Pretreatment with docosahexaenoic acid enhances the protective effect of angiopoietin-1 on rat brain microvascular endothelial cells during oxygen glucose deprivation
10.3760/cma.j.issn.1674-635X.2015.02.007
- VernacularTitle: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
- Author:
Xiaobo CHEN
;
Zhongyuan XIA
;
Fan LEI
;
Rui XUE
- Publication Type:Journal Article
- Keywords:
Docosahexaenoid acid;
Oxygen glucose deprivation;
Cell apoptosis;
Angiopoietin-1;
Brain microvascular endothelial cells
- From:
Chinese Journal of Clinical Nutrition
2015;23(2):103-109
- CountryChina
- Language:Chinese
-
Abstract:
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.
