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Published on: October 27, 2020
Mild hypothermic pretreatment alleviates hepatic ischemia-reperfusion injury by reducing CIRP-dependent
FuPing Cao1, XingJian Zhang1, QiFa Ye2
1Department of Transplantation, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang 330006, Jiangxi, China.
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.
Insights
Mild hypothermic pretreatment (MHP) protects against liver ischemia-reperfusion injury (HIRI) by blocking a novel inflammatory loop involving cold-inducible RNA-binding protein (CIRP), neutrophil extracellular traps (NETs), and pyroptosis. This discovery offers new therapeutic targets for HIRI.
Area of Science:
- Hepatology
- Immunology
- Cell Death Research
Background:
- Hepatic ischemia-reperfusion injury (HIRI) presents significant clinical challenges with limited treatment options.
- Mild hypothermic pretreatment (MHP) shows promise, but its underlying molecular mechanisms in sterile inflammation and programmed cell death require elucidation.
Purpose of the Study:
- To investigate the molecular mechanisms by which MHP protects against HIRI.
- To explore the role of cold-inducible RNA-binding protein (CIRP) in sterile inflammation and programmed cell death during HIRI.
Main Methods:
- A murine model of hepatic ischemia-reperfusion (IR) was established, with mice subjected to MHP or normothermia.
- In vitro studies utilized hepatocytes and neutrophils exposed to oxygen-glucose deprivation (OGD) or recombinant CIRP (rmCIRP).
- Pharmacological inhibitors targeting CIRP, TLR4, and PAD4 were employed, with assessments including liver injury, inflammatory markers, NETosis, and pyroptosis.
Main Results:
- MHP significantly reduced liver damage, oxidative stress, and inflammation post-IR.
- CIRP release from injured hepatocytes induced NETosis via the TLR4/PAD4 pathway, which MHP and targeted inhibitors suppressed.
- Neutrophil extracellular traps (NETs) were found to promote hepatocyte pyroptosis, forming a self-amplifying inflammatory loop.
Conclusions:
- MHP confers protection against HIRI by inhibiting CIRP-mediated NETosis and subsequent hepatocyte pyroptosis.
- The study identifies a novel CIRP-NETs-pyroptosis axis as a critical driver of HIRI.
- Targeting this axis presents a promising therapeutic strategy for liver I/R injury.
