脑红蛋白在肢体缺血预处理脑保护中的作用及其机制
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摘要
缺血性脑病具有发病率、致残率和死亡率高的特点,严重危害人类的健康。且近年来其发病率呈不断上升的趋势,随着我国老龄化社会的到来,形势将更加严峻。调动机体内源性保护机制提高神经细胞对缺血性损害的抵抗力作为缺血性脑病防治的新策略,受到医学界的高度重视。Kitagawa等在脑缺血模型中发现单次或两次的预缺血,可对后续缺血的海马CA1区锥体神经元产生保护作用,被称为脑缺血耐受,提前给与的轻微脑缺血称为脑缺血预处理。这表明脑缺血预处理诱导的脑缺血耐受能减轻缺血/再灌注所致的损伤。近年来提出的远程器官缺血预处理拓宽了预处理的途径,同时也为应用非重要生命器官的短暂缺血预处理对抗重要生命器官的缺血/再灌注损伤研究提供了新思路。我室近年研究证实肢体缺血预处理(limb ischemic preconditioning,LIP)可减轻大鼠海马的缺血/再灌注损伤。脑缺血预处理和LIP均可减轻脑的缺血/再灌注损伤,但从临床应用考虑,LIP具有较好的可操作性和安全性,更易为患者所接受,但这种保护作用的机制尚不完全清楚。
2000年,德国学者Burmester等在Nature上首次报道,在人和小鼠脑内存在一种特异的携氧球蛋白—脑红蛋白(neuroglobin,Ngb)。随后发现在大鼠、沙鼠等的脑内也存在Ngb表达。研究表明,Ngb可保护神经元对抗缺血/缺氧损伤,抑制神经元凋亡。Ngb过表达的转基因小鼠局灶性脑缺血后,脑梗体积减小;而应用反义寡核苷酸(antisense oligodeoxynucleotides, AS-ODNs)抑制Ngb表达,脑缺血损伤加重。同时在细胞水平发现,应用Ngb小干扰RNA降低H2O2诱导的PC12细胞Ngb表达,加剧了细胞损伤。Ngb作为一种内源性保护蛋白,它的发现为缺血缺氧性脑病的研究提出了新思路。我室应用免疫组化技术初步研究发现LIP可上调大鼠海马Ngb表达,但LIP是否是通过上调Ngb表达来发挥抗脑缺血作用尚有待于进一步证实。
研究发现,线粒体不仅在能量转换中扮演重要角色,在整合死亡信号刺激、决定细胞命运的过程中也起枢纽作用。线粒体成为脑缺血性损伤的亚细胞靶目标,在脑缺血/再灌注损伤和缺血预处理内源性脑保护中的作用越来越受到关注。资料显示,Ngb对神经元的保护作用可能与线粒体有关,如Ngb表达增加可改善H2O2诱导的PC12细胞线粒体膜电位的降低。线粒体介导的凋亡通路参与脑缺血后的细胞凋亡,凋亡信号的整合主要是通过Bcl-2家族蛋白在线粒体膜上的相互作用来实现的。Bcl-2和Bax是Bcl-2家族中已被发现的与细胞凋亡关系密切的两种基因,是一对在功能上相互对立的细胞凋亡调控基因。然而,LIP诱导的脑缺血耐受过程中,Ngb是否是通过调控线粒体的结构和功能及线粒体介导的凋亡途径等机制来发挥脑保护作用尚不清楚。
因此,本实验应用大鼠四血管闭塞全脑缺血整体动物模型,观察Ngb在LIP诱导的脑缺血耐受中表达的变化、以及上调或阻断Ngb表达对LIP脑保护作用的影响,探讨Ngb在LIP抗脑缺血/再灌注损伤中的作用;在此基础上,从线粒体的结构和功能以及线粒体介导的凋亡途径等方面探讨Ngb在LIP诱导的脑缺血耐受中的线粒体机制;并进一步观察青年和老年大鼠脑缺血后海马CA1区Ngb表达的变化及LIP对其影响,以及LIP对正常大鼠海马CA1区Ngb表达的影响,为全面深入地评价脑缺血后海马Ngb表达的变化及LIP的保护作用提供实验依据,为临床上脑血管疾病的防治研究提供新线索和思路。
1 Ngb参与LIP诱导的大鼠脑缺血耐受
目的:观察Ngb在LIP诱导的脑缺血耐受中表达的变化、以及应用Ngb诱导剂Hemin和Ngb AS-ODNs分别上调和抑制Ngb表达对LIP脑保护作用的影响,确定Ngb在LIP诱导的大鼠脑缺血耐受中的作用。
1.1 LIP上调脑缺血大鼠海马CA1区Ngb表达
动物分组及方法:雄性Wistar大鼠70只,随机分为5组:
①脑缺血sham组(n=14):永久凝闭双侧椎动脉,2 d后游离双侧颈总动脉,但不夹闭。
②LIP组(n=14):夹闭双侧股动脉10 min,再灌10 min,反复3次。
③脑缺血组(n=14):永久凝闭双侧椎动脉,2 d后夹闭双侧颈总动脉8 min致全脑缺血。
④LIP sham+脑缺血组(n=14):游离双侧股动脉,但不夹闭,持续60min,之后行全脑缺血8min。
⑤LIP+脑缺血组(n=14):夹闭双侧股动脉10 min,再灌10 min,反复3次,其余同第③组。
每组14只动物,用于3个指标的观察。①海马CA1区Ngb mRNA表达(n=3):于脑缺血sham手术或LIP或脑缺血后3 h取材,行RT-PCR检测;②海马CA1区Ngb蛋白表达(n=5):于脑缺血sham手术或LIP或脑缺血后6 h取材,行Western blot检测;③海马CA1区锥体神经元迟发性死亡(delayed neuronal death,DND)(n=6):于脑缺血sham手术或LIP或脑缺血后7 d取材,行硫瑾染色,在光学显微镜下观察海马CA1区组织形态,确定神经元密度(neuronal density,ND)(计数海马CA1区每1 mm区段内细胞膜完整、胞核饱满、核仁清晰的锥体细胞数目,每张切片双侧海马各计数6个区段,取平均数为神经元密度)和组织学分级(histological grade,HG)(0级:无神经元死亡;Ⅰ级:散在的神经元死亡;Ⅱ级:成片的神经元死亡;Ⅲ级:几乎全部的神经元死亡)。
结果:RT-PCR和Western blot分析显示,脑缺血sham组海马CA1区Ngb mRNA和蛋白有一定的表达。与脑缺血sham组相比,LIP组Ngb mRNA和蛋白表达明显增加(P<0.05)。脑缺血组海马CA1区Ngb mRNA和蛋白表达较脑缺血sham组明显下降(P<0.05)。与脑缺血sham组和脑缺血组相比,LIP+脑缺血组Ngb mRNA和蛋白表达明显增加(P<0.05),而LIP sham+脑缺血组Ngb表达则无明显变化。
硫瑾染色结果显示, LIP组大鼠海马CA1区锥体神经元无明显损伤,神经元排列整齐,轮廓完整,核饱满而核仁清晰,其HG及ND与脑缺血sham组相比无差异。脑缺血组大鼠海马CA1区DND明显,与脑缺血sham组和LIP组相比,HG显著升高,ND显著下降(P<0.05)。在LIP+脑缺血组,上述损伤被明显抑制,表现为与脑缺血组相比,HG明显降低,ND明显升高(P<0.05);而LIP sham+脑缺血组海马CA1区组织学特征则与脑缺血组无明显差异。这些结果表明,LIP具有抗脑缺血引起的海马CA1区DND的作用。
上述结果表明,LIP可诱导脑缺血耐受,同时上调脑内Ngb的表达。
1.2 Hemin上调Ngb表达诱导脑缺血耐受
动物分组及方法:选用永久凝闭双侧椎动脉的雄性Wistar大鼠84只,
随机分为:①脑缺血sham组(n=14):永久凝闭双侧椎动脉,2 d后游离双侧颈总动脉,但不夹闭。
②脑缺血组(n=14):永久凝闭双侧椎动脉,2 d后夹闭双侧颈总动脉8min致全脑缺血。
③Hemin溶剂+脑缺血组(n=14):腹腔注射Hemin溶剂持续2 d,每天1次,其余同第②组。
④Hemin+脑缺血组(n=42):腹腔注射Hemin持续2 d,每天1次,其余同第②组;根据Hemin的剂量,将该组进一步分为10 mg/kg、20 mg/kg和40 mg/kg三个亚组。
每组14只动物,用于3个指标的观察。①海马CA1区Ngb mRNA表达(n=3):于脑缺血sham手术或脑缺血后3 h取材,行RT-PCR检测;②海马CA1区Ngb蛋白表达(n=5):于脑缺血sham手术或脑缺血后6h取材,行Western blot检测;③海马CA1区锥体神经元DND(n=6):于脑缺血sham手术或脑缺血后7d取材,行硫瑾染色,在光学显微镜下观察海马CA1区组织形态,确定ND和HG。
结果:RT-PCR和Western blot分析显示,与脑缺血sham组相比,脑缺血组和Hemin溶剂+脑缺血组海马CA1区Ngb mRNA和蛋白表达显著下降(P<0.05)。与脑缺血组和Hemin溶剂+脑缺血组相比,Hemin+脑缺血组Ngb mRNA和蛋白表达显著增加,尤以20 mg/kg和40 mg/kg组明显(P<0.05),并呈现剂量依赖性。
硫瑾染色结果显示,与脑缺血sham组相比,脑缺血组大鼠海马CA1区锥体神经元有明显DND,HG明显升高,而ND则明显下降(P<0.05)。与脑缺血组相比,Hemin溶剂+脑缺血组HG和ND无明显差异。在Hemin+脑缺血组,DND被明显抑制,表现为与脑缺血组和Hemin溶剂+脑缺血组相比,HG明显降低,ND明显升高,并与Hemin的剂量呈现依赖性(P<0.05)。
上述结果表明,Hemin可上调Ngb表达而诱导脑缺血耐受。
1.3 Ngb AS-ODNs抑制LIP抗脑缺血引起的海马CA1区DND的作用
动物分组及方法:选用永久凝闭双侧椎动脉的雄性Wistar大鼠55只,随机分为:
①ACSF+LIP+脑缺血组(n=11):夹闭双侧股动脉10 min,再灌流10 min,反复3次,然后夹闭双侧颈总动脉8 min致全脑缺血;LIP前右侧脑室注射ODNs的溶剂人工脑脊液(artificial cerebrospinal fluid,ACSF) 2次,每天1次,每次8μl。
②S-ODNs+LIP+脑缺血组(n=11):LIP前右侧脑室注射1.6 nmol Ngb S-ODNs溶液2次,每天1次,每次8μl,其余同第①组。
③AS-ODNs+LIP+脑缺血组(n=33):LIP前右侧脑室注射Ngb AS-ODNs溶液2次,每天1次,每次8μl,其余同第①组。根据AS-ODNs的用量,进一步分为0.4 nmol、0.8 nmol和1.6 nmol三个亚组。
每组11只动物,用于2个指标的观察。①海马CA1区Ngb蛋白表达(n=5):于脑缺血后6h取材,行Western blot检测;②海马CA1区锥体神经元DND(n=6):于脑缺血后7d取材,行硫瑾染色,在光学显微镜下观察海马CA1区组织形态,确定ND和HG。
结果:Western blot分析显示,与ACSF+LIP+脑缺血组相比, AS-ODNs+LIP +脑缺血组海马CA1区Ngb蛋白表达显著降低(P<0.05),并与AS-ODNs的剂量呈现依赖性,而LIP前侧脑室注射S-ODNs,对CA1区Ngb蛋白表达无明显影响。
硫瑾染色结果显示,ACSF+LIP+脑缺血组和S-ODNs+LIP+脑缺血组,大鼠海马CA1区未见明显锥体神经元损伤,锥体神经元排列较整齐,轮廓完整,核饱满而核仁清晰。而AS-ODNs+LIP+脑缺血组CA1区锥体神经元损伤明显,并与AS-ODNs的剂量呈现依赖性。AS-ODNs 0.4 nmol组HG变化不明显,但ND明显降低(P<0.05);AS-ODNs 0.8 nmol和1.6 nmol组,HG明显增高,ND明显降低(P<0.05),表明Ngb AS-ODNs通过抑制Ngb表达,降低了LIP的脑保护作用。
上述结果表明,Ngb AS-ODNs可阻断Ngb表达,进而阻断LIP的脑保护作用。
1.4 LIP和Hemin降低脑缺血引起的血清Ngb蛋白含量增加动物分组及方法:雄性Wistar大鼠30只,随机分为6组:
①脑缺血sham组(n=5):永久凝闭双侧椎动脉,2 d后游离双侧颈总动脉,但不夹闭。
②LIP组(n=5):夹闭双侧股动脉10 min,再灌10 min,反复3次。
③脑缺血组(n=5):永久凝闭双侧椎动脉,2 d后夹闭双侧颈总动脉8 min致全脑缺血。
④LIP+脑缺血组(n=5):夹闭双侧股动脉10 min,再灌10 min,反复3次,其余同第③组。
⑤Hemin+脑缺血组(n=5):腹腔注射Hemin 20 mg/kg/d,持续2 d,其余同第③组。
⑥Hemin溶剂+脑缺血组(n=5):腹腔注射Hemin溶剂持续2 d,其余同第③组。
每组5只动物,用于检测血清Ngb蛋白含量:于脑缺血sham手术或LIP或脑缺血后6 h取血,行ELISA检测。
结果:ELISA显示,脑缺血sham组和LIP组血清Ngb蛋白含量较低,二者之间无统计学差异。与脑缺血sham组相比,脑缺血组血清Ngb蛋白含量显著增加(P<0.05)。而脑缺血前给予LIP预处理,血清Ngb蛋白含量明显下降(P<0.05)。与脑缺血组相比,Hemin(20 mg/kg)+脑缺血组血清Ngb蛋白含量明显下降(P<0.05);而Hemin溶剂+脑缺血组血清Ngb蛋白含量无明显变化。
以上结果表明,LIP和Hemin可明显降低脑缺血后血清Ngb蛋白含量升高。
2 Ngb通过线粒体机制参与LIP诱导的脑缺血耐受
目的:通过观察Ngb表达增加对海马CA1区神经细胞线粒体膜电位、线粒体Na+-K+-ATP酶活性、神经元及线粒体超微结构、Bcl-2和Bax mRNA表达的影响,探讨Ngb是否通过线粒体机制参与LIP诱导的脑缺血耐受。
动物分组及方法:选用永久凝闭双侧椎动脉的雄性Wistar大鼠126只,随机分为:
①脑缺血sham组(n=18):永久凝闭双侧椎动脉,2 d后游离双侧颈总动脉,但不夹闭。
②脑缺血组(n=18):永久凝闭双侧椎动脉,2 d后夹闭双侧颈总动脉8 min致全脑缺血。
③LIP+脑缺血组(n=18):夹闭双侧股动脉10 min,再灌10 min,反复3次,其余同第②组。
④AS-ODNs+LIP+脑缺血组(n=18):LIP前右侧脑室注射0.8 nmol Ngb AS-ODNs溶液2次,每天1次,每次8μl,其余同第③组。
⑤S-ODNs+LIP+脑缺血组(n=18):LIP前右侧脑室注射0.8 nmol Ngb S-ODNs溶液2次,每天1次,每次8μl,其余同第③组。
