The Delayed Liver Protection By Limb Ischemia Preconditioning And Its Mechanisms In Rabbits With Liver Ischemia-Reperfusion

Objectives(1) To investigate whether limb ischemia preconditioning could induce the late phase of preconditioning against hepatic ischemia-reperfusion (IR) injury in rabbits . (2) To test if the late phase of preconditioning induced by limb ischemia is involve with MAPK signaling pathway. (3) To investigate the changes of hepatic protein expression profiles 24 h after ischemia preconditioning, and to search for the proteins probably involved in the late phase of preconditioning induced by limb ischemia preconditioning with proteomics techniques.MethodsThe experiment consisted of three parts: (1) The late phase of preconditioning induced by limb ischemia preconditioning. Rabbits were divided into three groups including group limb ischemia preconditioning(group LIP) , group sham of limb ischemia preconditioning (group S) and group IR (group IR) . A two-day protocol was used. On day 1:Group LIP was performed by the bilateral hind limb femoral arteries and veins three alternate cycles of 5 min ischaemia followed by 10 min reperfusion under general anesthesia by pentobarbital i.p; bilateral hind limb femoral arteries and veins of group S was separated from the surrounding tissue without clamped ;any treatment was not done in rabbits in group IR. The rabbits twenty four hours after limb ischemia or sham limb ischemia pretreament were subjected to 25 min total inflow occlusions and were reperfused for 3h.Rabbits in the last one groups were subjected the same ischemia-reperfusion procedure. Serum alanine transaminase (ALT),tumor necrosis factor alpha (TNF-alpha) and systemic haemodynamics were measured for 3 h after reperfusion .The hepatic wet/dry weight ratios and conten of MDA,SOD were determined, the tissue and cell injury of liver was examined with optical and electron microscope. (2) Activation of JNK, p38-MAPK, and P44/P42 MAPK of hepatic tissue was assessed by western blotting after reperfusion 30 min in group S,group LIP and group C ( without ischemia-reperfusion). SB 203580 prior 24 h to sustained ischemia-reperfusion was treamented , serum ALT,TNF-alpha were measured during ischemia reperfusion . (3) Proteomic analysis of liver of limb ischemia pretreament. The Left lateral hepatic tissues of LIP (group LIP) or S (group S) preconditioned rabbits were sampled for proteomic analysis. The total proteins were extracted and separated by two dimensional gel electrophoresis(2-DE), and 12 differential expression protein spots were analyzed with matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry(MALDI-TOF-MS).Results(1) Serum alanine transaminase (ALT),tumor necrosis factor alpha (TNF-alpha),hepatic conten of MDA,the tissue and cell injury of liver examined with optical and electron microscope in group LIP were decreased compared with the other two groups, hepatic wet/dry weight ratios and the hepatic content of SOD in group LIP was highter than that in the other groups. (2) The phosphorylation of JNK, p38-MAPK, and P44/P42 MAPK was increased significantly after 30 min of reperfusion. The phosphorylation of JNK, p38-MAPK was reduced by limb ischemia pretreament. The release of plasma ALT,TNF-a was weakened by the treatment of SB 203580. (3) Analysis of 2-DE showed that 739±18% protein spots were seen in group S and 748±23 % protein spots in group LIP, and that the expression of 37 protein spots were different between the two groups. 12 protein spots were choosed to do MS analysis,and 7 proteins were preliminarily identified. six of the seven proteins were upregulated in group LIP. One protein, ADP-ribosylation factor-like 15, was newly expressed in group LIP. These proteins can be classified into three functional groups: ( i) stress proteins, (ii )signal transduction proteins,(iii)metabolism related proteins .Among these differential proteins, heat shock protein 27 which has been proved to be involved in the late phase of ischemic preconditioning,were firstly discovered to be involved in the late phase of limb ischemia preconditioning. Regucalcin, pyruvate dehydrogenase (lipoamide) beta isoform 2, guanine nucleotide binding protein beta 2, NADPH-dependent carbonyl reductase 1 and t aldehyde dehydrogenase were firstly discovered to be involved in the hepatic preconditioning. Conclusions(1) Limb ischemia pretreament can induce late phase of preconditioning in rabbit liver. (2) MAPK signaling pathway mediate limb ischemia pretreament-induced late phase of preconditioning.(3) Limb ischemia pretreament 24h before can result in the changes of hepatic protein expression profiles. The differential proteins identified by MALDI-TOF-MS might be involved in the delayed hepatic protection induced by Limb ischemia pretreament. So, this study provided important clues for elucidating the molecular mechanisms of late phase of limb ischemia preconditioning and lay foundations for searching for novel proteins related to liver preconditioning.