银杏叶提取物对神经细胞凋亡的保护作用

目的 研究银杏叶提取物（GbE)对神经细胞凋亡的影响。方法 对原代培养的大鼠脑皮质细胞进行细胞活力观察,并LDH释放量及琼脂糖凝胶电泳DNA结果。结果 GbE能够增强神经细胞活力,减少LDH释放,减轻细胞核形态的改变及DNA的断裂。结论 GbE对神经细胞凋亡有抑制作用 。     

Protective effect of L-deprenyl against apoptosis induced by okadaic acid in cultured neuronal cells

L-Deprenyl, an irreversible MAO-B (monoamine oxidase B, EC 1.4.3.4) inhibitor, is used for the treatment of Parkinson's disease and to delay the progression of Alzheimer's disease. L-Deprenyl also exhibits protective effects against neuronal apoptosis which are independent of its ability to inhibit MAO-B. The purpose of this study was to compare the antiapoptotic efficacy of L-deprenyl against different types of apoptotic inducers in three neuronal cell culture models. The level of apoptosis was quantified by measuring the activation of caspase-3 enzyme, which is the main apoptotic executioner in neuronal cells. MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] and LDH (lactate dehydrogenase, EC 1. 1.1.27) assays were used to demonstrate the cytotoxic response of apoptotic treatments. Our results showed that okadaic acid, an inhibitor of protein phosphatase 1 and 2A, induced a prominent increase in caspase-3 activity both in cultured hippocampal and cerebellar granule neurons as well as in Neuro-2a neuroblastoma cells. Interestingly, L-deprenyl offered a significant protection against the apoptotic response induced by okadaic acid in all three neuronal models. The best protection appeared at the concentration level of 10(-9) M. L-Deprenyl also provided a protection against apoptosis after AraC (cytosine beta-D-arabinoside) treatment in hippocampal neurons and Neuro-2a cells and after etoposide treatment in Neuro-2a cells. However, L-deprenyl did not offer any protection against apoptosis caused by serum withdrawal or potassium deprivation. Okadaic acid treatment in vivo is known to induce an Alzheimer's type of hyperphosphorylation of tau protein, formation of beta-amyloid plaques, and a severe memory impairment. Our results show that the okadaic acid model provides a promising tool to study the molecular basis of Alzheimer's disease and to screen the neuroprotective capacity of L-deprenyl derivatives.     

阿魏酸钠对硝普钠诱导的原代培养海马神经元凋亡的保护作用

目的:探讨阿魏酸钠(sodium ferulate,SF)对NO供体硝普钠(SNP)引起的大鼠海马神经元凋亡保护作用的可能机制.方法:原代培养的大鼠海马神经元,经终浓度为10～160 μmol·L-1的SF预处理6 h后,用50 μmol·L-1的SNP处理24 h,采用MTT法检测细胞存活率,Hochest 33258荧光染色及DNA琼脂糖凝胶电泳分析等方法检测凋亡,Western blot及RT-PCR检测bcl-2基因表达.结果:不同浓度SF(10～160μmol·L-1)预处理6 h可显著提高神经元的存活率,减少SNP引起的核固缩、凝聚和碎裂现象;DNA凝胶电泳图谱未见典型的"梯子状"改变;增加bcl-2 mRNA及蛋白的表达.结论:SF对NO供体SNP诱导的海马神经元凋亡具有明显的保护作用,其机制可能与增加抗凋亡基因bcl-2表达有关.     

