Excessive nonesterified fatty acids (NEFA) impair cellular metabolism and will induce fatty liver formation in dairy cows during the periparturient. Baicalin, an active flavonoid, has great potential efficacy in alleviating lipid accumulation and ameliorating the development of fatty liver disease. Nevertheless, its mechanism remains unclear. Here, the potential mechanism of baicalin on system levels was explored using network pharmacology and in vitro experiments. Firstly, the target of baicalin and fatty liver disease was predicted, and then the protein–protein interaction (PPI) network was constructed. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) (q-value) pathway enrichment is performed through the Database for Annotation, Visualization, and Integrated Discovery (DAVID) server. Finally, the results of the network analysis of the in vitro treatment of bovine hepatocytes by NEFA were confirmed. The results showed that 33 relevant targets of baicalin in the treatment of liver fatty were predicted by network pharmacology, and the top 20 relevant pathways were extracted by KEGG database. Baicalin treatment can reduce triglyceride (TAG) content and lipid droplet accumulation in NEFA-treated bovine hepatocytes, and the mechanism is related to inhibiting lipid synthesis and promoting lipid oxidation. The alleviating effect of baicalin on fatty liver may be related to the up-regulation of solute vector family member 4 (SLC2A4), Down-regulated AKT serine/threonine kinase 1 (AKT1), Peroxisome proliferator-activated receptor gamma (PPARG), Epidermal growth factor receptor (EGFR), tumor necrosis factor (TNF), Interleukin 6 (IL-6) were associated. These results suggested that baicalin may modulate key inflammatory markers, and lipogenesis processes to prevent fatty liver development in dairy cows.
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