⑥Hemin+脑缺血组(n=18):脑缺血前腹腔注射20mg/kg Hemin 2次,每天1次,其余同第②组。
⑦Hemin溶剂+脑缺血组(n=18):脑缺血前腹腔注射Hemin溶剂2次,每天1次,其余同第②组。
每组18只动物,用于4个指标的观察。①海马CA1区神经细胞线粒体膜电位(n=3):于脑缺血sham手术或脑缺血后24 h取材,行流式细胞检测;②海马CA1区线粒体Na+-K+-ATP酶活性(n=10):于脑缺血sham手术或脑缺血后6 h和24 h取材、检测;③海马CA1区Bcl-2和Bax mRNA表达(n=3):于脑缺血sham手术或脑缺血后24 h取材,行RT-PCR检测;④海马CA1区神经元及线粒体超微结构(n=2):于脑缺血sham手术或脑缺血后3 d取材,行透射电镜观察。
结果:流式细胞仪检测神经细胞线粒体膜电位显示,脑缺血sham组海马CA1区神经细胞线粒体膜电位较高。与脑缺血sham组相比,脑缺血后线粒体膜电位显著降低(P<0.05)。与脑缺血组相比,LIP+脑缺血组线粒体膜电位明显增加(P<0.05)。与LIP+脑缺血组相比,AS-ODNs+LIP+脑缺血组线粒体膜电位显著降低,而S-ODNs+LIP+脑缺血组则无明显变化。与脑缺血组相比,Hemin+脑缺血组线粒体膜电位明显增高(P<0.05),而Hemin溶剂+脑缺血组则无明显变化。
海马CA1区线粒体Na+-K+-ATP酶活性测定显示,在6 h时间点,脑缺血sham组该酶活性较高,而脑缺血后则较脑缺血sham组明显下降(P<0.05)。与脑缺血组相比,LIP+脑缺血组该酶活性明显下降(P<0.05)。与LIP+脑缺血组相比,AS-ODNs+LIP+脑缺血组该酶活性显著增加(P<0.05),而LIP前侧脑室注射Ngb S-ODNs,对该酶活性无影响。与脑缺血组相比,Hemin+脑缺血组该酶活性明显降低(P<0.05),而Hemin溶剂+脑缺血组则无明显变化。在24 h时间点,脑缺血sham组线粒体Na+-K+-ATP酶活性较高。与脑缺血sham组相比,脑缺血后该酶活性显著下降(P<0.05)。与脑缺血组相比,LIP+脑缺血组该酶活性明显增加(P<0.05)。与LIP+脑缺血组相比,AS-ODNs +LIP+脑缺血组该酶活性显著降低(P<0.05),而S-ODNs +LIP+脑缺血组则无明显变化。与脑缺血组相比,Hemin+脑缺血组该酶活性明显增加(P<0.05),而Hemin溶剂+脑缺血组则无明显变化。
RT-PCR结果显示,脑缺血sham组海马CA1区Bcl-2和Bax mRNA表达较低。与脑缺血sham组相比,脑缺血组Bcl-2和Bax mRNA表达均显著增加,而Bax增加更为明显(P<0.05)。与脑缺血组相比,LIP+脑缺血组Bcl-2 mRNA表达明显增加(P<0.05),而Bax mRNA表达明显下降(P<0.05)。与LIP+脑缺血组相比,AS-ODNs+LIP+脑缺血组Bcl-2 mRNA表达明显下降(P<0.05),Bax mNRA表达增加(P<0.05);而在LIP预处理前侧脑室注射Ngb S-ODNs干预,Bcl-2和Bax mRNA表达无明显变化。与脑缺血组相比,Hemin+脑缺血组Bcl-2 mRNA表达明显增加(P<0.05),而Bax mRNA表达下降(P<0.05);而Hemin溶剂+脑缺血组Bcl-2和Bax mRNA表达无明显变化。
透射电镜结果显示,①脑缺血sham组:海马CA1区神经元形态完整,结构清晰,核膜完整,核形态正常,胞质中细胞器丰富。胞质内有丰富的线粒体,呈圆形或椭圆形,嵴较多而清晰可见。②脑缺血组:海马神经元严重水肿变形。细胞核不规则,核膜皱缩,胞质高度水肿,细胞器数量明显减少。线粒体呈气球样肿胀,大量空泡化,嵴断裂,溶解,消失,同时有的线粒体致密化。③LIP+脑缺血组:与脑缺血组相比,LIP+脑缺血组神经元和线粒体损伤明显减轻。表现为神经元水肿明显减轻,核膜尚完整,核形态正常,染色质均匀分布,细胞器尚丰富。胞质内有丰富的线粒体,大多数线粒体嵴完整,排列紧密规律,基质颗粒极少量脱落,少量线粒体水肿,空泡化。④AS-ODNs+LIP+脑缺血组:与LIP+脑缺血组相比,AS-ODNs+LIP+脑缺血组神经元和线粒体损伤明显加重,但较脑缺血组损伤稍轻些。神经元明显水肿,细胞器数量明显减少。线粒体水肿、变形,部分膜溶解,同时有的线粒体致密化。⑤S-ODNs+LIP+脑缺血组:本组与LIP+脑缺血组比较,无显著差别。⑥Hemin+脑缺血组:与脑缺血组相比,Hemin+脑缺血组神经元和线粒体的损伤明显改善。神经元水肿明显减轻,核膜尚完整,核形态正常,细胞器尚丰富。胞质内有丰富的线粒体,多数线粒体嵴完整,排列紧密规律,基质颗粒极少量脱落,少量线粒体轻度水肿,或致密化。⑦Hemin溶剂+脑缺血组:本组与脑缺血组比较,无显著差别。
以上结果表明,Ngb表达增加可通过调控海马CA1区神经细胞线粒体膜电位、Na+-K+-ATP酶活性、线粒体超微结构、Bcl-2和Bax mRNA表达等线粒体机制参与LIP诱导的脑缺血耐受。
3青年和老年大鼠脑缺血后海马CA1区Ngb表达的变化及LIP对其影响
目的:观察比较青年和老年大鼠脑缺血时海马CA1区Ngb表达的变化及LIP对其影响,为进一步评价脑缺血后海马Ngb表达的变化及LIP的保护作用提供实验依据。
动物分组及方法:选用3月龄健康雄性Spague-Dawley大鼠30只,21~23月龄健康雄性Spague-Dawley大鼠30只。将凝闭双侧椎动脉2 d的青年和老年大鼠均随机分为脑缺血组和LIP+脑缺血组。脑缺血组:夹闭双侧颈总动脉8 min;LIP+脑缺血组:夹闭双侧股动脉10 min,再灌流10 min,反复3次,随后夹闭双侧颈总动脉8 min。
每组15只动物,用于3个指标的观察。①海马CA1区Ngb mRNA表达(n=3):于脑缺血后3 h取材,行RT-PCR检测;②海马CA1区Ngb蛋白表达(n=6):于脑缺血后6h取材,行Western blot检测;③海马CA1区锥体神经元DND(n=6):于脑缺血后7d取材,行硫瑾染色,在光学显微镜下观察海马CA1区组织形态,确定ND和HG。
结果:RT-PCR分析显示,Ngb和GAPDH扩增后的产物片段与设计相符。老年脑缺血组Ngb mRNA表达比青年脑缺血组明显降低(P<0.05)。LIP可明显上调青年和老年大鼠脑缺血后Ngb的mRNA表达(P<0.05),但老年大鼠(LIP+老年脑缺血组)的上调幅度小于青年大鼠(LIP +青年脑缺血组)(P<0.05)。
Western blot结果显示,各组均可在分子量相当于17KD和42KD的区域见Ngb和β-actin蛋白表达条带。Ngb蛋白表达在老年大鼠脑缺血组比青年大鼠脑缺血组显著降低(P<0.05),LIP后青年和老年脑缺血大鼠Ngb的蛋白表达均明显上调(P<0.05),但老年大鼠(LIP+老年脑缺血组)的上调幅度小于青年大鼠(LIP +青年脑缺血组)(P<0.05)。
硫瑾染色显示,青年脑缺血组有明显的组织损伤,表现为海马CA1区锥体细胞稀疏,排列紊乱,多数细胞胞核固缩,核仁欠清晰,尼氏小体减少或消失。老年脑缺血组海马CA1区锥体细胞几乎全部固缩或缺失,与青年脑缺血组相比ND显著下降(P<0.05),表明脑缺血后,老年大鼠的损伤比青年大鼠更严重。LIP+青年脑缺血组CA1区锥体细胞排列整齐致密,胞核饱满,核仁较清晰,仅个别锥体细胞胞核固缩或缺失,和青年脑缺血组相比,HG显著降低(P<0.05),ND明显升高(P<0.05)。LIP+老年脑缺血组与老年脑缺血相比,组织损伤明显减轻,部分锥体细胞固缩或缺失,HG显著降低(P<0.05),ND明显升高(P<0.05);但与LIP+青年脑缺血组比较,CA1区锥体细胞固缩或缺失仍较多,ND明显降低(P<0.05),表明LIP可减轻脑缺血损伤,但老龄降低了LIP的保护作用。
以上结果表明,老年大鼠全脑缺血后Ngb的表达及LIP对其上调作用较青年大鼠明显减弱,这可能是老年大鼠脑缺血后损伤较重和LIP对老年大鼠脑缺血保护作用较弱的原因之一。
4 LIP上调正常大鼠海马CA1区Ngb表达
目的:观察LIP后不同时间点正常大鼠海马CA1区Ngb表达的变化,为研究和评价LIP对正常机体的脑保护作用提供实验依据。动物分组及方法:雄性Wistar大鼠40只随机分为:
①sham组(n=8):游离双侧股动脉,但不夹闭。
②LIP组(n=32):夹闭双侧股动脉10 min,再灌10 min,反复3次。
以上动物于sham手术后30 min或LIP后30 min、1 h、3 h和6 h取材, RT-PCR检测海马CA1区Ngb mRNA表达(n=3),于sham手术后1 h或LIP后1 h、6 h、12 h和1 d取材,Western blot分析检测Ngb蛋白表达(n=5)。
结果:RT-PCR分析显示,在150bp和349bp处分别为Ngb和内参GAPDH的扩增条带。Sham组海马CA1区有一定的Ngb mRNA表达。与sham组相比,LIP后海马CA1区Ngb mRNA表达增加。这种增加于LIP后30 min最明显(P<0.05),随后逐渐下降,在6 h时接近sham组水平。
Western blot结果显示,各组均可在分子量相当于17KD和42KD的区域分别出现Ngb和β-actin蛋白表达条带。Sham组海马CA1区有一定的Ngb蛋白表达。与sham组相比,LIP后海马CA1区Ngb蛋白表达增加。这种增加于LIP后1 h达到高峰(P<0.05),随后逐渐下降,至1 d时接近sham组。
以上结果表明,LIP能上调正常大鼠海马CA1区Ngb mRNA和蛋白表达,Ngb mRNA表达高峰早于蛋白表达高峰。
5结论
⑴LIP能明显上调脑缺血大鼠海马CA1区Ngb mRNA和蛋白表达,同时LIP可明显抑制脑缺血后CA1区锥体神经元DND,降低血清Ngb蛋白含量;腹腔注射Ngb诱导剂Hemin能模拟LIP诱导的脑缺血耐受,而侧脑室注射Ngb AS-ODNs可部分阻断LIP的脑保护作用。这些结果表明,Ngb表达上调在LIP诱导脑缺血耐受中发挥重要作用。
⑵Ngb表达增加通过增加线粒体膜电位、改善线粒体Na+-K+-ATP酶活性、保护线粒体超微结构、调节Bcl-2和Bax mRNA表达等线粒体机制参与LIP诱导的脑缺血耐受。
⑶老年大鼠全脑缺血后Ngb的表达及LIP对其上调作用较青年大鼠明显减弱,这可能是老年大鼠脑缺血后损伤较重和LIP对老年大鼠脑缺血保护作用较弱的原因之一。
⑷LIP可上调正常大鼠海马CA1区Ngb表达。
Ischemic neuropathy is a leading cause of morbidity, mortality and disability rate. Activation of endogenous brain protection is a novel approach to prevent brain ischemia/reperfusion injury, and which has attracted extensive attention. Ischemic preconditioning is a phenomenon that brief episode of sub-lethal ischemia protects organs or tissues against a subsequent lethal ischemic insult. Brain ischemic preconditioning was first demonstrated by Kitagawa and colleagues. They showed in the gerbil that short periods of global cerebral ischemia can protect the brain against subsequent prolonged ischemia. Recent studies reported that a short sub-lethal ischemia and reperfusion in various organs can induce ischemic tolerance in another organ as well. This phenomenon is known as remote ischemic preconditioning. The finding opened up vast vista of the study to increase the resistance of important vital organs such as brain to ischemia with transient ischemic preconditioning in non-vital organs. Our recent study demonstrated that limb ischemic preconditioning (LIP) could attenuate delayed neuronal death (DND) or apoptosis of pyramidal neurons in the CA1 hippocampus induced normally by serious brain ischemia/reperfusion. However, mechanisms underlying the neuroprotection of LIP have not been well understood.
