白果内酯对谷氨酸兴奋毒性致神经损害的保护作用

目的 观察不同剂量白果内酯对谷氨酸致海马神经元损害的影响,以探讨白果内酯在抗兴奋毒性神经损害中的应用价值.方法 原代培养新生SD大鼠海马神经元,建立谷氨酸诱导的兴奋毒性模型;采用台盼蓝染色、TUNEL染色凋亡神经元测定及乳酸脱氢酶(LDH)活性测定的方法观察不同剂量白果内酯的神经保护作用,并与谷氨酸NMDA(N-甲基-D-天门冬氨酸盐)受体非竞争性拮抗剂MK-801的神经保护作用相比较.结果 在一定剂量范围内,白果内酯可提高谷氨酸损伤的海马神经元细胞的存活率、降低细胞凋亡率、减少细胞中LDH的漏出,具有剂量依赖性,于100 μmol/L剂量下呈现最佳神经保护效果,但弱于MK-801(浓度为10 μmol/L).结论 白果内酯对谷氨酸诱导的兴奋毒性神经损害具有保护作用.     

银杏叶提取物对Aβ诱导神经毒性的保护作用

目的:研究不同剂量银杏叶(ginkgo biloba extract,EGb761)对Aβ诱导神经元损伤的影响。方法:取高、中、低剂量的EGb761质量浓度分别为100,10,1 mg·L-1预处理Aβ1-42干预的神经元,检测神经元活力、凋亡率及NeuN染色的变化。结果:与Aβ组相比,高和低剂量EGb761组的神经元活力和NeuN+细胞数存在显著差异(P<0.05),而中剂量EGb761组神经元活力和NeuN+细胞数均存在极显著差异(P<0.01);中剂量EGb761组的对神经元凋亡的保护作用优于高、低剂量EGb761组。结论:高、中、低剂量组银杏叶制剂均可提高损伤海马神经元活力、提高NeuN+细胞数,降低神经元凋亡率;尤以中剂量组显著。提示采用预处理的给药方式可保护Aβ诱导的神经元损伤。     

银杏叶提取物EGb761对海马神经元氧糖剥夺后神经元凋亡及Bcl-2蛋白表达的影响

目的:通过观察银杏叶提取物(extract of ginkgo biloba,EGb761)对氧糖剥夺处理的原代培养海马神经元凋亡及Bcl-2蛋白表达的影响,探讨EGb761对脑缺血的神经保护作用。方法:采用海马神经元培养氧糖剥夺损伤(OGD)模型,分为正常对照组、OGD组及EGb761组,观察神经元凋亡及抗凋亡蛋白Bcl-2表达情况。结果:EGb761组较OGD组能明显减少神经元凋亡,上调Bcl-2表达(P<0.05)。结论:EGb761对氧糖剥夺处理后的海马神经元具有明显保护作用。     

银杏叶提取物对H2O2诱导的胰岛RIN-mβ细胞凋亡的影响

目的:探讨银杏叶提取物(Ginkgo biloba extract,EGb761)对过氧化氢(Hydrogen peroxide,H2O2)诱导的胰岛RIN-mβ细胞株凋亡的影响。方法:以500μmol/L H2O2作用于胰岛RIN-mβ细胞6 h建立凋亡模型;分别设空白对照组(Control)、阴性对照组(H2O2)、阳性对照组(槲皮素Que 100μmol/L)、EGb 761单用对照组(EGb 761100μmol/L),EGb 761给药组(EGb 761 10、30、100μg/ml);采用MTT检测细胞存活率,Hoechst 33258染色荧光显微镜观察细胞形态变化,PI单染色法和Annexin V-PI双染色法流式细胞术分析细胞凋亡情况。结果:与空白对照组比较,阴性对照组500μmol/L H2O2作用6 h后,细胞存活率明显降低、细胞凋亡率显著升高(P<0.01)。与阴性对照组相比,EGb 761显著降低H2O2诱导的细胞凋亡(P<0.01),且呈剂量依赖性。结论:过氧化氢可诱导胰岛RIN-mβ细胞凋亡,EGb 761对H2O2诱导的RIN-mβ细胞损伤和凋亡具有明显的保护作用。     

