Scutellarin protects PC12 cells from oxidative stress-induced apoptosis

The present study investigated the effects of scutellarin on oxidative stress-induced cell apoptosis in PC12 cells. Exposure of cells to hydrogen peroxide (H₂O₂) triggered a typical apoptosis, as evidenced by DNA fragmentation, DNA loss and externalization of phosphatidylserine (PS). This treatment also caused significant elevation of oxidative stress characterized by intracellular accumulations of reactive oxygen species (ROS) and malondialdehyde (MDA), a product of lipid peroxidation. Preincubation of cells with scutellarin significantly inhibited the fragmentation and loss of DNA, the externalization of PS, and decreased the percentage of cell apoptosis. Also, intracellular accumulations of ROS and MDA resulting from H₂O₂ exposure were significantly reduced by scutellarin. These findings suggest that scutellarin exerts significant protection against oxidative stress-induced apoptosis, which might be beneficial for the prevention and treatment of oxidative stress-mediated disorders.     

Scutellarin protects PC12 cells from oxidative stress-induced apoptosis

The present study investigated the effects of scutellarin on oxidative stress-induced cell apoptosis in PC12 cells. Exposure of cells to hydrogen peroxide (H₂O₂) triggered a typical apoptosis, as evidenced by DNA fragmentation, DNA loss and externalization of phosphatidylserine (PS). This treatment also caused significant elevation of oxidative stress characterized by intracellular accumulations of reactive oxygen species (ROS) and malondialdehyde (MDA), a product of lipid peroxidation. Preincubation of cells with scutellarin significantly inhibited the fragmentation and loss of DNA, the externalization of PS, and decreased the percentage of cell apoptosis. Also, intracellular accumulations of ROS and MDA resulting from H₂O₂ exposure were significantly reduced by scutellarin. These findings suggest that scutellarin exerts significant protection against oxidative stress-induced apoptosis, which might be beneficial for the prevention and treatment of oxidative stress-mediated disorders.     

2,4-dinitrophenol induces G1 phase arrest and apoptosis in human pulmonary adenocarcinoma Calu-6 cells.

2,4-dinitrophenol (DNP) is an uncoupler of oxidative phosphorylation in the mitochondria. Here, we investigated the effect of DNP on the growth of Calu-6 lung cancer cells in view of cell cycle, apoptosis, ROS production and GSH content. DNP dose-dependently decreased cell viability at 72 h (EC50 of about 200 microM) as measured by a MTT assay. The lower doses of DNP induced a G1 arrest of the cell cycle in Calu-6 cells. Analysis of the cell cycle regulatory proteins demonstrated that DNP decreased the steady-state levels of cyclin proteins and cyclin dependent kinase (CDK), but increased the protein levels of cyclin dependent kinase inhibitor (CDKI) p27. DNP also caused a marked increase in apoptosis, as evidenced by DNA fragmentation (sub-G1 DNA content), DAPI staining, the loss of mitochondrial membrane potential (DeltaPsim), externalization of phosphatidylserine (PS). In addition, DNP-treated cells significantly increased the intracellular H2O2 and O2.- levels. All of caspase inhibitors could markedly rescue Calu-6 cells from DNP-induced cell death and only pan-caspase inhibitor, Z-VAD-FMK, could slightly prevent the loss of mitochondrial membrane potential (DeltaPsim). However, none of the caspase inhibitors reduced the increased H2O2 levels, but the increased O2.- levels was slightly attenuated by pan-caspase inhibitor. In addition, the depletion of GSH content in DNP-treated cells was prevented by all of caspase inhibitors. In conclusion, DNP, which induced ROS and reduced GSH content, inhibited the growth of Calu-6 cells via cell cycle arrest at G1 phase and apoptosis.     

