溶血磷脂酸对β-淀粉样蛋白片段AβP31-35所致小鼠大脑皮层离体神经元凋亡的神经保护作用

已报道低浓度溶血磷脂酸(lysophosphatidic acid,LPA)对去血清培养所致的神经元凋亡有神经保护作用.为了进一步观察LPA是否对β-amyloid peptide fragment 31-35(AβP31-35)所致的神经元凋亡也起类似的作用,本研究应用DNA电泳分析、HO33342和TUNEL染色法等技术,对培养的小鼠大脑皮层神经元进行了观察.结果显示,只有使用较低浓度的LPA(1～10μmol/L)、并且将此剂量的LPA比AβP31-35提前12～24 h加入培养液时,才可看到LPA明显削弱了AβP31-35所致的神经元凋亡.以上结果表明,适当浓度的LPA在长时间预作用的条件下,可对AβP31-35所致的皮层神经元凋亡起保护因子或抗凋亡因子的作用,但其作用途径可能较在去血清培养所致的凋亡时更为复杂,因为在去血清的同时加入LPA就能制止去血清所致的凋亡.     

高同型半胱氨酸对大鼠神经元的损伤作用及其相关机制的研究

目的:研究同型半胱氨酸(Hcy)对体外培养神经元的损伤作用及其相关机制。方法:用Hcy处理体外培养的海马和皮层神经元,观察神经元的凋亡情况,以及神经元内游离钙浓度改变、DNA损伤和氧化损伤。结果:用250μmol/LHcy作用4h可使神经元凋亡率显著升高,并呈时间依赖性。甲基供体S-腺苷蛋氨酸(SAM)可显著抑制Hcy引起的神经元凋亡。Hcy(250μmol/L)可使神经元内游离钙浓度显著升高;导致神经元的彗星率明显增高;使神经元内MDA的产生增多,但并不影响神经元的总抗氧化能力(T-AOC)。结论:高浓度Hcy可通过神经兴奋作用、DNA损伤和氧化损伤等多方面机制对神经元造成损伤作用,从而诱导神经元发生凋亡。DNA损伤在Hcy导致神经元凋亡的作用中起着关键作用。     

MAPKs介导NMDA诱导的大鼠皮层神经元凋亡(英文)

NMDA诱导兴奋毒造成的神经损伤,包括细胞的凋亡和坏死。本研究旨在探讨神经元凋亡在NMDA兴奋毒所致大鼠皮层神经元死亡中的所占比例,并分析了NMDA致神经元凋亡的信号通路机制。通过使用Caspase抑制剂和测定乳酸脱氢酶活性,研究NMDA(100μmol/L,2h)兴奋毒所致的神经元凋亡;并使用MAPKs选择性抑制剂,分别采用Caspase-3活性检测,TUNEL和Annexin V染色方法,进一步观察MAPKs通路中细胞外信号调节激酶(ERK)、c-Jun N-末端激酶(JNK)和p38 MAPK三条不同途径在NMDA所致神经元凋亡中的作用。结果显示:(1)Caspase依赖的凋亡占NMDA所致细胞死亡总数的22.49%;(2)p38 MAPK抑制剂SB203580(10μmol/L)使NMDA诱导的caspase-3活性降低30.43%(P0.05);而ERK抑制剂PD98059(20μmol/L)和JNK抑制剂SP600125(20 μmol/L)不影响caspase-3的活性;(3)SB203580(10μmol/L)使NMDA所致的TUNEL阳性细胞数减少33.10%(P0.05);而PD98059(20μmol/L)或SP600125(20μmol/L)都没有作用;(4)Annexin V染色结果显示,SB203580(10μmol/L)使NMDA所致的早期凋亡细胞减少55.56%(P0.05);SP600125(20μmol/L)使NMDA所致的晚期凋亡/死亡细胞减少67.59%(P0.05);PD98059(20μmol/L)对细胞凋亡/死亡没有明显作用。以上结果表明,NMDA介导的大鼠皮层神经元死亡除坏死外,还包含有一小部分神经元凋亡;p38 MAPK途径,而非JNK和ERK途径,介导了NMDA诱导的神经元凋亡,抑制与此相关的凋亡信号通路可发挥神经保护作用;JNK途径可能介导了NMDA所致的神经元坏死而非凋亡。     

