咖啡因对苯妥英诱导的小脑颗粒神经元凋亡的保护作用及机制

目的　观察咖啡因 (caffeine)对苯妥英 (diphenylhy dantoin ,DPH) 10 0 μmol·L-1处理的大鼠小脑颗粒神经元(cerebellargranularneurons ,CGNs)存活率的影响 ,并探讨其作用机制。方法　体外培养 8d的CGNs,同时给予 10 0μmol·L-1苯妥英和 1 2 5～ 2 0mmol·L-1咖啡因 ,4 8h后行凋亡分析 ;采用dantrolene(2 0 μmol·L-1)、2APB (5 0 μmol·L-1)、nifedipine(10 0 μmol·L-1)和nimodipine(10 0 μmol·L-1)、MK80 1(4μmol·L-1)、KN93(1μmol·L-1)以及MEK1抑制剂PD980 5 9(5 0 μmol·L-1)分别预先孵育 30min ,再与10mmol·L-1咖啡因和 10 0 μmol·L-1苯妥英共孵育 4 8h ,测定CGNs存活率 ,观察咖啡因的作用与 [Ca2 + ]i 的关系 ;Westernblot法检测咖啡因对磷酸化c Jun和磷酸化ERK水平的影响。结果　① 1 2 5～ 2 0mmol·L-1咖啡因可浓度依赖性抑制 10 0 μmol·L-1苯妥英引起的CGNs凋亡 ,显著提高CGNs存活率 ;②dantrolene、2APB、nifedipine和nimodipine、KN93、MK80 1和PD980 5 9均不能取消 10mmol·L-1咖啡因对 10 0 μmol·L-1苯妥英引起的CGNs凋亡的保护作用。③咖啡因可明显抑制苯妥英诱导CGNs中c Jun磷酸化水平的升高 ,但不影响被苯妥英抑制的ERK的活性。结论　一定浓度的咖啡因可保护苯?     

以小鼠大脑皮质血管内皮细胞研究EGb761舒张血管机制

目的探讨银杏叶提取物EGb761对小鼠大脑皮质血管内皮细胞(bEnd3)一氧化氮合酶、前列腺素I_2、血栓素A_2活性和细胞内钙离子浓度等水平的影响。方法在体外培养的bEnd3系细胞培养液中加入EGb761(质量浓度分别为25、50、100、200 mg·L~(-1)),比较各用药组及空白对照组的一氧化氮合酶、6-酮-前列腺素Fla、血栓素B_2活性和细胞内钙离子浓度等水平的变化。结果与空白对照组比较,用药组细胞悬液中一氧化氮合酶的活性和细胞内钙离子浓度均有明显升高,并呈剂量依赖性;细胞培养液上清中的6-酮-前列腺素Fla水平略有升高,但无统计学意义;血栓素B_2水平略有下降,也无统计学意义。结论EGb761舒张血管机制与其增加血管内皮细胞一氧化氮合酶的活性,进而增加一氧化氮的生成有关;其进一步机制与其升高血管内皮细胞内钙离子浓度,进而增加内皮型一氧化氮合酶的活性有关。     

Enhanced caspase activity during ethanol-induced apoptosis in rat cerebellar granule cells

The effects of ethanol on cerebellar granule cell death were examined in cultures maintained for either 5 days in vitro (immature) or 8 and 12 days in vitro (mature). Ethanol did not alter cell survival under the usual growth conditions (i.e., 10% serum and 25 mM KCl). However, in mature cultures ethanol enhanced apoptosis induced by either serum withdrawal or incubation in non-depolarizing media. In immature cultures, serum deprivation, but not non-depolarizing media, resulted in granule cell death that was enhanced by ethanol. Serum removal increased both cleavage of the caspase-specific substrate N-acetyl-Asp-Glu-Val-Asp-7 amino-4-methylcoumarin (Ac-DEVD-amc) and the amount of active caspase-3. Inclusion of ethanol during the serum deprivation augmented Ac-DEVD-amc cleavage without further increasing the amount of active caspase-3. This study demonstrates that when neurotrophic factors are limiting, ethanol is toxic to cerebellar granule cells regardless of maturation status. The ability of ethanol to promote apoptosis involves an increase in caspase activity, but this does not entail an increase in the proteolytic activation of caspase-3.     

Glutathione levels modulate domoic acid induced apoptosis in mouse cerebellar granule cells.

