二十二碳六烯酸预处理增强血管生成素-1对氧糖剥夺条件下大鼠脑微血管内皮细胞的保护作用  

Pretreatment with docosahexaenoic acid enhances the protective effect of angiopoietin-1 on rat brain mi- crovascular endothelial cells during oxygen glucose deprivation

作　　者：陈小波[1] 夏中元[1] 雷凡[1] 薛锐[1] 

机构地区：[1]武汉大学人民医院麻醉科,430060

出　　处：《中华临床营养杂志》2015年第2期103-109,共7页Chinese Journal of Clinical Nutrition

摘　　要：目的评价二十二碳六烯酸（DHA）预处理是否增强血管生成素-1（Ang-1）对氧糖剥夺（OGD）条件下大鼠脑微血管内皮细胞（BMVECs）的保护作用。方法大鼠BMVECs传代培养，第3—4代用于实验。采用随机数字表法分为7组：正常对照组、正常对照＋Ang-1组、OGD组、OGD＋Ang-1组、OGD＋DHA组、OGD＋DHA＋Ang-1组、OGD＋DHA＋GW9662＋Ang-1组。在正常对照组和正常对照＋Ang-1组加人含血清、5mmol／L葡萄糖和1．25mmol／L丙酮酸盐的DMEM培养液；在各OGD组用无糖、无血清的DMEM液置换原培养液。在OGD＋DHA、OGD＋DHA＋Ang-1和OGD＋DHA＋GW9662＋Ang-1组加入40μmol／LDHA，同时在OGD＋DHA＋GW9662＋Ang-1组再加入5μmol／L GW9662[过氧化物酶体增殖物激活受体-1（PPAR-γ）的抑制剂]，以上3组在5％CO2和95％空气条件下预处理培养1h。预处理完成后，在正常对照＋Ang-1组、OGD＋Ang-1组、OGD＋DHA＋Ang-1组和OGD＋DHA＋GW9662＋Ang-1组加入浓度为250ng／ml的Ang-1。除正常对照组和正常对照＋Ang-1组在5％CO2和95％空气条件下培养外，其余各组均在94％N2：5％CO2：1％O2低氧条件下培养，培养时间均为24h。采用流式细胞术检测细胞凋亡情况，Westernblot检测Bax、Bel-2、半胱天冬酶-3（easpase-3）、血管生成素受体-1（Tie-1）、Tie-2、磷酸化的Tie-2（pTie-2）、磷酸化的蛋白激酶B（pAkt）和紧密连接蛋白-1（ZO-1）的表达水平。结果与正常对照组比较，OGD、OGD＋Ang-1、OGD＋DHA、OGD＋DHA＋Ang-1和OGD＋DHA＋GW9662＋Ang-1组细胞凋亡显著增加（P=0．000、0．000、0．000、0．004、0．000）；Bax、easpase．3、Tie-1表达显著增加（Bax：0．62±0．03、0．38±0．03、0．45±0．03、0．26±0．02、0．33±0．02比0．16±0．01；caspase．3：0．76±0．05、0．42±0．04、0．52±0．02、0．32±0．02、0．40±0．02比0．15±0．01；Tie-1：0．51±0．03、0．25±0．01、0．33±0．02、0．16±0．0Objective To evaluate the role of pretreatment with docosahexaenoic acid （DHA） in the protective effect of angiopoietin-1 （Ang-1 ） on rat brain microvascular endothelial cells （BMVECs） during oxy- gen glucose deprivation （OGD）. Methods BMVECs were sub-cultured in vitro and divided with a random number table into 7 groups ： normal control group, normal control ＋ Ang- 1 group, OGD group, OGD ＋ Ang- 1 group, OG]） ＋ ]）HA group, OGD ＋ DHA ＋ Ang-1 group, and OGD ＋ DHA ＋ GW9662 ＋ Ang-1 group. The normal control and normal control ＋ Ang- 1 groups were cultured in DMEM containing serum, 5 mmol/L glucose, and 1.25 mmol/L pyruvate; OGD groups were cultured in glucose- and serum-free ]）MEM. DHA （40 μmol/L） was added to OGD ＋ DHA, OGD ＋ DHA ＋ Ang- 1, and OGD ＋ DHA ＋ GW9662 ＋ Ang- 1 groups, and 5 μmol/L GW9662 [ inhibitor of peroxisome proliferator-activated receptor gamma （PPAR-γ） ] to OGD ＋ DHA ＋ GW9662 ＋ Ang-1 group, before pretreatment for 1 hour in 5% CO2 and 95% air. After the pretreatment, Ang-1 （250 ng/ml） was added to normal control ＋ Ang- 1, OGD ＋ Ang- 1, OGD ＋ DHA ＋ Ang- 1, and OGD ＋ DHA ＋ GW9662 ＋ Ang-1 groups. The cells were cultured in 94% N2： 5% CO2： 1% O2 for 24 hours, except for normal control and normal control ＋ Ang-1 groups, which were cultured in 5% CO2 and 95% air instead. The cell apoptosis rate was detected with flow cytometry, expressions of Bax, Bcl-2, caspase-3, Tie-1, Tie-2, pTie-2, pAkt, ZO-1 proteins with Western blot. Results Compared with normal control group; the cell apoptosis rate in OGD, OGD ＋ Ang- 1, OGD ＋ DHA, OGD ＋ DHA ＋ Ang- 1, and OGD ＋ ]）HA ＋ GW9662 ＋ Ang- 1 groups were signifi- cantly increased （P = 0. 000, 0. 000, 0. 000, 0. 004, 0. 000） ; the expression levels of Bax, caspase- 3, and Tie-1 were significantly enhanced （Bax： 0. 62 0. 03, 0. 38 0. 03,0.45 0. 03,0. 26 0. 02, 0. 33 0.02 vs. 0. 16 0. 01 ; caspase-3 ： 0. 76 0. 05, 0. 42 0. 04, 0. 52 0. 02, 0. 32 0.02, 0.

关 键 词：二十二碳六烯酸 氧糖剥夺 细胞凋亡 血管生成素-1 脑微血管内皮细胞 

分 类 号：R965[医药卫生—药理学]