基于网络药理学探究虎杖苷通过SIRT1/HMGB1对炎性巨噬细胞RAW264.7极化的调控机制

Mechanisms of polydatin in regulating the polarization of inflammatory macrophages RAW264.7 by SIRT1/HMGB1 based on network pharmacology

目的基于网络药理学和体外实验,验证巨噬细胞RAW264.7的极化调变规律,探究虎杖苷(PD)防治细菌性肺炎的可能作用机制。方法利用PharmMapper、SwissTargetPrediction、SEA和TCMSP数据库预测药物靶点,在GeneCards和DiseNET数据库里获取细菌性肺炎靶点,采用DAVID数据库进行GO和KEGG富集分析,以STRING数据库和Cytoscape软件构建蛋白质相互作用网络,再以体外实验予以验证。将细胞分为3组:以LPS刺激的为模型对照组(Model,M),同时予以虎杖苷干预的为PD实验组(PD),另设空白对照组(Control,C)。以CCK8法检测不同浓度虎杖苷对细胞活力的影响;以ELISA法检测细胞上清SIRT1/HMGB1信号通路终末效应分子NF-κB、IL-1β及巨噬细胞极化因子MCP-1、Arg1的分泌水平;以qPCR法检测细胞沉淀SIRT1/HMGB1信号通路相关分子SIRT1、HMGB1、TLR4及TRAF6的mRNA表达。结果网络药理学分析共得到61个虎杖苷与细菌性肺炎的交集靶点,结合GO和KEGG富集分析及蛋白质相互作用网络构建结果,确定了SIRT1、HMGB1、TLR4、TRAF6、NF-κB为后续实验靶点。体外实验进一步证实,PD组NF-κB、IL-1β及MCP-1分泌水平较M组显著降低,Arg1则明显升高;PD组SIRT1的mRNA表达较M组显著上调,但HMGB1、TLR4及TRAF6的mRNA表达显著下调。结论虎杖苷可通过SIRT1/HMGB1调控TLR4、NF-κB等信号通路分子及极化因子MCP-1、Arg1的表达,调变巨噬细胞极化,可能是治疗细菌性肺炎的潜在药物。

Based on network pharmacology and in vitro experimental verification,this study was designed to explore the potential mechanism of polydatin(PD)against bacterial pneumonia from the point of its polarization modulation on macrophages.Drug targets were predicted by SwissTargetPrediction,SEA,TCMSP and PharmMapper databases;disease targets were obtained in GeneCards and DisGeNET databases;GO and KEGG enrichment analysis were established by DAVID databases;protein interaction networks were constructed using STRING databases.And in vitro cell experiments were carried out to further verify the results of network pharmacology.The RAW264.7 cells were divided into three groups:model control group(model,M)stimulated with LPS,PD experimental group treated with PD,and blank control group(Control,C).CCK8 was used to detect the effect of different concentrations of PD on cell viability;ELISA was used to detect the secretion levels of NF-κB and IL-1β,the terminal effector molecules of SIRT1/HMGB1 signaling pathway,and the macrophage polarization factors MCP-1 and Arg1 in the cell supernatant;qPCR was used to detect the mRNA expression of SIRT1/HMGB1 signaling pathway related molecules SIRT1,HMGB1,TLR4 and TRAF6 in cell pellets.According to the results of network pharmacological analysis,there are 61 intersection targets were screened out,in which SIRT1,HMGB1,TLR4,TRAF6 and NF-κB were identified as subsequent experimental targets by combining the results of GO and KEGG enrichment analysis and protein interaction network construction.The results of cell viability experiments confirmed that the secretion levels of NF-κB,IL-1βand MCP-1 in the PD group were significantly lower,while Arg1 was significantly higher than those in the M group;the mRNA expression of SIRT1 in PD group was significantly up-regulated,but the mRNA expressions of HMGB1,TLR4 and TRAF6 were significantly down-regulated.In summary,polydatin can regulate the expression of signaling pathway molecules such as TLR4 and NF-κB and polarizing factors MCP-1 and Arg1 through SIRT1/HMGB1,and modulate macrophage polarization,thus may be a potential drug for the treatment of bacterial pneumonia.