Involvement of selenoprotein P in protection of human astrocytes from oxidative damage

Selenoprotein P (SeP) is a highly glycosylated, selenium-rich plasma protein. Aside from its role as selenium carrier protein, an antioxidative function of SeP has been suggested. Astrocytes, which detoxify reactive oxygen species in the brain, were described as potential target cells of SeP. We investigated the expression of SeP in human astrocytes and its involvement in the protection of these cells against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage. We show that primary human astrocytes and the human astrocytoma cell line MOG-G-CCM express SeP as an unglycosylated protein, which is not secreted. SeP expression in astrocytes is constitutive. Preincubation of astrocytes with hepatocyte-derived SeP mimicks the protective effect of low-molecular-weight selenocompounds such as sodium selenite or selenomethionine against oxidative damage, shielding astrocytes from t-BHP-induced cytotoxicity. Selenium supplementation of astrocytes counteracts oxidative stress via an increase in expression and activity of the selenoenzyme cytosolic glutathione peroxidase (cGPx). Furthermore, specific downregulation of SeP expression by small interfering RNA decreases cell viability of human astrocytes and makes them more susceptible to t-BHP-induced cytotoxicity. Our results implicate an antioxidant activity of constitutively expressed SeP in selenium-deficient astrocytes, while during adequate selenium supply the enhanced protection against oxidative stress is exerted by cGPx.     

Activation of HSP70 impedes tert-butyl hydroperoxide (t-BHP)-induced apoptosis and senescence of human nucleus pulposus stem cells via inhibiting the JNK/c-Jun pathway

The endogenous repair failure of degenerated intervertebral disk (IVD) is highly related to the exhaustion of nucleus pulposus stem cells (NPSCs). Excessive oxidative stress could induce apoptosis and senescence of NPSCs, thus, declining the quantity and quality of NPSCs. Heat shock protein 70 (HSP70) is a family of cytoprotective and antioxidative proteins. However, there is no report on the protective effects of HSP70 on oxidative stress-induced NPSC impairments and underlying mechanisms. In the present study, we treated NPSCs with tert-butyl hydroperoxide (t-BHP) in vitro to simulate an oxidative stress condition. HSP70 inducer TRC051384 was used to evaluate the cytoprotective effects of HSP70. The results suggested that HSP70 impeded t-BHP-mediated cell viability loss and protected the ultrastructure of NPSCs. Moreover, t-BHP could induce mitochondrial apoptosis and p53/p21-mediated senescence of NPSCs, both of which were significantly inhibited in HSP70 activation groups. Excessive oxidative stress and mitochondrial dysfunction reinforced each other and contributed to the cellular damage processes. HSP70 decreased reactive oxygen species (ROS) production, rescued mitochondrial membrane potential (MMP) collapse, and blocked ATP depletion. Finally, our data showed that HSP70 downregulated the JNK/c-Jun pathway. Taken together, activation of HSP70 could protect against t-BHP-induced NPSC apoptosis and senescence, thus, improving the quantity and quality of NPSCs. Therefore, HSP70 may be a promising therapeutic target for IVD degeneration.

     

FHC和Bim相互作用抵抗细胞凋亡

FHC和Bim参与细胞铁代谢和由ROS引起的细胞凋亡过程.但是其具体的分子机制还未阐明.用pLexA-Bim L作为诱饵,筛选了一个基于pBD42AD的胎脑cDNA文库,发现FHC是一个新的Bim相互作用蛋白.酵母杂交实验发现Bim的相互作用片段为BH3功能域.上述相互作用进一步用免疫共沉淀和荧光共定位得以证实.在HEK293细胞过表达FHC可以减轻由Bim过表达或ROS所引起的细胞凋亡,而用FHC特异性siRNA调低FHC表达,则增加Bim过表达或ROS引起的细胞凋亡.研究首次报道了Bim和FHC的相互作用以及对细胞凋亡和氧化应激的影响,为进一步阐明FHC和Bim参与凋亡和ROS反应提供了新的线索.     