灯盏花素对缺氧/复氧诱导的皮质神经元凋亡的影响

目的探讨灯盏花素对缺氧/复氧损伤大鼠皮质神经元的保护作用,并探讨其机制。方法培养大鼠皮质神经元,造成缺氧/复氧损伤模型,分为对照组、模型组、灯盏花素低浓度组及灯盏花素高浓度组,CCK-8法观察细胞活性,检测各组细胞上清液乳酸脱氢酶(LDH)及细胞超氧化物歧化酶(SOD)、丙二醛(MDA)含量变化,流式细胞技术检测细胞内活性氧(ROS)水平,Hoechst染色检测凋亡细胞比例,Western blot分析Bcl-2、Bax的变化。结果 CCK-8结果显示,用1、10μmol/L浓度的灯盏花素处理后,皮质神经元存活率分别提高到59.87%±4.53%和69.93%±5.27%,均高于模型组(36.68%±4.79%)(P<0.05);2′,7′-二氯二氢荧光素二乙酸酯(H2DCFDA)荧光探针染色结果显示,灯盏花素抑制了神经元细胞内ROS水平及细胞凋亡,增强了Bcl-2蛋白的表达,降低了Bax蛋白的表达。结论灯盏花素可以抑制缺氧/复氧诱导的神经元氧化损伤及凋亡的发生,为预防和治疗缺血性脑卒中及退行性神经疾病提供了理论支持。     

Protective effect of hopeahainol A, a novel acetylcholinesterase inhibitor, on hydrogen peroxide-induced injury in PC12 cells

In this study, we evaluated the effects of hopeahainol A, a novel acetylcholinesterase inhibitor (AChEI) from Hopea hainanensis, on H(2)O(2)-induced cytotoxicity in PC12 cells and the possible mechanism. Exposure of PC12 cells to 200μM H(2)O(2) caused cell apoptosis, reduction in cell viability and antioxidant enzyme activities, increment in malondialdehyde (MDA) level, and leakage of lactate dehydrogenase (LDH). Pretreatment of the cells with hopeahainol A at 0.1-10μM before H(2)O(2) exposure significantly attenuated those changes in a dose-dependent manner. Moreover, hopeahainol A could mitigate intracellular accumulation of reactive oxygen species (ROS) and Ca(2+), the loss of mitochondrial membrane potential (MMP), and the increase of caspase-3, -8 and -9 activities induced by H(2)O(2). These results show that hopeahainol A protects PC12 cells from H(2)O(2) injury by modulating endogenous antioxidant enzymes, scavenging ROS and prevention of apoptosis. There was potential for hopeahainol A to be used in treating Alzheimer's disease (AD) that involved acetylcholinesterase, free radical, oxidative damage and cell apoptosis.     

Cyclosporine A regulate oxidative stress-induced apoptosis in cardiomyocytes: mechanisms via ROS generation,iNOS and Hsp70

1. Previous study suggested that cyclosporine A (CsA) could partially reduce ischaemia/reperfusion-induced injury in isolated heart, but the mechanism was still unclear. In this study, the possible mechanisms of cyclosporine A in regulating oxidative stress-induced cardiomyocyte apoptosis were examined. 2. Morphological (cell shrinkage, apoptotic body formation, and DNA fragmentation) and biochemical (annexin-V staining for exposed phosphatidylserine residues) evidences showed that both hydrogen peroxide (H(2)O(2)) and hypoxia/reoxygenation could induce apoptotic change in the embryonal rat heart myoblast-derived cells (H9c2). These effects were inhibited by pre-treatment with CsA at concentration of 0.01-1.0 micro M for 24 h, but were increased with 10.0 micro M CsA. 3. While examining the mechanisms of CsA in protecting cardiomyocyte apoptosis, we found that the collapse of mitochondria membrane potential (DeltaPsim) induced by oxidative stress was partially reversed by CsA (0.01-1.0 micro M). 4. Compared to the control, CSA at the concentration of 0.1 and 10.0 micro M significantly increased the level of intracellular reactive oxygen species (ROS) to 117.2+/-12.4% and 234.4+/-9.3%, respectively. Co-incubating with the antioxidant, ascorbic acid (10.0 micro M), could partially reduce the protective effect of CsA (0.01-1.0 micro M) and the toxic effect of 10.0 micro M CsA. 5. Pre-treatment with CsA at concentration of 0.01-1.0 micro M for 24 h produced up-regulation of heat shock protein 70 (Hsp 70), inducible nitric oxide synthase (iNOS) and also induced NO production, indicating that these factors might be associated with the cell protective effects of CsA. 6. These results suggest that CsA could protect the oxidative stress-induced cardiomyocyte apoptosis not only by preventing the loss of DeltaPsim in mitochondria, but also through ROS generation, Hsp70, and iNOS up-regulation.     

