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Article Open Access

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

  • Authors:
    • Qin Deng
    • Zhongzhong Liu
    • Qifa Ye
    • Jia Liu
    • Zhihui Fu
    • Xingjian Zhang
    • Jun Luo
    • Zhongshan Lu
    • Pengpeng Yue
    • Jiansheng Xiao
    • Qi Xiao
  • View Affiliations / Copyright

    Affiliations: Department of Transplantation, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi 330006, P.R. China, National Quality Control Center for Donated Organ Procurement, Hubei Key Laboratory of Medical Technology on Transplantation, Hubei Clinical Research Center for Natural Polymer Biological Liver, Hubei Engineering Center of Natural Polymer‑Based Medical Materials, Zhongnan Hospital of Wuhan University, Institute of Hepatobiliary Diseases of Wuhan University, Transplant Center of Wuhan University, Wuhan, Hubei 430071, P.R. China
    Copyright: © Deng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 85
    |
    Published online on: February 5, 2026
       https://doi.org/10.3892/ijmm.2026.5756
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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.

View Figures

Figure 1

Experimental design and timeline. The
timeline of key procedural steps is illustrated, including the
initiation of the DCD process, organ procurement, preservation
period and reperfusion phase. NMP, normothermic machine perfusion;
CS, cold storage; HMP, hypothermic machine perfusion; DCD, donation
after circulatory death; PU, putrescine.

Figure 2

HMP improves donation after
circulatory death donor liver injury. Levels of (A) ALT and (B)
AST. (C) Histological scores were analyzed based on Suzuki's
criteria (n=6). (D) Hematoxylin and eosin staining of rat liver
tissue. (E) Hepatocyte microsomal modification was investigated via
transmission electron microscopy (n=3). ***P<0.001.
HMP, hypothermic machine perfusion; ALT, alanine aminotransferase;
AST, aspartate aminotransferase; CS, cold storage.

Figure 3

HMP inhibits hepatocyte apoptosis in
donation after circulatory death donor livers. (A) IF staining of
MPO was performed on paraffin-embedded rat liver tissues. (B) IF of
MPO was quantified. Expression levels of mRNA for (C) IL-1β, (D)
IL-6 and (E) TNF-α in liver tissue were quantified using reverse
transcription-quantitative PCR. (F) Hepatocyte apoptosis was
assessed using TUNEL assay. (G) Number of TUNEL-positive cells was
quantified. (H) Immunohistochemical staining of caspase-3 was
performed on paraffin-embedded rat liver tissues. (I)
Caspase-3-positive cells were quantified (n=6). (J) Expression
levels of (K) BAX/BCL2, (L) PUMA and (M) GADD45A in rat liver
tissue were assessed using western blotting (n=3).
*P<0.05, **P<0.01,
***P<0.001. HMP, hypothermic machine perfusion; IF,
immunofluorescence; MPO, myeloperoxidase; PUMA, P53 upregulated
modulator of apoptosis; GADD45A, growth arrest and DNA
damage-inducible alpha; CS, cold storage.

Figure 4

HMP-induced shear stress upregulates
KLF2 expression. (A) Representative fluorescence staining showing
the presence of KLF2 in rat liver tissue. (B) Number of
KLF2-positive cells (n=6). (C) Expression levels of KL2 in rat
liver tissue were assessed using western blot analysis and (D)
quantified using a Gel-Pro Analyzer. (E) Schematic diagram of an
in vitro parallel plate flow chamber simulating in
vivo laminar flow stimulation. (F) HUVECs were stained with
Calcein-AM/PI to assess (G) cell viability. (H) Expression levels
of KL2 protein in HUVECs subjected to laminar flow stimulation
in vitro were assessed using western blot analysis and (I)
quantified using a Gel-Pro Analyzer. (J) Representative flow
cytometry of apoptosis in CS/Rep model HUVECs (K) Apoptosis from
HUVECS of ov-control and ov-KLF2 groups was performed using
Image-pro plus 6.0. (n=3). (L) Statistical analysis of apoptosis
from HUVECS of sh-control and sh-KLF2 groups. was performed using
Image-pro plus 6.0 (n=3). *P<0.05,
**P<0.01, ***P<0.001. HMP, hypothermic
machine perfusion; KLF2, Kruppel-like Factor 2; HUVEC, human
umbilical vein endothelial cell; ov, overexpression; sh, short
hairpin ; CS, cold storage.

