Abstract：
BACKGROUND: Acute lung injury (ALI) has received considerable attention in the field of critical care as it can lead to high mortality rates. Polygala tenuifolia, a traditional Chinese medicine with strong expectorant properties, can be used to treat pneumonia. Owing to the complexity of its composition, the main active ingredient is not yet known. Thus, there is a need to identify its constituent compounds and mechanism of action in the treatment of ALI using advanced technological means.
OBJECTIVE: We investigated the anti-inflammatory mechanism and constituent compounds with regard to the effect of P. tenuifolia Willd. extract (EPT) in lipopolysaccharide (LPS)-induced ALI in vivo and in vitro.
METHODS: The UHPLC-Q-Exactive Orbitrap MS technology was used to investigate the chemical profile of EPT. Network pharmacology was used to predict the targets and pathways of action of EPT in ALI, and molecular docking was used to validate the binding of polygalacic acid to Toll-like receptor (TLR) 4. The main compounds were determined using LC-MS. A rat model of LPS-induced ALI was established, and THP-1 cells were stimulated with LPS and adenosine triphosphate (ATP) to construct an in vitro model. Pathological changes were observed using hematoxylin and eosin staining, Wright-Giemsa staining, and immunohistochemistry. The expression of inflammatory factors (NE, MPO, Ly-6 G, TNF-α, IL-1β, IL-6, and iNOS) was determined using enzyme-linked immunosorbent assay, real-time fluorescence quantitative polymerase chain reaction, and western blotting. The LPS + ATP-induced inflammation model in THP-1 cells was used to verify the in vivo experimental results.
RESULTS: Ninety-nine compounds were identified or tentatively deduced from EPT. Using network pharmacology, we found that TLR4/NF-κB may be a relevant pathway for the prevention and treatment of ALI by EPT. Polygalacic acid in EPT may be a potential active ingredient. EPT could alleviate LPS-induced histopathological lung damage and reduce the wet/dry lung weight ratio in the rat model of ALI. Moreover, EPT decreased the white blood cell and neutrophil counts in the bronchoalveolar lavage fluid and decreased the expression of genes and proteins of relevant inflammatory factors (NE, MPO, Ly-6 G, TNF-α, IL-1β, IL-6, and iNOS) in lung tissues. It also increased the expression of endothelial-type nitric oxide synthase expression. Western blotting confirmed that EPT may affect TLR4/NF-κB and NLRP3 signaling pathways in vivo. Similar results were obtained in THP-1 cells.
CONCLUSIONS: EPT reduced the release of inflammatory factors by affecting TLR4/NF-κB and NLRP3 signaling pathways, thereby attenuating the inflammatory response of ALI. Polygalacic acid is the likely compounds responsible for these effects.
OBJECTIVE: We investigated the anti-inflammatory mechanism and constituent compounds with regard to the effect of P. tenuifolia Willd. extract (EPT) in lipopolysaccharide (LPS)-induced ALI in vivo and in vitro.
METHODS: The UHPLC-Q-Exactive Orbitrap MS technology was used to investigate the chemical profile of EPT. Network pharmacology was used to predict the targets and pathways of action of EPT in ALI, and molecular docking was used to validate the binding of polygalacic acid to Toll-like receptor (TLR) 4. The main compounds were determined using LC-MS. A rat model of LPS-induced ALI was established, and THP-1 cells were stimulated with LPS and adenosine triphosphate (ATP) to construct an in vitro model. Pathological changes were observed using hematoxylin and eosin staining, Wright-Giemsa staining, and immunohistochemistry. The expression of inflammatory factors (NE, MPO, Ly-6 G, TNF-α, IL-1β, IL-6, and iNOS) was determined using enzyme-linked immunosorbent assay, real-time fluorescence quantitative polymerase chain reaction, and western blotting. The LPS + ATP-induced inflammation model in THP-1 cells was used to verify the in vivo experimental results.
RESULTS: Ninety-nine compounds were identified or tentatively deduced from EPT. Using network pharmacology, we found that TLR4/NF-κB may be a relevant pathway for the prevention and treatment of ALI by EPT. Polygalacic acid in EPT may be a potential active ingredient. EPT could alleviate LPS-induced histopathological lung damage and reduce the wet/dry lung weight ratio in the rat model of ALI. Moreover, EPT decreased the white blood cell and neutrophil counts in the bronchoalveolar lavage fluid and decreased the expression of genes and proteins of relevant inflammatory factors (NE, MPO, Ly-6 G, TNF-α, IL-1β, IL-6, and iNOS) in lung tissues. It also increased the expression of endothelial-type nitric oxide synthase expression. Western blotting confirmed that EPT may affect TLR4/NF-κB and NLRP3 signaling pathways in vivo. Similar results were obtained in THP-1 cells.
