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目的:研究离体肝脏缺血再灌注期间丝裂原活化蛋白激酶(mitogen activated protein kinase,p38MAPK)信号转导途径对肿瘤坏死因子α(tumor necrosis factorα,TNF-α)mRNA表达的影响。方法:建立兔离体肝脏缺血再灌注模型,对照组(n=12)灌注液中不加特异性p38MAPK抑制剂SB202190,抑制组(n=12)灌注液中加入SB202190(浓度为3μmol/L)。分别于肝脏离体前,冷保存末,再灌注10 min、30 min、60 min及120 min时获取离体肝组织标本。应用Western-blot法及免疫沉淀法检测离体肝组织中p38MAPK表达的水平及活性,RT-PCR法检测TNF-αmRNA表达水平。结果:对照组p38MAPK活性在冷保存末及再灌注10 min、30 min、60 min均较离体前和再灌注120 min显著升高(P<0.01),也显著高于同时相点的抑制组(P<0.01);抑制组p38MAPK活性在组内各时相点的变化无显著性差异(P>0.05)。两组肝脏于离体前、冷保存末及再灌注10 min及30 min,肝组织中仅有少量TNF-αmRNA表达,组间及组内比较无显著性差异(P>0.05);至再灌注60 min及120 min,对照组TNF-αmRNA的表达水平显著性高于组内其它时相点(P<0.01),而抑制组虽然也显著高于组内其它时相点(P<0.05),但却显著性低于同时相点对照组的表达水平(P<0.01)。离体再灌注期间供肝组织中p38MAPK活性与供肝组织内TNF-αmRNA的表达水平呈显著性正相关(r=0.996,P<0.01)。结论:p38MAPK对TNF-α生成的调节作用层次可能在转录水平,提示p38MAPK信号转导途径对TNF-αmRNA的调节可能是导致离体肝脏缺血再灌注损伤的重要机制之一。
Objective: To investigate the effect of p38 MAPK signal transduction pathway on tumor necrosis factor α (TNF-α) mRNA expression during ischemia-reperfusion in vitro. Methods: The model of isolated rabbit liver ischemia-reperfusion was established. In the control group (n = 12), SB202190, a specific inhibitor of p38MAPK, and SB202190 (3μmol / L ). Hepatic ex vivo specimens of ex vivo hepatic tissues were collected at the end of cold preservation, reperfusion at 10 min, 30 min, 60 min and 120 min respectively. Western blotting and immunoprecipitation were used to detect the expression of p38MAPK in ex vivo liver tissue. The expression of TNF-αmRNA was detected by RT-PCR. Results: The p38MAPK activity in the control group was significantly higher at the end of cold preservation and at 10 min, 30 min and 60 min than before and after reperfusion (P <0.01), and also significantly higher than that of the control group (P <0.01). There was no significant difference in the expression of p38MAPK between the two groups at each time point (P> 0.05). There was no significant difference between the two groups (P> 0.05), and only a small amount of TNF-αmRNA was found in the hepatic tissue before ex vivo, at the end of cold preservation and after 10 min and 30 min of reperfusion At 60 min and 120 min, the expression of TNF-αmRNA in the control group was significantly higher than that in other time points (P <0.01), while the inhibition group was significantly higher than other time points (P <0.05) But significantly lower than that of the control group at the same time point (P <0.01). There was a significant positive correlation between p38MAPK activity in hepatic tissue and the expression of TNF-αmRNA in donor tissue during reperfusion (r = 0.996, P <0.01). CONCLUSION: The regulatory effect of p38MAPK on TNF-α production may be at the transcriptional level, suggesting that the regulation of TNF-αmRNA by p38MAPK signal transduction pathway may be one of the important mechanisms leading to ischemia-reperfusion injury in isolated liver.