栝楼桂枝颗粒对谷氨酸诱导大鼠肾上腺嗜铬细胞瘤细胞兴奋性毒性损伤的影响

目的观察栝楼桂枝颗粒对谷氨酸诱导大鼠肾上腺嗜铬细胞瘤细胞(PC12)兴奋性毒性损伤的影响,并初步探讨其作用机制。方法采用谷氨酸诱导PC12兴奋性毒性损伤造模。将细胞随机分为正常组、谷氨酸组和栝楼桂枝颗粒低(200μg/m L)、中(400μg/m L)、高(800μg/m L)剂量组,MTT法和LDH法检测PC12活力;Caspase-3活性检测法、Annexine V/PI双染色法检测细胞凋亡,Western blot和RT-PCR分别检测Bcl-2、Bax蛋白和m RNA表达。结果与谷氨酸组比较,MTT法栝楼桂枝颗粒各剂量组PC12活力升高,LDH法栝楼桂枝颗粒各剂量组细胞活力降低;Annexine V/PI双染色法栝楼桂枝颗粒各剂量组PC12凋亡率降低;Caspase-3活性检测法栝楼桂枝颗粒各剂量组Caspase-3活性降低;RT-PCR及Western blot检测栝楼桂枝颗粒各剂量组Bax表达降低、Bcl-2表达升高。结论栝楼桂枝颗粒对谷氨酸诱导PC12兴奋性毒性损伤有一定的保护作用,其保护作用与其抗细胞凋亡作用有关。     

芍药苷预处理对皮质酮损伤皮层神经元保护作用机制研究

目的:采用逆转录-聚合酶链反应(RT-PCR)法检测芍药苷预处理对皮质酮损伤的大鼠皮层神经元凋亡相关基因以及营养因子BDNF mRNA表达的影响,初步阐明芍药苷对皮质酮损伤神经元的保护机制。方法:体外培养胎鼠皮层神经元,于原代第7天加入芍药苷低、中、高浓度(0.5,2,10μmol.L-1)预处理30 min后,加入皮质酮(200μmol.L-1)损伤,采用RT-PCR检测芍药苷预处理对神经元凋亡相关基因Bcl-2,Bax,Caspase-3以及营养因子BDNF mRNA表达的影响。结果:与模型组比较,芍药苷预处理组(2,10μmol.L-1)可显著下调Bax,Caspase-3 mRNA的表达,并显著上调Bcl-2和BDNF mRNA的表达(P0.05)。结论:芍药苷预处理对皮质酮损伤的大鼠皮层神经元的保护作用与芍药苷调控神经元凋亡相关基因Bcl-2,Bax,Caspase-3 mRNA的表达直接相关,同时可能与上调神经营养因子BDNF基因表达有关。     

丹酚酸B对谷氨酸诱导的 PC12 细胞兴奋毒保护作用的研究

目的:研究丹酚酸B对谷氨酸诱导PC12细胞兴奋毒的保护及作用机制。方法:以谷氨酸损伤PC12细胞24 h为模型,采用四甲基偶氮唑蓝法检测细胞存活率;LDH法检测乳酸脱氢酶的漏出率;AO/EB双染法荧光显微镜和PI单染流式细胞术检测细胞凋亡;流式细胞术检测细胞内活性氧的含量;Western blotting法检测细胞内Caspase-3蛋白的表达。结果:丹酚酸B可明显抑制谷氨酸诱导的PC12细胞的损伤,阻止细胞LDH的释放,在50～200μmol.L-1剂量呈一定的量效关系;同时,丹酚酸B明显降低谷氨酸诱导的活性Caspase-3蛋白的表达,抑制谷氨酸引起的ROS的累积,降低PC12细胞的凋亡率,在50～200μmol.L-1剂量呈量效相关性。结论:在一定剂量范围内,丹酚酸B对谷氨酸损伤的PC12细胞有保护作用,其保护的机制可能与丹酚酸B减少ROS的生成,阻止氧化损伤的发生,抑制Caspase-3途径依赖的凋亡相关。     