Exposure of mouse cerebellar granule neurons (CGNs) to domoic acid induced cell death, either by apoptosis or by necrosis, depending on its concentration. Necrotic damage predominated in response to domoic acid above 0.1 microM. In contrast, cell injury with apoptotic features (assessed by Hoechst staining and DNA laddering assay) was evident after exposure to lower concentrations of domoic acid (< or = 0.1 microM). The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate receptor antagonist 2,3-dihydroxy-6-nitro-sulfamoylbenzo [f] quinoxaline, but not the N-methyl-D-aspartate receptor antagonist MK-801, prevented domoic acid-induced apoptosis. To evaluate the role of oxidative stress in domoic acid-induced apoptosis, experiments were carried out in CGNs isolated from wild-type mice (Gclm (+/+)) and mice lacking the modifier subunit of glutamate-cysteine ligase, the first and rate-limiting step of glutathione (GSH) biosynthesis (Gclm (-/-)). CGNs from Gclm (-/-) mice have very low levels of GSH and were more sensitive to domoic acid-induced apoptosis and necrosis than Gclm (+/+) CGNs. The antioxidant melatonin (200 microM) and the membrane-permeant GSH delivery agent GSH ethyl ester (2.5 mM) prevented domoic acid-induced apoptosis. Domoic acid increased formation of reactive oxygen species but did not affect intracellular GSH levels. Domoic acid also increased cytosolic and mitochondrial calcium levels, increased oxidative stress in mitochondria, and altered mitochondrial membrane potential, which ultimately caused cytochrome c release, activation of caspase-3, and degradation of poly (ADP-ribose) polymerase. These results indicate that low concentrations of domoic acid cause apoptotic neuronal cell death mediated by oxidative stress.     

Cyclosporin A enhances colchicine-induced apoptosis in rat cerebellar granule neurons

1. Cyclosporin A (CsA, 1-50 microM), an immunosuppressive drug with known neurotoxic effects, did not decrease the viability of primary cultures of rat cerebellar granule neurons (CGN) or induce apoptotic features. However, CsA specifically enhanced the cytotoxicity and apoptosis induced by colchicine (1 microM). 2. Flavopiridol, an inhibitor of cyclin-dependent kinases (CDKs), prevented the neurotoxic effects of colchicine plus CsA. At 0.1-5 microM, it also showed antiapoptotic effects, as revealed by propidium iodide staining, flow cytometry and counting of cell nuclei. 3. Roscovitine (25-50 microM), a selective cdk1, 2 and 5 inhibitor, showed an antiapoptotic effect against colchicine- and colchicine plus CsA-induced apoptosis. 4. CsA increased the expression of cdk5 and cdk5/p25 mediated by colchicine, a CDK involved in neuronal apoptosis. After treatment of CGN with colchicine plus CsA, the changes in the p25/p35 ratio pointed to cdk5 activation. 5. Immunohistochemical results showed a nuclear localization of cdk5 after neurotoxic treatment, which was prevented by cdk inhibitors. Thus, we propose a new mechanism of modulation of CsA neurotoxicity mediated by cdk5.     

普卡霉素对大鼠小脑颗粒神经元低钾性凋亡的抑制作用(英文)

目的观察普卡霉素对大鼠小脑颗粒神经元低钾性凋亡的保护作用。方法建立大鼠小脑颗粒神经元低钾性凋亡模型,采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记,Hoechst 33258核染色,琼脂糖凝胶电泳以及醋酸荧光素染色等方法,根据细胞凋亡的形态学及生物化学等特征,分析普卡霉素对大鼠小脑颗粒神经元低钾性凋亡的影响。结果大鼠小脑颗粒神经元与普卡霉素预孵育1 h及持续作用下,普卡霉素浓度依赖性地抑制低钾诱导24 h所致的大鼠小脑颗粒神经元凋亡,其有效浓度在50~200 nmol.L-1之间,普卡霉素浓度为200nmol.L-1时达到的最大凋亡抑制率约为80%。结论普卡霉素对大鼠小脑颗粒神经元低钾性凋亡具有较强的抑制作用。     

缺氧复氧诱导的大鼠海马神经元凋亡和一氧化氮合酶表达变化及银杏叶提取物的作用

目的:观察银杏叶提取物(EGb)及其活性成份银杏总内酯(Gin)对缺氧再复氧诱导的大鼠海马神经元凋亡及一氧化氮合酶(nitricoxidesynthase,NOS)表达影响。方法:选用胎龄15d的SD胎鼠的大脑海马细胞进行原代培养,建立缺氧再复氧海马神经元损伤模型。实验分以下几组:正常组、缺氧复氧组、EGb组、Gin组、阳性对照组(MK801组)。应用TUNEL原位末端标记法定量分析细胞凋亡以及NADPHd组织化学染色法观察细胞NOS的表达。结果:(1)TUNEL免疫细胞化学染色结果显示,预先加入EGb和Gin的药物组神经元的凋亡率明显低于缺氧复氧组;(2)NADPHd组织化学染色结果显示,预先加入EGb和Gin的药物组抑制神经元NOS表达,NADPH阳性细胞减少。结论:EGb及其活性成份Gin对缺氧再复氧损伤的海马神经元具有保护作用,可部分拮抗缺氧再复氧条件下体外培养的海马神经元的凋亡,抑制NOS的表达。     

Mechanisms of the apoptotic and necrotic actions of trimethyltin in cerebellar granule cells.

