Oxidative stress-mediated early senescence contributes to the short replicative life span of human peritoneal mesothelial cells

The replicative life span of cells in culture is thought to be determined by the gradually rising pool of senescent cells rather than by the simultaneous loss of proliferative capacity by all cells in the population. We found that early-passage cultures of human peritoneal mesothelial cells (HPMCs) contained a significant fraction of senescent-like cells. Furthermore, early-passage populations with a high percentage of senescent cells had a reduced subsequent life span in culture compared with populations consisting of the same number of apparently young cells but containing no senescent cells. The exposure of early-passage HPMCs to the conditioned medium from cultures containing senescent cells resulted in the retardation of growth and the induction of senescence-associated beta-galactosidase (SA-beta-Gal). This effect could be partly reduced by neutralizing TGF-beta1 activity. The timely treatment with N-tert-butyl-alpha-phenylnitrone (PBN) reduced oxidative stress, the number of early senescent cells, TGF-beta1 secretion, and ultimately extended the population life span. The effect was evident only when PBN was introduced at a very early, but not at a late, phase of tissue culture history. These results indicate that a sudden onset of senescence in early-passage HPMCs is related to oxidative stress and may influence the replicative life span of the population as a whole.     

Angiopoietin-1 protects H9c2 cells from H2O2-induced apoptosis through AKT signaling

Loss of cardiomyocytes by apoptosis is proposed to cause ventricular remodeling and heart failure. Reactive oxygen species-induced apoptosis of cardiomyocytes has been reported to play an important role in many types of pathological processes of the heart. We investigated whether angiopoietin-1 (Ang1) has direct cytoprotective effects on cardiomyocytes against oxidative stress. Cultured H9c2 cells (cardiomyocytes) were treated with hydrogen peroxide (H(2)O(2)). Apoptosis was evaluated by flow cytometry, TUNEL assay and DNA laddering. The H(2)O(2) treatment caused typical apoptosis of H9c2 cells in a time-dependent manner. Transfection of recombinant adenovirus expressing Ang1 resulted in a sustained phosphorylation of AKT and inhibition of H(2)O(2)-induced apoptosis in H9c2 cells. This effect could be reversed by AKT inhibition. These results suggest that Ang1 protects cardiomyocytes from oxidative stress-induced apoptosis by regulating the activity of AKT.     

Protective effects of aloe-emodin on scopolamine-induced memory impairment in mice and H2O2-induced cytotoxicity in PC12 cells

Aloe-emodin (AE) is one of the most important active components of Rheum officinale Baill. The present study aimed to investigate that AE could attenuate scopolamine-induced cognitive deficits via inhibiting acetylcholinesterase (AChE) activity and modulating oxidative stress. Kunming (KM) mice were received intraperitoneal injection of scopolamine (2 mg/kg) to induce cognitive impairment. Learning and memory performance were assessed in the Morris water maze (MWM). After behavioral testing, the mice were sacrificed and their hippocampi were removed for biochemical assays (superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA), AChE and acetylcholine (ACh)). In vitro, we also performed the AChE activity assay and H₂O₂-induced PC12 cells toxicity assay. After 2 h exposure to 200 μM H₂O₂ in PC12 cells, the cytotoxicity were evaluated by cell viability (MTT), nitric oxide (NO)/lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS) production. Our results confirmed that AE showed significant improvement in cognitive deficit in scopolamine-induced amnesia animal model. Besides, it increased SOD, GPx activities and ACh content, while decreased the level of MDA and AChE activity in AE treated mice. In addition, AE was found to inhibit AChE activity (IC₅₀ = 18.37 μg/ml) in a dose-dependent manner. Furthermore, preincubation of PC12 cells with AE could prevent cytotoxicity induced by H₂O₂ and reduce significantly extracellular release of NO, LDH and intracellular accumulation of ROS. The study indicated that AE could have neuroprotective effects against Alzheimer’s disease (AD) via inhibiting the activity of AChE and modulating oxidative stress.     

