微小RNA-486靶向TRIM10抑制帕金森病细胞模型损伤

目的 探讨微小RNA（miR）-486对1-甲基-4-苯基吡啶离子(MPP+)诱导的体外帕金森病（PD）PC12细胞模型凋亡的影响和机制。 方法 实验分成对照（control）组、PD组（MPP+诱导PC12细胞）、miR-NC组［MPP+诱导PC12细胞转染模拟（mimics）对照（mimics control）］、miR-486组（MPP+诱导PC12细胞转染miR-486 mimics）、miR-486+载体（vector）组（共转染miR-486 mimics和pcDNA3.1）、miR-486+TRIM10组（共转染miR-486 mimics和pcDNA3.1-TRIM10）,每组n=9。CCK-8法分析细胞增殖变化,流式细胞术分析细胞凋亡水平变化,Western blotting分析Bax和Bcl-2蛋白表达变化,硫代巴比妥酸法检测细胞中丙二醛（MDA）含量,荧光探针法检测细胞中活性氧簇（ROS）水平,二硝基苯肼分析培养液上清中乳酸脱氢酶（LDH）水平。用生物信息学软件预测miR-486的靶基因,双荧光素酶报告基因实验检测miR-486、TRIM10靶向关系。 结果 与control组比较,PD组细胞存活率、Bcl-2蛋白表达降低,细胞凋亡率、Bax蛋白表达、MDA、ROS和LDH水平升高。与miR-NC组比,miR-486组细胞存活率、Bcl-2蛋白表达升高,细胞凋亡率、Bax蛋白表达、MDA、ROS和LDH水平降低。MiR-486靶向下调TRIM10表达。与miR-486+vector组比较,miR-486+TRIM10组细胞存活率、Bcl-2蛋白水平降低,细胞凋亡率、Bax蛋白水平、MDA、ROS和LDH水平升高。 结论 上调miR-486可通过靶向抑制TRIM10减少MPP+诱导的体外帕金森病PC12细胞模型的凋亡。     

左卡尼汀通过抑制钙/钙调素依赖蛋白激酶Ⅱ信号通路抑制过氧化氢诱导的大鼠心肌细胞凋亡

 目的： 观察左卡尼汀对过氧化氢（H2O2）诱导的大鼠心肌细胞凋亡的保护作用及其机制。方法：利用200 μmol/L H2O2刺激12 h,建立体外原代培养新生乳鼠心肌细胞凋亡模型。Ca2+螯合剂1,2-双(2-氨基苯氧基)乙烷-N, N, N′, N′-四乙酸(BAPTA)、钙调素依赖蛋白激酶II (CaMKII)特异性抑制剂KN93及左卡尼汀分别于加入H2O2前30 min或1 h加入,以检测这3种药物对H2O2刺激下心肌细胞活力、细胞凋亡、细胞内静息钙浓度（［Ca2+］i）及磷酸化CaMKII (p-CaMKII)表达的影响。利用MTT比色法检测心肌细胞活力;流式细胞仪检测细胞凋亡率;利用激光共聚焦扫描检测［Ca2+］i;蛋白质免疫印迹法检测cleaved caspase-3及p-CaMKII的表达。结果：模型组经200 μmol/L H2O2作用12 h后,细胞活力显著下降,细胞凋亡率显著增加。BAPTA、KN93及左卡尼汀预处理显著抑制上述细胞损伤。进一步研究发现,H2O2 诱导的 ［Ca2+］i水平升高、cleaved caspase-3及p-CaMKII的表达增加均可被上述3种药物不同程度地抑制。结论：左卡尼汀可抑制H2O2所致的心肌细胞凋亡,该心肌保护作用可能与其抑制Ca2+/CaMKⅡ信号通路有关。     