Neuroglobin (Ngb) first described in Nature by Burmester at 2000 is a globin with high affinity for oxygen and is widely expressed in neurons of central and peripheral nervous systems in mouse and human. Since then, Ngb have also been recognized in rat and gerbil, suggesting that Ngb is present in a broad range of vertebrate species. Recent studies suggested that Ngb plays an important role in the protection of brain neurons from ischemic and hypoxic injuries, and apoptosis. For example, Ngb overexpression can protect neurons against hypoxic/ischemic insults, and knocking down Ngb expression increases hypoxic neuronal injury in vitro and ischemic cerebral injury in vivo. Ngb acts as an endogenous neuroprotectant, which has provided a new strategy to the study of brain hypoxic/ischemic insults. Our preliminary study using immunohistochemistry staining showed that LIP up-regulated the Ngb expression in the rat CA1 hippocampus, but whether Ngb participates in the neuroprotection of LIP in the CA1 hippocampus against severe brain ischemia/reperfusion is still unclear and need to be clarified.
Mitochondria, besides playing a key role in energy metabolism within the cell, are involved in a cohort of other processes like integration of dead signal and determination of cell fate. Mitochondrial-mediated apoptosis pathway is considered to be important to neuronal death after cerebral blood flow arrest. Recent investigations have shown that mitochondrial-centered mechanisms are important mediators in promoting development of the preconditioning response. In the brain, ischemic preconditioning diminishes mitochondrial dysfunction induced by ischemia and confers neuroprotection. Targeting mitochondria may be a useful strategy to reduce ischemic brain injury. Bcl-2 gene families are identified as apoptosis regulating genes. Inhibiting effect of anti-apoptotic proteins Bcl-2 on apoptosis is mediated by its binding to pro-apoptotic proteins, e.g., Bax, inhibiting their oligomerization, and thus inhibiting mitochondrial outer membrane pore formation, through which other pro-apoptotic proteins, e.g., cytochrome c, are released to the cytosol. Whether mitochondrial mechanisms are involved in neuroprotection of Ngb induced by LIP remain unclear.
Therefore, the present study was primarily undertaken to investigate the role of Ngb in the neuroprotection of LIP against global brain ischemia by observing changes in expression of Ngb in the CA1 hippocampus after LIP in rat global brain ischemic model, influences of up- or down-regulating Ngb expression on the neuroprotection of LIP, and the involvements of mitochondria in the process. The present study also compared Ngb expression in the CA1 hippocampus after cerebral ischemia and the effect of LIP on it between young and aged rats, and in normal rats. The results obtained provided new and more direct evidence for illustrating the role of Ngb in the neuronal protection of LIP and new clues for the study in prevention and therapy of ischemic neuropathies.
1 Ngb participates in the neuroprotection of LIP in the CA1 hippocampus against severe brain ischemia/reperfusion
Using four-vessel occluding global brain ischemia model, the role of Ngb in the neuroprotection of LIP against severe ischemic insult was investigated by observing the influence of LIP on the expression of Ngb in the rat CA1 hippocampus, and the influence of Ngb inducer Hemin and Ngb (antisense oligodeoxynucleotides, AS-ODNs) on the neuroprotection of LIP.
1.1 LIP up-regulates Ngb expression in rat CA1 hippocampus and induces brain ischemic tolerance
Methods: Seventy male Wistar rats with permanently occluded bilateral vertebral arteries were randomly assigned to 5 groups:
①Sham of cerebral ischemia group: The rats were subjected to permanently occluding the bilateral vertebral arteries, and 2 d later the bilateral common carotid arteries were exposed, but without blocking the blood flow.
②LIP group: The bilateral femoral arteries of the rats were occluded for 10 min, 3 times, at 10-min intervals.
③Cerebral ischemia group: The bilateral vertebral arteries of the rats were permanently occluded, 2 d later the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia.
④Sham of LIP+cerebral ischemia group: The bilateral femoral arteries were exposed for 60 min but not clipped, and then global brain ischemia was induced as those in cerebral ischemia group.
⑤LIP+cerebral ischemia group: Global brain ischemic insult for 8 min was induced as those in cerebral ischemia group immediately after LIP as in LIP group.
Fourteen rats were included in each group, and assigned to 3 observations:①Ngb mRNA (n=3) (RT-PCR), and②Ngb protein (n=5) expression (Western blot), and③Pyramidal neurons DND in the CA1 hippocampus of the rats (n=6), in which the rats were, respectively, sampled for the observations at time point of 3 h, 6 h and 7 d after the sham operations of cerebral ischemia, or LIP, or cerebral ischemia in corresponding group. The profile of pyramidal neurons DND in the CA1 hippocampus was evaluated under light microscope by determining the neuronal density (ND) and histological grade (HG). The ND was determined by counting the number of surviving pyramidal neurons with intact cell membrane, full nucleus and clear nucleolus within 1 mm linear length of the CA1 hippocampus. The HG was divided into 4 grades according to the following standard: grade 0, no neuron death; gradeⅠ, scattered single neuron death; gradeⅡ, death of many neurons; gradeⅢ, death of almost complete neurons.
Results: RT-PCR and Western blot analysis showed that basal expression of Ngb mRNA and protein in the CA1 hippocampus could be observed in sham group of cerebral ischemia. Compared to sham group of cerebral ischemia, the expressions of Ngb mRNA and protein were obviously up-regulated after LIP (P<0.05), while significantly down-regulated after cerebral ischemia (P<0.05). LIP significantly up-regulated the expressions of Ngb mRNA and protein in LIP+cerebral ischemia group compared with cerebral ischemia group (P<0.05), while sham operation of LIP before the cerebral ischemia was without effect on the down-regulation of Ngb mRNA and protein expressions induced by cerebral ischemia.
Thionine staining showed that pyramidal neurons in the CA1 hippocampus in sham group of cerebral ischemia and LIP group were normal and lined up in order, and no DND was found. There were obvious DND of pyramidal neurons in cerebral ischemia group, and HD increased and ND decreased significantly compared with sham group of cerebral ischemia and LIP group (P<0.05). In LIP+cerebral ischemia group, the DND normally induced by cerebral ischemia was effectively prevented, which represented with increase in ND and decrease in HG compared with cerebral ischemia group (P<0.05), while, the sham operation of LIP (in sham of LIP+cerebral ischemia group) had no effect on the DND normally induced by cerebral ischemia.
The above results indicated that LIP could up-regulate the expression of Ngb, and induce brain ischemic tolerance in the rat CA1 hippocampus.
1.2 Hemin up-regulates Ngb expression in the rat CA1 hippocampus and induces brain ischemic tolerance
Methods: Eighty and four male Wistar rats with permanently occluded bilateral vertebral arteries were randomly divided into following groups:
①Sham of cerebral ischemia group: The rats were subjected to permanently occluding the bilateral vertebral arteries, and 2 d later the bilateral common carotid arteries were exposed, but without blocking the blood flow.
②Cerebral ischemia group: The bilateral vertebral arteries of the rats were permanently occluded, 2 d later the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia.
③Hemin vehicle+cerebral ischemia group: The rats were injected i.p. with Hemin vehicle once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia as those in cerebral ischemia group.
④Hemin+cerebral ischemia group: The rats were injected i.p. with Hemin once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia as those in cerebral ischemia group. According to doses of Hemin used, this group was divided into 3 subgroups: 10 mg/kg/d, 20 mg/kg/d and 40 mg/kg/d groups.
Fourteen rats were included in each group, and assigned to 3 observations:①Ngb mRNA (n=3) (RT-PCR), and②Ngb protein (n=5) expression (Western blotting analysis), and③Pyramidal neurons DND in the CA1 hippocampus of the rats (n=6)(thionine staining), in which the rats were, respectively, sampled for the observations at time point of 3 h, 6 h and 7 d after the sham operations of cerebral ischemia or cerebral ischemia in corresponding group.
Results: RT-PCR and Western blot analysis showed that compared to sham group of cerebral ischemia, the expressions of Ngb mRNA and protein were obviously down-regulated after cerebral ischemia (P<0.05). The administration of Hemin dose-dependently up-regulated the expressions of Ngb mRNA and protein in Hemin+cerebral ischemia group compared with cerebral ischemia group (P<0.05), while pretreatment with vehicle of Hemin had no influence on the Ngb mRNA and protein expressions.
Thionine staining showed that the outline of the pyramidal neurons was kept intact, and no neuronal loss was found in the CA1 hippocampus in sham group of cerebral ischemia. Obvious destruction of the CA1 hippocampus was found in cerebral ischemia group, the value of ND was decreased, and HG was increased compared with that in sham group of cerebral ischemia (P<0.05). Intraperitoneal injection with Hemin before cerebral ischemia showed a significant protective effect against the DND induced by cerebral ischemia, which was represented with dose-dependent increase in ND and decrease in HG after administration of Hemin, especially in large doses (P<0.05). Whereas, pretreated with Hemin vehicle before the cerebral ischemia had no significant effect on the DND normally induced by cerebral ischemia.
The above results indicated that Hemin could up-regulate the Ngb expression and induce brain ischemic tolerance.
1.3 Ngb AS-ODNs inhibit brain ischemic tolerance induced by LIP
Methods: Fifty and five male Wistar rats with permanently occluded bilateral vertebral arteries were randomly divided into following groups:
①ACSF+LIP+cerebral ischemia group: ACSF (artificial cerebrospinal fluid) was intraventricularly injected 30 min prior to LIP. LIP was preformed by occlusion of bilateral femoral arteries of the rats for 10 min 3 times in an interval of 10 min, and then the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia immediately after the LIP.
②S-ODNs+LIP+cerebral ischemia group: Ngb (sense oligodeoxynucleotides, S-ODNs) in a dose of 1.6 nmol was administered 30 min prior to LIP by intraventricular injection. Others were the same as ACSF+LIP+cerebral ischemia group.
③AS-ODNs+LIP+cerebral ischemia group: Ngb AS-ODNs was administered 30 min prior to LIP by intraventricular injection. Others were the same as ACSF+LIP+cerebral ischemia group. According to doses of Ngb AS-ODNs used, this group was divided into 3 subgroups: 0.4 nmol, 0.8 nmol, and 1.6 nmol groups.
Eleven rats were included in each group, and assigned to 2 observations:①Ngb protein expression (n=5()Western blotting analysis), and②Pyramidal neurons DND in the CA1 hippocampus of the rats (n=6)(thionine staining), in which the rats were, respectively, sampled for the observation at time point of 6 h and 7 d after the cerebral ischemia in corresponding group.
Results: Western blot analysis showed that compared with ACSF+LIP +cerebral ischemia group, the expression of Ngb protein in AS-ODNs+LIP+ cerebral ischemia group significantly down-regulated in a dose-dependent manner (P<0.05). While there was no influence on the Ngb protein expression induced by LIP after administration Ngb S-ODNs.
Thionine staining showed that pyramidal neurons in the CA1 hippocampus arranged in order, and the structure of most them was clear in ACSF+LIP+cerebral ischemia and S-ODNs+LIP+cerebral ischemia groups. Administration of Ngb AS-ODNs dose-dependently induced obvious DND of pyramidal neurons in the CA1 hippocampus, which was represented with the decrease in the value of ND, and increase in HG compared with ACSF+LIP+ cerebral ischemia group (P<0.05). The administration of Ngb S-ODNs had no effect on the histological characteristic of the CA1 hippocampus.
