【作者】 刘伟；

【导师】 常立文； Lee，Wei-Hua；

【作者基本信息】 华中科技大学 ， 儿科学， 2009， 博士

【摘要】 背景：临床上，缺氧缺血仍然是造成足月儿、近足月儿严重脑损伤的主要原因之一，其发生率（二和三级脑损伤）约为0．1-0．2％，其中约20％患儿死亡，高达40％幸存者遗留严重神经系统后遗症，如痉挛性麻痹，智力障碍，癫痫等。迄今为止，仍没有十分有效的临床手段能减轻脑损伤和改善此类患儿的预后。早期研究发现，缺氧缺血导致新生鼠和成年鼠神经细胞死亡的方式有显著差异。在新生鼠，由于神经细胞发育的不成熟，缺氧缺血早期阶段死亡以坏死为主，后期则以凋亡为主；而在成年鼠，神经细胞则主要发生坏死。这种差异很大程度上与新生脑组织具有较高的凋亡诱导激酶Caspase-3活性有关。由于对缺氧缺血十分敏感且受其增殖和再生能力的限制，一旦启动坏死、凋亡程序且未经干预，不成熟神经细胞终将死亡，即使动物幸存下来，其躯体感觉和记忆功能也受到极大影响。胰岛素样生长因子I（IGF-1）是一种多肽类神经营养因子，它对于哺乳动物神经系统的发育发挥十分重要的作用。IGF-1和其受体的表达在神经分化过程中达到顶峰，IGF-1通过激活其受体，促进神经细胞生存、分化、增殖、突触外伸以及髓鞘形成。研究表明，在7天的大鼠和胎羊，缺氧缺血后立即心腔内注射IGF-1能为神经细胞提供保护作用。我们前期的研究结果表明，缺氧缺血后24和48小时皮下注射IGF-1可显著减轻缺氧缺血诱导的不成熟鼠脑损伤，并且在2个月后能够明显改善它们的记忆和认知行为发展的潜能。然而，若干瓶颈阻碍了IGF-1进一步试用于临床：1．在新生大鼠缺氧缺血动物模型，即使在缺氧缺血发生后立即心腔内注射大剂量的IGF-1，三天后，脑损伤仅能减少40％左右；2．缺氧缺血后延迟（6小时以后）给予IGF-1治疗效果不确定；3．IGF-1对脑和行为学发展的远期效果不明了。在缺血再灌注诱导的脑损伤中，氧化损伤扮演了重要角色。相对于成熟脑组织而言，新生脑组织由于含有高浓度的铁离子，较低水平的谷胱苷肽过氧化物酶，并伴有H₂O₂的积累，导致其对缺血后氧化应激性损伤特别敏感。通过对小脑颗粒神经细胞的研究表明，高水平的H₂O₂除了本身的神经毒性以外，它还可诱导神经细胞产生对IGF-1保护作用的抵抗。那么，这种对IGF-1的抵抗是否是导致缺氧缺血后立即给予IGF-1治疗不佳的重要因素?目前尚不清楚。为此，我们希望采用一种抗氧化剂与IGF-1联合应用的方式以对缺血缺氧性神经细胞发挥足够保护作用。首先由抗氧化剂清除蓄积的ROS，消除神经细胞的氧化应激状态，恢复其对IGF-1神经营养作用的敏感性，然后，IGF-1得以发挥其保护功能和营养作用。丙酮酸钠正是这样一种药物，它具有：1．本身没有毒副作用，不会对细胞造成新的损伤；2．能够很容易的进入细胞内，在胞内胞外均能发挥作用；3．具有良好的抗氧化功能，能够清除ROS，消除缺氧缺血性发生后的氧化应激状态；4．与IGF-1有协同作用，能恢复细胞对IGF-1的反应性，增强IGF-1对缺氧缺血后细胞的保护作用。目的：尽管目前对缺氧缺血后脑损伤的机制有了进一步认识，但是我们对新生儿缺氧缺血性脑病仍然缺乏有效的临床治疗手段。IGF-1能够减少缺氧缺血性脑病动物模型的脑损伤，但是以一种时间依赖的方式。我们初期的数据显示，缺氧缺血后IGF-1在恢复的早期阶段疗效较低是由于氧化应激诱导了神经细胞的IGF-1抵抗。为此，我们进一步阐明：1、氧化应激诱导神经细胞IGF-1抵抗的机制；2、联合采用抗氧化剂治疗恢复神经细胞对IGF-1的敏感性后，是否能增强IGF-1对缺氧缺血诱导不成熟脑损伤的治疗作用。方法：1、原代培养的新生大鼠大脑皮层神经元细胞、少突胶质细胞和星形胶质细胞，待生长至第六天时，将培养液换成高糖DMEM，分别加入不同浓度的H₂O₂，4小时后，用Western-blot检测神经元细胞cleaved Caspase-3，Caspase-3，IGFIR，Gapdh的表达，24小时后，分别检测各种细胞LDH，MTT的变化。2、原代培养的新生大鼠大脑皮层神经元细胞，待生长至第六天时，将培养液换成高糖DMEM，分别加入不同浓度的H₂O₂，5分钟后，IGF-1组加入25ng／ml IGF-1，对照组不加IGF-1，45分钟后，Western-blot检测P-Akt（Ser473），Akt，P-P38，P38表达变化；2小时后，Western-blot检测P-JNK（Thr183／Tyr185）、JNK、P-FOXO3（Ser253）、P-FOXO3（Thr32）、FOXO3、P-P53（Ser392）、P53、Acetyl-H3、Acetyl-4、Gapdh等的表达；4小时后，用Western-blot检测两组细胞cleaved Caspase-3，Caspase-3，IGFIR，Gapdh，P-P53（Ser392），P53表达变化，用real-time PCR检测IGFIR，Gapdh的表达变化；24小时后，分别检测两组细胞LDH，MTT的变化。3、分别以Akt、JNK2、p38和P53特异性阻断剂LY294002（20uM）、SP600125（20uM）、SB239063（7．5uM）和Pifirin（10uM）作为干预方式，分别于相应的时间点运用相应的方法检测上述指标的改变。4、原代培养的新生大鼠大脑皮层神经元细胞，待生长至第六天时，将培养液换成高糖DMEM，分别加入不同浓度的H₂O₂，2小时后，用细胞裂解液提取细胞蛋白，分别以TBP和P53作为免疫共沉淀（IP）蛋白，用IB检测P-P53（Ser392）和HDAC1表达的变化。