Abstract: 【Objective】This study aimed to identify key targets through which metformin inhibits bovine mastitis using network pharmacology and molecular docking，with subsequent in vitro cellular validation of the underlying mechanisms.【Methods】Metformin?related targets were retrieved from the TCMSP database，while bovine mastitis– associated disease targets were obtained from the GeneCards and OMIM databases. The intersection of these two sets was determined using a Venn platform，and the resulting common targets were used to construct a protein–protein interaction （PPI） network. Gene Ontology （GO） functional enrichment and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analyses were performed via the DAVID database.Molecular docking was carried out using AutoDock software. The effect of metformin on bovine mammary epithelial cells was assessed using the CCK 8 assay. An in vitro inflammation model was established by stimulating MAC?T cells with lipopolysaccharide （LPS），and the expression levels of inflammation?related factors and selected targets genes were measured by RT qPCR.【Results】A total of 1 250 metformin ? associated targets and 2 304 bovine mastitis – related targets were identified，with 191 overlapping targets detected by the Venn platform. PPI network analysis revealed 10 key targets， including ALB，IL6，TNF，EGFR，and IL1B. These targets were predominantly enriched in the IL?17 ，TNF ，PI3K –Akt ，and NF?κB signaling pathway. In vitro experiments demonstrated that metformin significantly downregulated the mRNA expression of the pro?inflammatory cytokines TNF? α，IL?6，IL?1β，and IL?8，while concomitantly upregulating the anti?inflammatory cytokine IL?10. Furthermore，metformin significantly reduced the transcriptional levels of several upstream signaling adaptors/regulators（TRAF1，FADD，TRADD，TRAF6，ACT1）and decreased EGFR and ALB expression.【Conclusion】These findings suggest that metformin exerts anti?inflammatory effects in bovine mastitis primarily through stable interactions with ALB and EGFR，modulating IL ? 17，TNF and related signaling pathways. The mechanism likely involves downregulation of proinflammatory cytokine expression and upstream signaling adaptor transcription，together with upregulation of anti?inflammatory factor expression.

摘要： 【目的】该研究旨在运用网络药理学与分子对接技术，筛选二甲双胍抑制奶牛乳腺 炎的关键靶点，并通过体外细胞实验验证其可能的作用机制。【方法】通过TCMSP数据库获取 二甲双胍的作用靶点；利用GeneCards与OMIM数据库获取奶牛乳腺炎相关疾病靶点，取两者 交集得到共同靶点并构建PPI蛋白互作网络。借助DAVID数据库进行GO功能富集与KEGG通 路富集分析，采用AutoDock软件开展分子对接验证。采用CCK?8法检测二甲双胍对奶牛乳腺 上皮细胞的影响，以LPS诱导MAC?T细胞建立体外炎症模型，通过RT?qPCR检测炎症靶点、信 号通路关键因子和炎症相关因子的表达水平。【结果】筛选得到二甲双胍1 250个作用靶点及 2 304个奶牛乳腺炎相关靶点，通过Venn平台共找到191个共表达靶点。经PPI网络分析得到 ALB、IL6、TNF、EGFR、IL1B等10个关键靶点，主要富集于IL?17信号通路、肿瘤坏死因子信号 通路、PI3K?Akt信号通路、NF?κB信号通路等。体外细胞实验结果显示，二甲双胍可显著下调 促炎因子TNF?α、IL?6、IL?1β、IL?8的基因表达，同时上调抑炎因子IL?10的基因表达。此外，二 甲双胍可显著下调信号通路关键因子TRAF1、FADD、TRADD、TRAF6、ACT1的转录水平，并下 调了EGFR与ALB的表达。【结论】该研究结果表明二甲双胍通过与ALB、EGFR稳定结合调节 IL?17、TNF等信号通路，其作用机制可能通过下调促炎因子表达以及信号通路关键因子的转录 水平，上调抗炎因子的表达来抑制奶牛乳腺炎症反应。