Small molecule inhibitor DDQ-treated hippocampal neuronal cells show improved neurite outgrowth and synaptic branching

The process of neurite outgrowth and branching is a crucial aspect of neuronal development and regeneration.Axons and dendrites,sometimes referred to as neurites,are extensions of a neuron's cellular body that are used to start networks.Here we explored the effects of diethyl(3,4-dihydroxyphenethylamino)(quinolin-4-yl)methylphosphonate(DDQ)on neurite developmental features in HT22 neuronal cells.In this work,we examined the protective effects of DDQ on neuronal processes and synaptic outgrowth in differentiated HT22cells expressing mutant Tau(mTau)cDNA.To investigate DDQ chara cteristics,cell viability,biochemical,molecular,western blotting,and immunocytochemistry were used.Neurite outgrowth is evaluated through the segmentation and measurement of neural processes.These neural processes can be seen and measured with a fluorescence microscope by manually tracing and measuring the length of the neurite growth.These neuronal processes can be observed and quantified with a fluorescent microscope by manually tracing and measuring the length of the neuronal HT22.DDQ-treated mTau-HT22 cells(HT22 cells transfected with cDNA mutant Tau)were seen to display increased levels of synaptophysin,MAP-2,andβ-tubulin.Additionally,we confirmed and noted reduced levels of both total and p-Tau,as well as elevated levels of microtubule-associated protein 2,β-tubulin,synaptophysin,vesicular acetylcholine transporter,and the mitochondrial biogenesis protein-pe roxisome prolife rator-activated receptor-gamma coactivator-1α.In mTa u-expressed HT22 neurons,we observed DDQ enhanced the neurite characteristics and improved neurite development through increased synaptic outgrowth.Our findings conclude that mTa u-HT22(Alzheimer's disease)cells treated with DDQ have functional neurite developmental chara cteristics.The key finding is that,in mTa u-HT22 cells,DDQ preserves neuronal structure and may even enhance nerve development function with mTa u inhibition.

Neuroprotective effects of ginsenoside Rb1 on hippocampal neuronal injury and neurite outgrowth

Ginsenoside Rb1 has been reported to exert anti-aging and anti-neurodegenerative effects. In the present study, we investigate whether ginsenoside Rb1 is involved in neurite outgrowth and neuroprotection against damage induced by amyloid beta（25–35） in cultured hippocampal neurons, and explore the underlying mechanisms. Ginsenoside Rb1 significantly increased neurite outgrowth in hippocampal neurons, and increased the expression of phosphorylated-Akt and phosphorylated extracellular signal-regulated kinase 1/2. These effects were abrogated by API-2 and PD98059, inhibitors of the signaling proteins Akt and MEK. Additionally, cultured hippocampal neurons were exposed to amyloid beta（25–35） for 30 minutes; ginsenoside Rb1 prevented apoptosis induced by amyloid beta（25–35）, and this effect was blocked by API-2 and PD98059. Furthermore, ginsenoside Rb1 significantly reversed the reduction in phosphorylated-Akt and phosphorylated extracellular signal-regulated kinase 1/2 levels induced by amyloid beta（25–35）, and API-2 neutralized the effect of ginsenoside Rb1. The present results indicate that ginsenoside Rb1 enhances neurite outgrowth and protects against neurotoxicity induced by amyloid beta（25–35） via a mechanism involving Akt and extracellular signal-regulated kinase 1/2 signaling.

Ginsenoside Rg1 protects against neurodegeneration by inducing neurite outgrowth in cultured hippocampal neurons

