Role Of Notch Signaling Pathway In Hepatic Ischemia Reperfusion Injury And The Underlying Molecular Mechanisms

【Background】Ischemia reperfusion injury (IRI) is a pathophysiologic process in which hypoxic damage is aggravated following the return of blood flow and oxygen delivery into organs. I/R injury is involved in many clinical settings such as myocardial infarction, stroke, and trauma. In liver, IRI is an inevitable event after hepatic resection, liver transplantation, or hypotensive shock followed by recovery. It has been reported that during liver transplantation, hepatic I/R injury accounts for up to 10% of early organ dysfunction, leading to higher episodes of both acute and chronic rejection. The pathologic procedure of hepatic IRI is devided into two phases. The initial phase (< 6 h post reperfusion) of hepatic I/R injury is characterized by the generation of ROS which can induce apoptosis or necrosis of hepatocytes. In the second phase of hepatic I/R injury, it is characterized by intense infammatory response defined as infiltrition of inflammatory cells and production of inflammatory cytokines, which promote additional hepatocyte death directly or indirectly.The Notch signaling pathway is highly conserved through evolution and regulates cell proliferation, apoptosis, and cell fate decisions in a broad range of tissues. In mammals, five ligands (Jagged1, 2, and Delta-like [Dll] 1, 3,and 4) and four Notch receptors (Notch1-4) have been identified. Canonical Notch activation involves receptor cleavage within the transmembrane domain by ??-secretase mediated consecutive enzymatic reactions, releasing Notch intracellular domain (NICD) that subsequently translocates into the nucleus, where it interacts with the recombination signal binding protein J??(RBP-J). This protein-protein interaction leads to transactivate the transcription of target genes such as the Hes family basic helix-loop-helix (bHLH) factors. In recent years, it has also been suggested that Notch signaling participates in cell responses to extra cellular insults. Notch signal is reported to be involved in myocardial and cerebral ischemic injury. Does Notch signal participate in the process of hepatic IRI? What is the role of Notch signal in hepatic IRI? How about the molecular mechanisms of Notch regulating hepatic IRI? Answers to these questions will open a new window for research on IRI, accomplish related theory system and provide theoretical basis for prevention and therapy of hepatic IRI.【Aims】To investigate role of Notch signaling pathway in hepatic IRI and the underlying cellular and molecular mechanisms. To explore new melecular mechanisms regulating hepatic IRI and provide new theoretical basis for heaptic IRI prevention and therapy. 【Methods】1. An atraumatic clip was used to interrupt blood supply to the left lateral and median lobes of liver. After 90 min of hepatic ischemia, the clip was removed, initiating hepatic reperfusion. mRNA expression of Notch related molecules were detected by Real-time PCR. Expression of Notch1 intracellular domain was examined by Western blot.2. Mx-Cre-RBP-Jf/f mice and Mx-Cre-RBP-Jf/+ mice were genotyped by PCR. The Cre-mediated deletion of RBP-J was induced to block Notch signaling by using poly(I)-poly(C). We observed effects of Notch signaling blockade on hepatic IRI by detection of serum ALT and AST, HE staining, TUNEL staining, MPO immunol histochemistry staining, detection of inflammatory cells infiltrition by FACS and detction of inflammatory cytokines by Real-time PCR.3. We transplanted bone marrow cells from RBP-J KO and control mice to WT mice which were irradated lethally to obtain bone marrow cells RBP-J KO mice. Then we observed effects of blocking Notch signaling in bone marrow cells on hepatic IRI by detection of serum ALT and AST, HE staining, TUNEL staining.4. Hepatocytes Notch signaling was blocked by using GSI in vitro, Hepatocytes were incubated in a hypoxic chamber (0.5% O2) for 15 h. Subsequently, normaxic normal medium was used for culture further for 6 h to mimic IRI in vitro. We observed effects of blocking Notch signaling in hepatocytes on IRI by TUNEL staining, mRNA expression of Casepase-3, viable cells left after IRI, production of TNFαby macrophages stimulated by conditional media.5. We detected intracelluar ROS level by using DCFH-DA fluorescent probe and analyzed by FACS. We examined expression of iNOS and Bcl-xl downstrem molecules of ROS by Real-time PCR and Western blot. We observed role of ROS in Notch regulating hepatic IRI by using ROS scavenger.6. We examined mRNA expression of MnSOD by Real-time PCR, and protein expression of MnSOD and phospho-STAT3 by Western blot. Protein complex of STAT3 and Hes5 was detected by immunoprecipitation and Western blot. We observed role of STAT3 in Notch regulating hepatic IRI by overexpressing constitutively active STAT3 in hepatocytes. By these experiments the underlying molecular mechanisms of Notch regulating intracellular ROS of hepatocytes.7. We observed effects of Notch activation on hepatocytes IRI in vitro by co-culture with OP-9 overexpressing Dll1 or overexpressing Hes5.【Results】1. Notch signaling was activated in hepatic IRI. Notch1, Notch2, Dll4, Jagged2, Hes5 and Notch1 intracellular domain were upregulated in hepatic IRI.2. Blocking Notch signal led to aggravated hepatic IRI, as showed by elevated serum ALT and AST level; increased necrosis and apoptosis; more infiltrition of neutrophils; T lymphocytes and macrophages; increased production of proinflammatory cytokines: TNFα, IL1β, IL6 and IFNγ, chemokine: CCL3 and adhension moleclule ICAM1.3. Interruption of Notch signaling resulted in increased accumulation of intracellular ROS in hepatocytes, and a ROS scavenger cured exacerbated hepatic IRI showed as evlevated serum ALT and AST and increased apoptosis and necrosis after Notch signaling blockade, suggesting that Notch signal deficiency-mediated IRI aggravation could be attributed to increased ROS level.4. Further investigation demonstrated that this was due to that Notch signal blockade resulted in downregulation of Hes5 and hypophosphorylation of STAT3, leading to reduced MnSOD expression, because overexpression of a constitutively active STAT3 rescued MnSOD expression and IRI induced apoptosis in the absence of Notch signaling.5. Forced Notch activation by ligand stimulation or Hes5 overexpression reduced intracellular ROS and protected hepatocytes from apoptosis after I/R injury through the activation of STAT3 and MnSOD expression.【Conclusions】1. Notch signaling play critical role in hepatic IRI, and Notch signaling deficiency leads to exacerbated hepatic IRI.2. Notch regulating hepatic IRI depends on hepatocytes. Blocking Notch signaling in bone marrow cells exert no obvious effect on hepatic IRI but blocking Notch signaling in heaptocytes results in increased hepatocytes apoptosis.3. Notch signaling paticipate in hepatic IRI by regualting hepatocytes intracellular ROS. Notch signaling deficiency leads to increased intracellular ROS in hepatocytes in hepatic IRI.4. Notch regulates intracellular ROS of hepatocytes by expression of MnSOD through assisting activation of STAT3 by Hes5. Notch signaling deficiency leads to decreased fomation of complex by Hes5 and STAT3, then decreased activation of STAT3 and expression of MnSOD, finally increased ROS accumulation.5. Acitivation of Notch siganling protects hepatocytes from IRI. Activation of Notch signaling causes decreased apoptosis in hepatocytes IRI in vitro.Many kinds of cells and molecules participate in the pathology of hepatic IRI. And our results demonstrated that Notch signal protects hepatocytes from IRI by the expression of MnSOD through assisting the activation of STAT3 by Hes5. These results provide new insights into the molecular mechanisms controlling IRI, theoretical basis for clinical prevention and therapy and new clues for investigation of ROS related diseases such as tumor and autoimmune diseases.