Aims: To investigate the role of autophagy in the tight junction of human brain endothelial
cells during hypoxia and ischemia.
Background: Endothelial cells play an important role in the initiation, progression and recovery
from ischemic stroke. The role of autophagy on human brain endothelial cells (HBECs) subjected
to oxygen-glucose deprivation (OGD) is not fully elucidated.
Objective: The objective of this study was to investigate the effect of autophagy on HBECs during
Methods: HBECs were cultured in a 96-well plate and underwent 4 hours of OGD. For drug treatment,
3-Methyladenine (3-MA) (5mmol/L), an inhibitor of autophagy, was added at the start of
OGD. Cell viability and cytotoxicity were tested by cell counting kit-8 (CCK-8) and lactate dehydrogenase
(LDH) assays. Morphological changes in cells were examined by immunofluorescence
microscopy. The protein expression of light chain 3 (LC3) was measured. Autophagosomes and endothelial
cell tight junctions were observed using transmission electron microscopy.
Results: The results showed that OGD induced serious damage to HBECs. Cell viability was decreased
significantly and LDH release increased significantly (p<0.05) following OGD. 3-MA protected
HBECs from damage. Immunostaining further confirmed these results. Since 3-MA is an inhibitor
of autophagy, we chose to examine alterations in the amount of LC3, a marker of autophagy.
The ratios of LC3-II to LC3-I were significantly lower in the 3-MA treated OGD group than in
the non-3-MA treated OGD group (p<0.05). Electron microscopy showed that 3-MA inhibited the
formation of autophagolysosomes and revealed that the tight junction ultrastructure of HBECs,
which was destroyed by OGD, was significantly protected by treatment with 3-MA.
Conclusion: Autophagy is a key response to oxygen-glucose deprivation stress and its detrimental
effects are closely related to the destruction of tight junctions of human brain endothelial cells.
Strategies to inhibit autophagy could help to preserve tight junctions.