Differential regulation of phosphatidylserine externalization and DNA fragmentation by caspases in anticancer drug-induced apoptosis of rat mammary adenocarcinoma MTLn3 cells

Caspase activation is a central event in the execution phase of apoptosis and is associated with phosphatidylserine (PS) externalization and DNA fragmentation. We investigated the role of caspase activity in anticancer drug-induced PS externalization and DNA fragmentation in MTLn3 cells. Caspase activation (DEVD-AMC cleavage) occurred in a time- and concentration-dependent manner after exposure to doxorubicin, in association with cleavage of poly(ADP) ribose polymerase and protein kinase C delta, two caspase-3 substrates. Caspase activation was closely followed by oligonucleosomal DNA fragmentation and PS externalization as determined by flow cytometric analysis. Similar observations were made for etoposide and cisplatin. Inhibition of caspases with zVAD-fmk inhibited almost completely doxorubicin-induced DNA fragmentation as well as proteolysis of protein kinase C delta. In contrast, PS externalization induced by doxorubicin was only partly affected by caspase inhibition. Flow cytometric cell sorting demonstrated that DNA fragmentation in the remaining PS positive cells after doxorubicin treatment in the presence of zVAD-fmk was fully blocked. In conclusion, these data indicate that while DNA fragmentation in anticancer drug-induced apoptosis of MTLn3 cells is fully dependent on caspase activity, PS externalization is controlled by both caspase-dependent and caspase-independent pathways.     

The changes of intracellular H2O2 are an important factor maintaining mitochondria membrane potential of antimycin A-treated As4.1 juxtaglomerular cells

We investigated an involvement of ROS, such as H2O2 and O2- and GSH in the As4.1 cell death by antimycin A and examined whether ROS scavengers rescue antimycin A-induced As4.1 cell death and its mechanism. Levels of intracellular H2O2 and O2- were markedly increased in antimycin A-treated cells. Antimycin A reduced the intracellular GSH content. A ROS scavenger, Tiron down-regulated the production of intracellular H2O2. However, the reduction of intracellular H2O2 level did not change the apoptosis parameters, such as sub-G1 DNA content and annexin V binding. Interestingly, treatment of Tiron could partially prevent the loss of mitochondrial transmembrane potential (DeltaPsi(m)). Treatment of SOD and catalase also reduced the intracellular H2O2 and loss of mitochondrial transmembrane potential (DeltaPsi(m)) without reducing O2- level and apoptosis in antimycin A-treated As4.1 cells. All the ROS scavengers, SOD and catalase did not inhibit GSH depletion induced by antimycin A, resulting in failure of preventing the apoptosis. In addition, all the reagents including antimycin A did not induce any specific phase arrest of cell cycle in As4.1 cells. In summary, these results demonstrate that antimycin A generates potently ROS, H2O2 and O2- and induces the depletion of GSH content in As4.1 JG cells, and that Tiron, SOD and catalase inhibited partially the loss of mitochondrial transmembrane potential (DeltaPsi(m)) via the reduction of intracellular H2O2 level.     

