基于NOTCH1/DLL4通路的姜黄素联合生长因子促进ADSCs向血管内皮细胞分化

Curcumin Combined with Growth Factors Promotes the Differentiation of ADSCs into Vascular Endothelial Cells via Notch1/DLL4 Pathway

目的:通过网络药理学技术预测并探讨姜黄素联合生长因子促进脂肪间充质干细胞(ADSCs)向血管内皮细胞分化的靶点及相关机制。方法:利用网络药理学技术,预测姜黄素促进血管生成的潜在靶点及机制。对ADSCs加以不同浓度的姜黄素,筛选出最适浓度。根据姜黄素最适浓度联合生长因子最适浓度诱导ADSCs分化。ADSCs分为空白对照组、姜黄素20μmol/L组、生长因子(50μg/L血管内皮生长因子和10μg/L成纤维细胞生长因子)组、姜黄素20μmol/L联合生长因子组。观察各组细胞诱导第3 d、14 d时的形态学变化,使用流式细胞术测定血管内皮细胞标记物[CD31、第八因子(FⅧ)];蛋白质印迹法和qPCR验证网络药理学所得相关靶点。结果:网络药理学结果提示NOTCH1和DLL4是姜黄素促进血管分化的有效作用靶点。与空白ADSCs组相比,姜黄素20μmol/L组、生长因子组、姜黄素20μmol/L联合生长因子组CD31、FⅧ比例均显著升高(P<0.01);相较于空白对照组,姜黄素20μmol/L组NOTCH1、DLL4蛋白和mRNA表达均明显上调(P<0.05),而姜黄素20μmol/L联合生长因子组NOTCH1和DLL4表达相较于20μmol/L姜黄素组、生长因子组明显上调(P<0.05)。结论:姜黄素联合生长因子组可有效促进ADSCs向血管内皮细胞分化,且优于姜黄素组和生长因子组;NOTCH1-DLL4通路在该诱导分化中起重要作用,与网络药理学预测结果一致。

Objective:To predict the targets of curcumin combined with growth factors in promoting the differentiation of adipose-derived stromal cells(ADSCs)into vascular endothelial cells based on network pharmacology and explore the underlying mechanism.Methods:The potential targets and the underlying mechanism of curcumin in promoting angiogenesis were predicted by network pharmacology.Curcumin of different concentrations was applied to ADSCs to screen out the optimal induction concentration for the differentiation of ADSCs.The differentiation of ADSCs was then induced according to the optimal concentration of curcumin combined with the optimal concentration of growth factors.ADSCs were divided into a blank group,a 20μmol/L curcumin group,a growth factor group(50 ug/L vascular endothelial growth factor and 10 ug/L fibroblast growth factor),and a 20μmol/L curcumin combined with growth factor group.Morphological changes in each group were observed on the 3 rd and the 14 th day of induction.Vascular endothelial cell markers CD31 and factorⅧ(FⅧ)were determined by flow cytometry,and related targets obtained by network pharmacology were verified by qPCR and Western blot.Results:As revealed by network pharmacology,Notch1 and DLL4 were effective targets of curcumin in promoting vascular differentiation.Compared with the blank group,the 20μmol/L curcumin group,the growth factor group,and the 20μmol/L curcumin combined with growth factor group showed increased proportions of CD31 and FⅧ(P<0.05).Compared with the blank group,the 20μmol/L curcumin group showed up-regulated mRNA and protein expression of Notch1 and DLL4(P<0.05).Compared with the 20μmol/L curcumin group and the growth factor group,the 20μmol/L curcumin combined with growth factor group showed up-regulated expression of Notch1 and DLL4(P<0.05).Conclusion:Curcumin combined with growth factors effectively promoted the differentiation of ADSCs into vascular endothelial cells,and this effect was superior to curcumin or growth factors alone.The Notch1-DLL4 pathway played an important role in inducing differentiation,which was consistent with the prediction result of network pharmacology.