Doxorubicin compromises blood-brain barrier integrity by suppressing annexin A1 expression

Annexin A1 (ANXA1) is essential for blood-brain barrier (BBB) integrity, and its expression may be negatively regulated by the AP-1/miR-196a axis, while doxorubicin (DOX) is known to disrupt the BBB - though the underlying mechanism linking these factors remains unclear. To investigate how DOX impairs BBB integrity, we performed in vitro experiments on bEnd.3 brain microvascular endothelial cells and in vivo experiments on juvenile CD1 mice. In vitro, cells were treated with 1 μM DOX for 48 h, with or without pretreatment with 50 μM AP-1 inhibitor (SR11302) or 50 nM miR-196a inhibitor (LNA-antimiR-196a) for 24 h; in vivo, mice received weekly intraperitoneal injections of 4 mg/kg DOX for 5 weeks, with or without 1 h pretreatment with 10 mg/kg AP-1 inhibitor (SP600125) or 5 mg/kg LNA-antimiR-196a. Western blot was used to detect the expression of BBB tight junction proteins (occludin, claudin-5, ZO-1) and pathway molecules (AP-1, miR-196a, ANXA1), and Evans blue extravasation assay was employed to evaluate BBB permeability. Results showed that DOX increased BBB permeability by downregulating tight junction proteins, an effect associated with reduced ANXA1 expression. Further analyses revealed that miR-196a directly targets ANXA1 to suppress its expression, and AP-1 promotes miR-196a upregulation, thereby mediating DOX-induced ANXA1 downregulation and subsequent BBB damage. Inhibition of AP-1 or miR-196a reversed these DOX-induced abnormalities. Collectively, our findings demonstrate that DOX compromises BBB integrity by activating the AP-1/miR-196a axis, which suppresses ANXA1 and ultimately disrupts tight junctions - providing a novel mechanistic basis for DOX-induced neurotoxicity.