Humanin通过内源性途径抑制Aβ_(31-35)诱导的神经元凋亡(英文)

为了探讨Humanin(HN)对Aβ31-35诱导的培养皮层神经元凋亡的影响,本研究采用不同浓度(5、10、20μmol/L)的HN预孵育体外培养的皮层神经元不同时间(0、8、16h),加入Aβ31-35(25μmol/L)24h后,应用电子显微镜观察神经元的凋亡情况;流式细胞术、TUNEL法检测神经元的凋亡率;酶标仪检测caspase活性;Westernblot检测Bax蛋白的表达。结果显示:HN(20μmol/L)预孵育16h明显抑制了Aβ31-35诱导的神经元凋亡;HN降低了Aβ31-35诱导的caspase-3、9活性的升高;HN抑制了Aβ31-35诱导的Bax从胞浆到线粒体的转位。以上结果提示,HN通过阻断内源性凋亡途径抑制Aβ31-35诱导的神经元凋亡。     

利多卡因调节RhoA/ROCK轴对结直肠癌细胞生物学行为的影响

该文主要探讨利多卡因(lidocaine, Lido)通过调节Ras同源基因家族成员A(Rho A)/Rho相关的卷曲螺旋激酶(ROCK)轴对结直肠癌(CRC)细胞生物学行为的影响。该研究使用0～1 250μmol/L的利多卡因处理人结直肠癌细胞LS513, CCK-8法检测细胞活力筛选适宜药物浓度。将细胞分为对照组(Control组)、利多卡因低浓度组(Lido-L组, 500μmol/L Lido)、利多卡因中浓度组(Lido-M组, 750μmol/L Lido)、利多卡因高浓度组(Lido-H组, 1 000μmol/L Lido)和利多卡因高浓度+ROCK信号通路激活剂LPA组(Lido-H+LPA组, 1 000μmol/L Lido+10μmol/L LPA)。Edu检测细胞增殖;划痕愈合实验和Transwell小室实验分别检测细胞迁移和侵袭能力;流式细胞仪检测细胞凋亡情况;Westernblot检测PCNA、Bax、Bcl-2、RhoA、ROCK1、E-cadherin和N-cadherin蛋白表达情况。该研究得出与0μmol/L利多卡因相比, 500μmol/L、75...     

锌离子调节皮质酮对原代培养大鼠海马神经元的损伤

探讨皮质酮对原代培养大鼠海马神经元的损伤效应及锌的调节作用。用原位染色和RT-PCR方法,分别检测神经元的损伤情况及NMDA受体三种亚基(NRl、NR2A、NR2B)mRNA的表达。皮质酮(5μmol／L)作用2,4h可明显降低海马神经元的存活率,导致神经元凋亡,并随着作用时间的延长而加重;锌离子明显影响皮质酮对海马神经元的损伤效应：同时加入皮质酮和低、中浓度Zn^2 (10、100μmol／L),可明显降低神经元凋亡率,而加入高浓度Zn^2 (250μmol／L)则加重神经元损伤。皮质酮作用24h后,海马神经元NRl、NR2BmRNA的表达水平增高,而同时加入低、中浓度Zn^2 (10、100μmol／L)的海马神经元NRl、NR2BmRNA表达水平与对照组接近;NR2AmRNA表达无明显变化。这些结果表明,锌对皮质酮所致应激损伤的调节具有双向性;NMDA受体亚基水平的变化可能是其中重要环节之一。     

