Scu对大鼠脑缺血再灌注损伤的保护作用及对PARP/AIF介导的细胞凋亡途径的抑制研究

【作者】 张华锋；

【导师】 胡霞敏；

【作者基本信息】 武汉科技大学 ， 生理学， 2008， 硕士

【摘要】 脑血管意外是一种常见疾病。根据发病原因的不同,可分为缺血性和出血性脑血管意外两大类。前者发病率远较后者为高,尤以65岁以上的老年人多见。脑血管意外具有高死亡率和高致残率的特点。据世界卫生组织调查表明,脑血管意外引起的死亡占所调查的57个国家死亡总人数的11.3%,仅次于癌症和心肌梗死。脑血管意外的患者约有1/3在发病后不久死亡,幸存者则由于偏瘫、失语等后遗症而致残,丧失工作能力,甚至丧失生活自理能力。随着人口老龄化程度的加剧,脑血管疾病已成为一个倍受社会关注的问题。脑缺血是脑血管疾病最主要的病种。在我国,其死亡率仅次于恶性肿瘤,严重危害人类健康。大量文献报道,伴随着脑神经细胞缺血,机体的应激保护和缺血导致的损伤机制同时被激活,相关的细胞因子表达增高,信号通路被激活,最终导致神经细胞凋亡或坏死。因此,探寻缺血性脑血管疾病的有效治疗策略一直是世界医药学界共同关注的重大课题之一。灯盏花具有抗脑缺血、抗心肌缺血等药理作用。临床主要用于治疗心肌缺血损伤及脑血栓等引起的心脑血管疾病。灯盏花素是从灯盏花中提取的主要有效成分,是灯盏花甲素和灯盏花乙素(Scu)的混合物。其中Scu(黄芩素苷,scutellarin),即4, 5, 6-三羟基黄酮-7-葡糖醛酸苷,是灯盏花抗脑缺血损伤的主要有效成分。本研究采用大鼠局灶性脑缺血再灌注模型,旨在研究Scu对大鼠脑缺血再灌注过程中多聚ADP-核糖聚合酶(PARP) /凋亡诱导因子(AIF)介导的神经细胞凋亡途径的影响,为其在临床上防治脑血管疾病提供理论基础。第一部分灯盏花乙素(Scu)对大鼠脑缺血再灌注脑损伤的保护作用采用大脑中动脉阻塞(middle cerebral artery occulusion, MCAO)局灶性脑缺血再灌注模型,缺血前给予Scu25,50,75 mg·kg-1 7 d, ig。末次给药1 h后制备MCAO模型,缺血2 h,再灌注22 h后,分别用Longa’s法、TTC染色法来评价大鼠的神经功能状态及脑梗塞范围;用HE染色评价脑细胞损伤的程度。HE染色显示:模型组大鼠脑缺血再灌注后大鼠皮层和纹状体细胞排列紊乱,细胞间隙增宽,组织结构略显疏松,细胞形态异常,形状不规则,胞体缩小,胞质凝集,胞核固缩浓染,呈致密浓染块状或颗粒状,有的细胞界限不清,结构消失,并可见嗜神经现象;给予Scu50,75 mg·kg-1可明显减少MCAO后脑梗塞面积,改善神经功能症状,HE染色阳性染色细胞明显减少,与模型组比较,具有显著性差异(P<0.05)。第二部分灯盏花乙素(Scu)对大鼠脑缺血再灌注脑损伤所致神经细胞凋亡的影响大鼠在造模(MCAO)前1h给予7 d的Scu,ig。脑缺血2h再灌注22h后,用TUNEL法评价脑细胞凋亡的程度;用Western blot检测AIF蛋白在线粒体及细胞核中的表达水平,同时检测脑组织中NAD和PARP酶的活性。结果表明:模型组大鼠脑缺血再灌注后TUNEL阳性染色细胞明显增多(P<0.05,与伪手术组比较),脑组织中NAD水平明显降低,PARP酶过度活化,AIF在细胞核的表达明显增加。给予Scu(50,75 mg·kg-1)7 d后,TUNEL阳性染色细胞明显减少,凋亡程度减轻(P<0.05,与模型组比较);同时,Scu(50,75 mg·kg-1)能减少脑缺血再灌注损伤后大鼠脑组织中NAD的耗竭,抑制PARP酶的过度活化和AIF从线粒体向细胞核的易位效应。结论:Scu作为灯盏花的有效成分,具有抗脑缺血再灌注损伤的保护作用,其抗神经细胞凋亡的作用之一是通过抑制脑缺血损伤引起的PARP酶的过度活化及AIF的易位而实现的。更多还原

【Abstract】 Increasing evidence has shown that brain injury can develop as a result of cerebral ischemia-reperfusion due to stroke and other cardiovascular diseases. Cerebral ischemia/reperfusion(I/R) triggers a complex series of biochemical and molecular mechanisms that impairs neurological functions through breakdown of cellular integrity mediated by excitotoxic glutamatergic signaling, ionic imbalance, free-radical reactions.Scutellarin (Scu) is the major active principle (flavonoid) extracted from Erigeron breviscapus. Hand.-Mazz, a Chinese herbal medicine, which is a Ca2+ channels blocking agent used in the clinical therapy of cerebrovascular disorders. Studies have shown the protective effects of Scu on brain injury induced by cerebral ischemia /reperfusion through interaction with a wide variety of targets because of its anti-oxidation, anti-inflammatory and attenuating neuronal damage. The aim of this study is to investigate the effects of Scu on brain injury through the inhibition of AIF-mediated apoptosis induced by transient focal brain ischemia in rats.Part I The protective effects of Scu on cerebral injury induced by brain ischemia-reperfusionRats were pretreated with Scu 25, 50, 75 mg·kg-1 for 7 d and then subjected to cerebral ischemia/reperfusion injury induced by a middle cerebral artery occlusion (MCAO). After 2 h ischemia and 22 h reperfusion, the neurological outcome was evaluated by the Longa’s method; the infarct volume was assessed by TTC staining. Hematoxylin-Eosin (HE) staining was employed to determine the level of neuron damage. The results showed that Scu (50, 75mg·kg-1) significantly reduced the cerebral infarct volume and ameliorated the neurological deficit (P<0.05), and reduced the numbers of HE-positive staining cells after 22h reperfusion. These results showed that pretreatment with Scu could attenuate brain injury after cerebral ischemia/reperfusion.Part II The mechanism of Scu on apoptosis induced by transient focal cerebral ischemia in ratsRats were pretreated with Scu for 7 d and then subjected to brain ischemia/reperfusion injury induced by a middle cerebral artery occlusion (MCAO). After 2 h ischemia and 22 h reperfusion, in situ end-labeling of nuclear DNA fragmentation (TUNEL) were employed to determine the level of neuron apoptosis.NAD content and PARP activity in brain homogenate were determined. The expression of AIF in the mitochondria and in the nucleus were analyzed by western blot. The data showed that The increase numbers of HE-positive staining cells were significantly observed at 22 h after reperfusion, whiles the immunoreactivity was inhibited by Scu (50, 75 mg·kg-1) (P<0.05,vs vehicle-treated). At the meantime, Scu (50, 75 mg·kg-1)-treatment reverse brain NAD depletion, and inhibited PARP over activation and AIF translocation from the mitochondria to the nucleus following cerebral I/R.Conclusions:These findings suggested that the neuroprotective effects of Scu on brain ischemic injury-induced apoptosis were associated with inhibiting PARP-dependent mitochondrial dysfunction and subsequent translocation of AIF.更多还原