栀子苷联合美满霉素拮抗PC12细胞凋亡的作用

目的:观察栀子苷和美满霉素联合应用对H_2O_2诱导PC12细胞凋亡的作用,并对其机制进行初步探讨。方法:处于对数生长期的PC12细胞随机分为对照组、模型组、栀子苷组、美满霉素组以及栀子苷/美满霉素联合应用组。用300μmol/L H_2O_2诱导6 h建立PC12细胞凋亡模型。MTT法测定细胞存活率,比色法测定乳酸脱氢酶(LDH)活性,倒置相差显微镜观察细胞形态,Hoechest 33258荧光核染色观察细胞凋亡状况,Annexin V-FITC/PI双染法流式细胞仪检测细胞凋亡率,Western Blot测定Caspase-3蛋白表达。结果:与模型组相比,栀子苷组和美满霉素组PC12细胞存活率提高,LDH释放量减少,细胞凋亡率降低,同时Caspase-3蛋白表达减少(P均0.05);与栀子苷组和美满霉素组相比,栀子苷/美满霉素联合应用组PC12细胞的存活率进一步提高,LDH释放量进一步减少,细胞凋亡率进一步降低,同时Caspase-3蛋白表达进一步减少(P0.05或P0.01)。以上指标相比,差异均有统计学意义。结论:栀子苷与美满霉素联合应用较单一应用能更有效拮抗PC12细胞凋亡,其机制可能与抑制Caspase-3蛋白表达有关。     

p38应急信号通路参与一氧化氮诱导PC12细胞死亡

目的:探讨一氧化氮诱导PC12细胞死亡的信号转导途径。方法:将亚硝基铁氰化钠(sodiumnitroprusside,SNP)、caspase-3拮抗剂(caspase-3inhibitorⅡ)加SNP或p38拮抗剂(SB203580)加SNP与传代培养的PC12细胞一起孵育,观测细胞的存活率和caspase-3的活性;用MTT法测细胞存活率,caspase-3检测试剂盒测caspase-3活性。结果:SNP以浓度和时间依赖性方式诱导PC12细胞死亡,并增加caspase-3的活性;caspase-3拮抗剂Ⅱ和p38拮抗剂均明显减少细胞死亡且p38拮抗剂明显降低caspase-3的活性。结论:一氧化氮可能通过激活p38、caspase-3信号分子诱导PC12细胞死亡。     

多巴胺抑制Erastin诱导PC12细胞系铁死亡

目的探讨多巴胺在不同诱导剂导致的嗜铬细胞瘤PC12细胞死亡中的作用。方法应用RT-qPCR鉴定PC12的肿瘤特异性基因和神经相关基因的mRNA表达;通过细胞存活率评价生理浓度范围内(0~100μmol/L)不同浓度(0、6. 25、12. 5、25、50和100μmol/L)的多巴胺对PC12存活的影响,以及对药物诱导的铁死亡、凋亡和坏死的影响;流式细胞仪检测并分析PC12细胞铁死亡的发生和多巴胺保护PC12过程中PI、Annexin V和细胞周期的情况。结果 PC12表达肿瘤特异性基因和部分神经类基因。多巴胺在浓度25、50和100μmol/L时对PC12细胞有杀伤效果。生理浓度多巴胺特异性保护Erastin诱导的PC12细胞铁死亡。多巴胺能有效的降低Erastin造成的PI、Annexin V双阳性细胞上升和S期到G2/M期阻滞。结论生理浓度多巴胺对PC12有细胞毒性,并可以保护PC12抑制Erastin诱导的铁死亡。     

鱼藤酮对PC12细胞多巴胺相关转运体的影响

探讨鱼藤酮对PC12细胞多巴胺转运体(DAT)和突触囊泡单胺转运体2(VMAT2)的影响。我们将PC12细胞经不同浓度的鱼藤酮处理24h后,利用HE染色观察PC12细胞的形态学变化;利用免疫组织化学法观察不同浓度的鱼藤酮对PC12细胞酪氨酸羟化酶(TH),DAT和VMAT2蛋白表达的影响;利用RT-PCR法检测鱼藤酮对PC12细胞DAT和VMAT2基因表达的影响;通过生化实验检测Na+/K+-ATP酶活性。实验结果显示:鱼藤酮浓度为1.0mmol/L时,PC12细胞出现了明显的形态学改变,胞浆染色不均、胞体呈圆形、细胞核形状不规则、核浆比例增加;TH蛋白阳性细胞数量减少;DAT蛋白和基因表达增加;VMAT2蛋白和基因表达降低,与对照组比较均具有统计学意义(P<0.05),且随着鱼藤酮浓度的增加,变化趋势更为明显。鱼藤酮浓度为1.0mmol/L时,Na+/K+-ATP酶活力与对照组比较显著降低(P<0.05)。因此,DAT和VMAT2的表达异常可能参与了毒性作用。     

