乌头碱调控雌二醇合成抑制MCF7增殖机制研究  

Mechanism of aconitine regulating estradiol synthesis to inhibit MCF7 proliferation

作　　者：周春燕 杨富金[2] 江雪[2] 刘德明 ZHOU Chunyan;YANG Fujin;JIANG Xue;LIU Deming(Department of General Surgery,Chongqing Traditional Chinese Medicine Hospital,Chongqing 400011,China;Department of Dermatology,Chongqing Traditional Chinese Medicine Hospital,Chongqing 400011,China)

机构地区：[1]重庆市中医院普外科,重庆400011 [2]重庆市中医院皮肤科,重庆400011

出　　处：《中药与临床》2024年第4期32-38,共7页Pharmacy and Clinics of Chinese Materia Medica

基　　金：重庆市自然科学基金面上项目(cstc2020jcyjmsxm X1003);重庆市科研机构绩效激励引导专项(CSTC2022JXJL120005);成都中医药大学杏林学者项目(YYZX202160)。

摘　　要：目的:阐释附子乌头碱调控雌二醇生物合成抑制MCF7增殖的潜在机制。方法:通过CCK8检测不同浓度乌头碱对KGN、MCF7细胞增殖能力的影响;通过Elisa检测乌头碱对KGN细胞中雌二醇生物合成的影响;使用q RT-PCR检测乌头碱对芳香化酶及其启动子m RNA表达的影响;通过分子对接研究乌头碱与Na^(+)-K^(+)-ATP酶的相互作用;使用流式细胞仪检测细胞内Ca^(2+)浓度;Western bolt检测芳香化酶、Na^(+)-K^(+)-ATP、Ca M、Ca MKII蛋白表达。结果:乌头碱(1~80μM)处理KGN细胞48 h后对KGN细胞无毒性,而其浓度大于5μM时,对MCF7具有明显细胞毒性;乌头碱对芳香化酶及其启动子PⅠ.1、PⅠI、PⅠ.3、PⅠ.4均无影响;乌头碱与3A3Y受体蛋白有好的结合,结合能量分别为-7.8 kcal·mol^(-1),乌头碱能够下调Na^(+)-K^(+)-ATP酶蛋白表达;乌头碱处理KGN细胞24 h后,细胞内Ca^(2+)显著上调,Ca^(2+)-CaM-CaMKⅡ信号通路被激活,芳香化酶磷酸化水平升高。结论:乌头碱抑制细胞膜上Na^(+)-K^(+)-ATP酶,调控细胞内Na^(+)外流,激活Na^(+)/Ca^(2+)交换,胞内Ca^(2+)浓度升高,激活Ca^(2+)-Ca M-Ca MKⅡ信号通路,Ca MKⅡ调控芳香化酶ser118磷酸化,在转录后水平促进芳香化酶降解,抑制雌激素合成,从而抑制MCF7生长。Objective:To elucidate the potential mechanism of aconitine(AC)regulating estradiol biosynthesis and inhibiting the proliferation of MCF7.Methods:The effect of different concentrations of AC on KGN and MCF7 cells proliferation was detected by CCK8.The effect of AC on estradiol biosynthesis was detected by Elisa.After corresponding AC treatment,the expression of aromatase and its promoter mRNA were detected by qRT-PCR.The interaction between AC and Na^(+)-K^(+)-ATPase was studied by molecular docking.The intracellular Ca^(2+)concentration was measured by flow cytometry.The expressions of Na^(+)-K^(+)-ATP,CaM and CaMKII were detected by Western bolt.Results:There was no toxicity on KGN cells after 48 h AC(1^80μM)treatment,but obvious cytotoxicity on MCF7 was observed when its concentration was greater than 5μM.AC had no effects on aromatase and its promoters(PⅠ.1,PⅠI,PⅠ.3 and PⅠ.4)mRNA expression.AC targeted 3A3Y with affinity energy of-7.8 kcal·mol^(-1),and down-regulated Na^(+)-K^(+)-ATPase expression.After 24 h AC treatment,intracellular Ca^(2+)of KGN cells was significantly up-regulated,the Ca^(2+)-CaM-CaMKⅡsignaling pathway was activated,and aromatase phosphorylation was increased.Conclusion:AC inhibits Na^(+)-K^(+)-ATPase,regulates intracellular Na^(+)outflow,activates Na^(+)/Ca^(2+)exchange,increases intracellular Ca^(2+)concentration,and activates Ca^(2+)-CaM-CaMKⅡsignaling pathway.CaMKⅡregulates aromatase ser118 phosphorylation which promotes aromatase degradation post-transcriptional level and then reduce estrogen synthesis,finally inhibited MCF7 cell growth.

关 键 词：乌头碱 雌二醇 芳香化酶 雌激素受体阳性乳腺癌 

分 类 号：R285.5[医药卫生—中药学]