Ginsenoside Rg1（Rg1） has anti-aging and anti-neurodegenerative effects. However, the mechanisms underlying these actions remain unclear. The aim of the present study was to determine whether Rg1 affects hippocampal survival and neurite outgrowth in vitro after exposure to amyloid-beta peptide fragment 25–35（Aβ_（25–35））, and to explore whether the extracellular signal-regulated kinase（ERK） and Akt signaling pathways are involved in these biological processes. We cultured hippocampal neurons from newborn rats for 24 hours, then added Rg1 to the medium for another 24 hours, with or without pharmacological inhibitors of the mitogen-activated protein kinase（MAPK） family or Akt signaling pathways for a further 24 hours. We then immunostained the neurons for growth associated protein-43, and measured neurite length. In a separate experiment, we exposed cultured hippocampal neurons to Aβ_（25–35） for 30 minutes, before adding Rg1 for 48 hours, with or without Akt or MAPK inhibitors, and assessed neuronal survival using Hoechst 33258 staining, and phosphorylation of ERK1/2 and Akt by western blot analysis. Rg1 induced neurite outgrowth, and this effect was blocked by API-2（Akt inhibitor） and PD98059（MAPK/ERK kinase inhibitor）, but not by SP600125 or SB203580（inhibitors of c-Jun N-terminal kinase and p38 MAPK, respectively）. Consistent with this effect, Rg1 upregulated the phosphorylation of Akt and ERK1/2; these effects were reversed by API-2 and PD98059, respectively. In addition, Rg1 significantly reversed Aβ_（25–35）-induced apoptosis; this effect was blocked by API-2 and PD98059, but not by SP600125 or SB203580. Finally, Rg1 significantly reversed the Aβ_（25–35）-induced decrease in Akt and ERK1/2 phosphorylation, but API-2 prevented this reversal. Our results indicate that Rg1 enhances neurite outgrowth and protects against Aβ_（25–35）-induced damage, and that its mechanism may involve the activation of Akt and ERK1/2 signaling.

Role of chloride channels in nitric oxide-induced rat hippocampal neuronal apoptosis in vitro

BACKGROUND：Chloride channels participate in non-neuronal apoptosis.However,it remains unclear whether chloride channels are involved in ischemic neuronal apoptosis.OBJECTIVE：To explore the effects of 4-acetamido-4＇-isothiocyanatostilbene-2,2＇-disulfonic acid （SITS） and 4,4＇-diisothiocyanostilbene-2,2＇-disulfonic acid （DIDS）,two chloride channel blockers,on the hippocampal neuronal apoptosis induced by 3-morpholinosydnonimine （SIN-1） based on the nitric oxide toxicity theory of neuronal apoptosis following ischemic brain injury.DESIGN,TIME AND SETTING：Comparative observation and in vitro experiments were performed at the laboratory of Zhuhai Campus of Zunyi Medical College from January to May 2009.MATERIALS：SIN-1,SITS,and DIDS were purchased from Sigma,USA.METHODS：Hippocampal neurons from Sprague-Dawley rats,aged 1 day,were cultured In vitro for 12 days and randomly assigned to control,SIN-1,or chloride channel blocker groups.SIN-1 group neurons were induced by SIN-1 for 18 hours to establish a model of ischemic neuronal apoptosis.Neurons in chloride channel blocker groups were treated with SITS or DIDS plus SIN-1 for 18 hours.The controls were cultured in DMEM/Ham＇s F12 complete medium alone.MAIN OUTCOME MEASURES：The apoptotic neurons and nuclear appearance were detected by Hoechst 33258 fluorescence staining; neuronal viability was quantitatively determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide analysis.Caspase-3 activity was analyzed by Western blot.RESULTS：SIN-1 （1 mmol/L） dramatically induced apoptosis （50%-60%）.SITS and DIDS inhibited nitric oxide-induced neuronal injury in a dose-dependent manner,suppressed caspase-3 activation,reduced neuronal apoptosis,and improved neuronal survival.CONCLUSION：Chloride channel blockers can protect against neuronal injury induced by NO.Chloride channels might be involved in neuronal apoptosis following cerebral ischemia.

Protective mechanisms of micro RNA-27a against oxygen-glucose deprivation-induced injuries in hippocampal neurons

Hypoxic injuries during fetal distress have been shown to cause reduced expression of micro RNA-27a（mi R-27a）,which regulates sensitivity of cortical neurons to apoptosis.We hypothesized that miR-27 a overexpression attenuates hypoxia- and ischemia-induced neuronal apoptosis by regulating FOXO1,an important transcription factor for regulating the oxidative stress response.miR-27 a mimic was transfected into hippocampal neurons to overexpress miR-27 a.Results showed increased hippocampal neuronal viability and decreased caspase-3 expression.The luciferase reporter gene system demonstrated that mi R-27 a directly binded to FOXO1 3′UTR in hippocampal neurons and inhibited FOXO1 expression,suggesting that FOXO1 was the target gene for mi R-27 a.These findings confirm that mi R-27 a protects hippocampal neurons against oxygen-glucose deprivation-induced injuries.The mechanism might be mediated by modulation of FOXO1 and apoptosis-related gene caspase-3 expression.

