胰岛素受体磷酸化增加对MPP＋诱导的PC12细胞凋亡的影响

目的:研究胰岛素抗MPP 诱导PC12细胞凋亡中胰岛素受体(IR)的变化。方法:应用半定量逆转录聚合酶链式反应(RT-PCR)测定胰岛素抗MPP 诱导PC12细胞凋亡过程中IRmRNA的基因表达,免疫沉淀Western印迹分析技术测定相同条件下IR的蛋白表达,应用IR自身磷酸化的特异阻断剂HNMPA-AM3,通过MTT法观察PC12细胞生存率的改变、瞬转IR质粒后再次观察细胞生存率的改变及进一步通过免疫沉淀Western印迹分析技术检测IR蛋白质磷酸化水平。结果:IR磷酸化程度的变化与细胞生存率的变化有关,HNMPA-AM3可阻断胰岛素对MPP 诱导的PC12细胞的抗凋亡作用,IR可增强胰岛素的抗凋亡作用。结论:IR磷酸化增加可抵抗MPP 诱导的PC12细胞的凋亡。     

IGF-1通过PI3K/Akt通路抑制MPP~+诱导PC12细胞凋亡机制的研究

目的探讨胰岛素样生长因子-1(insulin-like growth factor-1,IGF-1)对1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium,MPP+)诱导的PC12细胞凋亡的抑制作用及其潜在的作用机制。方法以250μmol/L的MPP+损伤PC12细胞作为帕金森病(Parkinson disease,PD)细胞模型。实验分组如下:空白对照组、MPP+组、IGF-1组、IGF-1+MPP+组、IGF-1+MPP++LY294002组。孵育24 h后采用AO-EB法检测细胞凋亡率;采用MTT法检测细胞存活率;孵育4 h之后采用Western blot免疫印迹法检测Akt、磷酸化-Akt(phospho-Akt,p-Akt)蛋白表达。结果 (1)100 nmol的IGF-1能够显著抑制MPP+所致的细胞凋亡,对PC12细胞具有保护作用;(2)总Akt含量蛋白表达水平各处理组之间差别无统计学意义(P>0.05),但磷酸化的Akt在IGF-1+MPP+组表达高于MPP+单独处理组(P<0.05)。结论 IGF-1可减少MPP+所致的细胞凋亡,其保护作用与上调磷酸化的Akt的表达相关。     

磷脂酰肌醇3-激酶/蛋白激酶B磷酸化是胰岛素受体后信号转导通路控制PC12细胞凋亡的机制

目的:研究胰岛素抵抗1-甲基-4苯基砒啶(MPP )诱导的PC12细胞凋亡的信号转导途径中,磷脂酰肌醇3-激酶/蛋白激酶B(PI3-K/PKB)的活力变化。方法:应用Wortmannin(PI3-K抑制剂),比较用药前后细胞生存率的变化;应用Western印迹分析检测此间PKB及其磷酸化水平的变化。结果:Wortmannin预处理组细胞生存率较之胰岛素干预组明显下降;PKB的特异性磷酸化程度(Ser473磷酸化程度/激酶蛋白量)与细胞生存率的变化有关。结论:胰岛素主要通过调节PI3-K活性后再促进PKB的磷酸化,从而促进PKB的激活,并导致生物学效应的变化,但是尚不能排除其他机制的参与。     

