目的 采用网络药理学方法预测白屈菜红碱（CHE）抗黑色素瘤的作用机制，通过体外细胞实验验证作用机制。方法 通过中药系统药理学数据库与分析平台（TCMSP）数据库、Swiss Target Prediction数据库预测CHE作用的靶点集，GeneCards、OMIM和TTD数据库收集黑色素瘤的相关靶点，获得CHE抗黑色素瘤的交集靶点；应用STRING数据库构建靶点蛋白质-蛋白质相互作用（PPI）网络，并进行拓扑网络分析得到CHE抗黑色素瘤的核心靶基因；通过DAVID数据库进行基因本体（GO）注释及京都基因与基因组百科全书（KEGG）通路富集分析；借助AutoDock Vina软件将核心靶基因与CHE进行分子对接。运用CCK-8、乳酸脱氢酶（LDH）、流式细胞仪、Annexin V-FITC染色和Western blotting实验验证CHE抗黑色素瘤B16细胞的作用机制。结果 网络药理学获得CHE抗黑色素瘤的交集靶点46个，PPI网络核心蛋白为MTOR、GSK3B、MAPK1、PIK3CA、BRAF等；GO富集涉及228个条目，主要涉及信号转导、细胞凋亡和增殖调控等；KEGG通路富集分析得到137个条目，主要涉及癌症和磷脂酰肌醇-3-激酶-蛋白激酶B(PI3K-Akt)等信号通路；分子对接结果表明CHE与关键靶点具有较好的结合活性。体外实验表明，CHE能够明显抑制B16细胞增殖并诱导细胞凋亡，能够显著降低pPI3K、p-Akt、B细胞淋巴瘤/白血病-2(Bcl-2)蛋白的表达，能够显著增加Bax蛋白的表达（P<0.01）。结论 CHE具有抗黑色素瘤B16细胞增殖的作用，该作用与PI3K-Akt信号通路密切相关，其可通过下调p-PI3K、p-Akt及抗凋亡蛋白Bcl-2的表达，上调促凋亡蛋白Bax的表达，来抑制黑色素瘤B16细胞增殖并诱导其凋亡。

Objective To predict the mechanism of action of chelerythrine（CHE） against melanoma using network pharmacology, and to verify the mechanism of action through in vitro cell experiments. Methods The target sets of CHE were predicted through the TCMSP database and Swiss Target Prediction database. The relevant targets of melanoma were collected from the GeneCards, OMIM, and TTD databases to obtain the intersection targets of CHE against melanoma. The STRING database was applied to construct the protein-protein interaction network（PPI） of the targets, and topological network analysis was carried out to obtain the core target genes of CHE against melanoma. Gene Ontology（GO） function and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis was performed through the DAVID database. The Auto Dock Vina software was used to perform molecular docking between the core target genes and CHE. CCK-8 assay, LDH assay, flow cytometry, Annexin V-FITC staining assay, and Western blotting assay were used to verify the mechanism of action of CHE against melanoma. Results Network pharmacology obtained 46 intersection targets of CHE against melanoma. The core proteins in the PPI network were MTOR, GSK3B, MAPK1, PIK3CA, BRAF, etc. GO enrichment involved 228 items, mainly related to signal transduction, regulation of apoptosis and cell proliferation, etc. KEGG pathway enrichment analysis obtained 137 items, mainly related to cancer and signaling pathways such as PI3K-Akt. The results of molecular docking showed that CHE had good binding activity with key targets. In vitro experiments showed that CHE could significantly inhibit the proliferation of B16 cells, induce apoptosis, significantly reduce the expression of p-PI3K, p-Akt, and Bcl-2 proteins, and significantly increase the expression of Bax protein. Conclusion CHE can inhibit the proliferation and induce apoptosis of melanoma B16 cells by down regulating the expression of p-PI3K, p-Akt and anti-apoptotic protein Bcl-2, and up regulating the expression of Pro apoptotic protein Bax. This study laid a foundation for the research and development of CHE as a potential candidate drug for melanoma treatment.

R285.5

贵州省科技厅基础研究计划一般项目(黔科合基础-ZK[2022]一般472); 贵州中医药大学大学生创新创业训练计划项目(贵中医大创合字[2022]19号); 贵州省高等学校中药民族药(苗药)新剂型新制剂工程研究中心(黔教技[2022]022号); 地方病与少数民族性疾病教育部重点实验室(贵州医科大学)开放课题基金资助项目(黔教合KY字[2019]047号)