This study examined the effects of clinically relevant concentrations of isoflurane on the amplitude of NMDA receptor current （INMDA） and the expression of cytochrome C in cultured developing rat hippocampal neurons...This study examined the effects of clinically relevant concentrations of isoflurane on the amplitude of NMDA receptor current （INMDA） and the expression of cytochrome C in cultured developing rat hippocampal neurons. The hippocampi were dissected from newborn Sprague-Dawley rats. Hippocampal neurons were primarily cultured for 5 days and then treated with different concentrations of isoflurane [（0.25, 0.5, 0.75, 1 minimum alveolar concentration （MAC））]. The peak of INMDA was re- corded by means of the whole cell patch clamp technique. The cytochrome C level was detected by Western blotting and quantitative real-time PCR. Our results showed that isoflurane （0.25, 0.5, 0.75 and 1 MAC） potentiated the amplitude of INMDA by （116±8.8）%, （122±11.7）%, （135±14.3）% and （132~14.6）%, respectively, and isoflurane increased the mRNA expression of cytochrome C in a concentration-dependent manner. The cytochrome C mRNA expression reached a maximum after 0.5 MAC isoflurane stimulation for 6 h （P〈0.05）. It was concluded that isoflurane enhances the expression of cytochrome C in cultured rat hippocampal neurons, which may be mediated by facilitation of NMDA receptor.展开更多
In the mammalian central nervous system（CNS）coupling of neurons by gap junctions and the expression of neuronal gap junction protein,connexin 36（Cx36）rapidly increases（usually during 1–2 hours）following a wide ...In the mammalian central nervous system（CNS）coupling of neurons by gap junctions and the expression of neuronal gap junction protein,connexin 36（Cx36）rapidly increases（usually during 1–2 hours）following a wide range of neuronal injuries,including ischemia,traumatic brain injury（TBI）,spinal cord injury and epilepsy（reviewed in Belousov and Fontes,2013）.Pharmacological blockade or genetic elimi-nation of Cx36-containing gap junctions dramatically re- duce neuronal death in animal models of ischemia, TBI and epilepsy and prevent NMDA receptor （NMDAR）-mediated excitotoxicity （Belousov and Fontes, 2014）.展开更多
Summary: To investigate the exact mechanism of epileptogenesis induced by coriaria lactone (CL), the effect of CL on NMDA receptor mediated current (IAsp) in rat hippocampal CA1 neu- rons was investigated by using ny...Summary: To investigate the exact mechanism of epileptogenesis induced by coriaria lactone (CL), the effect of CL on NMDA receptor mediated current (IAsp) in rat hippocampal CA1 neu- rons was investigated by using nystatin perforated whole-cell patch clamp. 10-6-10-4 mol/L Asp acted on NMDA receptors and elicited an inward current (IAsp) at a holding potential (VH) of -40 mV in presence of 10-6 mol/L glycine and absence of Mg2+ extracellularly. CL enhanced NMDA receptor mediated current induced by Asp, but had no effect on threshold concentration, EC50, Hill coefficient as well as maximal-effect concentration and reversal potential of IAsp. The effect had no relationship with holding potential. These results showed that CL could enhance NMDA receptor mediated current to increase [Ca2+]i of neurons by acting on Gly site, thereby inducing epilepsy.展开更多
Objective To study the effect of salicylate on the expression and function of NMDA receptors in spiral ganglion neurons （SGNs）. Methods The mRNA of NR1 subunit of NMDA receptor in modiolus tissues were detected by R...Objective To study the effect of salicylate on the expression and function of NMDA receptors in spiral ganglion neurons （SGNs）. Methods The mRNA of NR1 subunit of NMDA receptor in modiolus tissues were detected by Real time fluorescence quantitative PCR （FQ-PCR）. NMDA receptor whole-cell currents were recorded using patch clamp in acute isolated SGNs. Results Compared with the control group, salicylate significantly increased the mRNA level of NR1 subunit in SGNs. NMDA of concentrations ranging from 0.1 mM to 10 mM evoked no current in SGNs. NMDA （0. 1mM and 0.5 mM） applied with salicylate （5 mM）, however, induced inward currents （212.6±15.2pA, n=5; 607.9±44.3pA, n=5） in a dose-dependent manner, which could be inhibited by APV. Salicylate alone did not produce any current in SGNs. Conclusion Salicylate increases the expression of NMDA receptors and facilitates the currents mediated by NMDA receptors in SGNs.展开更多
