Objective 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 oxygen 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,OGD + DHA 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 DMEM.DHA (40 μmol/L) was added to OGD + DHA,OGD + DHA + Ang-1,and OGD + DHA + GW9662 + Ang-1 groups,and 5 μmol/LGW9662 [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-1proteins 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 + DHA + GW9662 + Ang-1 groups were significantly 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 ± 0.02vs.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 vs.0.15 ±0.01;Tie-1:0.51 ±0.03,0.25 ±0.01,0.33 ±0.02,0.16±0.01,0.22±0.02 vs.0.12 ±0.01;all P=0.000);the expression levels of Bcl-2,Bcl-2/Bax,Tie-2,and pTie-2 were significantly decreased [Bcl-2:0.09±0.01,0.20±0.01,0.16±0.02,0.31±0.01,0.22±0.01 vs.0.34±0.01;Bcl-2/Bax:(14.93±1.86)％,(68.03±5.56)％,(36.93 ±2.22)％,(119.1 ±13.3)％,(64.23 ±6.07)％ vs.(208.33 ±7.37)％;Tie-2:0.07±0.01,0.16±0.02,0.11 ±0.01,0.21±0.01,0.18±0.01 vs.0.26±0.01;pTie-2:0.05±0.01,0.15 ±0.01,0.07 ±0.01,0.22 ±0.02,0.16±0.01 vs.0.27 ±0.01;allP=0.000],in addition,pAkt and ZO-1 in OGD,OGD + Ang-1,OGD + DHA,and OGD + DHA + GW9662 +Ang-1 groups were also significantly reduced (pAkt:0.13 ±0.01,0.26 ±0.01,0.14 ±0.01,0.28 ±0.02vs.0.39±0.02;ZO-1:0.08±0.01,0.18±0.01,0.10±0.01,0.19±0.01vs.0.23±0.02;allP=0.000).Compared with the OGD group,OGD + Ang-1 and OGD + DHA + Ang-1 groups demonstrated significantly reduced cell apoptosis (both P =0.000).Compared with normal control + Ang-1 group,the expression levels of pTie-2,pAkt,and ZO-1 were significantly decreased in OGD + Ang-1 and OGD + DHA +Ang-1 groups (pTie-2:0.15 ±0.01,0.22 ±0.02 vs.0.52 ±0.02;pAkt:0.26 ±0.01,0.37 ±0.04 vs.0.67 ± 0.05;ZO-1:0.18 ± 0.01,0.24 ± 0.02 vs.0.39 ± 0.05;all P =0.000).Compared with OGD +Ang-1 group,OGD + DHA + Ang-1 group had significantly decreased cell apoptosis and expression levels of Bax,caspase-3 and Tie-1,and significantly increased expressions of Bcl-2,Bcl-2/Bax,Tie-2,pTie-2,pAkt,and ZO-1 (all P =0.000),while OGD + DHA + GW9662 + Ang-1 group showed no significant differences in these indexes (P =0.202,0.770,0.382,0.448,0.233,0.736,0.143,0.526,0.495,0.670).Conclusion Pretreatment with DHA may enhance the protective effect of Ang-1 on rat BMVECs under the condition of OGD,possibly via activating PPAR-γ,suppressing Tie-1 expression,and hence amplifying the activation of Tie-2.%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组加入含血清、5 mmol/L葡萄糖和1.25 mmol/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40 μmol/L DHA,同时在OGD+ DHA+ GW9662+ Ang-1组再加入5 μmol/L GW9662[过氧化物酶体增殖物激活受体-γ(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250 ng/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,Western blot检测Bax、Bcl-2、半胱天冬酶-3(caspase-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、caspase-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±0.01、0.22±0.02比0.12±0.01;P均=0.000);Bcl-2、Bcl-2/Bax、Tie-2、pTie-2表达均显著降低[Bcl-2:0.09±0.01、0.20±0.01、0.16±0.02、0.31 ±0.01、0.22±0.01比0.34 ±0.01;Bcl-2/Bax:(14.93±1.86)％、(68.03±5.56)％、(36.93±2.22)％、(119.1±13.3)％、(64.23±6.07)％比(208.33 ±7.37)％;Tie-2:0.07±0.01、0.16±0.02、0.11 ±0.01、0.21±0.01、0.18±0.01比0.26±0.01;pTie-2:0.05±0.01、0.15±0.01、0.07±0.01、0.22±0.02、0.16±0.01比0.27±0.01;P均=0.000],OGD、OGD+ Ang-1、OGD+ DHA和OGD+ DHA+GW9662+ Ang-1组的pAkt、ZO-1表达亦显著降低(pAkt:0.13 ±0.01、0.26 ±0.01、0.14 ±0.01、0.28±0.02比0.39 ±0.02;ZO-1:0.08 ±0.01、0.18 ±0.01、0.10 ±0.01、0.19 ±0.01比0.23±0.02;P均=0.000).与OGD组比较,OGD+ Ang-1和OGD+ DHA+ Ang-1组细胞凋亡显著降低(P均=0.000).与正常对照+Ang-1组比较,OGD+ Ang-1和OGD+ DHA+ Ang-1组pTie-2、pAkt、ZO-1表达显著降低(pTie-2:0.15 ±0.01、0.22 ±0.02比0.52±0.02;pAkt:0.26±0.01、0.37±0.04比0.67±0.05;ZO-1:0.18±0.01、0.24±0.02比0.39 ±0.05;P均=0.000).与OGD+Ang-1组比较,OGD+ DHA+ Ang-1组细胞凋亡显著降低(P=0.000),Bax、caspase-3、Tie-1表达显著降低(P均=0.000),Bcl-2、Bcl-2/Bax、Tie-2、pTie-2、pAkt、ZO-1表达显著增加(P均=0.000);OGD+ DHA+ GW9662+ Ang-1组上述指标差异无统计学意义(P=0.202、0.770、0.382、0.448、0.233、0.736、0.143、0.526、0.495、0.670).结论 DHA预处理可提高Ang-1对OGD环境下大鼠BMVECs的保护作用,其机制可能与激活PPAR-γ、降低Tie-1表达,从而增强对Tie-2的激活作用相关.
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