Characterization of H2O2-induced acute apoptosis in cultured neural stem/progenitor cells

In the present study, we characterized hydrogen peroxide (H₂O₂)-induced cell apoptosis and related cell signaling pathways in cultured embryonic neural stem/progenitor cells (NS/PCs). Our data indicated that H₂O₂ induced acute cell apoptosis in NS/PC in concentration- and time-dependent manners and selectively, it transiently increased PI3K-Akt and Mek-Erk1/2 in a dose-dependent manner. Inhibition of PI3K-Akt with wortmannin, a PI3-K inhibitor, was found to significantly increase H₂O₂-induced acute apoptosis and dramatically decrease basal pGSK3β levels. The level of pGSK3β remained unchanged with H₂O₂ exposure. We conclude that the transient activation of PI3K-Akt signaling delays the H₂O₂-induced acute apoptosis in cultured NS/PCs in part through maintaining the basal pGSK3β level and activating other downstream effectors.     

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。     

Lipid constituents in oligodendroglial cells alter susceptibility to H2O2-induced apoptotic cell death via ERK activation

The present work examines the effect of membrane lipid composition on activation of extracellular signal-regulated protein kinases (ERK) and cell death following oxidative stress. When subjected to 50 microM docosahexaenoic acid (DHA, 22 : 6 n-3), cellular phospholipids of OLN 93 cells, a clonal line of oligodendroglia origin low in DHA, were enriched with this polyunsaturated fatty acid. In the presence of 1 mM N,N-dimethylethanolamine (dEa) a new phospholipid species analog was formed in lieu of phosphatidylcholine. Exposure of DHA-enriched cells to 0.5 mM H2O2, caused sustained activation of ERK up to 24 h. At this time massive apoptotic cell death was demonstrated by ladder and TUNEL techniques. H2O2-induced stress applied to dEa or DHA/dEa co-supplemented cells showed only a transient ERK activation and no cell death after 24 h. Moreover, while ERK was rapidly translocated into the nucleus in DHA-enriched cells, dEa supplements completely blocked ERK nuclear translocation. This study suggests that H2O2-induced apoptotic cell death is associated with prolonged ERK activation and nuclear translocation in DHA-enriched OLN 93 cells, while both phenomena are prevented by dEa supplements. Thus, the membrane lipid composition ultimately modulates ERK activation and translocation and therefore can promote or prevent apoptotic cell death.     

JNK1 activity lowers the cellular production of H2O2 and modulates the growth arrest response to scavenging of H2O2 by catalase

Hydrogen peroxide (H(2)O(2)) can interact with intracellular signaling pathways to regulate cell behavior. The c-Jun NH(2)-terminal kinase 1 (JNK1) signal, involved in diverse aspects of cellular functioning, is implicated as a cell sensor of redox stress. The growth-inhibitory effect of both high-level H(2)O(2) and H(2)O(2)-scavenging catalase treatments is accompanied by increased JNK1 activity. To investigate the role of this response in growth regulation, the JNK1 signal was increased by the introduction of ectopic HA-JNK1. HA-JNK1 expression correlated with increases in basal c-Jun phosphorylation in a dose-dependent manner. Transient expression of HA-JNK1 potentiated cell growth arrest by catalase; however, with stable expression a degree of resistance to this response was observed. Resistance was accompanied by a lowered endogenous production of H(2)O(2). Transient HA-JNK1 expression also reduced H(2)O(2) generation, and this effect was reversed by the JNK inhibitor SP600125. These results indicate that the JNK1 stress response contributes to growth inhibition by catalase treatment via inhibition of cellular H(2)O(2) production. Stable amplification of the JNK1 pathway leads to cellular adaptation to its signal, resulting in a diminished reliance upon H(2)O(2) for efficient growth.     

Salvia macilenta exhibits antiglycating activity and protects PC12 cells against H2O2-induced apoptosis

Salvia macilenta is a member of the genus Salvia (Laminaceae) whose antioxidant activity and neuroprotective effect has been shown previously. The present study aimed to examine the antiglycating and antiapoptotic abilities of methanolic extract of this plant. Moreover, the effect of S. macilenta on neurite outgrowth and complexity after exposure to H₂O₂ has been studied. Base on our results, S. macilenta has antiglycating activity and protects PC12 cells against oxidative stress-induced apoptotic cell death, as examined by Hoechst staining and Western blot analysis of caspase-3, Bax, Bcl-2 and PARP. We further showed that S. macilenta decreased neurite growth and complexity impairment in differentiated PC12 cells exposed to oxidative stress. It caused a decrease in cell body area, neurite width, and the proportion of bipolar cells, while significantly increasing neurite length, the number of primary neurites per cell and the ratio of nodes to primary neuritis. All around, the mentioned results open a new horizon for future works to use this plant as a potential neuroprotective agent.     

