拉莫三嗪对癫痫大鼠学习记忆及神经细胞凋亡的影响研究

目的 观察拉莫三嗪(lamotrigine,LTG)对戊四氮(PTZ)所致慢性癫痫大鼠学习记忆及神经细胞凋亡的影响,为LTG改善认知功能提供理论依据.方法 成年健康SD大鼠30只,随机分为对照组、模型组和LTG干预组各10只,采用35mg/kg PTZ溶液经腹腔注射制备癫痫模型.4w后对大鼠进行空间学习能力检测,应用免疫组化染色及原位细胞凋亡检测脑组织Bcl-2、Bax蛋白表达阳性细胞数及凋亡细胞数.结果 癫痫大鼠的学习记忆功能较正常大鼠明显下降(P＜0.05),LTG干预组海马区凋亡细胞明显减少(P＜0.01),Bcl-2蛋白阳性细胞数显著增加(P＜0.01),Bax蛋白阳性细胞数显著减少(P＜0.05).结论癫痫后大鼠的学习记忆功能受损,应用LTG可改善这一损害,可能与其能上调Bcl-2蛋白表达、下调Bax蛋白表达有关.     

右美托咪定调节MAPK/ERK-CREB通路对大鼠海马神经元凋亡的保护作用

目的探讨右美托咪定(DEX)调节MAPK/ERK-CREB通路对大鼠海马神经元凋亡的保护作用。方法通过腹腔注射氯化锂-毛果芸香碱构建癫痫持续状态(SE)大鼠模型,并随机分为4组,每组各10只。SE+DEX组在SE模型构建成功后腹腔注射DEX 1μmol/L,阳性对照组腹腔注射1μmol/L苯巴比妥,药物干预24 h后,通过Nissl法和TUNEL法检测大鼠海马神经元损伤及凋亡情况,Western blot法检测大鼠海马组织中MAPK、p ERK、p CREB蛋白和凋亡相关蛋白caspase-3、Bcl-2、Bax表达。结果与正常对照组相比,SE、阳性对照组、SE+DEX组大鼠Racine分值显著增加(P 0. 05),大鼠海马神经元数、Bcl-2蛋白表达量显著减少(P 0. 05),棕褐色TUNEL阳性细胞数、MAPK、p-ERK、p-CREB、caspase-3、Bax、Bax/Bcl-2蛋白表达量显著增加(P 0. 05)。与SE组相比,阳性对照组、SE+DEX组大鼠Racine分值显著降低(P 0. 05),大鼠海马神经元数、Bcl-2蛋白表达量显著增加(P 0. 05),棕褐色TUNEL阳性细胞、MAPK、p-ERK、p-CREB、caspase-3、Bax、Bax/Bcl-2蛋白表达量显著减少(P 0. 05)。结论 DEX可能通过抑制MAPK/ERK-CREB通路抑制海马神经元凋亡对其有保护作用。     

促红细胞生成素对血管性痴呆大鼠模型海马神经细胞凋亡的影响

目的:探讨促红细胞生成素(EPO)对血管性痴呆(VaD)模型大鼠海马神经细胞凋亡的影响。方法:将经行为学筛选合格的Wistar大鼠分为假手术组、VaD组、EPO组,采用永久性结扎双侧颈总动脉的方法建立VaD模型。应用TUNEL染色检测大鼠海马的凋亡细胞数;通过免疫组化学和RT-PCR方法,检测大鼠海马Bcl-2和Bax蛋白以及其mRNA表达水平的变化。结果:EPO能明显抑制海马神经细胞凋亡,上调Bcl-2表达,抑制Bax表达。结论:EPO可能通过调控VaD大鼠海马Bcl-2和Bax表达,从而抑制神经细胞凋亡。     

吸氧预处理对大鼠局灶脑缺血神经细胞凋亡及Bcl-2、Bax的影响

目的 观察吸氧预处理对大鼠局灶脑缺血再灌注后细胞凋亡及凋亡相关基因Bax、Bcl-2的关系,探讨吸氧预处理的脑保护作用.方法 采用线栓法阻塞大鼠大脑中动脉造成局灶脑缺血再灌注模型,用TUNEL染色标记神经细胞凋亡,免疫组化染色观察Bax,Bcl-2蛋白含量.结果 与模型组相比,吸氧预处理组半暗区的TUNEL阳性细胞数和Bax蛋白表达阳性细胞数明显下降,(P<0.05),Bcl-2蛋白表达细胞数明显上升(P<0.05).结论 吸氧预处理可通过抑制缺血再灌注后神经细胞凋亡和减少Bax的表达,增加Bcl-2蛋白的表达发挥脑保护作用.     

