Proteomic Identification of PFKFB3 and PFKFB4 Associated with Coenzyme Metabolism and Redox Imbalance in Dairy Cows with Clinical Mastitis

In this study, we identified a number of biological processes, pathways, and key protein targets associated with coenzyme metabolism in bovine clinical mastitis (CM). The expression patterns and subcellular localization of key proteins were examined to characterize their potential association with oxidative stress and inflammatory responses in mammary gland tissues. The CM group exhibited collapsed and atrophied mammary acini, inflammatory cell infiltration, increased reactive oxygen species fluorescence signals, and a significant reduction in glutathione content. Levels of key coenzymes, including nicotinamide adenine dinucleotide and flavin adenine dinucleotide, decreased significantly. Bioinformatic analysis identified four biological processes related to coenzyme metabolism and 20 key differentially expressed proteins associated with the glycolysis pathway. Among them, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and PFKFB4 were identified as key hub proteins involved in fructose and mannose metabolism and AMP-activated protein kinase (AMPK) signaling pathways. PFKFB3 and PFKFB4 were primarily localized in the cytoplasm of mammary epithelial cells, and the CM group showed significantly upregulated and downregulated expression at both the gene and protein levels. Molecular mechanism analysis based on pathway enrichment suggested a putative regulatory network in which pathogen-induced inflammation may be associated with ROS-AMPK-related signaling, potentially contributing to dysregulated PFKFB3/PFKFB4 expression, glycolytic imbalance, impaired coenzyme metabolism, and mammary epithelial cell injury.