The present study revealed the stimulatory effects of NMDA on intracellular ca 2+ concentration in rat dorsal root ganglion (DRG) neurons. Fura 3/AM, an intracellular calcium fluorescent indicator was used to monitor ...The present study revealed the stimulatory effects of NMDA on intracellular ca 2+ concentration in rat dorsal root ganglion (DRG) neurons. Fura 3/AM, an intracellular calcium fluorescent indicator was used to monitor the fluctuation of 〔ca 2+ 〕 i. Here we present the evidence that (1) Confocal microscopy resolved the cells of three different sizes, based on which a cell diameter distribution histogram was drawn. The fluorescence signals originated from the cells of different sizes, small size (diameter<30μm), medium size (diameter between 30 to 50μm),and large size (diameter>50μm); presumably intracellular Ca 2+ concentration was different in the cells of different sizes. (2) The time related variation of fluorescence intensity was observed. In particular, the fluorescence intensity in 0 Ca 2+ bath solution was affected by the application of high ca 2+ solution. (3) In 0 ca 2+ bath solution the intracellular Ca 2+ concentration nonlinear properties of distinct diameter cells was described. (4) A kind of SETAR model was fitted with a medium sized cell.(5)The residuals from the fitted model were tested to see whether they were plausibly Gaussian. These findings indicated that in distinct types of cells intracellular Ca 2+ concentration had different characteristics in different DRG neurons, and contributed to different functions of these neurons. Besides, the established threshold autoregressive model can share intracellular ca 2+ with the main nonlinear kinetic展开更多
The purpose of this study was to evaluate the roles of different housing environments in neurological function, cerebral metabolism, cerebral infarction and neuron apoptosis after focal cerebral ischemia. Twenty-eight...The purpose of this study was to evaluate the roles of different housing environments in neurological function, cerebral metabolism, cerebral infarction and neuron apoptosis after focal cerebral ischemia. Twenty-eight Sprague-Dawley rats were divided into control group （CG） and cerebral ischemia group, and the latter was further divided into subgroups of different housing conditions： standard environment （SE） subgroup, individual living environment （IE） subgroup, and enriched environment （EE） subgroup. Focal cerebral ischemia was induced by the middle cerebral artery occlusion （MCAO）. Beam walking test was used to quantify the changes of overall motor function. Cerebral infarction and cerebral metabolism were studied by in vivo magnetic resonance imaging and 1H-magnetic resonance spectra, respectively. Neuron necrosis and apoptosis were detected by hematoxylin-eosin and TUNEL staining methods, respectively. The results showed that performance on the beam-walk test was improved in EE subgroup when compared to SE subgroup and IE subgroup. Cerebral infarct volume in IE subgroup was significantly larger than that in SE subgroup （P〈0.05） and EE subgroup （P〈0.05） on day 14 after MCAO. NAA/Cr and Cho/Cr ratios were lower in MCAO groups under different housing conditions as compared to those in CG （P〈0.05）. NAA/Cr ratio was lower in IE subgroup （P〈0.05） and higher in EE subgroup （P〈0.05） than that in SE subgroup. NAA/ Cr ratio in EE was significantly higher than that in IE subgroup （P〈0.05）. Cho/Cr ratio was decreased in MCAO groups as compared to that in CG （P〈0.05）. A significant decrease in normal neurons in cerebral cortex was observed in MCAO groups as compared to CG （P〈0.05）. The amount of normal neurons was less in IE subgroup （P〈0.05）, and more in EE subgroup （P〈0.05） than that in SE subgroup after MCAO. The amotmt of normal neurons in EE subgroup was significantly more than that in IE subgroup after MCAO （P〈0.05）. The ratio of TUNEL-positive neurons in EE was significantly lower than that in SE subgroup （P〈0.05） and IE subgroup （P〈0.05）. Correlation analysis showed that the beam walking test was negatively correlated with NAA/Cr ratio （P〈0.05）. Cerebral infarct volume was negatively correlated with both NAA/Cr ratio （P〈0.01） and Cho/Cr ratio （P〈0.01）. The amount of normal cortical neurons was positively correlated with both NAA/Cr ratio （P〈0.0I） and Cho/Cr ratio （P〈0.05）. The TUNEL-positive neurons showed a negative correlation with both NAA/Cr ratio （P〈0.01） and Cho/Cr ratio （P〈0.01）. This study goes further to show that EE may improve neurological functional deficit and cerebral metabolism, decrease cerebral infarct volume, neuron necrosis and apoptosis, while IE may aggravate brain damage after MCAO.展开更多
