TRAIL's Role In Brain Death Liver Injury And Its Regulatory Mechanism

Obejectives:1.To verify the expression of TRAIL in the liver of brain-dead rats.2.To verify the damage effect of TRAIL by adding recombinant TRAIL on liver cell lines,and to preliminary explore the possible molecular mechanism.Methods:1.Establish the SD rat brain death model,and detect the location and expression of TRAIL in the liver of brain death rats by immunohistochemical staining,fluorescent quantitative PCR,western blot.2.Divide the LO2 cell line into control group and different concerntration of recombinant TRAIL intervention groups,use starvation hypoxia/reoxygenation method to establish a cell stress model,and use CCK-8,fluorescence quantitative PCR,western blot to detect the expression of apoptosis-related genes at the cellular level,and to verify the damage effect of TRAIL on liver cell lines.Results:1.From the results of immunohistochemical staining,it can be found that there is a large amount of inflammatory cells infiltration in rat liver tissues under brain death.TRAIL is ideally stained.It is strongly expressed in livers of brain dead rats.The positive stainnings are mostly distributed around blood vessels,liver TRAIL expression in brain-dead rat liver increased briefly after 1H,and then showed a general downward trend;Western blot results showed:TRAIL increased temporarily after brain death 1H,and then generally showed a downward trend;fluorescence quantitative PCR results showed:TRAIL mRNA expression level increased temporarily after brain death 1H,and then showed a general downward trend.Compared with brain death 1H,TRAIL mRNA significantly decreased after brain death 4H,6H(P<0.05)2.The results of CCK8 show that no matter whether it is normoxia or hypoxia-reoxygenation,the addition of rTRAIL at different concentrations and under different time of culture can damage LO2 cells.The damage of rTRAIL concentration to LO2 cell lines is 0.5ng/ml-1ng/ml,even if the concentration is increased,the cell damage will no longer increase significantly.Therefore,two concentrations of 0.5ng/ml-1ng/ml were used to extract protein and RNA.Western blot results showed that with the increase of rTRAIL concentration and the culture time,the expression level of BAX was up-regulated,and the expression of BCL2 was down-regulated.Fluorescence quantitative PCR results showed:Compared with the non-medicine group,TNFSF10C,TNFSF10D,TNFSF11B,BCL2 in the rTRAIL 1ng/ml group were significantly down-regulated at the mRNA level,and FADD,Caspase8,Caspase3,BAX were significantly up-regulated at the mRNA level.Conclusions:1.TRAIL expression follows a typical transient rise pattern in the brain death model.2.TRAIL has a damaging effect on hepatocytes.TRAIL reduces the protective receptor at the mRNA level by binding to a protective receptor,and increases apoptosis through the apoptosis pathway.