基于网络药理学、分子对接和体外实验探讨防己生物碱类活性成分逆转乳腺癌耐药的作用机制  

Mechanism of active components of Stephaniae Tetrandrae Radix alkaloids on reversing drug resistance in breast cancer based on network pharmacology,molecular docking and in vitro experiments

作　　者：杨燚 王毛毛 于淼 季宇彬[1,2] 李文兰 辛国松[1,2] 李海茹 YANG Yi;WANG Mao-mao;YU Miao;JI Yu-bin;LI Wen-lan;XIN Guo-song;LI Hai-ru(Engineering Research Center for Medicine,Harbin University of Commerce,Harbin 150076,China;Anti-tumor Natural Drug Engineering Research Center,Ministry of Education,Harbin 150076,China;Second Affiliated Hospital of Harbin Medical University,Harbin 150086,China)

机构地区：[1]哈尔滨商业大学药物工程技术研究中心,黑龙江哈尔滨150076 [2]国家教育部抗肿瘤天然药物工程研究中心,黑龙江哈尔滨150076 [3]哈尔滨医科大学附属第二医院,黑龙江哈尔滨150086

出　　处：《中草药》2023年第9期2841-2851,共11页Chinese Traditional and Herbal Drugs

基　　金：中国博士后面上项目(2021MD703828);黑龙江省自然科学基金优秀青年项目(YQ2022H002);黑龙江省博士后资助项目(LBHZ20172);2022年哈尔滨商业大学产业化项目支持计划(XL0086);哈尔滨商业大学研究生科研创新项目(YJSCX2022-753HSD)。

摘　　要：目的研究防己生物碱类活性成分逆转乳腺癌阿霉素耐药潜在的作用机制。方法首先通过TCMSP数据库对防己生物碱类活性成分进行筛选,采用SwissTargetPrediction数据库在线预测防己生物碱类活性成分潜在靶点,通过GeneCards数据库收集疾病靶点,取交集构建“成分-靶点”和蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络,然后进行基因本体(gene ontology,GO)功能及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析,最后对预测的防己生物碱类活性成分与核心靶点进行分子对接验证。进一步通过体外实验验证防己生物碱类活性成分抗肿瘤活性,采用CCK-8实验检测防己生物碱类活性成分对乳腺癌MCF-7、MCF-7/ADR细胞的增殖抑制作用,采用倒置光学显微镜、荧光显微镜检测防己生物碱类活性成分对MCF-7、MCF-7/ADR细胞形态的影响,采用流式细胞仪检测防己生物碱类活性成分对MCF-7、MCF-7/ADR细胞凋亡的影响,采用Western blotting检测防己生物碱类活性成分对MCF-7、MCF-7/ADR细胞核心靶点蛋白表达的影响。结果N-甲基毛果芸香碱、光千金藤碱、粉防己碱和甜菜碱为逆转乳腺癌阿霉素耐药的防己生物碱类活性成分,可能作用于原癌基因SRC、表皮生长因子受体(epidermal growth factor receptor,EGFR)、热休克蛋白90α家族A类成员1(heat shock protein 90 alpha family class A member 1,HSP90AA1)、蛋白激酶B1(protein kinase b1,AKT1)、有丝分裂原活化蛋白激酶1(mitogen-activated protein kinase 1,MAPK1)、磷脂酰肌醇激酶-3催化亚单位α基因(phosphatidylinositol 3-kinase catalytic alpha polypeptide gene,PIK3CA)、半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3,Caspase-3)等靶点,调控钙信号通路、环磷酸鸟苷(cyclic guanosine monophosphate,cGMP)-cGMP依赖性蛋白激酶(cGMP dependent protein kinase,PKG)信号通路、Apenlin信号通路、维甲酸诱导基�Objective To study the potential mechanism of active components of Fangji(Stephaniae Tetrandrae Radix)alkaloids on reversing adriamycin resistance in breast cancer.Methods Firstly,active components of Stephaniae Tetrandrae Radix alkaloids were screened by TCMSP database,potential targets were predicted online by SwissTargetPrediction database,and disease targets were collected by GeneCards database.Targets were taken by intersection,“component-target”and protein-protein interaction(PPI)networks were constructed,and then gene ontology(GO)function and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis were carried out.Finally,predicted active components of Stephaniae Tetrandrae Radix alkaloids were verified by molecular docking with core targets.Further,anti-tumor activity of active components of Stephaniae Tetrandrae Radix alkaloids was verified by in vitro experiments,the inhibitory effect of active components of Stephaniae Tetrandrae Radix alkaloids on proliferation of breast cancer MCF-7 and MCF-7/ADR cells was detected by CCK-8 experiment;Effect of active components of Stephaniae Tetrandrae Radix alkaloids on morphology of MCF-7 and MCF-7/ADR cells was detected by inverted optical microscope and fluorescence microscope;Flow cytometry was used to detect the effect of active components of Stephaniae Tetrandrae Radix alkaloids on apoptosis of MCF-7 and MCF-7/ADR cells;Western blotting was used to detect the effect of active components of Stephaniae Tetrandrae Radix alkaloids on core targets protein expressions in MCF-7 and MCF-7/ADR cells.Results N-methylflindersine,stepharine,tetrandrine and betaine were the active components of Stephaniae Tetrandrae Radix alkaloids in reversing adriamycin resistance in breast cancer,which may act on proto-oncogene SRC,epidermal growth factor receptor(EGFR),heat shock protein 90αfamily member 1(HSP90AA1),protein kinase B1(AKT1),mitogen-activated protein kinase 1(MAPK1),phosphoinositide 3-kinase catalyticαpolypeptide gene(PIK3CA),cysteine-aspartate pro

关 键 词：防己 生物碱类活性成分 乳腺癌 多药耐药 网络药理学 N-甲基毛果芸香碱 粉防己碱 光千金藤碱 甜菜碱 

分 类 号：R285.5[医药卫生—中药学]