death induced by K+ and serum deprivation was inhibited by cycloheximide, a CPP 32-like protease inhibitor (Ac-DEVD-CHO) and a pituitary adenylate cyclase-activating polypeptide (PACAP)-38. In addition, nuclear cyclic AMP responsive element (CRE)- and activator protein 1 (AP-1) DNA-binding activities were increased 2 h after K+ and serum withdrawal, and these increases were inhibited by cycloheximide, Ac-DEVD-CHO and PACAP 38. Although these agents also blocked HNE-induced cell death, consistent with their efficacy in preventing survival signal withdrawal-induced cells death, CRE and AP-1 DNA-binding activities were decreased in a time-dependent manner during HNE-induced cell death. These results suggest that mechanistic differences exist between apoptosis induced by HNE and that induced by withdrawal of survival signals in cerebellar granule neurons.     

Glucose enhances mesangial cell apoptosis

Mesangial cell apoptosis occurs in experimental diabetic nephropathy, and this correlates with worsening albuminuria. This study examines the mechanism by which glucose modulates mesangial cell apoptosis. Apoptosis was induced in mesangial cells by serum deprivation in the presence of 5 or 25 mM D-glucose, and examined by expression of Annexin-V and disruption of mitochondrial transmembrane potential. Involvement of Bax, Bcl-2 and NF-kappaB were examined by RT-PCR and EMSA. Involvement of TGF-beta1 was sought by determining the effect of recombinant TGF-beta1on apoptosis and the mediators of the apoptotic pathway (Bcl2/Bax and NF-kappaB). Culture of cells in the presence of 25 mM D-glucose (i) enhanced apoptosis stimulated by serum depletion, (ii) enhanced activation of caspase-3, (iii) inhibited NF-kappaB activation, and (iv) decreased Bcl-2:Bax ratio. Inhibition of NF-kappaB using SN50, also increased mesangial cell apoptosis, and decreased Bcl-2:Bax ratio. Addition of TGF-beta1 to mesangial cells mimicked the effect of high glucose reducing NF-kappaB expression and Bcl-2:Bax ratio. Furthermore glucose-mediated enhanced apoptosis was inhibited by the addition of a blocking antibody to TGF-beta1. Exposure of mesangial cells to 25 mM D-glucose stimulated the generation of both total and active TGF-beta1 in the cell culture supernatant, this increase was only significant after 48-72 h, that is at a time point later than enhanced apoptosis. Addition of 25 mM D-glucose, however, increased sensitivity of mesangial cells to TGF-beta1 as assessed by luciferase activity of a Smad sensitive reporter construct. The data suggest that elevated glucose concentration enhanced the pathway leading to apoptosis following serum deprivation. Furthermore, it is likely that this is dependent on glucose-mediated enhanced sensitivity to endogenous TGF-beta1 rather than glucose stimulated de novo TGF-beta1 synthesis.     

Differential sensitivity of naïve and subsets of memory CD4+ and CD8+ T cells to hydrogen peroxide-induced apoptosis

CD4+ and CD8+ memory T cells are identified into central and effector memory subsets, which are characterized by distinct homing patterns and functions. In this investigation, we show that na?ve and central memory CD4+ and CD8+ T cells are sensitive to hydrogen peroxide (H2O2)-induced apoptosis, whereas effector memory CD4+ and CD8+ T cells are relatively resistant to H2O2-induced apoptosis. Apoptosis in na?ve and central memory CD4+ and CD8+ is associated with the release of cytochrome c and activation of caspase-9 and caspase-3, upregulation of Bax and voltage-dependent anion channel (VDAC) expression, and decreased intracellular glutathione (GSH). In vitro GSH and a superoxide dismutase mimetic Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin inhibited H2O2-induced apoptosis in both na?ve and central memory CD4+ and CD8+ T cells. Furthermore, VDAC inhibitor 4,4'-diisothiocynostilbene-2,2'-disulfonic acid blocked H2O2-induced apoptosis. These data demonstrate that H2O2 induces apoptosis preferentially in human na?ve and central memory CD4+ and CD8+ T cells via the mitochondrial pathway by regulating intracellular GSH and the expression of Bax and VDAC.     