氯化钴对小鼠NIH3T3细胞增殖和凋亡的影响

目的:观察氯化钴(Cocl2)对体外培养的小鼠NIH3T3细胞增殖和凋亡的影响.方法:首先利用MTT法和流式细胞术检测不同浓度Cocl2对小鼠NIH3T3细胞增殖及凋亡的影响.然后利用免疫印迹检测HIF-1α和凋亡相关蛋白Bcl-2及Bax的表达.结果:(1)MTT结果显示,低于250μ mol/L的Cocl2作用NIH3T3细胞24 h对细胞的增殖活力影响不大,当浓度增加到500μ mol/L以上时会明显抑制细胞的增殖活力.(2)流式细胞仪检测结果显示低浓度Cocl2(≤ 250μ mol/L)并不会引起NIH3T3细胞明显的凋亡和坏死,增加浓度(≥500μmol/L)会导致细胞早期凋亡和坏死增加.3)不同浓度Cocl2作用NIH3T3细胞24 h均可诱导细胞中HIF-1α表达上调,同时Bax/Bcl-2的比值增高.结论:高浓度Cocl2可以抑制小鼠NIH3T3细胞增殖,促进凋亡发生.     

Sal B对肝癌细胞株HepG2的促凋亡作用

目的揭示丹酚酸B(Salvianolic Acid B,Sal B)对肝癌细胞株HepG2的杀伤作用。方法用不同浓度的丹酚酸B处理HepG2细胞,37℃培养24h。用RT-PCR检测促凋亡基因Bax的转录水平,并用流式细胞术检测细胞凋亡的水平。结果①100μmol/L、50μmol/L、25μmol/L等浓度的Sal B处理都能使HepG2细胞促凋亡基因bax的转录水平升高,其中100μmol/L处理组最为明显。②不同浓度的Sal B处理都能使HepG2细胞发生凋亡,其中100μmol/L处理组最为明显。结论 Sal B有促进HepG2细胞凋亡的作用。     

二苯乙烯苷抑制过氧化氢诱导的人脐静脉内皮细胞凋亡

目的:研究二苯乙烯苷(TSG)对过氧化氢(H2O2)诱导人脐静脉内皮细胞(HUVECs)凋亡的保护作用。方法:运用四甲基偶氮唑盐还原法(MTT法)和流式细胞术筛选建立细胞凋亡模型的H2O2合适浓度以及检测不同浓度TSG对H2O2诱导HUVECs的增殖率和凋亡率;Hoechst33258染色观察细胞凋亡形态。结果:MTT及流式法筛选300μmol/L为H2O2作用于细胞的最适凋亡浓度。MTT和流式结果显示,与H2O(2300μmol/L)损伤组比较,10μmol/L与100μmol/LTSG预处理组细胞的增殖率增加(P<0.05),凋亡率显著降低(P<0.01);Hoechst33258染色观察TSG能降低H2O2诱导的细胞凋亡,使细胞凋亡数减少。结论:TSG能抑制H2O2诱导的HUVECs凋亡,从而起到保护血管内皮细胞的作用。     

雷诺嗪对高糖高脂诱导的NIT-1细胞凋亡的保护作用

目的 研究雷诺嗪对高糖高脂诱导的NIT-1胰岛β细胞凋亡的保护作用及Cleaved caspase-3表达的影响,探讨雷诺嗪保护胰岛β细胞的机制.方法 采用CCK-8法测定不同浓度的雷诺嗪对体外培养及高糖高脂诱导的NIT-1胰岛β细胞的增殖能力的影响,同时应用流式细胞术检测NIT-1细胞凋亡,Western blot检测凋亡因子Caspase-3活化片段Cleaved caspase-3蛋白的表达.结果 不同浓度的雷诺嗪对NIT-1胰岛β细胞保护作用呈剂量依赖性:低浓度雷诺嗪对细胞凋亡无明显保护作用,随浓度升高保护作用明显.在培养基中加入高脂高糖及高浓度的雷诺嗪(5μmol/L)共同培养24h,雷诺嗪组细胞凋亡率明显低于高脂高糖单独作用组(P＜0.01),同时相对于高糖高脂组,激活型Caspase3表达明显降低(P＜0.05).结论 雷诺嗪能抑制高糖高脂诱导的NIT-1胰岛β细胞凋亡,其分子机制可能是雷诺嗪对Caspase-3的激活作用.     

