The Mechanism of DCA Regulation of NF-κB/NLRP3 Signaling Pathway in the Occurrence of Acute Mastitis
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 treat-ment 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 inflamma-tory response and the concentration of DCA that met the expectation were screened by CCK-8(cell counting kit-8),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(interleu-kin-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-αand 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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