BACKGROUND: Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and...BACKGROUND: Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE: To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN: A comparative experiment. SETTING: Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS: The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats (male or female) of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College (certification number was 021-97-03). METHODS: ① Preparation of cerebral cortical neurons of rats: The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons: After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages (1-20 g/L) was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③ Grouping: After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37 ℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours. ④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES: ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons; ② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining; ③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④ DNA agarose gel electrophoresis ladder-like strap appeared or not; ⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS: ① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons (P > 0.05). ② The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [(13.00±4.52)%,(12.72±2.15)%,(11.80±1.18)%,(38.03±1.05)%, P < 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture (36.77±1.45)%, (36.60±1.61)%, (36.37±2.02)%, (38.03±1.05)%, P > 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L (P < 0.01). Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation (P < 0.01). ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia; ⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased (P < 0.01), and those of Bax protein and Caspase-3 protein were reduced (P < 0.01), and the ratio of Bcl-2/Bax was increased (P < 0.01). CONCLUSION: Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.展开更多
The Sonic hedgehog (SHH) signaling pathway plays a pivotal role in neurogenesis and brain damage repair. Our previous work demonstrated that the SHH signaling pathway was involved in the neuroprotection of cortical ne...The Sonic hedgehog (SHH) signaling pathway plays a pivotal role in neurogenesis and brain damage repair. Our previous work demonstrated that the SHH signaling pathway was involved in the neuroprotection of cortical neurons against oxidative stress. The present study was aimed to further examine the underlying mechanism. The cortical neurons were obtained from one-day old Sprague-Dawley neonate rats. Hydrogen peroxide (H2O2, 100 μmol/L) was used to treat neurons for 24h to induce oxidative stress. Exogenous SHH (3μg/mL) was employed to activate the SHH pathway, and cyclopamine (20 μmol/L), a specific SHH signal inhibitor, to block SHH pathway. LY294002 (20 μmol/L) were used to pretreat the neurons 30 min before H2O2 treatment and selectively inhibit the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. The cell viability was measured by MTT and apoptosis rate by flow cytometry analysis. The expression of p38, p-p38, ERK, p-ERK, Akt, p-Akt, Bcl-2, and Bax in neurons was detected by immunoblotting. The results showed that as compared with H2O2 treatment, exogenous SHH could increase the expression of p-Akt by 20% and decrease the expression of p-ERK by 33%. SHH exerted no significant effect on p38 mitogen-activated protein kinase (p38 MAPK) pathway. Blockade of PI3K/Akt pathway by LY294002 decreased the cell viability by 17% and increased the cell apoptosis rate by 2-fold. LY294002 treatment could up-regulate the expression of the proapoptotic gene Bax by 12% and down-regulate the expression of the antiapoptotic gene Bcl-2 by 54%. In conclusion, SHH pathway may activate PI3K/Akt pathway and inhibit the activation of the ERK pathway in neurons under oxidative stress. The PI3K/Akt pathway plays a key role in the neuroprotection of SHH. SHH/PI3K/Bcl-2 pathway may be implicated in the protection of neurons against H2O2-induced apoptosis.展开更多
HECT, UBA and WWE domain-containing 1(Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, includin...HECT, UBA and WWE domain-containing 1(Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, including p53, Mcl-1, Cdc6 and N-myc, thereby playing a critical role in apoptosis and neurogenesis. However, the role of Huwe1 in brain ischemia and reperfusion injury remains unclear. Therefore, in this study, we investigated the role of Huwe1 in an in vitro model of ischemia and reperfusion injury. At 3 days in vitro, primary cortical neurons were transduced with a control or shRNA-Huwe1 lentiviral vector to silence expression of Huwe1. At 7 days in vitro, the cells were exposed to oxygen-glucose deprivation for 3 hours and reperfusion for 24 hours. To examine the role of the c-Jun N-terminal kinase(JNK)/p38 pathway, cortical neurons were pretreated with a JNK inhibitor(SP600125) or a p38 MAPK inhibitor(SB203508) for 30 minutes at 7 days in vitro, followed by ischemia and reperfusion. Neuronal apoptosis was assessed by TUNEL assay. Protein expression levels of JNK and p38 MAPK and of apoptosis-related proteins(p53, Gadd45 a, cleaved caspase-3, Bax and Bcl-2) were measured by western blot assay. Immunofluorescence labeling for cleaved caspase-3 was performed. We observed a significant increase in neuronal apoptosis and Huwe1 expression after ischemia and reperfusion. Treatment with the shRNA-Huwe1 lentiviral vector markedly decreased Huwe1 levels, and significantly decreased the number of TUNEL-positive cells after ischemia and reperfusion. The silencing vector also downregulated the pro-apoptotic proteins Bax and cleaved caspase-3, and upregulated the anti-apoptotic proteins Gadd45 a and Bcl-2. Silencing Huwe1 also significantly reduced p-JNK levels and increased p-p38 levels. Our findings show that downregulating Huwe1 affects the JNK and p38 MAPK signaling pathways as well as the expression of apoptosis-related genes to provide neuroprotection during ischemia and reperfusion. All animal experiments and procedures were approved by the Animal Ethics Committee of Sichuan University, China in January 2018(approval No. 2018013).展开更多
Brain-derived neurotrophic factor(BDNF)has robust effects on synaptogenesis,neuronal differentiation and synaptic transmission and plasticity.The maturation of BDNF is a complex process.Proprotein convertase 1/3(PC1/3...Brain-derived neurotrophic factor(BDNF)has robust effects on synaptogenesis,neuronal differentiation and synaptic transmission and plasticity.The maturation of BDNF is a complex process.Proprotein convertase 1/3(PC1/3)has a key role in the cleavage of protein precursors that are directed to regulated secretory pathways;however,it is not clear whether PC1/3 mediates the change in BDNF levels caused by ischemia.To clarify the role of PC1/3 in BDNF maturation in ischemic cortical neurons,primary cortical neurons from fetal rats were cultured in a humidified environment of 95%N_2 and 5%CO_2 in a glucose-free Dulbecco's modified Eagle's medium at 37℃for3 hours.Enzyme-linked immunosorbent assays and western blotting showed that after oxygen-glucose deprivation,the secreted and intracellular levels of BDNF were significantly reduced and the intracellular level of PC1/3 was decreased.Transient transfection of cortical neurons with a PC1/3 overexpression plasmid followed by oxygen-glucose deprivation resulted in increased PC1/3 levels and increased BDNF levels.When levels of the BDNF precursor protein were reduced,the concentration of BDNF in the culture medium was increased.These results indicate that PC 1/3 cleavage of BDNF is critical for the conversion of pro-BDNF in rat cortical neurons during ischemia.The study was approved by the Animal Ethics Committee of Wuhan University School of Basic Medical Sciences.展开更多
Objective To investigate the cytotoxic mechanism of cadmium(Cd) on cerebral cortical neurons.Methods The primary cultures of rat cerebral cortical neurons were treated with different concentrations of cadmium acetate(...Objective To investigate the cytotoxic mechanism of cadmium(Cd) on cerebral cortical neurons.Methods The primary cultures of rat cerebral cortical neurons were treated with different concentrations of cadmium acetate(0,5,10,and 20 μmol/L),and then the cell viability,apoptosis,ultrastructure,intracellular [Ca2+]i and reactive oxygen species(ROS) levels,mitochondrial membrane potential(ΔΨ),activities of catalase(CAT) and superoxide dismutase(SOD) were measured.Results A progressive loss in cell viability and an increased number of apoptotic cells were observed.In addition,Cd-induced apoptotic morphological changes in cerebral cortical neurons were also demonstrated by Hoechst 33258 staining.Meanwhile,ultrastructural changes were distortion of mitochondrial cristae and an unusual arrangement.Simultaneously,elevation of intracellular [Ca2+]i and ROS levels,depletion of ΔΨ were revealed in a dose-dependent manner during the exposure.Moreover,CAT and SOD activities in the living cells increased significantly.Conclusion Exposure of cortical neurons to different doses of Cd led to cellular death,mediated by an apoptotic mechanism,and the apoptotic death induced by oxidative stress may be a potential reason.And the disorder of intracellular homeostasis caused by oxidative stress and mitochondrial dysfunction may be a trigger for apoptosis in cortical neurons.展开更多