To investigate the role of mitochondria in neuronal apoptosis, ischemia-reperfusion mediated neuronal cell injury model was established by depriving of glucose, serum and oxygen in media. DNA fragmentation, cell viabi...To investigate the role of mitochondria in neuronal apoptosis, ischemia-reperfusion mediated neuronal cell injury model was established by depriving of glucose, serum and oxygen in media. DNA fragmentation, cell viability, cytochrome C releasing, caspase3 activity and mitochondrial transmembrane potential were observed after N2a cells suffered the insults. The results showed that N2a cells in ischemic territory exhibited survival damage, classical cell apoptosis change, DNA ladder and activation of caspase3. Apoptosis-related alterations in mitochondrial functions, including release of cytochrome C and depression of mitochondrial transmembrane potential (△Ψm) were testified in N2a cells after mimic ischemia-reperfusion. Moreover, activation of caspase3 occurred following the release of cytochrome C. However, the inhibitor of caspase3, Ac-DEVD-CHO, couldn't completely rescue N2a cells from apoptosis. Administration of cyclosporine A, an inhibitor of mitochondria permeability transition pore only partly inhibited caspase3 activity and reduced DNA damage. Interestingly, treatment of Z-IETD-FMK, an inhibitor of caspase8 could completely reverse DNA fragmentation, but can't completely inhibit caspase3 activity. It was concluded that there were caspase3 dependent and independent cellular apoptosis pathways in N2a cells suffering ischemia-reperfusion insults. Mitochondria dysfunction may early trigger apoptosis and amplify apoptosis signal.展开更多
BACKGROUND： Aggregation of β-amyloid peptide （A β ）, excitatory intoxication, oxidation injury and inflammation reaction are generally regarded as the main pathogenesis for Alzheimer disease （AD）. （ - ） claus...BACKGROUND： Aggregation of β-amyloid peptide （A β ）, excitatory intoxication, oxidation injury and inflammation reaction are generally regarded as the main pathogenesis for Alzheimer disease （AD）. （ - ） clausenamide is characterized by promoting intelligent development, resisting oxidation, cleaning free radicals, resisting A β neurotoxicity and nerve cell apoptosis, inhibiting over phosphorylation of tau protein, and improving central cholinergic system. However, whether （-） clausenamide has an effect on hippocampal neuron apoptosis or not need further study. OBJECTIVE： To observe the effect of （ - ） clausenamide on survival rate of hippocampal neurons due to sodium nitroprusside （SNP） and analyze the possible pathways. DESIGN： Contrast observation. SETTING： Institute of Biochemistry and Molecular Biology, Guangdong Medical College. MATERIALS： A total of 12 male SD rats of 24 hours old were provided by the Experimental Animal Center of Guangdong Medical College. The primer was synthesized by Beijing Huada Genetic Engineering Company and （-） clausenamide （99.6%） was provided by Pharmacological Department of Chinese Academy of Medical Sciences. SNP was provided by Sigma Company. METHODS： Bilateral hippocampus was collected from newborn rats to establish single cell suspension. On the 12th day, hippocampal neurons were pretreated with 0.2, 0.4, 0.8 and 1.6 la mol/L （ - ） clausenamide for 6 hours; the culture medium was gotten rid of and neurons were washed with non-serum DMEM solution for three times. In addition, non-serum DMEM solution was added with the above mentioned volume of （ - ） clausenamide and 50 μ mol/L SNP to culture neurons for 24 hours and the collected cells were prepared for the experiment. Neurons were equally divided into control group （culture medium control）, model group （SNP treatment） and experimental group [（ - ） clausenamide ＋ SNP]. Experiment of each group was done for three times at least. Survival rate of cells was measured with MTT chromatometry; levels of mRNA of hippocampal neuron bcl-2 and bax gene were detected with reverse transcription polymerase chain reaction （RT-PCR）; expression of hippocampal neuron Bcl-2 and Bax protein was measured with Western blot technique. MAIN OUTCOME MEASURES： ① Effect of （-） clausenamide on survival rate of SNP-induced hippocampal neuron apoptosis; ②bcl-2 and bax mRNA and protein expression ofhippocampal