Experimental Study On The Protective Effect Of Retrograde Reperfusion Method On The Function Of Liver Graft

Background Mitigating ischemia/reperfusion(I/R)injury of the liver graft is a significant challenge in the field of liver transplantation research.The I/R process leads to mitochondrial damage in graft hepatocytes to the extent that it exceeds their autophagic clearance capacity,triggering cell death(including necrosis and apoptosis).Maintaining or re-establishing the balance between mitochondrial dysfunction and cellular autophagic activity may become a new strategy to reduce graft liver I/R injury.Clinical studies have confirmed that,compared with the conventional initial portal reperfusion(IPR),the retrograde reperfusion(RTR)technique can alleviate graft liver I/R injury and show a protective effect on graft liver function.Still,the mechanisms involved have not been elucidated.Objective This study will observe RTR technology's effects on graft liver cytotoxicity,cellular autophagy activity,the formation of reactive oxygen species(ROS),Ca²⁺ overload,and mitochondrial dysfunction based on a modified rat liver transplantation model.Furthermore,we perform proteomics analysis to investigate the molecular networks involved.The goal is to elucidate the potential mechanism of the RTR approach to alleviate graft liver I/R injury.To provide substantial evidence and theoretical basis for promoting RTR technique in clinical liver transplantation and contribute new therapeutic ideas for liver I/R injury caused by other conditions(e.g.,hepatectomy,hemorrhagic shock).Methods(1)A in situ rat liver transplantation model that can flexibly mimic the IPR or RTR approach was established through a modified “double cuff” technique.In the IPR approach,the Portal Vein(PV)was reconstructed and opened first.Then the Inferior Vena Cava(IVC)flow was restored after 5 min of the PV's anterograde perfusion.Whereas in the RTR technique,the IVC was reconstructed and opened first.Then PV flow was restored after 5 min of retrograde perfusion of the IVC.The intraoperative graft liver ischemia time and the recipient rats' survival rate 7 days after liver transplantation were recorded to judge the model's validity and stability.(2)Compared with the conventional IPR approach,we assessed the effect of the RTR technique on graft liver I/R injury by observing alanine aminotransferase(ALT)activity,histopathological changes(hepatocyte necrosis,inflammatory cell infiltration,and microcirculatory disorders as the main criteria),in situ apoptosis(detected by TUNEL method),and activated caspase-3 expression level(examined by immunohistochemical technique)in recipient rats 7 days after liver transplantation.(3)To illustrate the effect of the RTR technique on autophagic activity in graft liver,we used immunoblotting assay to detect the microtubule-associated protein 1 light chain 3(LC3)conversion levels by combining chloroquine(CQ)pretreatment and p62/SQSTM1 protein expression at 1,2,and 6 h of graft liver reperfusion.Then,subcellular ultrastructure was examined by transmission electron microscopy(TEM)to assist in determining the impact of reperfusion modality on autophagy in graft liver from a morphological perspective.At the same time,differences in the degree of mitochondrial damage could be noted.(4)Calcein-AM/Calcein-AM+Co Cl2,JC-1,DCFH-DA,and Rhod-2/AM were used as fluorescent probes to detect and compare the effects of different reperfusion modes(IPR vs.RTR)on graft liver mitochondrial permeability pore(MPTP)opening,mitochondrial membrane potential(MMP),levels of intracellular ROS and Ca²⁺ by flow cytometry in real time,respectively.The initial perfusion blood stream's oxygen saturation was also measured using a blood gas analyzer for the different reperfusion methods.The effects and possible mechanisms of the RTR technique on cellular mitochondrial damage were synthesized.(5)We used tandem mass spectrometry tag(TMT)based liquid chromatography-tandem mass spectrometry(LC-MS/MS)proteomics and bioinformatics analysis(including subcellular localization,gene ontology(GO)function,Kyoto encyclopedia of genes and genomes(KEGG)pathway,protein interaction(PPI)network to detect differentially expressed proteins in graft's liver between different reperfusion modalities.Finally,selected target proteins were validated by the parallel reaction monitoring(PRM)method to fully elucidate the possible mechanism of the hepatoprotective effect of RTR technology on the graft's liver.Results(1)The 7-day postoperative survival rate of all rats was 100%.Consistent with the clinical outcomes,the RTR technique shortened the warm ischemic time(WIT)of the graft's liver.These results indicate that the modified rat liver transplantation model established in this study can stably and effectively mimic the IPR or RTR approach.(2)Compared with the conventional IPR approach,the RTR technique can reduce ALT activity,histopathological damage,and in situ apoptosis in recipient rats 7 days after liver transplantation,indicating that the RTR technique can alleviate graft liver I/R injury to a certain degree.(3)Using the conventional IPR method as a control,the LC3 conversion level of the graft's liver was raised,and the peak of p62 protein expression was pushed back after using the RTR technique.These results indicate that the RTR technique could effectively alleviate autophagic activity inhibition caused by I/R injury.Meanwhile,TEM examination results also showed that more mitochondrial autophagy occurred in the graft liver in the RTR group,and fewer mitochondria were damaged and degenerated.(4)Combined analysis of flow cytometry and blood gas analysis suggested that the RTR technique could effectively reduce ROS production and Ca²⁺ overload by perfusing the graft liver with lower oxygenated vena cava blood flow could alleviate MPTP opening and better maintained MMP levels.(5)TMT-based LCMS/MS proteomics assay and bioinformatics analysis screened three differentially expressed target proteins,namely fibrinogen ?-chain(Fgb),estrogen sulfotransferase(EST),and lysosome-associated membrane protein 1(Lamp1).The PRM assay confirmed that RTR technology could inhibit Fgb and EST expression and alleviate the decrease of Lamp1.It is speculated that these factors may be involved in the protective mechanism of RTR technology on graft liver function,but further studies are needed to confirm it.Conclusions The RTR technique reduces ROS production and Ca²⁺ overload by perfusing the graft liver with low oxygen saturation vena cava blood flow,alleviating the mitochondrial damage and inhibiting autophagic activity caused by I/R injury.So it facilitates the maintenance of a dynamic balance between mitochondrial dysfunction and autophagic clearance capacity.Therefore,it could mitigate graft liver I/R injury and have a protective effect on graft liver function.Meanwhile,inhibition of Fgb and EST expression and the reduction of Lamp1 decline may also protect the liver graft by the RTR technique,but further studies are needed to confirm this.