Quercetin Attenuates Cell Apoptosis in Focal Cerebral Ischemia Rat Brain Via Activation of BDNF–TrkB–PI3K/Akt Signaling Pathway

Many studies have demonstrated that apoptosis play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. Neuroprotective effect of quercetin has been shown in a variety of brain injury models including ischemia/reperfusion. It is not clear whether BDNF?CTrkB?CPI3K/Akt signaling pathway mediates the neuroprotection of quercetin, though there has been some reports on the quercetin increased brain-derived neurotrophic factor (BDNF) level in brain injury models. We therefore first examined the neurological function, infarct volume and cell apoptosis in quercetin treated middle cerebral artery occlusion (MCAO) rats. Then the protein expression of BDNF, cleaved caspase-3 and p-Akt were evaluated in either the absence or presence of PI3K inhibitor (LY294002) or tropomyosin receptor kinase B (TrkB) receptor antagonist (K252a) by immunohistochemistry staining and western blotting. Quercetin significantly improved neurological function, while it decreased the infarct volume and the number of TdT mediated dUTP nick end labeling positive cells in MCAO rats. The protein expression of BDNF, TrkB and p-Akt also increased in the quercetin treated rats. However, treatment with LY294002 or K252a reversed the quercetin-induced increase of BDNF and p-Akt proteins and decrease of cleaved caspase-3 protein in focal cerebral ischemia rats. These results demonstrate that quercetin can decrease cell apoptosis in the focal cerebral ischemia rat brain and the mechanism may be related to the activation of BDNF?CTrkB?CPI3K/Akt signaling pathway.     

Thimerosal-Induced Apoptosis in Mouse C2C12 Myoblast Cells Occurs through Suppression of the PI3K/Akt/Survivin Pathway

Background

Thimerosal, a mercury-containing preservative, is one of the most widely used preservatives and found in a variety of biological products. Concerns over its possible toxicity have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be markedly cytotoxic to neural tissue. However, little is known regarding thimerosal-induced toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells.

Methodology/Principal Findings

The study showed that C2C12 myoblast cells underwent inhibition of proliferation and apoptosis after exposure to thimerosal (125–500 nM) for 24, 48 and 72 h. Thimerosal caused S phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining and immunoblotting. The data revealed that thimerosal could trigger the leakage of cytochrome c from mitochondria, followed by cleavage of caspase-9 and caspase-3, and that an inhibitor of caspase could suppress thimerosal-induced apoptosis. Thimerosal inhibited the phosphorylation of Akt^ser473 and survivin expression. Wortmannin, a PI3K inhibitor, inhibited Akt activity and decreased survivin expression, resulting in increased thimerosal-induced apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF-I (50 ng/ml) increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of survivin expression by siRNA enhanced thimerosal-induced cell apoptosis, while overexpression of survivin prevented thimerosal-induced apoptosis. Taken together, the data show that the PI3K/Akt/survivin pathway plays an important role in the thimerosal-induced apoptosis in C2C12 cells.

Conclusions/Significance

Our results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and finally causes apoptosis via inhibition of PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic pathway.     

Estrogen Receptor and PI3K/Akt Signaling Pathway Involvement in S-(-)Equol-Induced Activation of Nrf2/ARE in Endothelial Cells

S-(-)equol, a natural product of the isoflavone daidzein, has been reported to offer cytoprotective effects with respect to the cardiovascular system, but how this occurs is unclear. Interestingly, S-(-)equol is produced by the human gut, suggesting a role in physiological processes. We report that treatment of human umbilical vein endothelial cells and EA.hy926 cells with S-(-)equol induces ARE-luciferase reporter gene activity that is dose and time dependent. S-(-)equol (10–250 nM) increases nuclear factor-erythroid 2-related factor 2 (Nrf2) as well as gene products of Nrf2 target genes heme oxygenase-1 (HO-1) and NAD(P)H (nicotinamide-adenine-dinucleotide-phosphate) quinone oxidoreductase 1 (NQO1). Endothelial cells transfected with an HA-Nrf2 expression plasmid had elevated HA-Nrf2, HO-1, and NQO1 in response to S-(-)equol exposure. S-(-)equol treatment affected Nrf2 mRNA only slightly but significantly increased HO-1 and NQO1 mRNA. The pretreatment of cells with specific ER inhibitors or PI3K/Akt (ICI182,780 and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) increased Nrf2, HO-1, and NQO1 protein, impaired nuclear translocation of HA-Nrf2, and decreased ARE-luciferase activity. Identical experiments were conducted with daidzein, which had effects similar to S-(-)equol. In addition, DPN treatment (an ERβ agonist) induced the ARE-luciferase reporter gene, promoting Nrf2 nuclear translocation. Cell pretreatment with an ERβ antagonist (PHTPP) impaired S-(-)equol-induced Nrf2 activation. Pre-incubation of cells followed by co-treatment with S-(-)equol significantly improved cell survival in response to H₂O₂ or tBHP and reduced apoptotic and TUNEL-positively-stained cells. Notably, the ability of S-(-)equol to protect against H₂O₂-induced cell apoptosis was attenuated in cells transfected with an siRNA against Nrf2. Thus, beneficial effects of S-(-)equol with respect to cytoprotective antioxidant gene activation may represent a novel strategy to prevent and treat cardiovascular diseases.     