5、原代培养的新生大鼠大脑皮层神经元细胞，待生长至第六天时，将培养液换成高糖DMEM，分别加入不同浓度的H₂O₂，分别于5分钟和1小时后加入IGF-1，45分钟后用Western-blot方法检测P-Akt，Akt表达的变化。6、原代培养的新生大鼠大脑皮层神经元细胞，待生长至第六天时，将培养液换成高糖DMEM，分别加入不同浓度的H₂O₂，5分钟后丙酮酸钠组加入2mM丙酮酸钠，IGF-1组加入25ng／ml IGF-1，丙酮酸钠+IGF-1组同时加入二者，45分钟后，Western-blot方法检测P-Akt（Ser473），Akt表达变化，4小时后，Western-blot方法检测IGFIR，Gapdh，cleaved Caspase-3，Caspase-3表达变化，并检测细胞培养液LDH的变化。结果：1、大脑皮层神经元细胞对氧化应激损伤异常敏感H₂O₂对神经元细胞，少突胶质细胞，星形胶质细胞LDH和MTT均有的影响，但对神经元细胞的影响更加显著。2、H₂O₂对皮层神经元细胞的毒性作用呈剂量依赖性H₂O₂使神经元细胞细胞培养液中LDH明显上升，MTT明显下降，cleavedCaspase-3表达明显上升，且呈明显的H₂O₂剂量依赖性。3、IGF-1对皮层神经元细胞的凋亡具有保护作用IGF-1使皮层神经元细胞LDH明显降低，cleaved Caspase-3表达显著降低。4、高浓度H₂O₂能够抑制IGF-1对皮层神经元细胞的保护作用高浓度的H₂O₂能够使IGF-1组和无IGF-1组皮层神经元细胞LDH，MTT和Caspase-3的表达无显著性差异。5、提高IGF-1的浓度并不能克服H₂O₂诱导的皮层神经元细胞IGF-1抵抗当H₂O₂浓度达到60uM时，无论何种浓度的IGF-1均不能使神经元细胞LDH降低。6、H₂O₂诱导皮层神经元细胞P38激活，加入IGF-1无法阻断这一过程H₂O₂显著升高神经元细胞磷酸化P38MAPK的表达，P38MAPK特异性的抑制剂SB239063能够抑制这种升高。而加入IGF-1并无影响。7、H₂O₂通过激活P38，从而阻断皮层神经元细胞Akt的磷酸化（Ser473）H₂O₂呈剂量依赖性的下调神经元细胞P-Akt（Ser473）的表达。8、IGF-1通过激活Akt（Ser473），从而钝化FOXO3（Thr32），但是这一过程能被高浓度的H2O2阻遏IGF-1显著增加神经元细胞P-Akt（Ser473）的表达，并且使P-FOXO3（Thr32）表达明显升高。但当加入高浓度的H₂O₂，IGF-1的作用即被抑制。9、H₂O₂对皮层神经元细胞Akt磷酸化（ser473）的抑制作用是可逆的，且其对IGF-1诱导的Akt（Ser473）磷酸化的阻遏作用亦可扭转当加入H₂O₂ 5分钟后加入IGF-1，可见P-Akt（Ser473）升高并不明显，而当加入H₂O₂ 1小时后加入IGF-1，则可见P-Akt（Ser473）显著升高。10、H₂O₂通过磷酸化JNK2（Thr183／Tyr185），从而激活FOXO3（Ser253）IGF-1可以显著增加P-JNK2（Thr183／Tyr185）的表达，并明显增加P-FOXO3（Ser253）的表达。11、H₂O₂对大脑皮层神经细胞IGF-1受体的影响是神经元细胞特异性的H₂O₂明显下调神经元细胞IGF-1受体的表达，但是对少突胶质细胞和星形胶质细胞IGF-1的表达没有显著影响。12、H₂O₂呈剂量依赖性诱导皮层神经元细胞P53的激活，并且这种激活作用具有时效性H₂O₂能够呈剂量依赖性的明显升高P-P53（Ser392）的表达，且这种作用在2小时达到高峰，4小时逐渐下降。13、H₂O₂通过激活P53，从而降低皮层神经元细胞IGF-1受体的表达H₂O₂明显减少神经元细胞IGF-1受体的表达，但当加入P53的抑制剂Pifithrin，IGF-1受体的表达明显升高14、磷酸化P53（Ser392）通过结合于TBP而影响IGF-1受体mRNA的转录H₂O₂能使P-P53（Ser392）与TBP的结合显著增加，从而影响转录15、H₂O₂诱导HDAC1与P53结合从而影响组蛋白3和组蛋白4乙酰化H₂O₂能使HDAC1与P53结合显著增加，并且明显下调乙酰化组蛋白4的表达，而对乙酰化组蛋白3却没有明显影响。16、在氧化应激状态下，丙酮酸钠能使大脑皮层神经细胞恢复对IGF-1的敏感性在H₂O₂存在的条件下，丙酮酸钠能使P-Akt（Ser473）表达明显升高，当同时加入丙酮酸钠和IGF-1，丙酮酸钠能显著上调IGF-1对P-Akt（Ser473）的刺激作用17、在氧化应激状态下，丙酮酸钠能减弱H₂O₂对大脑皮层神经细胞IGF-1受体的抑制作用在H₂O₂存在的条件下，丙酮酸钠能使神经元细胞IGF-1受体的表达明显升高。18、在氧化应激状态下，丙酮酸钠对大脑皮层神经元细胞LDH和活化Caspase-3表达的影响在H₂O₂存在的条件下，丙酮酸钠能使神经元细胞LDH明显下降，同时使激活的Caspase-3表达明显减少。表明丙酮酸钠能减少H₂O₂诱导的神经元细胞凋亡。结论：1、氧化应激首先激活应激酶P38，阻断了IGF-1／AKT信号通路的神经保护作用，随后激活另一应激酶JNK2，使AKT和JNK2两条独立通路之间的平衡被打破，从而激活神经转录因子FOXO3，触发了细胞凋亡级联反应；2、氧化应激状态下，P53首先被激活，激活的P53使IGF-1受体表达下调，是氧化应激诱导IGF-1抵抗的重要原因之一；3、氧化应激状态下，丙酮酸钠能够有效清除ROS，恢复神经细胞对IGF-1的敏感性，与IGF-1联合应用可增强IGF-1对神经细胞的保护作用。更多还原