Regulation of high glucose-induced apoptosis by mitochondrial NADP+-dependent isocitrate dehydrogenase

A high concentration of glucose has been implicated as a causal factor in initiation and progression of diabetic kidney complications, and there is evidence to suggest that hyperglycemia increases the production of free radicals and oxidant stress. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) to supply NADPH for antioxidant systems. In this report, we demonstrate that modulation of IDPm activity in HEK293 cells, an embryonic kidney cell line, regulates high glucose-induced apoptosis. When we examined the protective role of IDPm against high glucose-induced apoptosis with HEK293 cells transfected with the cDNA for mouse IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPm expressed in target cells and their susceptibility to apoptosis. The results suggest that IDPm plays an important protective role in apoptosis of HEK293 cells induced by a high concentration of glucose and may contribute to various pathologies associated with the long-term complications of diabetes.     

Accumulation of the SET protein in HEK293T cells and mild oxidative stress: cell survival or death signaling

SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 μM tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.     

Expression of the hyperphosphorylated tau attenuates ER stress-induced apoptosis with upregulation of unfolded protein response

The neural dysfunction in Alzheimer's disease (AD) could arise from endoplasmic reticulum (ER) stress and deficits of the unfolded protein response (UPR). To explore whether tau hyperphosphorylation, a hallmark of AD brain pathologies, plays a role in ER stress-induced alterations of cell viability, we established cell lines with stable expression of human tau (HEK293/tau) or the vector (HEK293/vec) and treated the cells with thapsigargin (TG), an ER stress inducer. We observed that the HEK293/tau cells were more resistant than the HEK293/vec cells to the TG-induced apoptosis, importantly, a time dependent increase of tau phosphorylation at Thr205 and Thr231 sites was positively correlated with the inhibition of apoptosis. We also observed that expression of tau upregulated phosphorylation of PERK, eIF2 and IRE1 with an increased cleavage of ATF6 and ATF4. The potentiation of UPR was also detected in HEK293/tau cells treated with other ER stress inducers, including staurosporine, camptothecin and hydrogen peroxide, in which a suppressed apoptosis was also shown. Our data suggest that tau hyperphosphorylation could attenuate the ER stress-induced apoptosis with the mechanism involving upregulation of UPR system.     

3-硝基酪氨酸对HepG2细胞氧化还原状态的影响

为了确定3-硝基酪氨酸是否能促进细胞内产生氧化应激,该文研究了不同浓度3．硝基酪氨酸对HepG2细胞作用不同时间（6-48h）后,其对细胞活力、细胞内ROS（H2O2、O2-）、细胞内总抗氧化能力、细胞内抗氧化酶活力和脂质氧化的影响。结果表明,在高浓度（300μmol／L）3．硝基酪氨酸作用48h后,细胞活力下降至48．5％。同时,3-硝基酪氨酸能显著提升细胞内ROS并降低细胞内抗氧化酶活力,同时造成细胞内脂质过氧化物大量积累,最终使细胞线粒体膜电位去极化,并导致SirT3表达下调。损伤随着3-硝基酪氨酸含量的增加和反应时间的延长而加重。结果发现,3．硝基酪氨酸不仅作为蛋白质氧化产物,还能进一步通过降低机体内抗氧化能力而导致细胞内氧化应激加剧,最终导致细胞凋亡。     

Kaempferol promotes melanogenesis and reduces oxidative stress in PIG1 normal human skin melanocytes

Vitiligo is an autoimmune disease characterized by depigmentation. Kaempferol is a flavonoid compound with broad anti-inflammatory and antioxidant properties. The purpose of this study was to investigate the effect of kaempferol on melanogenesis in PIG1 normal human skin melanocytes and its response to oxidative stress. The effect of kaempferol on melanin synthesis in PIG1 normal human skin melanocytes was explored by measuring tyrosinase activity, melanin content, mRNA and protein expression of key enzymes and expression of related pathway proteins. The effects of kaempferol pretreatment on cell viability, apoptosis, ROS level and HO-1 protein level under H₂O₂ stimulation were explored. When treated with kaempferol, the tyrosinase activity and melanin content of PIG1 cells increased, the mRNA and protein expressions of TYR, TRP1, TRP2 and MITF increased, and the phosphorylation level of ERK1/2 increased. Upon the stimulation of H₂O₂, kaempferol reduced the production of ROS, decreased apoptosis and increased the protein expression of HO-1 in PIG1 cells. In addition, kaempferol inhibited oxidative stress-induced melanin reduction and promoted melanin synthesis in PIG1 cells and protected against H₂O₂-induced oxidative stress damage.     

