| Objectives: To observe whether ischemic postconditioning (I-post) and ischemic proconditioning (IP) could exert protective effect on ischemia reperfusion (IR) injury of liver inexperimental animal model, to investigate the protective mechanism of I-post, and to provide more available method that can lesson IR injury.Methods: Thirty-two male wistar rats, weighing 250g-300g, were divided into four groups randomly, eight rats in each group, namely sham operation (SO) group, ischemia reperfusion (IR) group, ischemic preconditioning (IP) group and ischemic postconditioning group. (1) SO group: Cut off the abdomen after anaesthetize, only dissected out hepatoduodenal ligaments; (2) IR group: dissected out hepatoduodenal ligaments after anaesthetized, clipped caudate lobe and left lobe of liver blood, resulted in 70% livers ischemia, but didn't stop right lobe of liver blood, preventing from congestion of portal vein and stomach and intestinal tract, ischemia for 60 minutes and perfused for 120 minutes; (3) IP group: Treatmented as same as above, ischemia for 5 minutes and perfused for 5 minutes, ischemia for 60 minutes and perfused for 120 minutes; (4) I-post group: Treatmented as same as above, ischemia for 60 minutes, perfused for 2 minutes, ischemia for 2 minutes, perfused for 3 minutes, ischemia for 2 minutes, perfused for 5 minutes, ischemia for 2 minutes and perfused for 7 minutes, ischemia for 2 minutes, perfused for 95 minutes. Blood samples that taken from the heart were conserved in a freezer in order to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Liver samples, 1mm×1mm×3mm, were preserved in 4% glutaral, in order to observe through electron microscope. Another liver samples were conserved in paraformaldehyde in order to commit paraffin section. Differences were compared on hematoxylin and eosin(HE)-stained sections in each group. Expression of NF-κBp65 were examined useing immunohistochemistry and calculated positive index (PI). Apoptosis cell were studied useing terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling (TUNNEL) and apoptosis index (AI) was calculated according to that. Another liver sample was conserved in freezer in order to detect the superoxide dismutase (SOD). SPSS software package was used for date of statistical analysis. One-way ANOVA and SNK-q test were used for quantitative date analysis. P value that were less than 0.05 were considered statistical significance.Results: (1) The rat liver in SO group looked bright red; In the groups of IR group, IP group, I-post group, after stopping liver blood supply, the liver's color became dark red, the quality became soft. After perfuse 2 hours, the liver surface's color in IR group shallow, visceral surface had many dark and red spot piece; Compare with IR group, IP group and I-post group were obviously easer. (2) The result of HE-stainning manifestation: The SO group's structure of hepatic lobule outlined clear. The liver cell cord was integrity. Structure of parenchymal cell structure was normal, had no dropsy.Hepatic sinusoid had no obvious congestion, neutrophilic granulocyte spread at. The IR group: central veins of liver and hepatic sinusoid congested obvious. Hepatic sinusoid was narrow. Liver cell looked different degree of granular degeneration and vacuolus, accidentally punctiform necrosis. There was neutrophilic granulocyte aggregation and infiltration, gradually took central vein surroundings as very, in the liver tissue. Compare with IR grorp, the congestion of IP group and I-post group was easer, the hepatic lobule's structure was basic normal, and the cell degeneration was not obvious, and neutrophilic granulocyte infiltration easer. (3) The results of electron microscope: The IR proup looked, under the the mirror of×4 k, that greatly parts of double nuclear membrane were blend, unclear, and parts of perinuclear cisterna lost, and around the cell had an oval dropsy piece; under the mirror of×20 k, majority of mitochondrial crista and the greatly parts of mitochondria membrane were unclear or fusion and rough endoplasmic reticulum light degree extension, degranulation obvious. The IP group looked, under the the mirror of×4 k, one oval great electron density lipid droplet was inside the liver cell, greatly parts of double nuclear membrane were fusion, unclear, and parts of perinuclear cisterna imperfection or disappear. Under the mirror of×20 k, it looked greatly parts of mitochondrial crista and parts of mitochondria membrane were blend, unclear or imperfection, and rough endoplasmic reticulum had degranulation phenomenon. The I-post proup looked, under the the mirror of×4 k, that one oval and great electron density lipid droplet was inside the liver cell, and greatly parts of double nuclear membrane were fusion and unclear, and parts of perinuclear cisterna were imperfection or disappear, and the cell nucleus contained 3-4 nucleoli. Under the mirror of×20 k, part or greatly parts of mitochondrial crista, and parts of mitochondria membrane blend, were unclear or imperfection, and rough endoplasmic reticulum had degranulation phenomenon. (4) The levels of ALT in SO group, IR group, IP group and I-post group were 62.67±17.88U/L, 1312.83±96.95U/L, 730.67±198.80U/L, 753.67±105.73U/L; The levels of AST in SO group, IR group, IP group and I-post group were 130.17±52.24U/L, 1013.83±210.51U/L, 643.17±133.77U/L, 725.33±237.70U/L. Compare with SO group, the levelS of ALT, AST in IR group, IP group and I-post group were higher (P<0.05). Among these groups, the IR group was highest, and IP group and I-post group comparison, the difference had no statistical significance (P>0.05). (5) The PIs of NF-κB in SO group, IR group, IP group and I-post group were 6.83±2.27%, 55.65±5.77%, 35.17±6.17%, 33.88±5.54%. Compare with SO group, the PI values of NF-κB in IR group, IP group and I-post group were obviously higher (P<0.05). Among these groups, the IR group is highest. There were no statistics difference in IP group and I-post group (P>0.05). (6) The AIs of SO group, IR group, IP group and I-post were 1.00±1.26, 10.02±4.23, 5.03±1.06, 6.82±1.94. TUNNEL-positive cells could be seen rarely in SO group. Compare with SO group, the AI values of NF-κB in IR group, IP group and I-post group are obviously higher (P<0.05). The AI value was highest in IR group, and there were no statistics difference in IP group and I-post group (P>0.05). (7) The levels of SOD in SO group, IR group, IP group and I-post group were 14.56±2.44NU/mg, 8.53±1.28NU/mg, 10.64±2.35NU/mg, 9.62±2.86 NU/mg. Compare with SO group, the SOD values of NF-κB in IR group, IP group and I-post group were obviously higher (P<0.05). Among these groups, the IR group was highest, and the difference in IP group and I-post group was no statistical significance(P>0.05).Conclusion: (1) Ischemic postconditioning could depress the synthesis of oxygen-derived free radicals and reduce hepatocellular apoptosis and NF-κB after reperfusion,and thus produce a protective effect on hepatic ischemia reperfusion injury. (2) Ischemic proconditioning could depress the synthesis of oxygen-derived free radicals and reduce hepatocellular apoptosis and NF-κB after reperfusion, and thus produce a protective effect on hepatic ischemia-reperfusion injury. (3) Ischemia postconditioning produced the same protective effect on hepatic ischemia-reperfusion injury as ischemic procondtionning. (4) Ischemic postconditioning was more meaningful than ischemic proconditioning in the clinical Practice.
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