阿魏酸钠抑制纤维粘连蛋白和纤维蛋白原诱导的大鼠血管平滑肌细胞迁移和粘附

目的：探讨阿魏酸钠（SF）对纤维粘连蛋白(FN)和纤维蛋白原(Fg)诱导的血管平滑肌细胞(VSMCs)迁移和粘附的影响。 方法： VSMCs源于8周龄自发性高血压大鼠(SHR)的胸主动脉，组织块外生法体外培养VSMCs，在96孔聚苯乙烯平底培养板上进行细胞粘附实验；采用改良的Boyden微孔膜双槽法进行细胞迁移实验，荧光染料Fura-2/AM法测定细胞内游离钙离子(［Ca2+］i)浓度。 结果： (1) FN和Fg可诱导VSMCs的粘附，SF(10-7-10-3mol/L)呈浓度依赖性抑制上述物质诱导的细胞粘附，最大抑制率分别为67.12％和70.23％(P<0.01)。(2) FN和Fg可诱导VSMCs迁移，作用峰值浓度分别为40-60 mg/L和1-5 g/L。SF(10-7-10-3mol/L)呈浓度依赖性抑制上述物质诱导的细胞迁移，10-3mol/L SF对FN和Fg诱导的细胞迁移的抑制率达69.79%和87.06％。(3)Fg和FN显著促进［Ca2+］i升高(P<0.05)，SF明显抑制Fg和FN诱发的［Ca2+］i升高，峰抑制率分别为74.85％和71.57％。 结论： Fg和FN可诱导VSMCs粘附、迁移和［Ca2+］i浓度升高，SF可直接抑制VSMCs的粘附、迁移和［Ca2+］i浓度升高，抑制［Ca2+］i 浓度升高可能是它抑制上述物质诱导的VSMCs迁移的机制之一。     

二苯乙烯苷通过抑制氧化应激对MPP~+诱导的PC12细胞凋亡的保护作用

为了探讨2,3,5,4’-四羟基二苯乙烯-2-ο-β-D-葡萄糖苷(2,3,5,4’-tetrahydroxystibene-2-ο-β-D-glucoside,TSG)对1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium,MPP+)诱导PC12细胞凋亡的影响及其可能机制,本实验分为对照组,MPP+处理组和TSG(1、5和10μmol/L)预处理组。用Hoechst33258染色法和流式细胞术测定细胞凋亡,对活性氧(reactive oxygen species,ROS)敏感的荧光探针2,7-dichlorofluorescin dictate(DCF-DA),丙二醛(malondialdehyde,MDA)和总抗氧化能力(total anti-oxida-tion competence,T-AOC)检测试剂盒测定细胞的氧化应激的变化。结果显示:TSG三个浓度预处理后,对比MPP+处理组,观察到TSG在一定范围内以剂量依赖方式,使细胞核凝聚明显减少,细胞凋亡率降低。另外,TSG预处理后,PC12细胞中增高的ROS和MDA水平较MPP+处理组明显有所减低,T-AOC有所增强。以上结果提示,TSG可抑制MPP+诱导的PC12细胞的凋亡,其机制可能与TSG抑制氧化应激有关。     

促血小板生成素对化学性缺氧诱导的PC12细胞凋亡的影响

目的:研究促血小板生成素(TPO)对化学性缺氧诱导的大鼠嗜铬细胞瘤PC12细胞凋亡的影响及保护作用。方法:将PC12细胞进行相应实验处理,分为对照组、氯化钴(Co Cl2)处理组、Co Cl2+TPO组及TPO对照组。检测各组PC12细胞的存活率并用Annexin V/PI双染流式细胞术分别检测细胞凋亡率,线粒体膜电位的变化及细胞内活性氧簇的变化。结果:化学性缺氧模拟剂Co Cl2可以明显抑制PC12细胞的生长(P0.01);与对照组比较,Co Cl2组的细胞凋亡率明显升高(P0.05),而Co Cl2+TPO组的细胞凋亡率显著低于Co Cl2组(P0.05);TPO能减少细胞内活性氧簇生成以及抑制细胞线粒体膜电位的降低(P0.01)。结论:TPO能对抗Co Cl2缺氧所致的细胞凋亡,稳定线粒体膜电位,发挥细胞保护作用。     

