凋亡调控因子在凝血酶诱导海马神经元凋亡中的变化

目的探讨凝血酶（TM）对海马神经元凋亡调控凶子Bcl-2、Bax及c-jun氨基末端激酶（JNK）表达的影响，阐明TM诱导凋亡的机制。方法海马神经元经40U／mLTM作用0h、6h、12h、24h、48h、72h后终止培养，应用免疫细胞化学方法检测Bcl-2及Bax蛋白表达，Western blot检测磷酸化JNK（P—JNK）及JNK蛋白表达。结果TM作用6h后，Bcl-2蛋白表达增加，12h后开始下降，到48h达到最低值（P〈0．01），72h后Bcl-2蛋白表达开始回升。TM作用6h后，Bax蛋白表达增加，随作用时间的延长，Bax蛋白表达水平逐渐上升，到48h达到最高值（P〈0．01）。Bcl-2／Bax比值逐渐降低，在48h达最低。TM作用0h时P-JNK蛋白无表达，6h后开始表达，与0h组比较差异存在显著性（P〈0．011，12h时达高峰（P〈0．01），并持续到24h，48h后逐渐降低。0h组可见有少量JNK蛋白表达，6h后JNK蛋白表达显著增加（P〈0．01），随TM作用时间的延长，JNK蛋白表达无明显变化。结论TM可能是通过抑制Bcl-2的表达，增强Bax的表达，降低Bcl-2和Bax的比值，激活JNK信号传导通路导致海马神经元凋亡。     

JNK信号传导通路分子在破裂颅内动脉瘤中的表达

目前人们对颅内动脉瘤形成及其破裂的发病机制尚不清楚,研究表明C-Jun氨基末端激酶(c- Jun NH2-terminal kinase,JNK)信号传导通路参与了该病的发病机制,但是JNK介导颅内动脉瘤形成和破裂的具体通路缺乏深入研究,因此本课题采用免疫组化和荧光定量RT-PCR对JNK信号传导通路的关键分子进行相关检测,从而对颅内动脉瘤形成机制并采取可能的治疗措施提供实验依据。     

川芎嗪对脑梗死大鼠脑保护作用与ROCK-JNK信号通路的相关性研究

目的 探究Rho蛋白激酶(ROCK)-C-Jun氨基酸末端激酶(JNK)信号通路在川芎嗪对脑梗死大鼠脑保护作用机制.方法 将138只SPF级SD雄性大鼠随机分为假手术组、模型组、川芎嗪组和尼莫地平组(阳性对照),其中川芎嗪组再依据不同干预剂量分为川芎嗪25、50和100 mg·kg-1亚组.除假手术组外,其余各组大鼠均...     

MAPK信号通路在创伤性脑损伤中的作用

目的通过建立小鼠创伤性脑损伤(TBI)模型,研究丝裂原活化蛋白激酶(MAPKs)通路中的细胞外调节蛋白激酶1/2(ERK1/2)通路、JNK通路和p38通路的激活及在TBI中的作用及机制。方法建立小鼠TBI模型,通过Western blot检测ERK1/2、JNK和p38的相对磷酸化水平,确定TBI后MAPK通路的激活情况;分别加入ERK1/2通路抑制剂(PD98059,500μmol/L)、JNK通路抑制剂(SP600125,500μmol/L)和p38通路抑制剂(SB203580,500μmol/L),通过脑干湿重检测、神经功能学评分和TUNEL染色评估不同抑制剂对TBI的作用,并通过Western blot检测ERK1/2、JNK和p38的相对磷酸化水平,明确ERK1/2通路、JNK通路和p38通路之间的相互调节作用。结果 TBI可分别引起ERK1/2通路、JNK通路和p38通路的激活;抑制ERK通路和JNK通路可减轻TBI引起的脑水肿、神经功能损伤和细胞凋亡,而抑制p38通路则加重TBI引起的脑水肿、神经功能损伤和细胞凋亡;抑制JNK通路可减少ERK1/2的相对磷酸化水平,而抑制p38通路可增加ERK1/2的相对磷酸化水平。结论 TBI后,ERK1/2通路和JNK通路的激活发挥促进损伤形成的作用,而p38通路的激活则起到神经保护的作用;ERK1/2通路的激活受到JNK通路的促进和p38通路的抑制,表明MAPK通路之间存在相互调节。     