类叶升麻苷对鱼藤酮致SH-SY5Y细胞凋亡的保护作用

目的探讨类叶升麻苷对鱼藤酮致多巴胺能神经元SH-SY5Y细胞凋亡的保护作用及其机制。方法采用MTT法检测细胞存活率,以荧光染料Hoechst33342染色分析细胞核的形态学变化,用流式细胞仪定量分析细胞凋亡峰,以2,′7′-二氢二氯荧光黄双乙酸钠(DCFH-DA)为标记探针检测细胞内活性氧的产生。结果①0.5μmol.L-1的鱼藤酮处理SH-SY5Y细胞48 h能引起细胞存活率的显著下降;诱导细胞发生凋亡,凋亡率达47.39%;大部分细胞胞体皱缩,突起缩短消失或断裂;染色质皱缩、浓缩、断裂及形成凋亡小体;细胞内活性氧水平上升。②预先用盐生肉苁蓉提取物类叶升麻苷(10,20或40 mg.L-1)处理细胞6 h,可提高细胞存活率;明显改善鱼藤酮引起的细胞形态学变化;流式细胞仪检测凋亡率分别降低到25.87%,23.97%,10.45%;以DCFH-DA为标记探针检测到20 mg.L-1类叶升麻苷可明显抑制鱼藤酮引起的细胞内活性氧产生。结论类叶升麻苷能抑制鱼藤酮诱导的多巴胺能神经元SH-SY5Y细胞凋亡,其神经细胞保护作用可能与降低细胞内活性氧水平有关。     

银杏内酯B对内皮细胞的保护作用及分子机制研究

目的探讨银杏内酯B对氧化型低密度脂蛋白刺激脐静脉内皮细胞保护作用的分子机制。方法分别用银杏内酯B 0.2、0.4、0.6 g.L-1预孵育内皮细胞1 h,再加入ox-LDL(0.1 g.L-1)刺激细胞。用Western blot检测Lectin-likeoxidized LDL receptor-1(LOX-1)、PI3K、Nrf2、SIRT1表达及Akt磷酸化。结果 1.ox-LDL刺激内皮细胞LOX-1表达增加,银杏内酯B以剂量依赖方式抑制了LOX-1表达。2.银杏内酯B有效地抑制了ox-LDL刺激的Akt磷酸化,但对PI3K的表达无影响。3.ox-LDL刺激内皮细胞SIRT1表达降低,银杏内酯B有意义地增加了细胞SIRT1表达。4.ox-LDL刺激细胞抗氧化应激蛋白Nrf2表达明显增加,银杏内酯B下调了Nrf2的表达。结论银杏内酯B对内皮细胞保护作用与抑制LOX-1表达、Akt磷酸化,启动细胞内源性保护机制相关。     

丁苯酞对大鼠神经细胞凋亡的保护作用及其机制研究

目的 探讨丁苯酞对大鼠神经细胞凋亡的保护作用及其机制研究.方法 选用90只SD大鼠,随机分为给药组、对照组和健康组,每组30只.对给药组和对照组大鼠使用10％水合氯醛麻醉(剂量为0.5 mL/100 g),麻醉完成后,对大鼠双侧海马区位置进行准确定位后,将浓度为5μL(1 μg/μL)Aβ1-42注入造模.将配置的丁苯酞与食用麻油混合配制成悬浊液.造模完成后,分别给予不同的处理方法,给药组按75 mg/kg比例对大鼠进行灌胃给药,1次/d.对照组按同等比例灌胃给予生理盐水,1次/d.健康组为正常健康组,不给予任何手术和药物处理.取大鼠脑组织分为两部分,一部分经固定、脱水、石蜡包埋后制得厚度约5μm切片.采用TUNEL染色法对脑组织细胞凋亡进行检测;并使用H&E染色法观察各组大鼠脑组织细胞;采用Western Blot法检测各组脑组织MAPK、Erk和P38的蛋白表达水平,并使用RT-PCR法检测MAPK、Erk和P38的mRNA表达水平.结果 30 d后,给药组大鼠脑组织细胞凋亡明显少于对照组及健康组.给药组大鼠MAPK、Erk和P38的蛋白表达水平明显低于对照组但高于正常健康组(P＜0.05).使用RT-PCR法检测MAPK、Erk和P38的mRNA表达水平发现,给药组大鼠MAPK、Erk和P38的mRNA水平明显低于对照组但高于正常健康组(P＜0.05).对照组大鼠达到学会标准次数与健康组比较明显升高(P＜0.05).与对照组比较,给药组大鼠达到学会标准次数明显降低(P＜0.05).结论 丁苯酞对Aβ1-42处理的大鼠脑组织细胞凋亡具有保护作用,通过抑制大鼠脑组织MAPK,Erk和P38的表达发挥作用.     

