Abstract: To screen the traditional Chinese medicine formula with strong antibacterial effect on Riemerella anatipestifer（RA）and explore its antibacterial mechanism，the Oxford cup method was used to determine the diameter of antibacterial circle of 55 traditional Chinese medicines against RA，and the minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of highly sensitive and above traditional Chinese medicines were determined. Three traditional Chinese medicines with the best inhibitory and killing effect were selected，and L₉（ 3³）orthogonal test design was carried out with 1×MIC，5×MIC and 10×MIC as three levels.The traditional Chinese medicine formula with the best antibacterial effect was screened out，and the network pharmacology method was used to explore the antibacterial mechanism of the formula. The results showed that among the 55 tested traditional Chinese medicines，four induced extremely sensitive and highly sensitive responses in the RA model，respectively.The best inhibitory effect was from Coptidis Rhizoma，Sappan Lignum and Schisandrae Chinensis Fructus.MIC of Coptidis Rhizoma was 15.63 mg/mL，MBC was 31.25 mg/mL.The MIC and MBC of Schisandrae Chinensis Fructus and Sappan Lignum were both 31.25 mg/mL and 62.50 mg/mL，respectively.The results of orthogonal test showed that when Rhizoma Coptidis was 78.15 mg/mL，Schisandrae Chinensis Fructus was 312.5 mg/mL and Sappan Lignum was 312.5 mg/mL，the diameter of inhibition zone was 20.05 mm，which had the best antibacterial effect on RA and reached the extremely sensitive level. The results of network pharmacology showed that there were 13，12 and 18 chemical components related to antibacterial effect of Coptidis Rhizoma，Schisandrae Chinensis Fructus and Sappan Lignum，respectively，and the corresponding targets of chemical components of each Chinese medicine were 146，327 and 154，respectively. A total of 1 431 targets related to antibacterial effect were obtained by database retrieval，and there were 22 common intersection targets of three traditional Chinese medicines and antibacterial effect.The key proteins included RAC‑α serine/threonine protein kinase，focal adhesion kinase 1，epidermal growth factor receptor，etc.GO enrichment analysis showed that antibacterial‑related genes of the Coptidis Rhizoma‑Schisandrae Chinensis Fructus‑Sappan Lignum combination were enriched in three categories：biological process（BP），cellular component（CC）and molecular function（MF）.The BP terms were mainly related to PI3K/AKT signaling pathway regulation，protein autophosphorylation，and positive regulation of cell migration；the MF terms mainly centered on protein kinase activity，kinase activity and protein tyrosine kinase activity. KEGG enrichment analysis identified multiple key pathways including VEGF，Rap1，HIF‑1 signaling pathway and adherens junction pathway.These results indicated that the combination exerted antibacterial，anti‑inflammatory and immunoregulatory effects via synergistic regulation of multiple signaling pathways.In summary，the screened compound formula consisting of Coptidis Rhizoma，Schisandrae Chinensis Fructus and Sappan Lignum exerts potent antibacterial activity against RA.This study preliminarily elucidates the synergistic antibacterial mechanism of the three herbs.The findings are of great significance for reducing antibiotic consumption and lowering the risk of bacterial drug resistance，and also provide a green alternative strategy for the prevention and treatment of Riemerella anatipestifer.

Key words: Riemerella anatipestifer, Traditional Chinese medicine formula, Antibacterial effect, Network pharmacology

摘要： 为筛选对鸭疫里默氏杆菌（Riemerella anatipestifer，RA）具有较强抑菌作用的中药组方，并探究其抗菌作用机制，采用牛津杯法对55味中药进行抗RA抑菌圈直径的测定，对呈高度敏感及以上的中药进行最小抑菌浓度（MIC）和最低杀菌浓度（MBC）测定，选取抑杀效果最佳的3味中药，以其1×MIC、5×MIC和10×MIC作为3个水平进行L₉（3³）正交试验设计，筛选出抑菌效果最佳的中药组方，并采用网络药理学方法探究该组方的抗菌作用机制。结果表明，55味受试中药中，有4味可使RA达到极度敏感、高度敏感等级，其中抑杀效果最好的是黄连、苏木、五味子3味中药。黄连的MIC为15.63 mg/mL，MBC为31.25 mg/mL；五味子和苏木的MIC均为31.25 mg/mL，MBC均为62.50 mg/mL。正交试验组方筛选结果表明，当组方中黄连为78.15 mg/mL、五味子为312.5 mg/mL、苏木为312.5 mg/mL时，抑菌圈直径为20.05 mm，对RA的抑菌效果最佳，达极度敏感水平。网络药理学研究表明，黄连、五味子、苏木与抗菌作用相关的化学成分分别为13、12、18个，各味中药化学成分对应的作用靶点分别为146、327、154个；通过数据库检索获得抗菌作用相关靶点1 431 个，3 味中药与抗菌作用共同的交集靶点有22 个，关键蛋白包括RAC-α丝氨酸/苏氨酸蛋白激酶、黏着斑激酶1、表皮生长因子受体等。GO富集分析表明，黄连-五味子-苏木配伍的抗菌相关基因富集于生物过程（BP）、细胞组分（CC）、分子功能（MF）三大类别，BP以PI3K/AKT通路调控、蛋白质自身磷酸化、细胞迁移的正向调控为主，MF主要集中于蛋白激酶、激酶、蛋白酪氨酸激酶活性。KEGG通路分析表明其主要富集于血管内皮生长因子、Ras相关蛋白-1、缺氧诱导因子-1、黏附连接等多条关键通路，提示该配伍通过多通路协同调控发挥抗菌、抗炎及免疫调节作用。综上，筛选出的黄连、五味子、苏木中药组方对RA具有较强的抑菌作用，初步揭示了三者协同抗菌的作用机制，对减少抗生素使用及降低耐药性风险具有重要意义，也为防治鸭疫里默氏杆菌病提供了一种绿色替代方案。

关键词: 鸭疫里默氏杆菌, 中药组方, 抗菌作用, 网络药理学