B cell responses to oxidative stress

B-lymphocytes are exposed to a reduction/oxidation environment during activation or inflammatory process, and the antioxidant systems are functional to protect themselves against harmful reactive oxygen species (ROS). The crucial roles of thioredoxin-2 (Trx-2) and a DNA repair enzyme APE/Ref-1 in mitochondria are reported in B-lymphocytes. Furthermore, ROS stimulate different signaling pathways in many cellular responses. Their effects often cause some diseases or are utilized for the treatment of other diseases. For example, the cells derived from Fanconi anemia (FA) patients are intolerant of oxidative stress and the therapeutic effect of anti-CD20 monoclonal antibody rituximab on B cell lymphoproliferative disorders is due to the generation of ROS. To clarify the oxidative stress-induced signaling pathways, we stimulated a B cell line with various concentrations of H(2)O(2). As a result, a protein tyrosine kinase, Syk was involved in the induction of G2/M arrest and protection of cells from apoptosis. Syk might inhibit the activation of caspase-9 through Akt thereby protecting cells from oxidative stress-induced apoptosis. On the other hand, Syk-dependent PLC-gamma2 activation was required for acceleration towards apoptosis following oxidative stress. These findings suggest that oxidative stress-induced Syk activation triggers the activation of different pathways, such as pro-apoptotic or survival pathways, and that the balance of these pathways is a key factor in determining the fate of the cells exposed to oxidative stress. In contrast, the stimulation with the millimolar concentrations of H(2)O(2) rapidly led to necrosis in which tyrosine phosphorylation of FAK was involved at the downstream of Lyn and Syk.     

Cadmium induces caspase-mediated cell death: suppression by Bcl-2

Apoptosis is a process of active cell death and is characterized by activation of caspases, DNA fragmentation, and biochemical and morphological changes. To better understand apoptosis, we have characterized the dose- and time-dependent toxic effects of cadmium in Rat-1 fibroblasts. Staining of cells with phosphatidylserine (PS)-annexin V, Hoechst 33258 or Rhodamine 123 and Tunel assays showed that incubating cells with 10 microM cadmium induced a form of cell death exhibiting typical characteristics of apoptosis, including cell shrinkage, externalization of PS, loss of mitochondria membrane potential, nuclear condensation and DNA fragmentation. Expression of Bcl-2 or CrmA each suppressed cadmium-induced cell death although Bcl-2 was somewhat more effective than CrmA. In vitro assay of caspase activity carried out using poly(ADP-ribose) polymerase (PARP) as a substrate as well as intracellular caspase assays using a fluorigenic caspase-3 substrate confirmed that caspase-3 is activated in Rat-1 cells undergoing cadmium-induced apoptosis. Both Asp-Glu-Val-Asp-aldehyde (DEVD-cho) and Tyr-Val-Ala-Asp-chloromethylketone (YVAD-cmk), selective inhibitors of caspase-3 and caspase-1, respectively, suppressed significantly cadmium-induced cell death. However, the nonselective caspase inhibitor, z-Val-Ala-Asp-floromethylketone (zVAD-fmk), was the most efficacious agent, almost completely blocking cadmium-induced cell death. Taken together, these results demonstrate that as in other forms of apoptosis, caspases play a central role in cadmium-induced cell death.     

Tanshinone IIA protects cardiac myocytes against oxidative stress-triggered damage and apoptosis

Tanshinone IIA (tan), a derivative of phenanthrenequinone, is one of the key components of Salvia miltiorrhiza Bunge. Previous reports showed that tan inhibited the apoptosis of cultured PC12 cells induced by serum withdrawal or ethanol. However, whether tan has a cardioprotective effect against apoptosis remains unknown. In this study, we investigated the effects of tan on cardiac myocyte apoptosis induced both by in vitro incubation of neonatal rat ventricular myocytes with H(2)O(2) and by in vivo occlusion followed by reperfusion of the left anterior descending coronary artery in adult rats. In vitro, as revealed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay, treatment with tan prior to H(2)O(2) exposure significantly increased cell viability. Tan also markedly inhibited H(2)O(2)-induced cardiomyocyte apoptosis, as detected by ladder-pattern fragmentation of genomic DNA, chromatin condensation, and hypodioloid DNA content. In vivo, tan significantly inhibited ischemia/reperfusion-induced cardiomyocyte apoptosis by attenuating morphological changes and reducing the percentage of terminal transferase dUTP nick end-labeling (TUNEL)-positive myocytes and caspase-3 cleavage. These effects of tan were associated with an increased ratio of Bcl-2 to Bax protein in cardiomyocytes, an elevation of serum superoxide dismutase (SOD) activity and a decrease in serum malondialdehyde (MDA) level. Taken together, these data for the first time provide convincing evidence that tan protects cardiac myocytes against oxidative stress-induced apoptosis. The in vivo protection is mediated by increased scavenging of oxygen free radicals, prevention of lipid peroxidation and upregulation of the Bcl-2/Bax ratio.     

