Mechanism underlying docosahexaenoic acid-induced regulation of angiopoietin expression in human brain vascular pericytes subjected to oxygen-glucose deprivation and restoration: the relationship with SSeCKS

Abstract

Objective To evaluate the relationship between the mechanism underlying docosahexaenoic acid (DHA)-induced regulation of angiopoietin expression and Src-suppressed C kinase substrate (SSeCKS) in human brain vascular pericytes (HBVPs) subjected to oxygen-glucose deprivation and restoration (OGD/R). Methods HBVPs were seeded in 96-well or in 6-well plates at a density of 2×105 cells/ml and divided into 5 groups (n=18 each) using a random number table: control group (group C), OGD/R group, DHA group (group D), SSeCKS gene silencing group (group S) and SSeCKS gene silencing plus DHA group (group SD). The model of OGD/R injury was established as follows: the cells were subjected to O2-glucose deprivation for 24 h in glucose- and serum-free culture medium aerated with 94%N2-5%CO2-1%O2 followed by restoration of O2-glucose supply for 6 h in high-glucose DMEM culture medium in normal atmosphere.DHA was added at 1 h before hypoxia with the final concentration of 40 μmol/L in group D. Small interfering RNA induced SSeCKS gene silencing in S and SD groups.Subsequently, DHA with the final concentration of 40 μmol/L was added at 1 h before hypoxia in group SD.At 6 h of reoxygenation, the cell survival rate was determined by CCK-8 assay, the amount of LDH released was detected using ELISA, and the expression of SSeCKS, angiopoietin-1 (Ang-1) and Ang-2 was detected by Western blot. Results Compared with group C, the cell survival rate was significantly decreased, the amount of LDH released was increased, the expression of SSeCKS and Ang-1 was down-regulated, the expression of Ang-2 was up-regulated, and Ang-1/Ang-2 ratio was decreased in group OGD/R, and the expression of SSeCKS was down-regulated in group S (P 0.05). Compared with group D, the cell survival rate was significantly decreased, the amount of LDH released was increased, the expression of SSeCKS and Ang-1 was down-regulated, the expression of Ang-2 was up-regulated, and Ang-1/Ang-2 ratio was decreased in group SD (P<0.05). Conclusion The mechanism by which DHA increases the ratio of Ang-1/Ang-2 may be totally related to up-regulation of SSeCKS expression in HBVPs subjected to OGD/R. Key words: Docosahexaenoic acids; A kinase anchor proteins; Angiopoietins; Pericytes; Brain; Reperfusion injury