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TNFAIP3 and MLKL-Related Necroptosis Signatures in Liver Transplant Ischemia-Reperfusion Injury: Integrated
Weigao Pu1, Jianming Shi1, Jingyi Jiang1
1The Second Clinical Medical College, Lanzhou University, Lanzhou, Gansu, 730030, People's Republic of China.
Background And Aims:
Liver transplant ischemia-reperfusion injury (LIRI) is a major cause of early graft dysfunction. Necroptosis has been implicated in inflammatory tissue injury; however, its molecular characteristics and diagnostic relevance in LIRI remain unclear. This study aimed to identify necroptosis-related biomarkers and characterize their biological and immune-associated features.
Methods:
Five GEO datasets were integrated, with three used for training and two for external validation. Differential expression analysis, WGCNA, and a predefined necroptosis-related gene set were combined to identify candidate genes. Functional enrichment, protein-protein interaction, and immune infiltration analyses were performed. Machine learning approaches (SVM-RFE, LASSO, and random forest) were applied to identify diagnostic markers and construct a nomogram. An allogeneic DA-to-Lewis rat orthotopic liver transplantation model was used for in vivo validation via histological, ultrastructural, and molecular assays, including H&E staining, transmission electron microscopy, immunohistochemistry, RT-qPCR, and Western blotting.
Results:
Eight necroptosis-related candidate genes were identified and were enriched in inflammatory and immune-related pathways. Six of these genes were retained in the final nomogram, which achieved an apparent AUC of 0.908 in the training cohort and an externally validated AUC of 0.771. Among the candidates, TNFAIP3 showed the most consistent diagnostic performance, whereas MLKL/p-MLKL provided biologically relevant evidence consistent with necroptosis-related signaling changes. Immune analyses indicated substantial microenvironment remodeling associated with TNFAIP3 expression. In vivo, LIRI was confirmed by histopathological and ultrastructural alterations, elevated ALT and AST levels, increased mRNA levels of TNFAIP3 and MLKL, and increased TNFAIP3 protein expression, p-MLKL protein level, and p-MLKL/MLKL ratio.
Conclusion:
Necroptosis-related molecular alterations are closely associated with LIRI. TNFAIP3 and MLKL/p-MLKL may represent candidate necroptosis-associated markers in liver transplant ischemia-reperfusion injury, but their diagnostic utility, therapeutic relevance, and mechanistic relationship require further prospective clinical validation and functional experimental confirmation.
