PKM2 preconditioning protects endothelial cells from pyroptosis and BBB disruption via NRF2/TRX/TXNIP signaling in neonatal hypoxic-ischemic brain injury.

BACKGROUND

Neonatal hypoxic-ischemic brain damage (HIBD) is a leading cause of neurological deficits and death in neonates. In HIBD, the death of endothelial cells and disruption of the blood-brain barrier (BBB) are closely related to the severity of brain damage and long-term clinical outcomes. There is increasing evidence that a glycolytic enzyme, pyruvate kinase M2 (PKM2), is essential for managing metabolic processes in endothelial cells, but its role (and underlying molecular mechanism) in hypoxic-ischemic (HI)-associated endothelial cell metabolism, cell survival, and BBB function remains unknown.

METHODS

We established an in vivo HI-induced brain injury rat model and an in vitro model in which human cerebral microvascular endothelial cells (hCMECs) underwent oxygen-glucose deprivation (OGD). Infarct volume was measured and neurobehavioral tests were conducted to assess brain damage, and Evans blue extravasation and FITC-dextran were used to evaluate the BBB. RNA sequencing, qRT-PCR, western blotting, and immunofluorescence labeling were conducted to identify the molecular mechanisms underlying HIBD.

RESULTS

PKM2 expression was upregulated in the brains of HIBD rats and in OGD-treated hCMECs. The inhibition of PKM2 greatly upregulated the expression of pyroptosis-associated proteins, including NLRP3, cleaved caspase-1, GSDMD, IL-1β, and IL-18. In contrast, the activation of PKM2 preserved junctional proteins and maintained the integrity of the BBB, which together improved functional recovery in HIBD rats. Mechanistically, preconditioning of PKM2 contributed to lactate-mediated cellular defense mechanisms, including the activation of nuclear factor erythroid 2-related factor 2 (NRF2) and thioredoxin (TRX), and to the downregulation of thioredoxin-interacting protein (TXNIP) via a modest increase in reactive oxygen species.

CONCLUSIONS

Our analyses provide compelling evidence that PKM2 preconditioning attenuates endothelial cell pyroptosis and BBB disruption in neonatal HIBD by causing oxidative stress resistance and activating the NRF2/TRX/TXNIP pathway. Therefore, PKM2 represents a promising pharmacological target for treating HIBD.