Nerve growth factor pretreatment attenuates oxygen and glucose deprivation-induced c-Jun amino-terminal kinase 1 and stress-activated kinases p38alpha and p38beta activation and confers neuroprotection in the pheochromocytoma PC12 Model

Neurotrophins such as nerve growth factor (NGF) are considered putative neuroprotective compounds in the central nervous system. To investigate the cellular and molecular neuroprotective mechanisms of NGF under ischemia, we used a unique oxygen and glucose deprivation (OGD) device. In this system we used pheochromocytoma PC12 cells to elucidate NGF neuroprotective effect. PC12 cells were exposed to OGD, followed by addition of glucose and oxygen (OGD reperfusion). Neuronal cell death induced in this model was measured by the release of lactate dehydrogenase (LDH), activation of caspase-3 and mitogen-activated protein kinases (MAPKs), measured with specific anti-phospho-antibodies. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, conferred 30% neuroprotection. However, treatment of the cultures with NGF concomitantly with the OGD insult did not result in neuroprotection. Time-course experiments showed marked activation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase (JNK), and p38 MAPK isoforms during the OGD phase but not during OGD reperfusion. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, resulted in 50% attenuation of OGD-induced activation of JNK1, and 20% and 50% attenuation of OGD-induced activation of p38alpha and beta, respectively. These findings support the notion that NGF confers neuroprotection from OGD insult, a phenomenon coincidentally related to differential inhibition of MAPK stress kinase isoforms, and provide the PC12 model as an in vitro OGD system to investigate molecular mechanisms of neurotoxicity and neuroprotection.     

Nerve growth factor pretreatment attenuates oxygen and glucose deprivation-induced c-Jun amino-terminal kinase 1 and stress-activated kinases p38α and p38β activation and confers neuroprotection in the pheochromocytoma PC12 model

Neurotrophins such as nerve growth factor (NGF) are considered putative neuroprotective compounds in the central nervous system. To investigate the cellular and molecular neuroprotective mechanisms of NGF under ischemia, we used a unique oxygen and glucose deprivation (OGD) device. In this system we used pheochromocytoma PC12 cells to elucidate NGF neuroprotective effect. PC12 cells were exposed to OGD, followed by addition of glucose and oxygen (OGD reperfusion). Neuronal cell death induced in this model was measured by the release of lactate dehydrogenase (LDH), activation of caspase-3 and mitogen-activated protein kinases (MAPKs), measured with specific anti-phospho-antibodies. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, conferred 30% neuroprotection. However, treatment of the cultures with NGF concomitantly with the OGD insult did not result in neuroprotection. Time-course experiments showed marked activation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase (JNK), and p38 MAPK isoforms during the OGD phase but not during OGD reperfusion. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, resulted in 50% attenuation of OGD-induced activation of JNK1, and 20% and 50% attenuation of OGD-induced activation of p38α and β, respectively. These findings support the notion that NGF confers neuroprotection from OGD insult, a phenomenon coincidentally related to differential inhibition of MAPK stress kinase isoforms, and provide the PC12 model as an in vitro OGD system to investigate molecular mechanisms of neurotoxicity and neuroprotection.     

Magnolol protects against trimethyltin-induced neuronal damage and glial activation in vitro and in vivo

Trimethyltin (TMT), an organotin with potent neurotoxic effects by selectively damaging to hippocampus, is used as a tool for creating an experimental model of neurodegeneration. In the present study, we investigated the protective effects of magnolol, a natural biphenolic compound, on TMT-induced neurodegeneration and glial activation in vitro and in vivo. In HT22 murine neuroblastoma cells, TMT induced necrotic/apoptotic cell death and oxidative stress, including intracellular reactive oxygen species (ROS), protein carbonylation, induction of heme oxygenase-1 (HO-1), and activation of all mitogen-activated protein kinases (MAPKs) family proteins. However, magnolol treatment significantly suppressed neuronal cell death by inhibiting TMT-mediated ROS generation and activation of JNK and p38 MAPKs. In BV-2 microglial cells, magnolol efficiently attenuated TMT-induced microglial activation via suppression of ROS generation and activation of JNK, p38 MAPKs, and nuclear factor-κB (NF-κB) signaling. In an in vivo mouse study, TMT induced massive neuronal damage and enhanced oxidative stress at day 2. We also observed a concomitant increase in glial cells and inducible nitric oxide synthase (iNOS) expression on the same day. These features of TMT toxicity were reversed by treatment of magnolol. We observed that p-JNK and p-p38 MAPK levels were increased in the mouse hippocampus at day 1 after TMT treatment and that magnolol blocked TMT-induced JNK and p38 MAPK activation. Magnolol administration prevented TMT-induced hippocampal neurodegeneration and glial activation, possibly through the regulation of TMT-mediated ROS generation and MAPK activation.     

