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This research investigates the molecular mechanisms by which hypothermic machine perfusion protects DCD liver grafts from ischemia-reperfusion injury.

February 8, 2026Open Access

Hypothermic machine perfusion protects DCD graft liver from ischemia‑reperfusion injury by enhancing macrophage efferocytosis via KLF2‑NLRP3 signaling

Key Points

This research investigates the molecular mechanisms by which hypothermic machine perfusion protects DCD liver grafts from ischemia-reperfusion injury.
Used rat livers underwent DCD modeling and were subjected to either static cold storage or hypothermic machine perfusion.
Measured KLF2 expression, macrophage efferocytosis, and assessed injury via serum enzyme levels, histology, and immunofluorescence.
Performed in vitro studies on HUVECs and macrophages using simulated cold storage and reperfusion.
Utilized RNA sequencing and co-immunoprecipitation to explore underlying mechanisms.
HMP significantly reduced pathological injury, apoptosis, and inflammation in DCD livers compared to cold storage.
KLF2 expression was upregulated in the HMP group, while its knockdown negated protective effects in vitro.
Overexpression of KLF2 improved macrophage efferocytosis, with its suppression leading to impaired efferocytosis.
KLF2 inhibited the NLRP3 inflammasome, reducing pyroptosis and promoting inflammation resolution.

Abstract

Donation after circulatory death (DCD) is a key source of liver grafts but it is associated with more severe ischemia‑reperfusion injury (IRI) and poorer transplant outcomes compared with donation after brain death. Hypothermic machine perfusion (HMP) effectively decreases DCD graft injury, but its protective molecular mechanisms remain unclear. Kruppel‑like factor 2 (KLF2) is an endothelial protective transcription factor induced by hemodynamic mechanical stimulation. However, the role of KLF2 in IRI during HMP in DCD livers is unclear. Rat livers undergoing DCD modeling followed by static cold storage (CS) or HMP were used to assess KLF2 expression and macrophage efferocytosis. Injury was assessed by serum alanine transferase/aspartate transferase levels, histology, TUNEL apoptosis assay and immunofluorescence (IF) for in situ efferocytosis. Protein markers were analyzed via western blotting, immunohistochemistry and IF. In vitro, HUVECs and macrophages were subjected to simulated CS/reperfusion. Macrophages efferocytosis was quantified using fluorescently labeled apoptotic Jurkat cells. Mechanisms were explored by RNA sequencing and co‑immunoprecipitation. Compared with the CS group, HMP decreased pathological injury, apoptosis and inflammation in DCD liver injury. KLF2 expression was upregulated. However, knockdown of KLF2 abrogated these endothelial protective effects in vitro. Furthermore, overexpression of KLF2 enhanced macrophage efferocytosis, whereas suppression of KLF2 impaired this. Moreover, enhanced efferocytosis contributed to inflammation resolution, ultimately improving overall graft injury and decreasing apoptosis. Mechanistically, KLF2 inhibited the NOD‑like receptor protein 3 (NLRP3) inflammasome to suppress pyroptosis, thereby indirectly enhancing efferocytosis. HMP alleviated IRI in DCD liver grafts by upregulating endothelial KLF2, which inhibited NLRP3 inflammasome‑mediated pyroptosis, thereby improving the inflammatory microenvironment and promoting macrophage efferocytosis.

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