The iron chelator deferoxamine has been shown to inhibit ferroptosis in spinal cord injury.However,it is unclear whether deferoxamine directly protects neurons from ferroptotic cell death.By comparing the survival rat...The iron chelator deferoxamine has been shown to inhibit ferroptosis in spinal cord injury.However,it is unclear whether deferoxamine directly protects neurons from ferroptotic cell death.By comparing the survival rate and morphology of primary neurons and SH-SY5Y cells exposed to erastin,it was found that these cell types respond differentially to the duration and concentration of erastin treatment.Therefore,we studied the mechanisms of ferroptosis using primary cortical neurons from E16 mouse embryos.After treatment with 50μM erastin for 48 hours,reactive oxygen species levels increased,and the expression of the cystine/glutamate antiporter system light chain and glutathione peroxidase 4 decreased.Pretreatment with deferoxamine for 12 hours inhibited these changes,reduced cell death,and ameliorated cellular morphology.Pretreatment with the apoptosis inhibitor Z-DEVD-FMK or the necroptosis inhibitor necrostain-1 for 12 hours did not protect against erastin-induced ferroptosis.Only deferoxamine protected the primary cortical neurons from ferroptosis induced by erastin,confirming the specificity of the in vitro ferroptosis model.This study was approved by the Animal Ethics Committee at the Institute of Radiation Medicine of the Chinese Academy of Medical Sciences,China(approval No.DWLL-20180913)on September 13,2018.展开更多
BACKGROUND:Studies have reported the antioxidant effects of lead and cadmium in the central nervous system,but very few have addressed the combined toxicity of lead and cadmium.The mechanisms by which these combined h...BACKGROUND:Studies have reported the antioxidant effects of lead and cadmium in the central nervous system,but very few have addressed the combined toxicity of lead and cadmium.The mechanisms by which these combined heavy metals are toxic,as well as how to protect cells from these agents,remains poorly understood. OBJECTIVE:Primary cultured rat cortical neurons were used to determine the effects of combined lead and cadmium on levels of glutathione peroxidase(GSH-Px),superoxide dismutase(SOD), catalase(CAT),and acetylcholinesterase(AChE),as well as malondialdehyde(MDA),and to evaluate the neuroprotective effects of N-acetylcysteine(NAC). DESIGN,TIME AND SETTING:An in vitro toxicological observation was performed at the Comparative Medicine Center of Yangzhou University from August 2007 to April 2008. MATERIALS:Lead acetate,cadmium acetate,and NAC were purchased from Sigma-Aldrich(St. Louis,USA).Commercial kits of GSH-Px,SOD,CAT,AChE,and MDA were purchased from Nanjing Jiancheng Bioengineering Institute,Nanjing,China. METHODS:The cerebral cortical neurons were isolated from newborn Sprague dawley rats at 24 hours after birth and primary cultured for 6 days.Thereafter,the cells were treated with a range of cadmium doses(0,5.0,and 10.0μmol/L),lead doses(0,1.0,and 2.0μmol/L),or a combination of the two for 12 hours at 37℃in a 5%CO_2 incubator,respectively.In addition,the cells were incubated with different doses of cadmium and/or lead and(0 and 50μmol/L) NAC for 12 hours to assess the protective effects on cell survival. MAIN OUTCOME MEASURES:The activity of SOD,GSH-Px,CAT,and AChE,as well as MDA content,in the cell lysates was detected using commercial kits. RESULTS:At 12 hours after treatment,compared to the control group,activity of GSH-Px,SOD, and AChE in the lead,cadmium,or combined treated cells was significantly decreased with increasing doses of cadmium/or lead(P<0.05),but CAT activity and MDA levels were significantly increased(P<0.05).The combination of cadmium and lead led to higher levels of toxicity than individual exposure. CONCLUSION:The degree of oxidative damage increased when the two heavy metals were combined.NAC protected neonatal cortical neurons by increasing activity of anti-oxidative enzymes, and reducing lipid peroxidation,but the reduction was not statistically significant.展开更多
Estrogens and phytoestrogens have neuroprotective effect against neuronal damage induced by cerebral ischemia /reperfusion (I/R) injury. In preceding studies, the phytoestrogen effects have been assessed by administra...Estrogens and phytoestrogens have neuroprotective effect against neuronal damage induced by cerebral ischemia /reperfusion (I/R) injury. In preceding studies, the phytoestrogen effects have been assessed by administration previous to the ischemic period, conditions which are unusual to apply to the treatment of human stroke. Here we present a study on neuroprotection afforded by genistein on rat embryo cortical neurons subjected to oxygen and glucose deprivation (OGD) followed by re-oxigenation, when added after the stress stimulus. At 1 and 2 h of OGD times and after 24 h of reperfusion, cell viability, necrotic, apoptotic and autophagic cell death and different parameters related to oxidative stress and mitochondrial dysfunction were measured in the absence and presence of 1 μM genisteine. We found an in-creasing loss of neuronal viability after 1-5 h of OGD which was only reversed in part by 24 h of reperfusion. These changes were preceded by increases in ROS generation, caspase-3 activation, LDH release and increase in LC3B lipi-dation, indicative of autophagia. Treatment with 1 μM genistein during the 24 h reperfusion significantly attenuated neuronal necrosis and autophagia induced by 1 and 2 h of OGD exposure. Genistein also decreased ROS generation and lipid-peroxidation induced by 2 h of OGD. These results suggest an important neuroprotective effect of genistein against transient post-ischemic-like展开更多
To explore the roles of astrocytes in the epileptogenesis, astrocytes and neurons were isolated, purified and cultured in vitro from cerebral cortex of rats. The astrocytes were activated by ciliary neurotrophic facto...To explore the roles of astrocytes in the epileptogenesis, astrocytes and neurons were isolated, purified and cultured in vitro from cerebral cortex of rats. The astrocytes were activated by ciliary neurotrophic factor (CNTF) and astrocytic conditioned medium (ACM) was collected to treat neurons for 4, 8 and 12 h. By using Western blot, the expression of calmodulin dependent protein kinase Ⅱ (CaMKⅡ), inducible nitric oxide synthase (iNOS) and adenylate cyclase (AC) was de- tected in neurons. The results showed that the expression of CaMKⅡ, iNOS and AC was increased significantly in the neurons treated with ACM from 4 h to 12 h (P<0.05), and that of iNOS and AC peaked at 8 h and 12 h respectively. It was suggested that there might be some epileptogenic factors in the ACM and such signal pathways as NOS-NO-cGMP, Ca2+?CaM-CaMKⅡ and AC-cAMP-PKA might take part in the signal transduction of epileptogenesis.展开更多
Objective Low-intensity pulsed ultrasound(LIPUS) has been reported to enhance proliferation and to alter protein production in various kinds of cells.In the present study,we measured the neurites length after LIPUS tr...Objective Low-intensity pulsed ultrasound(LIPUS) has been reported to enhance proliferation and to alter protein production in various kinds of cells.In the present study,we measured the neurites length after LIPUS treatment to define the effectiveness of LIPUS stimulation on neurons,and then we examined the acticity of GSK-3βto study the intracellular mechanism of neurite's outgrowth.Methods LIPUS was applied to cultured primary rat cortical neurons for 5 minutes every day with spatial-and temporal average intensities(SATA) of 10 mW/cm^2,a pulse width of 200 microseconds,a repetition rate of 1.5 KHz,and an operation frequency of 1 MHz.Neurons were photographed on the third day after LIPUS treatment and harvested at third,seventh,and tenth days for immunoblot and semi-quantitative RT-PCR analysis.Results Morphology change showed that neurite extension was enhanced by LIPUS.There was also a remarkable decrease of proteins, including p-Akt,p-GSK-3β,and p-CRMP-2,observed on the seventh and tenth days,and of GSK-3βmRNA expression, observed on the seventh day,in neurons treated with LIPUS.Conclusion LIPUS can enhance elongation ofneurites and it is possible through the decreased expression of GSK-3β.展开更多
