目的 建立小鼠支氣管哮喘模型和人非小細胞肺癌細胞（A549）炎癥模型，研究金振口服液（Jinzhen Oral Liquid，JZOL）對支氣管哮喘的藥效作用和分子機制。方法 將 66只雌性 BALB/c小鼠隨機分為對照組和模型組，模型組小鼠采用卵清蛋白（OVA）致敏法建立哮喘模型后，將其分為模型組和 JZOL高、中、低劑量（4.4、2.2、1.1 mg·kg^-1）組和地塞米松組（Dex，2 mg·kg^-1）。全身體積描記系統(tǒng)測定小鼠氣道反應Penh值。HE染色觀察小鼠肺組織病理學情況。瑞氏-姬姆薩染色檢測肺泡灌洗液（BALF）中炎性細胞數(shù)量。ELISA檢測 BALF中腫瘤壞死因子-α（TNF-α）和血清中免疫球蛋白E（IgE）和免疫球蛋白G1（IgG1）濃度。采用20 ng·mL^-1白細胞介素（IL）-1β誘導A549細胞24 h建立細胞炎癥模型，隨機分為對照組、模型組以及4個質(zhì)量濃度JZOL（5.39、2.69、1.35、0.67 mg·mL^-1）處理組。MTS法測定JZOL對A549細胞的藥物毒性。采用 RNA-seq技術對 6個實驗組進行轉錄組學分析，篩選差異基因，對其進行 GSEA通路富集分析、京都基因與基因組百科全書（KEGG）通路富集分析和基因本體（GO）注釋通路富集分析，通過實時熒光定量 PCR（qRT-PCR）實驗對篩選出的關鍵基因進行實驗驗證。結果 體內(nèi)實驗表明，JZOL能夠改善小鼠肺組織炎性細胞浸潤，降低小鼠氣道高反應性Penh值，減少BALF中巨噬細胞、嗜酸性粒細胞和中性粒細胞的數(shù)量，降低BALF中TNF-α和血清中IgE和IgG1的濃度。體外實驗表明，JZOL對 A549細胞的最大無毒濃度為 5.39 mg·mL^-1?；蚋患治鼋Y果顯示，與對照組相比，模型組共獲得 46條炎癥模型相關通路，而 JZOL不同濃度處理組對這些通路的逆轉比例分別為 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）。差異表達基因（DEG）的KEGG富集分析顯示，與對照組相比，模型組共富集到58條通路；與模型組相比，JZOL處理組共富集到32條通路，逆轉了IL-17、TNF、核苷酸寡聚化結構域（NOD）樣受體信號通路等。對IL-17信號通路中趨化因子配體1（CXCL1）、趨化因子配體2（CXCL2）、脂質(zhì)運載蛋白2（LCN2）進行qRT-PCR驗證，與轉錄組測序結果一致。結論 JZOL對由OVA誘導的小鼠支氣管哮喘模型具有顯著的治療功效，能有效緩解哮喘小鼠的氣道高反應性并抑制肺部組織的炎癥反應。其抗炎效果是通過調(diào)節(jié)CXCL1、CXCL2和LCN2這幾個關鍵基因的表達，進而影響IL-17信號通路來實現(xiàn)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

國家中醫(yī)藥管理局“-岐黃學者”中醫(yī)藥領軍人才項目；江蘇省工業(yè)和信息產(chǎn)業(yè)轉型升級專項-多組分中藥關鍵技術研究