Estradiol prevents the injury-induced decrease of Akt activation and Bad phosphorylation

Estradiol prevents neuronal cell death through the inhibition of apoptotic signals and the activation of cell survival signals. This study investigated whether estradiol modulates the anti-apoptotic signal through the activation of Akt and its downstream targets, including Bad, Bcl-x(L), and 14-3-3. Adult female rats were ovariectomied and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 h after MCAO and infarct volumes were analyzed. We confirmed that estradiol significantly reduces infarct volume and decreases the positive cells of TUNEL staining in the cerebral cortex. Potential activation was measured by phosphorylation of Akt at Ser473 and Bad at Ser136 using Western blot analysis. Estradiol prevents the injury-induced decrease of pAkt, pBad, and Bcl-x(L). Further, in the presence of estradiol, the interaction of pBad and 14-3-3 increased, compared to that of oil-treated animals. Our findings suggest that estradiol prevents cell death due to brain injury and that Akt activation and Bad phosphorylation by estradiol mediated these protective effects.     

Estradiol attenuates the focal cerebral ischemic injury through mTOR/p70S6 kinase signaling pathway

We previously showed that estradiol prevents neuronal cell death through the activation of Akt and its downstream targets Bad and FKHR. This study investigated whether estradiol modulates the survival pathway through other downstream targets of Akt, including mammalian target of rapamycin (mTOR) and p70S6 kinase. It is known that mTOR is a downstream target of Akt and a central regulator of protein synthesis, cell growth, and cell cycle progression. Adult female rats were ovariectomied and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24h after MCAO and infarct volumes were analyzed. We confirmed that estradiol significantly reduces infarct volume and decreases the number of positive cells for TUNEL staining in the cerebral cortex. Brain injury-induced a decrease in phospho-mTOR and phospho-p70S6 kinase. Estradiol prevented the injury-induced decrease in Akt activation and phosphorylation of mTOR and p70S6 kinases, and the subsequent decrease in S6 phosphorylation. Our findings suggest that estradiol plays a potent protective role against brain injury by preventing the injury-induced decrease of mTOR and p70S6 kinase phosphorylation.     

Estradiol prevents the injury-induced decrease of Akt/glycogen synthase kinase 3beta phosphorylation

Estradiol prevents neuronal cell death through the activation of cell survival signals and the inhibition of apoptotic signals. This study investigated whether estradiol modulates the anti-apoptotic signal through the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3beta (GSK3beta). Adult female rats were ovariectomized and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 h after MCAO and infarct volumes were analyzed. Estradiol administration significantly reduced infarct volume and decreased the positive cells of TUNEL staining in the cerebral cortex. Potential activation was measured by phosphorylation of Akt at Ser(473) and GSK3beta at Ser(9) using Western blot analysis and immunohistochemistry. Estradiol prevented the injury-induced decrease of pAkt and pGSK3beta. Furthermore, pretreatment with estradiol decreased glutamate toxicity-induced cell death in a hippocampal cell line (HT22). Also, estradiol prevented the glutamate toxicity-induced decrease of pAkt and pGSK3beta in HT22 cells. Our findings suggest that estradiol plays a potent protective role against brain injury and that phosphorylation of Akt and GSK3beta by estradiol mediated these protective effects.     

Melatonin prevents ischemic brain injury through activation of the mTOR/p70S6 kinase signaling pathway

We previously reported that melatonin prevents neuronal cell death in ischemic brain injury through the activation of Akt and the inhibition of apoptotic cell death. We investigated whether melatonin inhibits the apoptotic signal through the activation of a mammalian target of rapamycin (mTOR) and p70S6 kinase and its downstream target, S6 phosphorylation. It is known that mTOR is a downstream target of Akt and a central regulator of protein synthesis, cell growth, and cell cycle progression. Adult male rats were treated with melatonin (5mg/kg) or vehicle prior to middle cerebral artery occlusion (MCAO). Brains were collected at 24h after MCAO and infarct volumes were analyzed. We confirmed that melatonin significantly reduces infarct volume and decreases the number of TUNEL-positive cells in the cerebral cortex. Brain injury induced a decrease in phospho-mTOR and phospho-p70S6 kinase. Melatonin prevented the injury-induced decrease in Akt activation and phosphorylation of mTOR and p70S6 kinases, and the subsequent decrease in S6 phosphorylation. Our results suggest that melatonin prevents cell death resulting from ischemic brain injury and that its neuroprotective effects are mediated by preventing the injury-induced decrease of mTOR and p70S6 kinase phosphorylation.     

