柴胡不同极性部位对肝癌SMMC-7721细胞的影响及机制研究  

Effect and mechanism of different polarity fractions of Bupleuri Radix on hepatocellular carcinoma SMMC-7721 cells

作　　者：王威宇 陈一笑 白帅东 冯建有 秦雪梅[1,2,3] 高晓霞 WANG Weiyu;CHEN Yixiao;BAI Shuaidong;FENG Jianyou;QIN Xuemei;GAO Xiaoxia(Modern Research Center for Traditional Chinese Medicine,Shanxi University,Taiyuan 030006,China;Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education,Shanxi University,Taiyuan 030006,China;Shanxi Key Laboratory of Active Constituents Research and Utilization of Traditional Chinese Medicine,Taiyuan 030006,China)

机构地区：[1]山西大学中医药现代研究中心,山西太原030006 [2]山西大学化学生物学与分子工程教育部重点实验室,山西太原030006 [3]地产中药功效物质研究与利用山西省重点实验室,山西太原030006

出　　处：《中草药》2024年第7期2271-2282,共12页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金面上项目(82174099);山西省基础应用项目面上项目(20210302123432);名优晋药再开发山西省重点实验室(202104010910001);地产中药功效物质研究与利用山西省重点实验室(201605D111004);山西省科技创新人才团队专项资助。

摘　　要：目的探究柴胡Bupleuri Radix不同极性部位对人肝癌SMMC-7721细胞的影响及相关机制。方法不同极性部位的柴胡作用于SMMC-7221细胞后,采用MTT法考察细胞增殖能力;采用划痕实验考察细胞迁移能力;采用流式细胞仪检测细胞周期及凋亡;采用qRT-PCR及Western blotting检测柴胡低极性部位(LPB)组和中极性部位(MPB)组干预后肝癌细胞与周期阻滞、凋亡及迁移相关的基因与蛋白表达;采用代谢组学技术检测LPB和MPB干预前后肝癌细胞内源性差异物的变化,采用京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)分析LPB和MPB调节的代谢通路。结果与对照组比较,LPB、MPB能够显著抑制SMMC-7721细胞增殖(P<0.05、0.01、0.001),LPB能够显著抑制细胞迁移(P<0.05、0.01)。LPB阻滞细胞于G1期和G2期,MPB阻滞细胞于G2期。LPB显著下调肝癌细胞中神经元PAS结构域蛋白2(neuronal pas domain protein 2,NPAS2)、细胞周期蛋白依赖性激酶1(cyclin dependent kinase 1,CDK1)、CDK2、细胞周期蛋白B1(Cyclin B1)、Cyclin E基因表达(P<0.05),MPB显著下调细胞分裂周期蛋白25B(cell division cycle 25B,CDC25B)、CDK1、Cyclin B1基因表达(P<0.05)。LPB显著下调NPAS2、CDC25A蛋白表达(P<0.01),LPB和MPB显著下调B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)蛋白表达(P<0.01、0.001),上调Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、细胞色素C(cytochrome C,Cyt-C)、半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3,Caspase-3)、基质金属蛋白酶-9(matrix metalloproteinase 9,MMP-9)、组织金属蛋白酶抑制因子-1(tissue inhibitor of metalloproteinase-1,TIMP-1)蛋白表达(P<0.05、0.01、0.001)。代谢组学结果显示肝癌的发生与甘油磷脂代谢、鞘磷脂代谢等7种代谢通路的紊乱密切相关。结论LPB和MPB能够抑制肝癌细胞的增殖,并促进其凋亡,肝癌细胞阻滞于G1期主要与Npas2-CDC25ACDK2-Cyclin E复合物有关,阻滞于G2期主要与CDC25B-CDK1-CycObjective To explore the effect and related mechanisms of different polarity parts of Chaihu(Bupleuri Radix)on human liver cancer SMMC-7721 cells.Methods After treating SMMC-7221 cells with different polarity parts of Bupleuri Radix,the cell proliferation ability was evaluated using MTT assay;Scratch assay was used to examine cell migration ability;Flow cytometry was used to detect cell cycle and apoptosis;qRT-PCR and Western blotting were used to detect the expressions of gene and protein related to cycle arrest,apoptosis and migration in liver cancer cells after intervention with low polarity part(LPB)and medium polarity part(MPB)of Bupleuri Radix;Metabolomics techniques was used to detect changes in endogenous foreign bodies in liver cancer cells before and after LPB and MPB intervention,and the metabolic pathways regulated by LPB and MPB were analyzed by Kyoto encyclopedia of genes and genomes(KEGG).Results Compared with control group,LPB and MPB could significantly inhibit the proliferation of SMMC-7721 cells(P<0.05,0.01,0.001),LPB could significantly inhibit the cell migration(P<0.05,0.01).LPB blocked cells in G1 and G2 phases,while MPB blocked cells in G2 phase.LPB significantly down-regulated the gene expressions of neuronal PAS domain protein 2(NPAS2),cyclin dependent kinase 1(CDK1),CDK2,Cyclin B1 and Cyclin E in liver cancer cells(P<0.05),while MPB significantly down-regulated the gene expressions of cell division cycle 25B(CDC25B),CDK1 and Cyclin B1(P<0.05).LPB significantly down-regulated the protein expressions of NPAS2 and CDC25A(P<0.01),LPB and MPB significantly downregulated the protein expressions of B-cell lymphoma-2(Bcl-2)(P<0.01,0.001),and up-regulated the protein expressions of Bcl-2 associated X protein(Bax),cytochrome C(Cyt-C),cysteine aspartate protease-3(Caspase-3),matrix metalloproteinase-9(MMP-9)and tissue inhibitor of metalloproteinase-1(TIMP-1)(P<0.05,0.01,0.001).The metabolomics results showed that the occurrence of liver cancer was closely related to the disruption of seven metaboli

关 键 词：柴胡 肝癌 凋亡 细胞周期 代谢组学 甘油磷脂代谢 鞘磷脂代谢 

分 类 号：R285.5[医药卫生—中药学]