士的宁对多巴胺能神经元的保护作用

目的:选择2种神经毒物1-甲基-4-苯基吡啶离子(MPP+)和谷氨酸在体外诱导多巴胺能神经元退行性损伤,并检测士的宁对此细胞的药理毒理作用,及对其退行性损伤的协同作用或拮抗作用。方法:采用孕14 d小鼠,取出胎鼠中脑,分离出多巴胺能神经元,调整细胞密度7.5×105/mL,于5%CO2,37℃,100%湿度条件培养,于体外培养的第10天分别加入士的宁0.1,1,5,10μmol.L-1,MPP+10μmol.L-1,谷氨酸500μmol.L-1作用48 h,第12天染色。选取细胞数,神经元树突长度和数量作为观察指标,并做出分析。结果:士的宁对未损伤的多巴胺能神经元在48 h培养过程中无毒性影响。在10μmol.L-1士的宁作用下,多巴胺能神经元数量明显高于对照组(P<0.05);在5～10μmol.L-1士的宁作用下,树突数量与对照组相比也有明显提高(P<0.05)。士的宁(10μmol.L-1)和MPP+共同培养48 h后,细胞计数明显高于MPP+单独培养组(P<0.05);这种保护作用更为明显的表现在细胞形态学变化方面,在10μmol.L-1的士的宁剂量下,神经树突长度明显高于MPP+单独培养组(P<0.05);神经树突的数量在5μmol.L-1士的宁作用下与MPP+单独培养组相比有明显增加(P<0.05)。在对谷氨酸(500μmol.L-1)诱导的神经元损伤模型中,与谷氨酸组相比,10μmol.L-1的士的宁对所有的观察指标的数值都有递增影响(P<0.05)。结论:首次证实士的宁具有可在体外刺激中脑分离出的多巴胺能神经元的生长,并且对此神经元退行性损伤有保护作用。     

比较丹参两种水溶性成分对谷氨酸诱导PC12细胞兴奋毒的保护作用

目的:比较丹参素、丹酚酸B对谷氨酸诱导的PC12细胞兴奋性毒的保护作用。方法:以PC12细胞为研究对象,将培养的细胞分为5组,分别为空白组,谷氨酸处理组(模型组),丹酚酸B组,丹参素组,维生素E组(20μmol·L~(-1)),除空白组外和模型组外,丹酚酸B,丹参素和维生素E预处理1 h后加谷氨酸共同孵育24 h。采用噻唑蓝(MTT)法检测细胞活性,乳酸脱氢酶(LDH)法检测乳酸脱氢酶的漏出率,流式细胞术检测细胞内活性氧的含量。结果:与空白组比较,丹参素、丹酚酸B能够明显抑制谷氨酸诱导的PC12细胞的神经毒性、阻止LDH的漏出(P0.05),在50,100,200μmol·L~(-1)剂量呈一定的量效关系,在100μmol·L~(-1)剂量浓度下,丹参素在抑制PC12细胞内活性氧(ROS)的堆积、减弱相对荧光强度方面,略好于丹酚酸B(约为12.5%),100μmol·L~(-1)剂量下,丹参素对PC12细胞的保护作用强于丹酚酸B。结论:在选择的实验模型和剂量条件下,丹参素、丹酚酸B均有潜在的保护谷氨酸损伤的PC12细胞的作用,且丹参素药效略强于丹酚酸B。     

银杏叶提取物对过氧化氢诱导的成纤维细胞氧化应激损伤的保护作用

目的:研究银杏叶提取物(EGb761)对过氧化氢诱导的大鼠真皮成纤维细胞氧化应激的保护作用。方法:原代培养大鼠真皮成纤维细胞,以不同浓度(20μg/mL、50μg/mL、100μg/mL)的EGb761对其生长进行干预后,H2O2诱导其损伤,MTT法观察细胞活力,硫代巴比妥酸(TBA)法和黄嘌呤氧化酶法分别测定丙二醛(Malondialdehyde,MDA)和超氧化物歧化酶(Superoxide dismutase,SOD)含量;荧光探针DCFH-DA进行活性氧(ROS)检测;流式细胞术(FCM)测定细胞凋亡。结果:不同浓度(20μg/mL、50μg/mL、100μg/mL)的EGb761均能提高H2O2诱导的成纤维细胞细胞存活率和SOD活力,降低MDA含量、活性氧水平和细胞凋亡率,且呈一定的剂量相关性。结论:EGB761在一定范围内能保护过氧化氢诱导的大鼠真皮成纤维细胞氧化应激损伤且呈一定的剂量相关性。     

