The Influence And Effect Of Hypothermic Machine Perfusion On PI3K/Akt Signaling Pathway In Cardiac Dead Liver Of Rats

Objective:The rat cardiac death model was established to investigate the influence of hypothermic machine perfusion and cold storage on the PI3 K / Akt signaling pathway in DCD rats and its effect.Methods: Eighteen Sprague-Dawley rats were randomly divided into normal control group(NC),cold storage group(CS)and hypothermic machine perfusion group(HMP),6 in each group.The animal model of cardiac death was established by cutting the diaphragm muscle,and the cardiac arrest time of each group was observed and recorded.The liver was obtained immediately after cardiac arrest in the NC group,then the liver was connected to isolated perfuse rat liver system via the hepatic portal vein cannulation to simulated liver transplantation for 2 hours;the CS and HMP groups waited for 30 minutes after cardiac arrest and then harvested the liver,followed by cold storage for 3 hours or hypothermic mechinel perfusion for 3 hours,then the liver was connected to isolated perfuse rat liver system via the hepatic portal vein cannulation to simulated liver transplantation for 2 hours.During the reperfusion,PE tubes were used to collect the amount of liver bile production in each group.Liver function tests were performed by collecting 1 ml of KHB perfusate from the superior vena cava eyery one hour intervals.Pathological examination was performed on liver tissues after simulating reperfusion for 2h;relative expression levels of Akt,p-Akt,Bcl-2,and Bax proteins in liver tissues were detected by western blot;livers were observed by TUNLE method for apoptosis.Results: The liver function of perfusate at each hour: the alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in CS group and HMP group were significantly higher than in NC group(p<0.001),but significantly lower in HMP group than in CS group(p<0.001).There was no significant difference in bile production between NC group,CS group and HMP group(p>0.05).Histopathological examination of liver: In the NC group,the morphology of the liver tissue was almost normal,and the structure of the hepatic cord was intact.There was no obvious cell swelling and necrosis.In the CS group,the hepatocyte damage was severe,the liver cells were swollen,the cytoplasm was loose,and the nucleus was dissolved.Peripheral inflammatory cell infiltration;Slightly dilated hepatic sinusoids in the HMP group,visible hepatic edema,and less necrosis than the CS group.Western blot results showed that: p-Akt/Akt in HMP group and CS group were significantly higher than those in NC group(p<0.001),but HMP group increased more significantly(p<0.001);HMP group Bcl-2/Bax than CS group was significantly elevated(p<0.001),but the HMP group was significantly lower than the NC group(p<0.001).TUNLE assay showed that the apoptosis rate of liver in CS group and HMP group was significantly higher than that in NC group(p<0.01),but HMP was significantly lower than CS group(p<0.01).Conclusion: Compared to cold storage,hypothermic machine perfusion of cardiac death rat livers can significantly activate the PI3K/Akt signaling pathway,up-regulate the expression of downstream Bcl-2 protein and inhibit the expression of Bax protein,thereby reducing the number of hepatocyte apoptosis and reducing the ischemia and reperfusiof donor liver and improves the quality of donor liver.