The above results indicated that Ngb AS-ODNs partly inhibited brain ischemic tolerance induced by LIP via inhibiting the expression of Ngb protein.
1.4 LIP and Hemin reduce the increase of the serum Ngb protein level induce by cerebral ischemia
Methods: Thirty male Wistar rats with permanently occluded bilateral vertebral arteries were randomly assigned to 6 groups:
①Sham of cerebral ischemia group: The rats were subjected to permanently occluding the bilateral vertebral arteries, and 2 d later the bilateral common carotid arteries were exposed, but without blocking the blood flow.
②LIP group: The bilateral femoral arteries of the rats were occluded for 10 min, 3 times, at 10-min intervals.
③Cerebral ischemia group: The bilateral vertebral arteries of the rats were permanently occluded, 2 d later the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia.
④LIP+cerebral ischemia group: LIP was preformed as those in LIP group, and then cerebral ischemia insult for 8 min was produced as those in cerebral ischemia group immediately after the LIP.
⑤Hemin+cerebral ischemia group: The rats were injected i.p. with Hemin 20 mg/kg once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia as those in cerebral ischemia group.
⑥Hemin vehicle+cerebral ischemia group: The rats were injected i.p. with Hemin vehicle once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia as those in cerebral ischemia group.
Five rats were included in each group, and the serum Ngb protein level was observed by ELISA at 6 h after the sham operation of cerebral ischemia, or LIP, or cerebral ischemia.
Results: The level of serum Ngb protein in sham group of cerebral ischemia and LIP group was low, and no significant differences between the two groups. Compared with the two groups, the level of serum Ngb was significantly increased in cerebral ischemia group (P<0.05). The increase in the level of serum Ngb after brain ischemia was significantly prevented by LIP or administration of Hemin prior to cerebral ischemia (P<0.05), while vehicle of Hemin had no evident effect on the level of serum Ngb protein.
The above results indicated that LIP and Hemin could significantly prevent the increase of the serum Ngb protein level induced by cerebral ischemia.
2 Ngb particapates in LIP-induced brain ischemic tolerance via mechanisms involving in mitochondria
To investigated whether Ngb particapates in LIP-induced brain ischemic tolerance via mitochondria mechanisms, the study observed the effect of up-regulated Ngb expression in the rat CA1 hippocampus on the mitochondria trans-membrane potential, Na+-K+-ATPase activity of mitochondria, Bcl-2 and Bax mRNA expression, neurons and mitochondrial ultrastructure in rat CA1 hippocampus.
Methods: One hundred and twenty-six male Wistar rats with permanently occluded bilateral vertebral arteries were randomly divided into 7 groups:
①Sham of cerebral ischemia group: The rats were subjected to permanently occluding the bilateral vertebral arteries, and 2 d later the bilateral common carotid arteries were exposed, but without blocking the blood flow.
②Cerebral ischemia group: The bilateral vertebral arteries of the rats were permanently occluded, 2 d later the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia.
③LIP+cerebral ischemia group: LIP was preformed by occlusion of bilateral femoral arteries of rats for 10 min 3 times in an interval of 10 min, then cerebral ischemia insult for 8 min was produced as those in cerebral ischemia group immediately after the last occlusion of the femoral arteries.
④AS-ODNs+LIP+cerebral ischemia group: Ngb AS-ODNs in a dose of 0.8 nmol was administered 30 min prior to LIP by intraventricular injection. Other procedures were the same as those in LIP+cerebral ischemia group.
⑤S-ODNs+LIP+cerebral ischemia group: Ngb S-ODNs in a dose of 0.8 nmol was administered 30 min prior to LIP by intraventricular injection. Other procedures were the same as those in LIP + cerebral ischemia group.
⑥Hemin+cerebral ischemia group: The rats were injected i.p. with Hemin 20 mg/kg once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia. Other procedures were the same as those in cerebral ischemia group.
⑦Hemin vehicle+cerebral ischemia group: The rats were injected i.p. with Hemin vehicle once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia. Other procedures were the same as those in cerebral ischemia group.
Eighteen rats were included in each group, and assigned to 4 observations:①mitochondria trans-membrane potential at 24 h (n=3),②Na+-K+-ATPase activity of mitochondria at 6 h and 24 h (n=10),③Bcl-2 and Bax mRNA expression at 24 h (n=3), and④neuronal and mitochondrial ultrastructure of the rats CA1 hippocampus on 3 d (n=2), in which the rats were, respectively, sampled at the corresponding time point after the sham operations of cerebral ischemia, or cerebral ischemia in each group.
Results: Flow cytometry analysis showed that mitochondrial trans- membrane potential significantly decreased in cerebral ischemia group compared with that of sham group of cerebral ischemia (P<0.05). The decrease was significantly ameliorated by LIP prior to cerebral ischemia insult (P<0.05). However, Pretreatment with Ngb AS-ODNs before LIP significantly inhibited the ameliorating effect of LIP on mitochondrial trans-membrane potential, which was represented with the decrease in the potential in AS-ODNs+LIP+cerebral ischemia group compared with that in LIP + cerebral ischemia group (P<0.05). In contrast, pretreatment with Ngb S-ODNs before LIP was without effect on the potential. Hemin significantly increased mitochondrial trans-membrane potential compared with that in cerebral ischemia group (P<0.05), whereas, Hemin vehicle administrated had no evident effect on it.
Results of Na+-K+-ATPase activity at 6 h time point showed that Na+-K+-ATPase activity was high in sham group of cerebral ischemia, but significantly decreased after cerebral ischemia (P<0.05). Compared with cerebral ischemia group, the enzyme activity was further decreased in LIP + cerebral ischemia group (P<0.05). Pretreated with Ngb AS-ODNs before LIP significantly increased the enzyme activity when compared to LIP + cerebral ischemia group (P<0.05). However, administration with Ngb S-ODNs before LIP was without effect upon the enzyme activity. Compared with cerebral ischemia group, Hemin significantly decreased the enzyme activity (P<0.05), and no difference of the enzyme activity was found at Hemin vehicle group. Results of Na+-K+-ATPase activity at 24 h time point showed that Na+-K+-ATPase activity was high in sham group of cerebral ischemia, which significantly decreased in cerebral ischemia group (P<0.05). The enzyme activity was significantly increased by LIP, when compared to cerebral ischemia group (P<0.05). Compared to LIP + cerebral ischemia group, the application of Ngb AS-ODNs before LIP significantly decreased the enzyme activity (P<0.05), but Ngb S-ODNs had no influence. When the animals were pretreated with the Hemin before the lethal ischemic insult, the enzyme activity was significantly increased compared with that in cerebral ischemia group (P<0.05), and no evident effect was observed in Hemin vehicle + cerebral ischemia group.
RT-PCR analysis showed that Bcl-2 and Bax mRNA are less in the rat CA1 hippocampus in the sham group of cerebral ischemia, which was significantly up-regulated after cerebral ischemia (P<0.05). Pretreated with LIP before cerebral ischemia dramatically up-regulated the expression of Bcl-2 mRNA, but down-regulated the expression of Bax mRNA (P<0.05). Administration of Ngb AS-ODNs before LIP+cerebral ischemia significantly inhibited Bcl-2 mRNA expression and increased Bax mRNA expression (P<0.05). However, Ngb S-ODNs had no effect on the Bcl-2 and Bax mRNA expression. Ngb inducer Hemin could also significantly up-regulate the expression of Bcl-2 mRNA, and down-regulate the expression of Bax mRNA (P<0.05). While, pretreated with vehicle of Hemin before cerebral ischemia had no effect on the Bcl-2 and Bax mRNA expression.
Transmission electron microscopic examination showed that the structure of neurons and mitochondria in sham group of cerebral ischemia was normal. Cerebral ischemia for 8 min induced obvious destruction of the neuronal and mitochondrial ultrastructure. The neuron was swollen and cellular organs were significantly decreased; meanwhile, the mitochondria were swollen or pyknosis and its cristae were broken, dissolved and disappeared. But in LIP + cerebral ischemia group, the above injury changes were prevented clearly. When the animals were pretreated with Ngb AS-ODNs prior to LIP + cerebral ischemia, the protective effect of LIP was prevented, while, Ngb S-ODNs had no effect on them. Ngb inducer Hemin significantly inhibited destruction of the neuronal and mitochondrial ultrastructure induced by cerebral ischemia, but Hemin vehicle had no effect on the cerebral ischemia induced destruction of the neuronal and mitochondrial ultrastructure.
The above results indicated that the up-regulation of Ngb expression participated in the brain ischemic tolerance induced by LIP via the mechanisms involving in modulated mitochondrial trans-membrane potential, Na+-K+-ATPase activity of mitochondria, Bcl-2 and Bax mRNA expression, neurons and mitochondrial ultrastructure.
3 The expression of Ngb in the CA1 hippocampus after cerebral ischemia and the effect of LIP on it in young and aged rats
Using four-vessel occluding global brain ischemia model, the role of age on the Ngb expression and the LIP neuroprotection against severe ischemic insult was investigated by observing the Ngb expression in the CA1 hippocampus after cerebral ischemia and the effect of LIP on it in young and aged rats.
Methods: Sixty male Spague-Dawley rats aged 3 months (thirty) and 21-23 months (thirty) with permanently occluded bilateral vertebral arteries were randomly divided into cerebral ischemic group and LIP + cerebral ischemic group, respectively. The expression of Ngb mRNA and protein in the CA1 hippocampus were investigated by RT-PCR and Western blot methods. The profile of DND of pyramidal neurons in the CA1 hippocampus was evaluated by using thionin staining.
Results: RT-PCR and Western blot analysis showed that the expression of Ngb mRNA and protein after cerebral ischemia for 8 min in aged rats was decreased compared with that in the young rats which suffered a identical cerebral ischemia with the aged rats (P<0.05). LIP upregulated Ngb mRNA and protein expression in both young and aged rats suffered cerebral ischemia (P<0.05). However, the upregulation of Ngb expression in aged rats was significantly less than that in young rats (P<0.05).
Neuropathological evaluation showed that obvious DND of pyramidal neurons was found in young and aged rats after cerebral ischemia. Although LIP effectively protected the pyramidal neurons in the CA1 hippocampus against DND normally induced by ischemic insult (P<0.05), the neuroprotection of LIP for aged rats was less effective than that for young rats.
The above results indicated that the expression of Ngb and the up-regulating effect of LIP on the expression in aged rats were significantly decreased compared to those in young rats when the rats suffered cerebral ischemia.
4 LIP up-regulate the expression of Ngb in the normal rat CA1 hippocampus
To investigate whether LIP could induce the up-regulation of Ngb expression in CA1 hippocampus of normal rats, the study observed the time courses of the Ngb expression induced by LIP in the normal rat CA1 hippocampus.
Methods: Forty male Wistar rats were randomly divided into 2 groups:
①Sham group (n=8): The bilateral femoral arteries of the rats were dissected out and exposed for 60 min, but not clamped.
②LIP group (n=32): The bilateral femoral arteries of the rats were dissected out and clamped for 10 min 3 times in an interval of 10 min.
The expression of Ngb mRNA in the CA1 hippocampus was measured by RT-PCR analysis, according to the timing of reperfusion after LIP, rats in LIP group were divided into 4 subgroups: 30min, 1h, 3h and 6h. The expression of Ngb protein was measured by Western blot analysis, rats in LIP group were divided into 4 subgroups: 1h, 6h, 12h and 1d.
Results: RT-PCR analysis showed that basal expression of Ngb mRNA in the CA1 hippocampus could be observed in sham group. The Ngb mRNA expression was significantly up-regulated by LIP. The expression of Ngb mRNA peaked at 30 min (P<0.05), then gradually decreased at 1 h and 3 h, but still higher than that of sham group (P<0.05). The expression of Ngb mRNA returned to sham level at 6h after LIP.
Western blot analysis showed that compared with sham group, the Ngb protein expression was significantly up-regulated after LIP, which peaked at 1h (P<0.05), then gradually decreased at 6 h and 12 h, but still higher than that of sham group (P<0.05). The expression of Ngb protein at 1d after LIP was still higher than that of sham group, but no statistic difference when compared to sham group.
The above results indicated LIP could induce up-regulation of Ngb expression in CA1 hippocampus of normal rats.
5 Conclusions
(1) LIP and Ngb inducer Hemin could significantly up-regulate the expression of Ngb in the CA1 hippocampus in cerebral ischemic rat, meanwhile protect the pyramidal neurons in the area against cerebral ischemia insults and decreased the serum Ngb protein level. The above effects of LIP could be inhibited partly by Ngb AS-ODNs. These results indicated that the up-regulation of the Ngb expression played an important role in the brain ischemic tolerance induced by LIP.
(2) LIP effectively improved the deterioration in the mitochondrial trans-membrane potential, Na+-K+-ATPase activity of mitochondria, Bcl-2 and Bax mRNA expression, neuronal and mitochondrial ultrastructure induced by cerebral ischemic insult. The above effects of LIP could be inhibited partly by Ngb AS-ODNs. Ngb inducer Hemin could mimic the above effects of LIP. The results suggested that LIP plays neuronal protection, at least, through improving structure and function of mitochondria.
