匍匐大戟醇提物抗肿瘤及抑制血管新生作用

In vivo anti-tumor effect and in vitro anti-angiogenic effect of alcohol extract from Euphorbia prostrata

选取未曾研究过的云南新平产匍匐大戟,系统研究其抗肿瘤血管生成作用机制。制备匍匐大戟醇提物,取其浸膏配制成合适浓度为受试药,采用经典小鼠的急性毒性实验方法测定最大耐受量(MTD)。同时取正常细胞及肿瘤细胞,给药后通过MTT法检测其IC50。原代培养大鼠胸主动脉内皮细胞(rat aortic endothelial cells,RAECs),通过体外i CELLigence实时细胞分析实验、细胞迁移实验、细胞小管形成实验分别检测匍匐大戟醇提物对RAECs的增殖、迁移和成管能力的影响,并通过应用Western blot方法检测Akt,p-Akt,eNOS,p-e NOS,TGF-β1,Smad3等血管新生相关信号通路蛋白的表达情况。通过建立人肝癌裸鼠移植瘤模型,给以匍匐大戟醇提物,检测裸鼠体重、肿瘤体积、瘤质量等,应用ELISA法检测各组裸鼠血清中血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)和血小板衍生生长因子BB(platelet-derived growth factor,PDGF-BB)的水平,并进行HE染色观察移植瘤组织形态。匍匐大戟醇提物对小鼠的最大耐受量MTD为94.29 g·kg^(-1)。匍匐大戟醇提物对大鼠成肌细胞(L6细胞株)无明显抑制作用,对人肝癌细胞株HepG-2、大鼠肾上腺嗜铬细胞瘤细胞株PC12、人非小细胞肺癌细胞株A549、人宫颈癌细胞细胞株Hela均有显著性的抑制作用,且匍匐大戟醇提物在体外能够显著抑制RAECs的增殖、迁移和小管形成,并且促进RAECs中AKT和e NOS的磷酸化及增加TGF-β1和Smad3蛋白表达。同时,匍匐大戟醇提物可显著性抑制裸鼠在体肿瘤的生长并降低肿瘤裸鼠血清中VEGF的产生,但可增加PDGF-BB因子的含量。匍匐大戟醇提物对肿瘤生长及血管生成具有显著的抑制作用,这可能是其发挥抗肿瘤作用的机制之一。

The study was designed to investigate the anti-tumor and anti-angiogenic effects of alcohol extract from Euphorbia prostrata. The alcohol extract of E. prostrata was prepared, and the tolerated dosage was determined in mice by the test for acute toxicity. Then, MTF method was used to study the anti-proliferation effect of E. prostrata on normal cells and tumor cells. The rat aortic endothelial cells （RAECs） were primarily cultured. Subsequently, in vitro cell proliferation, migration and tubule formation assays were per- formed to detect the effect of alcohol extract of E. prostrata on proliferation, migration and angiogenesis. Western blot analysis was per- formed to detect the protein expressions of Akt, p-Akt, eNOS, p-eNOS, TGF-β1 and Smad3 in RAECs treated with E. prostrata. In addition, an in vivo transplanted hepatocellular carcinoma model in nude mice was established to detect nude mass, tumor volume and tumor weight. The contents of vascular endothelial growth factor （VEGF） and the platelet-derived growth factor-BB （PDGF-BB） in blood serum were detected by using ELISA kits. HE staining was performed to study the morphology of tumor tissues. The tolerated dosage of alcohol extract of E. prostrata in mice was 94. 29 g · kg^- 1. Alcohol extract of E. prostrata showed no inhibitory effect on E6 ceils, but significantly inhibited the proliferations of HepG-2, PC12, A549, and Hela cells with the following order： HepG-2 〉 Hela 〉 PC12 〉 A549. Meanwhile, alcohol extract of E. prostrata markedly inhibited the proliferation, migration and tube formation of RAECs, and enhanced the expressions of phosphorylated Akt and eNOS and increased the expressions of TGF-β1 and Smad3. In addition, E. prostrata notably inhibited the tumor growth in mice, and decreased the amount of VEGF, but increased the amount of PDGF-BB factor in serum of nude mice. The alcohol extract of E. prostrata may show an inhibitory effect on tumor growth and angiogenesis, which may contribute to its anti-tumor effect.