Chrysophanol attenuates lead exposure-induced injury to hippocampal neurons in neonatal mice

Previous studies have shown that chrysophanol protects against learning and memory impairments in lead-exposed adult mice. In the present study, we investigated whether chrysophanol can alleviate learning and memory dysfunction and hippocampal neuronal injury in lead-exposed neonatal mice. At the end of lactation, chrysophanol（0.1, 1.0, 10.0 mg/kg） was administered to the neonatal mice by intraperitoneal injection for 15 days. Chrysophanol significantly alleviated injury to hippocampal neurons and improved learning and memory abilities in the lead-poisoned neonatal mice. Chrysophanol also significantly decreased lead content in blood, brain, heart, spleen, liver and kidney in the lead-exposed neonatal mice. The levels of malondialdehyde in the brain, liver and kidney were significantly reduced, and superoxide dismutase and glutathione peroxidase activities were significantly increased after chrysophanol treatment. Collectively, these findings indicate that chrysophanol can significantly reduce damage to hippocampal neurons in lead-exposed neonatal mice.

Real-time Microwave Exposure Induces Calcium Efflux in Primary Hippocampal Neurons and Primary Cardiomyocytes

Objective To detect the effects of microwave on calcium levels in primary hippocampal neurons and primary cardiomyocytes by the real-time microwave exposure combined with laser scanning confocal microscopy. Methods The primary hippocampal neurons and primary cardiomyocytes were cultured and labeled with probes, including Fluo-4 AM, Mag-Fluo-AM, and Rhod-2, to reflect the levels of whole calcium [Ca], endoplasmic reticulum calcium [Ca]ER, and mitochondrial calcium [Ca]MIT, respectively. Then, the cells were exposed to a pulsed microwave of 2.856 GHz with specific absorption rate(SAR) values of 0, 4, and 40 W/kg for 6 min to observe the changes in calcium levels. Results The results showed that the 4 and 40 W/kg microwave radiation caused a significant decrease in the levels of [Ca], [Ca]ER, and [Ca]MIT in primary hippocampal neurons. In the primary cardiomyocytes, only the 40 W/kg microwave radiation caused the decrease in the levels of [Ca], [Ca]ER, and [Ca]MIT. Primary hippocampal neurons were more sensitive to microwave exposure than primary cardiomyocytes. The mitochondria were more sensitive to microwave exposure than the endoplasmic reticulum. Conclusion The calcium efflux was occurred during microwave exposure in primary hippocampal neurons and primary cardiomyocytes. Additionally, neurons and mitochondria were sensitive cells and organelle respectively.

Astragalus injection inhibits c-Jun N terminal kinase mRNA expression following oxygen-glucose deprivation and reintroduction in rat hippocampal neurons