Berberine,a major constituent of Coptidis rhizoma,exhibits neural protective effects.The present study analyzed the potential protective effect of berberine against amyloid β-induced cytotoxicity in rat cerebral cort...Berberine,a major constituent of Coptidis rhizoma,exhibits neural protective effects.The present study analyzed the potential protective effect of berberine against amyloid β-induced cytotoxicity in rat cerebral cortical neurons.Alzheimer's disease cell models were treated with 0.5 and 2 μmol/L berberine for 36 hours to inhibit amyloid β-induced toxicity.Methyl thiazolyl tetrazolium assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining results showed that berberine significantly increased cell viability and reduced cell apoptosis in primary cultured rat cortical neurons.In addition,western blot analysis revealed a protective effect of berberine against amyloid β-induced toxicity in cultured cortical neurons,which coincided with significantly decreased abnormal up-regulation of activated caspase-3.These results showed that berberine exhibited a protective effect against amyloid β-induced cytotoxicity in cultured rat cortical neurons.展开更多
BACKGROUND:Numerous current studies have suggested that human telomerase reverse transcriptase(hTERT) gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses;however,the mechan...BACKGROUND:Numerous current studies have suggested that human telomerase reverse transcriptase(hTERT) gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses;however,the mechanism of action remains unknown. OBJECTIVE:To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 (Aβ_(25-35)). DESIGN,TIME AND SETTING:The randomized,controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research,Zhongshan School of Medicine,Sun Yat-sen University,China,from September 2005 to June 2008. MATERIALS:AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University,China.Human cortical neurons were derived from 12-20 week old aborted fetuses,obtained from the Guangzhou Maternal and Child Health Hospital,China.Mouse anti-Cdk5 and mouse anti-p16 monoclonal antibodies(Lab Vision,USA),and mouse anti-hTERT monoclonal antibody(Epitomics,USA),were used in this study. METHODS:(1) Recombinant adenovirus vectors,encoding hTERT(Ad-hTERT) and green fluorescent protein(Ad-GFP),were constructed using the AdEasy-1 Expression System.Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise,human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days.Human embryonic cortical neurons in the control group were cultured as normal.(2) Human embryonic cortical neurons in the Ad-hTERT group were treated with 10μmol/L Aβ_(25-35) for 24 hours.Normal human embryonic cortical neurons treated with 10μmol/LAβ_(25-35) for 24 hours served as a model group.Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ_(25-35). MAIN OUTCOME MEASURES:Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay.Telomerase activity was measured using a PcR-based telomeric repeat amplification protocol(TRAP) ELISA kit.Neural activity in human embryonic cortical neurons was examined by MTT assay;apoptosis was measured using TUNEL assay;and Cdk5 and p16 protein expressions were measured by Westem blot. RESULTS:Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group.No hTERT expression was detected in the Ad-GFP and control groups.Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups(P<0.01).Compared with the control group,cell activity was significantly decreased(P<0.05),and cell apoptotic rate,Cdk5 and p16 expression were significantly increased(P<0.01) in the model group.Compared with the model group,cell activity was increased in the Ad-hTERT group,and peaked at day 3 post-transfection(P<0.05). Neuroprotective effects also peaked at day 3 post-transfection;and the apoptotic rate,Cdk5 and p16 expression significantly decreased(P<0.01). CONCLUSION:Expression of hTERT in human embryonic cortical neurons can relieve Aβ_(25-35)-induced neuronal apoptosis.The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.展开更多
The aim of this study was to find a way to efficiently separate neuronal cells from the cerebral cortex of adult rats,providing a reference method for rapid acquisition of neuronal cells from the adult rat brain.Fifte...The aim of this study was to find a way to efficiently separate neuronal cells from the cerebral cortex of adult rats,providing a reference method for rapid acquisition of neuronal cells from the adult rat brain.Fifteen SD rats were randomly divided into three groups,with five SD rats in each group.Then,neuron cells were isolated from the adult rat cerebral cortex by the grinding method,the trypsin method,and the collagenase II method,respectively.The expression of anti-NeuN in the neurons of each group was analyzed by flow cytometry.The acquisition rates and morphology of neurons of each group were observed by immunofluorescence staining.The grinding or collagenase II method is more suitable for rapid acquisition of neuronal cells from an adult rat’s cerebral cortex.The number of neuron cells obtained by the trypsin method were very few,so it is not convenient for later experiments.展开更多
Advanced glycation end products lead to cell apoptosis, and cause cell death by increasing endoplasmic reticulum stress. Advanced glycation end products alone may also directly cause damage to tissues and cells, but t...Advanced glycation end products lead to cell apoptosis, and cause cell death by increasing endoplasmic reticulum stress. Advanced glycation end products alone may also directly cause damage to tissues and cells, but the precise mechanism remains unknown. This study used primary cultures of rat cerebral cortex neurons, and treated cells with different concentrations of glycation end products (50, 100, 200, 400 mg/L), and with an antibody for the receptor of advanced glycation end products before and after treatment with advanced glycation end products. The results showed that with increasing concentrations of glycation end products, free radical content increased in neurons, and the number of apoptotic cells increased in a dose-dependent manner. Before and after treatment of advanced glycation end products, the addition of the antibody against advanced glycation end-products markedly reduced hydroxyl free radicals, malondialdehyde levels, and inhibited cell apoptosis. This result indicated that the antibody for receptor of advanced glycation end-products in neurons from the rat cerebral cortex can reduce glycation end product-induced oxidative stress damage by suppressing glycation end product receptors. Overall, our study confirms that the advanced glycation end products-advanced glycation end products receptor pathway may be the main signaling pathway leading to neuronal damage.展开更多
Interleukin-6 has been shown to be involved in nerve injury and nerve regeneration, but the effects of long-term administration of high concentrations of interleukin-6 on neurons in the central nervous system is poorl...Interleukin-6 has been shown to be involved in nerve injury and nerve regeneration, but the effects of long-term administration of high concentrations of interleukin-6 on neurons in the central nervous system is poorly understood. This study investigated the effects of 24 hour exposure of interleukin-6 on cortical neurons at various concentrations(0.1, 1, 5 and 10 ng/m L) and the effects of 10 ng/m L interleukin-6 exposure to cortical neurons for various durations(2, 4, 8, 24 and 48 hours) by studying voltage-gated Na+ channels using a patch-clamp technique. Voltage-clamp recording results demonstrated that interleukin-6 suppressed Na+ currents through its receptor in a time- and dose-dependent manner, but did not alter voltage-dependent activation and inactivation. Current-clamp recording results were consistent with voltage-clamp recording results. Interleukin-6 reduced the action potential amplitude of cortical neurons, but did not change the action potential threshold. The regulation of voltage-gated Na+ channels in rat cortical neurons by interleukin-6 is time- and dose-dependent.展开更多
