六味地黄丸通过MAPKKK1、KLF4抑制三阴乳腺癌的作用机制  

Inhibitory Effect of Liuwei Dihuangwan on Triple-negative Breast Cancer Through MAPKKK1 and KLF4

作　　者：郑里翔[1] 刘婷婷 黄萍[1] 权威 俞志鹏 ZHENG Li-xiang;LIU Ting-ting;HUANG Ping;QUAN Wei;YU Zhi-peng(Jiangxi University of Chinese Medicine,Nanchang 330004,China)

机构地区：[1]江西中医药大学,南昌330004

出　　处：《中国实验方剂学杂志》2022年第11期16-25,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81160531);江西省科技厅重点研发项目(20203BBGL73205);江西省教育厅重点项目(GJJ180648)。

摘　　要：目的:探讨六味地黄丸通过丝裂原活化蛋白激酶激酶激酶1(MAPKKK1)、锌指转录因子4(KLF4)抑制三阴乳腺癌的作用机制。方法:400只SPF级11.5月龄昆明种雌性种鼠,每3 d触诊乳腺部位1次,自发瘤小鼠随机分为模型组(给予生理盐水)、紫杉醇组(0.025 g·kg^(-1)·d^(-1)腹腔注射21 d)、六味地黄丸高、中、低剂量组(7.2、3.6、1.8 g·kg^(-1)·d^(-1)灌胃),饲养至濒死期剥离瘤组织,测瘤体积、质量、计算抑瘤率及发瘤小鼠生存期(6个月后未发瘤小鼠设为正常组);选用SPF级SD大鼠制备不同浓度六味地黄丸含药血清用于培养细胞,沉默MDA-MB-231细胞中MAPKKK1,免疫荧光及蛋白免疫印迹法(Western blot)检测MAPKKK1和KLF4蛋白表达。结果:体内实验表明,与正常组比较,模型组肿瘤组织MAPKKK1、KLF4蛋白表达显著下降(P<0.01);与模型组比较,各用药组肿瘤组织体积明显减小、质量明显降低(P<0.05,P<0.01),抑瘤率升高,发瘤小鼠生存期明显延长(P<0.05),MAPKKK1蛋白表达显著升高(P<0.01),紫杉醇组及六味地黄丸高剂量组KLF4蛋白表达显著升高(P<0.01)。体外实验表明,与正常大鼠血清比较,各用药组MDA-MB-231细胞中MAPKKK1、KLF4荧光强度明显增强,紫杉醇组及六味地黄丸高、中剂量组MAPKKK1蛋白表达明显升高,紫杉醇组及六味地黄丸高剂量组KLF4蛋白表达显著升高(P<0.01)。沉默MAPKKK1后,与阴性质粒组(未沉默MAPKKK1)比较,阳性质粒组(沉默MAPKKK1)中MAPKKK1及KLF4荧光强度明显减弱(P<0.05),蛋白表达显著降低(P<0.01);与阳性质粒组比较,各用药组MAPKKK1及KLF4荧光强度均有明显增强,蛋白表达均有明显升高(P<0.05,P<0.01)。结论:六味地黄丸抑制三阴性乳腺癌的生长,其可能的分子机制是通过上调MAPKKK1的表达,从而激活KLF4的表达。Objective:To study the underlying mechanism of Liuwei Dihuangwan in inhibiting triplenegative breast cancer through mitogen-activated protein kinase kinase kinase 1(MAPKKK1)and Krüppel-like factor 4(KLF4).Method:Four hundreds SPF female Kunming mice aged 11.5 months were palpated once every 3 days.The model mice of spontaneous tumors were randomly divided into a model group(normal saline),a paclitaxel group(0.025 g·kg^(-1)·d^(-1),ip,21 days),and high-,medium-and low-dose Liuwei Dihuangwan groups(7.2,3.6,1.8 g·kg^(-1)·d^(-1),ig).Tumor tissues were separated until the moribund stage.The tumor volume and weight were measured,and the tumor inhibition rate and the survival time of the tumor mice were calculated(after 6 months,tumor-free mice were assigned into the normal group).SPF SD rats were selected to prepare serum samples containing Liuwei Dihuangwan of different concentrations for cell culture,and MAPKKK1 in MDA-MB-231 cells was silenced.The protein expression of MAPKKK1 and KLF4 was detected by immunofluorescence and Western blot.Result:The in vivo experimental results showed that compared with the conditions of the normal group,the protein expression of MAPKKK1 and KLF4 in tumor tissues of the model group dropped(P<0.01).Compared with the model group,all medication groups showed reduced tumor volume and weight(P<0.05,P<0.01),increased tumor inhibition rate,prolonged survival time of tumor mice(P<0.05),and increased protein expression of MAPKKK1(P<0.01).Additionally,the paclitaxel group and the high-dose Liuwei Dihuangwan group exhibited increased protein expression of KLF4(P<0.01).The in vitro experiments showed that compared with the conditions of the normal group,the fluorescence intensities of MAPKKK1 and KLF4 in MDA-MB-231 cells in all medication groups were potentiated,and the protein expression of MAPKKK1 in the paclitaxel group and the high-and medium-dose Liuwei Dihuangwan groups,and the protein expression of KLF4 in the paclitaxel group and high-dose Liuwei Dihuangwan group increased(P<0.01).Aft

关 键 词：六味地黄丸 三阴性乳腺癌 丝裂原活化蛋白激酶激酶激酶1 锌指转录因4 质粒沉默 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]