铜孵育对肝豆状核变性培养细胞内铜在不同分子量蛋白上分布的影响

研究不同浓度铜孵育后，肝豆状核变性（ＨＬＤ）患者和杂合子离体培养皮肤成纤维细胞内铜在不同分子量蛋白上分布的变化规律，提出了在ＨＬＤ患者细胞内，可能存在铜进入细胞后从大分子蛋白向金属硫蛋白（ＭＴ）转移的过程，并对ＭＴ异常在ＨＬＤ发病机理中的作用进行了讨论。     

肝豆状核变性患者肝细胞铜代谢的研究

肝豆状核变性(HLD)系常染色体隐性遗传性铜蓄积性疾病,是以肝脏和其他组织细胞内渐进性铜蓄积为发病基础。患者因肝铜蓄积,致急、慢性肝炎,肝硬化和暴发性肝功能衰竭等。在中枢神经系统,铜可致相应的损害,表现为多种神经、精神异常。此外,尚可致肾脏、心脏损害及多种内分泌紊乱等。资料和方法我们于2003年9月至2004年2月安徽中医学院神经病学研究所附属医院外科手术获取9例HLD患者和5例正常对照肝组织块,清洗、剪切并去除包膜、胶原酶温育消化、过筛、离心5min。接种于6孔培养板,37℃5%CO2培养箱中行细胞培养。定时换液,4～8d后,倾尽生…     

细胞外信号调节激酶在实验性脑缺血损伤皮层区的动态时空变化

目的探讨细胞外信号调节激酶(ERK)在局灶性脑缺血/再灌注损伤梗死灶周皮层区的动态时空变化及其作用机制。方法建立兔大脑中动脉阻断(MCAO)局灶性脑缺血再灌注模型,应用免疫组化检测ERK1在脑缺血2h再灌注不同时间灶周皮层的动态表达规律,同时应用免疫组化和流式细胞术检测细胞凋亡状态的动态变化。结果免疫组化分析显示,缺血2h再灌注灶周皮层区1hERK1表达开始增多,6h数目明显增多,3d达高峰,然后逐渐下降,14d回落到基线水平,其阳性细胞数分别为0.22±0.02、0.25±0.02、0.42±0.04、0.14±0.02。其表达时程变化与灶周皮层凋亡的变化相一致。结论脑缺血损伤诱导ERK表达增强,ERK在介导神经细胞凋亡和缺血性损伤中起重要作用。这为脑缺血治疗提供了新的思路。     

ERK1/2信号通路在弥漫性脑损伤中激活规律的实验研究

目的探讨弥漫性脑损伤（DBI）中细胞外信号调节激酶1／2（ErtK1／2）信号传导通路激活的时空变化特征。方法参照Marmarou方法制作大鼠DBI模型，Westernblot法检测损伤脑组织磷酸化ERK1／2（pErtK1／2）蛋白的表达。免疫组织化学法检测pERK1／2阳性细胞在损伤脑组织不同区域的分布。结果大鼠DBI后脑组织pERK1／2表达显著增高，5min即达峰值，随即下降，但至12-24h又有回升，达到第二个峰值，并持续高水平表达至72h。DBI损伤后30min-3h主要见胞浆阳性染色，损伤后24h在胞核、胞浆均可见阳性染色，以胞核为主。pERK1／2免疫阳性细胞多见于脑皮质深层，其次为海马，室管膜和脉络丛也可见pERK1／2阳性染色。结论大鼠DBI后ERK1／2通路被迅速和持久激活，pERK1／2免疫阳性细胞多见于脑皮质深层，也可见于海马、室管膜和脉络丛。     

外源性bFGF抑制辐射诱导的C17.2神经干细胞凋亡的实验研究

目的 观察外源性碱性成纤维细胞生长因子(bFGF)对辐射诱导的C17.2神经干细胞(NSCs)凋亡的影响,探讨细胞外信号调节激酶1/2(ERK1/2)在bFGF对辐射诱导的C17.2 NSCs凋亡的抑制效应中的作用. 方法 以直线加速器照射C17.2NSCs建立离体放射性损伤模型.MTT法检测细胞活性,流式细胞仪检测不同浓度的外源性bFGF和U0126对细胞凋亡的影响. 结果 外源性bFGF浓度为0～100ng/mL时,细胞凋亡率可由(12.78±1.04)%降低至(4.83±0.31)%,组间比较差异有统计学意义(P<0.05);加入U0126后细胞凋亡率为(8.42±0.71)%.DMSO对照组为(4.71±0.42)%,差异有统计学意义(P<0.05). 结论 外源性bFGF对辐射引起的C17.2 NSCs凋亡具有抑制作用,ERK1/2参与该效应.     

