基于网络药理学探讨毒结清复方对多发性骨髓瘤的作用机制及实验验证

To Explore the Mechanism and Experimental Verification of Dujieqing Compound(毒结清复方)on Multiple Myeloma Based on Network Pharmacology

目的:基于网络药理学探讨毒结清复方对多发性骨髓瘤(MM)的作用机制,并通过体外实验验证其重要通路的参与。方法:利用TCMSP、本草组鉴数据库筛选得到毒结清复方有效成分及其对应的靶标蛋白,在GeneCards数据库中获得MM疾病靶点,将药物的标靶蛋白与MM疾病靶点取交集后得到交集靶点,并构建交集靶点交互网络(PPI),使用Cytoscape的Cytohubba插件对PPI网络分析得到核心靶点,然后对核心靶点进行在线富集分析。使用毒结清含药血清干预RPMI8226细胞,利用CCK8检测毒结清复方对RPMI8226细胞增殖的影响,ELISA和Western blotting检测各组RPMI8226细胞Wnt/β-catenin信号通路中Wnt1、β-catenin、cyclinD1蛋白表达水平。结果:共得到毒结清复方有效成分90个及其对应的靶标蛋白415个,多发性骨髓瘤疾病靶点3340个,药物-疾病交集靶点148个,核心靶点13个,KEGG富集得到155条,GO富集得到生物过程1682条、分子功能98条、细胞成分36条。CCK8结果显示:与对照组比较,低、中、高剂量组RPMI8226细胞的OD值均明显降低(P<0.05),且存在剂量依赖性。ELISA结果显示:与对照组比较,低、中、高剂量组RPMI-8226细胞Wnt1、β-catenin、cyclin D1蛋白含量均明显降低(P<0.05),且呈剂量依赖性。Western blotting结果显示:与对照组比较,低、中、高剂量组RPMI-8226细胞β-catenin蛋白相对表达量均明显降低(P<0.05),且高剂量组RPMI-8226细胞β-catenin蛋白相对表达量明显低于低剂量组(P<0.05)。与对照组比较,中、高剂量组RPMI-8226细胞Wnt1、cyclin D1蛋白相对表达量均明显降低(P<0.05),且高剂量组RPMI-8226细胞Wnt1、cyclin D1蛋白相对表达量明显低于低剂量组(P<0.05),高剂量组RPMI-8226细胞Wnt1蛋白相对表达量明显低于中剂量组(P<0.05)。结论:毒结清复方可以抑制MM细胞增殖,其作用机制之一为通过抑制MM细胞中的经典Wnt信号通路来实现。

Objective:To explore the mechanism of Dujieqing Compoundon multiple myeloma(MM)based on network pharmacology,and to verify the participation of important pathways through in vitro experiments.Methods:The effective components of Dujieqing compound and their corresponding target proteins were screened by TCMSP and herbal group identification database.The MM disease targets were obtained in the GeneCards database.The target proteins of the drug were intersected with the MM disease targets to obtain the intersection targets,and the intersection target interaction network(PPI)was constructed.The Cytohubba plug-in of Cytoscape was used to analyze the PPI network to obtain the core targets,and then the core targets were enriched online.RPMI8226 cells were treated with serum containing Dujieqing.The effect of Dujieqing Compound on the proliferation of PMI8226 cells was detected by CCK8.The expression levels of Wnt1,β-catenin and cyclinD1 in Wnt/β-catenin signaling pathway of RPMI8226 cells in each group were detected by ELISA and Western blotting.Results:A total of 90 effective components of Dujieqing Compound and 415 corresponding target proteins,3340 disease targets of multiple myeloma,148 drug-disease intersection targets,13 core targets,155 KEGG enrichment,1682 biological processes,98 molecular functions and 36 cellular components were obtained with GO enrichment.CCK8 results showed that compared with the control group,the OD values of RPMI8226 cells in the low,medium and high dose groups were significantly decreased(P<0.05),and there was a dose-dependent manner.The results of ELISA showed that compared with the control group,the protein contents of Wnt1,β-catenin and cyclin D1 in RPMI-8226 cells in the low,medium and high dose groups were significantly decreased(P<0.05),and there was a dose-dependent manner.Western blotting results showed that compared with the control group,the relative expression ofβ-catenin protein in RPMI-8226 cells in the low,medium and high dose groups was significantly decreased(P<0.05),and the relative expression ofβ-catenin protein in RPMI-8226 cells in the high dose group was significantly lower than that in the low dose group(P<0.05).Compared with the control group,the relative expression levels of Wnt1 and cyclin D1 protein in RPMI-8226 cells in the middle and high dose groups were significantly decreased(P<0.05),and the relative expression levels of Wnt1 and cyclin D1 protein in RPMI-8226 cells in the high dose group were significantly lower than those in the low dose group(P<0.05).The relative expression of Wnt1 protein in RPMI-8226 cells in the high dose group was significantly lower than that in the middle dose group(P<0.05).Conclusion:Dujieqing Compound can inhibit the proliferation of MM cells,and one of its mechanisms is to inhibit the classical Wnt signaling pathway in MM cells.