狼毒大戟配伍大枣水煎液体外抗乳腺癌作用及机制研究

Study of the anti-breast cancer effects and mechanisms of decoction of Euphorbia fischeriana Steud.and jujuba in vitro

目的探讨狼毒大戟配伍大枣水煎液(decoction of Euphorbia fischeriana Steud.and jujuba,DEFSJ)体外抑制乳腺癌MCF-7细胞增殖并诱导其凋亡的可能分子机制。方法通过血清药理学方法制备DEFSJ载药血清(containing serum,CS);光镜及电镜下观察细胞形态学变化;DEFSJ-CS联合LY294002(PI3k信号通路抑制剂)作用后,采用CCK-8法检测细胞增殖抑制情况,流式细胞仪结合Annexin V-FITC/PI染色检测细胞凋亡情况,流式细胞仪结合免疫染色检测PI3k/Akt转导途径及Bcl-2家族相关蛋白的表达变化。结果光镜及电镜下可见细胞形态学变化及典型的细胞凋亡形态学特征;与阴性对照组比较,DEFSJ-CS对MCF-7细胞的增殖有明显的抑制作用,联合LY294002后抑制作用更加显著(P<0.01);Annexin V-FITC/PI染色结果显示,与阴性对照组比较,随着DEFSJ-CS含量的增加,细胞凋亡率明显增加,联合LY294002后凋亡作用更加显著(P<0.05,P<0.01);免疫染色检测结果显示,PI3k、p-Akt、p-FoxO3a、Bcl-2蛋白表达下调,Bax、Bim蛋白表达上调(P<0.05,P<0.01),联合LY294002后p-Akt、p-FoxO3a蛋白表达下调更加显著。结论PI3k/Akt转导途径参与了DEFSJ-CS抑制MCF-7细胞的增殖并诱导其凋亡作用的调控。

Objective To explore the proliferation-inhibition and inducement of apoptosis efficacy of decoction of Euphorbia fischeriana Steud.and jujuba(DEFSJ)on the human breast cancer cell line MCF-7 and reveal the underlying molecular mechanisms.Methods DEFSJ-containing serum(CS)was prepared via a serum pharmacology method.Cell morphology was observed by optical microscope and transmission electron microscope(TEM).After MCF-7 cells were treated with DEFSJ-CS and LY294002(PI3k signaling pathway inhibitor),the CCK-8 method was used to detect proliferation,the apoptosis rate was detected with a flow cytometer,and the expression of PI3k/Akt signaling pathway and Bcl-2 family-related proteins were detected by immunostaining.Results Morphological changes and typical features of apoptosis were observed by optical microscope and TEM.DEFSJ-CS had a significant inhibitory effect on the proliferation of MCF-7 cells(P<0.01),and the inhibitory effect was more significant when combined with LY294002(P<0.05,P<0.01).Annexin V-FITC/PI staining result showed that,compared with the negative control group,the apoptosis rate significantly increased with the increase of DEFSJ-CS,and the apoptosis rate was more significant when combined with LY294002.Immunostaining result showed that the protein expression levels of PI3k,p-Akt,p-FoxO3a,and Bcl-2 were down-regulated and the levels of Bax and Bim were up-regulated(P<0.05,P<0.01),and p-Akt and p-FoxO3a were down-regulated more significantly after combined treatment with LY294002.Conclusions The PI3k/Akt signaling pathway is involved in the effect of DEFSJ-CS on proliferation inhibition and inducing apoptosis in MCF-7 cells.