蓝莓提取物对H2O2诱导的体外培养的大鼠海马神经元氧化损伤的保护作用

目的：探讨蓝莓提取物对过氧化氢致大鼠海马神经元氧化应激损伤的减缓作用。方法：将培养7d的海马神经细胞分为8组：①过氧化氢组（H2O2）：培养液中加入50μmol/L的H2O2,作用24h。②不同剂量蓝莓提取物预处理组（BE＋H2O2）：在加入H2O2前24h,分别加入0.01、0.1、1.0、10.0、20.0和40.0μg/mlBE。③空白对照组（Control）：处理步骤同上组,但每次处理物质均为等量的培养液。通过测定细胞存活率和上清液中乳酸脱氢酶（LDH）的活性确定减轻海马神经元损伤的蓝莓提取物的适宜浓度。并检测细胞内丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性以及细胞凋亡率的变化。结果：①蓝莓提取物组（0.1,1.0和10.0μg/mlBE）LDH活性显著低于H2O2组,细胞存活率由H2O2组的57.44%分别上升至78.42%、87.71%、72.40%;1μg/ml蓝莓提取物组对H2O2诱导的海马神经细胞氧化应激损伤的保护作用最好。②1μg/ml蓝莓提取物组海马神经细胞培养上清液中MDA含量及细胞凋亡率显著低于H2O2组,SOD活性显著高于H2O2组。结论：适宜剂量的蓝莓提取物对氧化应激损伤的海马神经元有一定的保护作用,其机制可能与抑制海马神经元凋亡、增强神经细胞的抗氧化功能有关。     

Nucleolin enhances internal ribosomal entry site (IRES)-mediated translation of Sp1 in tumorigenesis

Our previous study indicated that specificity protein-1 (Sp1) is accumulated during hypoxia in an internal ribosomal entry site (IRES)-dependent manner. Herein, we found that the Sp1 was induced strongly at the protein level, but not in the mRNA level, in lung tumor tissue, indicating that translational regulation might contribute to the Sp1 accumulation during tumorigenesis. A further study showed that the translation of Sp1 was dramatically induced through an IRES-dependent pathway. RNA immunoprecipitation analysis of proteins bound to the 5′-untranslated region (5′-UTR) of Sp1 identified interacting protein — nucleolin. Knockdown of nucleolin significantly inhibited IRES-mediated translation of Sp1, suggesting that nucleolin positively facilitates Sp1 IRES activation. Further analysis of the interaction between nucleolin and the 5′-UTR of Sp1 mRNA revealed that the GAR domain was important for IRES-mediated translation of Sp1. Moreover, gefitinib, and LY294002 and MK2206 compounds inhibited IRES-mediated Sp1 translation, implying that activation of the epithelial growth factor receptor (EGFR) pathway via Akt activation triggers the IRES pathway. In conclusion, EGFR activation-mediated nucleolin phosphorylated at Thr641 and Thr707 was recruited to the 5′-UTR of Sp1 as an IRES trans-acting factor to modulate Sp1 translation during lung cancer formation.     

Protection by exogenous pyruvate through a mechanism related to monocarboxylate transporters against cell death induced by hydrogen peroxide in cultured rat cortical neurons

In cortical neurons cultured for 3 or 9 days in vitro (DIV), exposure to hydrogen peroxide (H(2)O(2)) led to a marked decrease in cell viability in a concentration-dependent manner at a concentration range of 10 microm to 1 mm irrespective of the duration between 6 and 24 h. However, H(2)O(2) was more potent in decreasing cellular viability in cortical neurons cultured for 9 DIV than in those for 3 DIV. Pyruvate was effective in preventing the neuronal cell death at 1 mm even when added 1-3 h after the addition of H(2)O(2). Semi-quantitative RT-PCR and western blotting analyses revealed significantly higher expression of both mRNA and protein for a particular monocarboxylate transporter (MCT) in neurons cultured for 9 DIV than in those for 3 DIV. A specific inhibitor of MCT significantly attenuated the neuroprotection by pyruvate in neurons cultured for 9 DIV, without markedly affecting that in neurons cultured for 3 DIV. These results suggest that vulnerability to H(2)O(2) may at least in part involve expression of particular MCT isoforms responsible for the bi-directional transport of pyruvate across cell surfaces in cultured rat cortical neurons.     