Oxygen-glucose deprivation activates 5-lipoxygenase mediated by oxidative stress through the p38 mitogen-activated protein kinase pathway in PC12 cells

5-Lipoxygenase (5-LOX) is a key enzyme catalyzing arachidonic acid to form leukotrienes. We have reported that ischemic-like injury activates 5-LOX in PC12 cells; however, the mechanisms are unknown. To determine whether ischemic-like injury activates 5-LOX mediated by oxidative stress through the p38 MAPK pathway, we transfected GFP-5-LOX into PC12 cells and induced ischemic-like injury by oxygen-glucose deprivation (OGD). We found that the transfected GFP-5-LOX was localized primarily in the nuclei and translocated to the nuclear envelope after OGD/recovery reaching a maximum 2 hr after a 2-hr exposure to OGD. The nonselective 5-LOX inhibitor caffeic acid, 5-LOX-activating protein inhibitor MK886, and selective 5-LOX inhibitor zileuton attenuated the cell injury and reduced the production of 5-LOX products, cysteinyl leukotrienes, after OGD/recovery. However, only caffeic acid inhibited OGD/recovery-induced 5-LOX translocation. OGD/recovery also increased reactive oxygen species (ROS), which was inhibited by caffeic acid only. Hydrogen peroxide, an exogenous ROS, evoked similar cell injury and 5-LOX translocation, and the inhibitors had effects on the changes after H(2)O(2) similar to those after OGD/recovery. Both OGD/recovery and H(2)O(2) increased the phosphorylated p38 MAPK level, which was inhibited by caffeic acid and the ROS scavenger edaravone, but not by MK886 or zileuton. Moreover, SB203580 (a p38 MAPK inhibitor) and edaravone inhibited the cell injury and 5-LOX translocation induced by OGD/recovery and H(2)O(2). Thus, we conclude that OGD/recovery-induced ischemic-like injury induces 5-LOX activation, which is mediated by oxidative stress through activating the p38 MAPK pathway.     

An increase in voltage‐gated sodium channel current elicits microglial activation followed inflammatory responses in vitro and in vivo after spinal cord injury

Inflammation induced by microglial activation plays a pivotal role in progressive degeneration after traumatic spinal cord injury (SCI). Voltage‐gated sodium channels (VGSCs) are also implicated in microglial activation following injury. However, direct evidence that VGSCs are involved in microglial activation after injury has not been demonstrated yet. Here, we show that the increase in VGSC inward current elicited microglial activation followed inflammatory responses, leading to cell death after injury in vitro and in vivo. Isoforms of sodium channel, Na_v1.1, Na_v1.2, and Na_v1.6 were expressed in primary microglia, and the inward current of VGSC was increased by LPS treatment, which was blocked by a sodium channel blocker, tetrodotoxin (TTX). TTX inhibited LPS‐induced NF‐κB activation, expression of TNF‐α, IL‐1β and inducible nitric oxide synthase, and NO production. LPS‐induced p38MAPK activation followed pro‐nerve growth factor (proNGF) production was inhibited by TTX, whereas LPS‐induced JNK activation was not. TTX also inhibited caspase‐3 activation and cell death of primary cortical neurons in neuron/microglia co‐cultures by inhibiting LPS‐induced microglia activation. Furthermore, TTX attenuated caspase‐3 activation and oligodendrocyte cell death at 5 d after SCI by inhibiting microglia activation and p38MAPK activation followed proNGF production, which is known to mediate oligodendrocyte cell death. Our study thus suggests that the increase in inward current of VGSC appears to be an early event required for microglia activation after injury. GLIA 2013;61:1807–1821     