Beta-amyloid increases somatostatin expression in cultured cortical neurons

In beta-amyloid (Abeta)-induced neurotoxicity, activation of the NMDA receptor, increased Ca2+ and oxidative stress are intimately associated with neuronal cell death as normally seen in NMDA-induced neurotoxicity. We have recently shown selective sparing of somatostatin (SST)-positive neurons and increased SST expression in NMDA agonist-induced neurotoxicity. Accordingly, the present study was undertaken to determine the effect of Abeta25-35-induced neurotoxicity on the expression of SST in cultured cortical neurons. Cultured cortical cells were exposed to Abeta25-35 and processed to determine the cellular content and release of SST into medium by radioimmunoassay and SST mRNA by RT-PCR. Abeta25-35 induces neuronal cell death in a concentration- and time-dependent fashion, increases SST mRNA synthesis and induces an augmentation in the cellular content of SST. No significant changes were seen on SST release at any concentration of Abeta25-35 after 24 h of treatment. However, Abeta25-35 induces a significant increase of SST release into medium only after 12 h in comparison with other time points. Most significantly, SST-positive neurons are selectively spared in the presence of a lower concentration of Abeta25-35, whereas, in the presence of higher concentrations of Abeta25-35 for extended time periods, SST-positive neurons decrease gradually. Furthermore, Abeta25-35 induces apoptosis at lower concentrations (5 and 10 micromol/L) and necrosis at higher concentrations (20 and 40 micromol/L). Consistent with the increased accumulation of SST, these data suggest that Abeta25-35 impairs cell membrane permeability. Selective sparing of SST-positive neurons at lower concentrations of Abeta25-35 at early time points directly correlates with the pathophysiology of Alzheimer's disease.     

溶血磷脂酸促进离体大鼠胚胎神经干细胞的增殖及其向胆碱能神经元分化

溶血磷脂酸（1ysophosphatidic acid,LPA）是一种细胞外磷脂信号。本研究用[^3H]-胸腺嘧啶掺入法、免疫细胞化学和Western blot等技术,观察了LPA对体外培养的大鼠胚胎神经干细胞（neural stem cells,NSCs）的增殖以及向MAF2标记的一般神经元和ChAT标记的胆碱能神经元的分化的影响。结果显示：（1）在特殊的无血清培养基中加入低浓度的LPA（0．01-1．0μmol/L）后,NSCs对【^3H】-胸腺嘧啶的摄入呈剂量依赖性增加,表明LPA对NSCs有显著的促增殖作用;（2）在培养基中加入胎牛血清以诱导NSCs的分化,发现低浓度的LPA增加MAF2阳性和ChAT阳性神经元的比例,0．1μmol／L LPA引起的增加达到峰值;（3）Western blot分析显示LPA促进了MAP2和ChAT的表达;（4）在诱导NSCs出现分化早期,用倒置显微镜观察到低浓度的LPA明显促进细胞突起的生长和细胞的迁移。以上结果表明,低浓度LPA在一定范围内可以促进NSCs的增殖、并分化为一般的MAP2阳性神经元和特殊的胆碱能神经元,而且LPA可以促进在分化早期出现的神经元或神经胶质细胞前体细胞的迁移和突起生长。     

谷氨酸转运抑制剂对器官型培养脊髓片的影响

观察谷氨酸转运体抑制剂苏一羟天冬氨酸(Threo-hydroxyaspartate,THA)对器官型培养的脊髓片的影响,探讨谷氨酸在运动神经元损伤中的作用。取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,在培养液中加入不同浓度THA(50μmol／L、100μmol／L、5001μmol／L),用神经元的特异性免疫组化染色剂SMI-32,非磷酸化神经丝标记物,对脊髓腹角α运动神经元进行鉴定,用单克隆抗钙网膜蛋白(calretinin)抗体对背角中间神经元进行记数,测定培养液中乳酸脱氢酶(LDH)的含量,并与对照组比较。结果显示对照组α运动神经元数目恒定,THA可以引起剂量依赖性的培养液中LDH含量增高和α运动神经元数目减少,而脊髓背角的中间神经元损伤相对较轻,其中THA100μmol／L组在体外培养4周后出现类似于肌萎缩侧索硬化(ALS)的病理改变：α运动神经元数目较对照组明显减少,而脊髓背角的中间神经元数目无显著变化。细胞外谷氨酸增高主要对运动神经元造成损伤,脊髓运动神经元较感觉神经元对谷氨酸的兴奋毒作用更加敏感。     