neurons. RESULTS： ①Survival rate of hippocampal neurons： Survival rate of hippocampal neurons affected by 0.4 - 1.6 μ mol/L （ - ） clausenamide was higher in the experimental group than the model group （P 〈 0.01）, and the survival rate was increased with the larger volume of （ - ） clausenamide. Survival rate was the highest when hippocampal neurons were induced by 1.6 μ mol/L, and it had obvious dosage dependence （P 〈 0.01）. ②Expression of bcl-2 and bax mRNA： Hippocampal neurons were pretreated with 0.2 - 1.6 μ mol/L （ - ） clausenamide for 6 hours in the experimental group and strap of PCR product of bcl-2 gene was brightened gradually. This suggested that, with the increase of concentration, expression of bcl-2 mRNA was increased simultaneously. However, when strap of PCR product of bax gene was darkened, expression of bax was decreased with the increase of concentration. ③Expression of Bcl-2 and Bax protein： Hippocampal neurons were pretreated with 0.2 - 1.6 μ mol/L （ - ） clausenamide for 6 hours in the experimental group and strap of PCR product of Bcl-2 protein was thickened gradually. This suggested that, with the increase of concentration, expression of Bcl-2 protein was increased simultaneously. However, when strap of PCR product of Bax protein was thinned, expression of Bax protein was decreased with the increase of concentration. CONCLUSION： （ - ） clausenamide can resist neurotoxic effect of SNP through dosage dependence, and the mechanism may be related to promoting expression of anti-apoptotic bcl-2 gene and inhibiting expression of pro-apoptotic bax gene.展开更多
Previous studies have shown that 5-hydroxymethylfurfural, a compound extracted from wine- processed Fructus corni, has a protective effect on hippocampal neurons. The present study was designed to explore the related ...Previous studies have shown that 5-hydroxymethylfurfural, a compound extracted from wine- processed Fructus corni, has a protective effect on hippocampal neurons. The present study was designed to explore the related mechanisms. Our study revealed that high and medium doses （10, 1 μmol/L） of 5-hydroxymethylfurfural could improve the morphology of H2O2-treated rat hippocampal neurons as revealed by inverted phase-contrast microscopy and transmission electron microscopy. MTT results showed that incubation with high and medium doses of 5-hydroxymethylfurfural caused a significant increase in the viability of neuronal cells injured by H2O2. Flow cytometry assays con- firmed that H2O2 could induce cell apoptosis, while high and medium doses of 5-hydroxymethylfurfural had a visible protective effect on apoptotic rat hippocampal neurons. Real-time PCR and western blot analysis showed that high and medium doses of 5-hydroxymethylfurfural prevented H2O2-induced up-regulation of p53, Bax and caspase-3 and an- tagonized the down-regulation of Bcl-2 induced by H2O2 treatment. These results suggested that 5-hydroxymethylfurfural could inhibit apoptosis of cultured rat hippocampal neurons injured by H2O2 via increase in Bcl-2 levels and decrease in p53, Bax and caspase-3 protein expression levels.展开更多
Objective: To explore the expression of nuclear factor-kappa B (NF-κB) in Schwann cells (SCs) and its effect on motor neuron apoptosis in spinal cord following sciatic nerves injury in adult rats. Methods: Thirty-six...Objective: To explore the expression of nuclear factor-kappa B (NF-κB) in Schwann cells (SCs) and its effect on motor neuron apoptosis in spinal cord following sciatic nerves injury in adult rats. Methods: Thirty-six adult Sprague-Dawley (SD) rats were divided randomly into normal control group (n=6), and sciatic nerves crushing group (n=30). and the later was further equally randomized into 5 subgroups: 1. 3. 7. 11. and 21 d post-injury groups. The expression of NF-κB of normal and injured nerves were examined by immunohistochemistry staining, and the apoptosis of motor neurons in spinal cord of lumbar 4 to lumbar 6 (L4-L6) was investigated by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNED assay. Both were quantitated by image analysis. Results: In crushing group, except 21 d post-injury group, the expression of NF-κB was markedly higher than that in the normal control group (P<0. 05,P<0. 01). At 1 d after sciatic nerves crushing, the expression of NF-κB was obviously up-regulated, reached peak at 3 d. and recovered at 21 d. The same trend was observed in the time-course on motor neuron apoptosis after sciatic nerves injury. Correlation analyses revealed that motor neuron apoptosis was significantly and positively correlated with the expression of NF-κB following sciatic nerves injury (r=0. 976 0,P<0. 01). Conclusion: After injury of sciatic nerves, the presence and up-regulation of NF-κB in SCs may be involved in motor neuron apoptosis in L4-L6 spinal cord.展开更多
