柴胡皂甙D对实验性大鼠肝癌血管形成的抑制作用

Inhibitory effect of saikosaponin-d on the angiogenesis of experimental hepatocarcinoma in rats

目的:观察柴胡皂甙D(saikosaponin-d,SSd)对大鼠肝脏癌变过程中环氧化酶-2(cycloxygenase-2,COX-2)、血管内皮细胞生长因子(vascular endothelial cell growth factor,VEGF)及CD34表达的调节作用,并探讨其与SSd抗肝肿瘤作用的关系.方法:清洁级雄性SD大鼠90只,随机分为5组:模型组(n=20),对照组(n=10)与SSd大、中、小剂量治疗组(各组n=20).对照组给予(ig)等量生理盐水,其余各组大鼠均给予(ig)2g/L二乙基亚硝胺(DEN,10mg/kg),每周5次,同时各治疗组每天腹腔注射不同浓度SSd(2.0,1.5,1.0mg/kg),至16wk停药.分别于第6、12、16周处死大鼠,HE染色观察实验大鼠各期肝组织病理学结构的改变,免疫组化检测大鼠肝脏癌变过程中COX-2、VEGF及CD34的表达.结果:实验第6、12、16周,单纯造模组大鼠呈现典型的肝细胞损伤、增生硬化和肝癌形成的病理变化.SSd各治疗组大鼠癌结节数及灶的大小均小于模型组.镜下单纯造模组癌细胞呈多形性,异形性明显;相反,SSd各干预组癌细胞分化程度高,异形性较低.免疫组化结果显示COX-2、VEGF及CD34均在诱癌早期表达较少,肝癌期表达明显增强.SSd干预后,肝癌形成期COX-2、VEGF及CD34表达的抑制作用最明显,与单纯造模组相比差异显著(P<0.05或P<0.01);COX-2与VEGF(r=0.815,P<0.01),VEGF与MVD(r=0.862,P<0.01)以及COX-2与MVD(r=0.726,P<0.01)的表达均明显正相关.结论:SSd对实验性大鼠肝癌形成具有一定的抑制作用,其作用机制可能与SSd下调肝肿瘤中COX-2的表达,抑制VEGF的活性有关.

AIM： To investigate the effect of saikosaponin-d （SSd） on the expressions of cycloxygenase-2 （COX-2）, vascular endothelial cell growth factor （VEGF） and CD34 during hepatocellular carcinogenesis induced by diethylinitrosamine （DEN）. METHODS： Ninety male SD rats were randomly divided into 5 groups： control group （n = 10）, model group （n = 20） and three treatment groups （n = 20 in each group）. Except for the control group, the other groups were administered （ig） with 2 g/L DEN at a dose of 10 mg/kg body weight （5 times/wk for 16 wk）. At the same time, the rats in the treatment groups were injected intraperitoneally with SSd at different doses （1.0, 1.5 and 2.0 mg/kg） once per day for 16 wk. The rats were anesthetized and killed at 6, 12, and 16 wk. Hematoxylin and eosin （HE） staining was used to examine the changes of liver pathology and the expression of COX-2, VEGF and CD34 in liver tissues were dynamically monitored by SABC immunohistochemistry. RESULTS： During 6, 12, and 16 wk of experiment, typical pathological changes such hepatocyte injury, hyperplasia, cirrhosis, and carcinogenesis appeared in the rats of model group. Both the volume and the number of tumors were significantly reduced in 3 SSd treatment groups. Under microscope, the cancer cells in the rats of model group showed polymorphic and abnormal shape. In contrast, the cancer cells in the rats of SSd treatment groups were well differentiated. Immunohistochemistry demonstrated that the expression of COX-2 and VEGF and the value of microvessel density （MVD） in the model group were low in the early stage of carcinogenesis, but they were all remarkably increased in the stage of carcinoma. Whereas, the expression of COX-2 and VEGF and MVD value were all reduced in every stage of carcinogenesis after SSd treatment, especially in the stage of carcinoma after high-dose SSd treatment. There was a highly positive correlation between COX-2 and VEGF expression （r = 0.815, P 〈 0.01）, VEGF expression and MVD value （r = 0.862, P 〈 0.01） and between COX-2 expression and MVD value （r = 0.726, P 〈 0.01）. CONCLUSION： SSd has inhibitory effect on the angiogenesis during DEN-induced hepatocarcinogenesis in rats, which may be related to the down-regulation of COX-2 expression and VEGF activity.