中国科技论文统计源期刊 中文核心期刊
美国《化学文摘》《国际药学文摘》
《乌利希期刊指南》
WHO《西太平洋地区医学索引》来源期刊
日本科学技术振兴机构数据库（JST）
第七届湖北十大名刊提名奖
美国《化学文摘》《国际药学文摘》
《乌利希期刊指南》
WHO《西太平洋地区医学索引》来源期刊
日本科学技术振兴机构数据库（JST）
第七届湖北十大名刊提名奖
, 吴庆文, 贾玉凤, WU Qingwen, JIA Yufeng
帕金森病(Parkinson's disease,PD)是一种常见于中老年人神经系统变性疾病,临床主要表现为静止性震颤、运动迟缓、肌强直和姿势平衡障碍。PD主要有两大病理特征,其一是黑质多巴胺能神经元及其他含色素的神经元大量变性丢失,尤其是黑质致密区多巴胺能神经元丢失最为严重,其二是在残留的神经元胞质内出现嗜酸性包涵体,即路易小体(Lewy bodies)。但为何会引起黑质多巴胺能神经元变性死亡尚未完全明了[1]。有研究认为凋亡可能在PD发病过程中起重要作用。药物治疗是PD整个治疗过程的首选方案,然而目前应用的治疗手段,无论药物或手术,只能改善症状,不能有效地阻止病情的发展,更无法治愈。丁苯酞(butylphthalide)具有抗氧化应激、线粒体保护作用、抑制炎症反应、减少细胞的凋亡等多种神经保护作用[2]。现阶段关于丁苯酞对PD模型的保护作用主要是通过增强抗氧化能力、提高黑质多巴胺能神经元残存率以及线粒体保护等方面进行研究[3]。关于丁苯酞降低C-Jun氨基末端激酶(c-N-terminal kinase,JNK)的表达有相关研究[4],但关于丁苯酞通过改善JNK通路死亡受体途径相关因子,降低凋亡的研究报道笔者未见,故在本实验主要研究丁苯酞通过调控JNK通路上死亡受体途径相关凋亡因子磷酸化C-Jun氨基末端激酶(p-c-N-terminal kinase,p-JNK)、凋亡蛋白-1(Apopotisis-1,Fas)、Fas配体(Fas ligand,FasL)的表达,抑制细胞凋亡发生,从而达到对PD模型保护作用。
SH-SY5Y细胞株购自中国科学院上海细胞库。
丁苯酞(相对分子质量为190.24,中国食品药品检定研究院,含量:99%)、JNK抑制剂SP600125(Cayman公司,批号:0445814-21); 1-甲基-4-苯基-吡啶离子(MPP+,Sigma公司,批号:014M4704V);噻唑蓝(MTT,TCI公司,批号:D0801);Annexin-V/PI试剂盒(贝博生物,批号:BB120086);胰酶(Sigma公司,批号:SLBJ6247V);DMEM/F12=1∶1培养基(Gibico公司,批号:8114359);胎牛血清(BI公司,批号:1408038);GADPH抗体(SANTA CRUZ公司,批号:sc-25778),p-JNK(abcam公司,批号:YK031401CS)、Fas、FasL(Affinity公司,批号:19U71)。
二氧化碳(CO2)培养箱(Thermo公司)、酶标仪(意大利Bio-Rad公司),倒置相差显微镜(Olympus公司),超净工作台(中国苏州净化设备厂),低温离心机(日本日立公司),流式细胞仪(FACS Calibuar,BD公司)。
SH-SY5Y细胞接种于培养瓶中,DMEM/F12培养基(含10%胎牛血清,青霉素100 U·mL-1,链霉素100 U·mL-1),置于37 ℃、5%CO2培养箱培养。实验分为正常对照组(细胞正常培养,不加任何药物干预),MPP+组(培养的细胞中加入MPP+),丁苯酞+MPP+组(培养的细胞中加入NBP作用3 h后,再加入MPP+),SP600125+MPP+组(培养的细胞中加入JNK抑制药SP600125作用3 h后再加入MPP+)。
以每孔(2~5)×10 5个·mL-1的活细胞悬液100 μL接种于96孔板中,10 μmol·L-1丁苯酞或10 μmol·L-1SP600125预先处理细胞3 h后加入1 mmol·L-1 MPP+继续培养24 h后,各组分别加入50 mg·mL-1的MTT 15 μL,继续孵育3 h,吸去培养基,加入二甲亚砜(DMSO)150 μL,摇床10 min,酶标仪490 nm波长检测吸光度值(
以(2~5)×10 5个·mL-1接种于6孔板中,用10 μmol·L-1丁苯酞或10 μmol·L-1SP600125作用3 h,加入1 mmol·L-1MPP+继续培养24 h后,胰酶消化,收集细胞,磷酸盐缓冲溶液(PBS)清洗,加入Annexin V-FITC结合液400 μL重悬细胞后,加入AnnexinV-FITC5 μL,室温避光孵育15 min,加入碘化丙啶(propidium iodide,PI)染色液10 μL,混匀,室温避光孵育5 min,随后进行流式细胞仪检测分析。