Ferulic acid prevents the cerebral ischemic injury-induced decrease of Akt and Bad phosphorylation

Ferulic acid protects neuronal cells from glutamate-induced excitotoxicity and focal cerebral ischemia. This study investigated whether ferulic acid exerts a neuroprotective effect through the activation of Akt and its downstream targets, Bad and 14-3-3. Adult male rats were immediately treated with ferulic acid (100 mg/kg, i.v.) after middle cerebral artery occlusion (MCAO). Brains were collected 24 h after MCAO and infarct volumes were analyzed using triphenyltetrazolium chloride staining. It was found that ferulic acid treatment significantly reduced infarct volume during MCAO. Ferulic acid attenuated the MCAO injury-induced decrease of phospho-PDK1, phospho-Akt and phospho-Bad levels. However, ferulic acid did not affect the expression of 14-3-3 and Bcl-xL, which exerts an anti-apoptotic effect through interaction with phospho-Bad. Immunoprecipitation analysis demonstrated that the interaction between phospho-Bad and 14-3-3 decreased during MCAO, whereas ferulic acid prevented the injury-induced decrease in these interaction levels. Moreover, ferulic acid prevented the injury-induced increase in cleaved caspase-3 levels. These findings suggest that ferulic acid attenuates cell death during MCAO and that these protective effects are due to inhibition of Akt signaling pathway inactivation and maintenance of the interaction between phospho-Bad and 14-3-3.     

Nicotinamide attenuates the ischemic brain injury-induced decrease of Akt activation and Bad phosphorylation

Nicotinamide protects cortical neuronal cells against cerebral ischemic injury through activation of various cytoprotective mechanisms. Here, this study confirmed the neuroprotective effects of nicotinamide in focal cerebral ischemic injury and investigated whether nicotinamide modulates a crucial survival pathway, Akt and its downstream targets. Adult male rats were treated with vehicle or nicotinamide (500 mg/kg) 2 h after the onset of middle cerebral artery occlusion (MCAO). Brains were collected 24 h after MCAO and infarct volumes were analyzed. Nicotinamide significantly reduced the infarct volume in the cerebral cortex. Potential activation was measured by phosphorylation of PDK1 at Ser²⁴¹, Akt at Ser⁴⁷³, and Bad at Ser¹³⁶ using Western blot analysis. Nicotinamide prevented the injury-induced decrease of pPDK1, pAkt, and pBad levels. 14-3-3 levels were not different between vehicle- and nicotinamide-treated animals. However, pBad and 14-3-3 interaction levels decreased during MCAO, but were maintained in the presence of nicotinamide, compared to levels in control animals. These findings suggest that nicotinamide attenuates cell death due to focal cerebral ischemic injury and that neuroprotective effects are mediated through the Akt signaling pathway, thus enhancing neuronal survival.     

Identification of proteins regulated by estradiol in focal cerebral ischemic injury—A proteomics approach