Wnt信号传导通路在阿尔茨海默病中的研究进展

阿尔茨海默病(Alzheimer's disease,AD)是一种中枢神经系统的退行性变性疾病,主要表现为进行性的认知功能障碍和精神神经异常.AD的病因与发病机制至今尚未阐明.由于在神经系统的发育过程中,Wnt信号传导通路在细胞间黏附及细胞分化上起着重要作用,所以它越来越受到人们的关注.近来有研究表明Wnt信号传导通路中多个信号分子的改变均与AD的发生、发展密切相关.     

乌司他丁对脑缺血再灌注大鼠脑组织JNK及细胞凋亡的影响

目的探讨乌司他丁对脑缺血再灌注大鼠缺血侧脑组织c-Jun氨基末端激酶(JNK)蛋白表达及凋亡细胞数的影响。方法 36只雄性清洁SD大鼠按随机平均原则分成3组:假手术组(12只)、脑缺血再灌注组(对照组,12只)、脑缺血再灌注+乌司他丁治疗组(治疗组,12只)。大鼠局灶性脑缺血再灌注损伤模型采用大脑中动脉线栓法制作。采用RT-PCR法检测大鼠脑组织JNK的表达,采用TUNEL法检测大鼠脑组织凋亡细胞数。结果与假手术组相比,对照组和治疗组大鼠脑组织皮质区JNK的表达明显升高,凋亡细胞数均明显增加(P均0.05)。与对照组相比,治疗组大鼠脑组织JNK的表达明显下降,凋亡细胞数均明显减少(均P0.05)。结论乌司他丁可下调缺血再灌注大鼠脑组织JNK的表达并抑制其细胞凋亡,乌司他丁抑制细胞凋亡可能与抑制JNK传导通路相关。     

Aluminum-induced apoptosis in cultured cortical neurons and its effect on SAPK/JNK signal transduction pathway

Aluminum exposure and apoptotic cell death has been implicated in several neurodegenerative diseases. The mechanisms by which aluminum interacts with the nervous system are only partly understood. In this study, we used cultured cortical neurons to investigate the ability of aluminum to induce the apoptosis of neurons and to explore the role of SAPK/JNK (stress-activated protein kinase or c-jun N-terminal kinase) signal transduction pathway on the apoptosis induced by aluminum. We found that aluminum-induced degeneration of cortical neurons involved the DNA fragmentation characteristic of apoptosis, and staining of aluminum-treated neurons with the DNA-binding fluorochrome Hoechst 33258 revealed the typical apoptotic condensation and fragmentation of chromatin. The rate of apoptosis increased significantly (from 4.9 to 13.1, 21.4, and 59.8%, P<0.01), which was measured by TdT-mediated dUTP nick end labeling. Western blot analysis showed that SAPK/JNK activities of cortical neurons varies when the exposure time of AlCl(3) were different. The phosphorylation levels were 4.2, 3.3, 1.9 and 1.1 times greater compared to control cultures for 6, 12, 24, and 48 h, respectively (P<0.01). Furthermore, a JNK pathway inhibitor, CEP-11004 (KT8138) inhibited the activation of SAPK/JNK to protect cortical neurons from apoptosis induced by aluminum chloride. Our study demonstrates that aluminum can induce the apoptosis of cortical neurons and SAPK/JNK signal transduction pathway may play an important role in the apoptosis.     

癫痫与JNK信号转导通路关系

信号通路是目前癫痫研究的主要方向,尤其是丝裂原活化蛋白激酶家族(mitogen activated protein kinases,MAPKs).该家族包括细胞外信号激酶(extraeellular signal-regulated protein kinase,ERK)、c-Jun氨基端激酶(c-Jun N-terminal kinase,JNK)、p38等,其在神经系统中广泛表达,受各种细胞外刺激,影响突触传递、神经重塑、形态分化和存活等,造成神经元的凋亡、苔藓纤维出芽、胶质增生等病理改变.     