碱性成纤维细胞生长因子对脑出血大鼠神经细胞凋亡和BaX、Bcl-2基因表达的影响

目的观察碱性成纤维细胞生长因子(bFGF)对大鼠脑出血后神经细胞凋亡及血肿周围大脑皮质和出血侧海马Bax、Bcl-2基因表达的影响。方法采用脑内注射胶原酶建立大鼠脑出血模型,应用TUNEL法测定大鼠大脑皮质、海马神经细胞凋亡数目。及应用采用半定量逆转录酶聚合链反应(RT-PCR),检测脑出血后血肿周围大脑皮质和出血侧海马的Bax mRNA、Bcl-2 mRNA表达。结果(1)bFGF组的皮质和海马神经细胞凋亡数比生理盐水(NS)对照组明显减少(P<0.05)。(2)bFGF组的血肿周围大脑皮质和出血侧海马Bax mRNA表达比NS对照组明显减少(P<0.05);而Bcl-2 mRNA表达比NS对照组明显增高(P<0.05)。结论bFGF提高大鼠脑出血后大脑组织和海马Bcl-2 mRNA的表达,降低Bax mRNA的表达,抑制脑出血后神经细胞凋亡。     

雌激素对血管性痴呆大鼠认知功能、海马Bcl-2、Bax蛋白表达及神经元凋亡的影响

目的 探讨雌激素对血管性痴呆(VD)大鼠认知功能、海马Bcl-2、Bax蛋白表达和神经元凋亡的影响.方法 60只雄性大鼠随机分为假手术组、VD组和雌激素组,每组20只.采用双侧颈总动脉结扎法制备VD大鼠模型;雌激素组腹腔注射17-β雌二醇(花生油溶解)200 μg/kg,同时假手术组和VD组腹腔注射等量的花生油,均为隔日1次,共30次.60 d后,Y-型迷宫试验检测大鼠学习记忆能力;HE染色观察大鼠海马神经元形态;免疫组化染色检测海马Bcl-2、Bax蛋白的表达,原位缺口末断标记(TUNEL)法检测神经元凋亡程度.结果 与假手术组相比,VD组及雌激素组认知能力明显下降, 海马CA1区Bcl-2、Bax蛋白表达增加,TUNEL 阳性细胞数明显增多(均P<0.001);与VD组比较,雌激素组认知能力改善,海马CA1区Bcl-2蛋白表达明显增加,Bax蛋白的表达明显减少,TUNEL 阳性细胞明显减少 (均P<0.001).结论 17-β雌二醇可调节Bcl-2、Bax蛋白表达而抑制神经元凋亡,有助于改善VD大鼠的认知能力.     

