Biliverdin alleviates cerebral ischemia-reperfusion injury by regulating the P4hb/MAPK/mTOR pathway to inhibit autophagy.

Background: Biliverdin (BV) exhibits anti-inflammatory and antioxidative effects. Autophagy activation is crucial in the pathogenesis of cerebral ischemia-reperfusion injury (CIRI). This study aimed to investigate whether BV could ameliorate CIRI by regulating autophagy.

Methods: A middle cerebral artery occlusion-reperfusion (MCAO/R) model in Sprague-Dawley (SD) rats and an oxygen-glucose deprivation/reoxygenation (OGD/R) model in PC12 cells were employed to explore the neuroprotective effects of BV and its underlying mechanisms. In these rats, once BV was administered post-MCAO/R, its treatment efficacy and underlying mechanisms were evaluated through behavioral, morphological, and molecular analyses. Alternatively, for PC12 cells, following successful OGD/R modeling, BV, autophagy activator rapamycin, prolyl 4-hydroxylase beta (P4hb) knockdown or overexpression, and the specific inhibitors of three classic autophagy pathways were applied. Cell viability (using CCK8 assay), Calcein/PI staining, autophagosome staining (using MDC assay), reverse transcription quantitative polymerase chain reaction, and western blot were subsequently carried out to investigate the mechanisms by which BV ameliorates CIRI.

Results: BV alleviated CIRI by inhibiting autophagy. Further investigation suggested that BV downregulated P4hb expression. In vitro experiments showed that P4hb knockdown reduced autophagy in post-CIRI cells, while its overexpression reversed the effects of BV. Rescue experiments indicated that MAPK pathway inhibitors counteracted the effects of P4hb overexpression on autophagy post-CIRI.

Conclusions: BV improves CIRI by regulating the P4hb/MAPK/mTOR signaling pathway to inhibit autophagy, offering a novel therapeutic strategy for ischemic stroke.