In evaluating mechanisms of trimethyltin (TMT)-initiated neuronal damage, the present study focused on involvement of reactive oxygen species, protein kinase C (PKC), and glutamate receptors. Exposure of cerebellar granule cells to TMT (0.01-0.1 microM) produced primarily apoptosis, but higher concentrations were associated with cellular lactate dehydrogenase efflux and necrosis. TMT increased generation of cellular reactive oxygen species, which was inhibited by either L-NAME (inhibitor of nitric oxide synthase, NOS) or catalase, indicating that both NO and H(2)O(2) are formed on TMT exposure. Since chelerythrine (selective PKC inhibitor) also inhibited oxidative species generation, PKC appears to play a significant role in TMT-induced oxidative stress. The metabotropic glutamate receptor antagonist, MCPG, (but not MK-801) prevented oxidative species generation, indicating significant involvement of metabotropic receptors (but not NMDA receptors) in TMT-induced oxidative stress. NOS involvement in the action of TMT was confirmed through measurement of nitrite, which increased concentration dependently. Nitrite accumulation was blocked by L-NAME, chelerythrine, or MCPG, showing that NO is generated by TMT and that associated changes in NOS are regulated by a PKC-mediated mechanism. Oxidative damage by TMT was demonstrated by detection of elevated malondialdehyde levels. It was concluded that low concentrations of TMT (0.01-0.1 microM) cause apoptotic cell death in which oxidative signaling is an important event. Higher concentrations of TMT initiate necrotic death, which involves both an oxidative and a non-oxidative component. TMT-induced necrosis but not apoptosis in granule cells is mediated by glutamate receptors.     

Genistein and daidzein prevent low potassium-dependent apoptosis of cerebellar granule cells

We have investigated the ability of certain dietary flavonoids, known to exert beneficial effects on the central nervous system, to affect neuronal apoptosis. We used cerebellar granule cells undergoing apoptosis due to potassium deprivation in a serum-free medium in either the absence or presence of the flavonoids genistein and daidzein, which are present in soy, and of catechin and epicatechin, which are present in cocoa. These compounds were used in a blood dietary concentration range. We found that genistein and daidzein, but not catechin and epicatechin, prevented apoptosis, with cell survival measured 24 h after the induction of apoptosis being higher than that of the same cells incubated in flavonoid free medium (80% and 40%, respectively); there was no effect in control cells. A detailed investigation of the effect of these compounds on certain mitochondrial events that occur in cells en route to apoptosis showed that genistein and daidzein prevented the impairment of glucose oxidation and mitochondrial coupling, reduced cytochrome c release, and prevented both impairment of the adenine nucleotide translocator and opening of the mitochondrial permeability transition pore. Interestingly, genistein and daidzein were found to reduce the levels of reactive oxygen species, which are elevated in cerebellar granule cell apoptosis. These findings strongly suggest that the prevention of apoptosis depends mainly on the antioxidant properties of genistein and daidzein. This could lead to the development of a flavonoid-based therapy in neuropathies.     

茶碱对低钾诱导的小脑颗粒神经元凋亡的保护作用

目的　研究茶碱对复极化浓度钾离子 (5mmol·L-1KCl,又称低钾 )诱导的小脑颗粒神经元凋亡是否具有保护作用。方法　体外培养大鼠小脑颗粒神经元 ,定性定量检测细胞凋亡 :①用FDA (fluoresceindiacetate)染色检测细胞存活率 ,用Hoechst 332 5 8染色 ,分析细胞核的形态变化 ;②琼脂糖凝胶电泳分析DNA断裂 ;③免疫荧光法检测细胞内cAMP水平。结果　茶碱使低钾培养的小脑颗粒神经元的存活率增加 ,使低钾引起的细胞核固缩、凝聚和断裂现象消失 ;低钾使神经元的DNA电泳图谱出现明显的“梯子状” ,而茶碱使此现象消失 ;敏感性内钙释放阻断剂、L 型钙通道阻断剂及NMDA受体阻断剂均不能抑制茶碱对神经元的保护作用 ;茶碱对细胞内cAMP水平没有明显的影响。结论　茶碱对复极化浓度钾离子诱导的小脑颗粒神经元凋亡具保护作用 ,此作用不依赖胞内钙离子浓度 ,也不通过升高胞内cAMP水平来实现     