下调GABA受体基因对癫痫模型大鼠海马神经细胞凋亡的影响及作用机制

目的 探讨下调GABA受体基因对癫痫模型大鼠海马神经细胞凋亡的影响及作用机制。方法 选择30只SD健康雄性大鼠,建立癫痫模型,建模成功后分为模型组、上调组、下调组各10只; 进行细胞转染建立稳定转染的GABA上调组、GABA下调组; 检测3组大鼠海马神经细胞凋亡及PI3K、AKt、mTOR、Bax、Caspase-3蛋白表达水平。结果 上调组大鼠各时间点海马神经细胞凋亡率高于模型组(P<0.05); 下调组大鼠各时间点海马神经细胞凋亡率低于模型组(P<0.05); 下调组大鼠各时间点海马神经细胞凋亡率低于上调组(P<0.05)。上调组大鼠各时间点海马神经细胞增殖率、Bcl-2、p-PI3K、p-AKt、p-mTOR蛋白表达水平均低于模型组,而Bax、Caspase-3蛋白表达水平高于模型组(P<0.05); 下调组大鼠各时间点海马神经细胞增殖率、Bcl-2、p-PI3K、p-AKt、p-mTOR蛋白表达水平均高于模型组,而Bax、Caspase-3蛋白表达水平低于模型组(P<0.05); 下调组大鼠各时间点海马神经细胞增值率、Bcl-2、p-PI3K、p-AKt、p-mTOR蛋白表达水平均高于上调组,而Bax、Caspase-3蛋白表达水平低于上调组(P<0.05)。结论 下调GABA受体基因可能是通过调节PI3K/AKt/mTOR来作用于下游凋亡靶基因,最终抑制癫痫模型大鼠海马神经细胞凋亡     

颞叶癫痫患者致痫灶中凋亡调控基因Bcl-2和Bax的表达

目的 探讨神经元凋亡与颞叶癫痫患者海马硬化的关系.方法 取16例颞叶癫痫患者手术切除标本,用光镜、电镜及原位末端标(TUNEL)方法检测神经细胞凋亡;应用免疫组化方法检测细胞凋亡相关基因表达产物Bax和Bcl-2蛋白的表达.结果 对照组患者光镜、电镜检查及TUNEL染色均未发现凋亡的神经细胞;癫痫组患者光镜检查及TUNEL染色未发现凋亡的神经细胞,但在电镜检查的3例标本中,1例有少量早期凋亡征象的神经元.免疫组化染色结果表明,对照组患者脑组织内Bcl-2蛋白无表达,癫痫组患者脑内Bcl-2蛋白表达明显增强(P<0.001);Bax蛋白在癫痫组与对照组中均微弱表达,两者差异无统计学意(P>0.05).结论 神经元凋亡部分参与了癫痫患者海马硬化的形成,Bcl-2和Bax 蛋白在这一过程中可能发挥了作用.     

局灶性脑缺血再灌注大鼠海马CA1区神经元凋亡研究

目的 观察大鼠局灶性脑缺血再灌注(ischemia reperfusion,I/R)损伤后海马CA1区神经元凋亡、TUNEL阳性细胞变化,以及凋亡相关蛋白Bcl-2与Bax蛋白的表达情况.方法 将健康雄性SD (Sprague-Dawley)大鼠随机分为假手术组和I/R组,每组再分为缺血再灌注后3、6、12、24、48、72 h亚组.应用免疫组化方法检测再灌注后不同时间点大鼠海马CA1区神经元凋亡基因Bcl-2和Bax蛋白的表达及Bcl-2/Bax比值变化,采用原位细胞凋亡检测(TUNEL)技术检测凋亡阳性细胞数.结果 各组非缺血侧相应区域神经元胞质中Bcl-2均有微弱表达.I/R组缺血侧海马CA1区于再灌注3 h开始出现Bcl-2和Bax蛋白微弱表达,随再灌注时间延长神经元内Bcl-2表达逐渐增强,再灌注24 h后Bcl-2表达达高峰,假手术组与I/R组比较差异有统计学意义(P<0.05).结论 I/R损伤后海马CA1区神经元不仅存在变性坏死,还存在明显的细胞凋亡且细胞凋亡在大鼠I/R损伤中发挥重要作用;I/R可诱导海马CA1区细胞凋亡基因Bcl-2和Bax蛋白表达,且其表达呈一定规律.     