基金supported by grants from the National Natural Science Foundation of China(No.30772086No.30901390)
文摘This study examined the effects of clinically relevant concentrations of isoflurane on the amplitude of NMDA receptor current （INMDA） and the expression of cytochrome C in cultured developing rat hippocampal neurons. The hippocampi were dissected from newborn Sprague-Dawley rats. Hippocampal neurons were primarily cultured for 5 days and then treated with different concentrations of isoflurane [（0.25, 0.5, 0.75, 1 minimum alveolar concentration （MAC））]. The peak of INMDA was re- corded by means of the whole cell patch clamp technique. The cytochrome C level was detected by Western blotting and quantitative real-time PCR. Our results showed that isoflurane （0.25, 0.5, 0.75 and 1 MAC） potentiated the amplitude of INMDA by （116±8.8）%, （122±11.7）%, （135±14.3）% and （132~14.6）%, respectively, and isoflurane increased the mRNA expression of cytochrome C in a concentration-dependent manner. The cytochrome C mRNA expression reached a maximum after 0.5 MAC isoflurane stimulation for 6 h （P〈0.05）. It was concluded that isoflurane enhances the expression of cytochrome C in cultured rat hippocampal neurons, which may be mediated by facilitation of NMDA receptor.
基金supported by NIH (R21 NS076925)the University of Kansas Medical Center funds to A.B.B+1 种基金supported in part by NIH P20 GM104936, P30 AG035982 and UL1 TR000001Core support was provided by NIH HD002528
文摘In the mammalian central nervous system（CNS）coupling of neurons by gap junctions and the expression of neuronal gap junction protein,connexin 36（Cx36）rapidly increases（usually during 1–2 hours）following a wide range of neuronal injuries,including ischemia,traumatic brain injury（TBI）,spinal cord injury and epilepsy（reviewed in Belousov and Fontes,2013）.Pharmacological blockade or genetic elimi-nation of Cx36-containing gap junctions dramatically re- duce neuronal death in animal models of ischemia, TBI and epilepsy and prevent NMDA receptor （NMDAR）-mediated excitotoxicity （Belousov and Fontes, 2014）.
基金This project was supported by a grant from the National Natural Science Foundation of China !(No. 39330210).
文摘Summary: To investigate the exact mechanism of epileptogenesis induced by coriaria lactone (CL), the effect of CL on NMDA receptor mediated current (IAsp) in rat hippocampal CA1 neu- rons was investigated by using nystatin perforated whole-cell patch clamp. 10-6-10-4 mol/L Asp acted on NMDA receptors and elicited an inward current (IAsp) at a holding potential (VH) of -40 mV in presence of 10-6 mol/L glycine and absence of Mg2+ extracellularly. CL enhanced NMDA receptor mediated current induced by Asp, but had no effect on threshold concentration, EC50, Hill coefficient as well as maximal-effect concentration and reversal potential of IAsp. The effect had no relationship with holding potential. These results showed that CL could enhance NMDA receptor mediated current to increase [Ca2+]i of neurons by acting on Gly site, thereby inducing epilepsy.
基金supported by a grant from National Nature Science Fund of China(No.81060082,30860098)Nature Science Fund of Guangxi(No.2011jjA40056)to Jiping Su
文摘Objective To study the effect of salicylate on the expression and function of NMDA receptors in spiral ganglion neurons （SGNs）. Methods The mRNA of NR1 subunit of NMDA receptor in modiolus tissues were detected by Real time fluorescence quantitative PCR （FQ-PCR）. NMDA receptor whole-cell currents were recorded using patch clamp in acute isolated SGNs. Results Compared with the control group, salicylate significantly increased the mRNA level of NR1 subunit in SGNs. NMDA of concentrations ranging from 0.1 mM to 10 mM evoked no current in SGNs. NMDA （0. 1mM and 0.5 mM） applied with salicylate （5 mM）, however, induced inward currents （212.6±15.2pA, n=5; 607.9±44.3pA, n=5） in a dose-dependent manner, which could be inhibited by APV. Salicylate alone did not produce any current in SGNs. Conclusion Salicylate increases the expression of NMDA receptors and facilitates the currents mediated by NMDA receptors in SGNs.
