Screening of Traditional Chinese Medicine Formula against Riemerella anatipestifer and Study on Its Antibacterial Mechanism Based on Network Pharmacology

doi:10.15933/j.cnki.1004-3268.2026.09.015

Abstract

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

CLC Number:

S852.6

Qiao Qingmei, Zhang Yao, Cheng Maogao. Screening of Traditional Chinese Medicine Formula against Riemerella anatipestifer and Study on Its Antibacterial Mechanism Based on Network Pharmacology[J]. Journal of Henan Agricultural Sciences, 2026, 55(9): 162-171.

乔卿梅, 张瑶, 程茂高. 抗鸭疫里默氏杆菌中药组方筛选及其抗菌作用网络药理学机制研究[J]. 河南农业科学, 2026, 55(9): 162-171.

Figures/Tables 7

References

［1］冯雅婷，朱敏，刘丹，等. 鸭疫里默氏杆菌流行菌株的分离鉴定及生物学特性［J］. 微生物学通报，2022，49（11）：4778‑4785.
Feng Y T，Zhu M，Liu D，et al. Isolation，identification，and biological characterization of Riemerella anatipestifer epidemic strains［J］. Microbiology China，2022，49（11）：4778‑4785.
［2］孟欣，陈红梅，江南松，等. 一株5型鸭疫里默氏杆菌的耐药性与毒力分析及全基因组测序［J］. 微生物学通报，2025，52（11）：5253‑5266.
Meng X，Chen H M，Jiang N S，et al. Whole genome sequence，antibiotic resistance，and virulence of astrain of Riemerella anatipestifer serotype 5［J］. Microbiology China，2025，52（11）：5253‑5266.

［3］卢金号. 鸭疫里默氏菌流行病学调查及里默氏菌属新种的鉴定［D］. 成都：四川农业大学，2022.

Lu J H. Epidemiological investigation of Riemerella anatipestifer and identification of new specie of Riemerella ［D］. Chengdu： Sichuan Agricultural University，2022.

［4］李晨阳，张传美，惠宇韬，等. 15种血清型的鸭疫里默氏杆菌毒力基因检测和耐药性分析［J］. 畜牧兽医学报，2026，57（1）：360‑368.
Li C Y，Zhang C M，Hui Y T，et al. Detection of virulence genes and drug resistance analysis of 15 serotypes of Riemerella anatipestifer［J］. Acta Veterinaria et Zootechnica Sinica，2026，57（1）：360‑368.

［5］Nowaczek A，Dec M，Stępień‑Pyśniak D，et al.Characterization of Riemerella anatipestifer strains isolated from various poultry species in Poland［J］.Antibiotics，2023，12（12）：1648.

［6］Yang Z S，Wang M S，Jia R Y，et al. Genome‑based assessment of antimicrobial resistance reveals the lineage specificity of resistance and resistance gene profiles in Riemerella anatipestifer from China［J］.Microbiology Spectrum，2024，12（2）：e03132‑e03123.

［7］沈扬扬，马芹，汪建华，等. 4型鸭疫里默氏杆菌的分离鉴定及其不同感染途径致病性和传播方式的研究［J］. 中国预防兽医学报，2025，47（2）：124‑129.
Shen Y Y，Ma Q，Wang J H，et al. Isolation and identification of Riemerella anatipestifer serotype 4 strain and its pathogenicity and transmission mode in different infection routes［J］. Chinese Journal of Preventive Veterinary Medicine，2025，47（2）：124‑129.

［8］高阳，马雪，杜鑫. 黄柏提取物的生物学功能及其在动物生产中的应用［J］. 饲料工业，2024，45（1）：61‑66.
Gao Y，Ma X，Du X. Biological functions of Phellodendri extract and its application prospects in animal productions［J］. Feed Industry，2024，45（1）：61‑66.

［9］黄艳红，堂格斯，廖景轩，等. 苦参素的生物学功能及其在动物生产中的应用［J］. 饲料研究，2024，47（21）：177‑180.
Huang Y H，Tang G S，Liao J X，et al. Biological function of oxymatrine and its application in animal production［J］. Feed Research，2024，47（21）：177‑180.

［10］杜珍美，孙佳琦，郭富强，等. 中药组方与生物体“多点嵌套调控”机制的研究路径探索［J］. 时珍国医国药，2023，34（9）：2201‑2204.
Du Z M，Sun J Q，Guo F Q，et al. Exploring research pathways into the “multi‑point nested regulatory”mechanisms of traditional Chinese medicine（TCM）formulas in biological systems［J］. Lishizhen Medicine and Materia Medica Research，2023，34 （9）：2201‑2204.

