Prevention of tight-junction disruption via enhancement of ROBO4 attenuates influenza-induced blood-brain barrier injury

Abstract This study sought to elucidate the protective role of Roundabout 4 (ROBO4) in maintaining blood-brain barrier (BBB) integrity during influenza virus-induced barrier dysfunction. The functional role of ROBO4 was examined using both in vivo and in vitro influenza infection models. Specifically, mechanistic investigations focused on the viral-induced down-regulation of ROBO4 and its subsequent impact on the vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) signaling axis and key tight junction (TJ) proteins, including Claudin-1, Occludin, and Claudin-5. Additionally, lentiviral-mediated ROBO4 overexpression was utilized to validate its potential protective efficacy. The results showed that influenza virus infection significantly down-regulated ROBO4 expression, which in turn enhanced the interaction between VEGF and its receptor, VEGFR2. Consequently, this molecular event triggered the PI3K/AKT/eNOS signaling cascade, leading to the depletion of TJ proteins and subsequent impairment of BBB integrity. Notably, ROBO4 overexpression effectively inhibited the VEGF-VEGFR2 interaction, suppressed the activation of the PI3K/AKT/eNOS pathway, and restored the expression levels of Claudin-1 and Occludin, thereby preserving barrier function. Overall, by identifying ROBO4 as a critical regulator of BBB integrity, this study highlights its potential as a novel therapeutic target. Furthermore, these findings provide theoretical support for the development of preventative and interventional strategies to mitigate the neurological complications associated with influenza virus infections.