文摘The present study revealed the stimulatory effects of NMDA on intracellular ca 2+ concentration in rat dorsal root ganglion (DRG) neurons. Fura 3/AM, an intracellular calcium fluorescent indicator was used to monitor the fluctuation of 〔ca 2+ 〕 i. Here we present the evidence that (1) Confocal microscopy resolved the cells of three different sizes, based on which a cell diameter distribution histogram was drawn. The fluorescence signals originated from the cells of different sizes, small size (diameter<30μm), medium size (diameter between 30 to 50μm),and large size (diameter>50μm); presumably intracellular Ca 2+ concentration was different in the cells of different sizes. (2) The time related variation of fluorescence intensity was observed. In particular, the fluorescence intensity in 0 Ca 2+ bath solution was affected by the application of high ca 2+ solution. (3) In 0 ca 2+ bath solution the intracellular Ca 2+ concentration nonlinear properties of distinct diameter cells was described. (4) A kind of SETAR model was fitted with a medium sized cell.(5)The residuals from the fitted model were tested to see whether they were plausibly Gaussian. These findings indicated that in distinct types of cells intracellular Ca 2+ concentration had different characteristics in different DRG neurons, and contributed to different functions of these neurons. Besides, the established threshold autoregressive model can share intracellular ca 2+ with the main nonlinear kinetic
文摘The purpose of this study was to evaluate the roles of different housing environments in neurological function, cerebral metabolism, cerebral infarction and neuron apoptosis after focal cerebral ischemia. Twenty-eight Sprague-Dawley rats were divided into control group （CG） and cerebral ischemia group, and the latter was further divided into subgroups of different housing conditions： standard environment （SE） subgroup, individual living environment （IE） subgroup, and enriched environment （EE） subgroup. Focal cerebral ischemia was induced by the middle cerebral artery occlusion （MCAO）. Beam walking test was used to quantify the changes of overall motor function. Cerebral infarction and cerebral metabolism were studied by in vivo magnetic resonance imaging and 1H-magnetic resonance spectra, respectively. Neuron necrosis and apoptosis were detected by hematoxylin-eosin and TUNEL staining methods, respectively. The results showed that performance on the beam-walk test was improved in EE subgroup when compared to SE subgroup and IE subgroup. Cerebral infarct volume in IE subgroup was significantly larger than that in SE subgroup （P〈0.05） and EE subgroup （P〈0.05） on day 14 after MCAO. NAA/Cr and Cho/Cr ratios were lower in MCAO groups under different housing conditions as compared to those in CG （P〈0.05）. NAA/Cr ratio was lower in IE subgroup （P〈0.05） and higher in EE subgroup （P〈0.05） than that in SE subgroup. NAA/ Cr ratio in EE was significantly higher than that in IE subgroup （P〈0.05）. Cho/Cr ratio was decreased in MCAO groups as compared to that in CG （P〈0.05）. A significant decrease in normal neurons in cerebral cortex was observed in MCAO groups as compared to CG （P〈0.05）. The amount of normal neurons was less in IE subgroup （P〈0.05）, and more in EE subgroup （P〈0.05） than that in SE subgroup after MCAO. The amotmt of normal neurons in EE subgroup was significantly more than that in IE subgroup after MCAO （P〈0.05）. The ratio of TUNEL-positive neurons in EE was significantly lower than that in SE subgroup （P〈0.05） and IE subgroup （P〈0.05）. Correlation analysis showed that the beam walking test was negatively correlated with NAA/Cr ratio （P〈0.05）. Cerebral infarct volume was negatively correlated with both NAA/Cr ratio （P〈0.01） and Cho/Cr ratio （P〈0.01）. The amount of normal cortical neurons was positively correlated with both NAA/Cr ratio （P〈0.0I） and Cho/Cr ratio （P〈0.05）. The TUNEL-positive neurons showed a negative correlation with both NAA/Cr ratio （P〈0.01） and Cho/Cr ratio （P〈0.01）. This study goes further to show that EE may improve neurological functional deficit and cerebral metabolism, decrease cerebral infarct volume, neuron necrosis and apoptosis, while IE may aggravate brain damage after MCAO.