鼠骨髓间充质干细胞对神经元凋亡的影响

目的探讨体外培养鼠骨髓间充质干细胞对谷氨酸诱导大脑皮质神经元凋亡的影响。方法分离、培养、传代Wistar大鼠骨髓间充质干细胞，细胞融合达90％时更换培养基，继续培养24h，收集细胞培养液即为骨髓间充质干细胞条件培养基；培养新生Wistar大鼠大脑皮质神经元，第8d随机分为对照组、谷氨酸损伤组和骨髓间充质干细胞条件培养基处理组。采用台盼蓝染色计算神经元存活率，同时用流式细胞仪和透射电镜技术检测各组神经元凋亡情况。结果谷氨酸（0．8mmol／L）可诱导细胞凋亡，骨髓间充质干细胞条件培养基处理组较谷氨酸损伤组细胞成活率明显升高（P〈0．001）；流式细胞仪检测见骨髓间充质干细胞条件培养基处理组细胞凋亡率明显降低（P〈0．001）；而电镜检测发现对照组无明显的凋亡细胞，谷氦酸组有典型凋亡神经元，骨髓间充质干细胞条件培养基处理组凋亡细胞数明显减少。结论骨髓间充质干细胞条件培养基对谷氨酸神经元毒性具有拮抗作用。     

Activation of caspase-3 in β-amyloid-induced apoptosis of cultured rat cortical neurons

Amyloid β protein (Aβ) has been thought to participate in the neurodegeneration associated with Alzheimer's disease. We here report on caspase-3 activation by Aβ-treatment of cultured neurons. Treatment of rat primary cortical culture with Aβ 25–35, an active fragment of Aβ, induced neuronal death as determined by a decrease in neuron-specific microtubule-associated protein 2 (MAP2)-like immunoreactivity and by the release of cellular lactate dehydrogenase (LDH). Aβ 25–35 also induced elevation of caspase-3-like Ac-DEVD-MCA cleavage activity in advance of neuronal death with similar concentration-dependency for neuronal death. Inhibitor sensitivity of the Aβ-induced proteolytic activity was similar to that of human recombinant caspase-3. Cleavage of pro-caspase-3 and cleavage of its endogenous substrates, poly (ADP-ribose) polymerase (PARP) and α-fodrin, were produced by Aβ-treatment. A caspase-3 inhibitor, Ac-DEVD-CHO, prevented Aβ-induced DNA fragmentation and cleavage of α-fodrin, but not of PARP. Caspase inhibitor of broad specificity, Z-VAD-CH₂-DCB, additionally prevented Aβ-induced cleavage of PARP and some early loss of cell membrane integrity measured by LDH release. However, Aβ-induced condensation of nuclear chromatin and most of the late disintegration of cell membranes were not prevented in the presence of these caspase inhibitors. These results suggest that activation of both caspase-3 and caspase(s) other than caspase-3 play distinct roles in Aβ-induced apoptosis of rat cortical neurons. Furthermore, in the presence of caspase inhibitors, Aβ-induced neuronal death still occurred with different morphological features.     

Signal transduction pathways regulating cyclooxygenase-2 in lipopolysaccharide-activated primary rat microglia

Inflammatory processes play a key role in the pathogenesis of a number of common neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). Abnormal iron accumulation is frequently noted in these diseases and compelling evidence exists that iron is involved in inflammatory reactions. Histochemical stains for iron repeatedly demonstrate that oligodendrocytes, under normal conditions, stain more prominently than any other cell type in the brain. Therefore, we examined the hypothesis that cytokine toxicity to oligodendrocytes is iron mediated. Oligodendrocytes in culture were exposed to interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha). Toxicity was observed in a dose-dependent manner for IFN-gamma and TNF-alpha. IL-1beta was not toxic in the concentrations used in this study. The toxic concentration of IFN-gamma, and TNF-alpha was lower if the cells were iron loaded, but iron loading had no effect on the toxicity of IL-1beta. These data provide insight into the controversy regarding the toxicity of cytokines to oligodendrocytes by revealing that iron status of these cells will significantly impact the outcome of cytokine treatment. The exposure of oligodendrocytes to cytokines plus iron decreased mitochondrial membrane potential but activation of caspase 3 is limited. The antioxidant, TPPB, which targets mitochondria, protected the oligodendrocytes from the iron-mediated cytotoxicity, providing further support that mitochondrial dysfunction may underlie the iron-mediated cytokine toxicity. Therapeutic strategies involving anti-inflammatory agents have met with limited success in the treatment of demyelinating disorders. A better understanding of these agents and the contribution of cellular iron status to cytokine toxicity may help develop a more consistent intervention strategy.     