心梗大鼠通过有氧运动激活PI3K-Akt-PKG-1/p-PLN-SERCA2a通路抑制心肌细胞凋亡改善心功能

为探讨有氧运动对心梗大鼠心功能的影响,将3月龄SD雄性大鼠适应性喂养1周后随机分为正常组(C组)、假手术组(S组)、心梗安静组(MI组)、正常+运动组(CE组)、心梗+运动组(ME组),每组8只. MI组结扎左冠状动脉前降支制备心梗模型;S组只穿线不结扎;CE组与ME组术后1周开始有氧训练,运动方式为依次以10 m/min×10 min,13 m/min×10 min,16 m/min×40 min进行跑台训练,60 min/d,每周5 d,连续4周.训练结束后次日,采用血流动力学检测左室收缩压(left ventricular systolic pressure,LVSP)、左室舒张末压(left ventricular end-diastolic pressure,LVEDP)和收缩/舒张速率(±dp/dtmax)等心功能相关指标,单细胞可视化动缘探测系统(IonOptix)测定[Ca²⁺]i变化百分数([Ca²⁺]iamplitude)、[Ca²⁺]i荧光比率(ratio)、达峰速率(departure veloc...     

Proliferation and Survival Signaling from Both Jak2-V617F and Lyn Involving GSK3 and mTOR/p70S6K/4EBP1 in PVTL-1 Cell Line Newly Established from Acute Myeloid Leukemia Transformed from Polycythemia Vera

The gain of function mutation JAK2-V617F is very frequently found in myeloproliferative neoplasms (MPNs) and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for JAK2-V617F from a 73-year-old female patient with acute myeloid leukemia (AML) transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),−16,+21,−22,+mar1. Sequence analysis of JAK2 revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1) or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in evolution of Jak2-V617F-expressing MPN to AML and to develop novel therapies against this intractable condition.     

Neuroprotective Effect of Fucoidan on H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Apoptosis in PC12 Cells Via Activation of PI3K/Akt Pathway

One of the plausible ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of endogenous antioxidant defense capacity. In this study, we investigated the neuroprotective effect of fucoidan on H(2)O(2)-induced apoptosis in PC12 cells and the possible signaling pathways involved. The results showed that fucoidan inhibited the decrease of cell viability, scavenged ROS formation and reduced lactate dehydrogenase release in H(2)O(2)-induced PC12 cells. These changes were associated with an increase in superoxide dismutase and glutathione peroxidase activity, and reduction in malondialdehyde. In addition, fucoidan treatment inhibited apoptosis in H(2)O(2)-induced PC12 cells by increasing the Bcl-2/Bax ratio and decreasing active caspase-3 expression, as well as enhancing Akt phosphorylation (p-Akt). However, the protection of fucoidan on cell survival, p-Akt, the Bcl-2/Bax ratio and caspase-3 activity were abolished by pretreating with phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. In consequence, fucoidan might protect the neurocytes against H(2)O(2)-induced apoptosis via reducing ROS levels and activating PI3K/Akt signaling pathway.     

Protracted Upregulation of Leptin and IGF1 is Associated with Activation of PI3K/Akt and JAK2 Pathway in Mouse Intestine after Ionizing Radiation Exposure

Ionizing radiation is a known risk factor for gastrointestinal (GI) pathologies including cancer. Hormones and related signaling crosstalk, which could contribute to radiation-induced persistent pathophysiologic changes in the small intestine and colon, remain to be explored. The current study assessed perturbation of GI homeostasis-related hormones and signaling pathways at the systemic as well as at the tissue level in small intestine and colon. Mice (6-8 week old C57BL/6J) were exposed to 2 Gy γ radiation, serum and tissue samples were collected, and insulin like growth factor 1 (IGF-1) and leptin signaling were assessed two or twelve months after radiation exposure. Serum levels of IGF-1, IGF binding protein 3 (IGFBP3), leptin, and adiponectin were altered at these times after irradiation. Radiation was associated with increased IGF1 receptor (IGF1R) and obesity (leptin) receptor (Ob-R), decreased adiponectin receptor 1 (Adipo-R1) and 2 (Adipo-R2), and increased Ki-67 levels in small intestine and colon at both time points. Immunoblot analysis further showed increased IGF1R and Ob-R, and decreased Adipo-R2. Additionally, upregulation of PI3K/Akt and JAK2 signaling, which are downstream of IGF1 and leptin, was also observed in irradiated samples at both time points. These results when considered along with increased cell proliferation in the small intestine and colon demonstrate for the first time that ionizing radiation can persistently increase IGF1 and leptin and activate downstream proliferative pathways, which may contribute to GI functional alterations and carcinogenesis.     

4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway

4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G₂/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested.