Figure 8. Broad-spectrum AR agonist NECA induces gradual and delayed downmodulation of P-gp expression and function in brain vascular endothelial cells in WT mice.

(A) Western blot analysis of P-gp and BCRP1 from mouse brain at 2 and 18 hours after NECA treatment. (B) Enumeration of expression intensity of P-gp (top graph) or BCRP1 (bottom graph) bands from NECA treatment from Western blot analysis. Intensity of bands from NECA treatment group was divided by that of DMSO control. Acquired values were normalized by GAPDH and graphed. *P < 0.05 (n = 3, 2-tailed Student’s t test). (C) IFA of P-gp in NECA-treated mouse brain at 2 and 18 hours after treatment. For IFA, brain frozen section were stained with GLUT1 (red) or P-gp (green) and counterstained with DAPI (blue). Scale bar: 100 μm. (D) Epirubicin brain accumulation assays in NECA-treated mice were performed after 0.08 mg/kg of NECA was injected intravenously for indicated time and, subsequently, 10 mg/kg of epirubicin was intravenously injected. At 15 minutes after epirubicin treatment, mice were perfused with ice-cold PBS and sacrificed at different time points. The accumulation of epirubicin in the brain was measured using fluorometry, with excitation at 488 nm and emission at 590 nm. *P < 0.05 (n = 4, 2-tailed Student’s t test).