人参皂甙Rg1对抗多巴胺诱导的PC12细胞凋亡

目的:探讨外源性多巴胺诱导PC12细胞凋亡以及人参皂甙Rg1保护作用的分子机制。方法:流式细胞仪定量测定PC12细胞的凋亡和Bcl-2、Bax蛋白的表达;电子显微镜观察PC12细胞的形态;凝胶电泳评价DNA的断裂;荧光分光光度计法测定caspase-3的活力;半定量RT-PCR分析bcl-2和bax mRNA的表达。结果:多巴胺(浓度为0.15、0.30、0.45和0.60mmol/L)诱导PC12细胞凋亡,各剂量组细胞凋亡率分别从对照组1.1％±0.4％增加到41％±3％,46.4％±2.7％,53％±3％和64.5％±2.7％;人参皂甙Rg1 10μmol/L预处理24h后,较多巴胺0.45mmol/L单独处理时,PC12细胞的凋亡率和caspase-3的活力分别从53％±3％和683±8(平均荧光强度)下降到1.9％±0.6％和325±5,Bcl-2蛋白阳性率从14.3％±1.1％增加到25.9％±1.6％,Bax蛋白阳性率从48％±3％下降到35％±3％。结论:人参皂甙Rg1通过抑制cas-pase-3的激活并调节Bcl-2和Bax两者间蛋白的比值对抗多巴胺对PC12细胞凋亡的诱导作用。     

Protective effect of vinconate on ischemia-induced neuronal damage in the rat hippocampus.

The protective effect of vinconate, a vinca alkaloid derivative, on ischemia-induced neuronal damage was investigated using a model of rat forebrain ischemia caused by occlusion of four vessels. Hippocampal cell loss was observed histologically and neurochemically 5 days after 10 min of ischemia. Treatment with vinconate (50 and 200 mg/kg i.p.) before cerebral ischemia significantly suppressed neuronal cell loss in the hippocampal CA1 region and the decrease in the content of neuroactive amino acids in the hippocampus. The release of neuroactive amino acids in the hippocampus was significantly increased by cerebral ischemia. Pretreatment with vinconate (50 and 200 mg/kg i.p.) significantly attenuated the increased release of glutamic acid and aspartic acid, but not the release of gamma-aminobutyric acid (GABA), taurine and glycine. This suppressive effect of vinconate was antagonized by scopolamine (10(-5) M). The addition of vinconate (10(-11)-10(-4) M) had no effect on the binding of [3H]MK-801. These results indicate that pretreatment with vinconate attenuates the ischemia-induced release of excitatory amino acids into the extracellular space of the hippocampus via the stimulation of presynaptic muscarinic acetylcholine receptors. The present results also suggest that this suppressive effect of vinconate on the release of excitatory amino acids (glutamic acid and aspartic acid) may play a crucial role in the protective action of this agent against ischemia-induced neuronal damage in the hippocampus.     

Protective effect of selaginellin on glutamate-induced cytotoxicity and apoptosis in differentiated PC12 cells

l-glutamate plays a key role in neuronal cell death associated with many neurodegenerative conditions such as cerebral ischemia, hypoxia, Alzheimer's, Huntington’s, and Parkinson’s diseases. Selaginellin, a component extracted from Saussurea pulvinata (Hook.et Grev.) Maximo, was assessed for its ability to protect rat pheochromocytoma (PC12) cells against oxidative toxicity induced by glutamate. The differentiated PC12 cells were pretreated with various concentrations (10⁻⁷, 3 × 10⁻⁷, or 10⁻⁶ M) of selaginellin for 1 h prior to exposure to l-glutamate. Selaginellin was shown to protect PC12 cells against glutamate toxicity, as determined by characteristic morphological features, lactate dehydrogenase release and cell viability, and apoptosis as evaluated by Hoechst 33342 staining assay and caspase-3 activity. In addition, the increase in levels of reactive oxygen species and decrease in klotho gene expression induced by glutamate were significantly reversed by selaginellin. Our study suggests that selaginellin has a neuroprotective effect against l-glutamate-induced neurotoxicity through mechanisms related to anti-oxidation and anti-apoptosis via scavenging reactive oxygen species and up-regulating the expression of klotho gene.     