Curcumin Protects Microglia and Primary Rat Cortical Neurons against HIV-1 gp120-Mediated Inflammation and Apoptosis

Curcumin is a molecule found in turmeric root that has anti-inflammatory, antioxidant, and anti-tumor properties and has been widely used as both an herbal drug and a food additive to treat or prevent neurodegenerative diseases. To explore whether curcumin is able to ameliorate HIV-1-associated neurotoxicity, we treated a murine microglial cell line (N9) and primary rat cortical neurons with curcumin in the presence or absence of neurotoxic HIV-1 gp120 (V3 loop) protein. We found that HIV-1 gp120 profoundly induced N9 cells to produce reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1). HIV-1 gp120 also induced apoptosis of primary rat cortical neurons. Curcumin exerted a powerful inhibitory effect against HIV-1 gp120-induced neuronal damage, reducing the production of ROS, TNF-α and MCP-1 by N9 cells and inhibiting apoptosis of primary rat cortical neurons. Curcumin may exert its biological activities through inhibition of the delayed rectification and transient outward potassium (K⁺) current, as curcumin effectively reduced HIV-1 gp120-mediated elevation of the delayed rectification and transient outward K⁺ channel current in neurons. We conclude that HIV-1 gp120 increases ROS, TNF-α and MCP-1 production in microglia, and induces cortical neuron apoptosis by affecting the delayed rectification and transient outward K⁺ channel current. Curcumin reduces production of ROS and inflammatory mediators in HIV-1-gp120-stimulated microglia, and protects cortical neurons against HIV-1-mediated apoptosis, most likely through inhibition of HIV-1 gp120-induced elevation of the delayed rectification and transient outward K⁺ current.     

Continuous heat shock enhances translational initiation directed by internal ribosomal entry site

Many cellular mRNAs contain internal ribosomal entry sites (IRES) that become functional under conditions of cellular stress, when the rate of protein synthesis for most cellular mRNA is reduced. Internal ribosomal entry increases in response to hypoxia, cell differentiation, apoptosis, gamma irradiation, and heat shock. Heat shock is the principal cellular stress in which general cap-dependent translation is inhibited. On the other hand, heat shock induces the preferential translation of a small class of mRNA, called heat shock protein (HSP) mRNAs, which probably occurs because little or no eIF4F activity is required for their translation. In this study, we found that continuous heat stress enhances expression of the heat shock protein BiP at the level of translation. Interestingly, heat stress also enhanced the viral IRES-dependent translation of encephalomyocarditis virus and hepatitis C virus but not poliovirus. Although several BiP inducers increased BiP protein expression, BiP IRES-dependent translation was enhanced only during heat shock, suggesting that heat shock is a specific inducer for BiP IRES-dependent translation. Taken together, these results indicate that the mechanism of IRES-dependent translation can be used during heat shock and suggest that this translational mechanism may be critical to the survival and proliferation of cells under stress.     

Huperzine A attenuates apoptosis and mitochondria-dependent caspase-3 in rat cortical neurons

The neuroprotection of huperzine A against apoptosis was investigated. In cultures of rat primary cortical neurons, neuronal apoptosis was induced by serum deprivation for 24 h, which was accompanied by enhanced caspase-3 activity and the release of cytochrome c into the cytosol from mitochondria. Pretreating the neurons for 2 h with huperzine A (0.1-10 microM) improved neuronal survival. Huperzine A at a concentration of 1 microM significantly attenuated apoptosis by inhibiting the mitochondria-caspase pathway directly and indirectly.     

Human Bcl-2 protects against AMPA receptor-mediated apoptosis

Dysfunctions of the (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of ionotropic receptor for the brain's major excitatory neurotransmitter, L-glutamate, occur in various neurological conditions. We have previously demonstrated that AMPA receptor-mediated excitotoxicity occurs by apoptosis and here examined the influence of the expression of cell death repressor gene Bcl-2 on this excitotoxic insult. Using neuronal cortical cultures prepared from transgenic mice expressing the human Bcl-2 gene, the influence of Bcl-2 on AMPA receptor-mediated neuronal death was compared with that seen with staurosporine and H2O2. At day 6 cultures were exposed to AMPA (0.1-100 microM), and cellular injury was analyzed 48 h after insult using phase-contrast microscopy, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay, and DNA staining with 4,6-diamidino-2-phenylindole and Sytox Green. AMPA produced a concentration-dependent increase in cell death that was significantly attenuated by human Bcl-2. AMPA (3 microM) increased the number of apoptotic nuclei to 60% of control in wild-type cultures, and human Bcl-2 significantly decreased the number of apoptotic nuclei to 30% of AMPA-treated cultures. Human Bcl-2 only provided significant neuroprotection against neuronal injury induced by low concentrations of staurosporine (1-10 nM) and H2O2 (0.1-30 microM) and where neuronal death was by apoptosis, but not against H2O2-induced necrosis. Our findings indicate that overexpression of Bcl-2 in primary cultured neurons protects in an insult-dependent manner against AMPA receptor-mediated apoptosis, whereas protection was not seen against more traumatic insults. This study provides new insights into the molecular therapeutics of neurodegenerative conditions.     