Crocin and Quercetin protect HCT116 and HEK293 cells from Zearalenone-induced apoptosis by reducing endoplasmic reticulum stress

Mycotoxins are considered to be significant contaminants of food and animal feed. Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium in cereals and agricultural products. ZEN has been shown to be cytotoxic, genotoxic, and mutagenic in different cell types. In the present study, we investigated the involvement of endoplasmic reticulum (ER) stress in ZEN-mediated toxicity in human intestine (HCT116) and kidney (HEK293) cells and evaluated the effects of the two common dietary compounds Quercetin (QUER) and Crocin (CRO). We show that ZEN treatment induces ER stress and activates the unfolded protein response (UPR) as evidenced by XBP1 mRNA splicing and upregulation of GRP78, ATF4, GADD34, PDIA6, and CHOP. Activation of the ER stress response is associated with activation of the mitochondrial pathway of apoptosis. This apoptotic process is characterized by an increase in ROS generation and lipid peroxidation, a loss of mitochondrial transmembrane potential (ΔΨm), and an activation of caspases and DNA damages. We also demonstrate that the antioxidant properties of QUER and CRO help to prevent ER stress and reduce ZEN-induced apoptosis in HCT116 and HEK293 cells. Our results suggest that antioxidant molecule might be helpful to prevent ZEN-induced ER stress and toxicity.     

伯氏肩孔南极鱼dusp1基因在冷应激中的功能研究

为了研究极地鱼类双特异性磷酸酶1 (dual-specificity phosphatase 1, dusp1)基因在低温胁迫下的作用,实验采用RT-PCR技术从Trematomus bernacchii中克隆获得了编码区含有1128个核苷酸的dusp1同源基因,可编码376个氨基酸残基。将其通过同源重组的方法构建真核表达载体pcDNA3.1-dusp1并转染至人胚肾293T(HEK293T)细胞中,同时以pcDNA3.1空载质粒作为对照。使用荧光探针DCFH-DA检测了细胞活性氧(Reactive oxygen species assay, ROS)含量,采用流式细胞术检测了低温胁迫下细胞的存活率,采用Western Blot检测了P38/MAPK的磷酸化水平和RT-qPCR技术分析了半胱氨酸天冬氨酸蛋白酶3(caspase-3)的mRNA表达水平。结果表明,伯氏肩孔南极鱼dusp1基因能在293T细胞中大量表达,并定位于细胞核;在低温胁迫下,与对照组相比,伯氏肩孔南极鱼dusp1基因的过表达能显著减少细胞ROS的含量和细胞凋亡率,并抑制促凋亡基因P38/MAPK的过度磷酸化和凋亡效...     

Altered Antioxidant System Stimulates Dielectric Barrier Discharge Plasma-Induced Cell Death for Solid Tumor Cell Treatment

This study reports the experimental findings and plasma delivery approach developed at the Plasma Bioscience Research Center, Korea for the assessment of antitumor activity of dielectric barrier discharge (DBD) for cancer treatment. Detailed investigation of biological effects occurring after atmospheric pressure non-thermal (APNT) plasma application during in vitro experiments revealed the role of reactive oxygen species (ROS) in modulation of the antioxidant defense system, cellular metabolic activity, and apoptosis induction in cancer cells. To understand basic cellular mechanisms, we investigated the effects of APNT DBD plasma on antioxidant defense against oxidative stress in various malignant cells as well as normal cells. T98G glioblastoma, SNU80 thyroid carcinoma, KB oral carcinoma and a non-malignant HEK293 embryonic human cell lines were treated with APNT DBD plasma and cellular effects due to reactive oxygen species were observed. Plasma significantly decreased the metabolic viability and clonogenicity of T98G, SNU80, KB and HEK293 cell lines. Enhanced ROS in the cells led to death via alteration of total antioxidant activity, and NADP⁺/NADPH and GSH/GSSG ratios 24 hours (h) post plasma treatment. This effect was confirmed by annexin V-FITC and propidium iodide staining. These consequences suggested that the failure of antioxidant defense machinery, with compromised redox status, might have led to sensitization of the malignant cells. These findings suggest a promising approach for solid tumor therapy by delivering a lethal dose of APNT plasma to tumor cells while sparing normal healthy tissues.     

