*Purpose: Pituitary adenylate cyclase-activating polypeptide (PACAP) is from vasoactive intestinal peptide-glucagon-secretin family. Our group was the first to demonstrate that neuropeptide PACAP, which innervates the hepatic cells, suppressed liver innate immune activation in liver warm ischemia and reperfusion injury (IRI).

*Methods: This study evaluated the pivotal role of PACAP promoted autophagy signaling in a murine model of extended hepatic cold storage and followed syngeneic orthotopic liver transplantation (OLT).

*Results: We first studied whether administration of PACAP neuropeptide protected liver against extended cold storage-mediated IRI and prolonged graft survival in a mouse syngeneic OLT model. Compared with 41.7% survival in control group, 91.7% of recipients conditioned with PACAP remained alive at 14 days post-OLT. Unlike PBS controls, PACAP treatment significantly diminished IRI-OLT, as shown by decreased sALT levels and preserved liver histology (without necrosis or congestion). PACAP therapy in IR-stressed livers enhanced hepatic autophagy induction, as assessed by expression of several key components (LC3, Beclin-1 and Atg5). Consistently, PACAP neuropeptide augmented LC3 expression, as well as promoted the LC3 I to LC3 II conversion, which was identified as the critical active step in autophagy pathway. We then determined the significance of PACAP-mediated hepatic autophagy in liver homeostasis by using an autophagy inhibitor 3-MA, which blocks the autophagosome formation in IR-stressed OLT. Autophagy inhibition diminished the induction of LC3 I, LC3 II and Beclin-1 and restored cardinal IRI-OLT features: increased sALT levels and deteriorated hepatic architecture. Of note, PACAP monotherapy-mediated autophagy amplified downstream hepatocellular regenerative genes: EGF, HGF and their receptor c-Met; whereas autophagy inhibition suspended hepatocyte repairing programs (EGF, HGF and c-Met) induced otherwise by PACAP-mediated autophagy. We then analyzed neural modulation of PACAP-mediated autophagy in a well-controlled primary hepatocyte culture stimulated by hydrogen peroxide, a system designed to mimic liver IRI in vivo. PACAP pre-treatment preferentially enhanced LC3 induction and accumulation in hepatocellular nucleus and cytoplasm, protected hepatocytes against oxidative stress: lower supernatant releasing of ALT/LDH, and prevented hepatocyte death. As 3-MA abolished LC3 accumulation in hepatocytes, autophagy inhibition exacerbated hepatocellular damage, evidenced by higher ALT/LDH and abundant dead hepatocytes.

*Conclusions: Our novel findings document the critical role of PACAP mediated autophagy in hepatic homeostasis and cytoprotection in liver IRI. Because the enhancement of hepatocellular autophagy differentially regulates local inflammation and hepatocyte survival, these results provide the rationale for emerging approaches to manage liver IRI in transplant patients.

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