磷脂酰肌醇3激酶／蛋白激酶B通过上调内皮型一氧化氮合酶参与远端缺血后处理的脑保护作用  

Phosphoinositide-3 kinase/protein kinase B pathway participates in the neuroprotection of remote ischemic postconditioning by up-regulating endothelial nitric oxide synthase

作　　者：彭蓓[1,2] 郭曲练[1] 叶治[1] 王娜[1] 郑利民[2] 

机构地区：[1]中南大学湘雅医院麻醉科,长沙410008 [2]北京大学深圳医院麻醉科

出　　处：《国际麻醉学与复苏杂志》2012年第10期668-673,共6页International Journal of Anesthesiology and Resuscitation

摘　　要：目的 探讨内皮型一氧化氮合酶（endothelial nitric oxide synthase, eNOS）及磷脂酰肌醇3激酶/蛋白激酶B（phosphatidylinositol-3 kinase/protein kinase B，PI3K/Akt）通路在远端缺血后处理（remote ischemic postconditioning, RIPoC）减少大鼠全脑缺血/再灌注（ischemia/reperfusion, I/R）损伤中的作用。方法 成年雄性SD大鼠100只，体重为200 g～250 g，按随机数字表法随机分为5组（每组20只）：假手术组（S组）、缺血/再灌注组（I/R组）、缺血/再灌注＋远端缺血后处理组（I/R＋RIPoC组）、左旋硝基精氨酸甲酯（L-NAME）＋缺血、再灌注＋远端缺血后处理组（L-NAME＋I/R＋RIPoC组），以及LY294002＋缺血、再灌注＋远端缺血后处理组（LY＋I/R＋RIPoC组）。采用四动脉阻断法建立大鼠全脑I/R模型。S组不制备全脑I/R模型；I/R＋RIPoC组、L-NAME＋I/R＋RIPoC组及LY＋I/R＋RIPoC组于再灌注开始行双侧股动脉缺血15 min，再灌注15 min，共3个循环。L-NAME＋I/R＋RIPoC组于脑缺血前10 min腹腔注射非选择性一氧化氮合酶（nitric oxide synthase, NOS）抑制剂L-NAME，LY＋I/R＋RIPoC组于脑缺血前10 min侧脑室注射PI3K特异性抑制剂LY294002。脑再灌注48 h时行海马CA1区NDA原位末端缺口标记技术（terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, TUNEL）阳性细胞计数，测定海马CA1区抗磷酸化的eNOS抗体（p-eNOS）、eNOS、p-Akt及Akt的蛋白水平，再灌注4 d时行Morris水迷宫实验，再灌注7 d时计算海马CA1区神经元密度。结果 与S组比较，I/R组、I/R＋RIPoC组、L-NAME＋I/R＋RIPoC组及LY＋I/R＋RIPoC组再灌注时海马CA1区凋亡细胞[（0.8±0.8）、（84.7±6.8）、（52.8±7.8）、（74.3±9.0）、（79.5±7.3）个/mm]增加（P〈0.01），行为学损伤增加（P〈0.01），神经元密度[（193±7）、（10±7）、（91±11）、（38±7）、（26±7）个/mm]降低（P〈0.01）。与I/R组比较，I/R＋RIPoC组再Objective To investigate the roles of endothelial nitric oxide synthase（eNOS） and phosphatidylinositol-3 kinase/protein kinase B（PI3K/Akt） pathway in the neuroprotection of remote ischemic postconditioning （RIPoC） on global cerebral ischemia/reperfusion （I/R） injury in rats. Methods One hundred male adult SD rats weighing 200 g -250 g were randomly divided into 5 groups （n=20 each）： group sham, group I/R, group I/R＋RIPoC, group L-NAME＋I/R＋RIPoC, group LY＋I/R＋RIPoC. Global cerebral I/R was induced by four-vessel occlusion. Group I/R＋RIPoC, group L-NAME＋I/R＋RIPoC and group LY＋I/R＋RIPoC received 3 cycles of 15 min ischemia in bilateral femoral arteries at the beginning of cerebral reperfusion followed by 15 min reperfusion. The group L-NAME＋I/R＋RIPoC：4-VO with coinjection of N-nitro-L-arginine methyl eater（L-NAME）, 5 mg/kg, in normal saline, intraperitoneslly, 10 min before 4-VO, then followed by RIPOC and 48 h and 7 d of reperfusioin. The group LY＋I/R＋RIPoC：I/R＋RIPoC with injection of LY294002（a highly selective inhibitor of PI3K, LY, 10 uL, 10 mol/L,in 3% DMSO,intracerebroventricularly, 10 min before 4-VO）, then followedby RIPOC and 48 h and 7 d of reperfusion. The rats were sacrificed at 48 h of cerebral reperfusion, and brains were removed for determination of neuronal apoptosis [by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） method] in hippocampal CA1 region and p-eNOS, eNOS, p-Akt and Akt expression （by Western blot） in hippocampal CA1 region. Morris water maze task was used to test the learning and memory function at 4 d of cerebral reperfusion, and the rats were sacrificed at 7 d of cerebral reperfusion, and brains were removed for determination of neuronal density in hippocampal CA1 region. Results Compared with group sham, the number of apoptotic neurons in CA1 region [（0.8±0.8）, （84.7±6.8）, （52.8±7.8）, （74.3±9.0）, （79.5±7.3）/mm] increased （P〈0.01）, learning and memory fu

关 键 词：再灌注损伤 脑 远端缺血后处理 一氧化氮合酶 磷脂酰肌醇3激酶/蛋白激酶B 

分 类 号：R614[医药卫生—麻醉学]