六味地黄二至丸合剂影响髓源性抑制细胞与乳腺癌肺转移的相关性  

Liuwei Dihuang Erzhiwan Combination Regulate Myeloid-derived Suppressor Cells to Inhibit Breast Cancer Lung Metastasis

作　　者：郑里翔[1] 郭子锋 郭慧文 王晓敏[1] 许传铭 胡玉亮 ZHENG Lixiang;GUO Zifeng;GUO Huiwen;WANG Xiaomin;XU Chuanming;HU Yuliang(Jiangxi University of Chinese Medicine,Nanchang 330004,China;Xinjian District People's Hospital,Nanchang 330100,China)

机构地区：[1]江西中医药大学,南昌330004 [2]江西省南昌市新建区人民医院,南昌330100

出　　处：《中国实验方剂学杂志》2024年第20期37-45,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81160531);江西省自然科学基金重点项目(20232ACB206053);江西省科技厅重点研发计划项目(20203BBGL73205)。

摘　　要：目的:探讨六味地黄二至丸合剂(合剂)通过调节髓源性抑制细胞(MDSCs)募集抑制自发乳腺癌小鼠肺转移进程的机制。方法:SPF级10月龄的昆明种小鼠雌种鼠380只,每3 d触诊乳腺部位1次。饲养6个月后仍未触摸到肿块的小鼠作为空白组,发瘤后的小鼠随机分为模型组、紫杉醇组(0.01 g·kg^(-1),腹腔注射,隔日1次,22 d)、六味地黄丸组(0.65 g·kg^(-1)·d^(-1),灌胃)、二至丸组(5.41 g·kg^(-1)·d^(-1),灌胃)及六味地黄丸二至丸合剂组(6.05 g·kg^(-1)·d^(-1),灌胃),小鼠肿块直径达到15 mm左右时,进行安乐死,剥离肿瘤和肺组织。记录发瘤小鼠生存期、瘤组织质量及肺转移率。苏木素-伊红(HE)染色观察小鼠肿瘤与肺组织病理形态变化,免疫荧光(IF)检测淋巴细胞抗原6复合物位点G6D(Ly6G)、CD11抗原样家族成员B(CD11b)双染MDSCs细胞检测各组小鼠MDSCs在组织中的分布。蛋白免疫印迹法(Western blot)检测小鼠肿瘤组织中基质金属蛋白酶-9(MMP-9)、转化生长因子-β(TGF-β)、锌指转录因子1(Snail1)、E-钙黏蛋白(E-cadherin)与小鼠肺组织中CC基序趋化因子9(CCL9)、CC基序趋化因子受体1(CCR1)的蛋白表达。结果:造模期间,与模型组比较,紫杉醇组与中药给药组小鼠存活中位天数增多,瘤重、肺转移率和肺部结节评分均明显减低(P<0.05,P<0.01),HE染色可见紫杉醇组和合剂组肿瘤细胞坏死增多。在原位肿瘤组织中,与模型组比较,紫杉醇组与中药给药组MDSCs荧光强度均明显下降(P<0.05,P<0.01)。在转移肺组织中,与空白组比较,模型组MDSCs荧光强度显著增加(P<0.01);与模型组比较,紫杉醇组与中药给药组MDSCs荧光强度均显著下降(P<0.01)。在原位肿瘤组织中,与空白组比较,模型组MMP-9、TGF-β、Snail1蛋白表达量均显著增多(P<0.01),E-cadherin表达量显著较少(P<0.01);与模型组比较,紫杉醇组与中药给药组MMP-9、TGF-β、Snail1蛋白表达量均明显减低(P<0.05,P<0.01Objective:To investigate the mechanism by which Liuwei Dihuang Erzhiwan combination inhibit the lung metastasis of spontaneous breast cancer in mice by regulating the recruitment of myeloid-derived suppressor cells(MDSCs).Method:Three hundred and eighty SPF-grade 10-month-old female breeders of Kunming mouse were palpated at the mammary gland site once every 3 days.Mice that have not had a lump touched after being raised for 6 months are used as control group.After tumor development,the mice were randomized into model,positive control(paclitaxel,intraperitoneal injection at 0.01 g·kg^(-1) every other day for 22 d),Liuwei Dihuangwan(0.65 g·kg^(-1)·d^(-1) by gavage),Erzhiwan(5.41 g·kg^(-1)·d^(-1) by gavage),and Liuwei Dihuang Erzhiwan combination(6.05 g·kg^(-1)·d^(-1) by gavage)groups.The mice were euthanised when the tumor reached a diameter of about 15 mm,and the tumor and lung tissues were collected.The survival time,tumor mass,and lung metastasis rate of tumor-bearing mice were recorded.Hematoxylin-eosin(HE)staining was used to observe the histopathological and morphological changes of mouse tumor and lung tissues.Immunofluorescence(IF)was used to detect the distribution of MDSCs in tissues of mice in each group by double-staining of MDSCs cells with lymphocyte antigen 6 complex site G6D(Ly6G)and CD11 antigen-like family member B(CD11b).Western blot was employed to determine the protein levels of matrix metalloproteinase-9(MMP-9),transforming growth factor-β(TGF-β),zinc finger transcription factor 1(Snail1),and E-cadherin in the tumor tissue and CC motif chemokine 9(CCL9)and CC motif chemokine receptor 1(CCR1)in the lung tissue.Result:During the modelling period,the paclitaxel group and Chinese medicine intervention groups had longer median number of days of survival and lower tumor weight,lung metastasis rate,and lung nodule than the model group(P<0.05,P<0.01).HE staining showed an increase in tumor cell necrosis in the paclitaxel group and the Liuwei Dihuang Erzhiwan combination group.The paclitaxel

关 键 词：六味地黄二至丸合剂 乳腺癌 肺转移 髓源性抑制细胞(MDSCs) 上皮间充质转化(EMT)相关蛋白表达 CC基序趋化因子9(CCL9)/CC基序趋化因子受体1(CCR1)蛋白表达 

分 类 号：R2-0[医药卫生—中医学] R33R289R737.9