| Objectives: To observe whether limb ischemic precondi- tioning(LIPC) can exert protective effect on reperfusion injury of liver following ischemia in experimental animal model, through which a new method that can lessen ischemia reperfusion injury of liver clinically was explored. Meanwhile, to explore the protective mechanism primarily by observing the expression of Bcl-2 and NF-κB p65 in hepatic tissue.Methods: 40 male SD(Sprague-Dawley) rats, weighing 227±12.6g, were randomly divided into 4 groups, 10 rats in each group: sham operation group(SO group), ischemia reperfusion group(IR group), hepatic ischemic preconditioning group(HIPC group), and limb ischemic preconditioning group(LIPC group). (1)SO group: hepatoduodenal ligament was dissected out, but not clamped, then the abdomen was closed temporarily. (2)IR group: hepatoduodenal ligament was occluded by a microvascular clamp, 30min later, the microvascular clamp was taken out allowing reperfusion, then the abdomen was closed temporarily. (3)HIPC group: hepatoduodenal ligament was occluded by a microvascular clamp for 10min in an interval of 10min, the same programe would last three times, up to 60min, the following treatments were same with the IR group. (4)LIPC group: bilateral femoral arteries were dissected and occluded for 10min in an interval of 10min, the same programe would last three times, up to 60min, the following treatments were same with the IR group. 6h after reperfusion, blood samples that taken from the heart were conserved in a freezer in order to detect the levels of serum alanine tranaminase(ALT) and aspartate tranaminase(AST) in each group. Liver samples were conserved in 4% paraformaldehyde in order to commit paraffin section. Differences were compared on hematoxylin and eosin (HE)-stained sections in each group. Expression of Bcl-2 and NF-κB p65 were examined by immunohistochemistry. Apop- tosis cell was studied by TUNEL(terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling) and AI was calculated according to that. SPSS software package was used for date of statistical analysis. One-way ANOVA and SNK-q test were used for quantitative date analysis. P values that were less than 0.05 were considered statistical significance.Results: (1)The rat liver in IR group looked bright red, but it turned soft and looked dark red after the occlusion of bloodstream in IR group, HIPC group and LIPC group. 6h after reperfusion, the rat liver in HIPC group and LIPC group became bright red again, but it turned hard and some punctiform or lamellar no-reflow region could be seen in the liver of IR group. (2)The level of ALT in SO, IR, HIPC and LIPC groups were 52.95±6.95U/L, 654.06±104.45U/L, 395.52±38.18U/L, 437.25±48.41U/L. The level of AST in SO, IR, HIPC and LIPC groups were 79.66±10.63 U/L, 782.49±112.40U/L, 431.75±40.89U/L, 481.53±58.17U/L. The levels of serum ALT and AST in IR, HIPC and LIPC groups increased significantly than that in SO group(P<0.05), and that in IR group was significantly higher compared with those in HIPC and LIPC groups(P<0.05). There was no statistical significance between HIPC and LIPC groups(p>0.05). (3) In the sections of SO group, histological examination showed that hepatic lobule could be seen clearly. Parenchymal cell looked normal, sinus hepaticus did not exist obvious congestion, few neutrophilic granulocyte could be seen; In the IR group, sinus hepaticus existed obvious congestion, parenchymal cell degenerated seriously, endotheliocyte existed obvious hydropsia and separated from basement membrane. The bile duct existed obvious congestion. Many neutrophilic granulocytes could be seen; The hepatocytes in HIPC and LIPC groups were similar to those in SO group. Parenchymal cell degenerated slightly, few endotheliocyte existed obvious hydropsia and separated from basement membrane, few neutrophilic granulocyte could be seen. (4)Expression of Bcl-2(positive index) in SO, IR, HIPC and LIPC groups were 0.119±0.026, 0.108±0.022, 0.632±0.099, 0.591±0.050. Expression of Bcl-2 in HIPC and LIPC groups increased significantly than those in SO and IR groups(P<0.05). There was no statistical significance between SO and IR groups(p>0.05), similarly, there was no statistical significance between HIPC and LIPC groups(p>0.05). (5)Expression of NF-κB p65 (positive index) in SO, IR, HIPC and LIPC groups were 0.093±0.025, 0.402±0.046, 0.635±0.079, 0.583±0.047. Expression of NF-κB p65 in IR, HIPC and LIPC groups increased significantly than that in SO group (P<0.05), and those in HIPC, LIPC groups were significantly higher than that in IR group(P<0.05). There was no statistical significance between HIPC and LIPC groups(p>0.05). (6) AI(%) in SO, IR, HIPC and LIPC groups were 0.92±0.16, 9.64±1.57, 4.94±0.42, 5.02±0.44. TUNEL-positive cells could be seen rarely in SO group. The apoptosis index(AI) in IR, HIPC and LIPC groups increased significantly than those in SO group(P<0.05). But AI in IR group were significantly higher than those in HIPC and LIPC groups (P<0.05). There was no statistical significance between HIPC and LIPC groups(p>0.05).Conclusion: (1)Ischemic preconditioning can inhibit oncosis and apoptosis induced by ischemia reperfusion, attenuate ischemia reperfusion injury consequently. (2)Limb ischemic preconditioning has the same protective effect for the IR injured liver as the hepatic ischemic preconditioning on the liver and can effectively decrease the IR injury. Limb ischemic preconditioning is a safe, sufficient, economical and convenient stimulus. (3)The anti-apoptosis effect relates to expression of Bcl-2. (4)Activation of NF-κB may play an important role in the protective effect of ischemic preconditioning.
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