Blockade of calcium entry accelerates arsenite-mediated apoptosis in rat cerebellar granule cells

Arsenical exposure can cause defects in the central nervous system, yet the underlying cellular and molecular mechanisms are largely unknown. We have recently demonstrated that sodium arsenite induces apoptosis of cultured cortical and cerebellar neurons, suggesting that arsenite-induced neuronal apoptosis may contribute to at least some of its neurotoxic effects. Here we investigated the effect of Ca2+ on arsenite-mediated cerebellar granule neuron death. Sodium arsenite induced apoptosis in cerebellar neurons which were maintained in the presence of serum and depolarizing concentrations of potassium chloride (25 mM KCI). Under these conditions, inhibition of calcium entry by N-methyl-D-aspartate (NMDA) receptor blocker DL-aminophosphonovalerate (APV) or calcium channel antagonist nifedipine increased arsenite-induced apoptosis, while APV or nifedipine alone had little effect on cell viability. In cortical neurons or cerebellar neurons maintained at low potassium (5 mM), arsenite also induced apoptosis. However, the addition of APV or nifedipine did not alter levels of arsenite-induced apoptosis. These data suggest that arsenite-mediated apoptosis is regulated by intracellular calcium levels.     

Combining ursodeoxycholic acid or its NO-releasing derivative NCX-1000 with lipophilic antioxidants better protects mouse hepatocytes against amiodarone toxicity

Nonalcoholic steatohepatitis (NASH) is a common and potentially severe form of liver disease. This study aimed to determine the effect of ursodeoxycholic acid and its NO-releasing derivative NCX-1000 alone or in combination with antioxidants on cultured mouse hepatocytes treated with amiodarone to mimic certain aspects of hepatocyte injury found in NASH. Isolated mouse hepatocytes were incubated with ursodeoxycholic acid or NCX-1000 (0-100 micromol/L) combined or not combined with the hydrophilic antioxidants butylated hydroxytoluene and ascorbic acid (0-100 micromol/L) or with the lipophilic antioxidant alpha-tocopherol (0-100 micromol/L) 15 min before adding amiodarone (50 micromol/L) to the culture medium. Twenty hours later, necrosis, apoptosis, superoxide anion production, and malondialdehyde levels were assessed in cultured cells. Amiodarone led to a dose-dependent decrease in cell viability with an LD50 of 50 micromol/L and increased production of superoxide anion and lipid peroxidation. NCX-1000 showed a better protective potential than ursodeoxycholic acid against the toxic effects of amiodarone. The hydrophilic antioxidants had no effect on the toxicity of amiodarone, whereas alpha-tocopherol at a concentration >100 micromol/L almost completely suppressed it. Ursodeoxycholic acid and NCX-1000 protection was additive only when they were combined with alpha-tocopherol, not with butylated hydroxytoluene or ascorbic acid. In addition, all the antioxidants tested reduced the superoxide anion detected, but only alpha-tocopherol prevented lipid peroxidation induced by amiodarone. The combination of lipophilic antioxidants with ursodeoxycholic acid or NCX-1000 enhances their protective potential and could represent an interesting therapeutic approach to explore for the treatment of NASH.     

Lysophosphatidic acid receptor-dependent secondary effects via astrocytes promote neuronal differentiation

Lysophosphatidic acid (LPA) is a simple phospholipid derived from cell membranes that has extracellular signaling properties mediated by at least five G protein-coupled receptors referred to as LPA(1)-LPA(5). In the nervous system, receptor-mediated LPA signaling has been demonstrated to influence a range of cellular processes; however, an unaddressed aspect of LPA signaling is its potential to produce specific secondary effects, whereby LPA receptor-expressing cells exposed to, or "primed," by LPA may then act on other cells via distinct, yet LPA-initiated, mechanisms. In the present study, we examined cerebral cortical astrocytes as possible indirect mediators of the effects of LPA on developing cortical neurons. Cultured astrocytes express at least four LPA receptor subtypes, known as LPA(1)-LPA(4). Cerebral cortical astrocytes primed by LPA exposure were found to increase neuronal differentiation of cortical progenitor cells. Treatment of unprimed astrocyte-progenitor cocultures with conditioned medium derived from LPA-primed astrocytes yielded similar results, suggesting the involvement of an astrocyte-derived soluble factor induced by LPA. At least two LPA receptor subtypes are involved in LPA priming, since the priming effect was lost in astrocytes derived from LPA receptor double-null mice (LPA(1)((-/-))/LPA(2)((-/-))). Moreover, the loss of LPA-dependent differentiation in receptor double-null astrocytes could be rescued by retrovirally transduced expression of a single deleted receptor. These data demonstrate that receptor-mediated LPA signaling in astrocytes can induce LPA-dependent, indirect effects on neuronal differentiation.     