Protective effects of salidroside on hydrogen peroxide-induced apoptosis in SH-SY5Y human neuroblastoma cells

Oxidative stress plays an important role in Alzheimer's disease and other neurodegenerative disorders. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L, shows potent antioxidant property. In this paper, the neuroprotective effects of salidroside on hydrogen peroxide (H2O2)-induced apoptosis in SH-SY5Y cells were investigated. Pretreatment with salidroside markedly attenuated H2O2-induced cell viability loss and apoptotic cell death in a dose-dependent manner. The mechanisms by which salidroside protected neuron cells from oxidative stress included the induction of several antioxidant enzymes, thioredoxin, heme oxygenase-1, and peroxiredoxin-I; the downregulation of pro-apoptotic gene Bax and the upregulation of anti-apoptotic genes Bcl-2 and Bcl-X(L). Furthermore, salidroside dose-dependently restored H2O2-induced loss of mitochondrial membrane potential as well as the elevation of intracellular calcium level. These results suggest that salidroside has protective effects against oxidative stress-induced cell apoptosis, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases implicated with oxidative stress.     

JWA as a novel molecule involved in oxidative stress-associated signal pathway in myelogenous leukemia cells

Previous data showed that JWA might be a novel environmental responsive gene regulated by environmental stressors such as heat shock and oxidative stress. However, the molecular mechanism underlying JWA gene function involved in oxidative stress is still unknown. In this study, the potential role of JWA was further investigated in hydrogen peroxide (H2O2) induced DNA damage and cell apoptosis in K562 cells. Series of the oxidative stress models were established to observe if JWA was involved in DNA damage or cell apoptosis induced by H2O2 exposure. These results indicated that the inhibitory effect on K562 cells' viability induced by H2O2 was concentration and time dependent. JWA was more sensitive to H2O2 (0.01 mmol/L) than the heat-shock proteins (hsp70 and hsp27), and its expression pattern was similar to that of hsp70. In addition, JWA, hsp70, hsp27, and p53 were overexpressed and the expression patterns of JWA, hsp70, and p53 were similar during cell apoptosis. H2O2 led to the cleavage and activation of procaspase-3. In conclusion, these results suggested that JWA might be an effective environmental responsive gene that functions as a parallel with hsp70 in oxidative stress-responsive pathways in K562 cells. Like hsp70, JWA might enhance intracellular defenses and function against H2O2-induced oxidative stress in leukemia cells. At the same time, JWA was involved in the p53-associated signal pathways of oxidative stress-induced apoptosis, which is also caspase-3 dependent.     

珠子参总皂苷对H2O2诱导新生大鼠心肌细胞凋亡的抑制作用

目的:观察珠子参总皂苷对H2O2诱导新生大鼠心肌细胞凋亡的抑制作用,并探讨其作用机制.方法:Wistar乳鼠心肌细胞原代培养,用H2O2建立氧化应激损伤模型,然后用珠子参总皂苷(100,200 μg/mL)孵育24 h后,MTT法检测珠子参总皂苷对细胞活力的影响,流式细胞仪和Hoechst33258染色检测珠子参总皂苷对氧化应激诱导细胞凋亡及胞内ROS含量的影响,比色法测定心肌细胞Caspase-3、Caspase-9的活性,荧光定量PCR测定心肌细胞Bcl-2和Bax mRNA表达,并计算Bcl-2与Bax的比值.结果:珠子参总皂苷(100,200 μg/mL)能显著改善心肌细胞活性,有效保护线粒体膜电位的稳定,抑制心肌细胞凋亡和改善细胞形态,降低细胞内活性氧(ROS)含量;下调Bax mRNA表达,上调Bcl-2mRNA表达及Bcl-2与Bax的比值;降低H2O2所致新生大鼠心肌细胞中Caspase-3、Caspase-9的活性.结论:珠子参总皂苷对H2O2诱导心肌细胞凋亡有显著的抑制作用,其机制可能与其稳定心肌细胞膜、清除ROS及调节心肌细胞Bcl-2、Bax和Caspase-3、Caspase 9表达有关.     