Oxidative neurotoxicity in rat cerebral cortex neurons: synergistic effects of H2O2 and NO on apoptosis involving activation of p38 mitogen-activated protein kinase and caspase-3

Oxidative stress in the brain has been increasingly associated with the development of numerous human neurological diseases. Microglia, activated upon neuronal injury or inflammatory stimulation, are known to release superoxide anion (*O(2) (-)), hydrogen peroxide (H(2)O(2)), and nitric oxide (NO), thereby further contributing to oxidative neurotoxicity. The reaction of NO and *O(2) (-), forming the toxic peroxynitrite (ONOO(-)), has been proposed to play a pathogenic role in neuronal injury. However, the interactions between H(2)O(2) and NO during oxidative stress, which may promote or diminish cell death, is less clear. In this study, we explored oxidative neurotoxicity induced by H(2)O(2) plus NO in primary cultures of rat cerebral cortex neurons. As the mechanisms may involve reactions between H(2)O(2) and NO, we monitored the production of ONOO(-)and reactive oxygen species (ROS) throughout the experiments. Results indicated that the NO donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and H(2)O(2) by themselves elicited neuronal death in a concentration- and time-dependent manner. Sublytic concentrations of H(2)O(2) plus SNAP were sufficient to induce neuronal apoptosis as determined by DNA laddering and fluorescent staining of apoptotic nuclei. Transient ONOO(-)increase was accompanied by rapid H(2)O(2) decay and NO production, whereas ROS slowly decreased following treatment. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation and the cleavage of caspase-3 were observed. Conversely, inhibition of p38 MAPK and caspase-3 significantly reduced apoptotic death induced by H(2)O(2) plus SNAP. These data suggest that H(2)O(2) and NO act synergistically to induce neuronal death through apoptosis in which activation of p38 MAPK and caspase-3 is involved.     

Bax shuttling after rotenone treatment of neuronal primary cultures: Effects on cell death phenotypes

Neonatal (P7) brain hypoxia–ischemia (HI) induces intracellular Bax protein shifts to the nucleus, mitochondria, and endoplasmic reticulum (ER), where it triggers the activation of the respective cell death signaling cascades. When compared with HI‐treated rat pups, 100% O₂ resuscitation of HI‐treated rat pups increases HI‐induced ER Bax levels, ER‐mediated cell death signaling, and resultant lesion volume and inflammation due to increased necrotic‐like cell death. To better characterize the role of Bax intracellular shuttling ER cell death signaling and necrotic‐like cell death, we used rotenone‐treated P5 neuronal cortical cultures to increase ER Bax levels and subsequent cell death signaling. We treated P5 primary cortical neurons with 25 μM and 100 μM rotenone as an apoptotic or necrotic‐like stimulus, respectively, and measured intracellular organelle Bax levels and the subsequent activation of ER/mitochondrial cell death signaling. The 25 μM rotenone treatment promptly increased nuclear Bax levels followed by a later increase in mitochondrial Bax levels and caspase‐mediated cleavage of α‐fodrin. The 100 μM rotenone treatment also resulted in an early increase in nuclear Bax levels followed by a subsequent increase in ER Bax levels and calpain‐mediated cleavage of α‐fodrin. After pretreatment with the immunosuppressive and neuroprotective FK506, there was a delay in Bax intracellular shifts and cell death signaling for both the 25 and 100 μM rotenone treatments. These results suggest that the different outcomes of apoptotic‐like vs. necrotic‐like cell death resulting from the treatment of neuronal cultures with rotenone at 25 and 100μM rotenone reflect changes in the intracellular trafficking of Bax among different organelles. © 2009 Wiley‐Liss, Inc.     

