Abstract:

This study aimed to explore the effects of curcumin (Curcumin, Cur) on the inflammatory response of lipopolysaccharide (LPS)-induced bovine mammary epithelial cells (MAC-T) from the ferroptosis pathway. MAC-T cells in the logarithmic growth phase were randomly grouped according to the experimental requirements and underwent corresponding treatments, with three replicates in each group. The MTT method was used to determine the effects of different concentrations of LPS/Cur on the survival rate of MAC-T cells; RT-PCR was employed to detect the transcription levels of inflammation-related genes (TNF-α, IL-6, IL-1β, iNOS, CXCL2, CCL2) and ferroptosis-related genes (PTGS2, SLC7A11, GPX4, FTH1, ACSL4); BODIPY 581/591 C11 staining was utilized to detect the lipid peroxidation level of cells; kits were used to measure the contents of MDA, LPO, GSH, and the GSH/GSSG ratio in cells; Ferro-Orange staining and the Fe²⁺ detection kit were used to determine the Fe²⁺ content in cells; ELISA kits were used to detect the concentrations of TNF-α, IL-6, and IL-1β in cells; Western blot was used to detect the relative expression levels of TNF-α, IL-6, and IL-1β proteins. The results revealed that compared with the CON group, treatments with 10 μg·mL^-1 LPS or 20 μmol·L^-1 Cur had no significant effect on the survival rate of MAC-T cells. After LPS treatment, the expression levels of pro-inflammatory related genes (TNF-α, IL-6, IL-1β, iNOS, CXCL2, CCL2), ferroptosis-related genes (PTGS2, ACSL4), and the related protein (ACSL4) in cells were significantly elevated (P < 0.01); the expression levels of anti-ferroptosis genes (SLC7A11, GPX4, FTH1) and protein (GPX4) were significantly decreased (P < 0.01); the MDA content and LPO concentration were all significantly increased (P < 0.01); the GSH content and GSH/GSSG ratio were both extremely decreased (P < 0.01); the Fe²⁺ content was extremely significantly increased (P < 0.01); the concentrations and protein expression levels of TNF-α, IL-6, and IL-1β were all extremely increased (P < 0.01). Compared with the LPS alone treatment group, the expression levels of pro-inflammatory related genes, ferroptosis-related genes and related proteins in the LPS and Cur-H co-treatment group were extremelydecreased (P < 0.01); the expression levels of anti-ferroptosis genes and proteins were extremely increased (P < 0.01); the lipid peroxidation level, MDA content, and LPO concentration were all extremely decreased (P < 0.01); the GSH content and GSH/GSSG ratio were both significantly increased (P < 0.01); the Fe²⁺ content was significantly decreased (P < 0.01); the concentrations and protein expression levels of TNF-α, IL-6, and IL-1β were all extremely decreased (P < 0.01). Additionally, the ferroptosis activator Erastin significantly inhibited the alleviating effect of Cur on LPS-induced inflammation. In conclusion, Cur can alleviate LPS-induced inflammatory responses in MAC-T cells by inhibiting ferroptosis.

Key words: ferroptosis, curcumin, lipopolysaccharide, bovine mammary epithelial cell (MAC-T)