IGF-1对MPP~+诱导PC12细胞凋亡的保护作用及其机制

目的探讨胰岛素样生长因子-1(IGF-1)对1-甲基-4-苯基吡啶离子(MPP+)诱导的PC12细胞凋亡的保护作用及机制。方法以250μmol/L的MPP+损伤PC12细胞作为帕金森病(PD)细胞模型,实验分组如下:空白对照组、MPP+组,IGF-1+MPP+组和抑制剂组。抑制剂组再分为:(1)空白对照组;(2)MPP+组;(3)IGF-1组;(4)IGF-1+MPP+组;(5)MPP++LiCL组;(6)IGF-1+MPP++LiCL组。孵育24h后采用AO-EB法检测细胞凋亡率;采用MTT法检测细胞存活率;孵育4h之后采用Western Blot免疫印迹法检测糖原合成酶激酶-3β(GSK-3β)、phospho-GSK-3β蛋白表达。结果(1)100nmol的IGF-1对MPP+处理的PC12细胞有保护作用,减少了MPP+所致的细胞凋亡。(2)LiCL起到了与IGF-1相似的对PC12细胞的保护作用。(3)总GSK-3β含量各处理组没有太大改变,但磷酸化的GSK-3β含量IGF-1组高于与MPP+单独处理组。结论IGF-1可减少MPP+所致的细胞凋亡,其保护作用与上调磷酸化的GSK-3β的表达相关。     

胰岛素可抵抗MPP^＋诱导的PC12细胞的凋亡

目的观察胰岛素在MPP+诱导PC12细胞凋亡中的干预作用.方法应用MTT法研究细胞活性的改变,应用HOECHST33258染色结合荧光显微镜技术及流式细胞技术检测不同药物对PC12细胞的凋亡诱导作用,应用半定量逆转录聚合酶链式反应(RT-PCR)测定胰岛素受体(insulin receptor,IR)mRNA的改变.结果①MPP+诱导PC12细胞凋亡,胰岛素可以抵抗此凋亡作用;②以上两种处理,均未见到胰岛素受体mRNA的改变,推测胰岛素受体的自身磷酸化有改变.结论胰岛素可以抵抗MPP+诱导的PC12细胞的凋亡.     

β分泌酶抑制剂对冈田酸诱导PC12细胞淀粉样蛋白前体蛋白代谢的影响

目的 探索β分泌酶抑制剂对冈田酸(OA)诱导的PC12细胞淀粉样蛋白前体蛋白(APP)代谢的影响. 方法 10 nmol/LOA作用4 h、8 h、16 h、24h及48 h诱导PC12细胞tau磷酸化,加β分泌酶抑制剂干预,MTT法测定细胞抑制率,免疫细胞化学法及Western blot检测全长APP和β-C末端片段(βCTF). 结果 10 nmol/L OA对PC12细胞的抑制作用呈时间依赖性,β分泌酶抑制剂可明显减轻该作用.OA诱导PC12细胞内全长APP和13CTF含量增加,B分泌酶抑制剂进一步增加细胞内全长APP含量,并减少βCTF含量. 结论 OA诱导PC12细胞中APP主要经β分泌酶途径代谢,生成具有神经毒性的βCTF.β分泌酶抑制剂通过维持细胞存活和减少13CTF从而减轻OA诱导的神经毒性,但使细胞内APP进一步增多.     

有氧运动对小鼠骨骼肌结节性硬化复合物蛋白2基因表达及胰岛素受体底物1丝氨酸磷酸化的影响**☆

背景：研究表明,胰岛素受体底物蛋白1(insulin receptor substrate 1,IRS1)的丝氨酸磷酸化和结节性硬化复合物蛋白2的表达及哺乳动物雷帕霉素靶蛋白/核糖体S6激酶1信号通路的异常可诱导机体产生胰岛素抵抗。 目的：观察有氧运动对小鼠骨骼肌结节性硬化复合物蛋白2基因表达及IRS1丝氨酸磷酸化的影响。 方法：将C57BL/6小鼠随机分为安静组和运动组,运动组进行6周的75%VO2max强度的有氧跑台运动,安静组不做任何干预。采用RT-PCR,Western blot和免疫荧光组织化学染色等方法检测各组大鼠骨骼肌结节性硬化复合物蛋白2,IRS1,pIRS1-Ser307和pIRS1-Ser636/639的表达和定位。 结果与结论：运动组结节性硬化复合物蛋白2 mRNA及蛋白表达高于安静组(P < 0.05),两组间胰岛素受体底物蛋白1 mRNA及蛋白表达差异无显著性意义。运动组胰岛素受体底物蛋白1的丝氨酸307和丝氨酸636/639位点的磷酸化明显低于安静组(P < 0.05)。结果提示,有氧运动可增强骨骼肌组织结节性硬化复合物蛋白2基因表达,进而抑制胰岛素受体底物蛋白1的丝氨酸磷酸化。     