Neuroprotective effect of Ginkgo biloba L. extract in a rat model of Parkinson's disease

The neuroprotective effects of a standardized extract of Ginkgo biloba L. (EGb 761) were investigated on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in the nigrostriatal dopaminergic system of the rat brain. Rats were given a week of pretreatment with daily administrations of EGb 761. Unilateral striatal injection of 6-OHDA was followed by treatment with EGb 761 for a week. Serial measurement of contralateral forepaw adjusting steps revealed a progressive deficit in motor activity. At 8 weeks after 6-OHDA lesion the number of contralateral forepaw adjusting steps was significantly higher in rats that were treated with high doses of EGb 761 (100 mg/kg daily) than in those treated with low doses (50 mg/kg) or with the vehicle. Dopamine neuron loss in the substantia nigra and a depletion in striatal dopamine corresponded with behavioural deficit. These data suggest that the neuroprotective effects of EGb 761 reduce the behavioural deficit in 6-OHDA lesions in rat and also indicates a possible role for the extract in the treatment of Parkinson's disease.     

银杏叶提取物EGb761预处理对小鼠大脑微梗死干预效应的影响

目的观察银杏叶提取物EGb761预处理对小鼠大脑微梗死(CMI)模型的影响。方法28只雄性C57BL/6J小鼠随机分为假手术组[n=6,PBS 100 mg(kg·d)]、磷酸缓冲液(PBS)组[n=11,PBS 100 mg/(kg·d)]和实验组[n=11,EGb761100 mg/(kg·d)]。PBS组和实验组小鼠均被双光子激光照射诱导建立CMI模型。HE染色计算微梗死体积、免疫荧光染色观察小鼠CMI区域神经元凋亡、小胶质细胞激活、星形胶质细胞过度活化及3-硝基酪氨酸(3-NT)沉积。结果假手术组未发现CMI病灶,大脑皮层组织的星形胶质细胞和小胶质细胞均呈现正常状态。与PBS组比较,实验组梗死体积、活化小胶质细胞、过度活化星形胶质细胞计数及3-NT沉积量减小(P<0.01)。结论EGb761预处理具有减少小鼠CMI区域神经元死亡,减小梗死体积的作用,机制可能与抑制小胶质细胞和星形胶质细胞活化,并降低硝化应激水平有关。     

Gingko biloba extract (EGb 761) attenuates the focal cerebral ischemic injury-induced decrease in astrocytic phosphoprotein PEA-15 levels

EGb 761 is an extract of Gingko biloba that is neuroprotective against focal cerebral ischemic injury. PEA-15 (phosphoprotein enriched in astrocytes 15) modulates cell proliferation and apoptosis. In this study, we investigated whether EGb 761 regulates the expression of PEA-15 and two phosphorylated forms of PEA-15 (Ser 104 and Ser 116) in middle cerebral artery occlusion (MCAO)-induced injury. Adult male rats were treated with vehicle or EGb 761 (100 mg/kg) prior to MCAO and cerebral cortices were collected 24 h after MCAO. A reduction in expression of PEA-15 and its phosphorylated forms induced by MCAO injury was detected using a proteomic approach. EGb 761 pretreatment prevented the ischemic injury-induced decrease in PEA-15 expression. Western blot analysis demonstrated that EGb 761 attenuates the injury-induced reduction in PEA-15, phospho-PEA-15 (Ser 104), phospho-PEA-15 (Ser 116). Phosphorylation of PEA-15 influences its anti-apoptotic function; a decrease in PEA-15 phosphorylation induces apoptotic cell death. The maintenance of PEA-15 phosphorylation by EGb 761 pretreatment during cerebral ischemic injury indicates that EGb 761 is a neuroprotective against cerebral ischemic injury.     

