自噬在熊果酸抑制人脐静脉内皮细胞增殖中的作用

Role of autophagy in inhibition of proliferation of human umbilical vein endothelial cells by ursolic acid

目的研究自噬在熊果酸(Ursolic acid,UA)抑制人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)增殖中的作用,探讨UA抑制血管生长的机制。方法体外培养HUVECs,分别采用不同浓度的UA和自噬特异性抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)+UA联合处理,采用MTT法检测UA对HUVECs增殖的抑制作用及其联合3-MA对HUVECs存活率的影响;透射电镜观察细胞的超微结构;微管相关蛋白1轻链3(Microtubule-associated protein 1 light chain 3,MAP1-LC3)免疫荧光和流式细胞术检测UA对HUVECs自噬表达水平的影响;RT-PCR检测自噬相关基因Beclin1和MAP1-LC3B转录水平的变化。结果 UA可抑制HUVECs增殖,且呈剂量依赖性;HUVECs经UA处理后,自噬泡数量明显增加;经UA处理后,可上调HUVECs中MAP1-LC3蛋白的表达水平,MAP1-LC3阳性细胞的荧光强度较对照组明显增强;UA处理HUVECs不同时间可上调MAP1-LC3B及Beclin1 mRNA的转录水平;而UA联合3-MA处理后,HUVECs的增殖抑制作用明显增强。结论UA抑制HUVECs增殖,并诱导其发生自噬,自噬在此过程中起保护性作用,抑制自噬可明显增强UA诱导的HUVECs死亡。

Objective To investigate the role of autophagy in inhibition of proliferation of human umbilical vein endothelial cells（HUVECs） by ursolic acid（UA） as well as the relevant mechanism.Methods HUVECs were cultured in vitro and treated with various concentrations of UA and 3-methyladenine（3-MA,an autophagy-specific inhibitor） ＋ UA respectively.The effects of UA on proliferation as well as 3-MA ＋ UA on survival of HUVECs were determined by MTT method.The ultrastructure of HUVECs was observed by transmission electron microscopy.The expressions of autophagy-associated proteins in HUVECs treated with UA were determined by fluorescent staining of microtubule-associated protein 1 light chain 3（MAP1-LC3） and flow cytometry.The transcription levels of autophagy-associated gene Beclin1 and MAP1-LC3B were determined by RT-PCR.Results UA showed dose-dependent inhibitory effect on proliferation of HUVECs.Autophagic vacuoles increased in the HUVECs after treatment with UA.UA treatment up-regulated the expression level of MAP1-LC3 protein in HUVECs.The fluorescent density of MAP1-LC3 positive HUVECs increased significantly as compared with those in control group.Treatment with UA for various hours up-regulated the transcription levels of MAP1-LC3B and Beclin1 mRNAs in HUVECs.Treatment with 3-MA ＋ UA enhanced the inhibitory effect on proliferation of HUVECs.Conclusion UA inhibited the proliferation and induced the autophagy of HUVECs,in which autophagy played a protective role.The inhibition of autophagy significantly promoted the death of HUVECs induced by UA.