清肝活血方及其拆方对酒精性肝纤维化大鼠尿激酶型纤溶酶原激活物途径的影响

Effects of Qinggan Huoxue Recipe and its separated recipes on urokinase-type plasminogen activator and plasminogen activator inhibitor-1 fibrinolytic system in rats with alcoholic liver fibrosis

目的:通过观察清肝活血方及其拆方对酒精性肝纤维化大鼠尿激酶型纤溶酶原激活物(urokinase-type plas-minogen activator,uPA)和纤溶酶原激活物抑制物1(plasminogen activator inhibitor-1,PAI-1)途经的调控作用,探讨清肝活血方及其拆方治疗酒精性肝纤维化的作用机制。方法:雄性SD大鼠随机分为空白组、四氯化碳(carbon tetrachloride,CCl4)组和造模组。造模组采用56度二锅头酒、玉米油、吡唑混合物灌胃联合腹腔注射CCl4橄榄油溶液(CCl4∶橄榄油=1∶3)的方法造模。8周后造模组再随机分为模型组、清肝活血方组、清肝方组及活血方组。各治疗组分别加用相应药物(4.75、1.5、3.25g/kg)灌胃,其他各组灌胃等体积生理盐水。12周末处死大鼠,观察大鼠肝功能变化和肝组织病理学改变;采用蛋白印迹法、实时荧光定量聚合酶链式反应、免疫荧光法检测uPA、PAI-1蛋白和mRNA表达。结果:与空白组比较,CCl4组肝纤维化程度未见明显加重;模型组肝纤维化程度则明显加重(P<0.01),血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)和门冬氨基氨转移酶(aspartate aminotransferase,AST)活性明显升高,肝组织uPA和PAI-1表达明显升高,并且PAI-1升高幅度高于uPA。与模型组比较,清肝活血方及其拆方均能不同程度地减轻酒精性肝纤维化大鼠肝纤维化程度(P<0.01),降低血清ALT和AST活性,显著降低PAI-1表达,提高uPA表达。全方对上述环节的调控优于拆方。结论:清肝活血方及其拆方均能改善酒精性肝纤维化大鼠肝功能,减轻纤维化程度,且全方作用优于拆方,其机制可能与调控uPA/PAI-1纤溶途径有关。

Objective： To study the action mechanisms of Qinggan Huoxue Recipe （QGHXR）, a compound traditional Chinese herbal medicine, and its separated recipes by observing their effects on expressions of urokinasetype plasminogen activator （uPA） and plasminogen activator inhibitor-1 （PAl-1） in rats with alcoholic liver fibrosis （ALF）. Methods： A total of 150 SD rats were divided into three groups： blank group, carbon tetrachloride （CCl4） group and ALF-inducing group. Rats in the ALF-inducing group were administered with a mixture diet （56% alcohol 10 mL/kg, corn oil 2 mL/kg, pyrazole 25 mg/kg） once daily and intraperitoneally injected with 0.3 mL/kg 25% solution of CCl4 in olive oil twice weekly. TheCCl4 group was intraperitoneally injected with equal volume of CCl4 and olive oil as the ALF-inducing group and ingested normal saline （12 mL/kg per day）. The blank group was intraperitoneally injected with and ingested saline in equal volumes of the above. At the end of the eighth week, the survived rats in the ALF-inducing group were divided into four subgroups： untreated group, QGHXR group, Qinggan Recipe （QGR） group and Huoxue Recipe （HXR） group. The three treated groups were given corresponding drugs respectively （4.75, 1.5, 3.25 g/kg）. The blank group, CCl4 group and untreated group were given normal saline in equal volume （5 mL/kg per day）. All rats were anaesthetized and killed at the end of the twelfth week. The activities ofalanineaminotransferase （ALT） and aspartate aminotransferase （AST） in the serum were analyzed. Pathological changes in liver tissues were observed by hematoxylin and eosin staining and Masson staining. The expressions of uPA and PAI-1 were evaluated with Western blotting, immunofluorescence, and real-time polymerase chain reaction. Results： There existed obvious liver fibrosis in liver tissues in the untreated group as compared with the blank group （ P 〈0.01）, and the activities of ALT and AST and the expressions of uPA and PAI-1 also increased in the untreated group. QGHXR and its separated recipes could improve the degree of liver fibrosis （ P 〈 0.01）. QGHXR and its separated recipes could degrade the activity of ALT as compared with the untreated group; QGHXR and its separated recipes advanced the expression of uPA, and decreased the expression of PAI-1 significantly as compared with the untreated group. The effect of QGHXR was the best among the three recipes. Conclusion： QGHXR and its separated recipes may improve ALF in rats by decreasing the expression of PAI- l and advance the expression of uPA. The effect of QGHXR is the best among them.