Estradiol protects neuronal cells against permanent and focal ischemic brain damage. We identified the proteins that are expressed following estradiol administration during cerebral ischemia in an animal model. Adult female rats were ovariectomized and treated with oil or estradiol prior to middle cerebral artery occlusion (MCAO) to induce cerebral ischemia, and brains were collected 24 h after MCAO. Protein analysis was performed on the cerebral cortex using two-dimensional gel electrophoresis. Protein spots with difference in intensity between oil- and estradiol-treated groups were identified by mass spectrometry. Among these proteins, levels of protein phosphatase 2A (PP2A) and astrocytic phosphoprotein PEA-15 were significantly decreased in the oil-treated group in comparison to the estradiol-treated group. Moreover, Western blot analysis demonstrated that estradiol treatment prevents injury-induced decrease of PP2A and PEA-15 levels during both MCAO-induced injury and glutamate exposure in HT22 cells. In contrast, levels of the 60 kDa heat shock protein (Hsp 60) were significantly increased in oil-treated animals, while estradiol prevented the injury-induced increase of Hsp 60. The results of this study provide an evidence that estradiol protects neuronal cells against ischemic brain injury through the up- and down-modulation of specific proteins.     

Ferulic acid attenuates the focal cerebral ischemic injury-induced decrease in parvalbumin expression

Ferulic acid exerts a neuroprotective effect through its anti-oxidant and anti-inflammation properties. Parvalbumin has calcium buffering capacity and protects neuronal cells from cytotoxic Ca(2+) overload. This study investigated whether ferulic acid regulates parvalbumin expression in cerebral ischemia and glutamate toxicity-induced neuronal cell death. Male Sprague-Dawley rats were immediately treated with vehicle or ferulic acid (100 mg/kg, i.v.) after middle cerebral artery occlusion (MCAO), and cerebral cortex tissues were collected 24 h after MCAO. A proteomics approach elucidated the decrease of parvalbumin in MCAO-operated animals, and ferulic acid treatment attenuated the injury-induced decrease in parvalbumin expression. Moreover, RT-PCR and Western blot analyses clearly showed that ferulic acid treatment prevents the injury-induced decrease in parvalbumin levels. The number of parvalbumin-positive cells also decreased in MCAO-operated animals, and ferulic acid attenuated this injury-induced decrease in parvalbumin-positive cells. In cultured hippocampal cells, glutamate toxicity significantly increased the intracellular Ca(2+) concentration, whereas this increase in Ca(2+) levels was inhibited by ferulic acid treatment. In addition, ferulic acid treatment attenuated the glutamate exposure-induced decrease in parvalbumin levels. These results suggest that ferulic acid exerts a neuroprotective effect by attenuating the injury-induced decrease of parvalbumin and modulating intracellular Ca(2+) levels.     

Extracellular progranulin protects cortical neurons from toxic insults by activating survival signaling

To reduce damage from toxic insults such as glutamate excitotoxicity and oxidative stresses, neurons may deploy an array of neuroprotective mechanisms. Recent reports show that progranulin (PGRN) gene null or missense mutations leading to inactive protein, are linked to frontotemporal lobar degeneration (FTLD), suggesting that survival of certain neuronal populations needs full expression of functional PGRN. Here we show that extracellular PGRN stimulates phosphorylation/activation of the neuronal MEK/extracellular regulated kinase (ERK)/p90 ribosomal S6 kinase (p90RSK) and phosphatidylinositol-3 kinase (PI3K)/Akt cell survival pathways and rescues cortical neurons from cell death induced by glutamate or oxidative stress. Pharmacological inhibition of MEK/ERK/p90RSK signaling blocks the PGRN-induced phosphorylation and neuroprotection against glutamate toxicity while inhibition of either MEK/ERK/p90RSK or PI3K/Akt blocks PGRN protection against neurotoxin MPP⁺. Inhibition of both pathways had synergistic effects on PGRN-dependent neuroprotection against MPP⁺ toxicity suggesting both pathways contribute to the neuroprotective activities of PGRN. Extracellular PGRN is remarkably stable in neuronal cultures indicating neuroprotective activities are associated with full-length protein. Together, our data show that extracellular PGRN acts as a neuroprotective factor and support the hypothesis that in FTLD reduction of functional brain PGRN results in reduced survival signaling and decreased neuronal protection against excitotoxicity and oxidative stress leading to accelerated neuronal cell death. That extracellular PGRN has neuroprotective functions against toxic insults suggests that in vitro preparations of this protein may be used therapeutically.     