Peroxide-triggered erythrocytes haemolysis as a model for the study of oxidative damage in marine fishes

The haemolysis of sea bass Dicentrarchus labrax red blood cells (RBC) was initiated by tert -butyl-hydroperoxide (t-BHP). The onset of the haemolytic process was accelerated by increasing t-BHP concentration. This process was preceded by a drop in the RBC glutathione content followed by the production of lipid peroxidation products. Also t-BHP induced DNA fragmentation in RBC nuclei as measured by COMET assay. The addition of the antioxidant Trolox C® dose-dependently delayed the onset of both lipid peroxidation and haemolysis, and protected GSH stores against t-BHP-induced depletion. DNA fragmentation was also pre-vented by Trolox C®. These results indicate that t-BHP induces haemolysis in sea bass RBC through the induction of oxidative stress. Such a simple model could prove useful for both fundamental and applied studies on marine fish antioxidant mechanisms.     

Cellular defense against heat shock-induced oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDPm) produces NADPH, an essential reducing equivalent for the antioxidant system. The protective role of IDPm against heat shock in HEK293 cells, an embryonic kidney cell line, was investigated in control and cells transfected with the cDNA for IDPm, where IDPm activity was 6–7 fold higher than that in the control cells carrying the vector alone. Upon exposure to heat shock, the viability was lower and the protein oxidation, lipid peroxidation and oxidative DNA damage were higher in control cells as compared to HEK293 cells in which IDPm was over-expressed. We also observed the significant difference in the cellular redox status reflected by the endogenous production of reactive oxygen species, NADPH pool and GSH recycling between two cells. The results suggest that IDPm plays an important role as an antioxidant defense enzyme in cellular defense against heat shock through the removal of reactive oxygen species.     

Stimulation of GSH synthesis to prevent oxidative stress-induced apoptosis by hydroxytyrosol in human retinal pigment epithelial cells: activation of Nrf2 and JNK-p62/SQSTM1 pathways

The Nrf2-Keap1 pathway is believed to be a critical regulator of the phase II defense system against oxidative stress. By activation of Nrf2, cytoprotective genes such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase (NQO-1) and γ-glutamyl-cysteine ligase (GCL) are induced. GCL-induced glutathione (GSH) production is believed to affect redox signaling, cell proliferation and death. We here report that tert-butyl hydroperoxide (t-BHP)-induced GSH reduction led to mitochondrial membrane potential loss and apoptosis in cultured human retinal pigment epithelial cells from the ARPE-19 cell line. Hydroxytyrosol (HT), a natural phytochemical from olive leaves and oil, was found to induce phase II enzymes and GSH, thus protect t-BHP-induced mitochondrial dysfunction and apoptosis. Depletion of GSH by buthionine-[S,R]-sulfoximine enhanced t-BHP toxicity and abolished HT protection. Overexpression of Nrf2 increased GSH content and efficiently protected t-BHP-induced mitochondrial membrane potential loss. Meanwhile, HT-induced GSH enhancement and induction of Nrf2 target gene (GCLc, GCLm, HO-1, NQO-1) messenger RNA (mRNA) were inhibited by Nrf2 knockdown, suggesting that HT increases GSH through Nrf2 activation. In addition, we found that HT was able to activate the PI3/Akt and mTOR/p70S6-kinase pathways, both of which contribute to survival signaling in stressed cells. However, the effect of HT was not inhibited by the PI3K inhibitor LY294002. Rather, c-Jun N-terminal kinase (JNK) activation was found to induce p62/SQSTM1 expression, which is involved in Nrf2 activation. Our study demonstrates that Nrf2 activation induced by the JNK pathway plays an essential role in the mechanism behind HT's strengthening of the antiapoptotic actions of the endogenous antioxidant system.     

Long-term mitochondrial stress induces early steps of Tau aggregation by increasing reactive oxygen species levels and affecting cellular proteostasis

Accumulating evidence indicates that mitochondrial dysfunction is involved in the pathogenesis of neurodegenerative diseases. Both of these conditions are often associated with an increase in protein aggregation. However, still unknown are the specific defects of mitochondrial biology that play a critical role in the development of Alzheimer’s disease, in which Tau protein aggregates are observed in the brains of some patients. Here, we report that long-term mitochondrial stress triggered Tau dimerization, which is the first step of protein aggregation. Mitochondrial dysfunction was induced in HEK293T cells that received prolonged treatment with rotenone and in HEK293T cells with the knockout of NDUFA11 protein. To monitor changes in Tau protein aggregation, we took advantage of the bimolecular fluorescence complementation assay using HEK293T cells that were transfected with plasmids that encoded Tau. Inhibition of the ISR with ISRIB induced Tau dimerization, whereas ISR activation with salubrinal, guanabenz, and sephin1 partially reversed this process. Cells that were treated with ROS scavengers, N-acetyl-l-cysteine or MitoQ, significantly reduced the amount of ROS and Tau dimerization, indicating the involvement of oxidative stress in Tau aggregation. Our results indicate that long-term mitochondrial stress may induce early steps of Tau protein aggregation by affecting oxidative balance and cellular proteostasis.     

Cellular defense against heat shock-induced oxidative damage by mitochondrial NADP+ -dependent isocitrate dehydrogenase

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) produces NADPH, an essential reducing equivalent for the antioxidant system. The protective role of IDPm against heat shock in HEK293 cells, an embryonic kidney cell line, was investigated in control and cells transfected with the cDNA for IDPm, where IDPm activity was 6-7 fold higher than that in the control cells carrying the vector alone. Upon exposure to heat shock, the viability was lower and the protein oxidation, lipid peroxidation and oxidative DNA damage were higher in control cells as compared to HEK293 cells in which IDPm was over-expressed. We also observed the significant difference in the cellular redox status reflected by the endogenous production of reactive oxygen species, NADPH pool and GSH recycling between two cells. The results suggest that IDPm plays an important role as an antioxidant defense enzyme in cellular defense against heat shock through the removal of reactive oxygen species.     

Oxidative stress induces inactivation of protein phosphatase 2A,promoting proinflammatory NF-κB in aged rat kidney

The molecular inflammation hypothesis of aging proposes that redox dysregulation causes an age-related activation of NF-κB and its signaling to upregulate various proinflammatory genes. In the present study, we focused on the inactive form of the protein phosphastase 2 A (PP2A). More specifically, we aimed to define the correlation between PP2A inactivation and NF-κB activation by age-related oxidative stress. Experimentations were designed to determine the effect of oxidative stress-induced PP2A inactivation on NF-κB activity, utilizing prooxidants t-BHP and AAPH, the PTP inhibitor Na₃VO₄, and the PP2A inhibitor Calyculin A and PP2A siRNA, in HEK293T cells. We also assessed the phosphorylation of PP2A catalytic subunit (PP2Ac) and the activities of PP2A and NF-κB in aged rat kidney, utilizing aging-retarding 40% calorie restriction (CR) −60% of food intake and inflammation-triggering LPS paradigms. Results revealed that an oxidative stress-induced PTK/PTP imbalance led to phosphorylation of PP2Ac, following exposures to t-BHP, AAPH, and Na₃VO₄ in HEK293T cells. Subsequently, we found that Calyculin A and PP2A siRNA activates NIK/IKK and MAPKs, leading to upregulation of NF-κB and its dependent oxidative stress. Also, the contrasting relation between PP2A inactivation and NF-κB activation was confirmed by AAPH-induced oxidative status in mice, and non-induced normal status or LPS-induced inflammatory status in aged rats while the antioxidative, anti-inflammatory, anti-aging effects of CR significantly blunted these actions. Thus, we present evidence that PP2A inactivation via PTK/PTP imbalance provoked by oxidative stress causes NF-κB activation, which contributes to the accumulation of oxidative stress in aged rat kidney.     

TAT-mediated PRDX6 protein transduction protects against eye lens epithelial cell death and delays lens opacity

A diminished level of endogenous antioxidant in cells/tissues is associated with reduced resistance to oxidative stress. Peroxiredoxin 6 (PRDX6), a protective molecule, regulates gene expression/function by controlling reactive oxygen species (ROS) levels. Using PRDX6 protein linked to TAT, the transduction domain from human immunodeficiency virus type 1 TAT protein, we demonstrated that PRDX6 was transduced into lens epithelial cells derived from rat or mouse lenses. The protein was biologically active, negatively regulating apoptosis and delaying progression of cataractogenesis by attenuating deleterious signaling. Lens epithelial cells from cataractous lenses bore elevated levels of ROS and were susceptible to oxidative stress. These cells harbored increased levels of active transforming growth factor (TGF)-beta 1 and of alpha-smooth muscle actin and beta ig-h3, markers for cataractogenesis. Importantly, cataractous lenses showed a 10-fold reduction in PRDX6 expression, whereas TGF-beta1 mRNA and protein levels were elevated. The changes were reversed, and cataractogenesis was delayed when PRDX6 was supplied. Results suggest that delivery of PRDX6 can postpone cataractogenesis, and this should be an effective approach to delaying cataracts and other degenerative diseases that are associated with increased ROS.