基金ThisprojectwassupportedbyagrantfromNaturalScienceFoundationofHygienicCommitteeofHubeiProvince (No .WJ0 1 5 1 0 ) .
文摘To investigate the role of mitochondria in neuronal apoptosis, ischemia-reperfusion mediated neuronal cell injury model was established by depriving of glucose, serum and oxygen in media. DNA fragmentation, cell viability, cytochrome C releasing, caspase3 activity and mitochondrial transmembrane potential were observed after N2a cells suffered the insults. The results showed that N2a cells in ischemic territory exhibited survival damage, classical cell apoptosis change, DNA ladder and activation of caspase3. Apoptosis-related alterations in mitochondrial functions, including release of cytochrome C and depression of mitochondrial transmembrane potential (△Ψm) were testified in N2a cells after mimic ischemia-reperfusion. Moreover, activation of caspase3 occurred following the release of cytochrome C. However, the inhibitor of caspase3, Ac-DEVD-CHO, couldn't completely rescue N2a cells from apoptosis. Administration of cyclosporine A, an inhibitor of mitochondria permeability transition pore only partly inhibited caspase3 activity and reduced DNA damage. Interestingly, treatment of Z-IETD-FMK, an inhibitor of caspase8 could completely reverse DNA fragmentation, but can't completely inhibit caspase3 activity. It was concluded that there were caspase3 dependent and independent cellular apoptosis pathways in N2a cells suffering ischemia-reperfusion insults. Mitochondria dysfunction may early trigger apoptosis and amplify apoptosis signal.
文摘BACKGROUND： Aggregation of β-amyloid peptide （A β ）, excitatory intoxication, oxidation injury and inflammation reaction are generally regarded as the main pathogenesis for Alzheimer disease （AD）. （ - ） clausenamide is characterized by promoting intelligent development, resisting oxidation, cleaning free radicals, resisting A β neurotoxicity and nerve cell apoptosis, inhibiting over phosphorylation of tau protein, and improving central cholinergic system. However, whether （-） clausenamide has an effect on hippocampal neuron apoptosis or not need further study. OBJECTIVE： To observe the effect of （ - ） clausenamide on survival rate of hippocampal neurons due to sodium nitroprusside （SNP） and analyze the possible pathways. DESIGN： Contrast observation. SETTING： Institute of Biochemistry and Molecular Biology, Guangdong Medical College. MATERIALS： A total of 12 male SD rats of 24 hours old were provided by the Experimental Animal Center of Guangdong Medical College. The primer was synthesized by Beijing Huada Genetic Engineering Company and （-） clausenamide （99.6%） was provided by Pharmacological Department of Chinese Academy of Medical Sciences. SNP was provided by Sigma Company. METHODS： Bilateral hippocampus was collected from newborn rats to establish single cell suspension. On the 12th day, hippocampal neurons were pretreated with 0.2, 0.4, 0.8 and 1.6 la mol/L （ - ） clausenamide for 6 hours; the culture medium was gotten rid of and neurons were washed with non-serum DMEM solution for three times. In addition, non-serum DMEM solution was added with the above mentioned volume of （ - ） clausenamide and 50 μ mol/L SNP to culture neurons for 24 hours and the collected cells were prepared for the experiment. Neurons were equally divided into control group （culture medium control）, model group （SNP treatment） and experimental group [（ - ） clausenamide ＋ SNP]. Experiment of each group was done for three times at least. Survival rate of cells was measured with MTT chromatometry; levels of mRNA of hippocampal neuron bcl-2 and bax gene were detected with reverse transcription polymerase chain reaction （RT-PCR）; expression of hippocampal neuron Bcl-2 and Bax protein was measured with Western blot technique. MAIN OUTCOME MEASURES： ① Effect of （-） clausenamide on survival rate of SNP-induced hippocampal neuron apoptosis; ②bcl-2 and bax mRNA and protein expression ofhippocampal neurons. RESULTS： ①Survival rate of hippocampal neurons： Survival rate of hippocampal neurons affected by 0.4 - 1.6 μ mol/L （ - ） clausenamide was higher in the experimental group than the model group （P 〈 0.01）, and the survival rate was increased with the larger volume of （ - ） clausenamide. Survival rate was the highest when hippocampal neurons were induced by 1.6 μ mol/L, and it had obvious dosage dependence （P 〈 0.01）. ②Expression of bcl-2 and bax mRNA： Hippocampal neurons were pretreated with 0.2 - 1.6 μ mol/L （ - ） clausenamide for 6 hours in the experimental group and strap of PCR product of bcl-2 gene was brightened gradually. This suggested that, with the increase of concentration, expression of bcl-2 mRNA was increased simultaneously. However, when strap of PCR product of bax gene was darkened, expression of bax was decreased with the increase of concentration. ③Expression of Bcl-2 and Bax protein： Hippocampal neurons were pretreated with 0.2 - 1.6 μ mol/L （ - ） clausenamide for 6 hours in the experimental group and strap of PCR product of Bcl-2 protein was thickened gradually. This suggested that, with the increase of concentration, expression of Bcl-2 protein was increased simultaneously. However, when strap of PCR product of Bax protein was thinned, expression of Bax protein was decreased with the increase of concentration. CONCLUSION： （ - ） clausenamide can resist neurotoxic effect of SNP through dosage dependence, and the mechanism may be related to promoting expression of anti-apoptotic bcl-2 gene and inhibiting expression of pro-apoptotic bax gene.