Tacrolimus (FK506) attenuates biphasic cytochrome c release and Bad phosphorylation following transient cerebral ischemia in mice

Tacrolimus (FK506) has a neuroprotective action on cerebral infarction produced by cerebral ischemia, however, detailed mechanisms underlying this action have not been fully elucidated. We examined temporal profiles of survival-and death-related signals, Bad phosphorylation, release of cytochrome c (cyt.c), activation of caspase 3 and DNA fragmentation in the brain during and after middle cerebral artery occlusion (MCAo) in mice, and then examined the effect of tacrolimus on these signals. C57BL/6J mice were subjected to transient MCAo by intraluminal suture insertion for 60 min. Tacrolimus (1 mg/kg, i.p.) was administered immediately after MCAo. There were biphasic increases in the release of cyt.c in the ischemic core and penumbra; with the first increase toward the end of the occlusion period and the second increase 3-12 h after reperfusion. Tacrolimus significantly inhibited the increase of cytosolic cyt.c during ischemia and reperfusion. Phosphorylated Bad, Ser-136 (P-Bad(136)) and Ser-155 (P-Bad(155)) were detected 30 min after MCAo and after reperfusion in the ischemic cortex, respectively. Tacrolimus increased P-Bad(136) during ischemia and prolonged P-Bad(155) expression after reperfusion. Tacrolimus also decreased caspase-3 and terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling-positive cells, and reduced the size of infarct 24 h after reperfusion. Our study provided the first evidence that the neuroprotective action of tacrolimus involved inhibition of biphasic cyt.c release from mitochondria, possibly via up-regulation of Bad phosphorylation at different sites after focal cerebral ischemia and reperfusion.     

Estrogen limits ischemic cell death by modulating caspase-12-mediated apoptotic pathways following middle cerebral artery occlusion

Estrogen has received considerable attention as a potential therapeutic agent against various forms of neurodegenerative diseases including stroke. Experimental data in animal models of stroke have provided exhaustive evidence of the neuroprotective properties of this steroid hormone. Our laboratory in particular has demonstrated that acute estrogen treatment in male rats significantly reduced (approximately 50%) ischemic cell death within 4 h following permanent occlusion of the middle cerebral artery occlusion (MCAO). However, the cellular and molecular mechanisms implicated in the protective actions of estrogen in this experimental model have yet to be elucidated. Accumulating evidence suggests that in various in vivo and in vitro models, estrogen can be pro-apoptotic and that this effect may be mediated by an estrogen-induced up-regulation of the Fas/FasL system and the subsequent activation of caspase-12. We therefore hypothesized that under ischemic conditions following MCAO, estrogen would up-regulate protective endoplasmic reticulum (ER) stress pathways leading to caspase-12 activation, thus limiting infarct volume. Our results showed that estrogen significantly increased activated caspase-12 at 2, 3 and 4 h post-MCAO. Immunostaining of brain sections showed a significantly higher number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling positive cells in estrogen-treated animals at 4 h, but not at 2 h, post-MCAO. These findings correlate with previous observations that differences in infarct volume between saline and estrogen-treated animals are not seen until 3 and 4 h post-MCAO. A decrease in m-calpain expression was observed in the infarct region only at 4 h post-MCAO following estrogen pre-treatment, suggesting m-calpain may not be involved in regulating estrogen-induced caspase-12 activation. Based on these cellular changes correlated to estrogen pretreatment, we conclude that estrogen may up-regulate ER-specific apoptotic pathways, thus limiting the extent of necrotic cell death which is responsible for the spreading depression and growth of the infarct volume following MCAO.     