BACKGROUND： In studies concerning cell injury induced by cerebral ischemia-reperfusion, current experiments have primarily focused on altered protein levels. In addition, the apoptotic proteins Bax and Bcl-2 have been thoroughly studied with regard to initiating neuronal apoptosis. OBJECTIVE： To establish an in vitro model of oxygen-glucose deprivation and reintroduction in the rat hippocampus to simulate cerebral ischemia-reperfusion injury; to observe c-Jun N-terminal kinase 3 （JNK3） mRNA expression in hippocampal neurons following Astragalus injection; and thus to determine changes in the signaling and downstream pathways of neuronal apoptosis at the cellular and molecular level. DESIGN, TIME AND SETTING： A randomized, controlled, cellular and molecular experiment was performed at the Department of Central Laboratory, Chengde Medical College from February to June 2008. MATERIALS： Astragalus injection, the main ingredient of astragaloside, was purchased from Chengdu Di＇ao Jiuhong Pharmaceutical Manufactory, China. JNK3 mRNA probe and in situ hybridization kit were purchased from Tianjin Haoyang Biological Technology, China, and JNK3 RT-PCR primers were designed by Shanghai Bio-engineering, China. METHODS： Primary cultures of hippocampal neurons derived from Sprague Dawley rats, aged 1 2 days, were established. After 8 days, the hippocampal neurons were assigned to the following interventions： model group, Astragalus group, and vehicle control group, cells were subjected to oxygen-glucose reintroduction after oxygen-glucose deprivation for 30 minutes in sugar-free Earle＇s solution and a hypoxia device, which contained high-purity nitrogen. The normal control group was subjected to primary culture techniques and was not treated using above-mentioned interventions. In addition, the Astragalus and vehicle control groups were treated with Astragalus injection （0.5 g/L raw drug） or sterile, deionized water at 2 hours prior to oxygen-glucose deprivation, respectively. MAIN OUTCOME MEASURES： JNK3 mRNA expression was measured by in situ hybridization and RT-PCR at 0, 0.5, 2, 6, 24, 72, and 120 hours after oxygen-glucose reintroduction. RESULTS： Hippocampal neuronal morphology was normal in the normal control group. Hippocampal neurons exhibited apparent apoptosis-like pathological changes in the model, as well as the vehicle control, groups. The apoptosis-like pathological changes in the hippocampal neurons were less in the Astragalus group. Results from in situ hybridization and RT-PCR showed that JNK3 mRNA expression significantly increased in hippocampal neurons from model group, as well as the vehicle control group, compared with the normal control group （P 〈 0.05）. In addition, JNK3 mRNA expression significantly decreased in hippocampal neurons of the Astragalus group, compared with the model group and vehicle control group （P 〈 0.05）. CONCLUSION： Astragalus injection inhibited apoptosis-related JNK3 mRNA expression following oxygen-glucose deprivation and reintroduction, and accordingly played a role in inhibiting hippocampal neuronal apoptosis.

Oxidative damage of primary cultured hippocampal neurons Does androgen have an antagonistic effect?

BACKGROUND： Evidence illustrates that androgen has a neuroprotective role. However, whether androgen also has the protective effect on hippocampal neurons during free radical mediated injury remains unclear. OBJECTIVE： To investigate the neuroprotective effect of androgen on hippocampal neurons during free radical damage. DESIGN, TIME AND SETTING： A controlled in vitro experiment was performed at the Department of Human Anatomy, Cell Culture Lab, and Neuroendocrinology Lab, Basic Medical School, Hebei Medical University from February to June 2009. MATERIALS： Testosterone was provided by Tianjin Jinyao Amino Acid Company, China. METHODS： Primary cultured neurons from 24 Sprague Dawley rats were randomly assigned into four groups： control, H202, testosterone, and testosterone （pre-added） plus H2O2 groups. MAIN OUTCOME MEASURES： The positive cell ratio of microtubule associated protein-Ⅱ and neuron specific enolase was determined by immunocytochemistry. Neuronal morphology was observed by hematoxylin-eosin staining and Nissl staining. Cell vitality and viability were determined using an inverted phase contrast microscope. The content of nitric oxide synthase, malondialdehyde, and superoxide dismutase were measured with a spectrophotometer. RESULTS： As compared with the control group, cell vitality and viability, and superoxide dismutase level were significantly decreased in the H202 group （P 〈 0.05）, while nitric oxide synthase and malondialdehyde levels were significantly increased （P 〈 0.05）. Neuronal vitality and viability as well as superoxide dismutase level in the testosterone plus H2O2 group were significantly greater than in the H2O2 group （P 〈 0.05）, and nitric oxide synthase and malondialdehyde levels were significantly less than in the H2O2 group （P〈 0.05）. CONCLUSION： Androgen partially reversed H2O2-induced neuronal damage and protected neurons.

Inhibition of TYRO3/Akt signaling participates in hypoxic injury in hippocampal neurons

In this study, we investigated the role of the TYRO3/Akt signaling pathway in hypoxic injury to hippocampal neurons. 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay showed that hypoxia inhibited the proliferation and viability of hippocampal neurons. Terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling assay demonstrated that hypoxia induced neuronal apoptosis in a time-dependent manner, with a greater number of apoptotic cells with longer hypoxic exposure. Immunofluorescence labeling revealed that hypoxia suppressed TYRO3 expression. Western blot assay showed that hypoxia decreased Akt phosphorylation levels in a time-dependent manner. Taken together, these findings suggest that hypoxia inhibits the proliferation of hippocampal neurons and promotes apoptosis, and that the inhibition of the TYRO3/Akt signaling pathway plays an important role in hypoxia-induced neuronal injury.