Heterogeneous nuclear ribonucleoprotein A1 is a novel internal ribosome entry site trans-acting factor that modulates alternative initiation of translation of the fibroblast growth factor 2 mRNA

Alternative initiation of translation of the human fibroblast growth factor 2 (FGF-2) mRNA at five in-frame CUG or AUG translation initiation codons requires various RNA cis-acting elements, including an internal ribosome entry site (IRES). Here we describe the purification of a trans-acting factor controlling FGF-2 mRNA translation achieved by several biochemical purification approaches. We have identified the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a factor that binds to the FGF-2 5'-leader RNA and that also complements defective FGF-2 translation in vitro in rabbit reticulocyte lysate. Recombinant hnRNP A1 stimulates in vitro translation at the four IRES-dependent initiation codons but has no effect on the cap-dependent initiation codon. Consistent with a role of hnRNP A1 in the control of alternative initiation of translation, short interfering RNA-mediated knock down of hnRNP A1 specifically inhibits translation at the four IRES-dependent initiation codons. Furthermore, hnRNP A1 binds to the FGF-2 IRES, implicating this interaction in the control of alternative initiation of translation.     

Preconditioning with hyperbaric oxygen induces tolerance against oxidative injury via increased expression of heme oxygenase-1 in primary cultured spinal cord neurons

Hyperbaric oxygen (HBO) preconditioning can induce ischemic tolerance in the spinal cord. The effect can be attenuated by the administration of an oxygen free radical scavenger or by inhibition of antioxidant enzymes. However, the mechanism underlying HBO preconditioning of neurons against ischemic injury remains enigmatic. Therefore, in the present study primary cultured spinal cord neurons were treated with HBO and then subjected to a hydrogen peroxide (H(2)O(2)) insult. The results show that H(2)O(2) stimulation of the cultured spinal neurons caused severe DNA damage and decreased cell viability, and that these neurons were well protected against damage after a single exposure to HBO preconditioning (0.35 MPa, 98% O(2), 37 degrees C, 2 h). The protective effect started 4 h after pretreatment and lasted for at least 24 h. The cultured neurons after HBO treatment also exhibited increased heme oxygenase-1 (HO-1) expression at both the protein and mRNA levels, which paralleled the protective effect of HBO. Treatment with tin-mesoporphyrin IX (SnMP), a specific HO-1 inhibitor, before HBO pretreatment abolished the HBO-induced adaptive protection noted in the cultured spinal neurons. In conclusion, HBO preconditioning can protect primary cultured spinal cord neurons against oxidative stress, and the upregulation of HO-1 expression plays an essential role in HBO induced preconditioning effect.     

Valproic acid induces functional heat-shock protein 70 via Class I histone deacetylase inhibition in cortical neurons: a potential role of Sp1 acetylation

Neuroprotective properties of the mood stabilizer valproic acid (VPA) are implicated in its therapeutic efficacy. Heat-shock protein 70 (HSP70) is a molecular chaperone, neuroprotective and anti-inflammatory agent. This study aimed to investigate underlying mechanisms and functional significance of HSP70 induction by VPA in rat cortical neurons. VPA treatment markedly up-regulated HSP70 protein levels, and this was accompanied by increased HSP70 mRNA levels and promoter hyperacetylation and activity. Other HDAC inhibitors – sodium butyrate, trichostatin A, and Class I HDAC-specific inhibitors MS-275 and apicidin, – all mimicked the ability of VPA to induce HSP70. Pre-treatment with phosphatidylinositol 3-kinase inhibitors or an Akt inhibitor attenuated HSP70 induction by VPA and other HDAC inhibitors. VPA treatment increased Sp1 acetylation, and a Sp1 inhibitor, mithramycin, abolished the induction of HSP70 by HDAC inhibitors. Moreover, VPA promoted the association of Sp1 with the histone acetyltransferases p300 and recruitment of p300 to the HSP70 promoter. Further, VPA-induced neuroprotection against glutamate excitotoxicity was prevented by blocking HSP70 induction. Taken together, the data suggest that the phosphatidylinositol 3-kinase/Akt pathway and Sp1 are likely involved in HSP70 induction by HDAC inhibitors, and induction of HSP70 by VPA in cortical neurons may contribute to its neuroprotective and therapeutic effects.     