Chromosomal DNA fragmentation in apoptosis and necrosis induced by oxidative stress

Chromosomal DNA dysfunction plays a role in mammalian cell death. Oxidative stress producing reactive oxygen species (ROS) induces chromatin dysfunction such as single- and double-strand DNA fragmentation leading to cell death through apoptosis or necrosis. More than 1 Mbp giant DNA, 200-800 or 50-300 kbp high molecular weight (HMW) DNA and internucleosomal DNA fragments are produced by oxidative stress and by some agents producing ROS during apoptosis or necrosis in several types of mammalian cells. Some nucleases involved in the chromosomal DNA fragmentation in apoptosis or necrosis are classified. ROS-mediated DNA fragmentation is caused and enhanced by polyunsaturated fatty acids (PUFAs) or their hydroperoxides through lipid peroxidation. A reduction of intracellular GSH levels induced by the inhibition of cystein transport or GSH biosynthesis leads to cell death through over production and accumulation of ROS in some types of mammalian cells. The ROS accumulation system has been used as a model of oxidative stress to discuss whether ROS-mediated DNA fragmentation associated with cell death is based on apoptosis or necrosis.     

柚皮素对氧化应激所致成骨细胞凋亡的影响及机制研究

目的：观察柚皮素对氧化应激导致的成骨细胞凋亡的影响,并探讨其机制。方法体外分离培养成骨细胞,细胞分空白对照组、单纯 H2 O2作用组、H2 O2＋0 J．1μmol／L 柚皮素组、H2 O2＋1μmol／L柚皮素组、H2 O2＋10μmol／L柚皮素组。 MTT法检测成骨细胞活力;流式细胞术检测成骨细胞凋亡率;荧光显微镜观察活性氧（ ROS）含量;比色法检测丙二醛（ MDA）含量;Real time-PCR和Western-blot检测凋亡相关蛋白Bcl-2、Bax、Caspase-3表达情况。结果与空白对照组比较,单纯H2 O2处理组细胞活性明显降低,凋亡率及ROS、MDA含量明显增加;同时Bcl-2 mR-NA和蛋白表达明显下调,Bax、Caspase-3 mRNA和蛋白表达明显上调（ P ＜0．05）。与单纯H2 O2处理组比较,柚皮素预处理24 h能够明显增强细胞活性,降低细胞凋亡率及ROS、MDA含量,上调Bcl-2 mRNA和蛋白表达,下调Bax、Caspase-3 mRNA和蛋白表达,呈剂量依赖性（ P ＜0．05）。结论柚皮素在氧化应激条件下能够促进成骨细胞增殖,抑制成骨细胞凋亡,其机制与上调Bcl-2表达,下调Bax、Caspase-3表达有关。     

Quercetin, but not rutin and quercitrin, prevention of H2O2-induced apoptosis via anti-oxidant activity and heme oxygenase 1 gene expression in macrophages