磷酸化蛋白酪氨酸激酶及信号转导子和转录激活子在谷氨酸诱导的PC12细胞凋亡中的表达研究

目的探讨谷氨酸诱导PC12细胞凋亡后磷酸化蛋白酪氨酸激酶2（P-JAK2）、磷酸化信号转导子与转录激活子3（P-STAT3）表达的变化及意义。方法采用谷氨酸诱导PC12细胞发生凋亡，实验分为6组，分别为正常对照组、500μmol／L谷氨酸作用5、10、20、30、60min组，应用流式细胞仪观察PC12细胞凋亡．Western blot定量观测PC12细胞P-JAK2与P-STAT3蛋白表达的变化。结果对照组PC12细胞的凋亡率为（2．71±0．32）％；经500μmol／L谷氨酸作用PC12细胞20min，凋亡率增加到（61．20±4．60）％，与对照组相比差异有显著性意义（P=0．000）；Westem blot检测结果表明P-JAK 25 min在胞浆中开始表达，20min达最高峰，是5min组（3．52±0．20）倍（P=0．002）；P-STAT35min表达增强，30min达高峰，为5min组的（4．76±0．17）倍（P=0．000）。结论细胞损伤激活了JAK／STAT信号转导通路，该通路参与了神经细胞凋亡的过程。     

miR-221靶向PTEN介导PI3K/AKT信号通路调节细胞自噬参与帕金森病的机制研究

目的探讨微小RNA-221(miR-221)靶向磷酸脂酶与张力蛋白同源物基因(PTEN)介导磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(PKB也称AKT)信号通路调节细胞自噬参与帕金森病(PD)进展的机制研究。方法采用6-羟基多巴胺(6-OHDA)作用于肾上腺嗜铬细胞瘤(PC12)细胞建立PD模型;转染miR-221模拟物或抑制剂来调节miR-221的表达;细胞计数试剂盒-8(CCK-8)检测PC12细胞存活率;电镜观察自噬体;实时荧光定量PCR(qRT-PCR)分析确定miR-221的表达;蛋白印迹法(Western blot)检测自噬蛋白标志物(LC3-II)和PTEN/PI3K/AKT信号蛋白的表达。结果在PD细胞模型中,miR-221的表达水平下降(P0.01),LC3-II蛋白表达水平上调(P0.01),自噬体增加;与6-OHDA组相比,过表达miR-221降低了LC3-II蛋白的表达水平(P0.05),并增加了细胞存活率(P0.001),而沉默miR-221增加了LC3-II蛋白的表达水平(P0.05),并降低了细胞存活率(P0.001);此外,与6-OHDA组相比,过表达miR-221降低了PTEN蛋白的表达(P0.01),增加了PI3K蛋白的表达(P0.001)以及AKT蛋白磷酸化程度(P0.01),而沉默miR-221增加了PTEN蛋白的表达(P0.01),降低了PI3K蛋白的表达(P0.05)以及AKT蛋白磷酸化程度(P0.05)。结论 miR-221在6-OHDA诱导的PC12细胞中表达降低,上调miR-221可靶向PTEN介导PI3K/AKT信号通路来调节自噬,从而发挥对PD细胞模型的保护作用。     