(3) The expression of Ngb and the up-regulating effect of LIP on it in aged rats were significantly decreased compared to those in young rats when the rats suffered cerebral ischemia. These differences might be one of underlying reasons why the aged rats exhibited severe DND after cerebral ischemia and why the neuroprotective effect of LIP was less in the aged rats than that in the young rats.
(4) LIP could induce up-regulation of Ngb expression in CA1 hippocampus in normal rats.
2000年,德国学者Burmester等在Nature上首次报道,在人和小鼠脑内存在一种特异的携氧球蛋白—脑红蛋白(neuroglobin,Ngb)。随后发现在大鼠、沙鼠等的脑内也存在Ngb表达。研究表明,Ngb可保护神经元对抗缺血/缺氧损伤,抑制神经元凋亡。Ngb过表达的转基因小鼠局灶性脑缺血后,脑梗体积减小;而应用反义寡核苷酸(antisense oligodeoxynucleotides, AS-ODNs)抑制Ngb表达,脑缺血损伤加重。同时在细胞水平发现,应用Ngb小干扰RNA降低H2O2诱导的PC12细胞Ngb表达,加剧了细胞损伤。Ngb作为一种内源性保护蛋白,它的发现为缺血缺氧性脑病的研究提出了新思路。我室应用免疫组化技术初步研究发现LIP可上调大鼠海马Ngb表达,但LIP是否是通过上调Ngb表达来发挥抗脑缺血作用尚有待于进一步证实。
研究发现,线粒体不仅在能量转换中扮演重要角色,在整合死亡信号刺激、决定细胞命运的过程中也起枢纽作用。线粒体成为脑缺血性损伤的亚细胞靶目标,在脑缺血/再灌注损伤和缺血预处理内源性脑保护中的作用越来越受到关注。资料显示,Ngb对神经元的保护作用可能与线粒体有关,如Ngb表达增加可改善H2O2诱导的PC12细胞线粒体膜电位的降低。线粒体介导的凋亡通路参与脑缺血后的细胞凋亡,凋亡信号的整合主要是通过Bcl-2家族蛋白在线粒体膜上的相互作用来实现的。Bcl-2和Bax是Bcl-2家族中已被发现的与细胞凋亡关系密切的两种基因,是一对在功能上相互对立的细胞凋亡调控基因。然而,LIP诱导的脑缺血耐受过程中,Ngb是否是通过调控线粒体的结构和功能及线粒体介导的凋亡途径等机制来发挥脑保护作用尚不清楚。
因此,本实验应用大鼠四血管闭塞全脑缺血整体动物模型,观察Ngb在LIP诱导的脑缺血耐受中表达的变化、以及上调或阻断Ngb表达对LIP脑保护作用的影响,探讨Ngb在LIP抗脑缺血/再灌注损伤中的作用;在此基础上,从线粒体的结构和功能以及线粒体介导的凋亡途径等方面探讨Ngb在LIP诱导的脑缺血耐受中的线粒体机制;并进一步观察青年和老年大鼠脑缺血后海马CA1区Ngb表达的变化及LIP对其影响,以及LIP对正常大鼠海马CA1区Ngb表达的影响,为全面深入地评价脑缺血后海马Ngb表达的变化及LIP的保护作用提供实验依据,为临床上脑血管疾病的防治研究提供新线索和思路。
1 Ngb参与LIP诱导的大鼠脑缺血耐受
目的:观察Ngb在LIP诱导的脑缺血耐受中表达的变化、以及应用Ngb诱导剂Hemin和Ngb AS-ODNs分别上调和抑制Ngb表达对LIP脑保护作用的影响,确定Ngb在LIP诱导的大鼠脑缺血耐受中的作用。
1.1 LIP上调脑缺血大鼠海马CA1区Ngb表达
动物分组及方法:雄性Wistar大鼠70只,随机分为5组:
①脑缺血sham组(n=14):永久凝闭双侧椎动脉,2 d后游离双侧颈总动脉,但不夹闭。
②LIP组(n=14):夹闭双侧股动脉10 min,再灌10 min,反复3次。
③脑缺血组(n=14):永久凝闭双侧椎动脉,2 d后夹闭双侧颈总动脉8 min致全脑缺血。
④LIP sham+脑缺血组(n=14):游离双侧股动脉,但不夹闭,持续60min,之后行全脑缺血8min。
⑤LIP+脑缺血组(n=14):夹闭双侧股动脉10 min,再灌10 min,反复3次,其余同第③组。
每组14只动物,用于3个指标的观察。①海马CA1区Ngb mRNA表达(n=3):于脑缺血sham手术或LIP或脑缺血后3 h取材,行RT-PCR检测;②海马CA1区Ngb蛋白表达(n=5):于脑缺血sham手术或LIP或脑缺血后6 h取材,行Western blot检测;③海马CA1区锥体神经元迟发性死亡(delayed neuronal death,DND)(n=6):于脑缺血sham手术或LIP或脑缺血后7 d取材,行硫瑾染色,在光学显微镜下观察海马CA1区组织形态,确定神经元密度(neuronal density,ND)(计数海马CA1区每1 mm区段内细胞膜完整、胞核饱满、核仁清晰的锥体细胞数目,每张切片双侧海马各计数6个区段,取平均数为神经元密度)和组织学分级(histological grade,HG)(0级:无神经元死亡;Ⅰ级:散在的神经元死亡;Ⅱ级:成片的神经元死亡;Ⅲ级:几乎全部的神经元死亡)。
结果:RT-PCR和Western blot分析显示,脑缺血sham组海马CA1区Ngb mRNA和蛋白有一定的表达。与脑缺血sham组相比,LIP组Ngb mRNA和蛋白表达明显增加(P<0.05)。脑缺血组海马CA1区Ngb mRNA和蛋白表达较脑缺血sham组明显下降(P<0.05)。与脑缺血sham组和脑缺血组相比,LIP+脑缺血组Ngb mRNA和蛋白表达明显增加(P<0.05),而LIP sham+脑缺血组Ngb表达则无明显变化。
硫瑾染色结果显示, LIP组大鼠海马CA1区锥体神经元无明显损伤,神经元排列整齐,轮廓完整,核饱满而核仁清晰,其HG及ND与脑缺血sham组相比无差异。脑缺血组大鼠海马CA1区DND明显,与脑缺血sham组和LIP组相比,HG显著升高,ND显著下降(P<0.05)。在LIP+脑缺血组,上述损伤被明显抑制,表现为与脑缺血组相比,HG明显降低,ND明显升高(P<0.05);而LIP sham+脑缺血组海马CA1区组织学特征则与脑缺血组无明显差异。这些结果表明,LIP具有抗脑缺血引起的海马CA1区DND的作用。
上述结果表明,LIP可诱导脑缺血耐受,同时上调脑内Ngb的表达。
1.2 Hemin上调Ngb表达诱导脑缺血耐受
动物分组及方法:选用永久凝闭双侧椎动脉的雄性Wistar大鼠84只,
随机分为:①脑缺血sham组(n=14):永久凝闭双侧椎动脉,2 d后游离双侧颈总动脉,但不夹闭。
②脑缺血组(n=14):永久凝闭双侧椎动脉,2 d后夹闭双侧颈总动脉8min致全脑缺血。
③Hemin溶剂+脑缺血组(n=14):腹腔注射Hemin溶剂持续2 d,每天1次,其余同第②组。
④Hemin+脑缺血组(n=42):腹腔注射Hemin持续2 d,每天1次,其余同第②组;根据Hemin的剂量,将该组进一步分为10 mg/kg、20 mg/kg和40 mg/kg三个亚组。
每组14只动物,用于3个指标的观察。①海马CA1区Ngb mRNA表达(n=3):于脑缺血sham手术或脑缺血后3 h取材,行RT-PCR检测;②海马CA1区Ngb蛋白表达(n=5):于脑缺血sham手术或脑缺血后6h取材,行Western blot检测;③海马CA1区锥体神经元DND(n=6):于脑缺血sham手术或脑缺血后7d取材,行硫瑾染色,在光学显微镜下观察海马CA1区组织形态,确定ND和HG。
结果:RT-PCR和Western blot分析显示,与脑缺血sham组相比,脑缺血组和Hemin溶剂+脑缺血组海马CA1区Ngb mRNA和蛋白表达显著下降(P<0.05)。与脑缺血组和Hemin溶剂+脑缺血组相比,Hemin+脑缺血组Ngb mRNA和蛋白表达显著增加,尤以20 mg/kg和40 mg/kg组明显(P<0.05),并呈现剂量依赖性。
硫瑾染色结果显示,与脑缺血sham组相比,脑缺血组大鼠海马CA1区锥体神经元有明显DND,HG明显升高,而ND则明显下降(P<0.05)。与脑缺血组相比,Hemin溶剂+脑缺血组HG和ND无明显差异。在Hemin+脑缺血组,DND被明显抑制,表现为与脑缺血组和Hemin溶剂+脑缺血组相比,HG明显降低,ND明显升高,并与Hemin的剂量呈现依赖性(P<0.05)。
上述结果表明,Hemin可上调Ngb表达而诱导脑缺血耐受。
1.3 Ngb AS-ODNs抑制LIP抗脑缺血引起的海马CA1区DND的作用
动物分组及方法:选用永久凝闭双侧椎动脉的雄性Wistar大鼠55只,随机分为:
①ACSF+LIP+脑缺血组(n=11):夹闭双侧股动脉10 min,再灌流10 min,反复3次,然后夹闭双侧颈总动脉8 min致全脑缺血;LIP前右侧脑室注射ODNs的溶剂人工脑脊液(artificial cerebrospinal fluid,ACSF) 2次,每天1次,每次8μl。
②S-ODNs+LIP+脑缺血组(n=11):LIP前右侧脑室注射1.6 nmol Ngb S-ODNs溶液2次,每天1次,每次8μl,其余同第①组。
③AS-ODNs+LIP+脑缺血组(n=33):LIP前右侧脑室注射Ngb AS-ODNs溶液2次,每天1次,每次8μl,其余同第①组。根据AS-ODNs的用量,进一步分为0.4 nmol、0.8 nmol和1.6 nmol三个亚组。
每组11只动物,用于2个指标的观察。①海马CA1区Ngb蛋白表达(n=5):于脑缺血后6h取材,行Western blot检测;②海马CA1区锥体神经元DND(n=6):于脑缺血后7d取材,行硫瑾染色,在光学显微镜下观察海马CA1区组织形态,确定ND和HG。
结果:Western blot分析显示,与ACSF+LIP+脑缺血组相比, AS-ODNs+LIP +脑缺血组海马CA1区Ngb蛋白表达显著降低(P<0.05),并与AS-ODNs的剂量呈现依赖性,而LIP前侧脑室注射S-ODNs,对CA1区Ngb蛋白表达无明显影响。
硫瑾染色结果显示,ACSF+LIP+脑缺血组和S-ODNs+LIP+脑缺血组,大鼠海马CA1区未见明显锥体神经元损伤,锥体神经元排列较整齐,轮廓完整,核饱满而核仁清晰。而AS-ODNs+LIP+脑缺血组CA1区锥体神经元损伤明显,并与AS-ODNs的剂量呈现依赖性。AS-ODNs 0.4 nmol组HG变化不明显,但ND明显降低(P<0.05);AS-ODNs 0.8 nmol和1.6 nmol组,HG明显增高,ND明显降低(P<0.05),表明Ngb AS-ODNs通过抑制Ngb表达,降低了LIP的脑保护作用。
上述结果表明,Ngb AS-ODNs可阻断Ngb表达,进而阻断LIP的脑保护作用。
1.4 LIP和Hemin降低脑缺血引起的血清Ngb蛋白含量增加动物分组及方法:雄性Wistar大鼠30只,随机分为6组:
①脑缺血sham组(n=5):永久凝闭双侧椎动脉,2 d后游离双侧颈总动脉,但不夹闭。
②LIP组(n=5):夹闭双侧股动脉10 min,再灌10 min,反复3次。
③脑缺血组(n=5):永久凝闭双侧椎动脉,2 d后夹闭双侧颈总动脉8 min致全脑缺血。
④LIP+脑缺血组(n=5):夹闭双侧股动脉10 min,再灌10 min,反复3次,其余同第③组。
⑤Hemin+脑缺血组(n=5):腹腔注射Hemin 20 mg/kg/d,持续2 d,其余同第③组。
⑥Hemin溶剂+脑缺血组(n=5):腹腔注射Hemin溶剂持续2 d,其余同第③组。
每组5只动物,用于检测血清Ngb蛋白含量:于脑缺血sham手术或LIP或脑缺血后6 h取血,行ELISA检测。
结果:ELISA显示,脑缺血sham组和LIP组血清Ngb蛋白含量较低,二者之间无统计学差异。与脑缺血sham组相比,脑缺血组血清Ngb蛋白含量显著增加(P<0.05)。而脑缺血前给予LIP预处理,血清Ngb蛋白含量明显下降(P<0.05)。与脑缺血组相比,Hemin(20 mg/kg)+脑缺血组血清Ngb蛋白含量明显下降(P<0.05);而Hemin溶剂+脑缺血组血清Ngb蛋白含量无明显变化。
以上结果表明,LIP和Hemin可明显降低脑缺血后血清Ngb蛋白含量升高。
2 Ngb通过线粒体机制参与LIP诱导的脑缺血耐受
目的:通过观察Ngb表达增加对海马CA1区神经细胞线粒体膜电位、线粒体Na+-K+-ATP酶活性、神经元及线粒体超微结构、Bcl-2和Bax mRNA表达的影响,探讨Ngb是否通过线粒体机制参与LIP诱导的脑缺血耐受。
动物分组及方法:选用永久凝闭双侧椎动脉的雄性Wistar大鼠126只,随机分为:
①脑缺血sham组(n=18):永久凝闭双侧椎动脉,2 d后游离双侧颈总动脉,但不夹闭。
②脑缺血组(n=18):永久凝闭双侧椎动脉,2 d后夹闭双侧颈总动脉8 min致全脑缺血。
③LIP+脑缺血组(n=18):夹闭双侧股动脉10 min,再灌10 min,反复3次,其余同第②组。
④AS-ODNs+LIP+脑缺血组(n=18):LIP前右侧脑室注射0.8 nmol Ngb AS-ODNs溶液2次,每天1次,每次8μl,其余同第③组。
⑤S-ODNs+LIP+脑缺血组(n=18):LIP前右侧脑室注射0.8 nmol Ngb S-ODNs溶液2次,每天1次,每次8μl,其余同第③组。