Differentiation and neural integration of hippocampal neuronal progenitors: Signaling pathways sequentially involved

In the context of their potential implication in regenerative strategies, we characterized cell mechanisms underlying the fate of embryonic rat hippocampal H19–7 progenitors in culture upon induction of their differentiation, and tested their capacities to integrate into a neuronal network in vitro. Without addition of growth factors, nearly 100% of cells expressed various neuronal markers, with a progressive rise of the expression of Synapsin I and II, suggesting that cells developed as mature neurons with synaptogenic capacities. Fully differentiated neurons were identified as glutamatergic and expressed the receptor‐associated protein PSD‐95. Quantification of ATP showed that 60% of cells died within 24 h after differentiation. Cell death was shown to imply Erk1/2‐dependent intrinsic mitochondrial apoptosis signaling pathway, with activation of caspase‐9 and ‐3, finally leading to single‐strand DNA. Surviving neurons displayed high levels of Akt, phospho‐Akt, and antiapoptotic proteins such as Bcl‐2 and Bcl‐XL, with decreased caspase activation. In the absence of trophic support, the proapoptotic death‐associated protein (DAP) kinase was dramatically stimulated by 24 h postdifferentiation, along with increased levels of p38 and phospho‐p38, and caspase reactivation. These findings show that different signaling pathways are sequentially triggered by differentiation, and highlight that ultimate cell death would involve p38 and DAP kinase activation. This was supported by the improvement of cell survival at 24‐h postdifferentiation when cells were treated by PD169316, a specific inhibitor of p38. Finally, when seeded on rat hippocampal primary cultured neurons, a significant number of differentiated H19–7 cells were able to survive and to develop cell–cell communication. © 2009 Wiley‐Liss, Inc.     

Protective effects of placental growth factor on retinal neuronal cell damage

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor family. Although it has been reported that PlGF protects against neuronal damage in the brain, little is known about the effects of PlGF in the retina. Therefore, we investigated the effects of PlGF on retinal neuronal cells. To evaluate the effects of PlGF against L‐buthionine‐(S,R)‐sulfoximine (BSO)/glutamate cell death, oxygen–glucose deprivation (OGD)‐induced cell death, and light‐induced cell death, RGC‐5 and 661W cells were used. We evaluated the mechanism responsible for the protective effects of PlGF against retinal neuronal cell death by performing the examinations with U1026, which is a mitogen‐activated protein kinase (MEK) inhibitor, and LY294002, which is a phosphoinositide 3‐kinase (PI3K) inhibitor. In addition, we measured caspase‐3/7 activity in RGC‐5 cells and 661W cells. PlGF protected against RGC‐5 cell death induced by BSO/glutamate and OGD and against 661W cell death induced by light irradiation. Moreover, an anti‐PlGF antibody negated these protective effects. The protective effects of PlGF against OGD‐induced RGC‐5 cell death and light‐induced 661W cell death were suppressed by using an anti‐PlGF antibody, U1026, and LY294002. Treatment with PlGF suppressed caspase‐3/7 activity in both cell lines. We demonstrated for the first time that PlGF exerts a protective effect by inhibiting the activation of caspase‐3/7 through the MEK and PI3K pathway in retinal neuronal cells. These data suggest that PlGF may be an important protective factor in the retina. © 2013 Wiley Periodicals, Inc.     

Differential vulnerability of immature murine neurons to oxygen-glucose deprivation