［11］郭闫闫，赵沛东，陈海燕，等. 熟地黄及其药对的研究进展［J/OL］. 中华中医药学刊，2026‑06‑22.https：//link.cnki.net/urlid/21.1546.R.20260622.1428.002.
Guo Y Y，Zhao P D，Chen H Y， et al. Research Progress on Prepared Rehmannia Root and Its Herb Pairs［J］.Chinese Archives of Traditional Chinese Medicine，2026‑06‑22. https：//link.cnki.net/urlid/21.1546.R.20260622.1428.002.

［12］黎智，吉艺宽，李美娣，等. 鸭疫里氏杆菌分离鉴定及紫锥菊等中药体外抑菌试验［J］. 中兽医医药杂志，2025，44（5）：59‑62.
Li Z，Ji Y K，Li M D，et al. Isolation and identification of Riemerella anatipestifer and in vitro antibacterial test of traditional Chinese medicine such as Echinacea purpurea［J］. Journal of Traditional Chinese Veterinary Medicine，2025，44（5）：59‑62.

［13］左春生，徐光科，焦凤超，等. 五种植物精油对鸭疫里默氏菌体外抑菌效果观察［J］. 中国饲料，2020（15）：55‑58.
Zuo C S，Xu G K，Jiao F C，et al. Antibacterial effect of five plant essential oils on Riemerella anatipestifer in vitro［J］. China Feed，2020（15）：55‑58.

［14］冯灿，张祺毅，陈菲，等. 大口黑鲈源迟缓爱德华氏菌的分离鉴定及中草药的体外抑菌试验［J］. 中国预防兽医学报，2025，47（8）：796‑804.
Feng C，Zhang Q Y，Chen F，et al. Isolation and identification of Edwardsiella tarda from largemouth bass and the in vitro antibacterial effect of Chinese herbal medicine on the bacteria［J］. Chinese Journal of Preventive Veterinary Medicine，2025，47（8）：796‑804.

［15］王慧，郝莹，董志国，等. 青蛤源溶藻弧菌的分离鉴定、药敏分析以及中草药体外抑菌研究［J］. 中国预防兽医学报，2026，48（1）：8‑17.
Wang H，Hao Y，Dong Z G，et al. Isolation，identification，and antibiotic susceptibility analysis of Vibrio alginolyticus from Cyclina sinensis and in vitro antibacterial effect of Chinese herbal medicine［J］.Chinese Journal of Preventive Veterinary Medicine，2026，48（1）：8‑17.

［16］郑连宝，陈文祥，嵇林风，等. 一种在菌落总数测定中防止芽胞杆菌菌落弥漫生长的方法［J］. 中国卫生检验杂志，2019，29（22）：2715‑2717.
Zheng L B，Chen W X，Ji L F，et al. A method for preventing colony diffuse growth of Bacillus spp.in bacterial plate counting［J］. Chin J Health Lab Tec，2019，29（22）：2715‑2717.

［17］姜小雨，汪海东，郭金杰，等. 基于网络药理学探究加味白头翁汤治疗溃疡性结肠炎的潜在机制［J］. 山西农业科学，2025，53（6）：166‑175.
Jiang X Y，Wang H D，Guo J J，et al. Evaluation of the potential mechanism of supplemented pulsatilla decoction in the treatment of ulcerative colitis by network pharmacology analysis［J］. Journal of Shanxi Agricultural Sciences，2025，53（6）：166‑175.

［18］Qin X，Wu Y L，Pang X，et al. Potential of traditional Chinese medicine‑antibiotic combinations in combating multidrug‑resistant organisms：advances，mechanisms，and clinical implications［J］. Journal of Ethnopharmacology，2026，361：121261.

［19］Man T，Yu H Y，Li L，et al. Integrative network pharmacology and experimental validation of multi‑target synergy against multidrug‑resistant Klebsiella pneumoniae via PI3K/AKT‑MAPK pathway disruption［J］.Naunyn‑Schmiedeberg’s Archives of Pharmacology，2026，399（4）：5157‑5171.

［20］Wang J，Liang Q. Beyond bactericidal：targeting plasmid‑mediated antibiotic resistance with natural product‑based plasmid curing agents［J］. Frontiers in Microbiology，2026，17：1802582.

［21］Yang Y Q，Jia X T，Qu M Y，et al. Exploring the potential of treating chronic liver disease targeting the PI3K/Akt pathway and polarization mechanism of macrophages［J］. Heliyon，2023，9（6）：e17116.

［22］ Kim L W，Castillo V，Barbieri A. PI3K/PKB signaling network as a host target to enhance bacterial ingestion and clearance［J］. Biologia Futura，2026，77（1）：29‑37.

