中药独活及其单体蛇床子素体外抗血管生成的实验研究

Experimental study of Angelica Pubescens and Osthole isolated from Angelica Pubescens inhibiting angiogenesis in vitro

目的:探讨独活醇提物及其单体蛇床子素对体外血管生成的抑制作用及其可能的机制。方法:采用MTT法观察中药独活醇提物及蛇床子素对人脐静脉血管内皮细胞(human umbilical vein endothelial cell,HUVEC)和人肠癌LoVo细胞增殖的影响,Transwell小室趋化实验、体外小管形成实验以及流式细胞术,观察并比较独活醇提物及蛇床子素对HUVEC迁移、小管形成、凋亡及周期的影响。结果:3.75-30μg/ml的独活醇提物及蛇床子素作用48h时对HUVEC的细胞增殖抑制率分别在5.16%-10.15%和22.64%-65.56%之间,对LoVo细胞增殖抑制率分别在2.86%-7.29%和5.15%-24.39%之间。体外小管及小管迁移实验显示,3.75-30μg/ml的蛇床子素作用24h时HUVEC小管形成数目减少,且管腔不完整。3.75-30μg/ml的独活醇提物和蛇床子素处理12h对HUVEC迁移抑制率分别在-2.16%至8.00%和13.70%至63.04%之间。3.75-30μg/ml的独活醇提物和蛇床子素诱导HUVEC细胞凋亡率分别在6.1%-14.4%和18.8%-89.5%之间。独活醇提物和蛇床子素作用HUVEC 24h后,使内皮细胞周期主要阻滞在G0-G1期,蛇床子素对细胞周期影响强于独活醇提物。结论:蛇床子素在体外抑制血管生成作用强于独活醇提物,说明蛇床子素可能是独活醇提物中发挥抗血管生成作用的主要成分,其作用机制可能与抑制HUVEC增殖、迁移和小管形成,诱导HUVEC凋亡,阻滞HUVEC细胞周期有关。

Objective：To study the effect and mechnism of alcohol extracts from Angelica Pubescens and Osthole on anti-angiogenesis in vitro.Methods：MTT assay was used to observe the effect of alcohol extracts from Angelica Pubescens and Osthole on the proliferation of human umbilical vein endothelial cells （HUVECs） and human colorectal carcinoma cancer cell LoVo.Transwell migration assay and tube formation assay were used to observe the impact of alcohol extracts from Angelica Pubescens and Osthole on cell migration and tube forming ability of HUVECs.The rate of alcohol extracts from Angelica Pubescens and Osthole inducing HUVEC apoptosis and the effect of alcohol extracts from Angelica Pubescens and Osthole on cell cycle of HUVEC was calculated by flow cytometry.Results：Alcohol extracts from Angelica Pubescens（3.75-30μg/ml） inhibited the proliferation of HUVECs （5.16％-10.15％） and LoVo cells （2.86％-7.29％） after 48h treatment.Osthole （3.75-30μg/ml） inhibited the proliferation of HUVECs （22.64％-65.56％） and LoVo cells （5.15％-24.39％） after 48h treatment.When HUVECs were incubated with Osthole（3.75-30μg/ml）,the number of tubules was reduced and the lumen lost its integrity after 24h;The inhibition rate of migration was-2.16％ to 8.00％ when treated with alcohol extracts from Angelica Pubescens （3.75-30μg/ml）,the inhibition rate of migration was 13.70％-63.04％ when treated with Osthole （3.75-30μg/ml）.The rate of HUVEC apoptosis after treated with alcohol extracts from Angelica Pubescens （3.75-30 μg/ml） for 48h was 6.1％-14.4％.The rate of HUVEC apoptosis after treat with Osthole （3.75-30μg/ml） for48h was 18.8％-89.5％.Alcohol extracts from Angelica Pubescens （3.75-30 μg/ml） and Osthole （3.75-30μg/ml） may block the HUVEC cell cycle and arrest at G0-G1 phase after 24h,and the effect of the latter better than the former.Conclusion：The effect of Osthole on anti-angiogenesis in vitro is better than the effect of alcohol extracts from Angelica Pubescens,it may conclude that the main antiangiogenic constitution of Angelica Pubescens is Osthole.Osthole effectively suppresses angiogenesis in vitro,which may be through inhibiting cell proliferation,migration and tube-formation,inducing apoptosis and suppressing cell cycle of HUVECs.