In vivo studies support selective neuronal vulnerability to hypoxia-ischemia (HI) in the developing brain. Since differences in intrinsic properties of neurons might be responsible, pure cultures containing immature neurons (6-8 days in vitro) isolated from mouse cortex and hippocampus, regions chosen for their marked vulnerability to oxidative stress, were studied under in vitro ischemic conditions-oxygen-glucose deprivation (OGD). Twenty-four hours of reoxygenation after 2.5 h of OGD induced significantly greater cell death in hippocampal than in cortical neurons (67.8% vs. 33.4%, P = 0.0068). The expression of neuronal nitric oxide synthase (nNOS) protein, production of nitric oxide (NO), and reactive oxygen species (ROS), as well as glutathione peroxidase (GPx) activity and intracellular levels of reduced glutathione (GSH), were measured as indicators of oxidative stress. Hippocampal neurons had markedly higher nNOS expression than cortical neurons by 24 h of reoxygenation, which coincided with an increase in NO production, and significantly greater ROS accumulation. GPx activity declined significantly in hippocampal but not in cortical neurons at 4 and 24 h after OGD. The decrease in GSH level in hippocampal neurons correlated with the decline of GPx activity. Our data suggest that developing hippocampal neurons are more sensitive to OGD than cortical neurons. This finding supports our in vivo studies showing that mouse hippocampus is more vulnerable than cortex after neonatal HI. An imbalance between excess prooxidant production (increased nNOS expression, and NO and ROS production) and insufficient antioxidant defenses created by reduced GPx activity and GSH levels may, in part, explain the higher susceptibility to OGD of immature hippocampal neurons.     

H2O2 and 4-hydroxynonenal mediate amyloid beta-induced neuronal apoptosis by activating JNKs and p38MAPK

Amyloid beta peptides (Abeta) may be neurotoxic during the progression of Alzheimer's disease by eliciting oxidative stress. Exposure of neuronally differentiated SK-N-BE cells to Abeta(25-35) fragment as well as to full-length Abeta(1-40) and Abeta(1-42) induces early and time-dependent generation of oxidative stress that has been evaluated by carefully monitoring generation of hydrogen peroxide (H(2)O(2)), 4-hydroxynonenal (HNE), thiobarbituric acid reactive substances (TBARS), and fluorescent chromolipids. Abeta treatment also results in the activation of c-Jun aminoterminal kinases (JNKs) and p38(MAPK) and is followed by characteristic nuclear changes of apoptosis as evaluated by DAPI staining and TUNEL technique. To reproduce the relationships between oxidative stress and Abeta apoptosis we found that only the simultaneous administration of HNE and H(2)O(2), at concentrations similar to those generated within the first 3 h of Abeta exposure, can fully mimic Abeta-dependent activation of JNKs and p38(MAPK) and occurrence of apoptosis. Antioxidants such as alpha-tocopherol and N-acetylcysteine prevent completely either neuronal apoptosis or activation of JNKs and p38(MAPK) elicited by Abeta or by simultaneous HNE and H(2)O(2) addition. Finally, direct evidence that activation of these kinases is required for cell death induced by Abeta has been obtained by pretreating cell with specific inhibitors of JNKs and p38(MAPK). These results suggest the existence of a sequence of events in Abeta-induced apoptosis involving simultaneous generation of HNE and H(2)O(2) and oxidative stress-dependent activation of JNKs and p38(MAPK).     

Heat shock induces neurite outgrowth in PC12m3 cells via the p38 mitogen-activated protein kinase pathway

We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in heat-shock-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to heat stress at 44 degrees C for 10 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The neurite extension was inhibited by the p38 MAPK inhibitor BS203580. Longer heat treatment of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that heat-induced activation of p38 MAPK is responsible for the neurite outgrowth and survival of PC12m3 cells.     

Reovirus-induced neuronal apoptosis is mediated by caspase 3 and is associated with the activation of death receptors

Reovirus infection of the central nervous system (CNS) is an important experimental system for understanding the pathogenesis of neurotropic viral infection. Infection of neonatal mice with T3 reoviruses causes lethal encephalitis in which injury results from virus-induced apoptosis. We now show that this apoptosis in vivo is associated with activation of caspase 3, and use neuroblastoma and primary neuronal cultures to identify the cellular pathways involved. Reovirus-induced apoptosis in neuronal cultures is initiated by activation of the tumor necrosis factor (TNF) receptor superfamily death receptors and is inhibited by treatment with soluble death receptors (DRs). The DR-associated initiator caspase, caspase 8, is activated following infection, this activation is inhibited by a cell-permeable peptide inhibitor (IETD-CHO). In contrast to our previous findings in non-neuronal cell lines, reovirus-induced neuronal apoptosis is not accompanied by significant release of cytochrome c from the mitochondria or with caspase 9 activation following infection. This suggests that in neuronal cells, unlike their non-neuronal counterparts, the mitochondria-mediated apoptotic pathway associated with cytochrome c release and caspase 9 activation does not play a significant role in augmenting reovirus-induced apoptosis. Consistent with these results, peptide caspase inhibitors show a hierarchy of efficacy in inhibiting reovirus-induced apoptosis, with inhibitors of caspase 3 > caspase 8 > caspase 9. These studies provide a comprehensive profile of the pattern of virus-induced apoptotic pathway activation in neuronal culture.     

