绿原酸联合连翘苷调控细胞因子风暴的网络药理分析及基于TLR4/TRAF6/PI3KC3通路的协同抗炎作用

Network pharmacology analysis of chlorogenic acid combined with forsythoside in regulating cytokine storm and synergistic anti-inflammatory effect based on TLR4/TRAF6/PI3KC3 pathway

目的基于网络药理学和体外实验探究绿原酸联合连翘苷调控细胞因子风暴的机制。方法利用Swiss Target Prediction、PharmMapper、SEA和TCMSP数据库预测绿原酸和连翘苷的靶点,在GeneCards、OMIM、DRUGBANK和DisGeNET数据库获取细胞因子风暴的靶点,筛选交集靶点绘制Venn图,以STRING数据库和Cytoscape软件构建蛋白质相互作用网络,采用DAVID数据库进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析。以脂多糖(LPS)诱导RAW264.7巨噬细胞构建细胞因子风暴模型;采用细胞计数试剂-8(CCK-8)法检测巨噬细胞存活率;采用Griess法检测细胞上清液中一氧化氮(NO)含量;以酶联免疫吸附试验(ELISA)法测定肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、诱导型一氧化氮合酶(iNOS)和环氧化酶-2(COX-2)的水平;蛋白免疫印迹法(Western blot)检测TNF受体相关因子(TRAF4)、TNF受体相关因子6(TRAF6)、磷酸肌醇-3-激酶3(PIK3C3)蛋白表达。结果网络药理分析获得绿原酸联合连翘苷治疗细胞因子风暴的8个核心网络靶点[丝氨酸/苏氨酸蛋白激酶(AKT1)、白蛋白(ALB)、低氧诱导因子1α(HIF1A)、IL-6、基质金属蛋白酶-9(MMP-9)、过氧化物酶体增殖物激活受体γ(PPARG)、酪氨酸激酶(SRC)、Toll样受体4(TLR4)];富集关键通路包括PI3K/AKT信号通路等。体外实验结果显示,与对照组比较,模型组中NO、TNF-α、IL-6的释放量和iNOS、COX-2蛋白表达量均显著升高(P<0.001)。与模型组比较,给药后NO、TNF-α、IL-6、iNOS、COX-2的水平均显著降低(P<0.001)。与单药给药组比较,绿原酸和连翘苷(1∶1)联合用药组炎症指标下降更明显(P<0.001)。与模型组比较,给药组TLR4、TRAF6、PI3K3C的蛋白表达和PI3K3C的磷酸化水平显著下降。结论绿原酸联合连翘苷能协同抑制促炎细胞因子的表达,调控细胞因子风暴,可能通过TLR4/TRAF6/PI3K3C信号通路实现。

Objective To explore the mechanisms by which chlorogenic acid combined with forsythoside regulates cytokine storms through network pharmacology and in vitro experiments.Methods The targets of chlorogenic acid and forsythoside were predicted using Swiss Target Prediction,PharmMapper,SEA,and TCMSP databases.Targets related to cytokine storms were retrieved from GeneCards,OMIM,DRUGBANK,and DisGeNET databases.Intersection targets were identified and depicted in a Venn diagram.A protein interaction network was constructed using the STRING database and Cytoscape software.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed using the DAVID database.A cytokine storm model was established in RAW264.7 macrophages induced by lipopolysaccharide(LPS).Cell viability was assessed using the Cell Counting Kit-8(CCK-8)method.Nitric oxide(NO)levels in the supernatant were measured by the Griess method.Levels of inflammatory cytokines,tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6),inducible nitric oxide synthase(iNOS),and cyclooxygenase-2(COX-2)were determined by enzyme-linked immunosorbent assay(ELISA).The expression of TNF receptor-associated factor 4(TRAF4),TNF receptor-associated factor 6(TRAF6),and phosphatidylinositol 3-kinase 3(PIK3C3)proteins were analyzed by Western blot.Results Network pharmacology analysis identified eight core network targets for the treatment of cytokine storms with chlorogenic acid and forsythoside,including serine/threonine-protein kinase(AKT1),albumin(ALB),hypoxia-inducible factor 1-alpha(HIF1A),IL-6,matrix metalloproteinase-9(MMP-9),peroxisome proliferator-activated receptor gamma(PPARG),tyrosine kinase(SRC),and Toll-like receptor 4(TLR4).Key pathways included the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway.In vitro experiments showed that compared to the control group,levels of NO,TNF-α,IL-6,iNOS,and COX-2 were significantly higher in the model group(P<0.001).After treatment,levels of NO,TNF-α,IL-6,iNOS,and COX-2 significantly decreased compared to the model group(P<0.001).The combined treatment of chlorogenic acid and forsythoside(1∶1)more effectively reduced inflammatory indicators compared to single treatments(P<0.001).Protein expression of TLR4,TRAF6,PIK3C3,and phosphorylation levels of PIK3C3 significantly decreased in the treatment group compared to the model group.Conclusion Chlorogenic acid combined with forsythoside synergistically suppresses the expression of pro-inflammatory cytokines and regulates cytokine storms,potentially through the TLR4/TRAF6/PIK3C3 signaling pathway.