基于网络药理学的淫羊藿苷对氟尿嘧啶所致大鼠骨髓间充质干细胞凋亡抑制作用的机制研究

Research on the Mechanism of Icariin on the Inhibition of 5-FU-induced Apoptosis of Rat Bone Marrow Mesenchymal Stem Cells Based on Network Pharmacology

目的基于网络药理学的淫羊藿苷(ICA)对氟尿嘧啶(5-FU)所致大鼠骨髓间充质干细胞(BMSCs)凋亡抑制作用机制研究。方法在TCMSP数据库收集淫羊藿苷的药代动力学相关数据,利用CTD和GeneCards两个数据库,检索获取淫羊藿苷的所有潜在作用靶点,采用DAVID 6.8数据库对淫羊藿苷潜在靶点进行GO富集分析以及进一步的筛选收集关键靶点。将关键靶点导入STRING数据库,结合运用Cytoscape 3.8.2软件制作蛋白互作网络(PPI),并用STRING数据库进行通路富集分析(KEGG)。采用Western Blot法检测Akt/GSK3β/CyclinD1通路3个特征蛋白以及凋亡密切相关蛋白Bcl-2、Bax的表达情况。结果获得淫羊藿苷潜在作用靶点83个,进一步筛选得到关键靶点29个,通路富集分析结果按照FDR值从小到大排序,其前20条中具有重要作用的为PI3K-Akt通路、HIF-1信号通路以及FoxO信号通路等。基于KEGG富集分析结果,建立了PI3K-Akt核心作用通路的可视化通路网络图。Western Blot实验结果揭示,与模型组比较,淫羊藿苷处理组p-Akt(P<0.01)的表达升高,p-GSK3β水平和CyclinD1的表达有升高趋势(P>0.05),Bcl-2/Bax比值升高(P<0.001);LY294002抑制Akt磷酸化之后,相应p-Akt、p-GSK3β水平和CyclinD1的表达下降(P<0.05,P<0.001),Bcl-2/Bax比值减少(P<0.001)。结论淫羊藿苷对5-FU所致大鼠骨髓间充质干细胞凋亡抑制作用可能是通过激活Akt/GSK3β/CyclinD1信号通路以及Bcl-2途径介导的。

Objective To explore the mechanism of icariin(ICA)on the inhibition of 5-FU-induced apoptosis of rat bone marrow mesenchymal stem cells(BMSCs)based on network pharmacology. Methods The pharmacokinetic data of ICA was collected from the TCMSP database. The CTD and GeneCards databases were used to retrieve all potential targets of ICA. The DAVID 6.8 database was used to perform GO enrichment analysis of potential targets and subject to further screening and collect key targets. The key targets were imported into the STRING database,which was combined with Cytoscape 3.8.2 software to make Protein-Protein Interaction Networks(PPI), and pathway enrichment analysis(KEGG) was analyzed by STRING database. Western Blot was used to detect the expression of three characteristic proteins in the Akt/GSK3β/CyclinD1 pathway and apoptosis-regulatory proteins Bcl-2 and Bax. Results 83 potential targets of ICA were obtained,and 29 key targets were further screened. The results of KEGG were ranked by the FDR value from small to large. The top 20 pathways,including PI3 K-Akt pathway,HIF-1 signaling pathway and FoxO signaling pathway, etc., play important roles in the inhibitory process.Visualization of PI3 K-Akt core pathway was established according to the results of KEGG enrichment analysis.Western blot experiments revealed that compared with the model group,the levels of p-Akt(P<0.01),p-GSK3βand the expression of CyclinD1(P>0.05)in the ICA treatment group increased,and the ratio of Bcl-2/Bax also increased(P<0.001). After LY294002 inhibited Akt phosphorylation,the corresponding p-Akt,p-GSK3β levels and CyclinD1 expression decreased(P<0.001,P<0.05),and the Bcl-2/Bax ratio decreased obviously(P<0.001).Conclusion The inhibitory effect of ICA on 5-FU-induced apoptosis of rat bone marrow mesenchymal stem cells is most likely mediated by activating the Akt/GSK3β/CyclinD1 signaling pathway and the Bcl-2 pathway.