Fig. 2. 6’-SL inhibits LPS-induced inflammation by suppressing NF-κB translocation and transactivation in RAW 264.7 cells.

The RAW 264.7 cells were exposed to varying concentrations of 6’-SL for 1 h, followed by treatment with 1 µg/ml LPS for 1 h. (A) Detect NF-κB phosphorylation of total cell, and then, protein expression was evaluated by Western blot. (B) Cells were subjected to nucleus fractionation to determine the amount of NF-κB in the nucleus. (C) RAW 264.7 cells were co-transfected with pNF-κB-Luc and Renilla-Luc for 24 h. After transfection, cells were pre-treated with 100 and 200 µM of 6’-SL, 10 µM of SB203580, and 10 µM of LY294002 for 1 h followed by treatment with 1 µg/ml LPS for 12 h. NF-κB-Luc promoter activity was measured using a dual-luciferase reporter assay. (D) RAW 264.7 cells were treated with the indicated concentrations of 6’-SL for 2 h, followed by treatment with 1 µg/ml LPS for 18 h. Protein expression of MMP9 was detected by Western blot. (E, F) The RAW 264.7 cells were treated with 100 and 200 µM of 6’-SL, 10 µM of SB203580, or 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 12 h. Total RNA samples were subjected to qRT-PCR using IL-1β (E) and MCP-1 (F) primers. Data are presented as the mean ± SEM (n = 4). 6’-SL, 6’-sialyllactose; LPS, lipopolysaccharide; NF-κB, nuclear factor kappa-light chain enhancer of activated B cells; MMP9, matrix metalloproteinase-9; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; PARP, poly ADP-ribose polymerase. *p < 0.05, ***p < 0.001 compared to the control group, #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the LPS-treated group.