Selenocysteine antagonizes oxygen glucose deprivation-induced damage to hippocampal neurons

Designing and/or searching for novel antioxidants against oxygen glucose effective strategy for the treatment of human isdlemic stroke. Selenium is deprivation （OGD）-induced oxidative damage represents an an essential trace dement, which is beneficial in the chemo- prevention and chemotherapy of cerebral ischemic stroke. The underlying mechanisms for its therapeutic effects, however, are not well documented. Selenocysteine （SeC） is a selenium-containing amino acid with neuroprotective potential. Studies have shown that SeC can reduce irradiation-induced DNA apoptosis by reducing DNA damage. In this study, the in vitro protective potential and mechanism of action of SeC against OGD-induced apoptosis and neurotoxicity were evaluated in HT22 mouse hippocampal neurons. We cultured HT22 cells in a glucose-free medium containing 2 mM Na2S402, which formed an OGD environment, for 90 minutes. Findings from MTT, flow cytometry and TUNEL staining showed obvious cytotoxicity and apoptosis in HT22 cells in the OGD condition. The activation of Caspa se-7 and Caspase-9 further revealed that OGD-induced apoptosis of HT22 cells was mainly achieved by triggering a mitochondrial-medi- ated pathway. Moreover, the OGD condition also induced serious DNA damage through the accumulation of reactive oxygen species and superoxide anions. However, SeC pre-treatment for 6 hours effectively inhibited OGD-induced cytotoxicity and apoptosis in HT22 cells by inhibiting reactive oxygen species-mediated oxidative damage. Our findings provide evidence that SeC has the potential to suppress OGD-induced oxidative damage and apoptosis in hippocampal neurons.

ClC-3 chloride channel in hippocampal neuronal apoptosis

Over-production of nitric oxide is pathogenic for neuronal apoptosis around the ischemic area fol- lowing ischemic brain injury. In this study, an apoptotic model in rat hippocampal neurons was es- tablished by 0.5 mmol/L 3-morpholinosyndnomine （SIN-l）, a nitric oxide donor. The models were then cultured with 0.1 mmol/L of 4,4＇-diisothiocyanostilbene-2,2＇-disulfonic acid （DIDS; the chloride channel blocker）for 18 hours. Neuronal survival was detected using the 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, and apoptosis was assayed by Hoechst 33342-labeled neuronal DNA fluorescence staining. Western blot analysis and immunochemilumi- nescence staining were applied to determine the changes of activated caspase-3 and CIC-3 channel proteins. Real-time PCR was used to detect the mRNA expression of CIC-3. The results showed that SIN-1 reduced the neuronal survival rate, induced neuronal apoptosis, and promoted CIC-3 chloride channel protein and mRNA expression in the apoptotic neurons. DIDS reversed the effect of SIN-I. Our findings indicate that the increased activities of the CIC-3 chloride channel may be involved in hippocampal neuronal apoptosis induced by nitric oxide.

Influence of Ginkgo Biloba extract on beta-secretase in rat hippocampal neuronal cultures following chronic hypoxic and hypoglycemic conditions

