目的 采用网络药理学和分子对接方法,探究鼓槌石斛素治疗宫颈癌的作用机制并进行体外实验验证。方法 通过中药系统药理学数据库与分析平台(TCMSP)、PharmMaper和SwissTarget Prediction平台获得鼓槌石斛素的预测靶点;在GeneCards、TTD、OMIM、Disgenet、Drugbank疾病数据库筛选宫颈癌的潜在靶点;使用R软件Venn包获取鼓槌石斛素与宫颈癌的交集靶点基因,利用STRING网站与Cytoscape软件获取蛋白质-蛋白质相互作用(PPI)网络,筛得核心靶点,进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析;运用Autodock Vina 1.1.2软件和Pymol软件进行鼓槌石斛素和核心靶点之间的分子对接和可视化分析。体外培养宫颈癌SiHa细胞,用0、50、100 μmol·L^-1鼓槌石斛素处理细胞,采用MTT法、Transwell实验分别检测鼓槌石斛素对细胞增殖、迁移和侵袭的影响,Western blotting检测鼓槌石斛素对PI3K/Akt/mTOR信号通路中p-PI3K、PI3K、p-Akt、Akt、mTOR关键蛋白的调控作用。结果 共得到79个鼓槌石斛素调控宫颈癌的潜在交集靶点,按照网络拓扑分析中的度(degree)值筛选出HSP90AA1、ESR1、PIK3CA、mTOR、MAPK1、ABL1、PARP1等多个核心靶点;KEGG富集筛选出PI3K/Akt、Focal adhesion、细胞衰老、催乳素等30条信号通路;分子对接结果显示,其中靶蛋白HSP90AA1、ESR1、PIK3CA、mTOR、MAPK1与鼓槌石斛素结合能绝对值较高,说明可能是鼓槌石斛素治疗宫颈癌的作用位点。细胞实验验证表明,鼓槌石斛素处理SiHa细胞后,SiHa细胞增殖明显减少(P<0.01),且与鼓槌石斛素的浓度呈现负相关,同时SiHa细胞的迁移和侵袭能力下降。鼓槌石斛素处理SiHa细胞后,细胞中p-PI3K、Akt、p-Akt表达及p-PI3K/PI3K和p-Akt/Akt下调(P<0.05)。结论 鼓槌石斛素可通过多靶点、多通路发挥治疗宫颈癌的作用,鼓槌石斛素可能通过抑制PI3K/Akt/mTOR信号通路的表达来影响宫颈癌细胞的增殖、迁移和侵袭。

Objective To investigate the mechanism of chrysotoxine in the treatment of cervical cancer using network pharmacology and molecular docking and validate the results through cell-based experiments. Methods Predicted targets of chrysotoxine were obtained by TCMSP, PharmMaper and SwissTarget Prediction platform. The potential targets of cervical cancer were screened in GeneCards, TTD, OMIM, Disgenet and Drugbank disease databases. R software Venn package was used to obtain common target genes of chrysotoxine and cervical cancer. STRING website and Cytoscape software were used to obtain the PPI network, screened the core targets, and analyzed them for GO and KEGG enrichment. Molecular docking and visualization between chrysotoxine and the core targets were conducted using Autodock Vina 1.1.2 and Pymol software. Cervical cancer cells SiHa were cultured and treated with 0, 50, 100 μmol·L^-1 chrysotoxine in vitro, and the effects of chrysotoxine on cell proliferation, migration and invasion were detected by MTT assay and Transwell assay, respectively. The molecular mechanism of chrysotoxine against cervical cancer was performed by Western blotting, focusing on the regulatory proteins of p-PI3K, PI3K, p-Akt, Akt, and mTOR in the PI3K/Akt/mTOR signaling pathway. Results A total of 79 potential common targets of chrysotoxine against cervical cancer were obtained, and then screened multiple core targets according to the degree value, such as:HSP90AA1, ESR1, PIK3CA, mTOR, MAPK1, ABL1, PARP1, et al. KEGG enrichment screened 30 pathways, including PI3K/Akt signaling pathway, Focal adhesion signaling pathway, cellular senescence signaling pathway, prolactin signaling pathway, et al. Molecular docking results showed that chrysotoxine had a higher affinity for target proteins, such as HSP90AA1, ESR1, PIK3CA, mTOR, and MAPK1, suggesting their potential role in chrysotoxine's anti-cervical cancer effects. Cellular experiments demonstrated that chrysotoxine significantly reduced the proliferation of SiHa cells (P < 0.01) and decreased their migration and invasion ability. Western blotting results further confirmed the down-regulation (P < 0.05) of p-PI3K, Akt, p-Akt, p-PI3K/PI3K, and p-Akt/Akt expression after treatment with chrysotoxine. Conclusion Chrysotoxine can exert therapeutic effects on cervical cancer through multi-targets and multi-pathways, and chrysotoxine may affect the proliferation and migration of cervical cancer cells by inhibiting the expression of PI3K/Akt/mTOR signaling pathway. These provide a reliable theoretical basis for the treatment of cervical cancer with chrysotoxine, and provide a new idea for the traditional Chinese medicine treatment of cervical cancer in the future.

R285.5

湖南省教育厅项目(18B538,22C0682);长沙医学院ESI学科专项(2022CYY033);湖南省大学生创新创业训练计划(湘教通[2022]174号-一般项目-4587);湖南省卫生和计划生育委员会科研基金项目(C20180139)