In the present study, we examine the protective mechanism of quercetin (QE) on oxidative stress-induced cytotoxic effect in RAW264.7 macrophages. Results of Western blotting show that QE but not its glycoside rutin (RUT) and quicitrin-induced HO-1 protein expression in a time- and dose-dependent manner, and HO-1 protein induced by QE was blocked by an addition of cycloheximide or actinomycin D. Induction of HO-1 gene expression by QE was accompanied by inducing ERKs, but not JNKs or p38, proteins phosphorylation. Addition of PD98059, but not SB203580 or SP600125, significantly attenuates QE-induced HO-1 protein and mRNA expression associated with blocking the expression of phosphorylated ERKs proteins. H(2)O(2) addition reduces the viability of cells by MTT assay, and appearance of DNA ladders, hypodiploid cells, and an increase in intracellular peroxide level was detected. Addition of QE, but not QI or RUT, significantly reduced the cytotoxic effect induced by H(2)O(2) associated with blocking the production of intracellular peroxide, DNA ladders, and hypodiploid cells. QE protection of cells from H(2)O(2)-induced apoptosis was significantly suppressed by adding HO inhibitor SnPP or ERKs inhibitor PD98059. Additionally, QE protects cells from H(2)O(2)-induced a decrease in the mitochondrial membrane potential and a release of cytochrome c from mitochondria to cytosol by DiOC6 and Western blotting assay, respectively. Activation of apoptotic proteins including the caspase 3, caspase 9, PARP, D4-GDI proteins was identified in H(2)O(2)-treated cells by Western blotting and enzyme activity assay, and that was significantly blocked by an addition of QE, but not RUT and QI. Furthermore, HO-1 catalytic metabolites carbon monoxide (CO), but not Fe(2+), Fe(3+), biliverdin or bilirubin, performed protective effect on cells from H(2)O(2)-induced cell death with an increase in HO-1 protein expression and ERKs protein phosphorylation. These data suggest that induction of HO-1 protein may participate in the protective mechanism of QE on oxidative stress (H(2)O(2))-induced apoptosis, and reduction of intracellular ROS production and mitochondria dysfunction with blocking apoptotic events were involved. Differential anti-apoptotic effect between QE and its glycosides RUT and QI via distinct HO-1 protein induction was also delineated.     

Characterization of hydrogen peroxide-induced apoptosis in mouse primary cultured hepatocytes

The influence of oxidative stress by hydrogen peroxide (H2O2) was examined in mouse primary cultured hepatocytes. A change in morphology was observed in hepatocytes incubated for 30 min in saline A containing H2O2. The percentage of dead cells, as measured by the fluorescence method, was increased in a dose-dependent manner. In addition, a ladder-like DNA fragmentation pattern was detected by agarose gel electrophoresis 1 h after exposure to 3 mM H2O2. This phenomenon was prolonged for 24 h. Hydrogen peroxide-induced cell viability reduction and DNA fragmentation were dose-dependently protected by the addition of antioxidants (N-acetylcysteine, L-ascorbic acid), a metal-chelator (1,10-phenanthroline), iron-chelator (deferoxamine) and intracellular calcium ion chelator (quin 2-AM). No influence, however, was detected by endonuclease inhibitors (zinc, aurintricarboxylic acid) and poly (ADP-ribose) polymerase inhibitors (3-aminobenzamide, theophylline). These results following H2O2-induced cell viability reduction suggested that oxidative stress by H2O2 itself or H2O2-derived changes involved in ferrous or intracellular calcium ions resulted in apoptosis in mouse primary cultured hepatocytes. These phenomena are not likely to be associated with endonuclease or poly (ADP-ribose) polymerase.     

唐古特大黄多糖对过氧化氢诱导的肠上皮细胞凋亡的保护作用及其可能机制

目的探讨唐古特大黄多糖(Rheum tanguticumpoly-saccharide,RTP)组分1(RTP1)对过氧化氢(H2O2)诱导的肠上皮细胞IEC-6凋亡的保护作用及其可能的机制。方法以不同浓度的RTP1(10、30、100 mg.L-1)预处理体外培养的IEC-6细胞后,加入H2O2诱导IEC-6细胞凋亡,MTT法检测细胞活力,激光共聚焦显微镜荧光染色法检测细胞内活性氧的产生,DNA琼脂糖凝胶电泳、吖啶橙染色及流式细胞仪检测细胞凋亡的发生,Western blot测定Caspase-3酶活性。结果 100μmol.L-1H2O2处理细胞2 h明显降低细胞存活率并诱导细胞凋亡,凋亡率为31.3%,细胞内活性氧水平及Caspase-3的活性明显升高,不同浓度(10、30、100 mg.L-1)RTP1预处理细胞24 h,可明显提高细胞存活率,有效抑制DNAladder的发生,流式细胞仪检测凋亡率(30、100 mg.L-1)分别降低至24.4%和21.5%,Caspase-3酶活性降低,并呈现一定的剂量依赖性。结论 RTP1可明显抑制H2O2诱导的IEC-6细胞凋亡,其细胞保护作用可能与降低细胞内活性氧水平和抑制Caspase-3活性相关。