【Abstract】 BackgroundCerebral hypoxia-ischemia remains a leading cause of severe brain damage that occursin as many as 0.1-0.2% of term or near -term infants （GradesⅡ&Ⅲbrain injuries）, amongwhom approximately 20% die and up to 40% of the survivors often suffer devastatingdisabilities, such as cerebral palsy, mental retardation, and epilepsy. Because of highmortality and poor prognosis, hypoxic-ischemic damage in neonatal brains continues to bea major medical emergency for newborn patients. To damage in neonatal brains continuesto be a major medical emergency for newborn patients. To date, no effective clinicaltreatment is available to mitigate brain damage and improve the prognosis and well beingof these children. Previously, the cascade of neuropathological events that leads to neuronaldamage was best revealed in a rat model of neonatal hypoxia-ischemia. Hypoxia-ischemiaresults in neuronal death that has different patterns and forms in young rats than those seenin adult rats. Young neurons die of necrosis at the early stage of recovery and of delayedapoptosis, whereas adult neurons die of necrosis. This difference is mainly due to theup-regulation of NMDA receptors and increased Caspase 3 activity in young brains that areundergoing programmed cell death. These two factors make young neurons particularlyvulnerable to hypoxia-ischemia. From the onset of oxygen and nutrient deprivation, acascade of pathological events are initiated, such as glutamate excitotoxicity, caspaseactivation, and activation of nitric oxide synthase. Without intervention, young neurons will eventually die and the animals, if they survive, will develop impaired somatosensoryfunction and lateralized memory loss in adult life.Insulin-like growth factorⅠ（IGF-Ⅰ） is a pleiotrophic factor essential for the development ofthe mammalian nervous system. IGF-Ⅰand its receptor are expressed at the highest levelsduring the peak of neuronal differentiation. Through activating its receptors on all braincells, IGF-Ⅰpromotes neuronal survival and differentiation as well as oligodendrogenesis,and myelination. Studies of IGF-Ⅰtransgenic or gene deletion mice have clearly shown thatthe IGF system plays a key role in neuronal survival and neuro- and oligodendrogenesis invivo, which is also supported by its effects in a variety of animal models of brain injury andneuronal degeneration, in day 7 rat