A novel 90-kDa tyrosine-phosphorylated protein associated with TCR complex in thymocytes

Ligation of the TCR-CD3 complex initiates a cascade of tyrosine phosphorylation that results in T cell activation. Initial activation of tyrosine kinases depends on the phosphorylation of activation motifs on CD3 chains. We previously found that a 90-kDa protein was tyrosine phosphorylated upon TCR cross-linking and the induction of the phosphorylation was dependent on the structure of the CD3 complex. In this study, we further characterized p90 phosphorylation. Phosphorylation of p90 was induced only by stimulation through the TCR-CD3 complex but not by other kinds of stimulation including CD28- or hydrogen peroxide-mediated activation and was dynamically regulated. Phosphorylated p90 was associated with the TCR-CD3 complex upon T cell activation. In a normal T cell population, thymocytes but not splenic T cells induced the tyrosine phosphorylation of p90 upon TCR cross-linking. These results suggest that p90 is a novel phosphoprotein associated with the TCR-CD3 complex and may play a role in TCR signaling during thymocyte differentiation.     

17Beta-estradiol downregulates Kupffer cell TLR4-dependent p38 MAPK pathway and normalizes inflammatory cytokine production following trauma-hemorrhage

Although studies have shown that 17beta-estradiol (estradiol) normalized Kupffer cell function following trauma-hemorrhage, the mechanism by which E2 maintains immune function remains unclear. Activation of Toll-like receptor 4 (TLR4) initiates an inflammatory cascade, involving activation of p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and nuclear factor-kappaB (NF-kappaB). This leads to the release of proinflammatory cytokines. Thus, we hypothesized that the salutary effects of estradiol on Kupffer cell function following trauma-hemorrhage are mediated via negative regulation of TLR4-dependent p38 MAPK and NF-kappaB. TLR4 mutant (C3H/HeJ) and wild type (C3H/HeOuJ) mice were subjected to trauma-hemorrhage (mean BP 35+/-5 mmHg approximately 90 min, then resuscitation) or sham operation. Administration of estradiol following trauma-hemorrhage in wild type mice decreased Kupffer cell TLR4 expression as well as prevented the phosphorylation of p38 MAPK and NF-kappaB. This was accompanied by normalization of Kupffer cell production capacities of IL-6, TNF-alpha, macrophage inflammatory protein (MIP)-1alpha, and MIP-2 and the decrease in plasma cytokine levels. In contrast, TLR4 mutant mice did not exhibit the increase in Kupffer cell p38 MAPK and NF-kappaB activation, cytokine production, or the increase in circulating cytokine levels following trauma-hemorrhage. No difference was observed in activation of PI3K among groups. These results suggest that the protective effect of estradiol on Kupffer cell function is mediated via downregulation of TLR4-dependent p38 MAPK and NF-kappaB signaling following trauma-hemorrhage, which prevents the systemic release of cytokines.     

Isoflurane preconditioning improves short-term and long-term neurological outcome after focal brain ischemia in adult rats

Isoflurane preconditioning improved short-term neurological outcome after focal brain ischemia in adult rats. It is not known whether desflurane induces a delayed phase of preconditioning in the brain and whether isoflurane preconditioning-induced neuroprotection is long-lasting. Two months-old Sprague–Dawley male rats were exposed to or were not exposed to isoflurane or desflurane for 30 min and then subjected to a 90 min middle cerebral arterial occlusion (MCAO) at 24 h after the anesthetic exposure. Neurological outcome was evaluated at 24 h or 4 weeks after the MCAO. The density of the terminal deoxynucleotidyl transferase biotinylated UTP nick end labeling (TUNEL) positive cells in the penumbral cerebral cortex were assessed 4 weeks after the MCAO. Also, rats were pretreated with isoflurane or desflurane for 30 min. Their cerebral cortices were harvested for quantifying B-cell lymphoma-2 (Bcl-2) expression 24 h later. Here, we showed that pretreatment with 1.1% or 2.2% isoflurane, but not with 6% or 12% desflurane, increased Bcl-2 expression in the cerebral cortex, improved neurological functions and reduced infarct volumes evaluated at 24 h after the MCAO. Isoflurane preconditioning also improved neurological functions and reduced brain infarct volumes in rats evaluated 4 weeks after the MCAO. Isoflurane preconditioning also decreased the density of TUNEL-positive cells in the penumbral cerebral cortex. We conclude that isoflurane preconditioning improves short-term and long-term neurological outcome and reduces delayed cell death after transient focal brain ischemia in adult rats. Bcl-2 may be involved in the isoflurane preconditioning effect. Desflurane pretreatment did not induce a delayed phase of neuroprotection.     