S. choloroleuca, S. mirzayanii and S. santolinifolia protect PC12 cells from H(2)O (2)-induced apoptosis by blocking the intrinsic pathway

Several studies have shown that neuronal cell death due to apoptosis is the major reason for cognitive decline in Alzheimer's disease. In this study, we report the anti-apoptotic effects of three Salvia species from Iran-S. choloroleuca, S. mirzayanii and S. santolinifolia-against H(2)O(2)-induced cytotoxicity in neuron-like PC12 cells. We showed that these antioxidant species could interfere with the intrinsic pathway of apoptosis by attenuating Bax/Bcl-2 ratio, decreasing outer mitochondrial membrane break and decreasing cytochrome c release to cytoplasm. Interestingly, we found that these species were able to replenish reduced glutathione level which affects cellular redox status and cytochrome c activity. Moreover, the decreased level of caspase-3, the executioner caspase, resulted in decrease of PARP-1 cleavage. Anti-apoptotic effects of these species along with their antioxidant effects, may represent a promising approach for treatment of neurodegenerative diseases.     

Protective effects of cyclosativene on H2O2-induced injury in cultured rat primary cerebral cortex cells

Sesquiterpenes have attracted much interest with respect to their protective effect against oxidative damage that may be the cause of many diseases including several neurodegenerative disorders and cancer. Our previous unpublished work suggested that cyclosativene (CSV), a tetracyclic sesquiterpene, has antioxidant and anticarcinogenic features. However, little is known about the effects of CSV on oxidative stress induced neurotoxicity. We used hydrogen peroxide (H₂O₂) exposure for 6 h to model oxidative stress. Therefore, this experimental design allowed us to explore the neuroprotective potential of CSV in H₂O₂-induced toxicity in new-born rat cerebral cortex cell cultures for the first time. For this aim, MTT and lactate dehydrogenase release assays were carried out to evaluate cytotoxicity. Total antioxidant capacity (TAC) and total oxidative stress (TOS) parameters were used to evaluate oxidative changes. In addition to determining of 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels, the single cell gel electrophoresis (or Comet assay) was also performed for measuring the resistance of neuronal DNA to H₂O₂-induced challenge. Our results showed that survival and TAC levels of the cells decreased, while TOS, 8-OH-dG levels and the mean values of the total scores of cells showing DNA damage (Comet assay) increased in the H₂O₂ alone treated cultures. But pre-treatment of CSV suppressed the cytotoxicity, genotoxicity and oxidative stress which were increased by H₂O₂. On the basis of these observations, it is suggested that CSV as a natural product with an antioxidant capacity in mitigating oxidative injuries in the field of neurodegenerative disorders.     

Identification of p38MAPK-dependent genes with changed transcript abundance in H2O2-induced premature senescence of IMR-90 hTERT human fibroblasts

Premature senescence of IMR-90 human diploid fibroblasts expressing telomerase (hTERT) establishes after exposure to an acute sublethal concentration of H2O2. We showed herein that p38(MAPK) was phosphorylated after exposure of IMR-90 hTERT cells to H2O2. Selective inhibition of p38(MAPK) activity attenuated the increase in the proportion of cells positive for senescence associated beta-galactosidase activity. We generated a low density DNA array to study gene expression profiles of 240 senescence-related genes. Using this array, p38(MAPK) inhibitor and p38(MAPK) small interferent RNA, we identified several p38(MAPK)-target genes differentially expressed in H2O2-stressed IMR-90 hTERT fibroblasts.     

H2 O2 stimulates Cl- /HCO 3- exchanger activity through oxidation of thiol groups in immortalized SHR renal proximal tubular epithelial cells

Cl(-) /HCO (3)(-) exchanger and Na(+) /H(+) exchanger 3 are the main transporters responsible for NaCl reabsorption in kidney proximal tubules (PT). It is well accepted that membrane exchangers can be regulated by reactive oxygen species (ROS). In the kidney, ROS are known to contribute to decreases in Na(+) excretion and consequently increase blood pressure. The present study investigated mechanisms by which H(2) O(2) -induced stimulation of Cl(-) /HCO (3)(-) exchanger activity is enhanced in proximal tubular epithelial (PTE) cells immortalized from spontaneously hypertensive rats (SHR) as compared to normotensive Wistar Kyoto (WKY). H(2) O(2) decreased K(m) values for Cl(-) /HCO (3)(-) exchanger activity in SHR PTE cells, but had no effect on the kinetic parameters in WKY cells. DTDP stimulated in a concentration-dependent manner Cl(-) /HCO (3)(-) exchanger activity in both cell lines, but SHR PTE cells were 2.4-fold more responsive to this oxidant. In contrast, thimerosal had no effect on exchanger activity in both cell lines. The effects of H(2) O(2) and DTDP upon the exchanger activity were blocked by DTT in WKY and SHR PTE cells. Similar to H(2) O(2), DTDP decreased K(m) values for Cl(-) /HCO (3)(-) exchanger activity in SHR PTE cells. Basal content of free thiol groups was higher in WKY PTE cells than in SHR. Upon H(2) O(2) treatment the free thiol groups decreased in both cell lines; however, this decrease was more pronounced in WKY cells. In conclusion, in SHR PTE cells H(2) O(2) stimulates Cl(-) /HCO (3)(-) exchanger activity via modification of thiol groups of intracellular and/or transmembrane protein. Furthermore, the thiol oxidation-dependent pathway also increases the HCO (3)(-) affinity in SHR PTE cells.     

Protection afforded by quercetin against H2O2-induced apoptosis on PC12 cells via activating PI3K/Akt signal pathway

Cell damage and apoptosis induced by oxidative stress have been involved in various neurodegenerative diseases. This study aims to explore the neuro-protective effects of quercetin on PC12 cells apoptosis induced by hydrogen peroxide (H₂O₂) and the underlying mechanisms. The cell viability was detected, as well as the production of reactive oxygen species (ROS), lactate dehydrogenase (LDH) leakage, and the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) of the cells in control, H₂O₂ and quercetin groups. It finally turned out that quercetin might protect PC12 cells against the negative effect of H₂O₂ by decreasing of LDH release, ROS concentration and MDA level and regaining the GSH-Px and SOD activities. To investigate the mechanism, LY294002 was introduced, the phosphatidylinositol-3-kinase (PI3K) inhibitor. Bax/Bcl-2 ratio and Akt phosphorylation (p-Akt) were examined by Western blot analysis. The data showed that LY294002 almost had the same effects with H₂O₂, which was also significantly reversed by quercetin could enhance Bax/Bcl-2 ratio and adjust the p-Akt expression, which indicated quercetin might protect PC12 cells against the negative effect of H₂O₂ via activating the PI3K/Akt signal pathway.     

Antioxidant activity of vasoactive intestinal peptide in HK2 human renal cells

Oxidative stress is a major mediator of tissue and cell injuries. The injury in chronic nephrotic syndrome, acute renal failure, myeloma kidney injury and other kidney diseases is initiated by oxidative stress. We have previously demonstrated that vasoactive intestinal peptide (VIP) acts as an antiproliferative agent in renal cancer cells. This study was designed to evaluate the renoprotective activity of VIP against H₂O₂-induced oxidative damage in a proximal tubule kidney cell line (human, non-tumor, HK2 cells) in order to investigate the potential usefulness of this peptide in the treatment of oxidative-stress related kidney diseases. HK2 cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Propidium iodide was used to identify cells undergoing apoptosis. Western blotting was performed with anti-Bcl-2, anti-Bax and anti-formyl peptide receptor (low-affinity variant FPRL-1) monoclonal antibodies whereas 2,7-dichlorofluorescein diacetate was used for measurement of levels of intracellular reactive oxygen species (ROS). HK2 cells were injured with H₂O₂ in order to induce apoptosis: the effect was time- and dose-dependent. VIP increased the levels of the antiapoptotic protein Bcl-2 and decreased those of the proapoptotic protein Bax. VIP decreased the intracellular ROS levels reached by H₂O₂-induced oxidative stress. VIP effect on ROS levels involved FPLR-1 but not VPAC_1,2 receptors as evidenced by the use of the respective antagonists WRW4 and JV-1-53. Thus, VIP protects HK2 cells from apoptosis by increasing Bcl-2 levels and this effect is initiated through FPLR1 receptor. In conclusion, VIP might exert a renoprotective effect by the suppression of oxidative stress.     

Expression of human myoglobin in H9c2 cells enhances toxicity to added hydrogen peroxide

Hydrogen peroxide (H2O2) is implicated in cardiac myocyte (CM) damage during myocardial ischemia-reperfusion (IR) injury. Myoglobin (Mb) is present in CM at significant concentrations and reacts with H2O2 to yield one- and two-electron oxidants that may promote myocardial injury. Paradoxically, hearts from mice lacking Mb are more susceptible to H2O2-induced dysfunction than the corresponding controls [U. Flogel, A. Godecke, L.O. Klotz, J. Schrader, Role of myoglobin in the anti-oxidant defense of the heart, FASEB J. 18 (2004) 1156-1158]. We have overexpressed wild-type or Y103F variant of human Mb in cultured CMs to test whether Mb protects against H2O2 insult. Contrary to expectation, cells expressing WT or the Y103F Mb show increased mitochondrial dysfunction and apoptosis, and decreased ATP in response to H2O2 that follows the order native < Y103F Mb < WT human Mb consistent with the increasing pro-oxidant activity for these proteins. These data indicate that (i) Mb promotes oxidative damage to cultured CM and (ii) Mb may be a useful target for the design of inhibitors of myocardial IR injury.     

Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe2+ in human monocytic U937 cells

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca²⁺-permeable channel. In monocytes/macrophages, H₂O₂-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H₂O₂ are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H₂O₂ were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30 °C to 37 °C enhanced H₂O₂-induced TRPM2-mediated increase in intracellular free Ca²⁺ ([Ca²⁺]_i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H₂O₂-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe²⁺-accumulation following pretreatment with FeSO₄. Thus intracellular Fe²⁺-accumulation sensitizes H₂O₂-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe²⁺-accumulation increased poly(ADP-ribose) levels in nuclei by H₂O₂ treatment, and the sensitization of H₂O₂-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe²⁺-accumulation enhances H₂O₂-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO₄ stimulated H₂O₂-induced TRPM2 activation at 37 °C in U937 cells and enhanced H₂O₂-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H₂O₂ to cells under conditions of intracellular Fe²⁺-accumulation caused cell death, concentration of H₂O₂ required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe²⁺-accumulation sensitizes TRPM2 channels to H₂O₂ and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H₂O₂-induced TRPM2 channels by Fe²⁺ may implicated in hemorrhagic brain injury via aggravation of inflammation, since Fe²⁺ is released by heme degradation under intracerebral hemorrhage.     

A molecular understanding of d‐homoestrone‐induced G2/M cell cycle arrest in HeLa human cervical carcinoma cells

2‐Methoxyestradiol (ME), one of the most widely investigated A‐ring‐modified metabolites of estrone, exerts significant anticancer activity on numerous cancer cell lines. Its pharmacological actions, including cell cycle arrest, microtubule disruption and pro‐apoptotic activity, have already been described in detail. The currently tested d ‐ring‐modified analogue of estrone, d ‐homoestrone, selectively inhibits cervical cancer cell proliferation and induces a G2/M phase cell cycle blockade, resulting in the development of apoptosis. The question arose of whether the difference in the chemical structures of these analogues can influence the mechanism of anticancer action. The aim of the present study was therefore to elucidate the molecular contributors of intracellular processes induced by d ‐homoestrone in HeLa cells. Apoptosis triggered by d ‐homoestrone develops through activation of the intrinsic pathway, as demonstrated by determination of the activities of caspase‐8 and ‐9. It was revealed that d ‐homoestrone‐treated HeLa cells are not able to enter mitosis because the cyclin‐dependent kinase 1‐cyclin B complex loses its activity, resulting in the decreased inactivation of stathmin and a concomitant disturbance of microtubule formation. However, unlike 2‐ME, d ‐homoestrone does not exert a direct effect on tubulin polymerization. These results led to the conclusion that the d ‐homoestrone‐triggered intracellular processes resulting in a cell cycle arrest and apoptosis in HeLa cells differ from those in the case of 2‐ME. This may be regarded as an alternative mechanism of action among steroidal anticancer compounds.