细胞干预情况同上,每孔加入裂解液100 μL,用细胞刮收集细胞,放入EP管中,超声破碎细胞,15 435×
实验数据输入Excel建立数据库,采用SPSS17.0版统计软件进行统计分析,计量资料用均数±标准差(
MTT结果显示,与正常对照组细胞存活率(100.00±0.00)%比较,MPP+组细胞存活率降低,仅为(49.30±2.07)%(
显微镜下观察细胞形态,正常对照组细胞贴壁数量多,并且细胞轴突明显;MPP+组细胞大部分变圆,突起结构减少,贴壁细胞数目减少;丁苯酞预处理组,细胞虽有变圆的现象,但与MPP+组比较,贴壁细胞增多,细胞间的突起联系增多且细胞形态与正常对照组相似;SP600125预处理组细胞与丁苯酞预处理组的细胞形态接近,贴壁细胞的数目相似。见
流式细胞术Annexin-V/PI双荧光染色检测细胞凋亡,结果显示,与正常对照组细胞凋亡率(10.63±2.07)%比较,MPP+组细胞凋亡率升高,为(32.27±2.26)%,差异有统计学意义(
结果显示,与正常对照组比较,MPP+组p-JNK、FAS、FasL蛋白表达量升高(
图3 4组细胞p-JNK、Fas、FasL蛋白表达强度 1.正常对照组;2. MPP+组;3. 丁苯酞+MPP+组;4. SP600125+MPP+组
Fig.3 Protein expression of p-JNK, Fas and FasL in four groups of cells 1.normal control group;2. MPP+ group;3. NBP+MPP+ group ;4. SP600125+MPP+ group
表1 4组细胞p-JNK、Fas、FasL蛋白相对表达量
Tab.1
Relative protein expression of p-JNK, Fas and FasL in four groups of cells
PD的发病并非是单因素所致,而是多因素交互作用的结果。只有在环境因素、神经系统老化等因素的共同作用下,通过氧化应激、线粒体功能紊乱、钙稳态失衡、兴奋性毒性、细胞凋亡等机制导致黑质多巴胺能神经元大量变性丢失,才会导致发病[1]。JNK是细胞内重要的应激调节蛋白,未激活时主要存在于细胞质,激活后迅速表达,环境应激和细胞因子均能导致JNK的激活,激活的JNK发生磷酸化,p-JNK在介导神经细胞死亡中发挥着重要作用[5],此过程和PD发病密切相关。p-JNK能通过两种途径介导多巴胺神经元凋亡:一条通路是死亡受体途径,有研究者在大鼠PD模型中发现,p-JNK能通过促进FasL的表达介导死亡受体途径的多巴胺神经元凋亡[6],Fas与FasL结合后引起Fas死亡结构域蛋白(Fas-assosiated death domain protein, FADD)交联,通过细胞内的死亡功能区发挥作用启动死亡信号传导,导致细胞凋亡,Fas和FasL表达的增加可促进细胞凋亡[7];另一条通路是线粒体途径,活化后的JNK通过参与介导caspase-3,促进环氧化酶2(cycloxygenase 2,COX-2)的表达,增加Bim蛋白的表达激活Bax表达增多,调节释放细胞色素C,减少线粒体复合体Ⅰ,增加活性氧等途径来造成纹状体运动区多巴胺能神经元凋亡。陈娜等[8]研究显示,异常蛋白质聚集可激活JNK通路,介导的细胞凋亡可能造成PD的发病。FERRER等[9]在PD患者尸检脑组织中发现p-JNK,说明p-JNK可能是造成PD发病的原因之一。潘静等[10]实验研究显示JNK通路参与并促进PD引发的凋亡样细胞死亡。
SP600125是JNK通路的特异性抑制剂,笔者在本研究发现使用抑制剂后,MTT法结果提示SP600125可以改善细胞增殖能力,倒置显微镜观察结果提示SP600125可以改善细胞形态,减少MPP+对细胞的损伤,流式细胞术结果提示SP600125可以降低细胞凋亡情况,Western blotting结果提示SP600125可以降低p-JNK蛋白的表达,同时降低死亡受体蛋白Fas、FasL的表达情况。综上结果,SP600125能有效阻断JNK的磷酸化,降低细胞凋亡的发生,与文献[11-12]研究一致,提示PD细胞模型的凋亡可能是因为JNK通路的激活,造成JNK磷酸化,引起死亡受体途径上相关因子表达增高。
笔者在本研究发现,在MPP+诱导的PD细胞模型中,经过丁苯酞处理后,细胞增殖能力提高,细胞形态和数量改善,细胞凋亡下降,死亡受体上相关因子蛋白表达减少,结果与使用JNK通路抑制剂SP600125组大致一致,提示丁苯酞对PD模型的保护作用可能是通过降低JNK通路的激活,减少通路磷酸化,从而抑制细胞凋亡实现的。
The authors have declared that no competing interests exist.
