目的 探究柴胡注射液抑制肝癌细胞增殖及诱导其凋亡的作用与机制。方法 体外培养人肝癌SMMC-7721细胞与人正常肝细胞（LO-2），给予不同质量浓度的柴胡注射液干预，MTT法与划痕实验检测细胞增殖和迁移；采用流式细胞仪检测细胞周期及凋亡；采用qRT-PCR及Western blotting技术检测与周期阻滞、凋亡及迁移相关的基因与蛋白表达；代谢组学技术检测柴胡注射液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）分析柴胡注射液调节的代谢通路。结果 柴胡注射液（100～450 mg/mL）显著抑制肝癌细胞增殖（P＜0.01、0.001），使细胞阻滞于S期与G₂期（P＜0.05），促进肝癌细胞凋亡（P＜0.001），抑制肝癌细胞迁移（P＜0.05、0.001），表明柴胡注射液具有显著的抗肝癌作用。柴胡注射液显著降低肝癌细胞中神经元PAS结构域蛋白2（neuronal pas domain protein 2，NPAS2）、细胞分裂周期蛋白25A（cell division cycle 25A，CDC25A）、周期蛋白依赖激酶2（cyclin-dependent kinase 2，CDK2）、细胞周期蛋白A（cyclin A）、CDC25B、CDK1、cyclin B1 mRNA表达（P＜0.01、0.001），下调NPAS2、CDC25A、B淋巴细胞瘤-2（B-cell lymphoma-2，Bcl-2）蛋白表达（P＜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）、金属蛋白酶组织抑制因子-1（tissue inhibitor of metalloproteinase-1，TIMP-1）蛋白表达（P＜0.01、0.001）。代谢组学结果显示，柴胡注射液干预核苷酸代谢、脂质代谢途径、糖酵解途径、氨基酸代谢途径、脂肪酸代谢途径等。结论 柴胡注射液对SMMC-7721细胞有阻滞及促凋亡的作用，其可能的机制为下调NPAS2-CDC25A使细胞阻滞于S期，下调CDC25B、CDK1、cyclin B1使细胞处于G₂期阻滞，下调MMP-9/TIMP-1值抑制肝癌细胞迁移，调节核苷酸、脂质与氨基酸代谢等通路，从而诱导肝癌细胞凋亡。

Objective To explore the effect and mechanism of Bupleurum Injection (柴胡注射液, BI) on inhibiting proliferation and inducing apoptosis of hepatocellular carcinoma cells. Methods Human hepatocellular carcinoma SMMC-7721 cells and normal human hepatocytes (LO-2) were cultured in vitro, and different concentrations of BI were given for intervention. MTT assay and scratch test were used to detect cell proliferation and migration. Cell cycle and apoptosis were detected by flow cytometry. qRT-PCR and Western blotting were used to detect the expressions of genes and proteins related to cycle arrest, apoptosis and migration. Metabolomics technology was used to detect the changes of endogenous differences in hepatocellular carcinoma cells before and after BI intervention, and the metabolic pathway regulated by BI was analyzed by Kyoto encyclopedia of genes and genomes (KEGG). Results BI (100—450 mg/mL) significantly inhibited the proliferation of hepatocellular carcinoma cells (P < 0.01, 0.001), arrested the cells in S phase and G₂ phase (P < 0.05), promoted the apoptosis of hepatocellular carcinoma cells (P < 0.001), and inhibited the migration of hepatocellular carcinoma cells (P < 0.05, 0.001), which indicated that BI had significant anti-hepatoma effect. BI significantly decreased neuronal pas domain protein 2 (NPAS2), cell division cycle 25A (CDC25A), cyclin-dependent kinase 2 (CDK2), cyclin A, CDC25B, CDK1 and cyclin B1 mRNA expression (P < 0.01, 0.001), down-regulated NPAS2, CDC25A and B-cell lymphoma-2 (Bcl-2) protein expressions (P < 0.001), up-regulated Bcl-2 associated X protein (Bax), cytochrome-C (Cyt-C), cystein-asparate protease-3 (Caspase-3), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) protein expressions (P < 0.01, 0.001). The results of metabolomics showed that BI interfered with nucleotide metabolism, lipid metabolism, glycolytic pathway, amino acid metabolism and fatty acid metabolism. Conclusion BI can block and promote apoptosis of SMMC-7721 cells. The possible mechanism is that down-regulation of NPAS2-CDC25A can block cells in S phase, down-regulation of CDC25B, CDK1 and cyclin B1 can block cells in G₂ phase, down-regulation of MMP-9/TIMP-1 can inhibit the migration of hepatocellular carcinoma cells, and regulate the metabolism of nucleotides, lipids and amino acids, thus inducing apoptosis of hepatocellular carcinoma cells.

R285.5

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）