| 摘要: |
| 目的：通过网络药理学挖掘金水六君煎治疗气道黏液高分泌的潜在靶点，并采用体外实验探究金水六君煎对人支气管上皮细胞黏液高分泌的干预作用及作用机制。方法：采用网络药理学预测金水六君煎治疗气道黏液高分泌潜在靶点。将24只雄性SPF大鼠随机分为2组，中药组与正常组分别用金水六君煎［5.26g/（kg·d）］和等体积0.9%氯化钠注射液灌胃，5 d后麻醉下取大鼠含药血清和正常血清；将第5代人支气管上皮细胞分为正常组、模型组、不同浓度（10%、15%、20%）含药血清组、阿奇霉素组；正常组予常规培养液培养12 h后，加入15%正常大鼠血清干预12 h；其余各组采用香烟提取物（CSE）联合脂多糖（LPS）干预12h构建人支气管上皮细胞黏液高分泌模型，模型组干预后，加入15%正常大鼠血清干预12 h；不同浓度含药血清组干预后，分别加入10%、15%、20%含药血清继续干预12 h；阿奇霉素组干预后，加入含25μmol/L阿奇霉素血清继续干预12 h。采用CCK8检测细胞活力；采用Western blot法验证潜在靶点。结果：网络药理学预测黄芩素、松柏苷是与疾病靶点连接最广泛的活性物质，蛋白质-蛋白质相互作用（PPI）网络与分子对接发现关键靶点可能为基质金属蛋白酶9（MMP9）、血管内皮生长因子（VEGF）。实验研究结果显示，干预后各组细胞活性较正常组降低（P<0.05），15%含药血清组和阿奇霉素组细胞活性较模型组升高（P<0.05），10%、20%含药血清组细胞活性较阿奇霉素组低（P<0.05）。黏蛋白5AC（MUC5AC）是黏液高分泌的重要蛋白，其表达增加代表模型建立成功。干预后各组MMP9、VEGF、MUC5AC蛋白相对表达量较正常组增加（P<0.05），15%含药血清组和阿奇霉素组MMP9、VEGF、MUC5AC蛋白相对表达量较模型组降低（P<0.05）。结论：金水六君煎对MUC5AC、MMP9、VEGF呈现抑制作用，揭示其机制可能是通过多靶点、多成分作用于气道黏液高分泌。 |
| 关键词: 金水六君煎 气道黏液高分泌 细胞实验 人支气管上皮细胞 |
| DOI： |
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| Effect of Jinshui Liujun decoction on mucus hypersecretion in human bronchial epithelial cells：A study based on network pharmacology and experiments |
| ZHANG Yu,LIU Yu,SONG Cheng,ZHOU Shengqiang,LIU Fang |
| (Hunan University of Chinese Medicine，Changsha 410208，Hunan，China;Hunan Academy of Chinese Medicine，Changsha 410006，Hunan，China;Hunan Hospital of Integrated Traditional Chinese and Western Medicine，Changsha 410006，Hunan，China) |
| Abstract: |
| Objective：To investigate the potential targets of Jinshui Liujun decoction in the treatment of airway mucus hypersecretion based on network pharmacology，as well as its intervention effect and mechanism of action in mucus hypersecretion of human bronchial epithelial cells by conducting an in vitro experiment.Methods：network pharmacology was used to predict the potential targets of Jinshui Liujun decoction in the treatment of airway mucus hypersecretion.A total of 24 specific pathogen-free male rats were randomly divided into traditional Chinese medicine (TCM) group ［treated with Jinshui Liujun decoction 5.26 g/（kg·d）by gavage］ and normal group (treated with an equal volume of 0.9% sodium chloride injection by gavage)，and the rats were anesthetized after 5 days to collect drug-containing serum and normal serum.The fifth-generation human bronchial epithelial cells were divided into normal group，model group，drug-containing serum groups at different concentrations (10%，15%，and 20%)，and azithromycin group.The cells in the normal group were cultured with a conventional medium for 12 hours，and then 15% normal rat serum was added for 12 hours.The cells in the other groups were treated with cigarette smoke extract combined with lipopolysaccharide for 12 hours to establish a mucus hypersecretion model of human bronchial epithelial cells.After modeling，the cells in the model group were added with 15% normal rat serum for 12 hours，those in the drug-containing serum groups at different concentrations were added with 10%，15%，and 20% drug-containing serum for 12 hours，and those in the azithromycin group were added with the serum containing 25 μmol/L azithromycin for 12 hours.CCK8 assay was used to measure cell viability，and Western blot was used to validate potential targets.Results：Network pharmacology predicted that baicalein and coniferin were the active substances with the most extensive connection to disease targets，and the protein-protein interaction network and molecular docking showed that matrix metalloproteinase-9 and vascular endothelial growth factor might be the key targets.The experimental study showed that after intervention，each group had a significant reduction in cell viability compared with the normal group (P<0.05)，and the 15% drug-containing serum group and the azithromycin group had a significant increase in cell viability compared with the model group (P<0.05)，compared with the azithromycin group, the 10% and 20% drug-containing serum groups had a significant reduction in cell viability（P＜0.05）.Mucin 5AC (MUC5AC) was an important protein for mucus hypersecretion，and the increase in its expression indicated successful modeling.Compared with the normal group after intervention，each group had significant increases in the relative protein expression levels of MMP9，VEGF，and MUC5AC (P<0.05)，and compared with the model group，the 15% drug-containing serum group and the azithromycin group had significant reductions in the relative protein expression levels of MMP9，VEGF，and MUC5AC (P<0.05).Conclusion：Jinshui Liujun decoction has an inhibitory effect on MUC5AC，MMP9，and VEGF，suggesting that it acts on airway mucus hypersecretion through multiple targets and components. |
| Key words: Jinshui Liujun decoction airway mucus hypersecretion cell experiment human bronchial epithelial cells |
