蚊母草提取物通过抑制Runx2活性抗乳腺癌破骨性骨转移作用  

Effect of Extracts from Veronica peregrine on Breast Cancer Cell-induced Osteoclastic Bone Metastasis via Inhibiting Runx2 Activation

作　　者：刘盛玲 吕红[1] 田会茹 姜思琴 袁萍 傅蔚然 高书亮[1] 付剑江[1] LIU Sheng-ling;LYU Hong;TIAN Hui-ru;JIANG Si-qin;YUAN Ping;FU Wei-ran;GAO Shu-liang;FU Jian-jiang(Jiangxi University of Chinese Medicine,Nanchang 330004,China;School of Basic Medical Sciences,Capital Medical University,Beijing 100069,China)

机构地区：[1]江西中医药大学,南昌330004 [2]首都医科大学基础医学院,北京100069

出　　处：《中国实验方剂学杂志》2022年第7期81-87,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然基金项目(81960736,81873043)。

摘　　要：目的:研究蚊母草提取物(EVP)的抗乳腺癌破骨性骨转移作用。方法:采用左心室注射肿瘤细胞法建立裸鼠骨转移模型,通过巢式聚合酶链式反应(PCR)检测裸鼠骨髓组织中人细胞角蛋白-19(Ck-19)基因表达量确定肿瘤细胞的骨转移程度及EVP的抑制作用;采用多核细胞计数法及组织蛋白酶K(Cathepsin K)分泌量检测法,确定小鼠破骨细胞前体骨髓巨噬细胞(BMMs)的活性,并观察EVP(40、80、160 g·L^(-1))的抑制作用;采用蛋白免疫印迹法(Western blot)观察EVP对BMMs细胞内核转录因子-κB受体活化因子(RANK)、Runt相关转录因子2(Runx2)、磷酸化Runx2(p-Runx2)及其下游蛋白基质金属蛋白酶-9(MMP-9)表达的影响;采用明胶酶谱法检测EVP对基质金属蛋白酶(MMPs)水解明胶活性的影响。结果:与空白组比较,裸鼠给予EVP 5.5、11、22 g·kg^(-1),其骨髓组织中人Ck-19水平明显降低(P<0.05),提示EVP具有抗骨转移作用;体外实验显示,与空白组比较,可溶性核转录因子-κB受体活化因子配基(sRANKL)可诱导BMMs分化为多核细胞(P<0.05),EVP(40、80、160 g·L^(-1))可显著抑制上述sRANKL诱导的多核细胞形成(P<0.01)。与空白组比较,sRANKL可显著增加BMMs的Cathepsin K分泌(P<0.01);与sRANKL组比较,EVP组Cathepsin K分泌量明显下调(P<0.05)。Western blot显示,与空白组比较,sRANKL可显著增加BMMs细胞RANK表达(P<0.01);与sRANKL组比较,EVP组(40、160 g·L^(-1))BMMs细胞表达RANK蛋白明显降低(P<0.05)。与空白组比较,sRANKL可明显增加BMMs细胞p-Runx2和MMP-9表达(P<0.05);与sRANKL组比较,EVP组(40、160 g·L^(-1))BMMs细胞表达的p-Runx2和MMP-9蛋白明显降低(P<0.05)。明胶酶谱显示,与空白组比较,sRANKL可明显增加pro-MMP-9、pro-MMP-2及64 kDa MMP-2水平(P<0.05);与sRANKL组比较,EVP组pro-MMP-9、64 kDa MMP-2水平明显降低(P<0.05),且160 g·L^(-1)EVP处理可明显降低pro-MMP-2水平(P<0.05)。结论:蚊母草提取物可抑制肿瘤细胞诱导的破骨性�Objective:To investigate inhibitory effect of extracts from Veronica peregrina(EVP)on the osteoclastic bone metastasis induced by breast cancer cells.Method:Bone metastasis model was established by injection of MDA-MB-231 cells,a human breast cancer cell line,into the left ventricle of BALB/c nude mice.The expression of human cytokeratin-19(Ck-19)gene in mouse bone marrow was determined by nested polymerase chain reaction(PCR)to assess the bone metastasis of MDA-MB-231 cells.To assess the effects of EVP on the activation of bone marrow macrophages(BMMs),we counted the multinuclear cells and measured the secretion of Cathepsin K.Western blot was adopted to assess the effects of EVP on receptor activator of nuclear factor-κB(RANK),Runt-related transcription factor 2(Runx2),phosphorylated Runx2(p-Runx2),and matrix metalloproteinase-9(MMP-9)in BMMs.Gelatin zymography was employed to determine the activities of matrix metalloproteinases(MMPs).Result:Compared with that in the blank group,Ck-19 expression was down-regulated in EVP groups(P<0.05).The multinucleated cells increased when the BMMs were induced by soluble receptor activator of nuclear factor-κB ligand(sRANKL),which was inhibited by EVP(P<0.05).The level of cathepsin K in the supernatant of sRANKL group increased compared with that of the blank group,while EVP groups had lower cathepsin K levels than sRANKL group(P<0.05).Compared with the blank group,the sRANKL group showed up-regulated RANK expression,Runx2 phosphorylation,and MMP-9 expression(P<0.05),while the expression levels of RANK,p-Runx2,and MMP-9 were down-regulated when the cells were incubated with EVP(P<0.05).Furthermore,exposure of BMMs to sRANKL resulted in an increase in gelatin hydrolyzation compared with the blank group(P<0.01),which,however,was reversed in EVP groups(P<0.05).Conclusion:EVP significantly inhibits bone marrow metastasis of MDA-MB-231 cells,which may be associated with the suppression of osteoclast activation by inhibiting Runx2 phosphorylation.

关 键 词：蚊母草 破骨性骨转移 乳腺癌 Runt相关转录因子2(Runx2) 核转录因子-κB受体活化因子 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]