⑥Hemin+脑缺血组(n=18):脑缺血前腹腔注射20mg/kg Hemin 2次,每天1次,其余同第②组。
⑦Hemin溶剂+脑缺血组(n=18):脑缺血前腹腔注射Hemin溶剂2次,每天1次,其余同第②组。
每组18只动物,用于4个指标的观察。①海马CA1区神经细胞线粒体膜电位(n=3):于脑缺血sham手术或脑缺血后24 h取材,行流式细胞检测;②海马CA1区线粒体Na+-K+-ATP酶活性(n=10):于脑缺血sham手术或脑缺血后6 h和24 h取材、检测;③海马CA1区Bcl-2和Bax mRNA表达(n=3):于脑缺血sham手术或脑缺血后24 h取材,行RT-PCR检测;④海马CA1区神经元及线粒体超微结构(n=2):于脑缺血sham手术或脑缺血后3 d取材,行透射电镜观察。
结果:流式细胞仪检测神经细胞线粒体膜电位显示,脑缺血sham组海马CA1区神经细胞线粒体膜电位较高。与脑缺血sham组相比,脑缺血后线粒体膜电位显著降低(P<0.05)。与脑缺血组相比,LIP+脑缺血组线粒体膜电位明显增加(P<0.05)。与LIP+脑缺血组相比,AS-ODNs+LIP+脑缺血组线粒体膜电位显著降低,而S-ODNs+LIP+脑缺血组则无明显变化。与脑缺血组相比,Hemin+脑缺血组线粒体膜电位明显增高(P<0.05),而Hemin溶剂+脑缺血组则无明显变化。
海马CA1区线粒体Na+-K+-ATP酶活性测定显示,在6 h时间点,脑缺血sham组该酶活性较高,而脑缺血后则较脑缺血sham组明显下降(P<0.05)。与脑缺血组相比,LIP+脑缺血组该酶活性明显下降(P<0.05)。与LIP+脑缺血组相比,AS-ODNs+LIP+脑缺血组该酶活性显著增加(P<0.05),而LIP前侧脑室注射Ngb S-ODNs,对该酶活性无影响。与脑缺血组相比,Hemin+脑缺血组该酶活性明显降低(P<0.05),而Hemin溶剂+脑缺血组则无明显变化。在24 h时间点,脑缺血sham组线粒体Na+-K+-ATP酶活性较高。与脑缺血sham组相比,脑缺血后该酶活性显著下降(P<0.05)。与脑缺血组相比,LIP+脑缺血组该酶活性明显增加(P<0.05)。与LIP+脑缺血组相比,AS-ODNs +LIP+脑缺血组该酶活性显著降低(P<0.05),而S-ODNs +LIP+脑缺血组则无明显变化。与脑缺血组相比,Hemin+脑缺血组该酶活性明显增加(P<0.05),而Hemin溶剂+脑缺血组则无明显变化。
RT-PCR结果显示,脑缺血sham组海马CA1区Bcl-2和Bax mRNA表达较低。与脑缺血sham组相比,脑缺血组Bcl-2和Bax mRNA表达均显著增加,而Bax增加更为明显(P<0.05)。与脑缺血组相比,LIP+脑缺血组Bcl-2 mRNA表达明显增加(P<0.05),而Bax mRNA表达明显下降(P<0.05)。与LIP+脑缺血组相比,AS-ODNs+LIP+脑缺血组Bcl-2 mRNA表达明显下降(P<0.05),Bax mNRA表达增加(P<0.05);而在LIP预处理前侧脑室注射Ngb S-ODNs干预,Bcl-2和Bax mRNA表达无明显变化。与脑缺血组相比,Hemin+脑缺血组Bcl-2 mRNA表达明显增加(P<0.05),而Bax mRNA表达下降(P<0.05);而Hemin溶剂+脑缺血组Bcl-2和Bax mRNA表达无明显变化。
透射电镜结果显示,①脑缺血sham组:海马CA1区神经元形态完整,结构清晰,核膜完整,核形态正常,胞质中细胞器丰富。胞质内有丰富的线粒体,呈圆形或椭圆形,嵴较多而清晰可见。②脑缺血组:海马神经元严重水肿变形。细胞核不规则,核膜皱缩,胞质高度水肿,细胞器数量明显减少。线粒体呈气球样肿胀,大量空泡化,嵴断裂,溶解,消失,同时有的线粒体致密化。③LIP+脑缺血组:与脑缺血组相比,LIP+脑缺血组神经元和线粒体损伤明显减轻。表现为神经元水肿明显减轻,核膜尚完整,核形态正常,染色质均匀分布,细胞器尚丰富。胞质内有丰富的线粒体,大多数线粒体嵴完整,排列紧密规律,基质颗粒极少量脱落,少量线粒体水肿,空泡化。④AS-ODNs+LIP+脑缺血组:与LIP+脑缺血组相比,AS-ODNs+LIP+脑缺血组神经元和线粒体损伤明显加重,但较脑缺血组损伤稍轻些。神经元明显水肿,细胞器数量明显减少。线粒体水肿、变形,部分膜溶解,同时有的线粒体致密化。⑤S-ODNs+LIP+脑缺血组:本组与LIP+脑缺血组比较,无显著差别。⑥Hemin+脑缺血组:与脑缺血组相比,Hemin+脑缺血组神经元和线粒体的损伤明显改善。神经元水肿明显减轻,核膜尚完整,核形态正常,细胞器尚丰富。胞质内有丰富的线粒体,多数线粒体嵴完整,排列紧密规律,基质颗粒极少量脱落,少量线粒体轻度水肿,或致密化。⑦Hemin溶剂+脑缺血组:本组与脑缺血组比较,无显著差别。
以上结果表明,Ngb表达增加可通过调控海马CA1区神经细胞线粒体膜电位、Na+-K+-ATP酶活性、线粒体超微结构、Bcl-2和Bax mRNA表达等线粒体机制参与LIP诱导的脑缺血耐受。
3青年和老年大鼠脑缺血后海马CA1区Ngb表达的变化及LIP对其影响
目的:观察比较青年和老年大鼠脑缺血时海马CA1区Ngb表达的变化及LIP对其影响,为进一步评价脑缺血后海马Ngb表达的变化及LIP的保护作用提供实验依据。
动物分组及方法:选用3月龄健康雄性Spague-Dawley大鼠30只,21~23月龄健康雄性Spague-Dawley大鼠30只。将凝闭双侧椎动脉2 d的青年和老年大鼠均随机分为脑缺血组和LIP+脑缺血组。脑缺血组:夹闭双侧颈总动脉8 min;LIP+脑缺血组:夹闭双侧股动脉10 min,再灌流10 min,反复3次,随后夹闭双侧颈总动脉8 min。
每组15只动物,用于3个指标的观察。①海马CA1区Ngb mRNA表达(n=3):于脑缺血后3 h取材,行RT-PCR检测;②海马CA1区Ngb蛋白表达(n=6):于脑缺血后6h取材,行Western blot检测;③海马CA1区锥体神经元DND(n=6):于脑缺血后7d取材,行硫瑾染色,在光学显微镜下观察海马CA1区组织形态,确定ND和HG。
结果:RT-PCR分析显示,Ngb和GAPDH扩增后的产物片段与设计相符。老年脑缺血组Ngb mRNA表达比青年脑缺血组明显降低(P<0.05)。LIP可明显上调青年和老年大鼠脑缺血后Ngb的mRNA表达(P<0.05),但老年大鼠(LIP+老年脑缺血组)的上调幅度小于青年大鼠(LIP +青年脑缺血组)(P<0.05)。
Western blot结果显示,各组均可在分子量相当于17KD和42KD的区域见Ngb和β-actin蛋白表达条带。Ngb蛋白表达在老年大鼠脑缺血组比青年大鼠脑缺血组显著降低(P<0.05),LIP后青年和老年脑缺血大鼠Ngb的蛋白表达均明显上调(P<0.05),但老年大鼠(LIP+老年脑缺血组)的上调幅度小于青年大鼠(LIP +青年脑缺血组)(P<0.05)。
硫瑾染色显示,青年脑缺血组有明显的组织损伤,表现为海马CA1区锥体细胞稀疏,排列紊乱,多数细胞胞核固缩,核仁欠清晰,尼氏小体减少或消失。老年脑缺血组海马CA1区锥体细胞几乎全部固缩或缺失,与青年脑缺血组相比ND显著下降(P<0.05),表明脑缺血后,老年大鼠的损伤比青年大鼠更严重。LIP+青年脑缺血组CA1区锥体细胞排列整齐致密,胞核饱满,核仁较清晰,仅个别锥体细胞胞核固缩或缺失,和青年脑缺血组相比,HG显著降低(P<0.05),ND明显升高(P<0.05)。LIP+老年脑缺血组与老年脑缺血相比,组织损伤明显减轻,部分锥体细胞固缩或缺失,HG显著降低(P<0.05),ND明显升高(P<0.05);但与LIP+青年脑缺血组比较,CA1区锥体细胞固缩或缺失仍较多,ND明显降低(P<0.05),表明LIP可减轻脑缺血损伤,但老龄降低了LIP的保护作用。
以上结果表明,老年大鼠全脑缺血后Ngb的表达及LIP对其上调作用较青年大鼠明显减弱,这可能是老年大鼠脑缺血后损伤较重和LIP对老年大鼠脑缺血保护作用较弱的原因之一。
4 LIP上调正常大鼠海马CA1区Ngb表达
目的:观察LIP后不同时间点正常大鼠海马CA1区Ngb表达的变化,为研究和评价LIP对正常机体的脑保护作用提供实验依据。动物分组及方法:雄性Wistar大鼠40只随机分为:
①sham组(n=8):游离双侧股动脉,但不夹闭。
②LIP组(n=32):夹闭双侧股动脉10 min,再灌10 min,反复3次。
以上动物于sham手术后30 min或LIP后30 min、1 h、3 h和6 h取材, RT-PCR检测海马CA1区Ngb mRNA表达(n=3),于sham手术后1 h或LIP后1 h、6 h、12 h和1 d取材,Western blot分析检测Ngb蛋白表达(n=5)。
结果:RT-PCR分析显示,在150bp和349bp处分别为Ngb和内参GAPDH的扩增条带。Sham组海马CA1区有一定的Ngb mRNA表达。与sham组相比,LIP后海马CA1区Ngb mRNA表达增加。这种增加于LIP后30 min最明显(P<0.05),随后逐渐下降,在6 h时接近sham组水平。
Western blot结果显示,各组均可在分子量相当于17KD和42KD的区域分别出现Ngb和β-actin蛋白表达条带。Sham组海马CA1区有一定的Ngb蛋白表达。与sham组相比,LIP后海马CA1区Ngb蛋白表达增加。这种增加于LIP后1 h达到高峰(P<0.05),随后逐渐下降,至1 d时接近sham组。
以上结果表明,LIP能上调正常大鼠海马CA1区Ngb mRNA和蛋白表达,Ngb mRNA表达高峰早于蛋白表达高峰。
5结论
⑴LIP能明显上调脑缺血大鼠海马CA1区Ngb mRNA和蛋白表达,同时LIP可明显抑制脑缺血后CA1区锥体神经元DND,降低血清Ngb蛋白含量;腹腔注射Ngb诱导剂Hemin能模拟LIP诱导的脑缺血耐受,而侧脑室注射Ngb AS-ODNs可部分阻断LIP的脑保护作用。这些结果表明,Ngb表达上调在LIP诱导脑缺血耐受中发挥重要作用。
⑵Ngb表达增加通过增加线粒体膜电位、改善线粒体Na+-K+-ATP酶活性、保护线粒体超微结构、调节Bcl-2和Bax mRNA表达等线粒体机制参与LIP诱导的脑缺血耐受。
⑶老年大鼠全脑缺血后Ngb的表达及LIP对其上调作用较青年大鼠明显减弱,这可能是老年大鼠脑缺血后损伤较重和LIP对老年大鼠脑缺血保护作用较弱的原因之一。
⑷LIP可上调正常大鼠海马CA1区Ngb表达。
Ischemic neuropathy is a leading cause of morbidity, mortality and disability rate. Activation of endogenous brain protection is a novel approach to prevent brain ischemia/reperfusion injury, and which has attracted extensive attention. Ischemic preconditioning is a phenomenon that brief episode of sub-lethal ischemia protects organs or tissues against a subsequent lethal ischemic insult. Brain ischemic preconditioning was first demonstrated by Kitagawa and colleagues. They showed in the gerbil that short periods of global cerebral ischemia can protect the brain against subsequent prolonged ischemia. Recent studies reported that a short sub-lethal ischemia and reperfusion in various organs can induce ischemic tolerance in another organ as well. This phenomenon is known as remote ischemic preconditioning. The finding opened up vast vista of the study to increase the resistance of important vital organs such as brain to ischemia with transient ischemic preconditioning in non-vital organs. Our recent study demonstrated that limb ischemic preconditioning (LIP) could attenuate delayed neuronal death (DND) or apoptosis of pyramidal neurons in the CA1 hippocampus induced normally by serious brain ischemia/reperfusion. However, mechanisms underlying the neuroprotection of LIP have not been well understood.