14-3-3γ过表达通过抑制促凋亡分子Bax转位保护多巴胺能细胞对抗MPP~+的毒性作用

目的:探讨14-3-3γ蛋白过表达能否抑制促凋亡分子Bax对抗MPP+对多巴胺能细胞的毒性作用。方法:用腺病毒载体携带人14-3-3γ基因感染PC12细胞,使14-3-3蛋白在细胞中过表达,通过Western Blot检测14-3-3γ、Bax、细胞色素c、caspase 3蛋白质的表达、DAPI染色法检测细胞凋亡、MTT法和自动生化检测法测定细胞活力及LDH酶的活性。结果:14-3-3γ蛋白的过表达能抑制Bax转位到线粒体,抑制LDH的活性(MPP+组41.18±2.71,m14-3-3γ组39.45±3.16,14-3-3γ组13.54±1.82),提高细胞活力(MPP+组49.86±6.27%,m14-3-3γ组52.35±5.59%,14-3-3γ组81.02±5.83%),从而抑制了MPP+诱导的PC12细胞的凋亡(MPP+组49.38±7.31%,m14-3-3γ组46.54±5.49%,14-3-3γ组8.76±3.59%)。结论:14-3-3γ蛋白可以通过对促凋亡分子Bax转位的抑制对抗MPP+对PC12细胞的毒性作用,最终达到细胞保护作用。     

线粒体损伤在创伤弧菌诱导树突状细胞凋亡中的作用

 目的：探讨线粒体损伤在创伤弧菌（Vibrio vulnificus,Vv）诱导树突状细胞（dendritic cell,DC）凋亡过程中的作用及其可能机制。方法：建立Vv 1.1758与DC2.4细胞混合培养模型。采用扫描电镜和透射电镜观察创伤弧菌侵入细胞方式和细胞线粒体病变情况。荧光探针DCFH-DA和Fluo-8/AM分别检测侵入细胞内活性氧（ROS）和Ca2+离子水平。流式细胞术检测细胞线粒体膜电位和细胞凋亡情况。采用Western blotting检测NF-κB p65和TNF-α蛋白的表达。结果：Vv 1.1758可诱导DC2.4细胞凋亡。Vv 1.1758以菌体一端与细胞表面结合的方式侵入细胞,侵入细胞的线粒体有明显病变,细胞内ROS和Ca2+水平升高,线粒体膜电位降低。共培育1 h,NF-κB p65蛋白即开始升高,5 h达高峰,6 h稍有下降;TNF-α蛋白则在共培育2 h开始增高,6 h达高峰。结论：线粒体损伤在Vv诱导DC凋亡中发挥作用,其作用机制可能与细胞内ROS和Ca2+水平升高、线粒体膜电位降低有关,NF-κB p65和TNF-α可能是细胞凋亡过程中的重要信号分子。     

MPP+对PC12细胞内源性H2S生成的影响

目的： 观察1-甲基4-苯基吡啶离子 (MPP+)对PC12细胞内源性硫化氢（H2S）生成的影响,以探讨MPP+损伤PC12细胞的新机制。方法: RT-PCR方法检测PC12细胞胱硫醚-β-合酶（CBS）mRNA 的表达;亚甲基蓝分光光度计法检测PC12细胞内源性H2S的含量及PC12细胞CBS活性;台盼蓝拒染色法观察PC12细胞的存活率。结果：MPP+ 可以抑制PC12细胞CBS的表达及其活性,减少内源性H2S的生成;MPP+可以明显地降低PC12细胞的存活率;H2S的供体硫氢化钠（NaHS）对MPP+诱导的PC12细胞损伤具有显著的拮抗作用。结论: MPP+能抑制CBS的表达和活性,减少内源性H2S生成,这可能与其损伤PC12细胞的机制有关。     

