Targeting the TLR-4/NF‐κB pathway by the plant-derived sulfur-containing Sulforaphane (SFN) and S-allyl-cysteine (SAC) is associated with the inhibition of pro-inflammatory gene expression induced by P. aeruginosa in Cystic Fibrosis IB3-1 cells

Sulforaphane (SFN) and S-allyl-cysteine (SAC) are potent sulfur-containing bioactive compounds derived from plants and exhibit significant antioxidant, anti-inflammatory, and anticancer properties. Sulforaphane is highly concentrated in broccoli sprouts (Brassica oleracea) and other cruciferous vegetables (cabbage, Brussels sprouts). S-allyl-cysteine is one of the major bioactive components present in garlic (Allium sativum). By performing docking and molecular dynamics (MD) simulations with SeeSAR 12.1.0 software (docking) and Gromacs 2021.1 (MD) software, we found that SFN stably interacts with the p60 NF‐κB monomer, forming a ternary NF‐κB/SFN/DNA complex and impairing the full interaction between the NF-kB and DNA molecules. Docking experiments performed on S-allyl-cysteine suggested that a possible mechanism of action of SAC is an interference with the activity of Toll-like receptors (TLRs), particularly TLR4, thereby inhibiting NF‐κB and NF‐κB-regulated genes. Considering the key role of NF‐κB in regulating the expression of pro-inflammatory genes in cystic fibrosis (CF), the main purpose of the present study was to determine the effects of SFN and SAC on the expression of the pro-inflammatory genes in cystic fibrosis cells infected with P. aeruginosa. The employed cellular model system was the human bronchial epithelial IB3-1 cell line, derived from a CF patient with a ΔF508/W1282X mutant genotype and immortalized with adeno12/SV40. After 48 hours of treatment with 5 µM SFN or 50 µM SAC, the cells were infected with the P. aeruginosa PAO1 strain for 4 hours. Changes in the expression of the pro-inflammatory genes such as IL-6, IL-8, IL-1b and G-CSF were studied by RT-qPCR using b-actin sequences as internal controls. Proteins released into culture supernatants were measured using Bio-Plex Human Cytokine 27-plex Assay. The results obtained demonstrated that PAO1 infection induced the expression of pro- inflammatory genes, and that this increase was inhibited by SFN and SAC with different efficiency depending on the interleukins studied. In particular, the Bio‐Plex data showed that SAC preferentially inhibits IL‐6 and G‐CSF, whereas SFN preferentially inhibits IL‐6 and IL‐8. Both compounds, however, are highly effective in inhibiting the IL‐1β transcripts, as indicated by RT-qPCR results. This finding suggests that plant-derivedsulfur-containing bioactive molecules should be considered to mitigate the inflammatory state in cystic fibrosis.