Inhibition of caspase-3 activation by SB 203580, p38 mitogen-activated protein kinase inhibitor in nitric oxide-induced apoptosis of PC-12 cells

Mitogen-activated protein kinase (MAPK) p38 plays pivotal role in cell proliferation, differentiation, and apoptosis when cysteine protease caspase induces apoptosis in different cell systems. SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1 H-imidazole) is widely used as a specific inhibitor of p38 MAPK, and prevents apoptosis induced by various agents. The effect of SB 203580 on nitric oxide(NO)- or peroxynitrite-induced cell death is not known. Western blotting results indicate that p38 MAPK was activated significantly in NO- or peroxynitrite-induced cell death in a time-dependent manner, and subsequently this cell death was markedly inhibited by SB 203580, as determined by fluorescence-activated cell sorting (FACS)-can analyzer. Furthermore, NO/peroxynitrite-induced caspase-3 activation was notably inhibited by SB 203580, however, phosphorylation of either p38 MAPK or p44/42 was not influenced by SB 203580. Thus, it is likely that SB 203580 prevents NO/peroxynitrite-induced cell death by inhibiting caspase-3 activation in PC-12 cells.     

c-Jun N-Terminal Kinase (JNK) and p38 Play Different Roles in Age-Related Purkinje Cell Death in Murine Organotypic Culture

Several studies have shown that Purkinje cells die by apoptosis in organotypic slice cultures from postnatal 3-day-old (P3) mice. This cell death is age-dependent and has been proposed as indirect evidence for the programmed Purkinje cell death occurring in in vivo cerebellum. Here, we studied whether c-jun N-terminal kinase (JNK) and p38 kinase pathways contribute to the Purkinje cell death observed in cerebellar slice cultures obtained from P3 mice. Slice culture treatment with D-JNKI1 or SB203580, respectively inhibitors of JNK and p38 MAP kinases, results in a better survival of Purkinje cells. Interestingly, the combined treatment with the two inhibitors potentiated single treatment effects. These results suggest that p38 and JNK pathways might be differently implicated in this Purkinje cell death. Time course experiments found p38 activation immediately post-slicing, whereas JNK activation was detected only 2 h after the culture. We hypothesize that p38 activation might be due to the “sliced condition,” and JNK activation might be more specific to P3 age-dependent cell death. The study of JNK and p38 activation in cerebellar lysates from P0 slice culture confirmed JNK activation being specific for the P3 explants, whereas p38 is activated both from P0 and P3 cerebellar slice culture lysates. These results suggest that p38 is activated by the slicing, whereas JNK activation is related to developmental Purkinje cell death.     

Induction of cellular stress and chaperone activation in organotypic slice cultures of hippocampus

Neurodegenerative diseases are often associated with the occurrence of misfolded proteins preceding neuronal cell death. Accumulation of misfolded proteins in the endoplasmic reticulum induces ER stress, which in consequence enhances chaperone expression to restore protein homeostasis. Here we used organotypic hippocampal slice cultures to analyze the time course of chaperone expression and neuronal death after induction of ER stress by tunicamycin treatment. Shortly after explantation many cells stain positive for Fluoro Jade B demonstrating neuronal cell death. While in control cultures the number of Fluoro Jade B labeled cells remarkably decrease over the total period of cultivation, neuronal death remains elevated in ER-stressed slice cultures. Caspase-3 staining revealed that neuronal death is primarily due to apoptosis in tunicamycin-treated slice cultures. The chaperone GRP78/BiP is expressed at low levels in control sections. Its expression is largely restricted to hippocampal neurons. Tunicamycin treatment resulted in upregulation of GRP78/BiP in the neuronal cells. Double-immunolabeling for GFAP shows a concomitant de novo expression of GRP78/BiP in astrocytes. The astrocytic GRP78/BiP upregulation might reflect an early, neuroprotective response. The increase of GRP78/BiP in neurons and astrocytes show successful induction of the ER stress response. The hippocampal slice cultures are, thus, a useful tool to examine the process of neurodegeneration and to investigate neuroprotective devices in an ER stress paradigm.     