Figure 5

Upregulation of the endothelial cell
protective molecule KLF2 promotes macrophage efferocytosis. (A)
In vivo assessment and (B) quantification of efferocytosis
(proportion of CD68-positive cells engulfing TUNEL-positive
apoptotic cells relative to the total CD68-positive cell
population). Arrows represent macrophage efferocytosis events. (C)
Schematic diagram of the co-culture protocol for HUVECs with
varying KLF2 expression levels and macrophages. (D) Macrophages
(CMTPX+) were co-cultured with apoptotic Jurkat cells
(CMFDA+) for 45 min, and efferocytosis was
quantitatively assessed in vitro by fluorescence
co-localization analysis. (E) Statistical analysis of fluorescence
signals from HUVECS of ov-control and ov-KLF2 groups was performed
using Image-Pro Plus 6.0 software (n=3). (F) Statistical analysis
of fluorescence signals from HUVECS of sh-control and sh-KLF2
groups was performed using Image-Pro Plus 6.0 software (n=3). (G)
Representative staining of TIMD4. (H) Immunofluorescence of TIMD4
from HUVECS of ov-control and ov-KLF2 groups was quantified using
Image-pro Plus 6.0. (I) Immunofluorescence of TIMD4 from HUVECS of
ov-control and ov-KLF2 groups was quantified using Image-pro Plus
6.0 (n=3). *P<0.05, **P<0.01,
***P<0.001. HMP, hypothermic machine perfusion; KLF2,
Kruppel-like Factor 2; AC, apoptotic Jurkat cell; HUVEC, human
umbilical vein endothelial cells; CMTPX, CellTracker™ Red CMTPX
dye; CMFDA, 5-Chloromethylfluorescein Diacetate; CS, cold storage;
Rep, reperfusion; TIMD4, T-cell immunoglobulin and mucin
domain-containing protein 4; ov, overexpression; sh, short
hairpin.

Figure 6

Increased macrophage efferocytosis
improves the quality of donation after circulatory death donor
livers. (A) Evaluation and (B) quantification of in vivo
efferocytosis. Levels of (C) ALT and (D) AST in serum. (E)
Hematoxylin and eosin staining and (F) Suzuki score in rat liver
tissue (n=6). (G) Expression levels of (H) BCL2, BAX, (I) PUMA and
(J) GADD45A. **P<0.01, ***P<0.001. AST,
aspartate transferase; ALT, alanine transferase; PUMA, P53
upregulated modulator of apoptosis; GADD45A, growth arrest and DNA
damage-inducible α; CS, cold storage; PU, Putrescine.

Figure 7

KLF2 inhibits the NLRP3-mediated
pyroptosis pathway during CS/Rep in ECs. (A) Heatmap and (B)
volcano plot of differentially expressed genes between control and
ov-KLF2 HUVECs after CS/Rep treatment. mRNA levels of (C) KLF2 and
(D) NLRP3 in ov-control and ov-KLF2 group of HUVECs following
CS/Rep treatment were quantified using reverse
transcription-quantitative PCR. (E) Co-IP analysis of the
interaction between KLF2 and NLRP3. (F) Microstructure of HUVECS.
(G) Representative Western blot images for KLF2, NLRP3, GSDMD,
Caspase-1 and IL-18 in CS/Rep HUVECs. Quantitative analysis of KLF2
(H), NLRP3 (I), GSDMD (J), Caspase-1 (K) and IL-18 (L) protein
expression based on western blot results from HUVECS of ov-control
and ov-KLF2 groups. Quantitative analysis of KLF2 (M), NLRP3 (N),
GSDMD (O), Caspase-1 (P) and IL-18 (Q) protein expression based on
western blot results from HUVECS of sh-control and sh-KLF2 groups.
(n=3). *P<0.05, **P<0.01,
***P<0.001. KLF2, Kruppel-like Factor 2; CS/Rep, cold
storage/reperfusion; HUVEC, human umbilical vein endothelial cells;
ov, overexpression; IP, immunoprecipitation; GSDMD, gasdermin D;
sh, short hairpin;; FC, fold-change.