银杏内酯B对谷氨酸诱导脑皮质神经元凋亡及Ca2+超载的影响

目的：观察银杏内酯B对谷氨酸诱导培养脑皮质神经元凋亡的拮抗作用，并探讨这种作用与神经细胞内游离Ca2+浓度改变的关系。方法： 采用改良的方法原代培养胎小鼠脑皮质神经元，用噻唑兰（MTT）法检测神经元的存活情况；细胞凋亡采用形态学观察、DNA琼脂糖凝胶电泳法和Hoechst 33258核染色方法进行分析；用Fura-2/AM荧光指示剂法测定细胞内Ca2+浓度。结果： 谷氨酸（0.8 mmol/L）能诱导神经细胞凋亡和胞内Ca2+超载，银杏内酯B（10-250 μmol/L）能减轻谷氨酸所致的细胞损伤，表现为神经元存活率提高，细胞形态的恢复和DNA断裂减少。结论： 银杏内酯B可拮抗谷氨酸所致的神经细胞毒性作用，这可能与其能竞争PAF受体并降低神经细胞内［Ca2+］从而抑制谷氨酸诱导的神经元凋亡有关。     

iPSC-MSCs对CoCl_2诱导的PC12细胞损伤时线粒体功能的影响

目的:观察诱导性多能干细胞来源的间充质干细胞(i PSC-MSCs)对氯化钴(Co Cl2)诱导的PC12细胞损伤的影响,并探讨其可能机制。方法:用Co Cl2处理PC12细胞建立化学损伤模型,加入i PSC-MSCs共培养。用细胞计数试剂盒-8(CCK-8)比色法检测细胞存活率,Annexin V/PI双染流式细胞术检测细胞凋亡比率,JC-1染色流式细胞术检测细胞线粒体膜电位,免疫荧光观察i PSC-MSCs向PC12细胞转移线粒体的情况。结果:用Co Cl2(400μmol/L)处理PC12细胞24 h可使其凋亡明显增多,线粒体膜电位明显下降。与i PSC-MSCs共培养能减轻PC12细胞的凋亡,使其膜电位恢复。i PSC-MSCs可以与PC12细胞间形成隧道纳米管并向PC12细胞转移线粒体。结论:i PSC-MSCs可以减轻Co Cl2诱导的PC12细胞损伤,机制可能与其向PC12细胞转移线粒体有关。     

米诺环素抑制MPP+诱导的PC12细胞凋亡和线粒体功能损伤

目的: 探讨米诺环素(minocycline)对1-甲基-4-苯基吡啶离子(MPP⁺)诱导的PC12细胞凋亡和线粒体功能损伤的保护作用。方法: 将MPP⁺加入体外培养的的 PC12 细胞中, 建立多巴胺能神经元凋亡模型, 实验过程中用minocycline进行预处理,四甲基偶氮唑盐法(MTT法)检测细胞存活率,Hoechst染色检测细胞凋亡,DCFH-DA检测ROS聚集,JC-1检测细胞线粒体膜电位变化。结果: 0.5 mmol/L MPP⁺处理PC12细胞24 h,能明显抑制细胞生长(抑制率80.8%),诱导细胞发生凋亡(凋亡率5.22%),同时ROS浓度提高230.0%,线粒体膜去极化(绿/红荧光强度比为11.95)。而加入10 μmol/L minocycline预处理30 min可明显升高MPP⁺处理的 PC12 细胞活性,细胞凋亡率明显降低(P<0.01),ROS浓度明显下降,绿/红荧光强度比也明显降低(P<0.01)。结论: Minocycline抑制MPP⁺诱导的PC12细胞凋亡部分通过对抗其线粒体功能而发挥作用。     

Protection by puerarin against MPP+-induced neurotoxicity in PC12 cells mediated by inhibiting mitochondrial dysfunction and caspase-3-like activation

Puerarin, a main isoflavone glycoside distributed in Pueraria lobata (Willd.) Ohwi, showed inhibitory activity on H2O2-induced PC12 cells damage in our previous work. However, there is insufficient evidence in protective mechanism of puerarin, especially that relating to the mitochondrial function. In this study, when cells were pretreated with puerarin prior to 0.4 mM MPP+, protective roles were accompanied by a reduction of cell viability loss, morphological changes of apoptosis and apoptotic rate. To explore the protective mechanism of puerarin in MPP+-induced PC12 cells, mitochondrial function and caspase-3-like activity were measured. The results indicated that puerarin inhibited the release of mitochondrial cytochrome c to cytosol and the loss of mitochondrial membrane potentials. In addition, puerarin also reduced MPP+-induced caspase-3-like activation. Taken together, the above results suggest that pretreatment of PC12 cells with puerarin could block MPP+-mediated apoptosis by mitochondria-dependent caspase cascade.     

