基于转录组学、网络药理学及实验验证探讨半夏泻心汤防治胃癌的作用机制

Mechanism of Banxia Xiexin Decoction on Gastric Cancer Based on Network Pharmacology,RNA-seq Technology and Experimental Validation

关键词:  半夏泻心汤  胃癌  UPLC-Q-Orbitrap MS/MS技术  网络药理学  RNA-seq  PI3K-Akt信号通路

Key words:  banxia  xiexin decoction, gastric  cancer, UPLC-Q-Orbitrap  MS/MS  technology, network  pharmacology, RNA-seq, PI3K-Akt  signaling pathway

摘要:

目的 基于转录组学和网络药理学的方法,结合实验验证,探讨半夏泻心汤(BXD)防治胃癌(GC)的作用机制。方法 利用UPLC-Q-Orbitrap MS/MS技术对BXD的活性成分进行质量控制和鉴定,并通过TCMSP、DrugBank和SwissTarget Prediction 平台预测药物成分的相关靶点,利用Genecards、OMIM、DisGeNet等数据库对GC基因进行鉴定,STRING数据库构建蛋白相互作用网络,Metascape数据库进行KEGG和GO富集分析,cytoscape构建活性成分-靶点-通路“网络图。采用RNA-seq测序技术筛选胃癌细胞MGC-803的差异表达基因(DEGs),分析RNA-seq和网络药理学之间的重合靶点,获得BXD治疗GC的关键靶点,使用STRING和Metascape数据库进行PPI网络、GO和KEGG富集分析。采用CCK8、细胞凋亡检测、细胞迁移检测进行细胞分子功能检测,利用qPCR检测关键靶点的mRNA表达。结果 UPLC-Q-Orbitrap MS/MS结合网络药理学共鉴定出BCD中有29个化合物,859个潜在靶点。从Genecards、OMIM、DisGeNet等数据库中共获得7695个GC相关基因,确定BXD中569个潜在靶点参与治疗GC。RNA-seq测序结合网络药理学共显示328个DEGs,KEGG结果显示BXD可能通过PI3K-Akt信号通路防治GC,其中PIK3CA、Akt1、EGFR、TNF等被归类为枢纽基因,体外细胞实验证实,BXD含药血清和LY294002可以抑制胃癌细胞增殖,促进细胞凋亡,抑制胃癌细胞迁移的作用,通过降低胃癌细胞MGC803细胞中PI3K-Akt信号通路中EGFR、PIK3CA、TNF的表达发挥作用。结论 BXD具有抑制胃癌增殖,延缓胃癌发展的作用。其作用机制可能与调节PI3K-Akt信号通路,减轻炎症反应有关。

Abstract:

OBJECTIVE Exploring the mechanism of action of Banxia Xiexin Decoction(BXD)against gastric cancer (GC) based on transcriptomics and network pharmacology, combined with experimental validation. METHODS The active ingredient of BXD was quality controlled and identified by UPLC-Q-Orbitrap MS/MS technology, and the relevant targets of the drug ingredient were predicted by TCMSP, DrugBank and SwissTarget Prediction platforms. DisGeNet and other databases were used to identify GC genes, STRING databases were used to construct protein interaction networks, Metascape databases were used for KEGG and GO enrichment analysis, and cytoscape was used to construct active ingredient-target-pathway network diagrams. RNA-seq sequencing technology was used to screen the differentially expressed genes (DEGs) of gastric cancer cells MGC-803, and the coincident targets between RNA-seq and network pharmacology were analyzed, and the key targets of BXD for the treatment of GC were obtained, and the PPI network, GO and KEGG enrichment analysis were performed using STRING and Metascape databases. CCK8, apoptosis assay and cell migration assay were used to detect the molecular function of cells, and qPCR was used to detect the mRNA expression of key targets. RESULTS UPLC-Q-Orbitrap MS/MS combined with network pharmacology identified a total of 29 compounds and 859 potential targets in BCD. A total of 7695 GC-related genes were obtained from databases such as Genecards, OMIM, and DisGeNet, and 569 potential targets in BXD were identified for the treatment of GC. A total of 328 DEGs were detected by RNA-seq sequencing combined with network pharmacology, and the KEGG results showed that BXD may prevent GC through the PI3K-Akt signaling pathway, including PIK3CA, Akt1, EGFR, TNF and others were classified as hub genes, and in vitro cell experiments confirmed that BXD medicated serum and LY294002 could inhibit the proliferation of gastric cancer cells, promote apoptosis, inhibit the migration of gastric cancer cells, and play a role in reducing the expression of EGFR, PIK3CA and TNF in the PI3K-Akt signaling pathway in gastric cancer cells MGC803 cells. CONCLUSION BXD has the effect of inhibiting the proliferation of gastric cancer and delaying the development of gastric cancer. Its mechanism of action may be related to the regulation of PI3K-Akt signaling pathway and the alleviation of inflammatory response.