基于网络药理学与细胞实验的山柰酚抗结直肠癌作用机制研究

Mechanisms of kaempferol against colorectal cancer based on network pharmacology and cellular experiments

摘要:

背景与目的 结直肠癌（CRC）发病率持续上升，晚期患者疗效有限，迫切需要开发新的抗肿瘤药物。山柰酚是一种天然黄酮类化合物，具有抗氧化、抗炎及抗癌活性。本研究结合网络药理学与实验验证，系统探讨山柰酚抗CRC的关键靶点及潜在机制。方法 通过SwissTargetPrediction、SEA、PharmMapper、TargetNet等数据库筛选山柰酚潜在靶点，从GeneCards、OMIM、CTD、DrugBank数据库获取CRC相关靶点，取交集构建“化合物-靶点-疾病”网络。利用STRING数据库和Cytoscape软件分析蛋白质-蛋白质相互作用（PPI）并筛选核心靶点，进一步进行GO与KEGG富集分析。采用人CRC HCT-116细胞进行CCK-8、克隆形成、划痕、Transwell实验及Western blot检测，验证山柰酚对细胞增殖、迁移、侵袭及凋亡相关蛋白表达的影响。结果 共筛得山柰酚作用靶点492个、CRC相关靶点5 078个，交集269个。PPI分析鉴定出Akt1、HSP90AA1、ESR1、SRC、CASP3、NFKB1等53个核心靶点。GO分析提示这些靶点主要参与细胞应激反应、凋亡与氧化应激调控；KEGG分析显示主要涉及化学致癌-受体活化、催乳素、雌激素及PI3K/Akt信号通路。细胞实验证实山柰酚能明显抑制HCT-116细胞的增殖、克隆形成、迁移与侵袭能力（均P<0.05），呈浓度依赖关系，并下调Bcl-2和cyclin D1、上调Bax的表达（均P<0.05）。结论 山柰酚可通过多靶点、多通路协同作用抑制CRC细胞的增殖、迁移与侵袭，并诱导凋亡，其机制可能与调控PI3K/Akt及雌激素信号通路等密切相关。本研究为山柰酚作为CRC潜在治疗药物提供了理论依据与实验支持。

Abstract:

Background and Aims Colorectal cancer (CRC) has a high incidence and mortality rate, and the effectiveness of current therapies for advanced cases remains limited. Kaempferol, a natural flavonoid compound, exhibits antioxidant, anti-inflammatory, and antitumor properties. This study aimed to elucidate the key molecular targets and underlying mechanisms of kaempferol against CRC through an integrated approach combining network pharmacology and experimental validation.Methods Potential targets of kaempferol were identified via SwissTargetPrediction, SEA, PharmMapper, and TargetNet databases, and CRC-related targets were retrieved from GeneCards, OMIM, CTD, and DrugBank. The intersection targets were used to construct a compound-target-disease network. Protein-protein interaction (PPI) analysis using the STRING database and Cytoscape software identified core targets, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In vitro experiments with human CRC HCT-116 cells evaluated cell proliferation (CCK-8 assay), colony formation, migration (scratch assay), invasion (Transwell assay), and expression of apoptosis- and cell cycle-related proteins (Western blot).Results A total of 492 kaempferol targets and 5 078 CRC-related targets were obtained, with 269 overlapping targets identified as potential therapeutic candidates. PPI network analysis revealed 53 core targets, including Akt1, HSP90AA1, ESR1, SRC, CASP3, and NFKB1. GO analysis indicated enrichment in cellular stress response, apoptosis regulation, and oxidative stress processes; KEGG pathways were primarily related to chemical carcinogenesis-receptor activation, prolactin, estrogen, and PI3K/Akt signaling. Experimental validation demonstrated that kaempferol markedly inhibited HCT-116 cell proliferation, colony formation, migration, and invasion in a dose-dependent manner (all P<0.05), accompanied by downregulation of Bcl-2 and cyclin D1 and upregulation of Bax (all P<0.05).Conclusion Kaempferol exerts anti-CRC effects through multi-target and multi-pathway mechanisms, including inhibition of proliferation, migration, and invasion, and induction of apoptosis, potentially via modulation of the PI3K/Akt and estrogen signaling pathways. These findings provide theoretical and experimental evidence supporting kaempferol as a promising candidate for CRC therapy.