Cell viability and dopamine secretion of 6-hydroxydopamine-treated PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells

In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson＇s Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesenchymal stem cells against 6-hydroxydopamine-induced neurotoxicity and to verify whether the mechanism of action relates to abnormal a-synuclein accumulation in cells Results showed that co-culture with bone marrow-derived mesenchymal stem cells enhanced PC12 cell viability and dopamine secretion in a cell dose-dependent manner. MitoLight staining was used to confirm that PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells demonstrate reduced levels of cell apoptosis. Immunocytochemistry and western blot analysis found the quantity of α-synuclein accumulation was significantly reduced in PC12 cell and bone marrow-derived mesenchymal stem cell co-cultures. These results indicate that bone marrow-derived mesenchymal stem cells can attenuate 6-hydroxydopamine-induced cytotoxicity by reducing abnormal α-synuclein accumulation in PC12 cells.

Baicalein causes alternation in dopamine metabolism in PC12 cells by inhibiting the expression of COMT and DAT

Aim Baicalein is the major flavonoid obtained from the Scutellaria root. Our previous studies have dem- onstrated that baicalein has a clear positive effect on recovery in an experimental model of Parkinsonism. The put- pose of this study was to investigate the role of baicalein in modulating dopamine （DA） metabolism in PC12 cells and to explore possible mechanisms of its actions. Methods The intracellular content and extracellular release of DA in both rotenone-treated and untreated PC12 cells were examined. Second, PC12 cells were first pretreated with baicalein （ 10 μmol · L^-1 ） for 10 rain, and then incubated with or without ionomycin （5 μmol · L^-1 ） for 10 rain to test whether short-term exposure to baicalein affected calcium-dependent or spontaneous DA release. Third, the intracellular and extracellular contents of DA and its related metabolites were examined. After treatment with baica- lein for 24 h, the The tyrosine hydroxylase （TH）, monoamine oxidase B （MAOB）, catechol-O-methyltransferase （COMT） and dopamine transporter （DAT） were detected by immunoblot analysis. Results The results showed that baicalein prevented rotenone-induced cytotoxicity and significantly increased the DA content in both rotenone- treated and untreated PC12 cells. Furthermore, it had no effect on ionomycin-induced or spontaneous DA release after short-term exposure but significantly increased DA content in a time- and dose-dependent manner after treat- ment for 6 h. Baicalein also significantly decreased the intracellular and extracellular homovanillic acid （HVA） content but increased the intracellular 3,4-dihydroxy phenylacetic acid （DOPAC） content. Finally, baicalein sig- nificantly decreased the expression of COMT and DAT, but it had no effect on the expression of TH and MAOB. Conclusion These data suggest that bacalein has the ability to increase DA content and modulate DA metabolism by inhibiting the expression of COMT and DAT. Our study provides evidence that baicalein may be a potential anti- PD drug that merits further study.

Treatment time influences the effects of a low-frequency pulsed electric field on synthesis of tyrosine hydroxylase and dopamine in PC12 cells