Correlation of glutamate-induced apoptosis with caspase activities in cultured rat cerebral cortical neurons

In cultured rat cortical neurons lactate dehydrogenase (LDH) activity in the medium, a cell-death marker, increased gradually after exposure to glutamate (100 microM to 1 mM) for 60 min and reached a plateau at 24 to 30 h. Neuronal death was mainly apoptotic as suggested by typical electron microscopic findings, fluorescent double staining with membrane-permeating and nonpermeating chromatin dyes, nick end labeling, and assessment of DNA fragmentation by agarose gel electrophoresis. After 1 mM glutamate exposure, a rise of interleukin-1beta converting enzyme (ICE)-like protease activity in neurons was parallel to cysteine protease p32 (CPP32)-like protease activity and declined before CPP32-like protease activity reached the peak (at 6 h). LDH activity in the medium of glutamate-exposed neurons was decreased by specific ICE and/or CPP32 inhibitors, acetyl-L-tyrosyl-L-valyl-L-alanyl-L-aspart-1-al (Ac-YVAD-CHO) and acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspart-1-al (Ac-DEVD-CHO), respectively, in a dose-dependent manner. Fluorescent double staining of nuclei also demonstrated that at 100 microM each inhibitor prevented neuronal apoptosis and that this effect was additive. Among agonists corresponding to various glutamate receptor subtypes, N-methyl-D-aspartate (NMDA) and kainate induced apoptosis in cortical neuronal cultures while alpha-amino-3-hydroxy-5-methylisoxazole-4-propinate (AMPA) did not. The metabotropic glutamate receptor agonist, 1-aminocyclopentane-1S, 3R-dicarboxylate (ACPD) prevented apoptosis. Interestingly, apoptosis at 24 h after agonist or antagonist exposure correlated closely with caspase activity 6 h after exposure.     

木犀草素对实验性自身免疫性脑脊髓炎大鼠SPH/SPHK1/S1P1通路及神经元凋亡的影响

目的 探讨木犀草素(Luteolin)对实验性自身免疫性脑脊髓炎(Experimental autoimmune en-cephalomyelitis,EAE)大鼠神经酰胺-鞘氨醇(Sphingosine,SPH)/鞘氨醇激酶1(Sphingosine kinase 1,SPHK1)/1-磷酸鞘氨醇受体1(Sphing...     

Lactational zinc deficiency‐induced hippocampal neuronal apoptosis by a BDNF‐independent TrkB signaling pathway

It is well‐known that zinc deficiency leads to neuronal death in the brain. Here we tested the hypothesis that changes in the TrkB signaling pathway are involved in hippocampal neuronal apoptosis of suckling offspring with maternal zinc deficiency. Postpartum mice were fed a zinc‐deficient (0.85 ppm) diet and their offspring were used as a lactational zinc deficiency mouse model. At P7, P14, and P21, changes in hippocampal neuronal apoptosis were assessed by Nissl and TUNEL staining. BDNF levels and TrkB neurotrophic signaling were examined using immunoblotting assay. Lactational zinc deficiency resulted in lower levels of p‐TrkB and p‐ERK, and higher levels of Bax/Bcl‐2 and caspase‐3 in the hippocampus, suggesting that zinc deficiency‐induced low levels of TrkB phosphorylation would abrogate the downstream ERK signaling pathway, leading to hippocampal neuronal apoptosis. Most interestingly, our data showed that the activity of Src, a key molecule for zinc‐induced TrkB activation through the BDNF‐independent pathway, was inhibited significantly, and the expression levels of BDNF were significantly increased in the hippocampus of suckling mice. The present data indicate that zinc depletion‐induced hippocampal neuronal apoptosis is likely through modulation of the TrkB neurotrophic signaling pathway by a BDNF‐independent and Src‐dependent mechanism, whereas higher expression of BDNF is considered as a protective response, which cannot fully compensate for the injury caused by maternal zinc deficiency. © 2010 Wiley‐Liss, Inc.