Apelin-13 prevents the effects of oxygen–glucose deprivation/reperfusion on bEnd.3 cells by inhibiting AKT–mTOR signaling

Abstract

Autophagy plays works by degrading misfolded proteins and dysfunctional organelles and maintains intracellular homeostasis. Apelin-13 has been investigated as an agent that might protect the blood–brain barrier (BBB) from cerebral ischemia/reperfusion (I/R) injury. In this study, we examined whether apelin-13 protects cerebral microvascular endothelial cells, important components of the BBB, from I/R injury by regulating autophagy. To mimic I/R injury, the mouse cerebral microvascular endothelia l cell line bEnd 3 undergoes the process of oxygen and glucose deprivation and re feeding in the process of culture. Cell viability was detected using a commercial kit, and cell migration was monitored by in vitro scratch assay. The tight junction (TJ) proteins ZO-1 and occludin; the autophagy markers LC3 II, beclin 1, and p62; and components of the AKT–mTOR signaling pathway were detected by Western blotting and immunofluorescence. To confirm the role of autophagy in OGD/R and the protective effect of apelin-13, we treated the cells with 3-methyladenine (3-MA), a pharmacological inhibitor of autophagy. Our results demonstrated that OGD/R increased autophagic activity but decreased viability, abundance of TJs, and migration. Viability and TJ abundance were further reduced when the OGD/R group was treated with 3-MA. These results indicated that bEnd.3 upregulates autophagy to ameliorate the effects of OGD/R injury on viability and TJs, but that the autophagy induced by OGD/R alone is not sufficient to protect against the effect on cell migration. Treatment of OGD/R samples with apelin-13 markedly increased viability, TJ abundance, and migration, as well as autophagic activity, whereas 3-MA inhibited this increase, suggesting that apelin-13 exerted its protective effects by upregulating autophagy.