Neuroglobin (Ngb) first described in Nature by Burmester at 2000 is a globin with high affinity for oxygen and is widely expressed in neurons of central and peripheral nervous systems in mouse and human. Since then, Ngb have also been recognized in rat and gerbil, suggesting that Ngb is present in a broad range of vertebrate species. Recent studies suggested that Ngb plays an important role in the protection of brain neurons from ischemic and hypoxic injuries, and apoptosis. For example, Ngb overexpression can protect neurons against hypoxic/ischemic insults, and knocking down Ngb expression increases hypoxic neuronal injury in vitro and ischemic cerebral injury in vivo. Ngb acts as an endogenous neuroprotectant, which has provided a new strategy to the study of brain hypoxic/ischemic insults. Our preliminary study using immunohistochemistry staining showed that LIP up-regulated the Ngb expression in the rat CA1 hippocampus, but whether Ngb participates in the neuroprotection of LIP in the CA1 hippocampus against severe brain ischemia/reperfusion is still unclear and need to be clarified.
Mitochondria, besides playing a key role in energy metabolism within the cell, are involved in a cohort of other processes like integration of dead signal and determination of cell fate. Mitochondrial-mediated apoptosis pathway is considered to be important to neuronal death after cerebral blood flow arrest. Recent investigations have shown that mitochondrial-centered mechanisms are important mediators in promoting development of the preconditioning response. In the brain, ischemic preconditioning diminishes mitochondrial dysfunction induced by ischemia and confers neuroprotection. Targeting mitochondria may be a useful strategy to reduce ischemic brain injury. Bcl-2 gene families are identified as apoptosis regulating genes. Inhibiting effect of anti-apoptotic proteins Bcl-2 on apoptosis is mediated by its binding to pro-apoptotic proteins, e.g., Bax, inhibiting their oligomerization, and thus inhibiting mitochondrial outer membrane pore formation, through which other pro-apoptotic proteins, e.g., cytochrome c, are released to the cytosol. Whether mitochondrial mechanisms are involved in neuroprotection of Ngb induced by LIP remain unclear.
Therefore, the present study was primarily undertaken to investigate the role of Ngb in the neuroprotection of LIP against global brain ischemia by observing changes in expression of Ngb in the CA1 hippocampus after LIP in rat global brain ischemic model, influences of up- or down-regulating Ngb expression on the neuroprotection of LIP, and the involvements of mitochondria in the process. The present study also compared Ngb expression in the CA1 hippocampus after cerebral ischemia and the effect of LIP on it between young and aged rats, and in normal rats. The results obtained provided new and more direct evidence for illustrating the role of Ngb in the neuronal protection of LIP and new clues for the study in prevention and therapy of ischemic neuropathies.
1 Ngb participates in the neuroprotection of LIP in the CA1 hippocampus against severe brain ischemia/reperfusion
Using four-vessel occluding global brain ischemia model, the role of Ngb in the neuroprotection of LIP against severe ischemic insult was investigated by observing the influence of LIP on the expression of Ngb in the rat CA1 hippocampus, and the influence of Ngb inducer Hemin and Ngb (antisense oligodeoxynucleotides, AS-ODNs) on the neuroprotection of LIP.
1.1 LIP up-regulates Ngb expression in rat CA1 hippocampus and induces brain ischemic tolerance
Methods: Seventy male Wistar rats with permanently occluded bilateral vertebral arteries were randomly assigned to 5 groups:
①Sham of cerebral ischemia group: The rats were subjected to permanently occluding the bilateral vertebral arteries, and 2 d later the bilateral common carotid arteries were exposed, but without blocking the blood flow.
②LIP group: The bilateral femoral arteries of the rats were occluded for 10 min, 3 times, at 10-min intervals.
③Cerebral ischemia group: The bilateral vertebral arteries of the rats were permanently occluded, 2 d later the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia.
④Sham of LIP+cerebral ischemia group: The bilateral femoral arteries were exposed for 60 min but not clipped, and then global brain ischemia was induced as those in cerebral ischemia group.
⑤LIP+cerebral ischemia group: Global brain ischemic insult for 8 min was induced as those in cerebral ischemia group immediately after LIP as in LIP group.
Fourteen rats were included in each group, and assigned to 3 observations:①Ngb mRNA (n=3) (RT-PCR), and②Ngb protein (n=5) expression (Western blot), and③Pyramidal neurons DND in the CA1 hippocampus of the rats (n=6), in which the rats were, respectively, sampled for the observations at time point of 3 h, 6 h and 7 d after the sham operations of cerebral ischemia, or LIP, or cerebral ischemia in corresponding group. The profile of pyramidal neurons DND in the CA1 hippocampus was evaluated under light microscope by determining the neuronal density (ND) and histological grade (HG). The ND was determined by counting the number of surviving pyramidal neurons with intact cell membrane, full nucleus and clear nucleolus within 1 mm linear length of the CA1 hippocampus. The HG was divided into 4 grades according to the following standard: grade 0, no neuron death; gradeⅠ, scattered single neuron death; gradeⅡ, death of many neurons; gradeⅢ, death of almost complete neurons.
Results: RT-PCR and Western blot analysis showed that basal expression of Ngb mRNA and protein in the CA1 hippocampus could be observed in sham group of cerebral ischemia. Compared to sham group of cerebral ischemia, the expressions of Ngb mRNA and protein were obviously up-regulated after LIP (P<0.05), while significantly down-regulated after cerebral ischemia (P<0.05). LIP significantly up-regulated the expressions of Ngb mRNA and protein in LIP+cerebral ischemia group compared with cerebral ischemia group (P<0.05), while sham operation of LIP before the cerebral ischemia was without effect on the down-regulation of Ngb mRNA and protein expressions induced by cerebral ischemia.
Thionine staining showed that pyramidal neurons in the CA1 hippocampus in sham group of cerebral ischemia and LIP group were normal and lined up in order, and no DND was found. There were obvious DND of pyramidal neurons in cerebral ischemia group, and HD increased and ND decreased significantly compared with sham group of cerebral ischemia and LIP group (P<0.05). In LIP+cerebral ischemia group, the DND normally induced by cerebral ischemia was effectively prevented, which represented with increase in ND and decrease in HG compared with cerebral ischemia group (P<0.05), while, the sham operation of LIP (in sham of LIP+cerebral ischemia group) had no effect on the DND normally induced by cerebral ischemia.
The above results indicated that LIP could up-regulate the expression of Ngb, and induce brain ischemic tolerance in the rat CA1 hippocampus.
1.2 Hemin up-regulates Ngb expression in the rat CA1 hippocampus and induces brain ischemic tolerance
Methods: Eighty and four male Wistar rats with permanently occluded bilateral vertebral arteries were randomly divided into following groups:
①Sham of cerebral ischemia group: The rats were subjected to permanently occluding the bilateral vertebral arteries, and 2 d later the bilateral common carotid arteries were exposed, but without blocking the blood flow.
②Cerebral ischemia group: The bilateral vertebral arteries of the rats were permanently occluded, 2 d later the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia.
③Hemin vehicle+cerebral ischemia group: The rats were injected i.p. with Hemin vehicle once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia as those in cerebral ischemia group.
④Hemin+cerebral ischemia group: The rats were injected i.p. with Hemin once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia as those in cerebral ischemia group. According to doses of Hemin used, this group was divided into 3 subgroups: 10 mg/kg/d, 20 mg/kg/d and 40 mg/kg/d groups.
Fourteen rats were included in each group, and assigned to 3 observations:①Ngb mRNA (n=3) (RT-PCR), and②Ngb protein (n=5) expression (Western blotting analysis), and③Pyramidal neurons DND in the CA1 hippocampus of the rats (n=6)(thionine staining), in which the rats were, respectively, sampled for the observations at time point of 3 h, 6 h and 7 d after the sham operations of cerebral ischemia or cerebral ischemia in corresponding group.
Results: RT-PCR and Western blot analysis showed that compared to sham group of cerebral ischemia, the expressions of Ngb mRNA and protein were obviously down-regulated after cerebral ischemia (P<0.05). The administration of Hemin dose-dependently up-regulated the expressions of Ngb mRNA and protein in Hemin+cerebral ischemia group compared with cerebral ischemia group (P<0.05), while pretreatment with vehicle of Hemin had no influence on the Ngb mRNA and protein expressions.
Thionine staining showed that the outline of the pyramidal neurons was kept intact, and no neuronal loss was found in the CA1 hippocampus in sham group of cerebral ischemia. Obvious destruction of the CA1 hippocampus was found in cerebral ischemia group, the value of ND was decreased, and HG was increased compared with that in sham group of cerebral ischemia (P<0.05). Intraperitoneal injection with Hemin before cerebral ischemia showed a significant protective effect against the DND induced by cerebral ischemia, which was represented with dose-dependent increase in ND and decrease in HG after administration of Hemin, especially in large doses (P<0.05). Whereas, pretreated with Hemin vehicle before the cerebral ischemia had no significant effect on the DND normally induced by cerebral ischemia.
The above results indicated that Hemin could up-regulate the Ngb expression and induce brain ischemic tolerance.
1.3 Ngb AS-ODNs inhibit brain ischemic tolerance induced by LIP
Methods: Fifty and five male Wistar rats with permanently occluded bilateral vertebral arteries were randomly divided into following groups:
①ACSF+LIP+cerebral ischemia group: ACSF (artificial cerebrospinal fluid) was intraventricularly injected 30 min prior to LIP. LIP was preformed by occlusion of bilateral femoral arteries of the rats for 10 min 3 times in an interval of 10 min, and then the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia immediately after the LIP.
②S-ODNs+LIP+cerebral ischemia group: Ngb (sense oligodeoxynucleotides, S-ODNs) in a dose of 1.6 nmol was administered 30 min prior to LIP by intraventricular injection. Others were the same as ACSF+LIP+cerebral ischemia group.
③AS-ODNs+LIP+cerebral ischemia group: Ngb AS-ODNs was administered 30 min prior to LIP by intraventricular injection. Others were the same as ACSF+LIP+cerebral ischemia group. According to doses of Ngb AS-ODNs used, this group was divided into 3 subgroups: 0.4 nmol, 0.8 nmol, and 1.6 nmol groups.
Eleven rats were included in each group, and assigned to 2 observations:①Ngb protein expression (n=5()Western blotting analysis), and②Pyramidal neurons DND in the CA1 hippocampus of the rats (n=6)(thionine staining), in which the rats were, respectively, sampled for the observation at time point of 6 h and 7 d after the cerebral ischemia in corresponding group.
Results: Western blot analysis showed that compared with ACSF+LIP +cerebral ischemia group, the expression of Ngb protein in AS-ODNs+LIP+ cerebral ischemia group significantly down-regulated in a dose-dependent manner (P<0.05). While there was no influence on the Ngb protein expression induced by LIP after administration Ngb S-ODNs.
Thionine staining showed that pyramidal neurons in the CA1 hippocampus arranged in order, and the structure of most them was clear in ACSF+LIP+cerebral ischemia and S-ODNs+LIP+cerebral ischemia groups. Administration of Ngb AS-ODNs dose-dependently induced obvious DND of pyramidal neurons in the CA1 hippocampus, which was represented with the decrease in the value of ND, and increase in HG compared with ACSF+LIP+ cerebral ischemia group (P<0.05). The administration of Ngb S-ODNs had no effect on the histological characteristic of the CA1 hippocampus.
The above results indicated that Ngb AS-ODNs partly inhibited brain ischemic tolerance induced by LIP via inhibiting the expression of Ngb protein.
1.4 LIP and Hemin reduce the increase of the serum Ngb protein level induce by cerebral ischemia
Methods: Thirty male Wistar rats with permanently occluded bilateral vertebral arteries were randomly assigned to 6 groups:
①Sham of cerebral ischemia group: The rats were subjected to permanently occluding the bilateral vertebral arteries, and 2 d later the bilateral common carotid arteries were exposed, but without blocking the blood flow.
②LIP group: The bilateral femoral arteries of the rats were occluded for 10 min, 3 times, at 10-min intervals.
③Cerebral ischemia group: The bilateral vertebral arteries of the rats were permanently occluded, 2 d later the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia.
