| Background:The advent of successful liver transplantation conducted by Starzl et al,which shifted the hepatic failure paradigm as an end-stage disease into a triumph of long-term meaningful survival,had created a new dilemma that organ supply can not meet liver transplantation requirements.Better organ preservation methods are needed to expanded the donor pool to meet the growing organs demand by using of marginal grafts.Method and materials:Male SD rats(250-300 g)and heme oxygenase(HO)-1 heterozygous(HO-1+/-)and wild-type(HO-1+/+)rats were used in this study.Remote ischemic conditioning(RIC)was compared with the traditional ischemic postconditioning(IPostC),and the temperature,velocity and oxygenation parameters of machine perfusion(MP)were optimized.And studies involving the correlation between MP and liver regeneration was conducted to find the best strategy of reducing liver transplantation injury.Results:Liver graft protection by remote ischemic perconditioning(RIPerC)is similar to or better than that of IPostC,and involves inhibition of oxidative stress and upregulation of the PI3K/Akt/endothelial nitric oxide synthase(eNOS)/nitric oxide(NO)pathway.The 50%of the portal vein physiological flow rate group had lower shear damage compared to 150%of the physiological flow rate group and avoid insufficient perfusion compared to the 12.5%of the physiological flow rate group.The effect of hypothermic machine perfusion(HMP)to the graft was better than subnormothermic and normothermic machine perfusion(SMP and NMP)without oxygen.Adding oxygen to the preservation showed improved liver graft function.And our results suggest that inhibition of HO-1 mitigate HMP induced liver recovery effects due to proliferation but not apoptosis,in part by downregulation of an hepatocyte growth factor(HGF)-Akt axis instead of interleukin(IL)-6 and transforming growth factor(TGF)-? signaling in vivo and in vitro.Conclusion:RIC is the ideal intervention for reducing liver injury in liver transplantation.MP is better than SCS for liver graft preservation due to its higher liver regeneration ability.Hypothermic oxygen perfusion is a relatively simple and effective method and 50%of the physiological flow rate group is better than the others;but it is still need to further develop the ideal oxygen perfusion fluid.Part ? Remote ischemic perconditioning prevents liver transplantation-induced ischemia/reperfusion injury in rats:role of ROS/RNS and eNOSBackground:Our previous studies have successfully established a novel model of RIC in rat liver transplantation and have demonstrated its protection against I/R injury.In this study,we further investigated the underlying mechanisms.Methods:Sprague-Dawley rats were subjected to sham,orthotopic liver transplantation(OLT),IPostC or RIPerC.After 3 h reperfusion,blood samples were taken for measurement of ALT,AST,creatinine(Cr)and creatinine kinase-myocardial band(CK-MB).The liver lobes were harvested for the following measurements:ROS,hydrogen peroxide(H2O2),mitochondrial membrane potential(??m)and total NOx.These measurements were determined using an ROS/H2O2,JC1 and Total NOx Assay Kit,respectively.The eNOS was analyzed by reverse transcription polymerase chain reaction(RT-PCR)and western blotting,and peroxynitrite was semi-quantified by western blotting of 3-nitrotyrosine.Results:Compared with the OLT group,the grafts subjected to RIPerC showed significantly improved liver and remote organ functions(P<0.05).ROS(P<0.001)including H2O2(P<0.05)were largely elevated in the OLT group compared with the sham group,and RIPerC(P<0.05)reversed this trend.The collapse of ??m induced by OLT ischemia/reperfusion(I/R)injury was significantly attenuated in the RIPerC group(P<0.001).A marked increase of NO content and phosphoserine eNOS both in protein and mRNA levels was observed in liver graft of the RIPerC group compared with the OLT group(P<0.05).I/R-induced 3-nitrotyrosine content was significantly reduced in the RIPerC group compared with OLT group(P<0.05).There were no significant differences between the RIPerC and IPostC groups for all the results except for Cr.The Cr level was lower in the RIPerC group than in the IPostC group(P<0.01).Conclusion:Liver graft protection by RIPerC is similar to or better than that of IPostC,and involves inhibition of oxidative stress and upregulation of the PI3K/Akt/eNOS/NO pathway.Part ? Optimization the key parameters of machine perfusion for isolated liver graftBackground:LT has been considered the gold standard therapy for terminal liver failure patients.Plenty of studies have showed MP is superior to SCS,particularly with the increasingly usage of marginal livers from ECD and those from DCD donors.However,there is no consensus on the key parameters such as temperature,velocity and oxygenation for MP.Method and materials:The experiments were carried out based on our previous machine perfusion system.According to the different velocity,six groups were divided(SCS,12.5%,25%,50%,100%and 150%);according to the different temperature,three groups were divided(0-4,20,37?);According to extra oxygenation,two groups were divided(Air and 95%O2+5%CO2).After 6-24 hours preservation for isolated liver grafts,perfusates were collected for liver function detection,liver tissues for myeloperoxidase(MPO),malonaldehyde(MDA)and adenosine triphosphate(ATP)detection,then HE staining,meanwhile portal vein pressure and velocity were monitored.Results:The 50%of the portal vein physiological flow rate group had lower shear damage compared to 150%of the physiological flow rate group and avoid insufficient perfusion compared to the 12.5%of the physiological flow rate group.The effect of HMP to the graft was better than SMP and NMP without oxygen.Adding oxygen to the preservation showed improved liver graft function,however the UW and HTK oxygen-carrying abilities were still not ideal.Conclusion:Hypothermic oxygen-carrying perfusion is still relatively simple and effective method,and 50%physiological flow rate is the optimum portal vein velocity.Part ? Partial inhibition of HO-1 attenuates HMP-induced hepatic regeneration against liver injury in ratsBackground:We found a higher recovering liver graft regeneration in HMP compared with SCS for the first time in our pilot study with unknown underlying mechanisms.Upregulated HO-1 expression have been reported to play a pivotal role in promotion of hepatocyte proliferation.Herein we evaluated the novel role of HO-1 in HMP's liver graft of half-size liver transplantation(HSLT)protection.Method and materials:Rats with heterozygous knocking out of HO-1(HO-1+/-)were generated and subjected to 3h SCS or HMP pre-HSLT in vivo or 6h SCS or HMP in vitro the same with control rats(HO-1+/+).At 0,1,3,and 6 h during the perfusion process in vitro groups and 1,3 and 7d after HSLT in vivo groups,perfusate and plasma samples were collected from for the analysis of liver function.At 6h of the end of the preservation in vitro and at 7d after HSLT in vivo,liver tissues were obtained and fixed in 10%neutral formalin for later histological and immunohistochemical analyses.The liver was collected for determination of the regeneration ratio(RR).Results:We found that HSLT induces a significant elevation of HO-1 protein level in the regenerated liver and HO-1 haplodeficiency results in decreased proliferation post-HSLT.Compared with SCS,HMP induces a significant elevation of HO-1 protein level along with better liver recovery both in vivo and in vitro,which were reduced by HO-1 haplodeficiency.HO-1 haplodeficiency induced decreased proliferation but not apoptosis was responsible for the attenuated HMP's regenerative ability both in vivo and in vitro.Mechanistically,HO-1 haploinsufficiency resulted in the suppression of HGF/Akt activity.Conclusion:Our results suggest that inhibition of HO-1 mitigate HMP induced liver recovery effects after HSLT due to proliferation but not apoptosis,in part by downregulation of an Hgf-Akt axis instead of IL-6 and TGF-? signaling in vivo and in vitro. |
PDF Full Text Request