水蛭素对凝血酶所致脑微血管内皮细胞损伤的保护作用

目的探讨凝血酶对体外培养的原代大鼠脑微血管内皮细胞(BMVEC)的损伤作用及凝血酶抑制剂水蛭素的保护作用。方法分离新生大鼠脑组织微血管段,原代培养BMVEC并鉴定。四唑盐(MTT)比色法检测不同浓度凝血酶及水蛭素处理后BMVEC生长抑制状况,并用TUNEL法、透射电镜、核DNA琼脂糖凝胶电泳等3种方法检测BMVEC凋亡。结果原代培养细胞经鉴定为BMVEC。150、300 U.mL-1凝血酶可明显抑制BMVEC生长并诱导BMVEC凋亡,这种作用可被水蛭素阻断。结论病理状态下异常增加的凝血酶可导致BMVEC及血脑屏障损伤。水蛭素对凝血酶所致的BMVEC损伤有明显的阻断作用。     

五味子乙素抗顺铂所致HK-2凋亡及其机制的研究

目的研究五味子乙素(SchB)对顺铂所致HK-2(肾小管上皮细胞)损伤的保护作用,并探讨其可能的机制。方法不同浓度的SchB和顺铂作用于HK-2,在倒置显微镜下观察细胞形态变化,流式细胞仪检测HK-2细胞凋亡,并检测细胞LDH(乳酸脱氢酶)、MDA(丙二醛)、LPO(脂质过氧化物)的改变,探讨SchB对HK-2保护作用的机制。结果顺铂有抑制HK-2生长、诱导其凋亡的作用IC50为18.39μmol·L-1,而20μmol·L-1的SchB能够明显减少顺铂引起的HK-2的凋亡,保护作用最强;SchB能使顺铂导致的细胞MDA、LPO、LOH升高有明显降低。结论五味子乙素能够有效的抑制顺铂引起HK-2的凋亡。机制可能是通过其抗氧化作用来抑制顺铂引起的氧化应激反应。     

Protective effect of erythropoietin against aristolochic acid-induced apoptosis in renal tubular epithelial cells

The goal of this research was to investigate the renoprotective effect of erythropoietin against aristolochic acid-induced apoptosis in cultured LLC-PK1 cells as well as underlying mechanism. LLC-PK1 cells impaired by aristolochic acid were used in this study as the cell model of aristolochic acid nephropathy. Apoptosis was studied by different methods (transmission electron microscopy, fluorescence-activated cell sorting, TUNEL, caspase-3 activation). Cells showed apoptotic morphology when exposed to 10 mug/ml aristolochic acid for 24 h, and the apoptotic index was increased (37.67%) compared with the control (6.09%). The presence of recombinant human erythropoietin (10, 20 U/ml, 24 h) significantly lowered the apoptotic index (22.41%, 14.63%) and the damage of cytoskeleton was also ameliorated. Studies on the apoptotic signaling showed that recombinant human erythropoietin inhibited the activation of caspase-3 and upregulated the expression of anti-apoptotic gene, Bcl-XL. Moreover, recombinant human erythropoietin (10, 20 U/ml, 24 h) promoted the expression of proliferating cell nuclear antigen in renal tubular cells stimulated by 10 mug/ml aristolochic acid (46.34%, 48.11% vs. 28.46%). Together, our data provide in vitro evidence that recombinant human erythropoietin mediated renoprotective effect against aristolochic acid injury in renal tubular cells by ameliorating the damage of cytoskeleton, reducing the number of apoptotic cells and promoting cell regeneration. So a possibility is suggested that recombinant human erythropoietin may be beneficial in preventing aristolochic acid-induced apoptosis and could be a new promising therapeutic strategy for aristolochic acid-induced kidney damage.     