BACKGROUND： Preparation of Ginkgo leaf has been widely used to improve cognitive deficits and dementia, in particular in Alzheirner＇s disease patients. However, the precise mechanism of action of Ginkgo leaf remains unclear. OBJECTIVE： To explore the effect of Ginkgo Biloba extract （Egb761）, Ginaton, on β -secretase expression in rat hippocampal neuronal cultures following chronic hypoxic and hypoglycemic conditions. DESIGN, TIME AND SETTNG： Completely by randomized, grouping study. The experiment was performed at the Laboratory of Molecular Imaging, Southeast University between August 2006 and August 2007. MATERIALS： A total of 128 Wistar rats aged 24 hours were selected, and hippocampal neurons were harvested for primary cultures. METHODS： On day 7, primary hippocampal neuronal cultures were treated with Egb761 （0, 25, 50, 100, 150, and 200μg/mL） under hypoxic/hypoglycemic or hypoglycemic culture conditions for 12, 24, and 36 hours, respectively. Hippocampal neurons cultured in primary culture medium served as control. MAIN OUTCOME MEASURES： Cell viability was assayed using 3-（4,5-Dimethylthiazol-2-yl）- 2,5-diphenyltetrazolium bromide （MTT）; fluorescence detection of β -secretase activity was performed; Western Blot was used to measure β -secretase expression. RESULTS： Cell viability under hypoxic/hypoglycemic or hypoglycemic culture conditions was significantly less than control cells （P 〈 0.05）. Under hypoxic/hypoglycemic or hypoglycemic culture conditions, treatment with 25 μg/mL Egb761 did not alter cell viability. However, 〉 25 μg/mL Egb761 induced greater cell viability （P 〈 0.05）. No differences were observed between hypoxic/hypoglycemic or hypoglycemic cells （P 〉 0.05）. α -secretase activity was increased after 12 hours in hypoxic/hypoglycemic culture （P 〈 0.01）. There were no significant differences between the 12-, 24-, or 36-hour Egb761 groups and the hypoxic/hypoglycemic groups （P 〉 0.05）. β -secretase activity was greater after 12, 24, and 36 hours in hypoxic/hypoglycemic culture conditions, compared with control conditions （P 〈 0.05）. β-secretase activity was significantly decreased in neurons treated with Egb761 for 12, 24, or 36 hours, compared with the hypoxichaypoglycemic group （P 〈 0.05）. β -secretase protein expression was significantly up-regulated in neurons cultured in hypoxic/hypoglycemic conditions for 12, 24, or 36 hours, compared to control cells （P 〈 0.05）, and was decreased compared to neurons treated with Egb761 （P 〈 0.05）. CONCLUSION： β -secretase expression and activity in rat neonatal hippocampal neurons were influenced by hypoxic and hypoglycemic culture. Egb761 played a protective role in hippocampal neurons damaged by chronic hypoxic and hypoglycemic culture conditions, possibly through its effect on β -secretase expression and activity.

Lead Can Inhibit NMDA-,K^+-,QA/KA-Induced Increases in Intracellular Free Ca^(2+) in Cultured Rat Hippocampal Neurons

Objective To examine the effects of Pb2+ on N-methyl-D-aspartate (NMDA)-, K+- and quisqualate(QA)/kainite(KA)-induced increases in intracellular free calcium concentration ([Ca2+],) in cultured fetal rat hippocampal neurons in order to explain the cognitive and learning deficits produced by this heavy metal. Methods Laser scanning confocal microscopy was used. Results The results clearly demonstrated that adding Pb2+ before or after NMDA/glycine stimulation selectively inhibited the stimulated increases in [Ca2+], in a concentration-dependent manner. In contrast, Pb2+ treatment did not markedly affect increases in [Ca2+], induced by an admixture of QA and KA. The minimal inhibitory effect of Pb2+ occurred at 1 μmol/L, and more than seventy percent abolition of the NMDA-stimulated increase in [Ca2+]; was observed at 100 Jμmoll/L Pb2+. Evaluation of Pb2+-induced increase in [Ca2+], response to elevating extracellular concentrations of NMDA, glycine or calcium revealed that Pb2+ was a noncompetitive antagonist of both NMDA and glycine, and a competitive antagonist of Ca2+ at NMDA receptor channels. In addition. Pb2+ inhibited depolarization-evoked increases in [Ca2+], mediated by K+ stimulation(30μmol/L). indicating that Pb2+ also depressed the voltage-dependent calcium channels. Also, the results showed that Pb2+ appeared to be able to elevate the resting levels of [Ca2+|, in cultured neurons, implying a reason for Pb2+-enhanced spontaneous release of several neurotransmitters reported in several previous studies. Conclusion Lead can inhibit NMDA-. K+-, QA/KA-jnduced increases in intracellular [Ca2+], in cultured hippocampal neurons.

MicroRNA-219 alleviates glutamate-induced neurotoxicity in cultured hippocampal neurons by targeting calmodulin-dependent protein kinase Ⅱ gamma

