【作者】 申红霞；

【导师】 陈俊； 曹国栋； 高艳琴； Ruth Anne Stetler；

【作者基本信息】 复旦大学 ， 神经生物学， 2008， 博士

【摘要】 新生儿缺氧缺血性脑病(hypoxic-ischemic encephalopathy,HIE)是围产期脑损伤最常见原因,不仅威胁新生儿的生命,而且有严重的神经系统后遗症。但对新生儿缺血缺氧性脑病的治疗,目前为止国内外尚无统一、有效的治疗方案。因此,寻找治疗新生儿缺血缺氧性脑病更为有效的方法已成为围产医学中的重要课题,越来越受到医学界广大医务工作者及学者的重视。新生大鼠脑缺血缺氧模型被广泛用于研究新生期脑缺血缺氧损伤的发病机理、早期诊断指标筛选和干预方法的评枯。丙酮酸乙酯是一种安全、稳定且极易被细胞摄取的物质。2001年,Sims等研究发现林格氏丙酮酸乙酯溶液治疗肠系膜缺血再灌,能大大减轻肠粘膜结构和功能的损伤;Yang等人于2002年首次提出了丙酮酸乙酯的抗炎作用,此后对丙酮酸乙酯保护作用抗炎机制研究倍受关注。本实验在7日龄Sprague-Dawley大鼠制备的新生大鼠脑缺血缺氧损伤动物模型基础上,研究丙酮酸乙酯对新生期脑缺血缺氧损伤的作用,并探讨其作用机制。实验结果如下:(一)丙酮酸乙酯对新生大鼠脑缺血缺氧损伤具有明显保护作用1.丙酮酸乙酯对新生大鼠脑缺血缺氧保护作用具有剂量依赖性2.丙酮酸乙酯对新生大鼠脑缺血缺氧保护作用具有时间依赖性,在缺血缺氧后30min腹腔注射依然有明显保护作用3.丙酮酸乙酯对损伤各个脑区均有明显保护作用(二)丙酮酸乙酯对新生大鼠脑缺血缺氧损伤的长时程保护作用1.丙酮酸乙酯在新生大鼠脑缺血缺氧损伤后8周依然有明显保护作用2.丙酮酸乙酯转归了新生大鼠生长发育(缺血缺氧后第2周～第8周)的行为变化(三)丙酮酸乙酯在新生大鼠脑缺血缺氧损伤保护中的抗凋亡作用1.抑制促凋亡因子caspase-3和AIF的激活1)丙酮酸乙酯抑制了新生大鼠脑缺血缺氧损伤后促凋亡因子caspase-3的激活2)丙酮酸乙酯抑制了新生大鼠脑缺血缺氧损伤后促凋亡因子AIF的核内水平2.升高抗凋亡因子Bcl-xl水平及抗凋亡信号通路PI3K/Akt中途径p-Akt的水平1)丙酮酸乙酯升高了新生大鼠脑缺血缺氧损伤后抗凋亡蛋白Bcl-xl的水平2)丙酮酸乙酯对新生大鼠脑缺血缺氧损伤保护作用机制中抗凋亡途径PI3K/Akt信号通路的作用a)新生大鼠脑缺血缺氧损伤后大脑皮层p-Akt表达水平降低b)丙酮酸乙酯升高了新生大鼠脑缺血缺氧损伤后大脑皮层降低的p-Akt水平c)侧脑室注射LY294002(PI3K/Akt信号通路抑制剂)后,不能抑制丙酮酸乙酯对新生大鼠脑缺血缺氧损伤的保护(四)丙酮酸乙酯降低了新生大鼠脑缺血缺氧损伤后致炎相关因子水平的升高1.丙酮酸乙酯降低了新生大鼠脑缺血缺氧损伤后升高的细胞因子表达水平1)丙酮酸乙酯降低了新生大鼠脑缺血缺氧损伤后升高的炎症因子COX-2,iNOS,TNF-a,IL-1a,IL-1b,IL-6的mRNA水平2)丙酮酸乙酯降低了新生大鼠脑缺血缺氧损伤后升高的炎症因子COX-2蛋白水平3)丙酮酸乙酯降低了新生大鼠脑缺血缺氧损伤后核内升高的转录因子NF-κB/p65的水平2.丙酮酸乙酯抑制了新生大鼠侧脑室注射LPS诱导的炎症反应中炎症因子COX-2,iNOS的表达水平总之,我们的研究证明了丙酮酸乙酯在新生大鼠脑缺血缺氧损伤中具有显著的保护作用。这种保护作用,至少部分,是由于抗炎作用,使NF-κB入核减少,降低其转录调控的炎症因子,如:COX-2,iNOS,TNF-α,IL-1α,IL-1β,IL-6;同时,丙酮酸乙酯具有抗凋亡作用,抑制促凋亡因子caspase-3、AIF的激活,升高抗凋亡因子Bcl-xl的表达水平,引起抗凋亡通路PI3K/Akt中p-Akt水平增高。我们的研究为临床治疗新生儿缺血缺氧性脑病提供了可能的手段和理论依据。更多还原

【Abstract】 Background and purpose:Hypoxic-ischemic(H-I) injury in the developing brain is a major cause of morbidity and mortality associated with periventricular leukomalacia and cerebral palsy.The way of neuronal cell death caused by H-I injury includes apoptosis and necrosis. Therefore,inhibiting necrosis-mediated inflammation is very important in the neuroprotection against H-I brain injury.Ethyl pyruvate(EP),a stable lipophilic pyruvate derivative identified recently by Fink and colleagues,is an experimental therapeutic that effectively protects animals from oxidative stress injury.The mechanism may be inhibiting the inflammation pathway,down-regulating expressions of various proinflammation factors and cytokines.However,whether EP attenuates neonatal brain damage following hypoxic ischemia(H-I) remains undemonstrated,and the mechanism should be further researched.Results:1.Administration of EP before and after H-I insult decreases cerebral tissue loss in a dose-dependent and time-dependent manner measured 7days later.Even within 30 rain after H-I insult,EP also protects cerebral tissue loss.2.In the long-term recovery experimental paradigms(8 weeks after H-I),the neuroprotective effect of EP remained.Administration of ethyl pyruvate also ameliorated behavioral deficits.The number of forelimb steps is different between vehicle and EP group from 3rd week.The number of backlimb steps is 4th week. 3.Ethyl pyruvate inhibited the activities of caspase-3 and AIF.And ethyl pyruvate rised the anti-apoptotic factors’ levels of phospho-Akt(p-Akt) and Bcl-xl.4.Ethyl pyruvate inhibited NF-κB activation.Then,ethyl pyruvate inhibited expressions of various proinflammation factors and cytokines which were regulated by NF-κB.Conclusion:These results demonstrate that EP confers potent neuroprotection against neonatal H-I brain injury and that the neuroprotective effect of EP is at least partially mediated via its anti-inflammatory actions.EP is thus a potential novel therapeutic agent for neonatal H-I brain injury.更多还原