The present study established a model of focal cerebral ischemia through heat-coagulation induced occlusion of the middle cerebral artery. Following electroacupuncture at Baihui (GV20) and Dazhui (GV14), or intracereb...The present study established a model of focal cerebral ischemia through heat-coagulation induced occlusion of the middle cerebral artery. Following electroacupuncture at Baihui (GV20) and Dazhui (GV14), or intracerebroventricular infusion of AG490, a Janus kinase 2 phosphorylation inhibitor, the amount of necrotic or degenerated neurons in the ischemic cerebral cortex decreased, neuronal swelling was ameliorated, and expression of phosphorylated Janus kinase 2 and signal transducer and activator of transcription 3 decreased. Results confirmed that electroacupuncture promoted neuronal repair in the cerebral cortex by reducing expression of phosphorylated Janus kinase 2 and signal transducer and activator of transcription 3, as well as weakening the phosphorylated activation, thereby blocking abnormal activation of the Janus kinase 2- signal transducer and activator of transcription 3 signal transduction pathway.展开更多
Aim This study is aimed to determine whether SPK2 （Sphingosine kinase-2） is involved in isoflurane preconditioning induced autophagy activation in primary cultured neurons. We also examined whether SPK2 pro- tects n...Aim This study is aimed to determine whether SPK2 （Sphingosine kinase-2） is involved in isoflurane preconditioning induced autophagy activation in primary cultured neurons. We also examined whether SPK2 pro- tects neurons from ischemic injury by activating autophagy, and explored the molecular mechanism of SPK2 contrib- uting to the autophagy activation in neurons. Methods Isoflurane preconditioning （ISO） and oxygen glucose dep- rivation （OGD） model was established in primary cultured murine cortical neurons. Neurons were transfected by siRNA to interfere SPK2 and Beclin 1, or lentivirus to overexpress SPK2. Protein expression of SPK2, LC3, and Beclinl were detected with immunofluorescence and Western blot analysis. The neurons were treated with lysosomal inhibitor ammonium chloride （NH4 C1） to test the autophagy flux. The protection of SPK2 on OGD/R induced neu- ronal death was detected with CCK-8 （cell counting kit-8） and LDH cytotoxicity assay kit. Autophagy inhibitor 3- MA （3-Methyladenine） was used to detect the protection of autophagy on SPK2 induced isehemie tolerance. Co-im- munoprecipitation was used to detect the interaction between Beclin 1 and Bel-2. Results In primary cultured neu- ISO enhanced SPK2 and LC3 immunofluorescenee. SPK2 siRNA inhibits LC3II upregulation induced by rons, ISO. Beclin 1 siRNA also inhibits LC3II upregulation induced by ISO. Lentivirus-indueed SPK2 overexpression in- creased LC3II/LC3I ratio and enhanced the autophagy flux in neurons. SPK2 overexpression also exerted neuropro- teetion against OGD model in cortical neurons, as evidenced by improvement of neuronal morphology, increased cellular viability and reduced LDH leakage, while 3-MA partly abolished the SPK2-induced neuroprotection. After SPK2 overexpression, Beclin 1 siRNA inhibited SPK2 induced LC3II upregulation, and the coimmunoprecipitation of Beclin 1 and Bcl-2 was reduced.. Conclusion ISO increases SPK2 and activates autophagy in neurons. SPK2 or Beclin 1 interference cancels ISO induced autophagy activation. SPK2 overexpression activates autophagy, and protects the neurons against ischemic injury. SPK2 may induce autophagy by disrupting Beclin 1/Bcl-2 interaction.展开更多
OBJECTIVE To explore the effect of icariside Ⅱ(ICS Ⅱ) on oxygen-glucose deprivation and reoxygenation(OGD/R)-induced injury in cerebral cortical neuronal cels.METHODS Primary cerebral cortical neuronal cells were de...OBJECTIVE To explore the effect of icariside Ⅱ(ICS Ⅱ) on oxygen-glucose deprivation and reoxygenation(OGD/R)-induced injury in cerebral cortical neuronal cels.METHODS Primary cerebral cortical neuronal cells were deprived of oxygen and glucose for 2 h to simulate ischemic stroke injury in vitro.The experiment was divided into 8 groups,which were control,control+ICSⅡ 25 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ(6.25,12.5,25 μmol·L^(-1)),OGD/R+3-methyladenine(3-MA) and OGD/R+Rapamycin(Rap).The protective effect of ICS Ⅱ were detected by MTT assay and lactate dehydrogenase(LDH),respectively.Autophagic flux and autophagy related proteins expressions were detected by using adenovirus harboring tf-LC3 and Western blotting,respectively.RESULTS Compared with OGD/R group,the cell viability treated with ICSⅡwas elevated in a concentration-dependent manner,and the leakage rate of LDH was lowed.Moreover,ICSⅡ not only suppressed OGD/R-induced autophagic flux,but also inhibited the increase of LC3-Ⅱ/LC3-Ⅰ ratio and Beclin 1 after OGD/R insulted.CONCLUSION ICS Ⅱ exerts protective effects on OGD/R-induced cerebral cortical neuronal cells through inhibiting excessive autophagy.展开更多
In sensorimotor cortical slices of guinea pig using local iontophoretic application of glutamate to the soma and dendrites it was found that a decrease of temperature of incubating fluid from 34 to 21°C - 22°...In sensorimotor cortical slices of guinea pig using local iontophoretic application of glutamate to the soma and dendrites it was found that a decrease of temperature of incubating fluid from 34 to 21°C - 22°C changes the somatic responses to the local injection of glutamate to the dendritic loci, while the responses to iontophoretic application of glutamate to the soma remain unchanged. Hypothermic changes in reactivity to dendritic stimulation start below 30°C and coincide with changes in the spontaneous activity of neurons, both in the direction of increasing and decreasing the frequency of firing in different nerve cells. On hypothermic decrease of spontaneous activity, the latencies of evoked dendritic responses on the soma became more longer, while on hypothermic increase of firing level, somatic spike responses to iontophoretic application of glutamate to dendritic loci appeared with shorter latencies. Hypothermic changes in the physiological parameters of neurons were accompanied by a drop in spike amplitude at the same temperature and with its further decrease. At the same time, there was a decrease of spike reaction to iontophoretic application of acetylcholine below 30°. It is proposed that the reason for hypothermic changes of neuronal activity is decreasing rate of M-cholinergic process at 27°C - 29°C which leads to opening K<sup>+</sup> channels of neuronal membranes and hence to attenuation of conductive function of dendrites and to imbalance of K<sup>+</sup> ion homeostasis. Peculiarities of hypothermic regulation of neuronal spike activity depend on individual functional properties of cortical neurons.展开更多
文摘BACKGROUND: Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE: To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN: A comparative experiment. SETTING: Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS: The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats (male or female) of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College (certification number was 021-97-03). METHODS: ① Preparation of cerebral cortical neurons of rats: The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons: After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages (1-20 g/L) was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③ Grouping: After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37 ℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours. ④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES: ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons; ② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining; ③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④ DNA agarose gel electrophoresis ladder-like strap appeared or not; ⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS: ① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons (P > 0.05). ② The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [(13.00±4.52)%,(12.72±2.15)%,(11.80±1.18)%,(38.03±1.05)%, P < 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture (36.77±1.45)%, (36.60±1.61)%, (36.37±2.02)%, (38.03±1.05)%, P > 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L (P < 0.01). Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation (P < 0.01). ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia; ⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased (P < 0.01), and those of Bax protein and Caspase-3 protein were reduced (P < 0.01), and the ratio of Bcl-2/Bax was increased (P < 0.01). CONCLUSION: Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.
