Role Of Retinoic-acid-inducible Gene â…  In Hepatic Ischemia Reperfusion Injury And The Underlying Immunological Mechanisms

Ischemia reperfusion （IR） injury is the damage caused when blood supply returns tothe tissue after a period of ischemia or lack of oxygen, which can even aggravate tissuestructure disruption and metabolic function disorders. It was first proposed by Jennings in1960. IR injury during the process of organ obtainment and transplantation can lead toprimary none function or delayed graft function, increase the incidence of acute andchronic rejection and affect the long-term graft survival. Therefore, how to effectivelyminimize IR injury and provide better protection for donor organs are subject to beurgently solved. It have been reported that the mechanisms of IR injury involves release offree radicals and reactive oxygen species, calcium overload, inflammation, impairedmitochondrial function and dysfunction of energy metabolism, apoptosis, and many otherfactors. Previous studies demonstrated that during the complex pathophysiologic processafter liver IR, the endogenous ligands or dangerous released from damaged cells whichexperienced direct damage by insufficient blood flow and lack of oxygen conditionsactivate pattern recognition receptors. And then pattern recognition receptors trigger innateimmune response which mediates further damage. However, the pathogen associatedpattern recognition receptors have been focused on TLRs （toll-like receptors, TLRs） andrelated signal pathways in recent years, while the other major pattern recognition receptors:RLRs （Retinoic acid-inducible gene （RIG）-I like receptors, RLRs） was not mentionedmuch. Current research shows that RLRs mediated antivirus innate immune response playa pivotal role against virus infection. Among which RIG-â… was the best characterized, butthere are still many unresolved issues need to be explored.RIG-â…  also known as DDX58A, containing925amino acid residues, belongs to theDExD/H family, mainly distributed in the cytoplasm. RIG-â…  primarily identify and directbind to viral RNA with phosphorylation at the5’end. After interacting with its adaptorprotein mitochondrial antiviral signaling protein （MAVS）, IFN-beta promoter stimulator（IPS-1） or CARD adaptor inducing IFN–beta （Cardif）, RIG-â…  initiates the gene expressionof type I interferon and proinflammatory cytokines by activating IRF3/7and NF-κB. Furthermore, studies of hepatitis C virus （HCV）-host interactions have revealed RIG-â… mediated signaling pathway may provide evasion targets for viral persistence. It have beenreported that serine protease NS3/4HCV encoded by HCV cleavage IPS-1and theninterrupt RIG-â…  mediated downstream antiviral immune response. In fact, proteaseinhibitors of HCV have already been applied for clinical treatment result fromstrengthening the innate immune response and inhibiting HCV replication. However, whatis the role of RIG-â…  and RIG-â…  mediated signaling in liver IR development remain unclear.We also wonder the complex contents released from damaged cells after IR injury whetherinclude the ligand of RIG-â… .In this study, we discussed the relationship between RIG-â…  and liver IR and relatedmechanism of liver IR injury regulation.PartI. The alteration of RIG-â…  expression during liver IR and itspossible regulation mechanismWe first assessed the expression of RIG-â…  during liver IR by employing a murinepartial hepatic IR model.IR significantly decreased the protein content of RIG-â…  in the liverfor a short period of time, and then increased gradually in the later period. The results ofimmunohistochemical also confirmed with RIG-â…  was located in cytoplasm mostly aroundthe infiltration of inflammatory cells after24h.Then we isolated liver cells and non-parenchymal cells at different time afterreperfusion and analyzed the expression of RIG-â…  respectively. The results revealed that IRmainly affects liver parenchymal cells while no measurable effects on non-parenchymalcells by Western blot analysis.We further investigated the underlying mechanism for the decreased expression ofRIG-â…  in the process of IR. A number of data emphasized the important role for oxidativestress generated within hours after liver IR. We used different doses of H₂O₂to stimulatehepatocytes and non-parenchymal cells, and found hepatocytes were sensitive to H₂O₂stimulation as compared to non-parenchymal cells. This inhibition also existed in a timedependent way by H₂O₂stimulation. We also considered using proteasome inhibitorMG132pretreating hepatocytes and the