RSK2与恶性肿瘤的关系及其抑制剂的研究进展

<正>核糖体S6激酶(ribosomal S6 kinase,RSK)2属于Ras-MAPK下游通路的90 k D的RSK家族,该家族包含4个成员(RSK1~4)及2个结构同系物MSK1/2。RSK家族的成员具有高度的序列同源性(75%~80%的氨基酸序列是一致的),只有2个激酶功能域不同。尽管结构相似,它们在组织内的分布及功能却不尽相同[1],而本文主要介绍RSK2。     

Misoprostol, an anti-ulcer agent and PGE2 receptor agonist, protects against cerebral ischemia

Induction of COX-2 activity in cerebral ischemia results in increased neuronal injury and infarct size. Recent studies investigating neurotoxic mechanisms of COX-2 demonstrate both toxic and paradoxically protective effects of downstream prostaglandin receptor signaling pathways. We tested whether misoprostol, a PGE(2) receptor agonist that is utilized clinically as an anti-ulcer agent and signals through the protective PGE(2) EP2, EP3, and EP4 receptors, would reduce brain injury in the murine middle cerebral artery occlusion-reperfusion (MCAO-RP) model. Administration of misoprostol, at the time of MCAO or 2h after MCAO, resulted in significant rescue of infarct volume at 24 and 72h. Immunocytochemistry demonstrated dynamic regulation of the EP2 and EP4 receptors during reperfusion in neurons and endothelial cells of cerebral cortex and striatum, with limited expression of EP3 receptor. EP3-/- mice had no significant changes in infarct volume compared to control littermates. Moreover, administration of misoprostol to EP3+/+ and EP3-/- mice showed similar levels of infarct rescue, indicating that misoprostol protection was not mediated through the EP3 receptor. Taken together, these findings suggest a novel function for misoprostol as a protective agent in cerebral ischemia acting via the PGE(2) EP2 and/or EP4 receptors.     

Ferulic acid prevents the cerebral ischemic injury-induced decreases of astrocytic phosphoprotein PEA-15 and its two phosphorylated forms

Ferulic acid protects neuronal cells against focal cerebral ischemic injury through its anti-oxidative and anti-inflammatory effects. Phosphoprotein enriched in astrocytes 15 (PEA-15) is known to modulate various cellular processes including cell proliferation, apoptosis, and survival. This study was investigated whether ferulic acid can regulate the levels of PEA-15 and its two phosphorylated forms (Ser 104 and Ser 116) in a cerebral ischemic injury model and in neuronal cells exposed to glutamate. A middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemic injury. Adult male rats were immediately treated with vehicle or ferulic acid (100 mg/kg) at the beginning of the MCAO, and then cerebral cortices were collected 24 h after MCAO. The decrease in PEA-15 level after ischemic injury was detected using a proteomic approach. Ferulic acid administration prevented the ischemic injury-induced decrease of PEA-15 level. Moreover, Western blot analysis clearly confirmed that ferulic acid attenuates the ischemic injury-induced decreases in PEA-15, phospho-PEA-15 (Ser 104), and phospho-PEA-15 (Ser 116) levels. Glutamate exposure induced significant reductions in the levels of PEA-15 and the two phospho-PEA-15 (Ser 104 and Ser 116) in cultured hippocampal neuron, while pretreatment with ferulic acid prevented the glutamate toxicity-induced decreases in these proteins levels. The decrease of phospho-PEA-15 protein level indicates that the anti-apoptotic function of PEA-15 was being inhibited. Thus, these results suggest that ferulic acid protects neuronal cells against ischemic injury by maintenance of phospho-PEA-15 protein levels.