基于网络药理学研究黄连解毒汤调控巨噬细胞极化的作用机制

Study on the Mechanism of Huanglian Jiedu Decoction Regulating Macrophage Polarization Based on Network Pharmacology

目的:研究黄连解毒汤调控巨噬细胞极化的作用机制,以期挖掘其抗炎机制。方法:通过中药系统药理学数据库(TCMSP)、Swiss Target Prediction数据库筛选出黄连解毒汤的活性成分及预测靶点;通过GeneCards、OMIM数据库获取巨噬细胞极化的相关靶点,利用Cytoscape 3.6.0软件绘制黄连解毒汤活性成分-巨噬细胞极化靶点网络图;通过String数据库构建蛋白互作网络并提取核心靶点;通过Cytoscape 3.6.0软件及DAVID网站进行基因本体(GO)富集分析和京都基因与基因组百科全书(KEGG)通路富集分析。结合网络药理学分析结果,将小鼠巨噬细胞RAW264.7细胞分为空白对照组、模型组、辛伐他汀组(10μmol/L)和黄连解毒汤含药血清组(将黄连解毒汤以10 g/kg灌胃大鼠并取血制得),除空白对照组和模型组加入培养基外,其余各组均加入相应药液或含药血清100μL,培养2 h;然后除空白对照组外,其余各组均加入脂多糖溶液(100μg/L)培养24 h以诱导炎症。采用Western blot法检测细胞中腺苷酸活化蛋白激酶(AMPK)的表达水平,采用实时荧光定量聚合酶链式反应(PCR)法检测细胞中M1型极化因子[白细胞介素1β(IL-1β)、一氧化氮合酶(iNOS)]和M2型极化因子[IL-10、缺氧诱导分化因子1(Fizz1)]mRNA的表达水平。结果:共筛选到黄连解毒汤的50种活性成分(如刺槐黄素、汉黄芩素、槲皮素、β-谷甾醇等),其可通过失巢凋亡负调节、星形胶质细胞活化等12个GO条目,以及雌激素信号通路、膀胱癌通路、AMPK信号通路等20条KEGG通路调控巨噬细胞极化。细胞试验结果显示,与空白对照组比较,模型组细胞中AMPK蛋白以及Fizz1、IL-10 mRNA的表达水平均显著降低(P<0.01),IL-1β、iNOS mRNA的表达水平均显著升高(P<0.01);与模型组比较,黄连解毒汤含药血清组和辛伐他汀组细胞中AMPK蛋白以及Fizz1、IL-10 mRNA的表达水平均显著升高(P<0.05或P<0.01),IL-1β、iNOS mRNA的表达水平均显著降低(P<0.01)。结论:黄连解毒汤可通过多个靶点、多个通路调控巨噬细胞极化;其可通过AMPK信号通路上调M2型极化因子的表达、下调M1型极化因子的表达,调控巨噬细胞极化,发挥抗炎作用。

OBJECTIVE:To study the mechanism of Huanglian jiedu decoction(HJD)regulating macrophage polarization in order to explore its anti-inflammatory mechanism.METHODS:The active components and predicted targets of HJD were screened through TCMSP and Swiss Target Prediction database;the related targets of macrophage polarization were obtained by GeneCards and OMIM database,and the network diagram of active ingredient-macrophage polarization target of HJD was drawn by using Cytoscape 3.6.0 software;protein interaction network was constructed by String database and core targets were extracted.Gene ontology(GO)enrichment analysis and Kyoto Encyclopedia of genes and genomes(KEGG)pathway enrichment analysis were carried out by using Cytoscape 3.6.0 software and DAVID website.Combined with the results of network pharmacology analysis,RAW264.7 macrophage cells were divided into blank control group,model group,simvastatin group(10μmol/L)and serum containing HJD group(obtained from the blood after the rats were given HJD at the dose of 10 g/kg).Except the blank control group and model group were added culture medium,the other groups were added with 100μL of relevant drug solution or serum containing drug.After 2 h of culture,except for the blank control group,LPS solution(100μg/L)was added to the other groups for 24 h to induce inflammation.Western blot assay was used to detect the expression of AMPK.mRNA expression of M1 type polarization factor(IL-1β,iNOS)and M2 type polarization factor(IL-10,Fizz1)were detected by RT-PCR.RESULTS:A total of 50 active components of HJD(such as acacetin,wogonin,quercetin,β-sitosterol)were screened,which could regulate macrophage polarization through 12 GO items(such as anoikis,astrocyte activation),and 20 KEGG pathways(such as estrogen signaling pathway,bladder cancer pathway,AMPK signaling pathway).The results of cell test showed that compared with blank control group,the expression of AMPK protein,Fizz1 and IL-10 mRNA in model group were significantly decreased(P<0.01),while the expression of IL-1βand iNOS mRNA were significantly increased(P<0.01);compared with model group,the expression of AMPK protein,Fizz1 and IL-10 mRNA in serum containing HJD group and simvastatin group were significantly increased(P<0.05 or P<0.01),while the expression of IL-1βand iNOS mRNA were significantly decreased(P<0.01).CONCLUSIONS:HJD can regulate macrophage polarization through multiple targets and pathways;it can up regulate the expression of M2-type polarization factors and down-regulate the expression of M1-type polarization factors through AMPK signaling pathway,regulate macrophage polarization and play an anti-inflammatory role.