摘要
目的探讨积雪草酸(Asiatic acid,AA)通过活性氧(ROS)对乳腺癌4T1细胞凋亡的影响及相关机制。方法通过MTT法和集落形成法检测不同浓度AA对4T1细胞增殖作用的影响;采用JC-1染色法检测细胞线粒体膜电位变化;Hoechst 33342染色法检测细胞凋亡情况;Annexin V-FITC/PI双染法检测细胞凋亡率;DCFH-DA探针法检测细胞ROS水平;RT-PCR法检测4T1细胞Bax、Caspase3、Bcl-2及细胞色素C(Cytochrome C)mRNA表达水平;Western Blot法检测4T1细胞Bax、cleaved-Caspase3、Bcl-2及Cytochrome C蛋白的表达。结果10~100μmol·L^(-1)AA呈浓度依赖性地抑制4T1细胞增殖(P<0.01),其作用24 h的IC_(50)值为41.01μmol·L^(-1)。与对照组比较,AA组的细胞集落形成能力显著下降(P<0.01);红/绿相对荧光强度(线粒体膜电位)显著降低(P<0.01);亮蓝色荧光强度(凋亡细胞数量)显著增加(P<0.01);细胞凋亡率显著升高(P<0.01);ROS水平显著升高(P<0.01);Bcl-2 mRNA表达显著下调(P<0.01),Bax、Caspase3、Cytochrome C mRNA表达明显上调(P<0.05,P<0.01);Bcl-2蛋白表达显著下调(P<0.01),Bax、cleaved-Caspase3、Cytochrome C蛋白表达显著上调(P<0.01)。AA与抗氧化剂NAC联用后,能够逆转AA对4T1细胞凋亡及凋亡相关蛋白的作用。结论AA能够促进乳腺癌4T1细胞内ROS水平升高,诱导4T1细胞凋亡。
Objective To investigate the effect and its related mechanisms of asiatic acid(AA)on apoptosis of breast cancer 4T1 cells via reactive oxygen species(ROS).Methods The effect of different concentrations of AA on the proliferation of 4T1 cells was detected by MTT and colony formation method;the change of mitochondrial membrane potential was detected by JC-1 staining method;the apoptosis was detected by Hoechst 33342 staining method;the rate of apoptosis was detected by Annexin V-FITC/PI double staining method;the level of ROS was detected by DCFH-DA probe method;the mRNA expression of Bax,Cleaved-Caspase3,Bcl-2 and Cytochrome C(Cytochrome C)in 4T1 cells were detected by RT-PCR method.The protein expression of Bax,cleaved-Caspase3,Bcl-2 and Cytochrome C in 4T1 cells was detected by Western Blot method.Results 10~100μmol·L^(-1) AA could inhibit the proliferation of 4T1 cells in a concentration-dependent manner(P<0.01),and the IC_(50) value of AA for 24-hour was 41.01μmol·L^(-1).Compared with the control group,colony formation ability of AA group was significantly decreased(P<0.01);the red/green relative fluorescence intensity(mitochondrial membrane potential)was significantly decreased(P<0.01);the bright blue fluorescence intensity(number of apoptotic cells)was significantly increased(P<0.01);the apoptosis rate was significantly increased(P<0.01);the ROS level was significantly increased(P<0.01);the mRNA expression of Bcl-2 was significantly down-regulated(P<0.01),and the mRNA expression of Bax,Caspase3 and Cytochrome C was significantly up-regulated(P<0.05,P<0.01);the protein expression of Bcl-2 was significantly down-regulated(P<0.01)and the protein expression of Bax,cleaved-Caspase3,Cytochrome C was significantly up-regulated(P<0.01).AA combined with antioxidant NAC could reverse the effect of AA on 4T1 cell apoptosis and apoptosis-related proteins.Conclusion AA is able to promote elevated ROS levels in breast cancer 4T1 cells and induces apoptosis in 4T1 cells.
作者
杨兴旺
田苗苗
马春宇
YANG Xingwang;TIAN Miaomiao;MA Chunyu(The First Affiliated Hospital of Jinzhou Medical University,Jinzhou 121000 Liaoning,China)
出处
《中药新药与临床药理》
CAS
CSCD
北大核心
2022年第6期770-776,共7页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
辽宁省自然科学基金项目(20170540368)。
关键词
积雪草酸
乳腺癌4T1细胞
活性氧
凋亡
asiatic acid(AA)
breast cancer 4T1 cells
reactive oxygen species(ROS)
apoptosis
