The Research Of PACAP Attenuates Liver Ischemia-reperfusion Injury In Mouse Orthotopic Liver Transplantation

Background: Liver ischemia reperfusion injury(IRI),an innate immunity-mediated sterile inflammation,represents a major complication in liver transplantation,hemorrhagic shock and partial hepatectomy.It may lead to primary graft dysfunction and acute rejection episodes,which further deteriorate the organ shortage.Liver IRI disturbs local blood circulation,generates reactive oxygen species(ROS),assembles peripheral neutrophils/macrophages,and inflicts tissue damage resulting in ultimate organ failure.Our group was the first to demonstrate that neuropeptide PACAP,which innervates the hepatic cells,suppressed liver innate immune activation in a mouse model of liver warm ischemia followed by reperfusion.The current study examined whether and how PACAP promoted hepatocellular autophagy to restore hepatocellular homeostasis in a clinically authentic model of hepatic cold IRI-OLT.Autophagy,an intracellular self-digesting progress degrading damaged organelles and malformed proteins by lysosomes,provides essential supply(amino acids,lipids and free fatty acid)to starved cells for energy metabolism and biosynthesis.As various cargo transported to the lysosome,three autophagy forms have been identified,i.e.,macroautophagy,microautophagy,and chaperone-mediated autophagy.Macroautophagy,the main autophagic pathway,could be divided into six principle steps to form the autophagosome: initiation,nucleation,elongation,closure,maturation and degradation or extrusion.Fusing with lysosome,the autophagosome turns to autolysosome,in which the enclosed cytoplasmic materials are eventually degraded into active monomers.As liver is rich in lysosome,dysregulation of macroautophagy(hereafter referred to as autophagy)is associated with multiple hepatic diseases,i.e.,IRI,non-alcoholic steatohepatitis and liver cancer.In liver transplantation,both warm and cold ischemia insults mainly interrupt the blood flow/oxygen supply,deplete hepatocellular adenosine triphosphate and produce ROS.This hepatocellular starvation significantly triggers autophagy signaling,which subsequently provides reused monomers to maintain cellular homeostasis.Although liver IRI is considered as a local sterile innate response,both immune and nervous systems are integrated to maintain dynamic communication.IR-triggered inflammation was shown to activate systemic neuroendocrine hypothalamus and regional neural-hormonal–stress responses.Neural regulation optimizes,monitors and adjusts immune response,as a pivotal feedback loop by stimulation of efferent vagus nerve activity.After elimination of invasion,the neural system modulates local immune response towards host homeostasis.PACAP,encoded by the ADCYAP1 gene,is a member of vasoactive intestinal peptide(VIP)/glucagon/secretin family.PACAP,contains two bioactive forms as 27 or 38 amino acid residues,functions as a neurotransmitter and neuromodulator in hypophysiotropic area,and involves in paracrine and autocrine regulation of gonadotrophs.Consistent with others' finding that PACAP exhibits neuroregulation on innate immunity,we have reported that partial warm IR triggered the inductions of hepatic PACAP and all three receptors in IR-stressed liver,whereas PACAP deficiency sensitized liver against IR-stress.Indeed,exogenous PACAP neuropeptide regulated the local innate immunity by diminishing neutrophil/macrophage sequestration in IR-liver and differentially modulating pro-inflammatory programs.CREB,binding to c AMP response elements,is a critical cellular transcription factor in neuroregulation and neuron survival.In innate immunity,CREB ameliorates TLR4 response by regulating macrophage polarization in infection and insulin resistance.Recently,CREB was identified as an important regulator in hepatic autophagy gene network,promoting autophagic degradation of lipids.We have recently shown that PACAP inhibited macrophage NF-?B activity by enhancing c AMP-PKA axis and CREB activity.KLF4,the crucial self-renewal transcriptional factor of KLF family,exerts diverse modulation in proliferation,differentiation,and somatic cell reprogramming.In addition to its crucial function in pluripotent stem cell induction,KLF4 is over-expressed in DNA-damaged non-dividing cells,and significant in inducing cell cycle arrest/preventing cell division.Indeed,KLF4 regulates macrophage polarization by cooperating with Stat6 to induce M2 gene profile and sequestering NF-?B activators to suppress M1 phenotype.Conversely,KLF4 extends the lifespan in nematode and modulates aged vascular dysfunction in mice by promoting autophagy.Putative synergy between KLF4 and CREB in modulation of innate immunity was discovered in