基于网络药理学探讨趋化因子-13对间充质干细胞增殖和迁移的影响

目的 以网络药理学技术探讨趋化因子-13（CXCL-13）对人骨髓间充质干细胞（BMSCs）增殖和迁移的影响。 方法 在线数据库预测CXCL-13作用于BMSCs的靶点。Metascape数据库对靶点的基因本体论和京都基因与基因组百科全书(KEGG）信号通路进行富集分析。STRING 11.0数据库进行蛋白质相互作用分析，Cytoscape 3.8的cytoHubba 0.1插件筛选核心基因编码的蛋白质。BMSCs分为对照组、CXCL-13组和PI3K抑制剂组。分别以MTT、流式细胞术和Transwell细胞小室迁移实验检测各组BMSCs的吸光度（A）值、细胞凋亡率和细胞迁移数目情况；ELISA检测各组BMSCs上清液表皮生长因子(EGF)和血管内皮生长因子(VEGF)蛋白含量。Western blotting检测各组BMSCs的Akt、磷酸化Akt（p-Akt）蛋白的表达。 结果 CXCL-13作用于BMSCs 21个靶点。与细胞增殖相关的生物学过程包括干细胞增殖、调节内皮细胞增殖、正向调控平滑肌细胞增殖等32条；与细胞迁移相关的生物学过程包括调节细胞迁移、阿米巴状细胞迁移、调节内皮细胞迁移等22条。KEGG通路包括癌症途径、PI3K-Akt信号通路、MAPK信号通路等40条。核心蛋白包括肿瘤蛋白P53（TP53）、表皮生长因子受体（EGFR）、90kD热休克蛋白αB1（HSP90AB1）、蛋白激酶Cα（PRKCA）、雌激素受体2（ESR2）及前列腺素E受体4（PTGER4）。与其他组相比，CXCL-13组BMSCs的吸光度（A）值和细胞迁移数目均显著增高（P＜0.01，n=15），细胞凋亡率明显降低（P＜0.01，n=15）；PI3K抑制剂组BMSCs的A值、细胞凋亡率和细胞迁移数目与CXCL-13组相比均呈相反变化（P＜0.01，n=15）。相对于对照组，CXCL-13组BMSCs的EGF和VEGF蛋白含量显著提高（P＜0.01，n=15），Akt和p-Akt相对表达均明显升高（P＜0.01，n=9）；而PI3K抑制剂组EGF和VEGF蛋白含量、Akt和p-Akt相对表达呈相反变化。 结论 CXCL-13激活PI3K-Akt通路促进BMSCs旁分泌EGF和VEGF蛋白，提高BMSCs增殖和迁移，抑制BMSCs凋亡。

Exploring the effect of C-X-C motif chemokine ligand-13 on the proliferation and migration of mesenchymal stem cells based on network pharmacology

Objective To explore the effect of C-X-C motif chemokine ligand-13 (CXCL-13) on the proliferation and migration of human bone marrow mesenchymal stem cells (BMSCs) by network pharmacology. Methods To predict that the targets of CXCL-13 on BMSCs by online database. Metascape was used to perform gene ontology (GO) of the targets and Kyoto encyclopedia of genes and genomes（KEGG）pathway was used to perform enrichment analysis. The protein interaction analysis was performed by STRING 11.0 database, and the protein module of core gene was screened by using the cytoHubba 0.1 of Cytoscape 3.8. We divided BMSCs into control group, CXCL-13 group and PI3K inhibitor group. MTT assay, flow cytometric analysis and Transwell cell migration assay were respectively used to detect the absorbance (A) value of BMSCs in each group, the apoptosis rate and the number of cell migration. The protein contents of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) in BMSCs supernatant were determined by ELISA. Western blotting was used to detect the protein expression of Akt and phosphorylated Akt (p-Akt) of BMSCs in each group. Results It was predicted that 21 targets of CXCL-13 effect on BMSCs. There were 32 biological processes related to cell proliferation include stem cell proliferation, regulation of endothelial cell proliferation and positive regulation of smooth muscle cell proliferation. There were 22 biological processes related to cell migration include regulating cell migration, amebic cell migration and endothelial cell migration. There were 40 KEGG pathways including cancer pathway, PI3K-Akt signaling pathway and MAPK signaling pathway. The core proteins included tumor protein P53 (TP53), epidermal growth factor receptor (EGFR), heat shock protein 90 kD alpha class B member 1 (HSP90AB1), protein kinase Cα (PRKCA), estrogen receptor 2 (ESR2) and prostaglandin E receptor 4 (PTGER4). Compared with other groups, the absorbance(A) value and cell migration number of BMSCs in CXCL-13 group increased significantly (P<0.01, n=15), and the apoptosis rate decreased significantly (P<0.01, n=15). However, absorbance value, apoptosis rate and migration number of BMSCs in PI3K inhibitor group were contrary to those in CXC-13 group (P<0.01, n=15). Compared with the control group, the protein contents of EGF and VEGF in BMSCs of CXCL-13 group increased significantly (P<0.01, n=15), and the relative expression of Akt and p-Akt increased significantly (P<0.01, n=9). However, the protein content of EGF and VEGF, and the relative expression of Akt and p-Akt in PI3K inhibitor group were opposite. Conclusion Through activating PI3K-Akt pathway, CXCL-13 may promote BMSCs paracrine EGF and VEGF proteins, and improve proliferation and migration of BMSCs, as well as inhibit BMSCs apoptosis.