④LIP+cerebral ischemia group: LIP was preformed as those in LIP group, and then cerebral ischemia insult for 8 min was produced as those in cerebral ischemia group immediately after the LIP.
⑤Hemin+cerebral ischemia group: The rats were injected i.p. with Hemin 20 mg/kg once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia as those in cerebral ischemia group.
⑥Hemin vehicle+cerebral ischemia group: The rats were injected i.p. with Hemin vehicle once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia as those in cerebral ischemia group.
Five rats were included in each group, and the serum Ngb protein level was observed by ELISA at 6 h after the sham operation of cerebral ischemia, or LIP, or cerebral ischemia.
Results: The level of serum Ngb protein in sham group of cerebral ischemia and LIP group was low, and no significant differences between the two groups. Compared with the two groups, the level of serum Ngb was significantly increased in cerebral ischemia group (P<0.05). The increase in the level of serum Ngb after brain ischemia was significantly prevented by LIP or administration of Hemin prior to cerebral ischemia (P<0.05), while vehicle of Hemin had no evident effect on the level of serum Ngb protein.
The above results indicated that LIP and Hemin could significantly prevent the increase of the serum Ngb protein level induced by cerebral ischemia.
2 Ngb particapates in LIP-induced brain ischemic tolerance via mechanisms involving in mitochondria
To investigated whether Ngb particapates in LIP-induced brain ischemic tolerance via mitochondria mechanisms, the study observed the effect of up-regulated Ngb expression in the rat CA1 hippocampus on the mitochondria trans-membrane potential, Na+-K+-ATPase activity of mitochondria, Bcl-2 and Bax mRNA expression, neurons and mitochondrial ultrastructure in rat CA1 hippocampus.
Methods: One hundred and twenty-six male Wistar rats with permanently occluded bilateral vertebral arteries were randomly divided into 7 groups:
①Sham of cerebral ischemia group: The rats were subjected to permanently occluding the bilateral vertebral arteries, and 2 d later the bilateral common carotid arteries were exposed, but without blocking the blood flow.
②Cerebral ischemia group: The bilateral vertebral arteries of the rats were permanently occluded, 2 d later the bilateral common carotid arteries of the rats were occluded for 8 min to induce global brain ischemia.
③LIP+cerebral ischemia group: LIP was preformed by occlusion of bilateral femoral arteries of rats for 10 min 3 times in an interval of 10 min, then cerebral ischemia insult for 8 min was produced as those in cerebral ischemia group immediately after the last occlusion of the femoral arteries.
④AS-ODNs+LIP+cerebral ischemia group: Ngb AS-ODNs in a dose of 0.8 nmol was administered 30 min prior to LIP by intraventricular injection. Other procedures were the same as those in LIP+cerebral ischemia group.
⑤S-ODNs+LIP+cerebral ischemia group: Ngb S-ODNs in a dose of 0.8 nmol was administered 30 min prior to LIP by intraventricular injection. Other procedures were the same as those in LIP + cerebral ischemia group.
⑥Hemin+cerebral ischemia group: The rats were injected i.p. with Hemin 20 mg/kg once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia. Other procedures were the same as those in cerebral ischemia group.
⑦Hemin vehicle+cerebral ischemia group: The rats were injected i.p. with Hemin vehicle once a day for 2 days, and the last time of the injection was administered 1 h prior to the global brain ischemia. Other procedures were the same as those in cerebral ischemia group.
Eighteen rats were included in each group, and assigned to 4 observations:①mitochondria trans-membrane potential at 24 h (n=3),②Na+-K+-ATPase activity of mitochondria at 6 h and 24 h (n=10),③Bcl-2 and Bax mRNA expression at 24 h (n=3), and④neuronal and mitochondrial ultrastructure of the rats CA1 hippocampus on 3 d (n=2), in which the rats were, respectively, sampled at the corresponding time point after the sham operations of cerebral ischemia, or cerebral ischemia in each group.
Results: Flow cytometry analysis showed that mitochondrial trans- membrane potential significantly decreased in cerebral ischemia group compared with that of sham group of cerebral ischemia (P<0.05). The decrease was significantly ameliorated by LIP prior to cerebral ischemia insult (P<0.05). However, Pretreatment with Ngb AS-ODNs before LIP significantly inhibited the ameliorating effect of LIP on mitochondrial trans-membrane potential, which was represented with the decrease in the potential in AS-ODNs+LIP+cerebral ischemia group compared with that in LIP + cerebral ischemia group (P<0.05). In contrast, pretreatment with Ngb S-ODNs before LIP was without effect on the potential. Hemin significantly increased mitochondrial trans-membrane potential compared with that in cerebral ischemia group (P<0.05), whereas, Hemin vehicle administrated had no evident effect on it.
Results of Na+-K+-ATPase activity at 6 h time point showed that Na+-K+-ATPase activity was high in sham group of cerebral ischemia, but significantly decreased after cerebral ischemia (P<0.05). Compared with cerebral ischemia group, the enzyme activity was further decreased in LIP + cerebral ischemia group (P<0.05). Pretreated with Ngb AS-ODNs before LIP significantly increased the enzyme activity when compared to LIP + cerebral ischemia group (P<0.05). However, administration with Ngb S-ODNs before LIP was without effect upon the enzyme activity. Compared with cerebral ischemia group, Hemin significantly decreased the enzyme activity (P<0.05), and no difference of the enzyme activity was found at Hemin vehicle group. Results of Na+-K+-ATPase activity at 24 h time point showed that Na+-K+-ATPase activity was high in sham group of cerebral ischemia, which significantly decreased in cerebral ischemia group (P<0.05). The enzyme activity was significantly increased by LIP, when compared to cerebral ischemia group (P<0.05). Compared to LIP + cerebral ischemia group, the application of Ngb AS-ODNs before LIP significantly decreased the enzyme activity (P<0.05), but Ngb S-ODNs had no influence. When the animals were pretreated with the Hemin before the lethal ischemic insult, the enzyme activity was significantly increased compared with that in cerebral ischemia group (P<0.05), and no evident effect was observed in Hemin vehicle + cerebral ischemia group.
RT-PCR analysis showed that Bcl-2 and Bax mRNA are less in the rat CA1 hippocampus in the sham group of cerebral ischemia, which was significantly up-regulated after cerebral ischemia (P<0.05). Pretreated with LIP before cerebral ischemia dramatically up-regulated the expression of Bcl-2 mRNA, but down-regulated the expression of Bax mRNA (P<0.05). Administration of Ngb AS-ODNs before LIP+cerebral ischemia significantly inhibited Bcl-2 mRNA expression and increased Bax mRNA expression (P<0.05). However, Ngb S-ODNs had no effect on the Bcl-2 and Bax mRNA expression. Ngb inducer Hemin could also significantly up-regulate the expression of Bcl-2 mRNA, and down-regulate the expression of Bax mRNA (P<0.05). While, pretreated with vehicle of Hemin before cerebral ischemia had no effect on the Bcl-2 and Bax mRNA expression.
Transmission electron microscopic examination showed that the structure of neurons and mitochondria in sham group of cerebral ischemia was normal. Cerebral ischemia for 8 min induced obvious destruction of the neuronal and mitochondrial ultrastructure. The neuron was swollen and cellular organs were significantly decreased; meanwhile, the mitochondria were swollen or pyknosis and its cristae were broken, dissolved and disappeared. But in LIP + cerebral ischemia group, the above injury changes were prevented clearly. When the animals were pretreated with Ngb AS-ODNs prior to LIP + cerebral ischemia, the protective effect of LIP was prevented, while, Ngb S-ODNs had no effect on them. Ngb inducer Hemin significantly inhibited destruction of the neuronal and mitochondrial ultrastructure induced by cerebral ischemia, but Hemin vehicle had no effect on the cerebral ischemia induced destruction of the neuronal and mitochondrial ultrastructure.
The above results indicated that the up-regulation of Ngb expression participated in the brain ischemic tolerance induced by LIP via the mechanisms involving in modulated mitochondrial trans-membrane potential, Na+-K+-ATPase activity of mitochondria, Bcl-2 and Bax mRNA expression, neurons and mitochondrial ultrastructure.
3 The expression of Ngb in the CA1 hippocampus after cerebral ischemia and the effect of LIP on it in young and aged rats
Using four-vessel occluding global brain ischemia model, the role of age on the Ngb expression and the LIP neuroprotection against severe ischemic insult was investigated by observing the Ngb expression in the CA1 hippocampus after cerebral ischemia and the effect of LIP on it in young and aged rats.
Methods: Sixty male Spague-Dawley rats aged 3 months (thirty) and 21-23 months (thirty) with permanently occluded bilateral vertebral arteries were randomly divided into cerebral ischemic group and LIP + cerebral ischemic group, respectively. The expression of Ngb mRNA and protein in the CA1 hippocampus were investigated by RT-PCR and Western blot methods. The profile of DND of pyramidal neurons in the CA1 hippocampus was evaluated by using thionin staining.
Results: RT-PCR and Western blot analysis showed that the expression of Ngb mRNA and protein after cerebral ischemia for 8 min in aged rats was decreased compared with that in the young rats which suffered a identical cerebral ischemia with the aged rats (P<0.05). LIP upregulated Ngb mRNA and protein expression in both young and aged rats suffered cerebral ischemia (P<0.05). However, the upregulation of Ngb expression in aged rats was significantly less than that in young rats (P<0.05).
Neuropathological evaluation showed that obvious DND of pyramidal neurons was found in young and aged rats after cerebral ischemia. Although LIP effectively protected the pyramidal neurons in the CA1 hippocampus against DND normally induced by ischemic insult (P<0.05), the neuroprotection of LIP for aged rats was less effective than that for young rats.
The above results indicated that the expression of Ngb and the up-regulating effect of LIP on the expression in aged rats were significantly decreased compared to those in young rats when the rats suffered cerebral ischemia.
4 LIP up-regulate the expression of Ngb in the normal rat CA1 hippocampus
To investigate whether LIP could induce the up-regulation of Ngb expression in CA1 hippocampus of normal rats, the study observed the time courses of the Ngb expression induced by LIP in the normal rat CA1 hippocampus.
Methods: Forty male Wistar rats were randomly divided into 2 groups:
①Sham group (n=8): The bilateral femoral arteries of the rats were dissected out and exposed for 60 min, but not clamped.
②LIP group (n=32): The bilateral femoral arteries of the rats were dissected out and clamped for 10 min 3 times in an interval of 10 min.
The expression of Ngb mRNA in the CA1 hippocampus was measured by RT-PCR analysis, according to the timing of reperfusion after LIP, rats in LIP group were divided into 4 subgroups: 30min, 1h, 3h and 6h. The expression of Ngb protein was measured by Western blot analysis, rats in LIP group were divided into 4 subgroups: 1h, 6h, 12h and 1d.
Results: RT-PCR analysis showed that basal expression of Ngb mRNA in the CA1 hippocampus could be observed in sham group. The Ngb mRNA expression was significantly up-regulated by LIP. The expression of Ngb mRNA peaked at 30 min (P<0.05), then gradually decreased at 1 h and 3 h, but still higher than that of sham group (P<0.05). The expression of Ngb mRNA returned to sham level at 6h after LIP.
Western blot analysis showed that compared with sham group, the Ngb protein expression was significantly up-regulated after LIP, which peaked at 1h (P<0.05), then gradually decreased at 6 h and 12 h, but still higher than that of sham group (P<0.05). The expression of Ngb protein at 1d after LIP was still higher than that of sham group, but no statistic difference when compared to sham group.
The above results indicated LIP could induce up-regulation of Ngb expression in CA1 hippocampus of normal rats.
5 Conclusions
(1) LIP and Ngb inducer Hemin could significantly up-regulate the expression of Ngb in the CA1 hippocampus in cerebral ischemic rat, meanwhile protect the pyramidal neurons in the area against cerebral ischemia insults and decreased the serum Ngb protein level. The above effects of LIP could be inhibited partly by Ngb AS-ODNs. These results indicated that the up-regulation of the Ngb expression played an important role in the brain ischemic tolerance induced by LIP.
(2) LIP effectively improved the deterioration in the mitochondrial trans-membrane potential, Na+-K+-ATPase activity of mitochondria, Bcl-2 and Bax mRNA expression, neuronal and mitochondrial ultrastructure induced by cerebral ischemic insult. The above effects of LIP could be inhibited partly by Ngb AS-ODNs. Ngb inducer Hemin could mimic the above effects of LIP. The results suggested that LIP plays neuronal protection, at least, through improving structure and function of mitochondria.
(3) The expression of Ngb and the up-regulating effect of LIP on it in aged rats were significantly decreased compared to those in young rats when the rats suffered cerebral ischemia. These differences might be one of underlying reasons why the aged rats exhibited severe DND after cerebral ischemia and why the neuroprotective effect of LIP was less in the aged rats than that in the young rats.
(4) LIP could induce up-regulation of Ngb expression in CA1 hippocampus in normal rats.
引文
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5 Zhang L, Li LH, Qu Y, et al. Neuroglobin and hypoxic-ischemic brain bamage. Zhongguo Dang Dai Er Ke Za Zhi, 2008, 10 (2):265-268
6 Wang X, Liu J, Zhu H, et al. Effects of neuroglobin overexpression on acute brain injury and long-term outcomes after focal cerebral ischemia. Stroke, 2008, 39 (6):1869-1874
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