黄芪三萜皂苷对CVB3病毒所致细胞凋亡的保护作用

目的 应用分子生物学技术探讨黄芪三萜皂苷(total extract of astragalus,TEA)对柯萨奇B组3型病毒(CVB3)致细胞凋亡的保护作用.方法 利用CVB3感染Vero细胞建立细胞凋亡模型并使用TEA对模型进行治疗,检测DNA Ladder以了解凋亡细胞DNA断裂情况;通过TUNNEL标记法、Annexin-V/PI染色、Hoechst33258染色观察细胞凋亡程度,比较各组间细胞凋亡的差异.结果 CVB3感染后,Vero细胞DNA的紫外成像呈现阶梯状条带,TEA组可观察到DNA断裂程度降低;TUNNEL检测可看到凋亡存在,病毒组显色较深,细胞凋亡程度较TEA组严重;Annexin-V/PI染色可见病毒组Vero细胞膜呈绿色荧光,细胞核显红色,核凝聚及裂解,并有凋亡小体存在,TEA组细胞状态改善;Hoechst33258染色结果表明,病毒组Vero细胞核显蓝色荧光,可看到凋亡中期核染色质凝聚、边缘化及晚期凋亡小体的典型变化.TEA组细胞状态改善,凋亡细胞数目减少.正常细胞对照组的细胞贴壁状态良好.流式细胞仪检测结果表明,黄芪三萜皂苷能够显著的降低试验组Vero细胞的凋亡率.结论 TEA对CVB3病毒感染的Vero细胞具有一定的保护作用,抑制细胞凋亡,其作用机制尚需做进一步探讨.     

Protective effect of Bojungbangdocktang on cisplatin-induced cytotoxicity and apoptosis in MCF-10A breast endothelial cells

Although cisplatin has been extensively used as a cancer chemotherapeutic agent for the treatment of various human cancers, it causes significant side effects such as nephrotoxicity and hepatotoxicity due to lethal bystander damage to normal cells. Thus, in the current study, we investigated the Oriental herbal medicine Bojungbangdocktang (BJBDT), as we reported previously its anti-angiogenic activity at nontoxic concentrations that could prevent cisplatin-induced toxicity and apoptosis in human normal breast epithelial cell MCF-10A, but not in MCF-7 and MDA MB-231 breast cancer cells. BJBDT protected cisplatin-induced cytotoxicity in MCF-10A cells and potentiated cytotoxicity and MMP loss in MCF-7 cells. Also, 4′,6-diamidino-2-phenylindole (DAPI) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed that BJBDT reduced cisplatin-induced apoptotic bodies in MCF-10A cells compared with cisplatin-treated control. Consistently, BJBDT attenuated the apoptotic portion sub-G1 DNA contents as well as blocked the activation of caspase-3 and -9 and poly(ADP-ribose)polymerase (PARP) cleavage in cisplatin-treated MCF-10A cells. Taken together, our findings suggest that BJBDT can protect cisplatin-induced cytotoxicity and apoptosis in normal MCF-10A breast cells as a cancer chemopreventive agent.     

SOD类似物阻断百草枯诱导的N27细胞凋亡的作用

目的探讨在离体培养的细胞中,SOD类似物[manganese(Ⅲ)meso-tetrakis(N,N′-diethylimidazolium-2-yl)porphyrin,MnTDM]阻断百草枯诱导的N27细胞凋亡的作用。方法以N27细胞为多巴胺能神经元的细胞模型,检测不同浓度的MnTDM对百草枯诱导的N27细胞凋亡的保护作用,四甲基偶氮唑盐(MTT)法检测细胞活性及代谢状态;电镜观察凋亡形态;免疫细胞化学染色法观察凋亡相关蛋白Bax、Bcl-2蛋白的表达。结果经百草枯作用后N27细胞活性下降,且其下降程度与百草枯浓度的升高呈线性关系,MnTDM明显阻断百草枯诱导的细胞活性下降(P<0.05),且该阻断作用与MnTDM的浓度成正相关;MnTDM预处理后,电镜观察到细胞凋亡形态好转,凋亡相关蛋白Bcl-2表达升高,Bax表达降低,Bcl-2/Bax值升高。结论MnTDM对百草枯介导的凋亡有一定的保护作用,提示其可能对帕金森病患者的临床防治有一定作用。     