Septic encephalopathy is a frequent complication of sepsis,but there are few studies examining the role of micro RNAs（mi Rs） in its pathogenesis.In this study,a mi R-219 mimic was transfected into rat hippocampal neurons to model mi R-219 overexpression.A protective effect of mi R-219 was observed for glutamate-induced neurotoxicity of rat hippocampal neurons,and an underlying mechanism involving calmodulin-dependent protein kinase II γ（Ca MKIIγ） was demonstrated.mi R-219 and Ca MKIIγ m RNA expression induced by glutamate in hippocampal neurons was determined by quantitative real-time reverse transcription-polymerase chain reaction（q RT-PCR）.After neurons were transfected with mi R-219 mimic,effects on cell viability and apoptosis were measured by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide（MTT） assay and flow cytometry.In addition,a luciferase reporter gene system was used to confirm Ca MKIIγ as a target gene of mi R-219.Western blot assay and rescue experiments were also utilized to detect Ca MKIIγ expression and further verify that mi R-219 in hippocampal neurons exerted its effect through regulation of Ca MKIIγ.MTT assay and q RT-PCR results revealed obvious decreases in cell viability and mi R-219 expression after glutamate stimulation,while Ca MKIIγ m RNA expression was increased.MTT,flow cytometry,and caspase-3 activity assays showed that mi R-219 overexpression could elevate glutamate-induced cell viability,and reduce cell apoptosis and caspase-3 activity.Moreover,luciferase Ca MKIIγ-reporter activity was remarkably decreased by co-transfection with mi R-219 mimic,and the results of a rescue experiment showed that Ca MKIIγ overexpression could reverse the biological effects of mi R-219.Collectively,these findings verify that mi R-219 expression was decreased in glutamate-induced neurons,Ca MKIIγ was a target gene of mi R-219,and mi R-219 alleviated glutamate-induced neuronal excitotoxicity by negatively controlling Ca MKIIγ expression.

Dose-dependent effects of lead on cell-cycle arrest, DNA damage, and cyclin D1 expression in primary cultured rat hippocampal neurons

BACKGROUND： Previous studies have suggested that the hippocampus is one of the neurotoxic target sites for lead. However, the molecular mechanisms of action, including the effect of lead on cell-cycle arrest, remain poorly understood. OBJECTIVE： To investigate the effects of different lead concentrations on cell-cycle arrest, DNA damage, and cyclin D1 expression in primary cultured rat hippocampal neurons. DESIGN, TIME AND SETTING： A randomized, controlled, in vitro experiment was performed at the China Medical University between July 2008 and May 2009. MATERIALS： Antibodies specific to cyclin D1 and actin were synthesized and purified by Santa Cruz Biotechnology, USA. FACStar flow cytometer was purchased from Becton Dickinson, San Jose, California, USA. METHODS： Wistar rat hippocampal neurons were primary cultured for 7 days. Neurons in the control group were treated with 0.01 mol/L phosphate buffered saline. Neurons in the 0.2, 1.0, and 10 umol/L lead acetate groups were subjected to 0.2, 1.0, and 10 umol/L lead acetate. Subsequently hippocampal neurons in each group were cultured for 24 hours. MAIN OUTCOME MEASURES： The effects of lead on cell cycle were measured by flow cytometry, DNA damage was measured using the comet assay, and cyclin D1 expression was measured using Western blot analysis. RESULTS： Treatment of hippocampal neurons with 0.2 umol/L lead acetate did not significantly alter cell cycle phase distribution, i.e., sub-G1, S, G0/G1, G2/M, whereas treatment with 1.0 and 10 umol/L lead acetate significantly increased the percentage of S and sub-G1 phase cells （P 〈 0.05）. Olive tail moment in all lead-treated groups and the percentage of DNA in the tail in 1.0 umol/L and 10 umol/L lead acetate groups were significantly greater compared with the control group （P 〈 0.05）. In addition, the percentage of tail DNA was greater in the 0.2 umol/L lead acetate group compared with the control group （P 〉 0.05）. Following incubation with 0.2, 1.0, and 10 umol/L lead acetate for 24 hours, cyclin D1 expression gradually decreased with exposure to increasing lead acetate concentrations （1.0-10 umol/L）. CONCLUSION： Lead exposure to primary cultured rat hippocampal neurons resulted in dose-dependently disturbed cellular homeostasis, including DNA damage, reduced cyclin D1 expression, and stagnation of cell-cycle progression.

