丹参注射液抑制血小板诱导的乳腺癌细胞体外转移作用  

Danshen Injection Inhibits Platelets-induced Metastasis of Breast Cancer Cells In Vitro

作　　者：田会茹 姜思琴 吕红[1] 卓董良 傅蔚然 付剑江[1] TIAN Huiru;JIANG Siqin;LYU Hong;ZHUO Dongliang;FU Weiran;FU Jianjiang(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》2023年第21期79-85,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81873043,81960736)。

摘　　要：目的:观察丹参注射液对血小板(PLT)诱导的乳腺癌细胞体外转移作用。方法:采用噻唑蓝(MTT)比色法观察丹参注射液对MDA-MB-231细胞体外生长的影响;采用OrisTM体外迁移试剂盒检测丹参注射液(终质量浓度分别为4、8、16 g·L^(-1))对PLT(1.5×10^(10)个/L)诱导的乳腺癌细胞体外迁移的影响;采用Transwell小室法检测丹参注射液对PLT诱导的细胞侵袭的影响;采用细胞免疫荧光法和蛋白免疫印迹法(Western blot)检测丹参注射液对PLT诱导的上皮间质转化(EMT)关键性转录因子Slug、Snail蛋白表达的影响;采用酶联免疫吸附测定法(ELISA)检测丹参注射液(终质量浓度为4、8、16、32、64 g·L^(-1))对转化生长因子-β_(1)(TGF-β_(1))分泌的影响;采用Western blot观察丹参注射液对PLT诱导MDA-MB-231细胞Podoplain(PDPN)蛋白表达的影响。结果:与空白组比较,丹参注射液组(32、64 g·L^(-1))细胞的吸光度A570均明显降低(P<0.05,P<0.01);丹参注射液组(4、8、16 g·L^(-1))A570差异无统计学意义。与空白组比较,PLT组迁移、侵袭细胞数明显增加,丹参注射液各组则可明显抑制PLT诱导的细胞迁移和侵袭。与空白组比较,PLT组细胞E-钙黏蛋白(E-cadherin)表达明显降低,丹参注射液可明显逆转PLT的这一作用。与空白组比较,PLT组Slug、Snail蛋白表达明显升高(P<0.05,P<0.01),丹参注射液则明显逆转PLT诱导的Snail蛋白表达(P<0.05,P<0.01);PLT组TGF-β_(1)含量显著升高(P<0.01),丹参注射液组(16、32、64 g·L^(-1))可明显降低PLT诱导的TGF-β_(1)分泌(P<0.05,P<0.01),其余丹参注射液组对TGF-β_(1)分泌无明显影响;PLT组细胞PDPN蛋白表达显著升高(P<0.01),丹参注射液可显著抑制PLT诱导的PDPN表达升高(P<0.01)。结论:丹参注射液可抑制PLT诱导的乳腺癌细胞的迁移、侵袭和EMT作用,其机制可能是通过下调乳腺癌细胞PDPN表达、干扰PLT与肿瘤细胞之间直接作用与PLT分泌TGF-β_(1)作用无关。Objective:To observe the effect of Danshen injection(DAN)on platelet(PLT)-induced metastasis of breast cancer cells in vitro.Method:The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay was used to observe the effect of DAN on the growth of MDA-MB-231 cells in vitro.Oris^(TM)migration assay was used to determine the effect of DAN(final mass concentrations 4,8,16 g·L^(-1))on PLT(1.5×10^(10) cells/L)-induced migration of breast cancer cells in vitro.The effect of DAN on PLT-induced cell invasion was detected by Transwell assay.Immunofluorescence and Western blot were used to detect the effect of DAN on the protein expression associated with PLT-induced epithelial-mesenchymal transition(EMT).In addition,enzyme-linked immune-sorbent assay(ELISA)was used to determine the effect of DAN(final mass concentrations 4,8,16,32,64 g·L^(-1))on the secretion of transforming growth factor-β_(1)(TGF-β_(1)).Western blot was used to observe the effect of DAN on the expression of podoplanin(PDPN)protein in MDA-MB-231 cells induced by PLT.Result:Compared with the blank group,the DAN groups(32 and 64 g·L^(-1))showed decreased A_(570)(P<0.05,P<0.01),and there was no significant difference in A570 between DAN groups(4,8,16 g·L^(-1)).Compared with the blank group,the PLT group showed increased cell migration and invasion,while DAN groups significantly inhibited PLT-induced cell migration and invasion.Compared with the blank group,the PLT group showed decreased expression of E-cadherin,while DAN could significantly reverse this effect of PLT.Compared with the blank group,the PLT group showed increased Slug and Snail protein expression(P<0.05,P<0.01),while DAN significantly reversed Snail protein expression induced by PLT(P<0.05,P<0.01).The content of TGF-β_(1) in the PLT group increased(P<0.01),while the secretion of TGF-β_(1) induced by PLT decreased in the DAN groups(16,32,and 64 g·L^(-1))(P<0.05,P<0.01),and the secretion of TGF-β_(1) was not significantly affected in other DAN groups.PDPN protein express

关 键 词：丹参注射液 血小板 乳腺癌 转移 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学] R2-031R285.5