小剂量左旋多巴上调pCREB及CD39蛋白表达发挥抗氧化应激损伤作用的研究

目的明确左旋多巴对PC12细胞生长及应激状态下存活的影响,探讨其抗氧化应激损伤的机制。方法不同浓度左旋多巴处理PC12细胞,用MTT法检测PC12细胞增长率及加入过氧化氢后细胞存活率;免疫荧光、Western blot方法测定磷酸化环单磷酸腺苷反应元件结合蛋白(pCREB)及CD39蛋白表达。结果低浓度左旋多巴(20μmol·L-1)促进PC12细胞生长,且可抗氧化应激损伤,而蛋白激酶抑制剂减弱此保护作用。免疫荧光及Western blot结果显示CD39及pCREB表达升高。结论低浓度左旋多巴可通过上调CD39及pCREB表达发挥抗氧化应激神经保护作用。     

PI3 K/Akt信号在阿尔茨海默病样细胞中的作用及依达拉奉对其影响

目的探讨PI3K/Akt信号在β样淀粉蛋白(Aβ1-40)引起的PC12细胞凋亡中的作用及依达拉奉(MCI-186)对其影响。方法采用流式细胞学检测细胞凋亡,Western blot法检测磷酸化Akt及总Akt水平,观察MCI-186对其保护作用。结果模型组中各时间点Akt Ser473的磷酸化水平与对照组比较均降低而保护组各时间点Akt Ser473的磷酸化水平均有明显的升高(P<0.05),保护组中细胞凋亡率较模型组显著降低(P<0.01)。结论Aβ1-40主要通过抑制磷酸化Akt水平,进而诱导PC12细胞凋亡。MC-186通过激活PI3K/Akt信号传导途径,发挥拮抗细胞凋亡的作用,最终达到保护神经细胞的目的。     

H2O2预处理通过ERK1/2和p38 MAPK信号通路上调14-3-3蛋白在PC12

OBJECTIVE: To investigate the protective effects of hydrogen peroxide preconditioning (HPP) on the pheochromocytoma (PC12) cells treated with 1-methyl-4-phenylpyridinium (MPP(+)) and to explore the potential mechanisms. METHODS: The viability and apoptosis of PC12 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 4',6'-diamidino-2-phenylindole (DAPI) staining, respectively. The expressions of 14-3-3 protein and phosphorylated p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. Enzyme-linked immunosorbent assay (ELISA) was used to measure the activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2). RESULTS: The cell viability decreased and the number of apoptotic cells increased dramatically in MPP(+) group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP(+)-treated PC12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC12 cells induced by HPP. CONCLUSION: HPP protects PC12 cells against MPP(+) toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.     

Alpha and beta estradiol protect neuronal but not native PC12 cells from paraquat-induced oxidative stress

Oxidative stress is currently considered a mediator of cell death in several neurodegenerative diseases. Notably, it may play an important role in the degeneration of dopamine neurons of the substantia nigra in Parkinson’s disease. We examined the effect of a strong oxidant, the herbicide paraquat, on cell distress using native and neuronal pheochromocytoma PC12 cells. Paraquat administration for 8 hours induced a significant cellular death in both native and in neuronal PC12 cells. Since the anti-oxidant properties of estrogens may promote neuroprotectionin vitro andin vivo, we then investigated the ability of estradiol stereoisomers, 17α-estradiol and 17β-estradiol, to rescue PC12 cells submitted to paraquat-induced oxidative stress. Our results show a protective effect of both estradiol stereoisomers in neuronal PC12 cells treated with paraquat, whereas this effect could not be observed in native PC12 cells. We also demonstrate that estrogen receptor β protein expression is modulated by paraquat administration in native PC12 cells, while paraquat does not change estrogen receptor β expression in neuronal PC12 cells. Paraquat also decreases estrogen receptor α in neuronal PC12 cells, thus suggesting new routes for paraquat to collapse cellular metabolism. Besides, the oxidation of dihydrodhodamine-123 into fluorescent rhodamine in the presence of paraquat but not in presence of paraquat and 17α-estradiol or 17β-estradiol,sustain a possible direct scavenging role of both estradiol stereoisomers.     

