| Objective:Hot liver ischemia-reperfusion Injury of rats model, the application of HO1specific revulsant cobalt protoporphyrin (CoPP) and inhibitors of zinc protoporphyrin (ZnPP) on experimental animals preprocessing, HO-1on the rat liver ischemia Reperfusion (IR) apoptosis and related gene, to choose effective pretreatment method, inhibiting cell apoptosis, can reduce the liver ischemia-reperfusion Injury (Hepatic Ischemic and Reperfusion Injury, HIRI) to provide theoretical basis.Methods:1. Adopt Nauta RJ introduced method of operation,70%of liver ischemia-reperfusion injury model was established.2. SPF health adult male SD rats, weight220～220g, were randomly divided into four groups:(1) control group (S):preoperative not vaccinating in reagent, only free after laparotomy hepatic Zhou Ren belt and then in the abdomen;(2) the ischemia reperfusion group (I group):preoperative not vaccinating in reagent, laparotomy revealed the first hepatic portal vein, GaBi middle of liver and the left lobe of liver artery and portal vein, dealing70%dry ischemia model, clamps, after60min restore blood flow to the liver;(3) cobalt protoporphyrin treatment group (group C): preoperative intraperitoneal injection of24hours revulsant HO-1CoPP, on ischemia-reperfusion;(4) zinc protoporphyrin treatment group (Z):preoperative intraperitoneal injection of24hours in HO-1revulsant ZnPP, ischemia-reperfusion. 3. Determination of index:in the liver ischemic reperfusion, portal,24h after reflow will kill rats,5ml inferior vena venous blood and get fresh liver tissue, detection in serum alanine aminotransferase (ALT), aspertate aminotransferase (AST) concentration in the evaluation of liver function and pathological change situation; Fluid cytology method to detect liver cell apoptosis rate; TUNEL method to detect the rat liver cell apoptosis; Western blot method to measure the Bcl-2, the Bcl-xl, Caspase3, NF-kB, the expression of p-IkBa conditions.Results:1. The total success for36cases of liver ischemia-reperfusion injury of rats model, the liver partial ischemia time for60min.2. The level of serum ALT and AST:group I, group C and Z group serum ALT and AST than S group (p<0.05); Serum ALT and AST below group I and group C Z group (p<0.05); Z group of serum ALT and AST was higher than group I (p<0.05), the difference was statistically significant.3. The liver histology inspection:S group of the hepatic lobule structure complete, normal liver cell and portal area structure; In group I, lobular central vein and hepatic blood sinus congestion, liver cell edema, degeneration degree of different cavity samples, part of the focal liver cell necrosis, eosinophilic increases; Z group, hepatic lobule structure disorder, liver cell cytoplasm osteoporosis, part into the balloon sample with eosinophilic necrosis, portal, surrounded by a large number of inflammatory cells infiltration, lesions compared with group I. And group C, hepatic lobule structure basic integrity, basic normal liver cell and portal area, a few mild edema, liver cells only.4. Check the liver cell apoptosis:(1) TUNEL detect liver cell apoptosis:at24h after reperfusion, few S set of visible liver nuclei were dyed tan; I set of visible dye lots of hepatic cells was tan; Group C compared with group I, the nucleus is a yellowing of the decrease in the number of liver cells, the color becomes weak; Z group increase in the number of liver cells with yellow, color burn.(2) fluid cytology test results:the liver cell apoptosis group I, group C and Z group apoptosis of rat liver tissue necrosis rate compared with S group increased significantly (P<0.05); Which group I apoptosis of rat liver tissue necrosis rate is significantly higher than group C (P<0.05); Z group rats liver necrosis tissue cell apoptosis rate was significantly higher than group I (P<0.05).5. Western Blotting test Caspase-3, p-IkBa and NF-kB seem protein expression level:(1) group I and Z Caspase-3, p-IκBa and NF-kB seem protein expression level was higher than group S (p<0.05); Caspase3group C and NF-kB seem protein expression levels lower than S group, the group I and Z group (p<0.05); Group C p-IκBa protein expression levels lower than group I and Z, higher than the group S (p<0.05); Z Caspase3, p-IκBa and NF-kB seem protein expression level was higher than group I (p<0.05), the difference was statistically significant.(2) S group and C group the Bcl-2, the Bcl-xl protein expression level was higher than group I (p<0.05); The Bcl-2Z group, the Bcl-xl protein expression level is lower than group I (p<0.05); The Bcl-2, the Bcl-xl group C protein expression level was higher than group S (p<0.05), the difference was statistically significant.Conclution:1. prevent the liver cells apoptosis, and eventually reduce reperfusion injury. The rat liver ischemia-reperfusion injury model is a stable, reliable, and simple model, the maneuverability is strong.2. Use using ZoPP pretreatment before reperfusion, after liver ischemia-reperfusion injury, caused the cohesion intracellular protein Caspase-3expression increased significantly, and use before reperfusion after pretreatment using the CoPP Caspase-3expression significantly reduce, prompt HO-1inhibit apoptosis in liver ischemia-reperfusion injury pathways may be related to Caspase-3.3. Use using ZoPP pretreatment before reperfusion, after liver ischemia-reperfusion injury, the Bcl-2gene, the Bcl-xl expression significantly reduced; And use before reperfusion after pretreatment using the CoPP gene the Bcl-2, the Bcl-xl expression increased significantly. Prompt HO, probably by increasing the Bcl-2system, the expression of Bcl-xl to inhibit apoptosis, so as to reduce the liver ischemia-reperfusion injury in rats.4. In the liver ischemia-reperfusion IkB kinase is activated, which can lead to IkB protein phosphorylation (p-IkBa), the nf-kappa B dimer was released, in cell nucleus, gene combination it with purpose, to promote the purpose gene transcription. And lead to an increased the expression of inflammatory factor, anti-inflammatory factor expression of HO-1; CoPP and use before infusion used for pretreatment of IkB kinase inhibition after activation, the nf-kappa B binding activity significantly decreased, the expression of inflammatory cytokines, increase anti-inflammatory factor expression of HO-1; HO-1inhibits IkB kinase activation, by inhibiting the activation of the nf-kappa B to further increase anti-inflammatory factors HO-1and the expression of inflammatory cytokines.5. HO-1high expression induced by CoPP can inhibit the liver ischemia-reperfusion injury after cell necrosis, so as to reduce reperfusion injury, liver may be associated with inhibition of cell apoptosis. |
PDF Full Text Request