PKCδ mediates Nrf2-dependent protection of neuronal cells from NO-induced apoptosis

A chemical inhibitor library of 84 compounds was screened to investigate the signaling pathway(s) leading to activation of Nrf2 in response to nitric oxide (NO). We identified the protein kinase C delta (PKCδ) inhibitor rottlerin as the only compound that reduced NO-induced ARE-luciferase reporter activity and diminished NO-induced up-regulation of two Nrf2/ARE-regulated proteins - NAD(P)H:quinone oxidoreductase-1 (NQO1) and hemeoxygenase-1 (HO-1) in SH-Sy5y cells. Rottlerin also sensitized neuroblastoma cells and mouse primary cortical neurons to NO-induced apoptosis. Stable over-expression of PKCδ augmented NO-induced, ARE-dependent gene expression of HO-1 in SH-Sy5y cells, which were more protected from NO killing. Conversely, NO-induced ARE-dependent gene expression was reduced in PKCδ-knockdown SH-EP cells, which displayed greater sensitivity to apoptosis. PKCδ^−/− cortical neurons exhibited increased NO-induced apoptosis and less HO-1 mRNA and protein induction compared with wild type neurons. Hence, PKCδ is an important positive modulator of NO-induced Nrf2/ARE-dependent signaling that counteracts NO-mediated apoptosis in neuronal cells.     

Thioredoxin peroxidase-1 (peroxiredoxin-1) is increased in thioredoxin-1 transfected cells and results in enhanced protection against apoptosis caused by hydrogen peroxide but not by other agents including dexamethasone, etoposide, and doxorubicin

Thioredoxin-1 (Trx-1) is a small redox oncoprotein whose expression is increased in a number of human primary cancers where it is associated with aggressive tumor growth, inhibition of apoptosis and decreased patient survival. We report that Trx-1-transfected MCF-7 human breast cancer cells have increased expression of thioredoxin peroxidase-1 (TrxP-1) a peroxiredoxin family member that scavenges H(2)O(2) using Trx-1 as a source of reducing equivalents. Our work shows that TrxP-1 is more effective than selenium-dependent glutathione peroxidase in protecting cells against H(2)O(2) damage. Transfection of mouse WEHI7.2 lymphoma cells with human TrxP-1 or TrxP-2, but not TrxP-4, protects the cells against H(2)O(2) induced apoptosis but does not protect against apoptosis induced by dexamethasone, etoposide, or doxorubicin. The results show that an increase in TrxP-1 expression contributes to the protection against H(2)O(2) induced apoptosis caused by Trx-1, but does not protect against apoptosis induced by other agents.     

Mitogen-activated protein kinases modulate H(2)O(2)-induced apoptosis in primary rat alveolar epithelial cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (ATII) cell apoptosis remain poorly defined. Here we investigated the role of MAPKs as modulators of oxidant-mediated ATII cell apoptosis using in vitro models of H(2)O(2)-stress. H(2)O(2), delivered either as a bolus or as a flux, lead to time- and concentration-dependent increases in ATII cells apoptosis. Increased apoptosis in primary rat ATII cells was detected at H(2)O(2) concentrations and production rates in the physiological range (1 microM) and peaked at 100 microM H(2)O(2). Immortalized rat lung epithelial cells (RLE), in contrast, required millimolar concentration of H(2)O(2) for maximal responses. H(2)O(2)-induced apoptosis was preceded by rapid activation of all three classes of mitogen-activated protein kinases (MAPKs): ERK, JNK, and p38. Specific inhibition of JNK using antisense oligonucleotides and ERK and p38 using PD98059 or SB202190, respectively, indicated a pro-apoptotic role for JNK pathway and an anti-apoptotic role for ERK- and p38-initiated signaling events. Our data show that the balance between the activation of JNK, ERK, and p38 is a critical determinant of cell fate, suggesting that pharmacological interventions on the MAPK pathways may be useful in the treatment of oxidant-related lung injury.