Xiao Shunli, Liu Lu, Sun Zhengxiao, Liu Xiaoqian, Xu Jing, Guo Zhongyuan, Yin Xiaojie, Liao Fulong, Xu Jun, You Yun, Zhang Tiejun
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.
College of Pharmacy, Henan University of Chinese Medicine, Zhengzhou, China.
Front Pharmacol. 2022 Jul 8;13:891889. doi: 10.3389/fphar.2022.891889. eCollection 2022.
Qing-Jin-Hua-Tan-Decoction (QJHTD), a classic famous Chinese ancient prescription, has been used for treatment of pulmonary diseases since Ming Dynasty. A total of 22 prototype compounds of QJHTD absorbed into rat blood were chosen as candidates for the pharmacological network analysis and molecular docking. The targets from the intersection of compound target and ALI disease targets were used for GO and KEGG enrichment analyses. Molecular docking was adopted to further verify the interactions between 22 components and the top 20 targets with higher degree values in the component-target-pathway network. experiments were performed to verify the results of network pharmacology using SPR experiments, Western blot experiments, and the PMA-induced neutrophils to produce neutrophil extracellular trap (NET) model. The compound-target-pathway network includes 176 targets and 20 signaling pathways in which the degree of MAPK14, CDK2, EGFR, F2, SRC, and AKT1 is higher than that of other targets and which may be potential disease targets. The biological processes in QJHTD for ALI mainly included protein phosphorylation, response to wounding, response to bacterium, regulation of inflammatory response, and so on. KEGG enrichment analyses revealed multiple signaling pathways, including lipid and atherosclerosis, HIF-1 signaling pathway, renin-angiotensin system, and neutrophil extracellular trap formation. The molecular docking results showed that baicalin, oroxylin A-7-glucuronide, hispidulin-7-O-β-D-glucuronide, wogonoside, baicalein, wogonin, tianshic acid, and mangiferin can be combined with most of the targets, which might be the core components of QJHTD in treatment of ALI. Direct binding ability of baicalein, wogonin, and baicalin to thrombin protein was all micromolar, and their K values were 11.92 μM, 1.303 μM, and 1.146 μM, respectively, revealed by SPR experiments, and QJHTD could inhibit Src phosphorylation in LPS-activated neutrophils by Western blot experiments. The experimental results of PMA-induced neutrophils to produce NETs indicated that QJHTD could inhibit the production of NETs. This study revealed the active compounds, effective targets, and potential pharmacological mechanisms of QJHTD acting on ALI.
清金化痰汤是中国古代经典名方，自明代以来一直用于治疗肺部疾病。选取清金化痰汤中22种吸收入大鼠血液的原型化合物作为药理网络分析和分子对接的候选物。将化合物靶点与急性肺损伤（ALI）疾病靶点交集得到的靶点用于基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析。采用分子对接进一步验证22种成分与成分 - 靶点 - 通路网络中度数较高的前20个靶点之间的相互作用。利用表面等离子体共振（SPR）实验、蛋白质免疫印迹（Western blot）实验以及佛波酯（PMA）诱导中性粒细胞产生中性粒细胞胞外诱捕网（NET）模型进行实验，以验证网络药理学的结果。化合物 - 靶点 - 通路网络包括176个靶点和20条信号通路，其中丝裂原活化蛋白激酶14（MAPK14）、细胞周期蛋白依赖性激酶2（CDK2）、表皮生长因子受体（EGFR）、凝血因子Ⅱ（F2）、原癌基因酪氨酸蛋白激酶（SRC）和蛋白激酶B（AKT1）的度数高于其他靶点，可能是潜在的疾病靶点。清金化痰汤治疗ALI的生物学过程主要包括蛋白质磷酸化、对损伤的反应、对细菌的反应、炎症反应调节等。KEGG富集分析揭示了多个信号通路，包括脂质与动脉粥样硬化、低氧诱导因子 - 1（HIF - 1）信号通路、肾素 - 血管紧张素系统以及中性粒细胞胞外诱捕网形成。分子对接结果表明，黄芩苷、木犀草素 - 7 - 葡萄糖醛酸苷、毛药黄素 - 7 - O - β - D - 葡萄糖醛酸苷、汉黄芩苷、黄芩素、汉黄芩素、甜石蒜碱和芒果苷可与大多数靶点结合，可能是清金化痰汤治疗ALI的核心成分。SPR实验显示，黄芩素、汉黄芩素和黄芩苷与凝血酶蛋白的直接结合能力均为微摩尔级，其解离常数（K值）分别为11.92 μM、1.303 μM和1.146 μM，Western blot实验表明清金化痰汤可抑制脂多糖（LPS）激活的中性粒细胞中Src的磷酸化。PMA诱导中性粒细胞产生NETs的实验结果表明，清金化痰汤可抑制NETs的产生。本研究揭示了清金化痰汤作用于ALI的活性成分、有效靶点和潜在药理机制。