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目的 探讨丁基苯酞(dl-3-n-Butylphthalide, NBP)对鱼藤酮诱导的帕金森病细胞模型的保护作用及其机制.方法 分别使用终浓度为0.1、1、10、100 μM NBP和溶剂二甲基亚砜(DMSO)预处理SH-SY5Y细胞24 h后,加入终浓度为200 nM的鱼藤酮处理24 h建立多巴胺能细胞损伤模型,观察各组细胞形态,采用四甲基偶氮唑盐(MTT)比色法检测细胞活性,流式细胞术检测细胞凋亡率(Annexin V-FITC/PI)、线粒体膜电位(JC-1)、细胞内活性氧水平(DCFH-DA).结果 200 nmol/L鱼藤酮处理SH-SY5Y细胞24 h能够诱导细胞活性下降和细胞凋亡,NBP预处理后SH-SY5Y细胞存活率明显升高,细胞凋亡率降低,线粒体膜电位显著升高(P<0.05),细 胞内活性氧水平显著降低(P<0.05),且随NBP浓度的增加对SH-SY5Y细胞的保护作用增强.结论 NBP对鱼藤酮诱导的SH-SY5Y细胞损伤具有良好的保护作用,线粒体保护可能是其作用机制之一.
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DL-3-n-Butylphthalide () has shown cytoprotective effects in animal models of stroke and has passed clinical trials as a therapeutic drug for stroke in China. Hence, as a potential clinical treatment for stroke, understanding the mechanism(s) of action of is essential. This investigation aimed to delineate the cellular and molecular mechanism of protection in neuronal cultures and in the ischemic brain. (10 渭M) attenuated serum deprivation-induced neuronal and the production of reactive oxygen species (ROS) in cortical neuronal cultures. Adult male 129S2/sv were subjected to permanent occlusion of the middle cerebral artery (). (100 mg/kg, i.p.) administrated 2 hrs before or 1 hr after reduced -induced infarct formation, attenuated caspase-3 and caspase-9 activation in the ischemic brain. TUNEL-positive cells and mitochondrial release of and () in the penumbra region were reduced by . The proapoptotic signaling mediated by phospho-and expression was downregulated by treatment in vitro and in vivo. It is suggested that protects against ischemic damage via multiple mechanisms including mitochondria associated caspase-dependent and -independent apoptotic pathways. Previous and current studies and recent clinical trials encourage exploration of as a neuroprotective drug for the treatment of .
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以JNK为中心的JNK信号通路是近20年来发现的与细胞分化、细胞凋亡、应激反应以及多种人类疾病的发生和发展关系非常密切的通路之一，其生物化学功能和对细胞生理、病理情况下的调节作用一直被国内外学者所关注。近年来，研究发现JNK信号通路在调控细胞凋亡中发挥重要作用，通过调节JNK信号通路有望成为治疗细胞凋亡引起疾病的重要靶点。本文就其生物学功能及其与细胞凋亡关系作一阐述。
DOI:
Magsci
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Our previous studies and those of others have strongly suggested that c-N-terminal kinase () signaling pathway plays a critical role in 6-hydroxydopamine (6-OHDA)-induced dopaminergic neuron injury in the substantia nigra. However, the downstream mechanism that accounts for the proapoptotic actions of in 6-OHDA lesion remains to be investigated in detail. , a member of the receptor family with proapoptotic functions, was reported to be elevated within the striatum and substantia nigra pars compacta (SNc) of () patients. In the present study, we examined the changes in the protein level of (FasL) and its interaction with in a model of . We demonstrate that the expression of FasL and not was increased after 6-OHDA lesion; additionally, the interaction of FasL and was increased due to 6-OHDA lesion. This indicates that the 6-OHDA-induced activation of signaling pathway is mediated by and that FasL may be a promising target in the therapeutic approach for patients.
