中药药理与临床

目的:探讨大黄甘遂汤对恶性腹水(malignant ascites, MA)小鼠腹膜肿瘤微血管生成的影响与作用机制。方法:通过腹腔注射H22肝癌细胞悬液建立MA小鼠模型，造模成功后随机分为模型对照组、恩度3×10^-3 g/kg组、华蟾素0.5 g/kg组、大黄甘遂汤5、10、20 g/kg组，每组10只，连续给予相应药物7 d。记录小鼠一般状态和死亡情况；测定腹水量；HE染色观察腹膜组织病理学变化；免疫组化和透射电镜检测腹膜微血管密度与超微结构；RT-PCR法检测腹膜组织缺氧诱导因子1α(Hif1α)、血管内皮生长因子(Vegf)、血管内皮细胞生长因子受体2(Vegfr2)、丝裂原活化蛋白激酶1(Mapk1) mRNA表达；Western blot法检测HIF1α、VEGF、VEGFR2、细胞外信号调节激酶(ERK)、磷酸化细胞外信号调节激酶(p-ERK)蛋白表达。结果:急性毒性实验显示，大黄甘遂汤在≤75 g/kg剂量范围内无小鼠死亡，提示其安全性良好。与正常对照组比较，模型对照组腹水量显著升高(P<0.01),腹膜组织肿瘤细胞增生及新生血管形成明显，微血管密度显著升高(P<0.01),血管结构异常，腹腔组织Hif1α、Vegf、Vegfr2、Mapk1 mRNA和HIF1α、VEGF、VEGFR2、ERK、p-ERK蛋白表达上调，pERK/ERK比值显著增高(P<0.01);与模型对照组比较，大黄甘遂汤各给药组腹水量明显降低(P<0.05),腹膜肿瘤细胞增生、血管新生和微血管密度有降低趋势，腹膜血管结构趋于正常，腹膜组织Hif1α、Vegf、Vegfr2、Mapk1 mRNA表达下调(P<0.05或P<0.01),大黄甘遂汤20 g/kg组HIF1α、VEGF、VEGFR2、ERK、p-ERK蛋白表达下调，pERK/ERK比值降低(P<0.01)。结论:大黄甘遂汤可能通过改善肿瘤缺氧环境中HIF1α的堆积，调节VEGF/VEGFR2/ERK信号通路，缓解肿瘤微血管生成，从而达到治疗MA的目的。

Objective:This paper aims to investigate the effects and action mechanism of Dahuang Gansui Decoction(大黄甘遂汤,DGD) on peritoneal tumor microangiogenesis in mice with malignant ascites(MA). Methods:First, the acute toxicity research of DGD was performed. Then, the MA mice models were established by intraperitoneal injection of H22 hepatocellular carcinoma cell suspension. After successfully modeling, mice were randomly divided into model control group, Endostar group(3×10^-3g/kg),Cinobufacini group(0.5 g/kg),and DGD groups(5,10,and 20 g/kg),with 10 mice in each group. All groups were administered with corresponding drugs for seven days. The general states and death conditions of mice were recorded, and the volume of ascites was measured. The peritoneal histopathological changes were observed by HE staining. The changes of peritoneal microvessel density and ultrastructure were detected by immunohistochemistry and transmission electron microscope. The mRNA levels of hypoxia-inducible factor 1α(Hif1α),vascular endothelial growth factor(Vegf),vascular endothelial cell growth factor receptor 2(Vegfr2),and mitogen-activated protein kinase 1(Mapk1) in peritoneal tissues were detected by RT-PCR. The protein expressions of HIF1α,VEGF,VEGFR2,extracellular signal-regulated kinase(ERK),and phosphorylated extracellular signal-regulated kinase(pERK) were detected by WB. Results:Acute toxicity test demonstrated that mortality was not observed in the mice administered with the dose range of 75 g/kg of DGD,suggesting its good safety. The results of pharmacological effect showed that compared with that in the normal control group, the volume of ascites in the model control group was significantly increased(P<0.01). The proliferation of tumor cells and neovascularization in peritoneal tissues was observed obviously, the microvessel density was significantly increased(P<0.01),the vascular structure was observed to be abnormal. The mRNA levels of Hif1α,Vegf,Vegfr2, and Mapk1 and the protein expression levels of HIF1α,VEGF,VEGFR2,ERK,and pERK were significantly increased, and the ratio of pERK/ERK was significantly increased(P<0.01). Compared with that in the model control group, the volume of ascites was remarkably reduced in all administered groups with DGD(P<0.05). The proliferation of peritoneal tumor cells, angiogenesis, and microvessel density showed a decreasing trend. The electron microscope results indicated that the peritoneal vascular structure tended to be normal, the mRNA expressions of Hif1α, Vegf,Vegfr2, and Mapk1 were markedly down regulated(P<0.01,P<0.05); the expressions of HIF1α,VEGF,VEGFR2,ERK,and pERK,as well as the ratio of pERK/ERK were down regulated in DGD group with 20 g/kg(P<0.01). Conclusion:DGD may achieve the therapeutic effect for MA by improving the accumulation of HIF1α in the hypoxic tumor environment, regulating the VEGF/VEGFR2/ERK signaling pathway, and then alleviating tumor microangiogenesis.