Gentianine protects hippocampal neurons in a rat model of recurrent febrile convulsion

Gentianine has been shown to have a protective effect on hippocampal CA1 neurons in rats subjected to recurrent febrile convulsion（FC）.The present study sought to explore the possible mechanism of gentianine by intraperitoneally injecting gentianine into rats with warm water-induced FC.The results revealed that neuronal organelle injury was slightly ameliorated in the hippocampal CA1 region.The level of glutamate was decreased,but the level of γ-aminobutyric acid was increased,as detected by ninhydrin staining.In addition,glutamate acid decarboxylase expression in hippocampal CA1 was increased,as determined by immunohistochemistry.The results demonstrated that gentianine can ameliorate FC-induced neuronal injury by enhancing glutamate acid decarboxylase activity,decreasing glutamate levels and increasing γ-aminobutyric acid levels.

Effect of Yiqi Bushen prescription on hippocampal neuronal apoptosis in diabetic rats

This study investigated the neuroprotective effect of Yiqi Bushen prescription （YQBS, supplementing qi and tonifying kidney） on neuronal cell apoptosis. Following YQBS treatment, the number of surviving hippocampal neurons increased, anti-apoptotic Bcl-2 expression increased and pro-apoptotic Bax expression decreased. In addition, diabetic rats exhibited improved learning and memory. YQBS treatment also increased Bcl-2 mRNA expression and the ratio of Bcl-2/Bax, but decreased levels of hypoxia-inducible factor-1α mRNA and Bax mRNA expression after high-glucose/hypoxia-induced injury. Results demonstrated that YQBS inhibited hippocampal neuronal apoptosis by decreasing hypoxia-inducible factor-1α expression and increasing Bcl-2 expression, thereby improving cognitive impairment in diabetic rats.

A Comparative Study About the Neuroprotective Effects of EPA-Enriched Phosphoethanolamine Plasmalogen and Phosphatidylethanolamine Against Oxidative Damage in Primary Hippocampal Neurons

s Oxidative stress is involved in the progression of neurodegenerative diseases.Previous evidences showed that plasma-logens could improve neurodegenerative diseases.In this study,we investigated the function of phosphoethanolamine plasmalogens enriched with EPA(EPA-pPE)and phosphatidylethanolamine enriched with EPA(EPA-PE)on oxidative damage prevention after hy-drogen peroxide(H2O2)and tert-butylhydroperoxide(t-BHP)challenge in primary hippocampal neurons.Results showed that neurons pretreated with EPA-pPE and EPA-PE demonstrated the ability to alleviate oxidative damage,which was proved by the in-creased cell viability.Moreover,the shape and number of neurons were more similar to those of the control group.Antioxidant acti-vity,apoptosis,as well as TrkB/ERK/CREB signaling pathway were investigated to explore the mechanisms.The results suggested that EPA-PE was superior to EPA-pPE in regulating mitochondrial apoptosis.EPA-pPE was more prominent than EPA-PE in upre-gulating TrkB/ERK/CREB signaling pathway.Phospholipids with EPA exerted neuroprotective effects via inhibiting oxidative stress,suppressing apoptosis,and regulating TrkB/ERK/CREB signaling pathway.Therefore,the results provide a scientific basis for utili-zation of phospholipids enriched with EPA on the treatment of neurodegenerative disease.

Effect of Thrombin on the Apoptosis of Hippocampal Neurons in vitro

Hippocampal neurons were treated by thrombin and thrombin receptor activatingpeptides (TRAP). Cell survival rate was decreased in a dose-dependent manner by MTT assay. Thenumbers of apoptotic cell and apoptotic rate of hippocampal neurons treated bydifferentconcentrations of thrombin were increased in a dose-dependent manner by terminal deoxynucleotidyltransferase (TdT) mediated dUTP-biotin nick end-labeling (TUNED method and Flow Cytometry. When theconcentration of thrombin is 40 U/mL, TUNEL positive cells and apoptotic rate of hippocampal neuronsreached peak value, were 27. 3 +- 4. 0 and (29. 333 +- 4. 633 ) % , respectively.Immunocytochemistry assay show that Bcl-2 protein expression was down- regulated and Bax proteinexpression was up-regulated with the concentration of thrombin increased. TRAP can mimic the effectof thrombin to induce apoptosis on hippocampal neurons. These data demonstrated that thrombininduced hippocampal neuron apoptosis in a dose-dependent manner through activatingprotease-acti-vated protein-1 (PAR-1). The change in expression of Bcl-2 and Bax was related withthe effect of high concentration thrombin induced apoptosis on hippocampal neurons.