BACKGROUND： Electromagnetic radiation can influence dopamine （DA） synthesis in brain tissues or ceils, but electromagnetic frequencies, intensities, and radiation time can produce different effects. In addition, the signal pathway by which electromagnetic radiation influences DA synthesis remains controversial. OBJECTIVE： To determine tyrosine hydroxylase （TH） expression in PC12 cells and DA levels in cell culture media after different periods of low-frequency pulsed electric field （LF-PEF） stimulation, and to determine how LF-PEF signaling stimulates TH synthesis using inhibitors. DESIGN, TIME AND SETTING： A parallel, controlled, cell experiment was performed at the Laboratory of Cell Biology, School of Life Science, East China Normal University, between January and October 2006. MATERIALS： PC12 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China. Nerve growth factor was purchased from PeproTech, USA. The protein kinase A inhibitor, H-89, and mitogen-activated protein kinase kinase inhibitor, U0126, were purchased from Sigma, USA. METHODS： （1） Following routine culture in Dulbecco＇s modified eagle medium, primary PC12 cells were stimulated under LF-PEF （pulse frequency 50.Hz, pulse width 20 μs, peak field strength 1 V/m） for 5, 10, 15, 20, and 30 minutes. （2） Inhibitors （H-89 or U0126, 1 μmol/L） were added 30 minutes before LF-PEF stimulation for 10 minutes. MAIN OUTCOME MEASURES： （1） TH expression was determined by Western blot in PC12 cells at 0.5, 1,2, 3, and 4 days after LF-PEF stimulation. Similarly, DA was measured by high-performance liquid chromatography in media at 2, 3, 4, or 5 days after LF-PEE （2） TH expression was detected 1 day after H-89 or U0126 treatment and LF-PEE RESULTS： （1） Short-term LF-PEF stimulation （5 and 10 minutes） increased TH expression and media DA levels after short-term culture （2 days） （P 〈 0.01）, but both parameters decreased with longer culture （3 4 days） （P 〈 0.01）. Long-term LF-PEF stimulation （15, 20, or 30 minutes） decreased TH and DA synthesis, followed by a rapid increase （P 〈 0.01）. （2） H89 could completely inhibit TH expression in PC12 cells stimulated by LF-PEF for 10 minutes, while the inhibition rate of U0126 was 53.2%. CONCLUSION： Short-term LF-PEF first promotes then inhibits, while long-term LF-PEF first inhibits then promotes, TH and DA synthesis. LF-PEF stimulation regulates TH expression primarily by activating protein kinase A to regulate DA synthesis.

Genistein and daidzein reduced chlorpyrifos induced damage in PC12 cell

The present study preliminarily evaluated neurotoxicity injuries induced by chlorpyrifos in PC12 cell,which were used as a model for nervous cell system.In cultured PC12 cell,application of soy isoflavones(genistein,daidzein,monomer and mixture)significantly reduced chlorpyrifos induced toxicity,a widely used pesticide,and resulted in a better cell survival rate.Treatments with isoflavones reduced malondialdehyde content,reactive oxygengeneration and acetylcholine level in medium,and maintained mitochondrial membrane potential integrity.Daidzein enhanced endogenous antioxidant system in PC12 cell with an increasing in superoxide dismutase perunit activity.Genistein reduced acetylcholine content in the medium.Daidzein and genistein showed different effects,and their combined effect were greater than individual.In conclusion,soy isoflavones as an antioxidant and neuroprotectant,enhanced choline metabolism,which effectively mitigated disadvantageous influence of PC12 cell caused by chlorpyrifos.

Effects of hypoxia, soman and their combination on PC12 cells

To investigate the effects of hypoxia, soman and their combined ones on PC12 cells. Methods: After the PC12 cells were exposed to an atmosphere containing different concentrations of oxygen and cultured in a medium containing different concentrations of soman, the amount of lactic dehydrogenase (LDH) released by the cells and their survival rate were determined to observe the dose-dependent and time-dependent cytotoxic effects. Student’s t test and two-way ANOVA were employed to determine the statistical differences and interaction between hypoxia and soman exposure. Results: 1) Both hypoxia and soman exposures exerted dose-dependent cytotoxic effects on PC12 cells and the interaction between the two injurious factors was significant; 2)The combined effects of the two factors were equal to the sum of those exerted by each one separately; and the combined application of the two factors resulted in a more severe cytotoxicity than that caused by either agent used singly; 3) The amount of LDH released from PC12 cells could serve as a more sensitive indicator of cytotoxicity than the survival rate of the cells. Conclusion: This study demonstrates the cytotoxic effects of the combined exposure to hypoxia and soman acted in a summative manner, which suggests that the two factors might induce intracellular release of LDH in PC12 cells through different mechanisms.