Survivin在过氧化氢预处理诱导的适应性细胞保护中的作用

目的探讨H2O2预处理对存活素(survivin)表达的影响及存活素在H2O2预处理的适应性细胞保护中的作用。方法在PC12细胞建立H2O2预处理对抗H2O2诱导细胞凋亡的实验模型,应用甲氮甲唑蓝(MTT)法检测细胞存活率,碘化丙啶(PI)染色流式细胞术检测细胞凋亡率,Hoechst染色检测细胞凋亡的形态学改变,免疫印迹法(Westernblot)测定存活素的表达水平。结果用100μmol/LH2O2预处理PC12细胞90min可明显地抑制300μmol/LH2O2作用12h后引起的细胞毒性和细胞凋亡,并可以显著地上调PC12细胞存活素的表达;JAK2抑制剂AG-490不仅可以抑制存活素的表达,而且拮抗H2O2预处理的适应性细胞保护作用。结论存活素是JAK-STAT通路的靶基因,可能在H2O2预处理诱导的适应性细胞保护机制中起着重要的作用。     

雌激素减轻H2O2诱导PC12细胞凋亡

目的观察雌激素对H2O2诱导细胞凋亡的作用,探讨雌激素保护作用机制。方法在PC12细胞建立H2O2诱导细胞凋亡的实验模型。用MTT法检测细胞存活率,比色法测定乳酸脱氢酶(LDH)活性,Hoechst染色检测细胞凋亡,碘化丙啶(PI)染色流式细胞术检测细胞凋亡率,比色法测定caspase-3活性。结果H2O2明显降低PC12细胞的存活率,使LDH释放增加,促进细胞凋亡,并能明显地升高caspase-3的活性。雌激素能显著地减轻上述变化。结论雌激素对抗H2O2诱导的细胞凋亡,抑制caspase-3的激活是其细胞保护机制之一。     

Ceramide-mediated apoptosis in lung epithelial cells is regulated by glutathione.

Reactive oxygen species (ROS) are mediators of lung injury, and glutathione (GSH) is the major nonprotein antioxidant that protects the cell from oxidative stress. We have recently shown that H(2)O(2) induces ceramide-mediated apoptosis in human lung epithelial cells. We hypothesized that ROS-mediated depletion of GSH plays a regulatory role in ceramide generation, and thus in the induction of apoptosis. Our present studies demonstrate that GSH at physiologic concentrations (1 to 10 mM) inhibits ceramide production in a time- and dose-dependent manner in A549 human alveolar epithelial cells. On the other hand, buthionine-sulfoximine-mediated depletion of intracellular GSH induces elevation of ceramide levels and apoptosis. In addition, GSH blocks H(2)O(2)-mediated induction of intracellular ceramide generation and apoptosis. These effects were not mimicked by oxidized GSH (GSSG) or other thiol antioxidants, such as dithiothreitol and 2-mercaptoethanol. Moreover, increase of intracellular H(2)O(2), mediated by inhibition of catalase by aminotriazole, also induces ceramide generation and apoptosis. These effects were blocked by N-acetylcysteine. Our results suggest that GSH depletion may be the link between oxidative stress and ceramide-mediated apoptosis in the lung.     

Autophagic cell death and its execution by lysosomal cathepsins

In the last decade, the molecular mechanisms of apoptosis, a major type of active cell death (type I cell death) have largely been clarified in mammalian cells. Particularly, the caspase family of proteinases has been shown to play crucial roles in the execution of apoptosis. Differing from apoptosis, type II cell death is known to be associated with autophagosomes/autolysosomes and appear in the developing nervous system (CLARKE, 1990). We have previously shown that delayed neuronal death occurring in the CA1 pyramidal layer of the gerbil hippocampus after brief forebrain ischemia is apoptotic in nature and autophagosomes/autolysosomes abundantly appear in the neurons before DNA fragmentation. To further understand the roles of autophagosomes/autolysosomes in active cell death, we examined the apoptosis of PC12 cells using morphological and biochemical techniques. PC12 cells are known to undergo apoptosis when cultured in the absence of serum. In such an environment, the mitochondrial pathway of apoptosis is activated; cytochrome c is released from mitochondria, and caspase-9/caspase-3 are activated. We have first examined morphological features of PC12 cells during the apoptotic process following serum deprivation, and found that autophagy is induced from the early stage of the process in the cells before typical nuclear changes. When autophagy is inhibited in the cells by 3-methyladenine, an autophagy inhibitor, they are largely protected from apoptosis. In relation to the induction of autophagy in PC12 cells following serum deprivation, immunoreactivity, protein amounts, and the proteolytic activity of lysosomal proteinases, particularly cathepsins B and D, are all greatly altered; those of cathepsin B drastically decrease in the cells from the early stage of serum-deprived cultures, whereas those of cathepsin D increase. Moreover, PC12 cells overexpressing cathepsin D undergo apoptosis more rapidly in serum-deprived cultures than wild-type cells, whereas those overexpressing cathepsin B increase the viability. These lines of evidence suggest that autophagy is involved in PC12 cell death following serum deprivation, this type of cell death being regulated by lysosomal proteinases, cathepsins B and D, downstream autophagy.     