CONCLUSIONS: EPT reduced the release of inflammatory factors by affecting TLR4/NF-κB and NLRP3 signaling pathways, thereby attenuating the inflammatory response of ALI. Polygalacic acid is the likely compounds responsible for these effects.
摘要：
背景：急性肺损伤（ALI）在重症监护领域受到了相当大的关注，因为它可以导致高死亡率。远志,一种具有强烈祛痰特性的中药，可用于治疗肺炎。由于其组成的复杂性，主要活性成分尚不清楚。因此，在使用先进的技术手段治疗ALI中需要确定其组成化合物和作用机制。
目的：我们研究了黄菊的抗炎机制和组成化合物的作用。脂多糖（LPS）诱导的ALI的体内和体外提取物（EPT）。
方法：使用UHPLC-Q-ExactiveOrbitrapMS技术研究EPT的化学特征。网络药理学用于预测EPT在ALI中的作用靶点和作用途径。和分子对接用于验证聚半乳糖与Toll样受体（TLR）4的结合。使用LC-MS测定主要化合物。建立LPS诱导的ALI大鼠模型,用LPS和三磷酸腺苷(ATP)刺激THP-1细胞构建体外模型。采用苏木精和伊红染色观察病理变化,Wright-Giemsa染色,和免疫组织化学。炎症因子（NE，MPO,Ly-6G,TNF-α,IL-1β,IL-6和iNOS)使用酶联免疫吸附测定，实时荧光定量聚合酶链反应,和西方印迹。采用LPS+ATP诱导的THP-1细胞炎症模型验证体内实验结果。
结果：从EPT中鉴定或初步推导出了99种化合物。使用网络药理学，我们发现TLR4/NF-κB可能是EPT预防和治疗ALI的相关通路。EPT中的聚半乳糖酸可能是潜在的活性成分。EPT可以减轻LPS诱导的肺组织病理学损伤，降低ALI大鼠模型的湿/干重比。此外，EPT降低支气管肺泡灌洗液中白细胞和中性粒细胞计数,降低相关炎症因子(NE,MPO,Ly-6G,TNF-α,IL-1β,IL-6和iNOS）在肺组织中。它还增加了内皮型一氧化氮合酶的表达。Westernblotting证实EPT可能在体内影响TLR4/NF-κB和NLRP3信号通路。在THP-1细胞中获得类似的结果。
结论：EPT通过影响TLR4/NF-κB和NLRP3信号通路减少炎症因子的释放，从而减弱ALI的炎症反应。聚半乳糖酸可能是导致这些作用的化合物。
目的：我们研究了黄菊的抗炎机制和组成化合物的作用。脂多糖（LPS）诱导的ALI的体内和体外提取物（EPT）。
方法：使用UHPLC-Q-ExactiveOrbitrapMS技术研究EPT的化学特征。网络药理学用于预测EPT在ALI中的作用靶点和作用途径。和分子对接用于验证聚半乳糖与Toll样受体（TLR）4的结合。使用LC-MS测定主要化合物。建立LPS诱导的ALI大鼠模型,用LPS和三磷酸腺苷(ATP)刺激THP-1细胞构建体外模型。采用苏木精和伊红染色观察病理变化,Wright-Giemsa染色,和免疫组织化学。炎症因子（NE，MPO,Ly-6G,TNF-α,IL-1β,IL-6和iNOS)使用酶联免疫吸附测定，实时荧光定量聚合酶链反应,和西方印迹。采用LPS+ATP诱导的THP-1细胞炎症模型验证体内实验结果。
结果：从EPT中鉴定或初步推导出了99种化合物。使用网络药理学，我们发现TLR4/NF-κB可能是EPT预防和治疗ALI的相关通路。EPT中的聚半乳糖酸可能是潜在的活性成分。EPT可以减轻LPS诱导的肺组织病理学损伤，降低ALI大鼠模型的湿/干重比。此外，EPT降低支气管肺泡灌洗液中白细胞和中性粒细胞计数,降低相关炎症因子(NE,MPO,Ly-6G,TNF-α,IL-1β,IL-6和iNOS）在肺组织中。它还增加了内皮型一氧化氮合酶的表达。Westernblotting证实EPT可能在体内影响TLR4/NF-κB和NLRP3信号通路。在THP-1细胞中获得类似的结果。
结论：EPT通过影响TLR4/NF-κB和NLRP3信号通路减少炎症因子的释放，从而减弱ALI的炎症反应。聚半乳糖酸可能是导致这些作用的化合物。
