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

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/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+ 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±0.02 vs. 0.16±0.01; caspase-3: 0.76±0.05, 0.42±0.04, 0.52±0.02, 0.32±0.02, 0.40±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; all P=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±0.02 vs. 0.39±0.02; ZO-1: 0.08±0.01, 0.18±0.01, 0.10±0.01, 0.19±0.01 vs. 0.23±0.02; all P=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. Key words: Docosahexaenoid acid; Oxygen glucose deprivation; Cell apoptosis; Angiopoietin-1; Brain microvascular endothelial cells