The Mechanism of DCA Regulation of NF-κB/NLRP3 Signaling Pathway in the Occurrence of Acute Mastitis
ZHAO Ting, ZHAO Nan*
The aim of this study was to investigate the regulatory mechanism of DCA-mediated NF-κB/NLRP3 signaling pathway in the development of acute mastitis, and to provide theoretical basis for the prevention and treatment of acute mastitis. Inflammation model was established by LPS stimulation of human mammary epithelial cell line DU4475, and the experiment was divided into a control group, a model group, a DCA low-concentration group (DCA-L, 50 μmol/L), a DCA-medium-concentration group (DCA-M, 100 μmol/L), and a DCA-high-concentration group (DCA-H, 200 μmol/L). The optimal concentration of LPS to stimulate DU4475 cells to produce inflammatory response and the concentration of DCA that met the expectation were screened by CCΚ-8 (cell counting kit8), and the viability of each group was determined by CCK-8 assay; the level of intracellular ROS (reactive oxygen species) production in cells was detected by fluorescence staining in each group; and Western blot was performed to detect the NF-κB/NLRP3 pathway-related proteins and apoptosis pathway-related proteins; ELISA (enzyme-linked immunosorbent assay) was used to detect TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), IL-6 (interleukin-6), and IL-18 (interleukin-18) levels in the cells of each group. It was found that compared with the control group, the cell viability of the model group was significantly decreased, and the levels of TNF-α, IL-1β, IL-6 and IL-18 were significantly increased, indicating that the inflammation model of DU4575 cells was successfully constructed under the treatment of 50 μg/mL LPS. The cell viability of each DCA-treated group showed a significant upward trend compared with that of the model group, with a decrease in ROS production and a decrease in the phosphorylation levels of IκB-α and NF-κB p65 proteins, and a significant downward trend in the expression of pyroptosis pathway proteins, such as NLRP3, GSDMD, Caspase-1, and NLRC4, was also observed. In addition, the levels of inflammatory factors such as TNF-α, IL-1 β, IL-6, and IL-18 in each group also significantly decreased. The DCA-M and DCA-H groups showed the most significant effects in improving cell viability, antioxidant stress, and reducing inflammation levels. DCA can play an anti-inflammatory role by inhibiting the activation of the NF-κB signaling pathway, reducing downstream ROS production, and the expression of inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-18. In addition, NF-κB inhibits the activity of NLRP3 inflammatory vesicles, which reduces the expression of inflammatory factors at another level. Therefore, DCA has potential therapeutic effects on acute mastitis
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