人参皂苷Rg1对抗多巴胺对PC12细胞凋亡的诱导作用

通过测定细胞的凋亡率 ,内源性NO的水平和iNOSmRNA的表达及半胱天冬酶 3的活性 ,探讨人参皂苷Rg1对抗多巴胺对PC12细胞凋亡诱导作用的可能机理 .结果表明多巴胺 (0 .15～ 0 .60mmol·L- 1)可诱导PC12细胞凋亡 ,预先经过 10 μmol·L- 1Rg 1处理后 ,PC12细胞的凋亡率显著下降 (P <0 .0 0 1) ,同时NO2- 水平和iNOSmRNA表达水平及半胱天冬酶 3活力较单纯多巴胺处理组明显降低(P <0 .0 0 1) .结果提示 ,Rg1减少细胞内源性NO的生成及抑制半胱天冬酶 3的活化可能是Rg1对抗多巴胺诱导PC12细胞凋亡的重要机理 .     

绿原酸对过氧化氢诱导内皮细胞凋亡的保护作用

目的探讨绿原酸对体外过氧化氢诱导人脐静脉内皮细胞凋亡的影响及机制。方法体外培养的人脐静脉内皮细胞株传代后进行实验,分为(A)正常对照组、(B)绿原酸组(30μmol.L-1)、(C)H2O2损伤组、(D)绿原酸+H2O2组。检测细胞线粒体膜电位;Hoechst33258荧光染色检测细胞凋亡;RT-PCR检测不同组内皮细胞Caspase-3和Bcl-2基因的表达。结果与正常对照组比较,过氧化氢(400μmol.L-1)能明显的造成内皮细胞的凋亡(P<0.05),绿原酸(30μmol.L-1)可降低过氧化氢引起的内皮细胞凋亡,表现为细胞凋亡率减少(P<0.05),线粒体膜电位增加,Bcl-2的表达增强,Caspase-3的表达减弱。结论绿原酸可抑制过氧化氢引起的内皮细胞凋亡,其作用机制可能与保护线粒体膜电位,促进凋亡抑制基因Bcl-2的表达及抑制Caspase-3的表达有关。     

甘肃黄芪黄酮对AngⅡ致内皮细胞凋亡的保护作用

目的探讨甘肃黄芪黄酮化合物芒柄花素与毛蕊异黄酮-7-O-β-D-吡喃葡萄糖对血管紧张素Ⅱ(AngⅡ)诱导人脐静脉内皮细胞(HUVECs)凋亡的影响。方法将不同浓度上述两种黄酮单体(0.01、0.1、1.0、10mg.L-1)分别与AngⅡ(10-6mol.L-1)和HUVECs作用24h后,MTT比色法检测细胞存活率,Hoechst33258荧光染色法观察细胞核形态变化,流式细胞仪及激光共聚焦仪检测细胞凋亡,并观察凋亡基因Fas表达的变化。结果甘肃黄芪黄酮化合物芒柄花素与毛蕊异黄酮-7-O-β-D-吡喃葡萄糖呈剂量依赖性地抑制AngⅡ引起的细胞存活率下降(P<0.05),AngⅡ(10-6mol.L-1)孵育24h后,内皮细胞凋亡率明显多于对照组(P<0.01);芒柄花素(1.0mg.L-1)与毛蕊异黄酮-7-O-β-D-吡喃葡萄糖(1.0mg.L-1)可抑制AngⅡ(10-6mol.L-1)诱导的内皮细胞凋亡(P<0.01),并减少Fas的表达(P<0.05);以上实验芒柄花素与毛蕊异黄酮-7-O-β-D-吡喃葡萄糖两化合物间比较无差异(P>0.05)。结论甘肃黄芪黄酮化合物可减轻AngⅡ诱导的HUVECs凋亡,其机制之一可能与其降低Fas的表达有关。