多巴胺对PC12细胞的双重影响

本实验运用PC12细胞,研究不同浓度多巴胺(dopamine,DA)对细胞的影响。同时利用兼具促进多巴胺释放和抑制多巴胺摄取双重作用的安非它命(amphetamine,AMP)观察胞内外多巴胺对细胞的不同作用。结果显示:胞外高浓度DA能引起细胞抗氧化能力下降,胞内游离Ca^(2+)浓度上升,细胞存活率大幅度降低,部分细胞出现凋亡;低浓度DA对细胞存活率无明显影响,而使细胞抗氧化能力有一定提高。长时间安非它命单独作用也可引起细胞存活率下降,并伴随胞内GSH水平降低;安非它命与多巴胺共同作用在一定程度上可导致细胞内抗氧化物质水平低于多巴胺单独作用,表明细胞内一定浓度DA可以维持或提高细胞抗氧化物质水平。结果提示,脑内同样存在的多巴胺神经元对DA重摄取功能下降,胞外氧化应激增强,可能是引起脑内多巴胺神经元退行性病变的重要原因之一。

Mechanisms of rotenone-induced neurotoxicity in PC 12 cells

BACKGROUND： Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson＇s disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal degeneration in Parkinson＇s disease remains unclear. OBJECTIVE： To establish rotenone-induced neurotoxicity in PC 12 cells, and to investigate the possible action pathways to rotenone-induced neural cell injury at the protein level. DESIGN, TIME AND SETTING： A controlled proteomics study was performed at the Department of Neurology, First Hospital, Jilin University between March 2006 and March 2007. MATERIALS： PC 12 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences, China. Rotenone was provided by Sigma, USA. METHODS： PC 12 cells in logarithmic growth phase were treated under experimental and control conditions, respectively. A total of 0.5 μmol/L rotenone, or the same amount of Dulbecco＇s modified eagle＇s medium （DMEM）, was added in the experimental and control conditions, respectively. MAIN OUTCOME MEASURES： Following 72 hours of rotenone treatment, cellular survival rate was determined by methyl thiazolyl tetrazolium assay, and apoptotic changes were detected by Hoechst 33342 staining. Total cellular protein was extracted to acquire differential protein expression data utilizing two-dimensional differential in-gel electrophoresis. To identify differential protein spots, matrix-assisted laser desorption/ionization-time of flight mass spectrometry （MALDI-TOF-MS） was used. RESULTS： In the MTT assay, the experimental condition induced significantly less cell survival compared to the control condition （P 〈 0.01）. Hoechst 33342 staining revealed a larger number of apoptotic cells under the experimental condition compared to the control condition （P 〈 0.01）, as determined by the presence of nuclear condensation, pyknosis, and nuclear fragmentation. Two-dimensional electrophoresis results showed that the differential expression of protein spots 1069 and 1538 was increased by 144% and 124%, respectively, while that of protein spot 1094 was decreased by 123% in the experimental condition compared to the control condition （P 〈 0.01）. By MALDI-TOF-MS analysis and database retrieval, γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A were confirmed to be involved in rotenone-induced neural cell injury. CONCLUSION： γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A might participate in rotenone-induced neurotoxicity in PC 12 cells.

Necrostatin-1 protection of dopaminergic neurons

Necroptosis is characterized by programmed necrotic cell death and autophagic activation and might be involved in the death process of dopaminergic neurons in Parkinson＇s disease. We hypothesized that necrostatin-1 could block necroptosis and give protection to dopaminergic neurons. There is likely to be crosstalk between necroptosis and other cell death pathways, such as apoptosis and autophagy. PC12 cells were pretreated with necroststin-1 1 hour before exposure to 6-hydroxydopamine. We examined cell viability, mitochondrial membrane potential and expression patterns of apoptotic and necroptotic death signaling proteins. The results showed that the autophagy/lysosomal pathway is involved in the 6-hydroxydopamine-induced death process of PC12 cells. Mitochondrial disability induced overactive autophagy, increased cathepsin B expression, and diminished Bcl-2 expression. Necrostatin-1 within a certain concentration range（5–30 μM） elevated the viability of PC12 cells, stabilized mitochondrial membrane potential, inhibited excessive autophagy, reduced the expression of LC3-II and cathepsin B, and increased Bcl-2 expression. These findings suggest that necrostatin-1 exerted a protective effect against injury on dopaminergic neurons. Necrostatin-1 interacts with the apoptosis signaling pathway during this process. This pathway could be a new neuroprotective and therapeutic target in Parkinson＇s disease.

