黄芪注射液及其有效成分对乳腺癌细胞增殖和Akt磷酸化的影响

目的：观察黄芪注射液、黄芪甲苷、芒柄花素对basal-like乳腺癌细胞株MDA—MB-468和MDA—MB-231增殖和磷酸化Akt（phospho-Akt，p-Akt）蛋白表达的影响。方法：用不同浓度的黄芪注射液、黄芪甲苷和芒柄花素对乳腺癌细胞株MDA—MB-468和MDA-MB-231进行干预，以不加药物干预的细胞作为空白对照。用甲基噻唑基四唑（methyl thiazolyl tetrazolium，MTT）法观察药物对细胞增殖的作用。再选取药物抑制细胞增殖的最佳浓度，用细胞内蛋、白质印迹法观察对p-Akt（Thr308）和p-Akt（Ser473）蛋白表达水平的影响。结果：根据MTT比色法实验结果确定黄芪注射液、黄芪甲苷、芒柄花素和黄芪甲苷配伍芒柄花素的最佳浓度，黄芪注射液终浓度为生药含量1g／mL，黄芪甲苷终浓度为80μg／mL，芒柄花素终浓度为40μg／mL，黄芪甲苷：芒柄花素为1：4。干预MDA—MB-468细胞1d后，各用药组p-Akt（Thr308）和p—Akt（Ser473）蛋白表达水平下调，与阴性对照组比较，差异有统计学意义（P〈0．05）；药物干预2d后，各用药组p-Akt（Thr308）水平显著下调（P〈0．05），其中以芒柄花素组、黄芪甲苷＋芒柄花素组下调最为明显；芒柄花素和黄芪甲苷＋芒柄花素明显下调P—Akt（Ser473）蛋白表达。干预MDA—MB-231细胞1d后，与阴性对照组比较，芒柄花素组和黄芪甲苷＋芒柄花素组p-Akt（Thr308）蛋白水平有明显的下调；在药物干预2d后，黄芪注射液、芒柄花素和黄芪甲苷＋芒柄花素组p-Akt（Thr308）蛋白水平都有明显下调。备用药组p—Akt（Ser473）蛋白水平与阴性对照组比较，差异无统计学意义。结论：黄芪注射液、黄芪甲苷、芒柄花素对乳腺癌细胞株MDA—MB-468和MDA—MB-231均有抑制增殖的作用，但抑制作用程度及其浓度存在差异，其抑制细胞增殖的机制可能与下调Akt的磷酸化有关。

Effects of Astragalus injection and its ingredients on proliferation and Akt phosphorylation of breast cancer cell lines

Objective： To observe the effects of Astragalus injection, astragaloside Ⅳ and formononetin on proliferation and Akt phosphorylation of basal-like human breast carcinoma cell lines MDA-MB-468 and MDA-MB-231. Methods： The effects of different concentrations of Astragalus injection, astragaloside Ⅳ and formononetin on proliferation of breast cancer cell lines were assayed by methyl thiazolyl tetrazolium （MTT） assay, and their effects on phospho-Akt were assayed by in-cell Western blot method. Results： The results of the MTT assay showed that the best concentrations of Astragalus injection. astragaloside Ⅳ formononetin and astragaloside Ⅳ plus formononetin were 1 g/mL, 80 μg/mL, 40 μg/mL and 10 μg/mL plus 40 μg/mL respectively. After 1- or 2-day culture, Astragalus injection, astragaloside Ⅳ,formononetin and astragaloside IV plus formononetin decreased the expressions of p-Akt （Thr 308） and p-Akt （Ser 473） in MDA-MB-468 ceils. Formononetin and astragaloside IV plus formononetin down-regulated the expression of p-Akt （Thr 308） protein in MDA-MB-231 cells after 1- and 2-day culture, but had no effects on the expression of p-Akt （Ser 473） protein in MDA-MB-231 cells. Conclusion： Astragalus injection, astragaloside Ⅳ and formononetin can inhibit proliferation of breast cancer cell lines MDA-MB-468 and MDA-MB-231, and the antiproliferation effects vary according to their concentrations. And the antiproliferation mechanisms may be related to their down-regulation effects on Akt phosphorylation.