槲皮素抑制鱼藤酮诱导的PC12细胞凋亡

目的: 探讨槲皮素对鱼藤酮诱导的PC12细胞凋亡的影响及其作用机制。方法: 运用鱼藤酮诱导损伤PC12细胞,经300 μmol/L槲皮素预处理后,观察细胞形态学改变,流式细胞术检测细胞凋亡率,Western blotting检测Bax和Bcl-2蛋白的表达,JC-1染色检测细胞线粒体膜电位,比较各组的差异。结果: 与鱼藤酮组相比,槲皮素加鱼藤酮处理组细胞形态明显改善,凋亡率降至6.3%(P<0.01),Bcl-2表达增加(P<0.01),Bax表达降低(P<0.01),线粒体膜电位上升(P<0.01)。结论: 槲皮素对鱼藤酮诱导的PC12细胞凋亡具有抑制作用,上调Bcl-2和下调Bax蛋白的表达,维持线粒体膜电位可能是其作用的机制之一。     

N-乙酰半胱氨酸保护PC12细胞对抗化学性低氧诱导的损伤

目的 探讨活性氧( ROS)清除剂N-乙酰半胱氨酸(NAC)能否保护PC12细胞对抗化学性缺氧引起的损伤.方法 应用化学性低氧模拟物氯化钴(CoCl2)处理PC12细胞,建立化学性缺氧损伤PC12细胞的实验模型.在CoCl2处理PC12细胞前60 min将NAC加入培养基中,作为预处理.应用CCK-8比色法检测细胞存活...     

Nodosin通过Apaf-1和caspase-3诱导HepG2细胞凋亡

目的:研究传统中药提取物nodosin对人肝细胞癌Hep G2细胞凋亡的影响,并探讨其作用机制。方法:将nodosin设置为1. 25μmol/L、2. 5μmol/L、5μmol/L、10μmol/L和20μmol/L不同浓度组,作用于Hep G2细胞24 h后,用Hoechst 33258染色和电镜观察不同浓度的药物对细胞形态学的影响,用流式细胞术检测细胞凋亡率,用RT-qPCR检测凋亡蛋白酶激活因子1 (Apaf-1) mRNA的表达,用Western blot检测caspase-3及其前体和活化体的蛋白水平。结果:形态学结果显示,随着用药剂量的增加,细胞皱缩和细胞核偏移越明显,凋亡小体在5μmol/L、10μmol/L和20μmol/L剂量组明显增多。Apaf-1 mRNA的表达增加,caspase-3及其前体的表达和cleaved caspase-3的蛋白水平随着用药剂量的增加逐渐增加(P 0. 01)。结论:Nodosin能够诱导Hep G2细胞凋亡。该作用可能是通过增加Apaf-1 mRNA的表达继之激活caspase-3实现的。     

PC12细胞缺糖损伤过程中凋亡相关基因的表达

目的探讨PC12细胞缺糖损伤时凋亡相关基因bcl-2、bcl-xl、c-myc的表达变化.方法无葡萄糖培养基培养PC12细胞建立细胞缺糖损伤细胞模型,用RT-PCR法分析PC12细胞有糖和无糖条件下bcl-2、bcl-xl、c-myc的表达情况.结果细胞缺糖损伤6-16hbcl-2基因、bcl-xl基因、c-myc基因表达量显著增高,此后表达量逐渐下降.结论缺糖损伤早期,凋亡抑制基因bcl-2、bcl-xl、c-myc上调表达产生细胞应激保护效应.     