基金supported by a grant from the National Natural Science Foundation of China(No.81070938)
文摘The Sonic hedgehog (SHH) signaling pathway plays a pivotal role in neurogenesis and brain damage repair. Our previous work demonstrated that the SHH signaling pathway was involved in the neuroprotection of cortical neurons against oxidative stress. The present study was aimed to further examine the underlying mechanism. The cortical neurons were obtained from one-day old Sprague-Dawley neonate rats. Hydrogen peroxide (H2O2, 100 μmol/L) was used to treat neurons for 24h to induce oxidative stress. Exogenous SHH (3μg/mL) was employed to activate the SHH pathway, and cyclopamine (20 μmol/L), a specific SHH signal inhibitor, to block SHH pathway. LY294002 (20 μmol/L) were used to pretreat the neurons 30 min before H2O2 treatment and selectively inhibit the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. The cell viability was measured by MTT and apoptosis rate by flow cytometry analysis. The expression of p38, p-p38, ERK, p-ERK, Akt, p-Akt, Bcl-2, and Bax in neurons was detected by immunoblotting. The results showed that as compared with H2O2 treatment, exogenous SHH could increase the expression of p-Akt by 20% and decrease the expression of p-ERK by 33%. SHH exerted no significant effect on p38 mitogen-activated protein kinase (p38 MAPK) pathway. Blockade of PI3K/Akt pathway by LY294002 decreased the cell viability by 17% and increased the cell apoptosis rate by 2-fold. LY294002 treatment could up-regulate the expression of the proapoptotic gene Bax by 12% and down-regulate the expression of the antiapoptotic gene Bcl-2 by 54%. In conclusion, SHH pathway may activate PI3K/Akt pathway and inhibit the activation of the ERK pathway in neurons under oxidative stress. The PI3K/Akt pathway plays a key role in the neuroprotection of SHH. SHH/PI3K/Bcl-2 pathway may be implicated in the protection of neurons against H2O2-induced apoptosis.
基金supported by the National Natural Science Foundation of China,No.81771642(to WMX)the New Bud Research Foundation of West China Second University Hospital of China(to GQH)
文摘HECT, UBA and WWE domain-containing 1(Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, including p53, Mcl-1, Cdc6 and N-myc, thereby playing a critical role in apoptosis and neurogenesis. However, the role of Huwe1 in brain ischemia and reperfusion injury remains unclear. Therefore, in this study, we investigated the role of Huwe1 in an in vitro model of ischemia and reperfusion injury. At 3 days in vitro, primary cortical neurons were transduced with a control or shRNA-Huwe1 lentiviral vector to silence expression of Huwe1. At 7 days in vitro, the cells were exposed to oxygen-glucose deprivation for 3 hours and reperfusion for 24 hours. To examine the role of the c-Jun N-terminal kinase(JNK)/p38 pathway, cortical neurons were pretreated with a JNK inhibitor(SP600125) or a p38 MAPK inhibitor(SB203508) for 30 minutes at 7 days in vitro, followed by ischemia and reperfusion. Neuronal apoptosis was assessed by TUNEL assay. Protein expression levels of JNK and p38 MAPK and of apoptosis-related proteins(p53, Gadd45 a, cleaved caspase-3, Bax and Bcl-2) were measured by western blot assay. Immunofluorescence labeling for cleaved caspase-3 was performed. We observed a significant increase in neuronal apoptosis and Huwe1 expression after ischemia and reperfusion. Treatment with the shRNA-Huwe1 lentiviral vector markedly decreased Huwe1 levels, and significantly decreased the number of TUNEL-positive cells after ischemia and reperfusion. The silencing vector also downregulated the pro-apoptotic proteins Bax and cleaved caspase-3, and upregulated the anti-apoptotic proteins Gadd45 a and Bcl-2. Silencing Huwe1 also significantly reduced p-JNK levels and increased p-p38 levels. Our findings show that downregulating Huwe1 affects the JNK and p38 MAPK signaling pathways as well as the expression of apoptosis-related genes to provide neuroprotection during ischemia and reperfusion. All animal experiments and procedures were approved by the Animal Ethics Committee of Sichuan University, China in January 2018(approval No. 2018013).
基金supported by the National Nature Science Foundation of China,No.81501053(to YC)
文摘Brain-derived neurotrophic factor(BDNF)has robust effects on synaptogenesis,neuronal differentiation and synaptic transmission and plasticity.The maturation of BDNF is a complex process.Proprotein convertase 1/3(PC1/3)has a key role in the cleavage of protein precursors that are directed to regulated secretory pathways;however,it is not clear whether PC1/3 mediates the change in BDNF levels caused by ischemia.To clarify the role of PC1/3 in BDNF maturation in ischemic cortical neurons,primary cortical neurons from fetal rats were cultured in a humidified environment of 95%N_2 and 5%CO_2 in a glucose-free Dulbecco's modified Eagle's medium at 37℃for3 hours.Enzyme-linked immunosorbent assays and western blotting showed that after oxygen-glucose deprivation,the secreted and intracellular levels of BDNF were significantly reduced and the intracellular level of PC1/3 was decreased.Transient transfection of cortical neurons with a PC1/3 overexpression plasmid followed by oxygen-glucose deprivation resulted in increased PC1/3 levels and increased BDNF levels.When levels of the BDNF precursor protein were reduced,the concentration of BDNF in the culture medium was increased.These results indicate that PC 1/3 cleavage of BDNF is critical for the conversion of pro-BDNF in rat cortical neurons during ischemia.The study was approved by the Animal Ethics Committee of Wuhan University School of Basic Medical Sciences.