Intravenous immunoglobulin preparations promote apoptosis in lipopolysaccharide-stimulated neutrophils via an oxygen-dependent pathway in vitro

Since prolonged survival of activated neutrophils has an autotoxic potential, neutrophil apoptosis plays an important role in the rapid resolution of inflammation. Intravenous immunoglobulin (IVIG) preparations, which are beneficial therapeutic agents for the treatment of autoimmune diseases and systemic inflammatory diseases, have been reported to induce apoptosis of lymphocytes and endothelial cells in vitro. In the present study, we investigated whether IVIG may induce apoptosis of neutrophils cultured in vitro. After neutrophils prestimulated with or without lipopolysaccharide (LPS) were cultured in the presence or absence of IVIG, the number of apoptotic cells, intracellular H2O2 and GSH were measured by a flow cytometer. IVIG induced apoptosis of LPS-stimulated neutrophils dose dependently, but not in unstimulated neutrophils. Although anti-Fas monoclonal antibodies (mAbs) had no effect on the IVIG-induced apoptosis in the LPS-stimulated neutrophils, anti-Fc gamma receptor (Fc gammaR) II- and III-blocking mAbs significantly inhibited the IVIG-induced apoptosis. IVIG increased the production of intracellular H2O2, while it decreased the production of GSH, in the LPS-stimulated neutrophils. Furthermore, a specific NADPH oxidase inhibitor and anti-oxidants inhibited the IVIG-induced neutrophil apoptosis. Therefore, these findings indicate that IVIG preparations induce apoptosis of LPS-stimulated neutrophils and suggest that the IVIG-induced apoptosis may be mediated by an oxygen-dependent pathway via Fc gammaRII and III.     

IL-6预处理保护H2O2致心肌细胞损伤作用机制初探

目的初步探讨IL-6预处理对H2O2致心肌细胞氧化应激损伤的作用机制。方法采用心肌细胞原代培养方法,以H2O2刺激心肌细胞,建立细胞氧化应激模型;采用MTT法检测细胞活力;AnnexinV-FITC染色法和流式细胞术检测细胞凋亡率;检测心肌细胞内谷胱甘肽（GSH）、超氧化物歧化酶（SOD）、丙二醛（MDA）的表达情况。结果 H2O2可降低心肌细胞存活率并能增加其凋亡,IL-6预处理后能显著改善细胞活力及凋亡情况,与模型组相比差异有统计学意义（P〈0.05）。低浓度IL-6能明显增加细胞内GSH与SOD的水平,并降低MDA的含量,随着IL-6浓度的增加,这种效应逐渐消失。结论 IL-6预处理能保护H2O2致心肌细胞损伤作用,这可能与IL-6调节细胞GSH、SOD、MDA表达有关。     

Nuclear factor-kappaB and caspases co-operatively regulate the activation and apoptosis of human macrophages

Accumulating evidence suggests that macrophages function as major effector cells in the pathological process of various human diseases. We examined here the role of nuclear factor-kappaB (NF-kappaB) and caspases in the regulation of activation and apoptosis of macrophages. Activation of the human monoblastic leukaemia cell line, U937, by phorbol 12-myristate 13-acetate (PMA) increased the expression of CD14/CD86, and cytokine production. PMA stimulation also increased the expression of both pro-caspase-8 and pro-caspase-3 in U937, but not apoptosis or intracellular caspase-3 activity. PMA also increased the expression of X-chromosome-linked inhibitor of apoptosis protein (XIAP) in U937, suggesting an inhibitory action for XIAP on the caspase cascade in PMA-stimulated U937. Electrophoretic mobility shift assay (EMSA) showed a significant increase of nuclear NF-kappaB activity in PMA-stimulated U937. When a potent NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), was added to U937 cell culture in the presence of PMA, apoptosis was triggered by activation of caspase-3, which was induced by caspase-8 activation. XIAP expression was markedly suppressed in PMA-treated U937 in the presence of PDTC. The inhibitors of caspase-8 and caspase-3 mostly inhibited apoptosis of U937 treated with PMA in the presence of PDTC. Furthermore, a phenotype of U937 treated with PMA and PDTC in the presence of caspase inhibitor was almost identical to that of unstimulated U937. Our results suggest that the signalling pathways involved in the activation and apoptosis of human macrophages could be co-operatively regulated by the use of NF-kappaB and caspase inhibitors, thus enabling the control of macrophage function and number.     

