Background: Autophagy (‘self-eating’) represents a critical mechanism for cellular homeostasis and survival response to the stress. We have shown that neuropeptide PACAP (Pituitary Adenylate Cyclase-Activating Polypeptides) regulates liver ischemia/reperfusion injury (IRI) by innervating the liver and immune cells. Here, we hypothesize that PACAP enhances parenchyma cell autophagy in liver IRI and promotes hepatocyte survival. Methods&Results: In our model of liver warm ischemia (90min) and reperfusion (0-24h), the kinetics of autophagic activity, represented by induction of LC-3a and Beclin-1, peaked at 6h of reperfusion, coinciding with the maximal hepatocellular damage. Treatment with PACAP ameliorated liver IRI, and enhanced hepatic autophagy, evidenced by increased LC-3a and Beclin-1 expression. Because of the functional linkage between cAMP-PKA signaling and CREB-KLF4 survival pathway, we focused at self-renewal KrÜppel-like transcription factor 4 (KLF4) that regulates proliferation, differentiation and cell survival. Indeed, KLF4 gene induction peaked in the early phase (1-2h) of liver IRI, and its frequency was enhanced by concomitant PACAP therapy. This effect was accompanied by up-regulation of CREB, enhanced autophagy, and HGF (Hepatocyte Growth Factor)/c-Met signaling. Strikingly, PACAP-facilitated autophagy was abolished and hepatocellular injury was restored after adjunctive treatment with CREB inhibitor (CAS92-78-4) or KLF4 inhibitor (Kenpaullone). Consistently, PACAP-induced autophagy further prevented hydrogen peroxide (H₂O₂)-triggered hepatocyte necrosis in primary hepatocyte cultures. The regulatory effect of PACAP on hepatocytes in vitro was confirmed by decreased cytoplasmic LDH release, increased conversion of LC3-I/LC3-II, and abundant visualized endogenous LC3 punctae. In contrast, addition of CREB or KLF4 inhibitors reversed PACAP-induced hepatocellular autophagy in vitro. Conclusion: This is the first study to provide evidence that harnessing neuroimmunomodulatory mechanisms by PACAP may: 1) promote hepatocellular autophagy against liver IRI; 2) enhance CREB-KLF4 signaling; 3) exert cytoprotrotection via integrated network of PACAP-cAMP/PKA axis and CREB/KLF4 binding protein in hepatocellular autophagy. Hence, PACAP-promoted hepatocyte survival may represent a refined therapeutic option to mitigate liver IRI in transplant recipients.

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