The effect of ginkgo biloba extract (EGb 761) pretreatment on intestinal epithelial apoptosis induced by intestinal ischemia/reperfusion in rats: role of ceramide

Apoptosis was demonstrated to be a major mode of intestinal epithelial cell death caused by intestinal ischemia/reperfusion (II/R). Ceramide has been proposed as a messenger for apoptosis. The present study was aimed to investigate the effect of Ginkgo biloba extract 761 (EGb 761) pretreatment on II/R-induced intestinal mucosal epithelial apoptosis in rats and the mechanism related to ceramide. The rat model of II/R injury was produced by clamping superior mesenteric artery for 60 min followed by reperfusion for 180 min. Twenty four rats were randomly allocated into Sham, II/R and EGb + II/R groups. In EGb + II/R group, EGb 761 (100 mg/kg per day) was administered intragastrically for 7 days before the surgery. Animals in II/R and sham groups were treated with equal volume of normal saline solution. Intestinal mucosal epithelial apoptosis was detected via electron microscopy and TUNEL method. Lipid peroxidation in intestinal mucosa was determined by detecting the malondialdehyde level and the activities of superoxide dismutase and peroxidase glutathione. The ceramide generation and sphingomyelinase (SMase) mRNA expression in intestinal mucosa were determined by high performance, thin layer chromatography, and RT-PCR, respectively. II/R caused intestinal mucosal epithelial apoptosis and over-production of the ceramide accompanied by up-regulation of SMase mRNA expression and increases of lipid peroxidation. EGb 761 pretreatment significantly decreased apoptosis index, and concurrently reduced the ceramide generation accompanied by down-regulation of SMase expression and inhibition of lipid peroxidation. The findings indicate that EGb 761 pretreatment attenuates II/R-induced intestinal epithelial apoptosis, which might be attributable to its antioxidant action of mediating ceramide pathway.     

Connexin 43在银杏叶提取物抗大鼠脑缺血损伤中的作用

目的通过体内体外实验研究缝隙连接蛋白43(Cx43)与EGb761脑缺血神经保护作用的关系,探讨银杏叶制剂EGb761治疗脑缺血的机制。方法在大鼠原代神经细胞中转染荷载Cx43-shRNA的慢病毒,干扰Cx43蛋白表达,同时给予EGb761 200μg/mL处理观察Cx43的表达以及EGb761对Cx43蛋白表达的影响。建立大鼠脑缺血模型,腹腔给予EGb761 50、100 mg/kg,使用HE染色,TTC染色,免疫组化和westernblot方法检测大鼠脑组织细胞的形态变化和大鼠海马Caspase-3,TUNEL阳性细胞,Cx43,p-Cx43蛋白的表达变化。结果 EGb761给药可以减少脑缺血大鼠脑组织梗死面积,显著性抑制脑缺血大鼠海马Caspase-3表达和TUNEL阳性凋亡细胞数目,显著性抑制大鼠海马神经细胞p-Cx43的表达水平。体外培养的大鼠神经细胞转染Cx43-shRNA慢病毒,给予EGb761处理,处理前后神经细胞Cx43/p-Cx43蛋白表达未见明显变化。结论 EGb761可以减轻脑缺血缺氧引起的细胞损伤,其神经保护作用与抑制缺血引起的缝隙连接蛋白Cx43的表达和激活有关。     

银杏叶提取物对星形胶质细胞诱导型一氧化氮合酶基因表达的调控

 目的研究银杏叶提取物(EGh761)对星形胶质细胞合成一氧化氮(NO)的作用以及对共培养的小脑颗粒神经元的作用。方法以脂多糖(LPS)和IFN-γ诱导体外培养的大鼠星形胶质细胞表达诱导型一氧化氮合酶(iNOS),产生NO作为病理条件下胶质细胞过度活化的实验模型,应用逆转录基因扩增技术和蛋白质印迹技术检测EGb761对星形胶质细胞中iNOS基因表达的影响,并探讨这一调控作用的分子机制。结果EGb761能明显降低星形胶质细胞中iNOS mRNA和蛋白质的表达、减少NO 的生成、防止活化的胶质细胞损伤共培养神经元,抑制IκB-α的降解和阻止p65/RelA进入细胞核,提示EGb761对iNOS基因表达的抑制作用依赖于NF-κB信号通路。结论EGb761可能对与胶质细胞过度活化有关的神经系统疾病具有治疗、预防的作用。     