基金supported by the National Nature Science Foundation of China (no.30972229 and 31101866)a project Funded by Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)
文摘Objective To investigate the cytotoxic mechanism of cadmium(Cd) on cerebral cortical neurons.Methods The primary cultures of rat cerebral cortical neurons were treated with different concentrations of cadmium acetate(0,5,10,and 20 μmol/L),and then the cell viability,apoptosis,ultrastructure,intracellular [Ca2+]i and reactive oxygen species(ROS) levels,mitochondrial membrane potential(ΔΨ),activities of catalase(CAT) and superoxide dismutase(SOD) were measured.Results A progressive loss in cell viability and an increased number of apoptotic cells were observed.In addition,Cd-induced apoptotic morphological changes in cerebral cortical neurons were also demonstrated by Hoechst 33258 staining.Meanwhile,ultrastructural changes were distortion of mitochondrial cristae and an unusual arrangement.Simultaneously,elevation of intracellular [Ca2+]i and ROS levels,depletion of ΔΨ were revealed in a dose-dependent manner during the exposure.Moreover,CAT and SOD activities in the living cells increased significantly.Conclusion Exposure of cortical neurons to different doses of Cd led to cellular death,mediated by an apoptotic mechanism,and the apoptotic death induced by oxidative stress may be a potential reason.And the disorder of intracellular homeostasis caused by oxidative stress and mitochondrial dysfunction may be a trigger for apoptosis in cortical neurons.
基金supported by the National Natural Science Foundation of China,Nos.81672171(to XY),81620108018(to SQF),81772342(to GZN)the State Key Laboratory of Medicinal Chemical Biology of Nankai University of China,No.2017027(to XY)
文摘The iron chelator deferoxamine has been shown to inhibit ferroptosis in spinal cord injury.However,it is unclear whether deferoxamine directly protects neurons from ferroptotic cell death.By comparing the survival rate and morphology of primary neurons and SH-SY5Y cells exposed to erastin,it was found that these cell types respond differentially to the duration and concentration of erastin treatment.Therefore,we studied the mechanisms of ferroptosis using primary cortical neurons from E16 mouse embryos.After treatment with 50μM erastin for 48 hours,reactive oxygen species levels increased,and the expression of the cystine/glutamate antiporter system light chain and glutathione peroxidase 4 decreased.Pretreatment with deferoxamine for 12 hours inhibited these changes,reduced cell death,and ameliorated cellular morphology.Pretreatment with the apoptosis inhibitor Z-DEVD-FMK or the necroptosis inhibitor necrostain-1 for 12 hours did not protect against erastin-induced ferroptosis.Only deferoxamine protected the primary cortical neurons from ferroptosis induced by erastin,confirming the specificity of the in vitro ferroptosis model.This study was approved by the Animal Ethics Committee at the Institute of Radiation Medicine of the Chinese Academy of Medical Sciences,China(approval No.DWLL-20180913)on September 13,2018.
基金the National Natural Science Foundation of China,No.305713647Natural Science Foundation of Jiangsu Province,No. BK2008214
文摘BACKGROUND:Studies have reported the antioxidant effects of lead and cadmium in the central nervous system,but very few have addressed the combined toxicity of lead and cadmium.The mechanisms by which these combined heavy metals are toxic,as well as how to protect cells from these agents,remains poorly understood. OBJECTIVE:Primary cultured rat cortical neurons were used to determine the effects of combined lead and cadmium on levels of glutathione peroxidase(GSH-Px),superoxide dismutase(SOD), catalase(CAT),and acetylcholinesterase(AChE),as well as malondialdehyde(MDA),and to evaluate the neuroprotective effects of N-acetylcysteine(NAC). DESIGN,TIME AND SETTING:An in vitro toxicological observation was performed at the Comparative Medicine Center of Yangzhou University from August 2007 to April 2008. MATERIALS:Lead acetate,cadmium acetate,and NAC were purchased from Sigma-Aldrich(St. Louis,USA).Commercial kits of GSH-Px,SOD,CAT,AChE,and MDA were purchased from Nanjing Jiancheng Bioengineering Institute,Nanjing,China. METHODS:The cerebral cortical neurons were isolated from newborn Sprague dawley rats at 24 hours after birth and primary cultured for 6 days.Thereafter,the cells were treated with a range of cadmium doses(0,5.0,and 10.0μmol/L),lead doses(0,1.0,and 2.0μmol/L),or a combination of the two for 12 hours at 37℃in a 5%CO_2 incubator,respectively.In addition,the cells were incubated with different doses of cadmium and/or lead and(0 and 50μmol/L) NAC for 12 hours to assess the protective effects on cell survival. MAIN OUTCOME MEASURES:The activity of SOD,GSH-Px,CAT,and AChE,as well as MDA content,in the cell lysates was detected using commercial kits. RESULTS:At 12 hours after treatment,compared to the control group,activity of GSH-Px,SOD, and AChE in the lead,cadmium,or combined treated cells was significantly decreased with increasing doses of cadmium/or lead(P<0.05),but CAT activity and MDA levels were significantly increased(P<0.05).The combination of cadmium and lead led to higher levels of toxicity than individual exposure. CONCLUSION:The degree of oxidative damage increased when the two heavy metals were combined.NAC protected neonatal cortical neurons by increasing activity of anti-oxidative enzymes, and reducing lipid peroxidation,but the reduction was not statistically significant.
文摘Estrogens and phytoestrogens have neuroprotective effect against neuronal damage induced by cerebral ischemia /reperfusion (I/R) injury. In preceding studies, the phytoestrogen effects have been assessed by administration previous to the ischemic period, conditions which are unusual to apply to the treatment of human stroke. Here we present a study on neuroprotection afforded by genistein on rat embryo cortical neurons subjected to oxygen and glucose deprivation (OGD) followed by re-oxigenation, when added after the stress stimulus. At 1 and 2 h of OGD times and after 24 h of reperfusion, cell viability, necrotic, apoptotic and autophagic cell death and different parameters related to oxidative stress and mitochondrial dysfunction were measured in the absence and presence of 1 μM genisteine. We found an in-creasing loss of neuronal viability after 1-5 h of OGD which was only reversed in part by 24 h of reperfusion. These changes were preceded by increases in ROS generation, caspase-3 activation, LDH release and increase in LC3B lipi-dation, indicative of autophagia. Treatment with 1 μM genistein during the 24 h reperfusion significantly attenuated neuronal necrosis and autophagia induced by 1 and 2 h of OGD exposure. Genistein also decreased ROS generation and lipid-peroxidation induced by 2 h of OGD. These results suggest an important neuroprotective effect of genistein against transient post-ischemic-like
基金a grant from Doctor Point Foundation of Ministry of Education of China (No. 2004 0487060)
文摘To explore the roles of astrocytes in the epileptogenesis, astrocytes and neurons were isolated, purified and cultured in vitro from cerebral cortex of rats. The astrocytes were activated by ciliary neurotrophic factor (CNTF) and astrocytic conditioned medium (ACM) was collected to treat neurons for 4, 8 and 12 h. By using Western blot, the expression of calmodulin dependent protein kinase Ⅱ (CaMKⅡ), inducible nitric oxide synthase (iNOS) and adenylate cyclase (AC) was de- tected in neurons. The results showed that the expression of CaMKⅡ, iNOS and AC was increased significantly in the neurons treated with ACM from 4 h to 12 h (P<0.05), and that of iNOS and AC peaked at 8 h and 12 h respectively. It was suggested that there might be some epileptogenic factors in the ACM and such signal pathways as NOS-NO-cGMP, Ca2+?CaM-CaMKⅡ and AC-cAMP-PKA might take part in the signal transduction of epileptogenesis.