results suggested MG132pretreating almostentirely blocked the RIG-â…  down-regulation in the early stage after H₂O₂treatment,indicating that activation of ubiquitin-proteasome pathway was directly involved in thedegradation of RIG-â… . Partâ…¡. Role of RIG-â…  pathways in the pathophysiological processduring liver IR injurySince the expression of RIG-â…  was correlated with liver IR in mice, which indicatedRIG-â…  participate in the complex pathophysiological process. So, whether in turn did RIG-â… actually play its role in liver IR? In this part, we first tested the levels of ALT and AST inserum in mice undergoing liver IR, ischemic lobes were also harvested at indicated time.The results showed that the levels of ALT and AST in RIG-â… -/-mice significantlydecreased than the WT controls and the Hï¹ E staining suggested there are a lot ofhepatocytes apoptosis, obvious necrosis areas in the WT controls as well. The pathologicaldamage in RIG-â… -/-mice was significantly reduced.Subsequently, we investigated macrophage infiltration in liver IR byimmunohistochemical staining. The results showed that the infiltrated macrophages （F4/80）was significantly decreased in RIG-â… -/-mice than in WT groups. To detect the apoptosis ofhepatocytes, we stained with TUNEL and similar results were observed as those ofmacrophage infiltration, showing further evidence for the reduced pathological injury inRIG-â… -/-mice.In order to further determine the contribution of RIG-â…  in hepatocytes andnon-parenchymal cells, we constructed marrow chimeric mice. There was no significantdifference of ALT and AST serum levels between transfer of deficiency bone marrow andRIG-â… -/-mice, but both significantly lower than the WT mice. The above results indicatedthat RIG-â…  both on hepatocytes and non-parenchymal cells are involved in mediating liverIR injury. But as a result of hepatocytes account for the majority of liver cells and also theexpression of RIG-â…  in hepatocytes can be directly regulated by liver IR, in the followingstudy, we focused on RIG-â…  expressed in hepatocytes and how the RIG-â…  signaling pathwayaffect liver IR process.Part â…¢. RIG-â…  pathway promotes apoptosis during liver IR injuryAs an important member of the RLRs family, RIG-â…  play a key role of triggeringantiviral innate immune response against virus infection. RIG-â…  also initiate a proapoptoticsignaling pathway that is independent of typeI IFNs. It has been reported that triggeringthis pathway led to efficient activation of apoptosis in human melanoma cells. In addition,apoptosis is closely related to liver IR injury, then we wonder what the effect of RIG-â…  deficiency is on the apoptotic signaling pathway after liver IR.First, liver samples were harvested2,4,8and12hours after reperfusion and weanalyzed the contributions of caspase-3,8. Western blot results revealed activation ofcaspase-3and8were strongly decreased in RIG-â…  deficiency groups, suggesting activationof RIG-â…  signaling pathway induced liver IR injury by promoting apoptosis. To identify themolecular mechanism for the pro-apoptotic effect of RIG-â… , we proved that RIG-â…  directlyinteracts with caspase8by immuno-precipitation.To figure out how did RIG-â…  activate in the process of liver IR, the followingexperiments were conducted: hepatocytes from WT or RIG-â… -/-mice were stimulated withliver homogenate from WT mice at24h after liver IR for indicated times. Expression ofIFN-βwas examined by real time PCR and the phosphorylation of IRF3was tested byWestern blot, apoptosis was also tested by flow cytometry. The results suggestedhomogenate from IR injured liver can activate RIG-â…  signaling pathway. Next, necrosissupernatant of hepatocytes and non-parenchymal cells were prepared and incubated withhepatocytes isolated from RIG-â… -/-and WT mice respectively as conditional culturemedium. The activity of ROS and cytokines levels of TNFa、IL-6、IL-1β in necrosissupernatant were previously measured. Therefore, we speculate that endogenous dangeroussubstances released by necrotic cells activate RIG-â…  pathway and then promote apoptosis tofurther mediate liver IR injury.In summary, we demonstrate that liver IR decrease RIG-â…  expression through amechanism involving oxidative stress, and ubiquitin-proteasome activation make RIG-â… directly degrade. Furthermore, we show that activation of RIG-â…  in hepatocytes induceliver IR injury more sensitively by promoting cell apoptosis. These findings encouragetargeting RIG-â…  in the development of novel molecular therapies for liver IR injury.