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<a name="Abs1"></a>There is increasing evidence that the active contribution of hepatocytes to liver disease is strongly dependent on local cytokine environment. It has been shown in vitro that TNFα can enhance hepatocyte FasLigand (FasL)-mediated cytotoxicity. Here, we demonstrate that TNFα-induced apoptosis was associated with Fas and FasL upregulation and that a FasL-neutralizing antibody prevented TNFα-induced apoptosis. We further examined in vivo the relevance of the Fas/FasL pathway to hepatocellular apoptosis in a TNFα-driven model of acute liver failure. Livers of galactosamine/lipopolysaccharide (Gal/LPS)-exposed Fas wild-type mice highly expressed both Fas and FasL and revealed marked hepatocellular apoptosis that was almost completely blocked by soluble TNFα-receptor; this was also almost absent in Gal/LPS-exposed Fas lymphoproliferation mutant mice. Our data provide evidence for a direct link between TNFα and Fas/FasL in mediating hepatocyte apoptosis. Fratricidal death by Fas–FasL interactions of neighbouring hepatocytes may actively contribute to acute liver failure.
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<a name="Abs1"></a> The expression of mitogen-activated protein kinases, extracellular signal-regulated kinases (MAPK/ERK), stress-activated protein kinases, c-Jun N-terminal kinases (SAPK/JNK), and p38 kinases is examined in Parkinson disease (PD), in Dementia with Lewy bodies (DLB), covering common and pure forms, and in age-matched controls. The study is geared to gaining understanding about the involvement of these kinases in the pathogenesis of Lewy bodies (LBs) and associated <i>tau</i> deposits in Alzheimer changes in the common form of DLB. Active, phosphorylation dependent MAPK (MAPK-P) is found as granular cytoplasmic inclusions in a subset of cortical neurons bearing abnormal <i>tau</i> deposits in common forms of DLB. Phosphorylated p-38 (p-38-P) decorates neurons with neurofibrillary tangles and dystrophic neurites of senile plaques in common forms of DLB. Phosphorylated SAPK/JNK (SAPK/JNK-P) expression occurs in cortical neurons with neurofibrillary tangles in the common form of DLB. Lewy bodies (LBs) in the brain stem of PD and DLB are stained with anti-ERK-2 antibodies, but they are not recognized by MAPK-P, SAPK/JNK-P and p-38-P. Yet MAPK-P, p-38-P and SAPK/JNK-P immunoreactivity is found in cytoplasmic granules in the vicinity of LBs or in association with irregular-shaped or diffuse α-synuclein deposits in a small percentage of neurons, not containing phosphorylated <i>tau</i>, of the brain stem in PD and DLB. MAPK-P, p-38-P and SAPK-P are not expressed in cortical LBs or in cortical neurons with α-synuclein-only inclusions in DLB. MAPK-P, p-38-P and SAPK/JNK-P are not expressed in α-synuclein-positive neurites (Lewy neurites) in PD and DLB as revealed by double-labeling immunohistochemistry. These results show that MAPKs are differentially regulated in neurons with α-synuclein-related inclusions and in neurons with abnormal <i>tau</i> deposits in DLB. Moreover, different kinase expression in brain stem and cortical LBs suggest a pathogenesis of brain stem and cortical LBs in LB diseases. Finally, no relationship has been observed between MAPK-P, p-38-P and SAPK/JNK-P expression and increased nuclear DNA vulnerability, as revealed with the method of in situ end-labeling of nuclear DNA fragmentation, and active, cleaved caspase-3 expression in neurons and glial cells in the substantia nigra in PD and DLB.
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c-JunN端蛋白激酶（JNK）是细胞功能的重要激酶,在各种刺激以及生理状态下导致神经 元凋亡中起着重要的作用。JNK主要与一些神经退行性疾病有着密切的关系。理解JNK信号通路能够为将来选择JNK作为靶点干预这些疾病条件。JNK参与 这些神经退行性疾病的发病机制,因此抑制其活性成为有效治疗的靶点。
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目的研究1-甲基-4-苯基-吡啶离子（MPP^＋）对PC12细胞的毒性作用及其机制。方 法 PC12细胞体外培养,以100、300、500μmol/L MPP^＋进行染毒。Western blot法检测JNK1/2磷酸化水平;使用JNK通路阻断剂SP60012预处理细胞,TUNEL法观察其对MPP^＋诱导的细胞凋亡的影响。结果 MPP^＋染毒可以引起细胞JNK1/2的磷酸化水平增高,使用JNK通路阻断剂SP600125可以抑制MPP^＋诱导的PC12细胞凋亡。结论激活 JNK通路可能是MPP^＋诱导PC12细胞凋亡、产生多巴胺能神经毒性的重要分子机制。
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