活化素A对百草枯所诱导的PC12细胞损伤的保护作用

目的:观察重组人活化素A(rhAct)对百草枯所诱导的PC12细胞损伤的保护作用。方法:将百草枯、活化素A、Ldeprenyl加入体外培养的PC12细胞中,用四甲基偶氮唑盐(MTT)法检测细胞活力的变化;免疫细胞化学法和RTPCR评价细胞的酪氨酸羟化酶和Bcl2蛋白及mRNA表达水平的变化,脱氧核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测细胞凋亡的变化,比较各组的差异。结果:预先给予活化素A和Ldeprenyl的两组细胞活力明显高于百草枯损害组,酪氨酸羟化酶和Bcl2蛋白及mRNA的表达强于损害组,同时两组的凋亡细胞明显减少,活化素A和Ldeprenyl两组间无显著差异。结论:活化素A和Ldeprenyl通过上调Bcl2的表达,抑制凋亡的发生,而对百草枯所诱导的PC12细胞损伤具有保护作用。     

Protein kinase B is involved in Nogo-66 inhibiting neurite outgrowth in PC12 cells

Nogo-A, a member of the reticulon family, is one of the most important myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the central nervous system. RhoA has been targeted pharmacologically to promote neurite outgrowth and functional recovery in the brain and spinal cord. However, the underlying mechanism of the inhibition of neurite outgrowth by Nogo-A has not yet been fully defined. Protein kinase B (PKB, also known as Akt) is a protein serine/threonine kinase that plays a key role in intracellular signaling and cellular homeostasis. This study reports the role of PKB signaling on Nogo-A-treated PC12 neuronal cells. An inhibitory fragment of Nogo-A (Nogo-66) activated RhoA and reduced the phosphorylation of PKB at Ser473 in a time-dependent manner. In contrast, pretreatment with Y27632, a specific inhibitor of Rho-A, resulted in an increase of the phosphorylation of PKB. Nogo-66 also inhibited the neurite outgrowth of PC12 cells, whereas pretreatment with LY294002, a specific inhibitor of PKB, ameliorated the neurite outgrowth. These data suggest that PKB is involved in the inhibition of neurite outgrowth by Nogo-A in PC12 cells.     

姜黄素通过诱导热休克蛋白70保护多巴胺能细胞

目的 探讨姜黄素通过诱导热休克蛋白70(Hsp70)高表达,抑制α-突触核蛋白的异常表达和聚集,促进蛋白酶体系统降解异常α-突触核蛋白对多巴胺能细胞的保护作用.方法 选用大鼠嗜铬细胞瘤细胞株(PCI2细胞),鱼藤酮诱导其损伤建立帕金森病细胞模型,利用姜黄素进行干预:采用MTT法检测细胞活力,荧光酶标仪检测蛋白酶体水解酶活性,Western blot检测Hsp70和α-突触核蛋白的表达,免疫荧光法检测细胞内Hsp70的表达和α-突触核蛋白的聚集.结果 鱼藤酮组PC12细胞活力及蛋白酶体水解酶活性明显降低,Hsp70的表达轻度增加,α-突触核蛋白表达和聚集明显增加,与对照组比较差异有统计学意义(P<0.05).经不同浓度姜黄素预处理4h后与0.1 μmol/L鱼藤酮共同孵育PC12细胞24 h,与鱼藤酮组比较,0.5 μmol/L和1.0 μmol/L姜黄素使细胞活力以及蛋白酶体水解酶活性明显升高,Hsp70表达明显升高,α-突触核蛋白的表达和聚集明显减少,差异有统计学意义(P<0.05);5.0 μmol/L和10 μmol/L姜黄素对鱼藤酮的拮抗作用明显减弱,细胞活力与鱼藤酮组比较差异均无统计学意义(P>0.05),胰蛋白酶、多肽-谷氨酰-多肽水解酶活性与鱼藤酮组比较差异均无统计学意义(P>0.05);10 μmol/L姜黄素组糜蛋白酶样水解酶活性进一步降低,与鱼藤酮组比较差异有统计学意义(P<0.05).结论 低浓度姜黄素能够通过诱导PC12细胞表达Hsp70,诱导蛋白酶体水解酶活性表达,进而抑制α-突触核蛋白的表达和聚集,从而拮抗鱼藤酮诱导的PC12细胞的损伤.     