二硫苏糖醇通过激活calpain-2/caspase-12信号通路诱导BRL-3A细胞凋亡

目的:探讨二硫苏糖醇(DTT)诱导大鼠正常肝细胞株BRL-3A发生内质网应激时细胞内葡萄糖调节蛋白78(GRP78)、钙蛋白酶2 (calpain-2)、caspase-12及caspase-3的表达变化及对细胞凋亡的影响。方法:采用2. 5 mmol/L DTT分别处理BRL-3A细胞12 h和24 h,应用real-time PCR检测细胞内GRP78、calpain-2、caspase-12及caspase-3的mRNA水平;采用细胞免疫荧光检测细胞内GRP78、calpain-2、caspase-12及caspase-3的蛋白表达;应用Western blot检测cleaved caspase-12及cleaved caspase-3的表达变化;采用流式细胞术检测细胞凋亡情况。结果:BRL-3A细胞经DTT处理12 h及24 h后,GRP78、calpain-2及caspase-12的mRNA表达较正常对照组显著升高(P 0. 01),而caspase-3的mRNA水平与正常对照组比较无显著变化;细胞免疫荧光及Western blot检测发现,DTT处理细胞12 h及24 h后,BRL-3A细胞内GRP78、calpain-2、caspase-12及caspase-3的蛋白表达均较正常对照组显著增高,同时,cleaved caspase-12及cleaved caspase-3的表达也较正常对照组明显增多(P 0. 05);流式细胞术检测发现,经DTT处理后BRL-3A细胞的凋亡率较正常对照组显著增加(P 0. 05)。结论:二硫苏糖醇诱导BRL-3A细胞凋亡可能与calpain-2/caspase-12信号通路激活有关。     

JNK信号转导通路在NGF抗6-OHDA诱导PC12细胞凋亡中的作用

目的： 以6-羟基多巴胺(6-OHDA)作用于大鼠肾上腺嗜铬细胞瘤细胞(PC12 cells)以诱导其凋亡,然后在其中分别加入神经生长因子（NGF）及c-Jun氨基端激酶（JNK）阻断剂SP600125,研究在加入NGF后JNK的活性与凋亡的关系。方法: 实验分为对照组、6-OHDA组、NGF组、6-OHDA+NGF组、6-OHDA+JNK阻断剂SP600125组,以流式细胞分析法检测各组PC12细胞的凋亡率,以免疫印迹（Western blotting）法检测各组PC12细胞JNK的活化情况。结果: 6-OHDA导致PC12细胞凋亡,JNK1活性提高;预孵SP600125或NGF15min后再加入6-OHDA则PC12细胞凋亡率及JNK1活性均降低。结论: JNK1参与了6-OHDA致PC12细胞凋亡作用,NGF抗6-OHDA所诱导的PC12细胞凋亡作用与其抑制JNK的活化有关。     

Epigallocatechin gallate inhibits nitric oxide-induced apoptosis in rat PC12 cells

Nitric oxide (NO) is associated with many pathophysiology of the central nervous system including brain ischemia, neurodegeneration and inflammation. Epigallocatechin gallate (EGCG) is a major compound of green tea polyphenol that has shown the protective activity against neuronal diseases. This study examined the effect of EGCG on NO-induced cell death in PC12 cells. The administration of sodium nitroprusside (SNP), a NO donor, decreased the cell viability and induced apoptosis showing characterization such as cell shrinkage and chromatin condensation as well as subG1 fraction of cell cycles. EGCG inhibited the cytotoxicity and apoptotic morphogenic changes induced by SNP. EGCG attenuated the production of reactive oxygen species (ROS) by SNP, and ameliorated the SNP-induced Bax to Bcl-2 expression ratio leading to apoptosis. In addition, EGCG prevented the release of cytochrome c from the mitochondria into the cytosol as well as the upregulation of the voltage-dependent anion channel (VDAC), a cytochrome c releasing channel, in the mitochondria of SNP-treated cells. EGCG abrogated the activation of caspase-9, caspase-8 and caspase-3 induced by SNP. These results demonstrate that EGCG has a protective effect against SNP-induced apoptosis in PC12 cells by scavenging ROS and modulating the signal molecules associated with cytochrome c, caspases, VDAC and the Bcl-2 family. These findings suggest that EGCG might be a natural neuroprotective substance.     

Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells

Hyperglycemia, which occurs under the diabetic condition, is widely recognized as the causal link between diabetes and its serious complications. Diabetic neuropathies, which are among the most frequent complications of diabetes, affect sensory, motor, and autonomic nerves. The exact molecular mechanisms of high glucose-induced toxicity on neuronal cells, is still unclear. We previously reported that high glucose can induce apoptosis in PC12 cells, as evidenced by DNA fragmentation and high Bax/Bcl-2 ratio. The present study examined the involvement of caspase-3, the executioner, and two initiators of apoptosis, caspase-8 and caspase-9, during high glucose-induced apoptosis in PC12 cells, a neuronal cell line. Cells were exposed to high glucose with or without z-VAD-fmk, a pan-caspase inhibitor. Cell viability was measured by MTT assay. Caspase activity was determined spectrophotometrically using enzyme specific substrates. To correlate and confirm the caspase activity with changes in protein expression, procaspase-8, -9, and -3 were evaluated by Western blot analysis. The DNA-fragmentation was determined by DNA ladder using gel electrophoresis. The PC12 cell viability on high glucose exposure was decreased compared to controls, which was reversed by z-VAD-fmk. The activities of caspase-8, -9, and -3 were significantly increased in treated cells compared to controls. Moreover, high glucose exposure induced a significant decrease in protein levels of procaspases, indicating conversion of pro-form into the mature caspases. Finally, DNA fragmentation (Ladder) was shown in treated cells by high glucose. Based on the current data, it could be concluded that high glucose-induced apoptosis in PC12 cells is mediated, in part, by activation of caspase-8, -9, and -3 dependent pathways.     

Thapsigargin-induced apoptosis was prevented by glycogen synthase kinase-3 inhibitors in PC12 cells

Uncontrolled calcium stress has been linked causally to a variety of neurodegenerative diseases, including ischemia, excitotoxicity and Alzheimer's disease. Thapsigargin, which increases [Ca²⁺]_i, induces apoptotic cell death (chromatin condensation and DNA fragmentation) accompanied by caspase-3 activation in PC12 cells. We examined whether GSK-3 is involved in thapsigargin-induced cell death by using GSK-3 inhibitors in PC12 cells. Cells treated with 0.1 μM thapsigargin for 24 h shrank. The injured cells underwent chromatin condensation and nuclear fragmentation, indicating apoptotic cell death. We assayed the effects of selective GSK-3 inhibitors, SB216763, azakenpaullone and alsteropaullone on thapsigargin-induced apoptosis. These inhibitors completely protected cells from thapsigargin-induced apoptosis. Alsterpaullone did not reduce the GRP78 protein expression induced by thapsigargin, suggesting that GSK-3 activation is not involved in induction of GRP78. In addition, GSK-3 inhibitors inhibited caspase-3 activation accompanied by thapsigargin-induced apoptosis. We showed in this report that thapsigargin-induced apoptosis is prevented by GSK-3 inhibitors, suggesting that thapsigargin induces caspase-dependent apoptosis mediated through GSK-3 activation in PC12 cells.     

没食子儿茶素没食子酸酯对1-甲基-4-苯基吡啶离子诱导的大鼠PC12细胞凋亡的作用

目的： 探讨没食子儿茶素没食子酸酯(EGCG)抑制1-甲基-4-苯基吡啶离子（MPP+）诱导大鼠PC12细胞凋亡及其抗氧化作用、调节胞浆钙离子稳态与其抑制细胞凋亡作用之间的关系。方法: 培养大鼠肾上腺嗜铬细胞瘤细胞株PC12细胞,给予MPP+诱导细胞凋亡。EGCG（10、50及100 μmol·L-1）预处理0.5 h,再加入MPP+使其终浓度为900 μmol·L-1处理24 h后,MTT法检测细胞存活率,Annexin V-PI双染流式细胞仪检测细胞凋亡,荧光酶标仪测定细胞内活性氧,激光共聚焦荧光显微镜通过检测细胞内钙的荧光强度、检测细胞胞浆［Ca2+］i的变化,透射电镜观察凋亡细胞线粒体结构形态变化,并测定细胞内超氧化物歧化酶（SOD）和丙二醛（MDA）的含量。结果: MPP+呈剂量依赖性损伤PC12细胞,诱导细胞凋亡发生率达到31.0％。与模型组比较,EGCG处理后,明显提高细胞活力,降低凋亡细胞率,同时增强SOD活性、减少MDA和ROS的含量,降低胞浆［Ca2+］i浓度,减轻MPP+诱致的细胞线粒体改变。结论: EGCG具有抑制MPP+诱导的PC12细胞凋亡的作用,其作用机制可能与其提高细胞抗氧化能力和减少胞浆［Ca2+］i有关。     

Acrylamide decreased dopamine levels and increased 3-nitrotyrosine (3-NT) levels in PC 12 cells