Can amino-functionalized carbon nanotubes carry functional nerve growth factor?

Carbon nanotubes can carry protein into cells to induce biological effects. Amino-functionalized carbon nanotubes are soluble and biocompatible, have high reactivity and low toxicity, and can help promote nerve cell growth. In this study, amino-functionalized ethylenediamine-treated multi-walled carbon nanotubes were used to prepare carbon nanotubes-nerve growth factor complexes by non-covalent grafting. The physicochemical properties, cytotoxicity to PC12 and chick embryo dorsal root ganglion, and biological activity of the carbon nanotubes-nerve growth factor complexes were investigated. The results showed that amino functionalization improved carbon nanotubes-nerve growth factor complex dispersibility, reduced their toxicity to PC12 cells, and promoted PC 12 cell differentiation and chick embryo dorsal root ganglion.

百草枯诱导PC12细胞损害和miR-133b表达的变化

目的探讨百草枯（P0）对大鼠肾上腺嗜铬细胞瘤（PC12）细胞毒性作用和对miR-133b表达的影响。方法以50、100、300μmol／LPQ分别处理PCI2细胞24h，用噻唑蓝（MTT）法检测PCI2细胞毒性；以100、300μmol／LP0分别处理PCI2细胞24h，用AnnexinV．FITC／PI法流式细胞仪检测细胞凋亡，用荧光定量反转录聚合酶链反应（RT—PCR）法检测miR-133b相对表达水平。结果与对照组比较，100、300μmol／LPQ处理细胞24h后细胞存活率明显下降，差异有统计学意义（P〈0．05或P〈0．01）。与对照组比较，100、300μmol／LPQ处理细胞24h后细胞凋亡率明显升高，差异有统计学意义（P〈0．01）。300μmol／LPQ处理细胞24h可诱导细胞miR-133b表达，差异有统计学意义（P〈0．05）.结论PQ致PCI2细胞损害和细胞凋亡时miR-133b的表达上调。

UPLC/Q Exactive MS检测肉苁蓉多糖促PC12细胞释放神经递质的方法研究

目的:建立UPLC/Q Exactive MS法测定PC12细胞释放的神经递质多巴胺、去甲肾上腺素和谷氨酸含量。方法:采用Ultima te 3000系列高效液相色谱仪和Q Exactive四极杆-静电场轨道阱高分辨质谱系统检测。标准品用超纯水溶解后,以乙腈-水(0.1%甲酸,10 mmol·L^(-1)醋酸铵)(4∶96)为流动相,Hypersil GOLD色谱柱分离,通过高分辨质谱的全扫描/选择离子监测(Full MS/SIM)模式进行定性筛查和定量检测。应用Sigma Plot12.0统计软件包进行数据分析。结果:多巴胺和谷氨酸在3~2 000 ng·mL^(-1)浓度范围内线性关系良好,最低定量限为3 ng·mL^(-1);去甲肾上腺素在100~2 000 ng·mL^(-1)浓度范围内线性关系良好,最低定量限为1 00 ng·mL^(-1);3种神经递质的加标回收率高,精密度RSD均小于5%;常温放置8 h,4℃冷藏7 d后以及-20℃冷冻1个月后稳定性良好。本实验同时在药物肉苁蓉多糖(CDPS)改善学习记忆功能已有研究的基础上,测定PC12细胞给药后3种神经递质不同时间点的释放浓度,结果显示CDPS对3种神经递质释放量有明显促增趋势。结论:UPLC/Q Exactive MS法适合同时测定PC12细胞分泌的多巴胺、去甲肾上腺素和谷氨酸的含量。