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

Abstract

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.

Conclusion

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