Figure 4.

The role of circ-FoxO3 in alleviation of BBB damage depends on autophagy

(A and B) Evans blue extravasation was detected in male mice with circ-FoxO3 knockdown or upregulation that were pretreated with Rapa or CQ for 1 week before MCAO/R. CQ, chloroquine, 5 mg/kg/daily. Rapa, rapamycin, 0.75 mg/kg/daily. Vehicle control, saline containing 5% polyethylene glycol 400 and 5% Tween 80. White arrowheads indicate cerebral hemorrhage. Four of five mice displayed cerebral hemorrhage. Scale bar, 2 mm. n = 5. (C) The level of BBB-related proteins such as ZO-1, Mfsd2a, and Cldn5 in BMECs (bEnd.3 and HBMEC) with circ-FoxO3 knockdown or overexpression was analyzed by immunoblotting when cells were pretreated with CQ (or Rapa) before OGD/R. Rapa, 50 nmol/L; CQ, 30 μmol/L. n = 4. p values indicate one-way ANOVA with Dunnett's multiple comparisons test. MCAO/R, middle cerebral artery occlusion for 2 h followed by reperfusion for 2 h; OGD/R, oxygen-glucose deprivation for 3 h followed by reoxygenation for 3 h. All data are presented as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.