mycobacterium tuberculosis.This study elucidated the cytoprotective role of PACAP-mediated autophagy in a clinically authentic mouse model of extended hepatic cold storage and followed syngeneic OLT.First,we determined whether and how PACAP neuropeptide protected liver grafts against extended cold storage in an autophagy-dependent manner.The question then arose whether PACAP-mediated autophagy could preserve hepatocyte viability,and promote hepatic regeneration/repair.Finally,we addressed whether PACAP-mediated CREB and KLF4 coactivation was essential for hepatic autophagy in liver IRI.Objectives: This study elucidated the cytoprotective role of PACAP-mediated autophagy in a clinically authentic mouse model of extended hepatic cold storage and followed syngeneic OLT.First,we determined whether and how PACAP neuropeptide protected liver grafts against extended cold storage in an autophagy-dependent manner.The question then arose whether PACAP-mediated autophagy could preserve hepatocyte viability,and promote hepatic regeneration/repair.Finally,we addressed whether PACAP-mediated CREB and KLF4 coactivation was essential for hepatic autophagy in liver IRI.Methods: 1.Animals.Male 8-12 weeks old wild-type(WT)mice were used.2.Liver extended cold storage followed by syngeneic OLT model.Liver grafts from C57BL/6 mice were stored in 4 °C UW solution for 20 h,and then transplanted orthotopically into syngeneic C57BL/6 mice.Groups of recipients were given PBS,PACAP38,or 3-Methyladenine(3-MA)via portal vein at the time of liver graft procurement and immediately prior to reperfusion.OLT survival was observed and the serum and OLT samples were collected at 6 h and 24 h post-transplant.3.Partial liver warm IRI model.The artery and portal vein of cephalad lobes(70% of whole liver)were clamped by an atraumatic clip for 90 min.A single dose of PACAP38,CRP-CREB Interaction Inhibitor(CREB inhibitor),or KLF4 inhibitor(Kenpaullone)was given 1 h before warm ischemia.Ischemic liver tissue and serum samples were harvested at 6 h post reperfusion.4.Liver function and histology.Serum alanine transaminase(s ALT)indicated liver function were assessed by IDEXX Lab(Sacramento,CA).Hematoxylin and eosin-stained liver sections and electron microscopy imagine were analyzed blindly for liver histology.5.Histopathology.Liver specimens(4?m),stained with hematoxylin and eosin(H&E),were analyzed blindly to measure the structure injury of liver.6.Propidium Iodide(PI)staining.PI staining(Thermo Fisher)was used to detect dead cells by binding DNA.Red fluorescence images were merged with bright-field images for blindly evaluation.7.Quantitative RT-PCR.Quantitative PCR was performed by using Platinum SYBR green quantitative PCR kit and Quant Studio3 Real-time PCR system(Thermo Fisher).The used primer sequences were published [31-37] or shown in Table S1: LC3 a,LC3b,Beclin-1,EGF,HGF,c-Met,KLF1,KLF2,KLF3 and KLF4.The ratios of target gene induction to housekeeping gene HPRT were calculated.8.Western blots.Ischemic liver tissue proteins,after electrophoretic separation and transfer,were detected by monoclonal antibodies of Atg5(12944),LC3(4108),p62(39749),proliferating cell nuclear antigen(PCNA)(13110),Ki67(9129),p CREB(9198),KLF4(12173),and ?-actin(4970)(Cell Signaling Technology,Danvers,MA).9.Immunofluorescence staining.Stressed hepatocytes were stained with LC3(4108),p CREB(9198)and KLF4(12173)monoclonal antibodies(Cell Signaling Technology)followed with Alexa Fluor 488 secondary antibody(A11008),Alexa Fluor 555 F-actin conjugate(A34055)for cellular skeleton,and DAPI counterstain(P36931)(Thermo Fisher).10.LDH/ALT Release Assay.LDH/ALT release into culture medium was measured to evaluate liver IRI.11.Cell culture and H2O2 stressed hepatocytes assay.After liver collagenase perfused digestion in situ,primary hepatocytes were separated by Percoll gradient centrifugation,and then cultured in collagen-coated plate for 24 h.Pre-treatment with PACAP38(10 n M),with 3-MA(10 ?M),CREB inhibitor(10 ?M),KLF4 inhibitor(10 ?M),DMSO for 1 h,KLF4 siRNA or NC siRNA(transfected by Lipofectamine 2000,Thermo Fisher),hepatocellular necrosis was triggered by hydrogen peroxide(H2O2: 0.4 m M,Millipore Sigma).Cells were processed for immunofluorescence and PI staining after 6 h,whereas supernatants were assessed by ALT/LDH kit(Thermo Fisher).12.Statistical analysis.Liver graft survival curves were plotted by Kaplan-Meier survival analysis and Log-rank comparison test.All results were indicated by mean ± standard deviation,and analyzed with Student's t test and ANOVA test.Statistical significant was identified as P<0.05.Results: 1.PACAP attenuates hepatic cold IRI and promotes OLT survival.First,we 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 PBS