Protective Effects And New Mechanism Of CO On Liver Ischemia And Reperfusion Injury

BackgroundWith the development of liver transplantation and prohibition of using organs from executed prisoners,the shortage of available liver donors has become a prominent global problem which is more severe in compared with the post-transplant rejection,surgical complications and postoperative infection that leads to an increasing use of marginal liver donors.However,the marginal liver donors are extremely sensitive to ischemia and reperfusion injury that carry poor prognosis in this type of liver transplant recipients,and in severe cases even could result in death.Current therapeutic methods against IRI provide limited efficacy which are not enough to meet the clinical demands,so it's urgent to find a new therapeutic regimen which is more effective.CO functions as a very important cell signaling molecule which has anti-inflammatory and anti-apoptotic effects,recent findings have shown that CO may participate in the process of autophagy.So far the role of autophagy in liver IRI is still indefinite and the researches are also little.ObjectiveTo explore the protective effects and new mechanism of CO on hepatic ischemia and reperfusion injury by establishing low concentration serum(4%)culture medium and hypoxia-reoxygenation injury model of hepatic parenchymal cells in vitro and hepatic ischemia reperfusion injury model in vivo.Methods1.Simulate the ischemic microenvironment of severe liver injury by establishing low concentration serum(4%)culture medium of WB-F344 in vitro,investigate the effects of CORM-2 therapy on cell viability and proliferation,primarily study the impact of autophagy on hepatic parenchymal cells.The experiment was divided into 5 groups: 10%FBS,4%FBS,4%FBS+iCORM-2,4%FBS+CORM-2 and 4%FBS+CORM-2+3-MA.Measure the cell viability and proliferation by CCK-8;detect the formation of acid cell autophagosome by acridine orange;measure the expression of AKT,p-AKT,p-mTOR,HO-1 and autophagy related protein LC3-I,LC3-II by Western-Blot;2.Isolate the primary rat hepatocytes,detect the effects of CORM-2 therapy on hypoxia-reoxygenation injury of hepatocytes and investigate the impact of autophagy on the process.The experiment was divided into 4 groups: blank control of DMSO,iCORM-2,CORM-2 and CORM-2+3-MA.Isolate the primary rat hepatocytes by two step collagenase perfusion method,observe the cell adhesion and detect cell's status by Trypan blue staining.Select well growth primary hepatocytes and followed by culturing under hypoxia-reoxygenation condition in that the hypoxia time last 4h.Collect supernatant from every culture medium and measure the values of AST and ALT by automatic biochemical analyzer;measure the expression of LC3-I and LC3-II by Western-blot;3.Establish 70% hepatic ischemia-reperfusion rat model to further investigate the impact of CORM-2 in liver IRI.The experiment was divided into 2 groups: sham group and IRI group,each group was treated with Vehicle and CORM-2 therapy respectively.Preoperative fasting in rats under ambient temperature of 26?,only abdominal incision in sham group and collect the blood and liver samples immediately;in IRI group,recover the liver blood supply after blocking-up the blood flow for 1h,then execute the rats at 1h and 6h respectively post reperfusion,collect the blood and liver samples,measure the values of AST,ALT;evaluate the liver injury degree by HE stain and TUNEL;measure the mRNA expression of TNF-?,IL-1? and IL-6 by qRT-PCR;measure the expression of LC3-I and LC3-II by Western-blot,evaluate activity of autophagy.4.Use the reversing verification by CORM-2 therapy combined with 3-MA to confirm the role of autophagy in process of IRI.Reconstruction of the 70% hepatic ischemia-reperfusion rat model,give CORM-2 therapy combined with 3-MA within the hour before operation but only CORM-2 therapy in control group.Execute the rats at 6h post reperfusion,collect the blood and liver samples,measure the values of AST,ALT;evaluate the liver injury degree by HE stain;measure the expression of LC3-I and LC3-II by Western-blot.Results1.4%FBS culture medium resulted in evident inhibitory effects on cell viability and proliferation of WB-F344;CCK-8 showed CORM-2 therapy could enhance cell viability of WB-F344 and promote its proliferation;acridine orange showed CORM-2 therapy could enhance orange red fluorescence intensity and formation of autophagosome;meanwhile Western-blot showed down-regulation of p-AKT,p-mTOR,but up-regulation of HO-1,the ratio of LC3-II/LC3-I was increased;after 3-MA intervention,the effect of CORM-2 therapy was reversed.2.Primary rat hepatocytes showed well state and adhesion after 24h;Trypan blue staining showed the cell survival rate was above 90%.Under the condition of hypoxia reoxygenation,measuring the supernatant showed release of AST and ALT which suguests stress injury appeared in cells;after CORM-2 therapy the biochemical detection showed the release of AST and ALT in supernatant were decreased,meanwhile Western-blot showed upregulation of LC3-II,but down-regulation of LC3-I.After 3-MA intervention,the effect of CORM-2 therapy was reversed.3.There were severe liver damage and evident increase of AST and ALT values after liver IRI.Gived CORM-2 therapy before operation,then the level of AST and ALT at 1h and 6h post reperfusion were decreased which suguest relief on liver injury;histological examinations showed IRI can lead to hepatic parenchymal cells' apoptosis,but CORM-2 therapy could significantly reduce the apoptosis;qRT-PCR showed up-regulation of TNF-?,IL-1? and IL-6,but opposite results in the group with CORM-2 therapy.In the group with CORM-2 therapy,Western-blot also showed upregulation of LC3-II and the ratio of LC3-II/LC3-I was increased.4.In compared with CORM-2 therapy,after giving 3-MA combined with CORM-2 therapy there were rebound elevated values of AST and ALT,suggest the protective effects of CORM-2 were inhibited;histological examination also confirmed that 3-MA could aggravate the apoptosis of parenchymal cells in liver tissue;meanwhile Western-blot showed up-regulation of LC3-I,but down-regulation of LC3-II,suggest autophagy was inhibited.ConclusionCORM-2 is beneficial to cell viability and proliferation of WB-F344 in the condition of lacking nutrition by releasing CO;meanwhile it could reduce hypoxia-reoxygenation injury of rat hepatocytes in vitro and prevent hepatic ischemia reperfusion injury in vivo.The mechanism may be related to the inhibition of AKT/mTOR signaling pathway which induces cell autophagy and inhibits cell apoptosis,HO-1 may also be involved in this process.