Involvement of Ca2+, CaMK II and PKA in EGb 761-induced insulin secretion in INS-1 cells

EGb 761, a standardized form of Ginkgo biloba L. (Ginkgoaceae) leaf extract, was recently reported to increase pancreatic beta-cell function. To determine whether EGb 761 elicits insulin secretion directly, we treated INS-1 rat beta cells with EGb 761 and then measured insulin release. Treatment of EGb 761 (50 microg/ml) significantly stimulated insulin secretion in INS-1 cells, compared with untreated control (p<0.05) and the stimulatory effect of EGb 761 on insulin secretion was dose-dependent. To elucidate the mechanism of EGb 761-induced insulin secretion, we investigated the involvement of calcium. The treatment with nifedipine, an L-type calcium channel blocker, prevented EGb 761-induced insulin secretion and furthermore, EGb 761 itself elevated [Ca(2+)](i), suggesting the involvement of calcium in this process. To identity the protein kinases involved in EGb 761-induced insulin secretion, INS-1 cells were treated with different kinase inhibitors and their effects on EGb 761-induced secretion were investigated. KN62 and H89, calium/calmodulin kinase (CaMK) II and protein kinase A (PKA) inhibitor, respectively, significantly reduced EGb 761-induced insulin secretion. Immunoblotting studies showed an increase in the phosphorylated-forms of CaMK II and of PKA substrates after EGb 761 treatment. Our data suggest that EGb 761-induced insulin secretion is mediated by [Ca(2+)](i) elevation and subsequent activation of CaMK II and PKA.     

银杏叶提取物的心肌延迟保护作用及其机制研究

目的观察银杏叶提取物(EGb761)对大鼠离体心脏心肌的延迟保护作用及其机制。方法离体大鼠心脏全心停灌缺血30min后再灌注30min产生缺血再灌损伤,观测心率(HR)、冠脉流量(CF)、左室内压(LVP)和左室内压变化最大速率( dp/dtmax),测定心肌组织中肌酸激酶(CK)释放量、心肌组织丙二醛(MDA)和一氧化氮(NO)的量。结果实验前24h单次ig给予EGb761(50或100mg/kg)可显著改善心肌缺血再灌注所致的心功能(LVP和 dp/dtmax)损伤,抑制心肌组织CK释放和MDA水平的增加以及NO水平的降低。预先给予NO合酶抑制剂L-NAME(5mg/kg)或心肌肌细胞膜ATP敏感钾通道(sarcKATP)阻断药HMR1883(3mg/kg),均可明显抑制EGb761对心肌缺血再灌注损伤的延迟保护作用。结论EGb761对缺血再灌注诱导大鼠心肌损伤具有延迟性保护作用,这一保护作用可能与增加NO合成和开放sarcKATP通道有关。     

Identification of proteins differentially expressed in cerebral cortexes of Ginkgo biloba extract (EGb761)-treated rats in a middle cerebral artery occlusion model--a proteomics approach

EGb 761 is a standardized extract of Ginkgo biloba that appears to have a neuroprotective effect against neurodegenerative diseases. Adult male rats were treated with EGb 761 (100 mg/kg) or vehicle prior to middle cerebral artery occlusion (MCAO), and brains were collected 24 h after MCAO. Proteins that were differentially expressed after EGb 761 treatment during cerebral ischemia were detected using two-dimensional gel electrophoresis. Protein spots with more than a 2.5-fold change in intensity between vehicle- and EGb 761-treated groups were identified by mass spectrometry. The levels of peroxiredoxin-2 and protein phosphatase 2A subunit B were significantly decreased in the vehicle-treated group in comparison to the EGb 761-treated group. In contrast, levels of the collapsing response mediator protein 2 (CRMP2) were significantly increased in vehicle-treated animals, while EGb 761 prevented the injury-induced increase of CRMP2. These results suggest that EGb 761 protects neuronal cells against ischemic brain injury through the specific up- and down-modulation of various proteins.