*Purpose: RXFP1 (RLX family peptide receptor 1) is a cognate receptor for relaxin (RLX), an insulin-like growth factor, with vasoactive/remodeling properties. We have documented cytoprotective function of RLX via glucocorticoid receptor (GR), another RLX receptor, in the mechanism of ischemia-reperfusion injury (IRI) in mouse and human orthotopic liver transplantation (OLT). However, whether/how RXFP1 may affect human OLT remains unknown. As the conventional murine OLT offers a limited translational utility, we have generated a novel humanized liver chimeric mouse model system in which human hepatocytes transplanted into a urokinase-type plasminogen activator/SCID mouse (PXB mouse) have replaced >90% of host hepatocytes. We reasoned the PXB mouse may serve as a powerful research tool for the molecular study of RXFP1 signaling in human IRI-OLT.

*Methods: Fifty-five adult primary human OLT patients were recruited under IRB protocol. Liver graft biopsies (2h post-reperfusion) were evaluated based on high (n=27) vs. low (n=28) RXFP1 levels (WB/RT-PCR). To evaluate the effect of RXFP1 signaling in hepatocytes, H₂O₂ (20mM)-stressed primary hepatocytes isolated from humanized PXB mice, were cultured/treated with: (i) recombinant human RLX; (ii) agonist of human RXFP1 (ML290); or (iii) RXFP1-inactivate RLX (DHCH-RLX).

*Results: The high-RXFP1 expressing clinical cohort showed longer warm ischemia time (p<0.05), lower anti-apoptotic Bcl-xL levels (p<0.05), increased ER-stress CHOP expression (p=0.06), with up-regulated macrophage/neutrophil activation, evidenced by TLR4, CXCL10, CD68, and Cathepsin G levels (p<0.05), as compared with low-RXFP1 cohort. This pro-inflammatory phenotype correlated with elevated serum AST levels at POD4-7. Unlike IR-stressed C57BL/6 mouse hepatocytes, humanized hepatocytes from PXB mice expressed RXFP1.H₂O₂-stressed humanized hepatocyte cultures suffered increased damage (AST level) with up-regulated RXFP1 gene expression profile. Adjunctive use of RLX and ML290 mitigated AST release in hepatocyte cultures, as compared to DHCH-RLX treatment. RLX and DHCH-RLX treatment decreased CHOP, while RLX and ML290 increased LC3B (autophagy marker) expression. These results are consistent with the efficacy of RLX to affect human hepatocyte function, consisting of ER stress regulation via GR signaling and autophagy enhancement via RXFP1 pathway.

*Conclusions: This is the first report to document RXFP1 function in human hepatocytes in a novel liver chimeric mouse system. Indeed, RXFP1 may serve not only as a biomarker of IR-stressed hepatocellular damage, but also as a novel target for homeostatic regulation in IRI-OLT human patients.

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