Summary It has been showed bioactive di‐phosphoserine peptide (SS‐2) possesses functions to ameliorate oxidative stress in vitro. This study aimed to substantiate the role of bioactive di‐phosphoserine peptide (SS‐2) in modulating inflammatory responses in TNF‐α‐stimulated HT‐29 cells, and its mechanism of action. SS‐2 significantly reduced IL‐8 secretion in TNF‐α‐induced HT‐29 cells, and also suppressed pro‐inflammatory cytokines, including IL‐8, IL‐12, MCP‐1 and TNF‐α. Moreover, SS‐2 inhibited TNF‐α initiated signalling cascades by suppressing phosphorylation of the ERK1/2, JNK, P38 and IκB those culminate in above cellular inflammatory responses. Differentially expressed genes analysis within NF‐κB signalling pathway revealed that SS‐2 blocks multiple sites of upstream NF‐κB signalling cascade, including FADD and MyD88, thereby preventing the signalling transduction involved in cellular inflammatory response. These results provide a new insight into molecular mechanism for anti‐inflammatory action of SS‐2, suggesting SS‐2 is a potential alternative approach to treat IBD by particular targeting TNF‐α driven inflammatory event.

SummaryPrevious studies have shown that phosvitin phosphopeptides could exert anti‐inflammatory activity in lipopolysaccharide (LPS)‐stimulated small intestine epithelial cells. However, the role of phosphopeptides in inflammation‐related cell signalling pathways remains to be elucidated. Here, the anti‐inflammation effect of a synthetic phosphoserine dimer (2PS) in cell signalling pathways were evaluated by PCR array and immunoblotting assays. Our results showed that 2PS led to reduced secretion of LPS‐induced IL‐8 in a dose‐dependent manner, and downregulation of the mRNA expression of pro‐inflammation cytokines in HT‐29 cell cultures, including IL‐8, IL‐6, IL‐12, TNF‐α and MCP‐1. Compared to LPS alone, treatment with 2PS + LPS exerted downregulation of gene expression in the TLR4‐related cell signalling pathway, including MyD88, IRAK1, IKKε and IKKγ, as well as transcription factors RELs, JUN, and FOS. At the protein level, cell treatment with 2PS led to a decline in LPS‐induced phosphorylation of IκB, ERK1/2, and p‐38 MAPKs. Our results indicate that 2PS attenuates LPS‐induced inflammation through down‐regulation of NF‐κB pathway and inhibition of MAPKs phosphorylation.