三氧化二砷诱导Hela细胞凋亡的机制研究

目的:了解三氧化二砷(As2O3)诱导的细胞凋亡是否与线粒体跨膜电位(Δψm)改变有关。方法:以Hela细胞为体外模型,应用碘化丙啶(PI)/Rhodamine123(Rh123)双重染色检测Δψm,通过测定细胞活力、流式细胞仪、HE染色及DNA电泳检测细胞凋亡。结果:As2O3处理Hela细胞后。台盼蓝拒染法测定细胞活力降低,流式细胞仪检测发现凋亡细胞明显增多,HE染色可见明显的细胞凋亡形态特征,琼脂糖凝胶电泳出现DNA梯形条带;As2O3使线粒体膜电位降低(P<0.01)。结论:As2O3在体外诱导Hela细胞凋亡的机制可能与降低线粒体膜电位有关。     

土槿乙酸衍生物PBA-D1诱导Hela细胞凋亡及其机制

目的研究土槿乙酸衍生物PBAD1体外能否诱导宫颈癌细胞株(Hela细胞)发生凋亡及其发生机制。方法采用MTT、SRB、细胞生长曲线、细胞集落、荧光显微镜检测凋亡细胞、DNAladder、流式细胞仪等方法进行凋亡检测,用Westernblot方法检测凋亡过程中相关基因表达的变化。结果土槿乙酸衍生物PBAD1在体外试验中对Hela细胞的杀伤力强,荧光显微镜观察到了凋亡小体;DNAladder法检测到凋亡时DNA降解形成的梯带,流式细胞仪检测到了细胞凋亡峰,同时观察到细胞周期的变化。在凋亡过程中,凋亡相关基因p53基因表达明显增高,而bcl2表达下调。结论PBAD1体外能诱导Hela细胞发生凋亡,其机制可能与p53基因表达上调及bcl2基因表达下调有关。     

Facilitative effects of EGb 761 on olfactory recognition in young and aged rats

The aim of the present study was to evaluate the effects of chronic and acute treatment by the Gingko biloba extract, EGb 761 (IPSEN, France) on olfactory short-term memory in rats, using a spontaneous recognition procedure. The effects of a daily EGb 761 treatment (30 or 60 mg/kg) over a period of 30 days (Experiment 1) were evaluated in young male rats. Those of a single injection of EGb 761 were assessed either in young male rats at 60 or 120 mg/kg (Experiment 2) or in aged female rats at 60 mg/kg (Experiment 3). Results showed that, at the highest dose (60 mg/kg), chronic EGb 761 treatment enhanced the recognition performances, allowing recognition at delays at which control animals did not show any recognition. Acute treatment enhanced recognition at both doses tested. The results of the third experiment showed that EGb 761 had an overall enhancement effect on the performances of aged rats. In summary, our results provide evidence for a short-term memory enhancement effect of EGb 761 in both young and aged rats.     

银杏叶提取物对缺氧复氧诱导的大鼠皮层神经元凋亡和Bcl-2蛋白表达的影响

目的 观察银杏叶提取物（EGb）及其活性成份银杏总内酯（Gin）对缺氧复氧诱导的大鼠皮层神经元凋亡的保护作用。方法 选用胎龄14～16d的SD胎鼠的大脑皮层细胞进行原代培养,建立缺氧复氧（H／R）神经元损伤模型。实验分为正常组、缺氧复氧（H／R）组、药物实验（EGb、Gjn）组、阳性对照（MK-801）组。应用MTF、TUNEL及Western blot方法。结果 EGb和Gin均能提高皮层神经元缺氧复氧后的存活率,EGb和Gin组神经元的凋亡率低于H／R组且Bcl-2蛋白表达量较H／R组升高。结论 EGb和Gin可部分拮抗缺氧复氧条件下体外培养的皮层神经元的凋亡,该保护作用与其诱导Bcl-2蛋白表达有关。     

Iron-induced oxidative damage and apoptosis in cerebellar granule cells: attenuation by tetramethylpyrazine and ferulic acid