经鼻给予TGFβ1对氯化锂-匹罗卡品诱导的SE大鼠海马神经元凋亡调控因子Bcl-2、Bax蛋白表达的影响

目的探讨经鼻给予TGFβ1(Transforming growth factor-beta1,TGFβ1)对氯化锂-匹罗卡品诱导的癫痫持续状态(status epilepticus,SE)大鼠海马神经元凋亡调控因子Bcl-2、Bax蛋白表达的影响。方法健康雄性SD大鼠60只,随机分为TGF组、Pilo组和正常对照组(Control)。建立氯化锂-匹罗卡品癫痫持续状态模型。采用免疫组化方法检测凋亡相关基因Bcl-2、Bax的蛋白表达。结果 (1)SE后24h、48h、72h,TGF组大鼠海马Bax阳性细胞均较Pilo组显著减少(P<0.05);72h最为明显(P<0.01)。HE染色是对各组大鼠海马神经元的形态结构变化的大体观察。(2)SE后24h、48h、72h,TGF组大鼠海马Bcl-2阳性细胞均较Pilo组显著增加(P<0.05);24h最为明显(P<0.01)。结论经鼻(IN)给予TGFβ1可以显著抑制癫痫持续状态诱导的大鼠海马神经元Bax蛋白的表达,上调Bcl-2蛋白表达,从而发挥神经保护作用。     

Influence of Ganoderma lucidum spores on the levels of neuropeptide Y and somatostatin in brains of seizure rats

BACKGROUND: Previous research has revealed that somatostatin can induce epilepsy, and that the levels of neuropeptide Y may increase and become more active in brain areas with epileptic seizures.OBJECTIVE: To observe the effect of Ganoderma lucidum spore powder on the neuropeptide Y and somatostatin content in the cerebral cortex and hippocampal regions of seizure rats induced by pentylenetetrazol (PTZ). Furthermore, to verify any effect of Ganoderma lucidum spore powder on inhibition of epileptic seizures.DESIGN, TIME AND SETTING: A randomized group animal study was performed in August 2007 in the School of Basic Medical Sciences, Jiamusi University (Jiamusi, Heilongjiang, China).MATERIALS: Thirty healthy, male, Wistar rats, aged 12 weeks and weighing 180-220g, were taken as the experimental animals. PTZ (Sigma Company, United States) was used to induce epilepsy. Ganoderma lucidum spores (Leyss, ex Fr variety) were purchased from Jiamusi City Wild Growing Case of the Ganoderma Lucidum (China). Rabbit anti-somatostatin antibodies and secondary antibodies were purchased from Wuhan Boster Company (China). Neuropeptide Y radioimmunoassay kit was purchased from Beijing Furui Biotechnology Company (China).METHODS: Thirty rats were randomly divided into three groups: a control group, an epilepsy model group and a Ganoderma lucidum spore-treated group. Each group contained 10 animals. Rats in the epilepsy model group were treated with intraperitoneal injections of PTZ and gastric perfusion of physiologic saline, In the Ganoderma lucidum spore-treated group, intraperitoneal injection of PTZ and gastric perfusion of Ganoderma lucidum spore powder were administered. The blank control group was only administered with the physiological saline by intraperitoneal injection and gastric perfusion.MAIN OUTCOME MEASURES: Immunohistochemical staining and radioimmunoassay methods were used to observe the changes of somatostatin and neuropeptide Y content in brain tissue of epileptic rats, as well as the morphology of neurons.RESULTS: All 30 rats were involved in the result analysis, without any loss. The number of somatostatin immunoreacted positive cells in the cerebral cortex and hippocampus was significantly increased in the epilepsy model group compared to the blank control group (P<0.01). The number of somatostatin immunoreacted positive cells in cerebral cortex and hippocampus was significantly decreased in the Ganoderma lucidum spore-treated group compared to the epilepsy model group (P<0.01). The contents of neuropeptide Y in cerebral cortex and hippocampus were significantly increased in the epilepsy model group compared to the blank control group (P<0.01). The contents of neuropeptide Y in the cerebral cortex and hippocampus were significantly decreased in the Ganoderma lucidum spore-treated group compared to the epilepsy model group (P<0.05). The epilepsy seizures in the Ganoderma lucidum spore-treated group were obviously reduced compared to the epilepsy model group.CONCLUSION: Ganoderma lucidum spore powder was able to reduce the somatostatin and neuropeptide Y content in the cerebral cortex and hippocampus effectively, so as to achieve an anti-epileptic function and protect neurons from being damaged.     

