目的 探讨清肝化瘀颗粒治疗肝癌的有效成分、作用靶点及可能作用机制。方法 通过中药与疾病数据库，运用网络药理学工具筛选清肝化瘀颗粒治疗肝癌的主要有效成分与作用靶点，分析作用靶点的分子机制，构建“中药-成分-靶点-疾病”关系网络。采用体内外实验验证清肝化瘀颗粒核心有效成分苦参碱对肝癌细胞学行为的作用，并检测苦参碱对核心靶点及主要信号通路的调节作用。结果 共筛选出清肝化瘀颗粒治疗肝癌的潜在作用靶点86个，富集分析结果显示作用靶点共涉及生物过程20种，细胞组成15种，分子功能20种，以及信号通路20条。利用构建的“中药-成分-靶点-疾病”网络发现清肝化瘀颗粒主要通过槲皮素、木犀草素及苦参碱对肿瘤蛋白p53（tumor protein p53，TP53）、血管内皮生长因子A（vascular endothelial growth factor A，VEGFA）及G₁/S期特异细胞周期蛋白D1（G₁/S-specific cyclin D1，CCND1）等核心靶点参与细胞凋亡及细胞自噬等与肝癌有关的肿瘤生物学行为发挥治疗肝癌的作用。体外实验发现，清肝化瘀颗粒有效成分苦参碱以剂量相关的方式抑制肝癌细胞的增殖，促进肝癌细胞凋亡，并能调节肝癌细胞氧化应激胁迫标志物，同时可对TP53、VEGFA及CCND1等核心靶点的表达量进行调节。肝癌荷瘤裸鼠实验发现苦参碱对人肝癌细胞HepG2荷瘤裸鼠具有抑瘤作用，并诱导肝癌细胞凋亡，同时能调节线粒体自噬及线粒体分裂2种线粒体稳态调节过程相关蛋白的表达量。结论 清肝化瘀颗粒治疗肝癌的主要机制包括以槲皮素、木犀草素及苦参碱为代表的多成分，以TP53、VEGFA及CCND1为代表的多靶点，以细胞凋亡、线粒体稳态及氧化应激为代表的多通路调节的系统性、协同性作用。

Objective To investigate the main active compounds, molecular target genes, and target signal pathways of Qinggan Huayu Granule (清肝化瘀颗粒) treating liver cancer. Methods Based on multiple traditional Chinese medicine and disease databases, the network pharmacology tool was used to screen the main active compounds and action targets of Qinggan Huayu Granule treating liver cancer, analyze the biological functions of potential targets, and construct a network of “herb-ingredient-target-disease”. The in vitro and in vivo experiment was used to verify the effect of matrine, the core component of Qinggan Huayu Granule, on cytological behaviors of liver cancer cells. The regulation of matrine on the candidate target protein was also detected. Results A total of 86 potential targets for the treatment of liver cancer were screened by traditional Chinese medicine and disease databases. Through enrichment analysis of potential targets, there were 20 biological processes, 15 cellular components, 20 molecular functions, and 20 signaling pathways were yielded. Using “herb-ingredient-target-disease” network, it was found that Qinggan Huayu Granule regulated the biological behaviors of tumors related to liver cancer, such as apoptosis and autophagy, by mainly targets tumor protein p53 (TP53), vascular endothelial growth factor A (VEGFA), G1/S-specific cyclin-D1(CCND1), and other hub genes through quercetin, luteolin, matrine and other active ingredients. In vitro experiments revealed that matrine could inhibit cell proliferation, promote apoptosis, and modulate the levels of molecular markers of oxidative stress in a dose-dependent manner in liver cancer cell model. At the same time, matrine can regulate expression of TP53, VEGFA, and CCND1 in protein level. In vivo experiments showed that matrine had a tumor suppression effect on HepG2 xenograft tumors in nude mice, and induced apoptosis of liver cancer cells. Meanwhile, matrine regulated the expression in protein levels of mitophagy and mitochondrial fission related molecules. Conclusion The main mechanism of Qinggan Huayu Granule in treating liver cancer was related to systematic synergy effect of multiple compounds as represented by quercetin, luteolin and matrine, multiple targets as represented by TP53, VEGFA and CCND1, multiple signal pathways as presented by the regulation effects of apoptosis, mitochondrial homeostasis and oxidative stress.

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北京市科委G20工程创新研究十病十药研发项目（Z171100001717008）