Nuclear Factor Kappa B Participates Hypoxia Preconditioning On JNK Pathway In Rat Orthotopic Liver Transplantation

[Objective]:This thesis is based on the orthopotic liver transplantation model constructed by portal vein perfusion,on the basis of preoperative rats goven hypoxic preconditioning(hypoxic preconditioning,HP). After operation, rats were sacrificed for detection of malondialdehyde and superoxide dismutase (SOD) expressions, cytochrome C、Cleaved-Caspase-3protein,. p-C-JUNmRNA、NF-κB mRNA content was detected by RT-PCR, and the ultrastructure changes of hepatocytes were observed under light microscopy and transmission electron microscopy,to assess the role and protective effects of NF-κB on JNK pathway mediated by hypoxia preconditioning in rat orthotopic liver transplantation.[Method]:1.96Sprague-Dawley rats were randomly divided into the following three groups: normal control group(NC):received no treatment; autotransplantation group: performed autotransplantation(AT); and hypoxia preconditioning group(HP):rats were given an8%oxygen mixed gas for90minutes before autotransplantation.2. At1,2,12and24hours after the operation, the ras were sacrificed and the following tests were conducted:(1) blood was drawn to test live function(ALT and AST);(2) Liver tissue was sampled for detection of malondialdehyde、superoxide dismutase (SOD) and cytochrome C expressions;(3) the expression of Caspase-3was observed by Western blot (4) C-JUN mRNA and NF-κB mRNA was detected by RT-PCR.(5) the ultrastructure changes of hepatocytes were observed under light microscope and transmission electron microscopy.[Results]: 1.Analysis of animals number:all rats was selected result analysis. There were32rats in each group. In each group, eight rats were killed at1,2,12and24hours after surgery. The criteria for successful operation after surgery, rats immediately regained consciousness, stood up, and to maintain effective breathing, heartbeat2.Serum levels of ALT and AST The serum levels of ALT and AST in hypoxia preconditioning group were significantly lower than that of autotransplantation group(P<0.05) at1,2,12and24hours after the orthopotic liver autotransplantation.3. Expressions of malondialdehyde and superoxide dismutase (SOD) Compare to the normal control group, the expression of malondialdehyde level was significant increased, but SOD was obviously decreased (P<0.05) in the transplantation groups. Compare to the autotransplantation group, the expression of malondialdehyde was decreased significantly, and SOD was increased significantly in the hypoxia preconditioning group (P<0.05).4. Expressions of cytochrome C The expression of cytochrome C protein in the transplantation groups were dramatically increased compared with normal control group (P<0.05). The cytochrome C protein expression decreased in the hypoxia preconditioning group compared with autotransplantation group (P<0.05).5.Expression of Cleaved-Caspase-3The expression of Cleaved-Caspase-3in the transplantation groups were dramatically increased compared with normal control group (P<0.05).The expression of Cleaved-Caspase-3protein was decreased significantly(P<0.05) in hypoxia preconditioning group compared with the autotransplantation group.6.Expression of p-C-JUN mRNA The expression of p-C-JUNmRNA in the transplantation groups were dramatically increased compared with normal control group (P<0.05) at lh,2h and12h. The expression of p-C-JUNmRNA were decreased significantly (P<0.05) at1h,2h and12h in hypoxia preconditioning group compared with the autotransplantation group,but there was no difference in autotransplantation, hypoxia preconditioning and normal control group at24h(P>0.05).7.Expression of NF-κB mRNA The expression of NF-κB mRNA in the transplantation groups were dramatically increased compared with normal control group (P<0.05). The NF-κB mRNA level was increased in the hypoxia preconditioning group compared with autotransplantation group (P<0.05).8. Histology Under the light microscope, the hepatic tissues in hypoxia preconditioning group were signifcantly less swollen than those in the autotransplantation group at1,2,12and24hours.9. TEM TEM showed that the hepatocytes in the AT group had typical signs of apoptosis: concentrated cells and organelles; increased electron density as the cells began to condense, condensed or split nuclei and nucleoli, and swollen mitochondria, as mitochondrial cristae, which became solubilized. Nevertheless, the appearance of hepatocytes in the HP group was much better than that in the AT group. There were no typical signs of hepatocellular damage in the HP group, but small signs of swelling in the mitochondria[Conclusion]Hypoxia preconditioning has marked inhibition to apoptosis by upregulating protein expression of NF-κB, preventing reactive oxygen species accumulation and suppressing prolonged JNK activation, downregulating protein expression of Caspase-3and cytochrome C after orthopotic liver transplantation.