普瑞巴林对慢性颞叶癫癎大鼠海马凋亡调控基因的影响

目的通过观察普瑞巴林对匹罗卡品慢性癫癎大鼠海马区Bcl-2和Bax表达的影响,探讨普瑞巴林治疗癫癎的药理学机制及对大鼠海马神经元的抗凋亡作用。方法采用氯化锂-匹罗卡品化学诱导方法建立慢性颞叶癫癎模型。经腹腔注射普瑞巴林40mg(/kg·d)连续治疗3周,免疫组织化学染色和Western blotting法检测不同处理组大鼠海马区Bcl-2和Bax表达变化。结果与生理盐水对照组比较,模型组大鼠海马区Bcl-2和Bax表达水平显著升高(均P=0.000);与模型组比较,普瑞巴林治疗组大鼠海马区Bcl-2表达水平升高、Bax表达水平降低,组间差异具有统计学意义(均P=0.000)。结论新型抗癫癎药物普瑞巴林可通过降低慢性颞叶癫癎大鼠海马区Bax表达、上调Bcl-2表达而抑制细胞凋亡,发挥神经元保护作用。     

大鼠脑创伤后海马CA3区细胞凋亡及相关基因表达研究

目的研究弥漫性脑损伤后不同时间,大鼠海马CA3区细胞凋亡及相关基因Bcl-2、Bax和Caspase-3蛋白的表达情况,探讨脑创伤后神经细胞凋亡的分子生物学机制.方法应用流式细胞仪和免疫组化法,分别检测脑创伤后不同时间海马CA3区细胞凋亡率及Bcl-2,Bax和Caspase-3基因在蛋白质水平的表达情况.结果脑创伤后海马CA3区存在不同程度细胞凋亡,Bcl-2在脑损伤后表达下降,而Bax和Caspase-3在脑创伤后表达升高;Caspase-3表达的峰值时间(72 h)出现在Bax之后(48 h).结论弥漫性脑损伤后,大鼠海马CA3区存在细胞凋亡及Bcl-2,Bax和Caspase-3的表达变化.Bcl-2/Bax表达比值下降早于Caspase-3的上升,Bcl-2/Bax表达比值改变可能与Caspase-3活化有关,进而启动并加重脑损伤后神经细胞凋亡.     

Neuroprotective effects of ganoderma lucidum polysaccharides against oxidative stress-induced neuronal apoptosis

Ganoderma lucidum polysaccharides have protective effects against apoptosis in neurons exposed to ischemia/reperfusion injury, but the mechanisms are unclear. The goal of this study was to investigate the underlying mechanisms of the effects of ganoderma lucidum poly-saccharides against oxidative stress-induced neuronal apoptosis. Hydrogen peroxide (H2O2) was used to induce apoptosis in cultured cerebellar granule cells. In these cells, ganoderma lucidum polysaccharides remarkably suppressed H2O2-induced apoptosis, decreased ex-pression of caspase-3, Bax and Bim and increased that of Bcl-2. These findings suggested that ganoderma lucidum polysaccharides regulate expression of apoptosis-associated proteins, inhibit oxidative stress-induced neuronal apoptosis and, therefore, have significant neuropro-tective effects.     

Neuroprotective effect of pretreatment with ganoderma lucidum in cerebral ischemia/reperfusion injury in rat hippocampus

Ganoderma lucidum is a traditional Chinese medicine,which has been shown to have both anti-oxidative and anti-inflammatory effects,and noticeably decreases both the infarct area and neuronal apoptosis of the ischemic cortex.This study aimed to investigate the protective effects and mechanisms of pretreatment with ganoderma lucidum(by intragastric administration)in cerebral ischemia/reperfusion injury in rats.Our results showed that pretreatment with ganoder-ma lucidum for 3 and 7 days reduced neuronal loss in the hippocampus,diminished the content of malondialdehyde in the hippocampus and serum,decreased the levels of tumor necrosis fac-tor-αand interleukin-8 in the hippocampus,and increased the activity of superoxide dismutase in the hippocampus and serum.These results suggest that pretreatment with ganoderma lucidum was protective against cerebral ischemia/reperfusion injury through its anti-oxidative and an-ti-inflammatory actions.     