Acrylamide is a chemical known to produce neurotoxicity in animals, as well as in humans. The mechanism of acrylamide-induced neurotoxicity is not fully known. However, recent studies have revealed that acrylamide affects the dopaminergic system. Therefore, the aim of this study was to investigate the effect of acrylamide on dopamine (DA) and the metabolites, 3,4-dihydroxy phenylacetic acid (DOPAC) and homovanillicacid (HVA), levels in Pheochromocytoma (PC 12) cells. In addition, the generation of peroxynitrite (ONOO⁻), measured by 3-nitrotyrosine (3-NT), was investigated as a possible mechanism in acrylamide-induced neurotoxicity. HPLC-coupled to electrochemical detection (ECD) was used to determine DA, DOPAC, HVA and 3-NT levels. Acrylamide (0.01–5 mM) exposure produced a dose- and time (1–42 h)-dependent decrease in DA levels. The decrease (P < 0.05) in DA levels was noted at 24 h after exposure to acrylamide. The study also revealed that 3-NT levels in PC 12 increased as a result of treatment with acrylamide. Thus, these data suggest that acrylamide-induced decrease in DA levels in PC 12 cells may be associated with peroxynitrite formation, measured as 3-NT levels.     

米诺环素通过调控PI3K/Akt通路抑制硝普钠诱导的PC12细胞凋亡

 目的： 探讨PI3K/Akt信号通路在米诺环素（minocycline,MC）抑制硝普钠(sodium nitoprusside,SNP)诱导的PC12细胞凋亡中的作用。方法：将体外培养的PC12细胞分为4组：空白对照组、SNP组、MC+SNP组和PI3K抑制剂LY294002+ MC+SNP组。用四甲基偶氮唑盐（MTT）法检测细胞活力,流式细胞术检测细胞凋亡;Western blotting检测不同时点（0.5、1、2、3 h）各处理组PI3K/Akt通路蛋白p-Akt和Akt的表达。结果：SNP处理PC12细胞24 h能抑制细胞生长,加入10 μmol/L MC预处理30 min可明显提高细胞活力,降低细胞凋亡率（P<0.05）,抑制SNP诱导的PC12细胞凋亡。MC组的p-Akt表达高于其它组,而加入LY294002后可阻断MC的上述效应。结论：MC可通过调控PI3K/Akt通路抑制SNP诱导的PC12细胞凋亡。     

同型半胱氨酸诱导PC12细胞凋亡的研究

目的：研究同型半胱氨酸（Hcy）在生理浓度铜离子（10 μmol/L Cu²⁺）作用下能否诱导PC12细胞凋亡及对bcl-2、bax基因表达的影响。 方法: 将细胞随机分成对照组和处理组，采用MTT法检测细胞活力；倒置相差显微镜、荧光显微镜和流式细胞仪检测细胞凋亡；半定量RT-PCR分析bcl-2和bax mRNA的表达水平。 结果: 0.125-1.0 mmol/L Hcy在Cu²⁺的作用下可以导致PC12细胞凋亡，具有明显的剂量-效应关系；bcl-2 mRNA表达降低，bax mRNA表达升高，且效应与Hcy浓度相关。 结论: 高浓度Hcy在生理浓度Cu²⁺作用下可诱导PC12细胞凋亡，且凋亡作用与Hcy浓度相关。其机制可能是通过调节bcl-2和bax mRNA的比值起作用的。     

美托洛尔对大鼠冠状动脉微栓塞后心肌细胞凋亡及caspase-12活化的影响及意义

目的： 探讨美托洛尔对大鼠冠状动脉微栓塞后心肌细胞凋亡及caspase-12活化的影响及意义。方法： 30只大鼠随机分为假手术组、微栓塞组、美托洛尔组（每组n＝10）,经左室注入42 μm微栓塞球,建立大鼠冠状动脉微栓塞模型,假手术组注射生理盐水代替微栓塞球,美托洛尔组为微栓塞术前30 min静脉注射美托洛尔。各组术后6 h分别心脏超声检测左室射血分数（LVEF）,TUNEL检测心肌细胞凋亡,Western blotting 检测凋亡蛋白caspase-12的活化。结果：① 与假手术组比较,微栓塞组LVEF显著下降（P＜0.05）;与微栓塞组比较,美托洛尔组LVEF没有显著差异。② 与假手术组比较,微栓塞组心肌细胞凋亡率、活化的caspase-12含量显著增加（均P＜0.05）;与微栓塞组比较,美托洛尔组心肌细胞凋亡率、活化的caspase-12含量显著减少（均P＜0.05）。结论： 美托洛尔抑制大鼠冠状动脉微栓塞后心肌细胞凋亡及caspase-12活化。