槲皮素抗急性加重慢性阻塞性肺疾病作用机制的网络药理学与细胞实验研究

Mechanism of Quercetin in the Treatment of Acute Exacerbation of Chronic Obstructive Pulmonary Disease Based on the Network Pharmacology and Cell Experiments

目的探讨槲皮素抗急性加重慢性阻塞性肺疾病(AECOPD)的分子作用机制。方法将槲皮素相关作用靶点与AECOPD相关疾病靶点取交集,获得槲皮素治疗AECOPD潜在作用靶点并导入String数据库,利用Cytoscape软件构建蛋白相互作用(PPI)网络;对潜在作用靶点进行基因本体论(GO)功能富集分析和京都基因与基因组百科全书(KEGG)通路富集分析;借助Autodock软件对槲皮素和核心靶点进行分子对接验证。以含不同体积分数(0,1%,2%,4%)香烟提取物(CSE)溶液的无血清培养基培养人支气管上皮BEAS-2B细胞24 h,检测白细胞介素(IL)-6和IL-8水平,确定最佳CSE体积分数;另设模型组(等体积无血清培养基)及槲皮素低、中、高剂量(1,3,10μmol/L)组,分别予相应处理12 h后,加入含2%CSE的无血清培养基继续孵育细胞24 h,检测磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(Akt)、半胱氨酸蛋白水解酶-3(Caspase-3)和B淋巴细胞瘤-2(Bcl-2)蛋白表达水平。结果共获得槲皮素潜在靶点854个,AECOPD相关疾病靶点1176个,槲皮素对AECOPD潜在作用靶点196个。槲皮素抗AECOPD机制可能与TNF,MMP9,EGFR,CASP3,MAPK8,TRP53,MTOR,AKT1,SRC,PTGS2等蛋白相关;富集分析显示,该作用可能涉及PI3K/Akt信号通路、趋化因子信号通路、细胞凋亡等信号通路。分子对接结果显示,槲皮素与Akt蛋白结合能最高。与模型组比较,槲皮素中、高剂量组BEAS-2B细胞中PI3K蛋白表达水平均显著降低,槲皮素高剂量组Akt蛋白表达水平显著降低(P<0.01);槲皮素高剂量组Caspase-3蛋白表达水平显著降低,槲皮素中、高剂量组Bcl-2蛋白表达水平均显著降低(P<0.05)。结论槲皮素抗AECOPD的作用机制可能与抑制PI3K/Akt信号通路、细胞凋亡等生物学过程有关。

Objective To investigate the molecular mechanism of quercetin in the treatment of acute exacerbation of chronic obstructive pulmonary disease(AECOPD).Methods The quercetin-related targets intersected with the AECOPD-related disease targets to obtain the potential targets of quercetin in the treatment of AECOPD,and these targets were imported into the String database.The protein-protein interaction(PPI)network was constructed by the Cytoscape software.Gene Ontology(GO)function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were carried out on potential targets.Molecular docking verification of quercetin and key targets was carried out by the Autodock software.Human bronchial epithelial BEAS-2B cells were cultured in serum-free medium containing different volume fractions(0,1%,2%,4%)of cigarette smoke extract(CSE)solution for 24 h.The levels of interleukin(IL)-6 and IL-8 were detected to determine the optimal volume fraction of CSE.In addition,the model group(equal volume of serum-free medium)and low-,medium-and high-dose quercetin groups(1,3,10μmol/L)were set.After 12 h of corresponding treatment,cells were incubated for 24 h with serum-free medium containing 2%CSE.The expression levels of phosphatidylinositol 3 kinase(PI3K),protein kinase B(Akt),cysteine proteolytic enzyme-3(Caspase-3)and B lymphoblastoma 2(Bcl-2)proteins were detected.Results A total of 854 potential targets of quercetin,1176 targets of AECOPD-related diseases and 196 potential targets of quercetin against AECOPD were obtained.The mechanism of quercetin in the treatment of AECOPD may be related to TNF,MMP9,EGFR,CASP3,MAPK8,TRP53,MTOR,AKT1,SRC,PTGS2 and other proteins.Enrichment analysis showed that this effect might involve PI3K/Akt signaling pathway,chemokine signaling pathway,apoptosis and other signaling pathways.Molecular docking results showed that quercetin had the highest binding energy with Akt1 protein.Compared with those in the model group,the expression level of PI3K protein in BEAS-2B cells in the medium-and high-dose quercetin groups significantly decreased,and the expression level of Akt protein in the high-dose quercetin group significantly decreased(P<0.05).Compared with those in the model group,the level of Caspase-3 protein in the high-dose quercetin group significantly decreased,and the level of Bcl-2 protein in medium-and high-dose quercetin groups significantly decreased(P<0.05).Conclusion The mechanism of quercetin in the treatment of AECOPD may be related to the inhibition of PI3K/Akt signaling pathway,cell apoptosis and other biological processes.