基金supported by Natural Science Foundation of Beijing,ChinaGrant number:5072020.
文摘Objective Low-intensity pulsed ultrasound(LIPUS) has been reported to enhance proliferation and to alter protein production in various kinds of cells.In the present study,we measured the neurites length after LIPUS treatment to define the effectiveness of LIPUS stimulation on neurons,and then we examined the acticity of GSK-3βto study the intracellular mechanism of neurite's outgrowth.Methods LIPUS was applied to cultured primary rat cortical neurons for 5 minutes every day with spatial-and temporal average intensities(SATA) of 10 mW/cm^2,a pulse width of 200 microseconds,a repetition rate of 1.5 KHz,and an operation frequency of 1 MHz.Neurons were photographed on the third day after LIPUS treatment and harvested at third,seventh,and tenth days for immunoblot and semi-quantitative RT-PCR analysis.Results Morphology change showed that neurite extension was enhanced by LIPUS.There was also a remarkable decrease of proteins, including p-Akt,p-GSK-3β,and p-CRMP-2,observed on the seventh and tenth days,and of GSK-3βmRNA expression, observed on the seventh day,in neurons treated with LIPUS.Conclusion LIPUS can enhance elongation ofneurites and it is possible through the decreased expression of GSK-3β.
基金the Key Project of Ministry of Education of China,No. 208009the National Science & Technology Major Project "Key New Drug Creation and Manufacturing Program",China,No. 2009ZX09308-005
文摘Berberine,a major constituent of Coptidis rhizoma,exhibits neural protective effects.The present study analyzed the potential protective effect of berberine against amyloid β-induced cytotoxicity in rat cerebral cortical neurons.Alzheimer's disease cell models were treated with 0.5 and 2 μmol/L berberine for 36 hours to inhibit amyloid β-induced toxicity.Methyl thiazolyl tetrazolium assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining results showed that berberine significantly increased cell viability and reduced cell apoptosis in primary cultured rat cortical neurons.In addition,western blot analysis revealed a protective effect of berberine against amyloid β-induced toxicity in cultured cortical neurons,which coincided with significantly decreased abnormal up-regulation of activated caspase-3.These results showed that berberine exhibited a protective effect against amyloid β-induced cytotoxicity in cultured rat cortical neurons.
基金the National Key Basic Research Program of China,No. 2006cb500700the National Natural Science Foundation of China,No.30470904the Natural Science and Technology Foundation of Guangdong Province,No. 04009356, 2008B030301320
文摘BACKGROUND:Numerous current studies have suggested that human telomerase reverse transcriptase(hTERT) gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses;however,the mechanism of action remains unknown. OBJECTIVE:To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 (Aβ_(25-35)). DESIGN,TIME AND SETTING:The randomized,controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research,Zhongshan School of Medicine,Sun Yat-sen University,China,from September 2005 to June 2008. MATERIALS:AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University,China.Human cortical neurons were derived from 12-20 week old aborted fetuses,obtained from the Guangzhou Maternal and Child Health Hospital,China.Mouse anti-Cdk5 and mouse anti-p16 monoclonal antibodies(Lab Vision,USA),and mouse anti-hTERT monoclonal antibody(Epitomics,USA),were used in this study. METHODS:(1) Recombinant adenovirus vectors,encoding hTERT(Ad-hTERT) and green fluorescent protein(Ad-GFP),were constructed using the AdEasy-1 Expression System.Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise,human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days.Human embryonic cortical neurons in the control group were cultured as normal.(2) Human embryonic cortical neurons in the Ad-hTERT group were treated with 10μmol/L Aβ_(25-35) for 24 hours.Normal human embryonic cortical neurons treated with 10μmol/LAβ_(25-35) for 24 hours served as a model group.Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ_(25-35). MAIN OUTCOME MEASURES:Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay.Telomerase activity was measured using a PcR-based telomeric repeat amplification protocol(TRAP) ELISA kit.Neural activity in human embryonic cortical neurons was examined by MTT assay;apoptosis was measured using TUNEL assay;and Cdk5 and p16 protein expressions were measured by Westem blot. RESULTS:Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group.No hTERT expression was detected in the Ad-GFP and control groups.Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups(P<0.01).Compared with the control group,cell activity was significantly decreased(P<0.05),and cell apoptotic rate,Cdk5 and p16 expression were significantly increased(P<0.01) in the model group.Compared with the model group,cell activity was increased in the Ad-hTERT group,and peaked at day 3 post-transfection(P<0.05). Neuroprotective effects also peaked at day 3 post-transfection;and the apoptotic rate,Cdk5 and p16 expression significantly decreased(P<0.01). CONCLUSION:Expression of hTERT in human embryonic cortical neurons can relieve Aβ_(25-35)-induced neuronal apoptosis.The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.
基金the National Natural Science Foundation of China(No.81960129)Qinghai basic Research Plan Project(No.2019-ZJ-922)Middle-aged and Youth Foundation of Qinghai university affiliated hospital(No.2018-QYY-13).
文摘The aim of this study was to find a way to efficiently separate neuronal cells from the cerebral cortex of adult rats,providing a reference method for rapid acquisition of neuronal cells from the adult rat brain.Fifteen SD rats were randomly divided into three groups,with five SD rats in each group.Then,neuron cells were isolated from the adult rat cerebral cortex by the grinding method,the trypsin method,and the collagenase II method,respectively.The expression of anti-NeuN in the neurons of each group was analyzed by flow cytometry.The acquisition rates and morphology of neurons of each group were observed by immunofluorescence staining.The grinding or collagenase II method is more suitable for rapid acquisition of neuronal cells from an adult rat’s cerebral cortex.The number of neuron cells obtained by the trypsin method were very few,so it is not convenient for later experiments.
文摘Advanced glycation end products lead to cell apoptosis, and cause cell death by increasing endoplasmic reticulum stress. Advanced glycation end products alone may also directly cause damage to tissues and cells, but the precise mechanism remains unknown. This study used primary cultures of rat cerebral cortex neurons, and treated cells with different concentrations of glycation end products (50, 100, 200, 400 mg/L), and with an antibody for the receptor of advanced glycation end products before and after treatment with advanced glycation end products. The results showed that with increasing concentrations of glycation end products, free radical content increased in neurons, and the number of apoptotic cells increased in a dose-dependent manner. Before and after treatment of advanced glycation end products, the addition of the antibody against advanced glycation end-products markedly reduced hydroxyl free radicals, malondialdehyde levels, and inhibited cell apoptosis. This result indicated that the antibody for receptor of advanced glycation end-products in neurons from the rat cerebral cortex can reduce glycation end product-induced oxidative stress damage by suppressing glycation end product receptors. Overall, our study confirms that the advanced glycation end products-advanced glycation end products receptor pathway may be the main signaling pathway leading to neuronal damage.