pups and fetal sheep, intraventricular infusion of IGF-Ⅰsoon after hypoxia-ischemia provided a certain degree of neuroprotection. However, severalconcerns prevented IGF-Ⅰfrom being further tested in a clinical trial: （1）. In awell-established neonatal hypoxia-ischemia rat model, even intraventricular infusion ofhigh doses of IGF-Ⅰimmediately following hypoxia-ischemia only reduced brain damageby 40% when evaluated 3 days later; （2）. The therapeutic effects of delayed IGF-Ⅰtreatmentfollowing hypoxia-ischemia （after 6 hours） are unknown; and （3）. The long-term effects ofIGF-Ⅰtreatment on brain and behavior development have not been evaluated. Our recentstudies answered these concerns and strongly support IGF-Ⅰas a potential effectivetreatment for HIE patients. Subcutaneous administration of IGF-Ⅰat 24 and 48 hours ofrecovery significantly reduced hypoxia-ischemia induced injury to immature rat brains andimproved their memory and cognitive behavior development evaluated 2 months later.However, this new evidence raised this question: why is immediate IGF-ⅠⅠtreatmentfollowing hypoxia-ischemia less effective than delayed treatment?Oxidative damage plays a major role in ischemia/reperfusion induced brain injuries,such as HIE. Compared to the adult brain, the neonatal brain is exceedingly vulnerable tooxidative stress due to high levels of free iron, lower levels of glutathione peroxidase, and aconcomitant accumulation of hydrogen peroxide （H₂O₂）. Besides its own neurotoxicity, high levels of H₂O₂ likely induce neuronal resistance to IGF-Ⅰby （1） inhibiting IGF-Ⅰsignaling, as recently shown in cerebellar granule neurons, and （2） decreasing IGF-Ⅰtreatment following hypoxia-ischemia is less effective than delayed treatment. Immediatelyfollowing hypoxia-ischemia, ROS rapidly accumulates and induces neuronal IGF-Ⅰresistance which deprives neurons of this trophic factor for their maturation and survival.The survival of immature neurons is further compromised by a decline in IGF-Ⅰcalculatinglevels and neuronal IGF-Ⅰexpression following hypoxia-ischemia. It is not surprising thatantioxidants alone had only limited efficacy in clinical trials, because young neurons stilllack essential trophic support even though ROS levels are lowered.Thus, we expect to find a way to resolve this problem. Because of hypoxia-ischemia,oxidative stress induces neuronal cells