Lysophosphatidic Acid Induces Necrosis and Apoptosis in Hippocampal Neurons

Abstract: A diverse body of evidence indicates a role for the lipid biomediator lysophosphatidic acid (LPA) in the CNS. This study identifies and characterizes the induction of neuronal death by LPA. Treatment of cultured hippocampal neurons from embryonic rat brains with 50 µ M LPA resulted in neuronal necrosis, as determined morphologically and by the release of lactate dehydrogenase. A concentration of LPA as low as 10 µ M led to the release of lactate dehydrogenase. In contrast, treatment of neurons with 0.1 or 1.0 µ M LPA resulted in apoptosis, as determined by chromatin condensation. In addition, neuronal death induced by 1 µ M LPA was characterized as apoptotic on the basis of terminal dUTP nick end-labeling (TUNEL) staining, externalization of phosphatidylserine, and protection against chromatin condensation, TUNEL staining, and phosphatidylserine externalization by treatment with N -benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad-spectrum inhibitor of caspases, i.e., members of the interleukin-1β converting enzyme family. Studies with antagonists of ionotropic glutamate receptors did not indicate a significant role for these receptors in apoptosis induced by 1 µ M LPA. LPA (1 µ M ) also induced a decrease in mitochondrial membrane potential. Moreover, pretreatment of neurons with cyclosporin A protected against the LPA-induced decrease in mitochondrial membrane potential and neuronal apoptosis. Thus, LPA, at pathophysiological levels, can induce neuronal apoptosis and could thereby participate in neurodegenerative disorders.     

In vitro and in vivo neuroprotective effect and mechanisms of glabridin, a major active isoflavan from Glycyrrhiza glabra (licorice)

Stroke is a life-threatening disease characterized by rapidly developing clinical signs of focal or global disturbance of cerebral function due to cerebral ischemia. A number of flavonoids have been shown to attenuate the cerebral injuries in stroked animal models. Glabridin, a major flavonoid of Glycyrrhiza glabra (licorice), possesses multiple pharmacological activities. This study aimed to investigate whether glabridin modulated the cerebral injuries induced by middle cerebral artery occlusion (MCAO) in rats and staurosporine-induced damage in cultured rat cortical neurons and the possible mechanisms involved. Our study showed that glabridin at 25mg/kg by intraperitoneal injection, but not at 5mg/kg, significantly decreased the focal infarct volume, cerebral histological damage and apoptosis in MCAO rats compared to sham-operated rats. Glabridin significantly attenuated the level of brain malonyldialdehyde (MDA) in MCAO rats, while it elevated the level of two endogenous antioxidants in the brain, i.e. superoxide dismutase (SOD) and reduced glutathione (GSH). Co-treatment with glabridin significantly inhibited the staurosporine-induced cytotoxicity and apoptosis of cultured rat cortical neurons in a concentration-dependent manner. Consistently, glabridin significantly reduced the DNA laddering caused by staurosporine in a concentration-dependent manner. Glabridin also suppressed the elevated Bax protein and caspase-3 proenzyme and decreased bcl-2 induced by staurosporine in cultured rat cortical neurons, facilitating cell survival. Glabridin also inhibited superoxide production in cultured cortical neurons exposed to staurosporine. These findings indicated that glabridin had a neuroprotective effect via modulation of multiple pathways associated with apoptosis. Further studies are warranted to further investigate the biochemical mechanisms for the protective effect of glabridin on neurons and the evidence for clinical use of licorice in the management of cerebral ischemia.