基金supported by a grant from the National Natural Science Foundation of China,No.30972766,31170852,81001322,81172795,81173048the Specialized Research Fund for the Doctoral Program of Colleges and Universities,No.20094402110004
文摘Interleukin-6 has been shown to be involved in nerve injury and nerve regeneration, but the effects of long-term administration of high concentrations of interleukin-6 on neurons in the central nervous system is poorly understood. This study investigated the effects of 24 hour exposure of interleukin-6 on cortical neurons at various concentrations(0.1, 1, 5 and 10 ng/m L) and the effects of 10 ng/m L interleukin-6 exposure to cortical neurons for various durations(2, 4, 8, 24 and 48 hours) by studying voltage-gated Na+ channels using a patch-clamp technique. Voltage-clamp recording results demonstrated that interleukin-6 suppressed Na+ currents through its receptor in a time- and dose-dependent manner, but did not alter voltage-dependent activation and inactivation. Current-clamp recording results were consistent with voltage-clamp recording results. Interleukin-6 reduced the action potential amplitude of cortical neurons, but did not change the action potential threshold. The regulation of voltage-gated Na+ channels in rat cortical neurons by interleukin-6 is time- and dose-dependent.
基金supported by the National Basic Research Program of China (973 Program), No.2010CB530500the National Natural Science Foundation of China, No. 30973788+1 种基金Guangdong Province Science and Technology Projects, No. 2010B031500016the Natural Science Foundation of Guangdong Province, No.9351040701000001
文摘The present study established a model of focal cerebral ischemia through heat-coagulation induced occlusion of the middle cerebral artery. Following electroacupuncture at Baihui (GV20) and Dazhui (GV14), or intracerebroventricular infusion of AG490, a Janus kinase 2 phosphorylation inhibitor, the amount of necrotic or degenerated neurons in the ischemic cerebral cortex decreased, neuronal swelling was ameliorated, and expression of phosphorylated Janus kinase 2 and signal transducer and activator of transcription 3 decreased. Results confirmed that electroacupuncture promoted neuronal repair in the cerebral cortex by reducing expression of phosphorylated Janus kinase 2 and signal transducer and activator of transcription 3, as well as weakening the phosphorylated activation, thereby blocking abnormal activation of the Janus kinase 2- signal transducer and activator of transcription 3 signal transduction pathway.
文摘Aim This study is aimed to determine whether SPK2 （Sphingosine kinase-2） is involved in isoflurane preconditioning induced autophagy activation in primary cultured neurons. We also examined whether SPK2 pro- tects neurons from ischemic injury by activating autophagy, and explored the molecular mechanism of SPK2 contrib- uting to the autophagy activation in neurons. Methods Isoflurane preconditioning （ISO） and oxygen glucose dep- rivation （OGD） model was established in primary cultured murine cortical neurons. Neurons were transfected by siRNA to interfere SPK2 and Beclin 1, or lentivirus to overexpress SPK2. Protein expression of SPK2, LC3, and Beclinl were detected with immunofluorescence and Western blot analysis. The neurons were treated with lysosomal inhibitor ammonium chloride （NH4 C1） to test the autophagy flux. The protection of SPK2 on OGD/R induced neu- ronal death was detected with CCK-8 （cell counting kit-8） and LDH cytotoxicity assay kit. Autophagy inhibitor 3- MA （3-Methyladenine） was used to detect the protection of autophagy on SPK2 induced isehemie tolerance. Co-im- munoprecipitation was used to detect the interaction between Beclin 1 and Bel-2. Results In primary cultured neu- ISO enhanced SPK2 and LC3 immunofluorescenee. SPK2 siRNA inhibits LC3II upregulation induced by rons, ISO. Beclin 1 siRNA also inhibits LC3II upregulation induced by ISO. Lentivirus-indueed SPK2 overexpression in- creased LC3II/LC3I ratio and enhanced the autophagy flux in neurons. SPK2 overexpression also exerted neuropro- teetion against OGD model in cortical neurons, as evidenced by improvement of neuronal morphology, increased cellular viability and reduced LDH leakage, while 3-MA partly abolished the SPK2-induced neuroprotection. After SPK2 overexpression, Beclin 1 siRNA inhibited SPK2 induced LC3II upregulation, and the coimmunoprecipitation of Beclin 1 and Bcl-2 was reduced.. Conclusion ISO increases SPK2 and activates autophagy in neurons. SPK2 or Beclin 1 interference cancels ISO induced autophagy activation. SPK2 overexpression activates autophagy, and protects the neurons against ischemic injury. SPK2 may induce autophagy by disrupting Beclin 1/Bcl-2 interaction.
基金National Natural Science Foundation of China(81560666)Program for Changjiang Scholarsand Innovative Research Team in University, China(IRT_17R113).
文摘OBJECTIVE To explore the effect of icariside Ⅱ(ICS Ⅱ) on oxygen-glucose deprivation and reoxygenation(OGD/R)-induced injury in cerebral cortical neuronal cels.METHODS Primary cerebral cortical neuronal cells were deprived of oxygen and glucose for 2 h to simulate ischemic stroke injury in vitro.The experiment was divided into 8 groups,which were control,control+ICSⅡ 25 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ(6.25,12.5,25 μmol·L^(-1)),OGD/R+3-methyladenine(3-MA) and OGD/R+Rapamycin(Rap).The protective effect of ICS Ⅱ were detected by MTT assay and lactate dehydrogenase(LDH),respectively.Autophagic flux and autophagy related proteins expressions were detected by using adenovirus harboring tf-LC3 and Western blotting,respectively.RESULTS Compared with OGD/R group,the cell viability treated with ICSⅡwas elevated in a concentration-dependent manner,and the leakage rate of LDH was lowed.Moreover,ICSⅡ not only suppressed OGD/R-induced autophagic flux,but also inhibited the increase of LC3-Ⅱ/LC3-Ⅰ ratio and Beclin 1 after OGD/R insulted.CONCLUSION ICS Ⅱ exerts protective effects on OGD/R-induced cerebral cortical neuronal cells through inhibiting excessive autophagy.
文摘In sensorimotor cortical slices of guinea pig using local iontophoretic application of glutamate to the soma and dendrites it was found that a decrease of temperature of incubating fluid from 34 to 21°C - 22°C changes the somatic responses to the local injection of glutamate to the dendritic loci, while the responses to iontophoretic application of glutamate to the soma remain unchanged. Hypothermic changes in reactivity to dendritic stimulation start below 30°C and coincide with changes in the spontaneous activity of neurons, both in the direction of increasing and decreasing the frequency of firing in different nerve cells. On hypothermic decrease of spontaneous activity, the latencies of evoked dendritic responses on the soma became more longer, while on hypothermic increase of firing level, somatic spike responses to iontophoretic application of glutamate to dendritic loci appeared with shorter latencies. Hypothermic changes in the physiological parameters of neurons were accompanied by a drop in spike amplitude at the same temperature and with its further decrease. At the same time, there was a decrease of spike reaction to iontophoretic application of acetylcholine below 30°. It is proposed that the reason for hypothermic changes of neuronal activity is decreasing rate of M-cholinergic process at 27°C - 29°C which leads to opening K<sup>+</sup> channels of neuronal membranes and hence to attenuation of conductive function of dendrites and to imbalance of K<sup>+</sup> ion homeostasis. Peculiarities of hypothermic regulation of neuronal spike activity depend on individual functional properties of cortical neurons.
