黄连解毒汤治疗金黄色葡萄球菌肺炎的机制研究

Mechanism study of Huanglian Jiedu Decoction in the treatment of Staphylococcus aureus pneumonia

关键词:  黄连解毒汤  金黄色葡萄球菌肺炎  网络药理学  分子对接

Key words:  Huanglian Jiedu Decoction  Staphylococcus aureus pneumonia  network pharmacology  molecular docking

摘要:

[目的] 运用分子对接技术探讨黄连解毒汤治疗金黄色葡萄球菌肺炎的作用机制。[方法] 依托中国天然产物化学成分库平台，筛选出黄连解毒汤的主要化学成分和相应靶点，利用Cytoscape软件构建“药物-疾病-靶点”的网络关系，将药物主要成分及疾病的交集靶点导入STRING平台构建蛋白质-蛋白质相互作用（PPI）网络，并利用AutoDock软件将黄连解毒汤的主要成分与金黄色葡萄球菌肺炎的关键靶点进行分子对接。结合网络药理学及分子对接结果，构建金黄色葡萄球菌肺炎大鼠模型，将90只大鼠随机分为正常组、模型组、万古霉素组、黄连解毒汤低剂量组、黄连解毒汤中剂量组、黄连解毒汤高剂量组，每组15只。除正常组外，其余各组制备金黄色葡萄球菌肺炎大鼠模型，各组灌胃相应药物4 d。模型组、万古霉素组和黄连解毒汤低、中、高剂量组构建金黄色葡萄球菌肺炎大鼠模型，正常组、模型组给予生理盐水2 mL灌胃，每天1次，万古霉素组给予0.2 g/mL万古霉素溶液2 mL灌胃，黄连解毒汤低、中、高剂量组分别给予0.5、1.0、2.0 g/mL黄连解毒汤2 mL灌胃，每天1次。给药4 d后，采用酶联免疫吸附实验（ELISA）检测白细胞介素1β（IL-1β）、白介素6（IL-6）、肿瘤坏死因子α（TNF-α）水平，蛋白质免疫印迹法（Western blot）检测雌激素受体1（ESR1）、糖原合成酶激酶3β（GSK3B）、大鼠β连接素（CTNNB）、低聚果糖（FOS）、Jun原癌基因（JUN）和过氧化物酶体增生激活受体γ（PPARG）蛋白表达。[结果] 筛选出黄连解毒汤有效成分167个，作用靶点125个，金黄色葡萄球菌肺炎相关靶点1 668个，其交集靶点50个。京都基因与基因百科全书（KEGG）分析得出主要涉及信号转导、氧化应激、细胞凋亡等一系列的生物学反应过程，主要参与ESR1、GSK3B、CTNNB、FOS、JUN和PPARG等靶点的调控。分子对接结果表明，筛选得到的主要活性成分与靶点有较强的结合。与模型组比较，黄连解毒汤低、中、高剂量组大鼠肺组织菌落计数、IL-1β、IL-6、TNF-α、CTNNB、FOS、JUN降低（P<0.05），ESR1、GSK3B、PPARG升高（P<0.05）。[结论] 黄连解毒汤对金黄色葡萄球菌肺炎的治疗具有积极作用，其作用机制可能与调节ESR1/NLRP3/Caspase-1信号通路、GSK3B/Nrf2信号通路、CTNNB信号通路、FOS信号通路、JUN信号通路和PPARG信号通路有关。

Abstract:

[Objective] To investigate the mechanism of action of Huanglian Jiedu Decoction in the treatment of Staphylococcus aureus pneumonia by molecular docking technology. [Methods] Relying on the platform of the Chinese natural products chemical composition library，we screened out the main chemical components and corresponding targets of Huanglian Jiedu Decoction，and constructed the network relationship of “drug-disease-target” by using Cytoscape software，and imported the main components of the drug and the intersecting targets of the disease into the STRING platform to construct the Protein-Protein-Interaction（PPI） network. The main components of the drug and the intersecting targets of the disease were imported into the STRING platform to construct a PPI network. Combined with the results of network pharmacology and molecular docking，a rat model of staphylococcus aureus pneumonia was constructed，and 90 rats were randomly divided into the normal group，the model group，the vancomycin group，the Huanglian Jiedu Decoction low-dose group，the Huanglian Jiedu Decoction medium-dose group，and the Huanglian Jiedu Decoction high-dose group，with 15 rats in each group. Except for the normal group，the other groups were prepared as Staphylococcus aureus pneumonia rat models，and each group was gavaged with the corresponding drugs for 4 d. The model group，the vancomycin group，and the Huanglian Jiedu Decoction low-，medium-，and high-dose groups were constructed as the staphylococcus aureus pneumonia rat models，and the normal group and the model group were gavaged with 2 mL of physiological saline，once a day，while the vancomycin group was gavaged with 0.2 g/mL vancomycin solution in 2 mL，and the Huanglian Jiedu Decoction low-，medium-，and high-dose group was gavaged with 2 mL of physiological saline. Huanglian Jiedu Decoction low，medium，and high dose groups were given 0.5，1.0，and 2.0 g/mL Huanglian Jiedu Decoction 2 mL by gavage once a day，respectively. After 4 d of drug administration，the levels of interleukin 1β（IL-1β），interleukin 6（IL-6），and tumor necrosis factor α（TNF-α） were detected by enzyme-linked immunosorbent assay（ELISA），and the levels of estrogen receptor 1（ESR1），gluconeogenic synthase kinase 3β（GSK3B），rat β-conjugated hormone（CTNNB），oligofructose（FOS），Jun proto-oncogene（JUN） and peroxisome proliferator-activated receptor γ（PPARG） protein expression. [Results] 167 active ingredients of Huanglian Jiedu Decoction were screened，with 125 active targets，1 668 targets related to Staphylococcus aureus pneumonia，and 50 intersecting targets KEGG analysis showed that the active ingredients of Huanglian Jiedu Decoction were mainly involved in a series of biological reactions，such as signal transduction，oxidative stress，apoptosis，etc.，and were mainly involved in the regulation of the targets，such as ESR1，GSK3B，CTNNB，FOS，JUN，and PPARG. And PPARG. The molecular docking results showed that the main active ingredients obtained from the screening had strong binding with the targets. Compared with the model group，the colony counts，IL-1β，IL-6，TNF-α，CTNNB，FOS，and JUN of rat lung tissues in the low，medium，and high dose groups of Huanglian Jiedu Decoction were decreased（P<0.05），and ESR1，GSK3B，and PPARG were increased（P<0.05）. [Conclusion] Huanglian Jiedu Decoction have a positive effect on the treatment of Staphylococcus aureus pneumonia，and its mechanism of action might be related to the regulation of the ESR1/NLRP3/Caspase-1 signaling pathway，GSK3B/Nrf2 signaling pathway，CTNNB signaling pathway，FOS signaling pathway，JUN signaling pathway，and PPARG signaling pathway.