Abstract：Puerarin (Pue), an isoflavone extracted from the root of Pueraria lobata of leguminous plant, has an anti-inflammatory effect. The aim of this experiment was to investigate anti-inflammatory mechanism of puerarin in the inflammation model of bovine(Bos taurus) mammary epithelial cells (bMECs) induced by lipopolysaccharide (LPS). In this study, the bMECs were primary cultured with the collagenase digestion and both cytokeratin-18 (CK-18) and β-casein of the cells were identified by immunofluorescence technique and Western blot, respectively. The cell counting kit-8 (CCK-8) method was used to determine the suitable stimulating does of LPS used for inducing inflammation of the bMECs. The enzyme-linked immunosorbent assay (ELISA) kits was used to detect the changes of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) content in bMECs culture supernatant. The bMECs were divided into control group, inflammatory model group, puerarin group and dexamethasone positive control group. The protein expressions of p-IκB-α, IκB-α, p-p65 and p65 were detected by Western blot in each group of cells. The results showed that the bMECs were proved with the presence of specific CK-18 in cytoplasm and could express β-casein. There was a significant difference in the contents of TNF-α and IL-1β between the cells treated with 10 μg/mL LPS for 24 h and the cells in the control group (P＜0.05). The data above demonstrated the establishment of inflammation model in the bMECs was successful. The results indicated that the p-IκB-α protein was obviously decreased (P＜0.05) in the puerarin group compared to that in the inflammatory model group. Meanwhile, p-p65 protein and the contents of TNF-α in the cell culture supernate of the puerarin group were significantly lower than that in inflammatory model group (P＜0.01). There was no significant difference between the puerarin group and the dexamethasone positive group. Taken together, our date indicate that the puerarin acted as an anti-inflammatory via blockading the activation of nuclear transcription factor-κappaB(NF-κB) signaling pathway and inhibited TNF-α expression. This study laid the foundation for the development of clinical anti-inflammatory agent and therapy of dairy cow mastitis.