基金financially supported by the National Natural Science Foundation of China,No.30772851a grant from the Six Talents Peaks Program of Jiangsu Province,Chinaa grant from the Priority Academic Program Development of Jiangsu Higher Education Institutions,PAPD(Traditional Chinese medicine combined with Western Medicine
文摘Previous studies have shown that 5-hydroxymethylfurfural, a compound extracted from wine- processed Fructus corni, has a protective effect on hippocampal neurons. The present study was designed to explore the related mechanisms. Our study revealed that high and medium doses （10, 1 μmol/L） of 5-hydroxymethylfurfural could improve the morphology of H2O2-treated rat hippocampal neurons as revealed by inverted phase-contrast microscopy and transmission electron microscopy. MTT results showed that incubation with high and medium doses of 5-hydroxymethylfurfural caused a significant increase in the viability of neuronal cells injured by H2O2. Flow cytometry assays con- firmed that H2O2 could induce cell apoptosis, while high and medium doses of 5-hydroxymethylfurfural had a visible protective effect on apoptotic rat hippocampal neurons. Real-time PCR and western blot analysis showed that high and medium doses of 5-hydroxymethylfurfural prevented H2O2-induced up-regulation of p53, Bax and caspase-3 and an- tagonized the down-regulation of Bcl-2 induced by H2O2 treatment. These results suggested that 5-hydroxymethylfurfural could inhibit apoptosis of cultured rat hippocampal neurons injured by H2O2 via increase in Bcl-2 levels and decrease in p53, Bax and caspase-3 protein expression levels.
基金Supported by the National Natural Science Foundation of China (No. 30600665)the Opening Project Foundation of State Key Laboratory of Trauma. Burns and Combined Injury (No. 2006A-3)the Youth Scientific Research Foundation of Third Military Medical University (No.06XG048)
文摘Objective: To explore the expression of nuclear factor-kappa B (NF-κB) in Schwann cells (SCs) and its effect on motor neuron apoptosis in spinal cord following sciatic nerves injury in adult rats. Methods: Thirty-six adult Sprague-Dawley (SD) rats were divided randomly into normal control group (n=6), and sciatic nerves crushing group (n=30). and the later was further equally randomized into 5 subgroups: 1. 3. 7. 11. and 21 d post-injury groups. The expression of NF-κB of normal and injured nerves were examined by immunohistochemistry staining, and the apoptosis of motor neurons in spinal cord of lumbar 4 to lumbar 6 (L4-L6) was investigated by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNED assay. Both were quantitated by image analysis. Results: In crushing group, except 21 d post-injury group, the expression of NF-κB was markedly higher than that in the normal control group (P<0. 05,P<0. 01). At 1 d after sciatic nerves crushing, the expression of NF-κB was obviously up-regulated, reached peak at 3 d. and recovered at 21 d. The same trend was observed in the time-course on motor neuron apoptosis after sciatic nerves injury. Correlation analyses revealed that motor neuron apoptosis was significantly and positively correlated with the expression of NF-κB following sciatic nerves injury (r=0. 976 0,P<0. 01). Conclusion: After injury of sciatic nerves, the presence and up-regulation of NF-κB in SCs may be involved in motor neuron apoptosis in L4-L6 spinal cord.