Serum-free culture conditions for serial subculture of undifferentiated PC12 cells

PC12 cells, a widely used model neuronal cell line, are usually cultured in serum-supplemented medium. This report describes a serum-free medium for the culture of PC12 cells. PC12 cells grown in the two media types had similar growth rates and released dopamine in response to high potassium-induced calcium elevation. However, the levels of dopamine and of dopamine release in cells cultured in the serum-free medium were less than 10% of that in cells cultured in serum-supplemented medium. Dopamine levels recovered within 10 days if cells were returned to serum-supplemented medium, but dopamine release could not be recovered. Nerve growth factor (NGF) induced similar responses in PC12 cells cultured in both media, including phosphorylation of extracellular signal-regulated protein kinases and neurite extension. Transferrin was necessary for survival of neurite-bearing PC12 cells subcultured in serum-free medium and insulin promoted the cells proliferation. Ten days culture with NGF produced a similar increase in neurofilament expression and acetylcholinesterase activity in both media. These results suggest that PC12 in the hormonally defined serum-free media are qualitatively the same as those cultured in serum-supplemented media, and therefore this new culture protocol should enable more precise studies of PC12 cells culture in the absence of confounding unknown factors.     

Multiple treatments with L‐3,4‐dihydroxyphenylalanine modulate dopamine biosynthesis and neurotoxicity through the protein kinase A‐transient extracellular signal‐regulated kinase and exchange protein activation by cyclic AMP‐sustained extracellular signal‐regulated kinase signaling pathways

Multiple treatments with L‐3,4‐dihydroxyphenylalanine (L‐DOPA; 20 µM) induce neurite‐like outgrowth and reduce dopamine biosynthesis in rat adrenal pheochromocytoma (PC) 12 cells. We therefore investigated the effects of multiple treatments with L‐DOPA (MT‐LD) on cell survival and death over a duration of 6 days by using PC12 cells and embryonic rat midbrain primary cell cultures. MT‐LD (10 and 20 µM) decreased cell viability, and both types of cells advanced to the differentiation process at 4–6 days. MT‐LD induced cyclic adenosine monophosphate (cAMP)‐dependent protein kinase A (PKA) phosphorylation and exchange protein activation by cAMP (Epac) expression at 1–3 days, which led to transient extracellular signal‐regulated kinase (ERK1/2) phosphorylation in both cells. In these states, MT‐LD activated cAMP‐response element binding protein (CREB; Ser133) and tyrosine hydroxylase (Ser40) phosphorylation in PC12 cells, which led to an increase in intracellular dopamine levels. In contrast, MT‐LD induced prolonged Epac expression at 4–5 days in both cells, which led to sustained ERK1/2 phosphorylation. In these states, the dopamine levels were decreased in PC12 cells. In addition, MT‐LD induced c‐Jun N‐terminal kinase1/2 phosphorylation and cleaved caspase‐3 expression at 4–6 days in both cells. These results suggest that MT‐LD maintains cell survival via PKA‐transient ERK1/2 activation, which stimulates dopamine biosynthesis. In contrast, at the later time period, MT‐LD induces differentiation via both prolonged Epac and sustained ERK1/2 activation, which subsequently leads to the cell death process. Our data demonstrate that L‐DOPA can cause neurotoxicity by modulating the Epac‐ERK pathways in neuronal and PC12 cells. © 2014 Wiley Periodicals, Inc.