*Purpose: Cold static preservation is a gold-standard in orthotopic liver transplantation (OLT). However, the role of hepatocyte-specific SIRT1 transcriptional regulation of cell death programs during cold liver preservation remains unclear. Recent studies show how apoptosis induces secondary pyroptosis via Gasdermin E (GSDME) regulation. Here we aimed to determine the mechanism by which hepatocyte SIRT1-mediated apoptosis-pyroptosis cell death affects OLT outcomes in mice and human.

*Methods: WT or hepatocyte-specific SIRT1-deficient (hSIRT1-KO) mouse livers, subjected to extended (18h) cold storage (CS; 4ºC/UW sol), were transplanted to WT mice (hSIRT1-KO>WT). Sample collection included liver/liver flush (after CS) and liver/serum samples (6h post-OLT). In vitro sampling of SIRT1-silenced hepatocytes (RNA interference) was conducted under CS (6h) conditions. In parallel, human liver biopsies (Bx) collected from OLT patients (n=60) before and after reperfusion (2h) were analyzed using Western immunoblot assays.

*Results: Donor livers deficient of SIRT1 showed significant evidence of aggravated IRI as compared to WT controls, and evidenced by sALT/sAST levels (n=12/12, p<0.05), Suzuki's histological grading of hepatocellular damage (p<0.05), and TUNEL-positive necrotic/apoptotic cells (p<0.05). These findings were supported by increased hepatic cleaved caspase-9 (cCasp9), cleaved caspase-3 (cCasp3), cleaved GSDME (cGSDME) (p<0.05) levels and impaired OLT survival (2-wks: 11% vs 44%, n=9/9, p=0.036). CS conditions triggered a decrease in anti-apoptotic Bcl-2 levels, and up-regulation of cCasp9, cGSDME, HMGB1 and IL18 in the liver flush, as compared to WT controls. In our in vitro study, SIRT1-silencing in CS primary hepatocyte cultures led to suppressed Bcl-2 but up-regulated BAX/cCasp9levels, and increased secretion of cCasp3/cGSDME/HMGB1/IL18. In the clinical arm, pre-reperfusion SIRT1 levels in human liver Bx positively correlated with Bcl-2 (r=0.4827, p=0.0003) protein expression, while post-reperfusion SIRT1 levels in OLT correlated negatively with cCasp3 (r=-0.3692, p=0.0187).

*Conclusions: This novel bench-to-bedside translational study documents how hepatocyte-specific SIRT1-Bcl-2 signaling axis regulates GSDME-mediated pyroptosis during cold-stress conditions. Our findings provide a putative therapeutic target against hepatocyte cell death programs in donor livers undergoing cold preservation, prior to OLT.

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