control group,91.7% of recipients conditioned with PACAP remained alive at 14 days post-OLT.As cold IR-exacerbated hepatocellular damage peaks at 6 h post-reperfusion,we then assessed OLT and sera samples at 6 h and 24 h.Similar as Rapamycin treated group,PACAP treatment significantly diminished IRI-OLT,as shown by decreased s ALT levels and preserved liver histology.2.PACAP promotes hepatic autophagy.PACAP therapy in IR-stressed livers enhanced hepatic autophagy induction,as assessed by expression of several key components(LC3,Beclin-1 and Atg5),as compared with controls.Consistently,PACAP neuropeptide augmented the expression level of LC3,diminished p62 level,as well as promoted the LC3 I to LC3 II conversion,which was identified as the critical active step in autophagy pathway.Electron micrographs of autophagosome in liver graft at 6 h post of OLT were tested.3.PACAP-mediated cytoprotection/regeneration in OLT is autophagy-dependent.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,i.e.,increased s ALT levels and deteriorated hepatic architecture.Of note,PACAP monotherapy-mediated autophagy amplified downstream hepatocellular regeneration,i.e.,PCNA,Ki67,epidermal growth factor(EGF),hepatocyte growth factor(HGF)and their receptor c-Met,as compared with controls;whereas autophagy inhibition suspended hepatocyte repairing programs(PCNA,Ki67,EGF,HGF and c-Met)induced otherwise by PACAP-mediated autophagy.4.PACAP-mediated autophagy prevents hepatocyte death.We then analyzed neural modulation of PACAP-mediated autophagy in a well-controlled primary hepatocyte culture stimulated by hydrogen peroxide(H2O2),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,as shown by lower supernatant releasing of ALT/LDH,and prevented hepatocyte death.As 3-MA abolished LC3 accumulation in hepatocytes(Figure 2A: no green fluorescence),autophagy inhibition exacerbated hepatocellular damage,evidenced by higher ALT/LDH and abundant PI positive hepatocytes.5.PACAP-CREB axis is critical for hepatic autophagy in liver IRI.As we have shown PACAP activated PKA-c AMP-CREB signaling in liver IRI,we consistently detected hepatic p CREB activation in cold-stored OLT;and oxidative stressed hepatocyte cultures.We then used a well-established murine model of partial liver warm ischemia(90 min)and reperfusion(6 h)to study the mechanism of PACAP-CREB axis-mediated autophagy in IR-damage.In agreement with our previous results,PACAP pre-treatment strongly depressed IR-mediated hepatocellular damage;while CREB inhibition restored hepatic IR-damage,as shown by enhanced s ALT levels and exacerbated IR-injured histology.In parallel,CREB inhibition enhanced pro-inflammatory immune profile while reducing IL-10 level.6.CREB is required for PACAP-mediated hepatic autophagy in liver IRI.As PACAP promoted hepatic autophagy in liver IRI,PACAP administration failed to activate autophagy-related gene expression program after CREB inhibition in vivo and in oxidative stressed hepatocyte cultures,implying that PACAP mediated hepatocellular autophagy was CREB-dependent.Furthermore,CREB inhibition abolished PACAP-mediated hepatocellular regeneration,recreated oxidation-mediated hepacellular death and ALT/LDH release in hepatocytes in vitro.7.PACAP-CREB signaling controls KLF4 in liver IRI.Compared with controls,PACAP enhanced KLF4 gene induction(but not other KLFs and protein expression in both liver IRI and in vitro hepatocyte cultures,which otherwise are diminished after CREB inhibition.We then employed KLF4 inhibitor in liver warm IRI.In analogy with PACAP-CREB ablation,KLF4 inhibition blunted PACAP-mediated cytoprotection,and restored IR-induced hepatocellular injury,as shown by increased s ALT levels and deteriorated liver pathology.In addition,KLF4 inhibition recreated PACAP-suspended pro-inflammatory cytokine program,and suppressed IL-10 expression.8.KLF4 is essential for PACAP-CREB-mediated hepatic autophagy in liver IRI.KLF4 suspension significantly diminished otherwise abundant hepatic autophagy related components and hepatocellular regeneration programs in PACAP-conditioned liver IRI.Consistently,KLF4 inhibition attenuated PACAP-mediated hepatocellular autophagy in stressed hepatocyte cultures,and restored hepatocyte damage,as shown by amplified cell death and ALT/LDH release.Conclusion: 1.In OLT model,PACAP mediated autophagy could improve OLT survival and ameliorates hepatic IRI.Meanwhile,PACAP could improve the hepatocyte repair/regeneration program,as well as decrease IR-Induced liver oxidative stress and necrosis/apoptosis.2.In liver IRI,PACAP mediates autophagy protect hepatocytes via CREB-KLF4 signaling.The integrality of PACAP-CREB-KLF4 pathway is essential in autophagy mediated cytoprotection in IR-stressed liver.