钙拮抗剂对大鼠缺血性脑损伤后Bcl-2和Bax基因表达的作用

目的探讨大鼠实验性脑缺血后海马组织Bcl-2和Bax基因的表达及尼莫地平预处理对Bcl-2和Bax基因表达的影响。方法采用线栓法建立大鼠脑缺血模型；尼莫地平预处理；逆转录聚合酶链式反应(RT-PCR)法测定其海马组织中Bcl-2和Bax mRNA。结果大鼠大脑中动脉阻断(MCAO)后，海马组织Bcl-2和Bax基因均被诱导表达，Bcl-2表达量持续升高，而Bax表达量在脑缺血24h达高峰，随后逐渐下降。在脑缺血后6h和24h，经尼莫地平预处理7d的大鼠，海马组织Bcl-2基因的表达水平较未经预处理的大鼠明显上调，而Bax基因表达水平则较未经预处理的大鼠明显下调。结论钙拮抗剂尼莫地平能有效地调控大鼠缺血性脑损伤后海马组织Bcl-2和Bax基因表达的水平，为干预脑卒中后基因表达提供依据。     

大鼠局灶性脑缺血再灌注后海马Bcl-2、Bax蛋白的表达

目的 探讨大鼠局灶性脑缺血再灌注后Bcl-2、Bax蛋白在海马表达的变化.方法 线栓法制作大鼠局灶性脑缺血再灌注模型,应用免疫组化染色检测Bcl-2、Bax蛋白表达,应用TUNEL法检测海马区细胞凋亡.结果 缺血再灌注2h后海马神经元Bcl-2、Bax蛋白开始表达,Bcl-2蛋白12h达高峰,Bax蛋白12h～24h达高峰,之后开始下降.再灌注2h后海马凋亡细胞开始表达,随着再灌注时间的延长,其表达不断增加.Bcl-2/ Bax的比率在再灌注开始时升高,再灌注12h达高峰,随后开始下降.结论 凋亡是脑缺血再灌注损伤的重要形式之一,Bcl-2/ Bax的改变与缺血再灌注后海马的神经元存亡有关,缺血再灌注可导致海马神经元凋亡.     

大鼠全脑缺血再灌注损伤后调控基因Bcl-2、Bax的表达及与细胞凋亡的关系

目的:本研究旨在探讨Bcl-2及Bax蛋白在大鼠全脑缺血再灌注损伤中的变化及与细胞凋亡的关系。方法:雄性Wistar大鼠56只,随机分为假手术组、缺血15分钟再灌注1、6、12、24、48、72小时组。采用大鼠四条血管阻断方法制备大鼠全脑缺血再灌注模型。采用TUNEL法观察不同再灌注时间组海马CAl区细胞凋亡的变化。采用免疫组化法观察Bcl-2及Bax蛋白表达水平的变化。结果:脑缺血损伤后随再灌注时间延长凋亡细胞逐渐增多,至再灌注48小时达到高峰,72小时后减少。Bcl-2表达至再灌注12小时达高峰,再灌注24~72小时组逐渐减弱。Bax表达至48小时达高峰,再灌注72小时减少。结论:Bcl-2于再灌注早期表达增强,Bax于再灌注中期表达增强,Bcl-2/Bax比例失衡可能是大鼠全脑缺血再灌注后神经细胞凋亡的机制之一。     

异丙酚预处理大鼠缺血再灌注脑皮质细胞的凋亡

The present study aimed to observe cortical expression of Bcl-2 and Bax,cysteine-dependent aspartate directed proteases-3 activity and apoptotic cell death in a rat model of middle cerebral artery occlusion pretreated with propofol.Results showed that,propofol pretreatment significantly reduced oxidative stress levels and attenuated neuronal apoptosis in the cortex of rats.Propofol pretreatment upregulated Bcl-2 expression,and downregulated Bax expression and cysteine-dependent aspartate directed proteases-3 activity.These findings indicate that propofol pretreatment inhibits cell apoptosis during focal cerebral ischemia/reperfusion injury.This neuroprotective effect is most likely achieved through the Bcl-2/Bax/cysteine-dependent aspartate directed proteases-3 pathway.