［23］Wu Y H，Kuo Y H，Lin Y Y，et al. Antcin K suppresses proinflammatory cytokines expression via the PI3K，Akt and NF‑κB pathways in human gingival fibroblasts：implications for periodontitis treatment［J］. Cell Death Discovery，2026，12：25.

［24］Zhang Y Y，Liang D Y，Li L L，et al. Relinqing Granules inhibiting lipopolysaccharide‑induced pneumonia injury through modulation of the PI3K/AKT/mTOR signaling pathway［J］. Journal of Ethnopharmacology，2025，353：120303.

［25］Zinkernagel A S，Johnson R S，Nizet V. Hypoxia inducible factor（HIF）function in innate immunity and infection［J］. Journal of Molecular Medicine，2007，85（12）：1339‑1346.

［26］李庆雯，南亚昀，姜希娟，等. 丹酚酸B对离体内皮祖细胞黏附、趋化和增殖的影响［J］. 天津中医药大学学报，2009，28（1）：20‑23.
Li Q W，Nan Y Y，Jiang X J，et al. Effects of salvianolic acid B on conglutination，migration and proliferation of endothelial progenitor cells（EPCs）in vitro［J］. Journal of Tianjin University of Traditional Chinese Medicine，2009，28（1）：20‑23.

［27］Andreata F，Clément M，Benson R A，et al. CD31 signaling promotes the detachment at the uropod of extravasating neutrophils allowing their migration to sites of inflammation［J］. ELife，2023，12：e84752.

［28］Vestweber D. Adhesion and signaling molecules controlling the transmigration of leukocytes through endothelium［J］. Immunological Reviews，2007，218 （1）：178‑196.

［29］Caron E，Self A J，Hall A. The GTPase Rap1 controls functional activation of macrophage integrin αMβ2 by LPS and other inflammatory mediators［J］. Current Biology，2000，10（16）：974‑978.

［30］Tang S Q，Chen T Y，Yu Z，et al. RasGRP3 limits toll‑like receptor‑triggered inflammatory response in macrophages by activating Rap1 small GTPase［J］.Nature Communications，2014，5：4657.
［31］闫子怡，白晓琳，李卓，等. DHA虾青素双酯抑制脂肪细胞与巨噬细胞共培养体系中炎症反应［J］. 中国海洋药物，2021，40（5）：35‑41.
Yan Z Y，Bai X L，Li Z，et al. Astaxanthin‑DHA diester inhibits inflammatory response in the co‑culture system of adipocytes and macrophages［J］.Chinese Journal of Marine Drugs，2021，40（5）：35‑41.
［32］Grant R W，Stephens J M. Fat in flames：influence of cytokines and pattern recognition receptors on adipocyte lipolysis［J］. American Journal of Physiology‑Endocrinology and Metabolism，2015，309（3）：E205‑E213.
［33］Rinnerthaler M，Sykora M，Christoforakos A，et al.STAT1/3/5 functions driving lipid and energy metabolism in cancer and immunity［J］. International Journal of Molecular Sciences，2026，27（6）：2828.
［34］Lee C，Kim M J，Kumar A，et al. Vascular endothelial growth factor signaling in health and disease：from molecular mechanisms to therapeutic perspectives［J］.Signal Transduction and Targeted Therapy，2025，10：170.
［35］Abhinand C S，Raju R，Soumya S J，et al. VEGF‑A/VEGFR2 signaling network in endothelial cells relevant to angiogenesis［J］. Journal of Cell Communication and Signaling，2016，10（4）：347‑354.

［36］Ma L，Li W Y，Zhang Y B，et al. FLT4/VEGFR3 activates AMPK to coordinate glycometabolic reprogramming with autophagy and inflammasome activation for bacterial elimination［J］. Autophagy，2022，18（6）：1385‑1400.
［37］Luo Z H，Gong K. Inflammatory signaling in targeted therapy resistance：focus on EGFR‑targeted treatment［J］. Cancer Biology & Medicine，2024，21（10）：831‑837.

[1]	GAO Tiantian, LIU Xuesong, LIU Yongti, QIU Zongsheng, LIU Yishan, LI Shuxin, QI Jingjing, LI Ya’nan, WEI Chengwei, MA Tianwen. Study on Mechanism of Feizhu Powder for Prevention and Treatment of Deficiency of Qi and Blood in Sows Based on Network Pharmacology and Animal Experiments [J]. Journal of Henan Agricultural Sciences, 2025, 54(4): 117-128.
[2]	SONG Shuhong, WANG Ziyu, SHAO Lizhu, ZHANG Boyuan, ZHAO Heng, LI Peng, ZHANG Li. Inhibiting Effect of Artemisia argyi Polysaccharide on Spoilage Bacteria of Strawberry Fruit and Its Preservation Effects [J]. Journal of Henan Agricultural Sciences, 2025, 54(11): 172-180.