Protective effects of atypical antipsychotic drugs against MPP(+)-induced oxidative stress in PC12 cells

Recent studies have suggested that some atypical antipsychotic drugs may have protective properties against oxidative stress. To confirm these findings, we investigated the protective effects of atypical antipsychotic drugs such as olanzapine, aripiprazole, and ziprasidone on oxidative stress induced by the N-methyl-4-phenylpyridinium (MPP(+)) ion in PC12 cells. Haloperidol, a typical antipsychotic drug, was used for comparison. We determined the antioxidant effects of atypical antipsychotic drugs using a number of measures, including cell viability, the formation of reactive oxygen species (ROS), superoxide dismutase (SOD) activity and Bax levels. MPP(+) treatment induced significant loss of cell viability, the formation of ROS, reduction of SOD activity, and up-regulation of Bax expression. However, olanzapine, aripiprazole and ziprasidone reversed these effects caused by MPP(+) treatment, but ziprasidone did not influence cell viability. In contrast, haloperidol did not affect all these effects. Moreover, haloperidol strongly increased the expression of Bax under MPP(+)-free conditions. Olanzapine, aripiprazole, and ziprasidone, but not haloperidol, may exert antioxidant effects through modulating ROS levels, SOD activity, and Bax expression to provide protective effects against MPP(+)-induced oxidative stress in PC12 cells. These results suggest that some atypical antipsychotic drugs have a useful therapeutic effect by reducing oxidative stress in schizophrenic patients.     

N-methylated beta-carbolines protect PC12 cells from cytotoxic effect of MPP+ by attenuation of mitochondrial membrane permeability change

Opening of the mitochondrial permeability transition pore has been recognized to be involved in cell death. The present study investigated the effect of beta-carbolines (harmaline and harmalol) on the MPP(+)-induced change in the mitochondrial membrane permeability and cell death in differentiated PC12 cells. beta-Carbolines and antioxidants (superoxide dismutase, catalase, ascorbate or rutin) prevented the loss of cell viability in PC12 cells treated with 250 microM MPP(+), while the effects of N-acetylcysteine and dithiothreitol were not observed. beta-Carbolines reduced the condensation and fragmentation of nuclei caused by MPP(+) in PC12 cells. beta-Carbolines alone did not exhibit a significant cytotoxic effect on PC12 cells. beta-Carbolines (50 microM) inhibited the decrease in mitochondrial transmembrane potential, cytochrome c release, activation of caspase-3, formation of reactive oxygen species (ROS) and depletion of GSH caused by MPP(+) in PC12 cells. beta-Carbolines reduced the hydrogen peroxide- or SIN-1-induced cell death in PC12 cells. The results suggest that beta-carbolines may attenuate the MPP(+)-induced viability loss in PC12 cells by inhibition of change in the mitochondrial membrane permeability and by antioxidant effect.     

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.     

