Mild hypothermic pretreatment alleviates hepatic ischemia-reperfusion injury by reducing CIRP-dependent NETosis-related hepatocyte pyroptosis.

BACKGROUND

Hepatic ischemia-reperfusion injury (HIRI) is a serious clinical complication with limited effective interventions. Although mild hypothermic pretreatment (MHP) has demonstrated protective potential, the precise molecular mechanisms, particularly its role in modulating sterile inflammation and programmed cell death, remain poorly understood.

METHODS

A murine model of 70% hepatic IR was established. Mice were pretreated with MHP (32 °C for 2 h) or normothermia. In vitro, AML-12 hepatocytes and isolated mouse neutrophils were subjected to oxygen-glucose deprivation (OGD) or stimulation with recombinant CIRP (rmCIRP). Inhibitors targeting CIRP (C23), TLR4 (TAK-242), and PAD4 (Cl-amidine) were used. Liver injury, inflammatory markers, NETosis markers (CitH3, PAD4), and pyroptosis (NLRP3, Caspase-1, GSDMD) were assessed via histology, ELISA, western blot, and immunofluorescence.

RESULTS

MHP significantly attenuated liver damage, oxidative stress, and systemic inflammation following IR. It reduced the release of CIRP, neutrophil infiltration, and NETosis markers (PAD4 and CitH3). In vitro, CIRP released from injured hepatocytes directly induced NETosis via the TLR4/PAD4 pathway, which was inhibited by C23, TAK-242, or Cl-amidine (Cl). Furthermore, NETs promoted hepatocyte pyroptosis by upregulating NLRP3, Caspase-1, and GSDMD. MHP or inhibition of the CIRP-TLR4-PAD4 axis effectively suppressed both NETosis and pyroptosis.

CONCLUSION

MHP protects against HIRI by inhibiting CIRP release, which subsequently reduces TLR4/PAD4-dependent NETosis and hepatocyte pyroptosis, thereby disrupting a self-amplifying inflammatory loop. These findings reveal a novel CIRP-NETs-pyroptosis axis as a key mechanism in HIRI and highlight potential therapeutic targets for liver I/R injury.