目的 建立小鼠支气管哮喘模型和人非小细胞肺癌细胞（A549）炎症模型，研究金振口服液（Jinzhen Oral Liquid，JZOL）对支气管哮喘的药效作用和分子机制。方法 将 66只雌性 BALB/c小鼠随机分为对照组和模型组，模型组小鼠采用卵清蛋白（OVA）致敏法建立哮喘模型后，将其分为模型组和 JZOL高、中、低剂量（4.4、2.2、1.1 mg·kg^-1）组和地塞米松组（Dex，2 mg·kg^-1）。全身体积描记系统测定小鼠气道反应Penh值。HE染色观察小鼠肺组织病理学情况。瑞氏-姬姆萨染色检测肺泡灌洗液（BALF）中炎性细胞数量。ELISA检测 BALF中肿瘤坏死因子-α（TNF-α）和血清中免疫球蛋白E（IgE）和免疫球蛋白G1（IgG1）浓度。采用20 ng·mL^-1白细胞介素（IL）-1β诱导A549细胞24 h建立细胞炎症模型，随机分为对照组、模型组以及4个质量浓度JZOL（5.39、2.69、1.35、0.67 mg·mL^-1）处理组。MTS法测定JZOL对A549细胞的药物毒性。采用 RNA-seq技术对 6个实验组进行转录组学分析，筛选差异基因，对其进行 GSEA通路富集分析、京都基因与基因组百科全书（KEGG）通路富集分析和基因本体（GO）注释通路富集分析，通过实时荧光定量 PCR（qRT-PCR）实验对筛选出的关键基因进行实验验证。结果 体内实验表明，JZOL能够改善小鼠肺组织炎性细胞浸润，降低小鼠气道高反应性Penh值，减少BALF中巨噬细胞、嗜酸性粒细胞和中性粒细胞的数量，降低BALF中TNF-α和血清中IgE和IgG1的浓度。体外实验表明，JZOL对 A549细胞的最大无毒浓度为 5.39 mg·mL^-1。基因富集分析结果显示，与对照组相比，模型组共获得 46条炎症模型相关通路，而 JZOL不同浓度处理组对这些通路的逆转比例分别为 34.8% （5.39 mg·mL^-1）、50%（2.69 mg·mL^-1）、32.6%（1.35 mg·mL^-1）和26%（0.67 mg·mL^-1）。差异表达基因（DEG）的KEGG富集分析显示，与对照组相比，模型组共富集到58条通路；与模型组相比，JZOL处理组共富集到32条通路，逆转了IL-17、TNF、核苷酸寡聚化结构域（NOD）样受体信号通路等。对IL-17信号通路中趋化因子配体1（CXCL1）、趋化因子配体2（CXCL2）、脂质运载蛋白2（LCN2）进行qRT-PCR验证，与转录组测序结果一致。结论 JZOL对由OVA诱导的小鼠支气管哮喘模型具有显著的治疗功效，能有效缓解哮喘小鼠的气道高反应性并抑制肺部组织的炎症反应。其抗炎效果是通过调节CXCL1、CXCL2和LCN2这几个关键基因的表达，进而影响IL-17信号通路来实现的。

Objective To establish a mouse bronchial asthma model and a human non-small cell lung cancer cell (A549) inflammation model, and to study the pharmacological efficacy and molecular mechanism of Jinzhen Oral Liquid (JZOL) on bronchial asthma.Methods Sixty-six female BALB/c mice were randomly divided into control group and model group. After the asthma model was established by the ovalbumin (OVA) sensitization method, mice in the model group were divided into model group, JZOL high, medium, and low dose group, and dexamethasone group (Dex). Whole-body volumetric tracing system was used to determine the penh value of airway response in mice. HE staining was used to observe the histopathology of lungs in mice. The number of inflammatory cells in alveolar lavage fluid (BALF) was detected by Riesling-Jimsa staining. ELISA was performed to detect tumor necrosis factor-α (TNF-α) in BALF and immunoglobulin E (IgE) and immunoglobulin G1 (IgG1) concentrations in serum. A cellular inflammation model was established by using 20 ng·mL^-1 interleukin (IL)-1β induced A549 cells for 24 h. The cells were randomly divided into control group, model group, and four mass concentration JZOL-treated groups (5.39, 2.69, 1.35, and 0.67 mg·mL^-1). The MTS assay was used to determine the pharmacotoxicity of JZOL on A549 cells. RNA-seq technology was used to perform transcriptomics analysis of six experimental groups to screen differential genes, which were subjected to GSEA pathway enrichment analysis, KEGG pathway enrichment analysis and GO pathway enrichment analysis, and the key genes screened were experimentally verified by qRT-PCR experiments.Results In vivo experiments showed that JZOL was able to improve inflammatory cell infiltration in mouse lung tissues, reduce the Penh value of airway hyperresponsiveness in mice, decrease the number of macrophages, eosinophils and neutrophils in BALF, and decrease the concentrations of TNF-α in BALF and IgE and IgG1 in serum. In vitro experiments showed that the maximum nontoxic concentration of JZOL on A549 cells was 5.39 mg·mL^-1. Gene enrichment analysis showed that a total of 46 inflammation model-related pathways were obtained in the model group compared with the control group, and the percentage of the reversal of these pathways in the groups treated with different concentrations of JZOL was 34.8% (5.39 mg·mL^-1), 50% (2.69 mg·mL^-1), 32.6% (1.35 mg·mL^-1) and 26% (0.67 mg·mL^-1), respectively. KEGG enrichment analysis of differentially expressed genes (DEGs) showed that a total of 58 pathways were enriched in model group compared with control group, and a total of 32 pathways were enriched in JZOL-treated group compared with the model group, reversing IL-17, TNF, and nucleotide oligomerization structural domain (NOD)-like receptor signaling pathways. The qRT-PCR validation of chemokine ligand 1 (CXCL1), chemokine ligand 2 (CXCL2), and lipid carrier protein 2 (LCN2) in the IL-17 signaling pathway was consistent with the transcriptome sequencing results.Conclusion JZOL has significant therapeutic efficacy in mouse model of bronchial asthma induced by OVA, effectively alleviating airway hyperresponsiveness and suppressing inflammatory responses in lung tissues in asthmatic mice. The anti-inflammatory effect was likely achieved by regulating the expression of several key genes, including CXCL1, CXCL2 and LCN2, which in turn affected the IL-17 signaling pathway.

R965

国家中医药管理局“-岐黄学者”中医药领军人才项目；江苏省工业和信息产业转型升级专项-多组分中药关键技术研究