蜂胶醇提物通过抑制caspase-12减轻氧化低密度脂蛋白诱导的巨噬细胞凋亡

 目的: 研究蜂胶醇取物(ethanol extract of propolis,EEP)对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导的巨噬细胞凋亡的抑制作用,并探讨可能的分子机制。 方法: 体外培养RAW264.7巨噬细胞,给予EEP(7.5、15和30 mg/L)、4-苯丁酸(4-phenylbutyric acid,PBA;5 mmol/L)或二亚苯基碘鎓(diphenyleneiodo-nium, DPI;5 μmol/L)预处理1 h,再加入ox-LDL(100 mg/L)或衣霉素(tunicamycin,TM;4 mg/L)继续培养24 h。分别采用MTT法和Annexin V-FITC双染法检测细胞活力和凋亡情况;试剂盒测定细胞内超氧化物歧化酶(superoxide dismutase,SOD)活性、活性氧簇(reactive oxygen species,ROS)和丙二醛(malondialdehyde,MDA)的水平。采用免疫印迹技术检测内质网应激(endoplasmic reticulum stress,ERS)凋亡途径关键蛋白caspase-12的表达变化。结果: 与ERS抑制剂PBA相似,EEP呈剂量依赖性减轻ox-LDL所致的巨噬细胞损伤,表现为细胞活力增加(P<0.01),凋亡率降低(P<0.05),且可抑制ERS诱导剂TM所引起的巨噬细胞活力下降和凋亡(P<0.05);与氧化应激抑制剂DPI相似,EEP可抑制ox-LDL诱导的氧化应激反应,表现为ROS和MDA生成减少(P<0.01),SOD活性增加(P<0.05);EEP显著抑制ox-LDL和TM所诱导的caspase-12活化(P<0.05);与ox-LDL组比较,PBA和DPI预处理组caspase-12活性也受到明显抑制(P<0.01)。结论: EEP可减轻 ox-LDL 所诱导的RAW264.7巨噬细胞凋亡,其机制可能与抑制氧化应激和caspase-12活化有关。     

利福平抑制α-synuclein的聚集及对MPP＋诱导细胞损伤的拮抗作用

目的： 探讨利福平对帕金森病致病蛋白α-突触共核蛋白（α-synuclein）聚集的影响，以及对1-甲基-4-苯基-吡啶离子(MPP+)诱导的细胞损伤的拮抗作用。 方法： 选用大鼠嗜铬细胞瘤株PC12细胞，利用MPP+诱导建立帕金森病细胞模型，利福平进行干预；采用MTT法检测细胞活性、Western blotting法检测α-synuclein表达及聚集，流式细胞仪检测细胞凋亡。 结果： 1 mmol/L MPP+组细胞活性明显低于对照组，细胞凋亡率和α-synuclein表达及聚集高于对照组；预先经过100、200和300 μmol/L各浓度利福平处理后，MPP++利福平组细胞活性明显高于1 mmol/L MPP+组，而细胞凋亡率和α-synuclein表达及聚集均低于1 mmol/L MPP+组，并具有明显的剂量-效应关系。 结论： 利福平能够抑制α-synuclein的表达及聚集，并对MPP+诱导的PC12细胞损伤具有拮抗作用。     

EGCG通过mTOR通路拮抗低氧诱导的PC12细胞的凋亡与自噬

 目的:氯化钴（cobalt chloride, CoCl2）诱导的低氧具有神经毒性,可以诱导神经细胞的凋亡和自噬。茶多酚活性成分表没食子儿茶素没食子酸酯（epigallocatechin gallate, EGCG）具有一定的抗细胞凋亡和自噬的作用,但作用机理尚未完全阐明。近年来有研究报道,雷帕霉素靶蛋白（mammalian target of rapamycin,mTOR）通路参与了多种神经功能的调节,如神经细胞的分化成熟、抗氧化应激等。为此,我们用CoCl2诱导低氧引起细胞的凋亡与自噬,从mTOR通路探讨EGCG拮抗CoCl2诱导低氧引起细胞凋亡与自噬的作用机制。方法:研究使用Wes-tern blotting方法测定CoCl2以及EGCG处理后mTOR和自噬蛋白beclin-1的表达,使用ELISA法检测细胞caspase-3表达,CCK-8检测细胞活力,免疫荧光法观察LC-3在胞核内的表达。结果:CoCl2诱导低氧引起了细胞凋亡与自噬,而EGCG通过mTOR通路拮抗了CoCl2诱导低氧引起的细胞凋亡与自噬,阻断mTOR通路则逆转了EGCG对神经细胞的保护作用。结论: EGCG通过mTOR通路拮抗了CoCl2诱导的低氧引起的PC12细胞的凋亡与自噬。