SB239063,新一代p38丝裂原活化蛋白激酶抑制剂在缺氧缺糖所致SHSY5Y细胞损伤中的作用

目的 探讨SB239063,新一代p38丝裂原活化蛋白激酶(p38MAPK)抑制剂在缺氧缺糖(OGD)所致SHSY5Y细胞损伤中的作用.方法 体外培养SHSY5Y神经母细胞瘤株,以OGD、SB239063(10μmol/L)处理,以MTT检测SHSY5Y细胞活性,以Western blot方法检测p38MAPK活性,应用HEt和fluo-3/AM的荧光强度测定OGD后SHSY5Y细胞超氧化物阴离子和钙离子的浓度.结果 SB239063能提高OGD后SHSY5Y细胞的活性(P<0.01),降低OGD后p38MAPK活性(P<0.05),HEt(P<0.05)和fluo-3/AM荧光强度(P<0.01).结论 SB239063通过抑制p38MAPK的激活,降低细胞内超氧阴离子的浓度,进而抑制细胞内钙超载,从而达到保护SHSY5Y细胞对抗OGD的细胞损害的作用.

Abstract：

Objective To investigate the neuroprotective effect of SB239063,the new p38 mitogen—activated protein kinase (MAPK) inhibitor on SHSY5Y neuronal cells against oxygen-glucose deprivation(OGD),and the mechanisms of the neuroprotective effect.Methods SHSY5Y neuroblastoma cells were exposed to OGD with or without SB239063 (10μmol/L),cell viability was measured by the MTF assay,activity of p38MAPK was measured by western blot,intracellular concentration of superoxide anion and calcium were evaluated via the fluorescence intensity of Het and fluo-3/AM. Resuits Compared with the OGD group.The MTT value was elevated significantly in the SB239063 group(P<0.01),the activitv of p38MAPK decreased significantly in the SB239063 group(P<0.01),the fluorescence intensity of Het(P<0.05)and fluo-3/AM(P<0.01)decreased significantly in the SB239063 group.Conclusion SB239063 protects SHSY5Y neuronal cells against OGD by inhibiting the activity of p38MAPK,then reducing the intracellular concentration of superoxide anion and calcium.     

Neurotoxicity of pneumolysin,a major pneumococcal virulence factor,involves calcium influx and depends on activation of p38 mitogen-activated protein kinase

Neuronal injury in bacterial meningitis is caused by the interplay of host inflammatory responses and direct bacterial toxicity. We investigated the mechanisms by which pneumolysin, a cytosolic pneumococcal protein, induces damage to neurons. The toxicity after exposure of human SH-SY5Y neuroblastoma cells and hippocampal organotypic cultures to pneumolysin was time- and dose-dependent. Pneumolysin led to a strong calcium influx apparently mediated by pores on the cell membrane formed by the toxin itself and not by voltage-gated calcium channels. Buffering of intracellular calcium with BAPTA-AM [1, 2-bis (o-aminophenoxy) ethane N, N, N', N'-tetraacetic acid tetra(acetomethoxyl) ester] improved survival of neuronal cells following challenge with pneumolysin. Western blotting revealed increased phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) as early as 30 min after challenge with pneumolysin. SB 203580, a potent and selective inhibitor of p38 MAPK, rescued human neuronal cells from pneumolysin-induced death. Inhibition of the mitochondrial permeability transition pore using bongkrekate and caspase inhibition also improved survival following challenge with the toxin. Modulation of cell death pathways activated by pneumolysin may influence the outcome of pneumococcal meningitis.