加味二至丸植物雌激素样作用及作用机制的实验研究  

Experimental Study on Estrogen-like Action and Action Mechanism of Modified Erzhi Pill

作　　者：苏婷 赵元辰 Su Ting;Zhao Yuanchen(Tianjin Tianjin Binhai New Area Chinese Medicine Hospital,300451;Tianjin Beichen District Chinese Medicine Hospital,300400)

机构地区：[1]天津市滨海新区中医医院,天津300451 [2]天津市北辰区中医医院,天津300400

出　　处：《中药药理与临床》2020年第2期201-205,共5页Pharmacology and Clinics of Chinese Materia Medica

基　　金：国家科技支撑计划项目(编号:2014BAI05B01)。

摘　　要：目的:建立基于荧光素酶报告基因的HEK293T细胞体外雌激素样物质检测方法,评估加味二至丸的雌激素样作用及作用机制。方法:通过荧光定量PCR方法扩增人雌激素受体(Estrogen receptor,ER)及雌激素反应原件(Estrogen response element,ERE)目的基因,构建PCMV-FLAG-His-hERa、PCMV-FLAG-His-hERβ重组质粒以及ERE调控的双荧光素酶报告基因质粒psiCHECK2-ERE-luc,将pERE-Luc和phERa/phERβ共转染HEK293T细胞,药物处理后,检测报告基因荧光素酶活性,分析加味二至丸的雌激素样作用及作用机制。结果:加味二至丸在雌激素受体阴性HEK293T细胞中对ERE调控的报告基因没有转录激活作用,而在过表达ERa或ERβ的HEK293T细胞中,加味二至丸诱导的荧光素酶活性显著高于空白对照组,而且对ERβ的选择性作用强于ERa,其雌激素活性能被ER抑制剂ICI182780拮抗。结论:加味二至丸通过ER途径发挥雌激素样作用,而且对ERβ的亲和力高于ERa,治疗绝经相关外阴阴道萎缩的同时无刺激子宫内膜过度增殖的不良反应。Objective:To establish an in vitro estrogen-like substance detection method for HEK293 T cells based on luciferase reporter gene,and to evaluate its estrogen-like effects and mechanism.Methods:Human estrogen receptors(ER)and Estrogen response element(ERE)genes were amplified by PCR,construct PCMV-FLAG-His-hERa,PCMV-FLAG-His-hERβrecombinant plasmids,and ERE-regulated dual luciferase reporter plasmids psiCHECK2-ERE-luc.were constructed.HEK293 T cells were co-transfected with pERE-Luc and phERa/phERβ.After treatment,reporter gene luciferase activity was determined to analyze estrogen-like effects of modified Erzhi pill and its mechanism.Results:Modified Erzhi Pills showed no activating transcription of ERE-regulated reporter genes in HEK293 T cells.After treatment,in HEK293 T cells with over-expressing ERa or ERβ,luciferase activity was significantly increased than that in the control group,the selective action of ERβwas stronger than that of ERa.Its estrogenic activity were inhibited by the estrogen receptor antagonist ICI182780.Conclusion:Modified Erzhi Pill can exert estrogen-like effects through the ER pathway,it has higher affinity for ERβthan ERa.No adverse reactions to stimulation of endometrial hyperproliferation when treating on vulvovaginal atrophy.

关 键 词：加味二至丸 外阴阴道萎缩 雌激素受体 植物雌激素 

分 类 号：R285[医药卫生—中药学]