5-脂氧合酶抑制剂齐留通抑制二乙基亚硝胺诱导大鼠肝细胞癌形成的实验研究

Zileuton,a 5-lipoxygenase specific inhibitor prevents hepatocarcinogenesis induced by diethylnitrosamine in rats and its mechanism

目的:研究5-脂氧合酶(5-lipoxygenase,5-LOX)在原发性肝癌中的表达及其抑制剂齐留通对肝癌的影响和机制。方法:实验大鼠随机分为正常对照组(n=5)、模型组(n=8)及齐留通组(n=8)。HE染色观察肝癌病理图片,免疫组织化学染色检测5-LOX蛋白,M2型丙酮酸激酶(M2-PK)与细胞角蛋白19(CK19)的表达,RT-PCR检测肝组织5-LOX的表达,TUNEL检测肝癌细胞凋亡。结果:二乙基亚硝胺(DEN)可以诱导大鼠肝癌模型形成。模型组大鼠肝脏可见中等强度5-LOX的蛋白表达,较对照组明显增强(P<0.01),而齐留通组5-LOX的表达较模型组明显降低(P<0.01)。免疫组织化学检测M2-PK在对照组阴性表达,模型组大鼠中等强度表达(P<0.01),齐留通组弱表达,较模型组差异显著(P<0.05)。CK19在对照组大鼠中阴性表达,模型组大鼠较强表达(P<0.01),齐留通组中等表达,较模型组差异显著(P<0.01)。对照组可检测出5-脂氧合酶mRNA的表达,但明显低于模型组,差异显著(P<0.01);齐留通组的表达介于二者之间,与模型组相比,有统计学意义(P<0.01)。TUNEL法检测对照组几乎未见凋亡细胞,模型组大鼠肝脏仅见少数肝癌细胞阳性表达(30%-50%),而齐留通治疗组大鼠肝脏见大量肝癌细胞阳性表达(70%-80%),与模型组相比有统计学意义(P<0.01)。结论:DEN诱发的大鼠原发性肝癌存在5-脂氧合酶表达,5-脂氧合酶抑制剂可防治DEN诱发的大鼠原发性肝癌的形成,其机制与诱导肝癌细胞凋亡相关。

Objective：To study the expression of 5-lipoxygenase in rat hepatocellular carcinoma（HCC） and to investigate whether its specific inhibitor Zileuton can prevent hepatocarcinogenesis.Methods： Male Wistar rats were randomly divided into three groups： normal control group（n=5）,model group（n=8） and Zileuton group（n=8）.HE staining was used for pathological examination of rat liver samples.The expression of 5-lipoxygenase protein,M2-PK and CK19 were detected by immunochemical staining,5-lipoxygenase mRNA was detected by reverse transcriptase-polymerase chain reaction（RT-PCR）.Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling（TUNEL） assay was used to detect apoptosis in the liver.Results： DEN successfully induced rat HCC model.In model group,5-lipoxygenase protein was middle or highly expressed,which is significant higher than the control group（P0.01）.However,Zileuton group was significant lower than model group（P0.05）.M2-PK and CK19 were negative expressed in control group and M2-PK was medium expressed in model group（P0.01）,while in Zileuton group M2-PK was poorly expressed.CK19 was strongly detected in model group,while in Zileuton group was slightly expressed（P0.05）.5-lipoxygenase mRNA in control group was expressed significant lower than model group（P0.05）,Zileuton group was between them（P0.05）.TUNEL assay showed that almost no apoptosis occurred in control group and the model group rats liver expressed minority hepatoma carcinoma cell masculine（30%-50%）.However in Zileuton group rats liver found multiplicity hepatoma carcinoma cell masculine expressed（70%-80%）statistically higher compared with the model group（P0.05）.Conclusion： DEN induced rat HCC have 5-lipoxygenase expressed.Inhibition of 5-lipoxygenase by Zileuton exerts inhibitory effect on the development of HCC in rats induced 5-lipoxygenase.The mechanism may be associated with induction of apoptosis.