IGF-Ⅰresistance. Theoretically speaking, we need anantioxidant and IGF-Ⅰin neuroprotection. Firstly, antioxidant scavenges accumulated ROS,in order to remove neuron cell in oxidant stress and to recover its sensitiveness to theneuronal trophic support of IGF-1. Then IGF-1 can exert its protective functions andtrophic effect. Sodiumpyruvate is just an reagent of this kind. It has the followingcharacteristics: 1. No by-effects. It will not damage cell. 2. It can easily enter cells andfunctions both in internal cell and external cell. 3. With excellent antioxidant functions, itcan scavenge ROS and remove the oxidative stress following hypoxia-ischemia. 4. Itcollaborates with IGF-1 and recovers the reactivity of IGF-1. It can also increase theprotective function of IGF-1 to hypoxia-ischemia. Applying IGF-1 or sodiumpyruvatealone in clinic does not achieve satisfactory results. However, sodiumpyruvate’scollaboration with IGF-1 resolves this problem. It sheds lights on the curation ofhypoxia-ischemia.Objective:Despite advances in our knowledge of neuronal injury, effective clinical treatments forHIE in newborns are still lacking. IGF-Ⅰreduces hypoxic-ischemic brain injuries in animal models of HIE, but in a time-dependent manner. Our preliminary data demonstrated that thelower rate of therapeutic efficacy in the early phase of recovery may result from oxidativestress induced neuronal IGF-Ⅰresistance. We will （1） investigate potential mechanisms ofthe oxidative stress induced neuronal IGF-Ⅰresistance; and （2） determine whether restoringneuronal sensitivity to IGF-Ⅰwill increase its therapeutic efficacy in the treatment ofhypoxia-ischemia induced injury to immature brains.Conclusions:1. Oxidative stress activated P38 blocks the IGF-1/Akt signaling pathway, which will inturn attenuate Akt’s inhibition on FOXO3. Moreover, oxidative stress will also activatethe JNK2 signaling pathway, which will induce FOXO3 activation. As a result, thesetwo signaling pathways work together to induce the neuronal apoptosis.2. Oxidative stress also activates P53, which suppresses IGF-Ⅰreceptor gene expression.This is an important mechanisms of oxidative stress induced neuronal IGF-Ⅰresistance.3. Under oxidative stress status, sodium pymvate scavenges ROS effectively and restoresneuronal sensitivity to IGF-1. Combined with IGF-Ⅰwill improve IGF-Ⅰ’sneuroprotective function.更多还原