Trypanosoma cruzi infection and nuclear factor kappa B activation prevent apoptosis in cardiac cells

Studies of cardiac pathology and heart failure have implicated cardiomyocyte apoptosis as a critical determinant of disease. Recent evidence indicates that the intracellular protozoan parasite Trypanosoma cruzi, which causes heart disease in chronically infected individuals, impinges on host apoptotic pathways in a cell type-dependent manner. T. cruzi infection of isolated neuronal cells and cardiomyocytes protects against apoptotic cell death, whereas apoptosis is triggered in T cells in T. cruzi-infected animals. In this study, we demonstrate that the ability of T. cruzi to protect cardiac cells in vitro from apoptosis triggered by a combination of tumor necrosis factor alpha and serum reduction correlates with the presence of intracellular parasites and involves activation of host cell NF-kappaB. We further demonstrate that the apoptotic block diminishes activation of caspase 3. The ability of T. cruzi to prevent apoptosis of infected cardiomyocytes is likely to play an important role in establishment of persistent infection in the heart while minimizing potential damage and remodeling that is associated with cardiomyocyte apoptosis in cardiovascular disease.     

葡萄糖调节蛋白75对缺糖诱导的凋亡相关基因Bax和NF κB改变的影响

目的 通过已获得稳定表达葡萄糖调节蛋白(Grp75)的PC12细胞株,检测Grp75对缺糖诱导的细胞凋亡过程中Bax和NF-κB的影响。 方法 Grp75过表达组和对照组细胞无糖培养6h、12h、24h和48h后,进行相关的实验。免疫印迹法检测缺糖状态下两组细胞中Grp75的表达水平和NF-κB的活性;应用半定量RT-PCR和免疫印迹法比较Bax表达水平的变化;免疫细胞化学通过构象特异性的Bax 6A7抗体检测Bax的活化。 结果 Bax活化和NF-κB活性的下降在缺糖诱导的PC12细胞凋亡过程中发挥了重要的作用。而Grp75通过阻止Bax的活化和NF-κB活性的下降抑制缺糖诱导的凋亡。缺糖状态下Grp75过表达组和对照组细胞中Bax表达水平均未发生改变。 结论 Bax活化和NF-κB活性下降与缺糖诱导的PC12细胞凋亡关系密切,Grp75通过阻止Bax的活化和维持NF-κB的活性保护PC12细胞。     

葡萄糖调节蛋白75对缺糖诱导的凋亡相关基因Bax和NF-κB改变的影响

目的 通过已获得稳定表达葡萄糖调节蛋白(Grp75)的PC12细胞株,检测Grp75对缺糖诱导的细胞凋亡过程中Bax和NF-κB的影响. 方法 Grp75过表达组和对照组细胞无糖培养6h、12h、24h和48h后,进行相关的实验.免疫印迹法检测缺糖状态下两组细胞中Grp75的表达水平和NF-κB的活性;应用半定量RT-PCR和免疫印迹法比较Bax表达水平的变化;免疫细胞化学通过构象特异性的Bax 6A7抗体检测Bax的活化. 结果 Bax活化和 NF-κB活性的下降在缺糖诱导的PC12细胞凋亡过程中发挥了重要的作用.而Grp75通过阻止Bax的活化和NF-κB活性的下降抑制缺糖诱导的凋亡.缺糖状态下Grp75过表达组和对照组细胞中Bax表达水平均未发生改变. 结论 Bax活化和NF-κB活性下降与缺糖诱导的PC12细胞凋亡关系密切,Grp75通过阻止Bax的活化和维持NF-κB的活性保护PC12细胞.