Tetramethylpyrazine and ferulic acid are two active ingredients of a Chinese herbal medicine Ligusticum wallichi Franchat. In the present investigation, iron-induced oxidative neuronal damage and the protective effects of tetramethylpyrazine and ferulic acid against this induction were studied in primary cultures of rat cerebellar granule cells. When neurons were treated with 200 microM of FeSO(4) for 1 h, lipid peroxidation in neurons increased time dependently, as measured with the thiobarbituric acid assay. Thirty-six hours after iron treatment, the cell viability decreased to 43.6% and the percentage of apoptotic cells increased to 50.6%. Transmission electron microscopic examination showed a disrupted nuclear envelope and condensed chromatin in iron-treated neurons. Analysis of DNA extracted from iron-treated cells by agarose gel electrophoresis showed the typical "ladder pattern", which indicated the formation of mono- and oligonucleosomes. After iron treatment, caspase 3 activity increased significantly, as measured in a fluoregenic assay. The results above suggested that iron treatment triggered oxidative stress and apoptosis in neurons. Western blot revealed that iron treatment up-regulated the apoptosis-related gene p53 as well as its effector gene p21(waf1/cip1). Pretreatment of the cells with 100 microM of tetramethylpyrazine or ferulic acid effectively decreased the activation of caspase 3 as well as the expression of p53 and p21(waf1/cip1), and attenuated iron-induced oxidative damage and apoptosis. The results suggest that tetramethylpyrazine and ferulic acid might be used as preventive agents against neuronal diseases associated with oxidative stress.     

Measurement of GABAA receptor function in rat cultured cerebellar granule cells by the Cytosensor microphysiometer

1. γ-Aminobutyric acid (GABA), acting via the GABA_A receptor, increased the extracellular acidification rate of rat primary cultured cerebellar granule cells, measured by the Cytosensor microphysiometer.
2. The optimal conditions for the measurement of GABA_A receptor function in cerebellar granule cells by microphysiometry were: cells seeded at 9–12×10⁵ cells/transwell cup and maintained in vitro for 8 days, GABA stimulation performed at 25°C, with a stimulation time of 33 s.
3. GABA stimulated a concentration-dependent increase in the extracellular acidification rate with an EC₅₀ of 2.0±0.2 μM (mean±s.e.mean, n=7 experiments) and maximal increase (E_max) over basal response of 15.4±1.2%.
4. The sub-maximal GABA-stimulated increase in acidification rate could be potentiated by the 1,4-benzodiazepine, flunitrazepam (100 nM). The 10 nM GABA response showed the maximal benzodiazepine facilitation (GABA alone, 1.4 μV s⁻¹, GABA+flunitrazepam, 3.8 μV s⁻¹, mean increment over basal, n=7).
5. The GABA-stimulated increase in acidification rate was inhibited by the GABA_A antagonist, bicuculline (100 μM) (90% inhibition at 1 mM GABA).
6. The results of this study show that activation of GABA_A receptors in rat cerebellar granule cells caused an increase in the extracellular acidification rate; an effect which was potentiated by benzodiazepines and inhibited by a GABA_A receptor antagonist. This paper defines the conditions and confirms the feasibility of using microphysiometry to investigate GABA_A receptor function in primary cultured CNS neurones. The microphysiometer provides a rapid and sensitive technique to investigate the regulation of the GABA_A receptor in populations of neurones.

     

诱导型HSP70过表达对低钾诱导的小脑颗粒神经元凋亡的保护作用

目的观察腺病毒介导的人诱导型HSP70过表达对低钾诱导的原代培养的大鼠小脑颗粒神经元(cerebellargranuleneurons,CGN)凋亡的影响。方法原代培养5d的CGN共感染含人诱导型HSP70和绿色荧光蛋白(GFP)的腺病毒(AdTR5/HSP70-GFP)和四环素调控的启动子(AdCMV/tTA),或共感染含GFP的腺病毒(AdTR5/GFP)和AdCMV/tTA(对照组)。48h后,采用细胞荧光免疫组织化学法、Westernblot法检测HSP70的表达,或者换成无血清含5mmol·L-1KCl的培养基以诱导神经元凋亡。24h后,采用相差显微镜观察细胞形态学变化,MTT法检测神经元存活率,Hoechst33258核染色和DNA琼脂糖凝胶电泳分析神经元凋亡,以观察HSP70过表达对低钾诱导的CGN凋亡的影响。结果共感染了AdCMV/tTA和AdTR5/HSP70-GFP的CGN过表达了HSP70,抑制了低钾诱导的CGN的凋亡:使神经元存活率由45·5%±5·2%提高至82·3%±5·2%(P<0·01),核固缩减少,DNA的片段化减轻。结论腺病毒介导的人诱导型HSP70的过表达抑制了低钾诱导的CGN凋亡。