标题：基于网络药理学和生物信息学的黄杨碱抑制肾癌细胞的作用机制及实验验证研究

      作者：马旭东 1，2，杨进 2，陈林 2，吴波 3，李嘉 1，2，王威威 1，2，梁国标 1    1.遵义医科大学，贵州 遵义 563000；2.成都大学附属医院泌尿外科，四川 成都 610081；3.郫都区人民医院泌尿外科，四川 成都 611730

      卷次： 2023年34卷4期

      【摘要】 目的 利用网络药理学和生物信息学研究黄杨碱(CVB-D)抑制肾癌细胞的作用机制，并通过人肾癌细胞系 786-O，Caki-1进行体外实验验证相关预测结果。方法 PharmMapper数据库检索CVB-D的作用靶点，DisGeNET数据库获得肾癌疾病靶点，通过 venny平台整合CVB-D治疗肾癌的潜在靶点，将潜在靶点数据导入String数据库构建蛋白质-蛋白质互作网络(PPI)，CytoScape软件可视化并修饰String数据，并用CytoHubba插件进一步分析网络中的Hub基因，得到的Hub基因用TCGA数据库作表达差异及生存分析。此外再用CVB-D与肾癌的交叉靶点数据进行GO和KEGG富集分析，得到CVB-D治疗肾癌潜在靶点的分子功能及相关作用信号通路等分析结果。最后采用CCK-8、克隆形成、细胞形态实验、流式细胞术、Western blot实验验证CVB-D对肾癌的作用。结果 分析得到CVB-D潜在作用靶点136个，疾病作用靶点2 085个，CVB-D治疗肾癌的预测作用靶点68个，核心靶点5个。靶点共涉及生物过程 238种，细胞组成30种，分子功能59种，以及参与肿瘤、PI3K-AKT、前列腺癌等细胞信号通路222条。CCK-8、克隆形成、细胞形态体外实验结果表明，CVB-D能显著抑制肾癌细胞的活力与增殖能力，呈浓度依赖性；流式细胞凋亡分析表明CVB-D能促进肾癌细胞凋亡，从而抑制肾癌细胞；Western blot 实验表明CVB-D作用后细胞中EGFR、p-PI3K、p-AKT相对表达水平降低，呈浓度依赖性。结论 黄杨碱可能通过EGFR靶点和PI3K-AKT信号通路诱导人肾癌细胞凋亡，从而在肾细胞癌的治疗中发挥重要作用。
      【关键词】 网络药理学；生物信息学；黄杨碱；肾细胞癌；表皮生长因子受体；PI3K-AKT通路
      【中图分类号】 R737.11 【文献标识码】 A 【文章编号】 1003—6350（2023）04—0462—08

Mechanism and experimental verification of Cyclovirobuxine D in inhibiting renal carcinoma cells based onnetwork pharmacology and bioinformatics.
MA Xu-dong1,2, YANG Jin 2, CHEN Lin 2, WU Bo 3, LI Jia 1,2, WANG Wei-wei 1,2,LIANG Guo-biao 1. 1. Zunyi Medical University, Zunyi 563000, Guizhou, CHINA; 2. Department of Urology, AffiliatedHospital of Chengdu University, Chengdu 610081, Sichuan, CHINA; 3. Department of Urology, Pidu District People’sHospital, Chengdu 611730, Sichuan, CHINA
【Abstract】 Objective To study the mechanism of Cyclovirobuxine D (CVB-D) in inhibiting renal carcinomacells based on network pharmacology and bioinformatics, and to verify the predicted results through human renal carci-noma cell lines 786-O, Caki-1 in vitro. Methods Firstly, the therapeutic targets of CVB-D were retrieved from Pharm-Mapper database, and the renal cancer targets were obtained from DisGeNET database. Then the potential targets ofCVB-D for the treatment of renal cancer were integrated through Venny platform, and the protein-protein interaction net-work (PPI) was constructed through String database. The CytoScale software was used to visualize and decorate theString data, and The CytoHubba plug-in in CytoScape was used to further analyze Hub genes in the network. After-wards, the expression differences and survival of Hub genes were analyzed by TCGA database. The data of the potentialtargets of CVB-D for the treatment of renal cancer were analyzed by GO and KEGG enrichment analysis, and the molec-ular functions and related signaling pathways of potential targets for the treatment of renal cancer by CVB-D were ob-tained. Finally, CCK-8, clonogenesis, cell morphology, and western blot were used to verify the effect of CVB-D on re-nal carcinoma. Results A total of 136 potential targets of CVB-D, 2 085 disease targets, 68 predictive targets ofCVB-D in the treatment of renal cancer, and 5 core targets were recognized after analysis. Targets are related to 238kinds of biological processes, 30 kinds of cell compositions, 59 kinds of molecular functions, and participated in 222 cellsignaling pathways such as tumor, PI3K-Akt, prostate cancer. The results of in vitro experiments on CCK-8, clonal for-mation, and cell morphology showed that CVB-D significantly inhibited the viability and proliferation of renal carcino-ma cells in a concentration-dependent manner. Flow cytometry analysis showed that CVB-D could promote the apopto-sis of renal carcinoma cells and inhibit the renal carcinoma cells. Western blot assay showed that the relative expres-sion level of EGFR, p-PI3K, p-AKT in cells treated with CVB-D decreased in a concentration-dependent manner.Conclusion CVB-D may play an important role in renal cell carcinoma by regulating cell proliferation and apoptosisthrough PI3K-Akt signaling pathway and EGFR.
      【Key words】 Network pharmacology; Bioinformatics; Cyclovirobuxine D; Renal cell carcinoma; Epidermalgrowth factor receptor; PI3K-AKT pathway