Figure 8

Pyroptosis of ECs inhibits the
occurrence of efferocytosis in macrophages. (A) Experimental
protocol of nigericin treatment. (B) Morphology and structure of
HUVECs treated with cell pyroptosis agonist nigericin were
investigated by bright-field imaging (n=3). (C) Schematic diagram
of the procedure for adding nigericin to the HUVEC-macrophage
co-culture system. (D) Representative staining images showing
efferocytosis in macrophages underwent non-contact co-culture with
HUVECs treated with nigericin. (E) Quantitative analysis of
efferocytosis in macrophages. (n=3). *P<0.05,
**P<0.01, ***P<0.001. AC, apoptotic
Jurkat cell; HUVEC, human umbilical vein endothelial cell; CMTPX,
CellTracker™ Red CMTPX dye; CMFDA, 5-Chloromethylfluorescein
Diacetate.
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Copy and paste a formatted citation
Spandidos Publications style
Deng Q, Liu Z, Ye Q, Liu J, Fu Z, Zhang X, Luo J, Lu Z, Yue P, Xiao J, Xiao J, et al: <p>Hypothermic machine perfusion protects DCD graft liver from ischemia‑reperfusion injury by enhancing macrophage efferocytosis via KLF2‑NLRP3 signaling</p>. Int J Mol Med 57: 85, 2026.
APA
Deng, Q., Liu, Z., Ye, Q., Liu, J., Fu, Z., Zhang, X. ... Xiao, Q. (2026). <p>Hypothermic machine perfusion protects DCD graft liver from ischemia‑reperfusion injury by enhancing macrophage efferocytosis via KLF2‑NLRP3 signaling</p>. International Journal of Molecular Medicine, 57, 85. https://doi.org/10.3892/ijmm.2026.5756
MLA
Deng, Q., Liu, Z., Ye, Q., Liu, J., Fu, Z., Zhang, X., Luo, J., Lu, Z., Yue, P., Xiao, J., Xiao, Q."<p>Hypothermic machine perfusion protects DCD graft liver from ischemia‑reperfusion injury by enhancing macrophage efferocytosis via KLF2‑NLRP3 signaling</p>". International Journal of Molecular Medicine 57.4 (2026): 85.
Chicago
Deng, Q., Liu, Z., Ye, Q., Liu, J., Fu, Z., Zhang, X., Luo, J., Lu, Z., Yue, P., Xiao, J., Xiao, Q."<p>Hypothermic machine perfusion protects DCD graft liver from ischemia‑reperfusion injury by enhancing macrophage efferocytosis via KLF2‑NLRP3 signaling</p>". International Journal of Molecular Medicine 57, no. 4 (2026): 85. https://doi.org/10.3892/ijmm.2026.5756
Copy and paste a formatted citation
x
Spandidos Publications style
Deng Q, Liu Z, Ye Q, Liu J, Fu Z, Zhang X, Luo J, Lu Z, Yue P, Xiao J, Xiao J, et al: <p>Hypothermic machine perfusion protects DCD graft liver from ischemia‑reperfusion injury by enhancing macrophage efferocytosis via KLF2‑NLRP3 signaling</p>. Int J Mol Med 57: 85, 2026.
APA
Deng, Q., Liu, Z., Ye, Q., Liu, J., Fu, Z., Zhang, X. ... Xiao, Q. (2026). <p>Hypothermic machine perfusion protects DCD graft liver from ischemia‑reperfusion injury by enhancing macrophage efferocytosis via KLF2‑NLRP3 signaling</p>. International Journal of Molecular Medicine, 57, 85. https://doi.org/10.3892/ijmm.2026.5756
MLA
Deng, Q., Liu, Z., Ye, Q., Liu, J., Fu, Z., Zhang, X., Luo, J., Lu, Z., Yue, P., Xiao, J., Xiao, Q."<p>Hypothermic machine perfusion protects DCD graft liver from ischemia‑reperfusion injury by enhancing macrophage efferocytosis via KLF2‑NLRP3 signaling</p>". International Journal of Molecular Medicine 57.4 (2026): 85.
Chicago
Deng, Q., Liu, Z., Ye, Q., Liu, J., Fu, Z., Zhang, X., Luo, J., Lu, Z., Yue, P., Xiao, J., Xiao, Q."<p>Hypothermic machine perfusion protects DCD graft liver from ischemia‑reperfusion injury by enhancing macrophage efferocytosis via KLF2‑NLRP3 signaling</p>". International Journal of Molecular Medicine 57, no. 4 (2026): 85. https://doi.org/10.3892/ijmm.2026.5756
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