Anti-inflammatory effect of sweetfish-derived protein and its enzymatichydrolysate on LPS-induced RAW264.7 cells via inhibition of NF-κB transcription

The present study examined the anti-inflammatory effect of a sweetfish-derived protein and its hydrolysates on lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells. Hydrolysates of the sweetfish-derived protein were obtained on enzymatic hydrolysis by pepsin, trypsin, and alpha-chymotrypsin. The anti-inflammatory activity was determined based on the production of nitric oxide (NO), inflammatory cytokine [tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6)] and prostaglandin E-2 (PGE(2)), mRNA expression levels of inflammation mediated proteins, and the inhibition of nuclear factor (NF)-kappa B. The fish protein and its enzymatic hydrolysates were not found to exert a cytotoxic effect on RAW264.7 macrophage cells; they inhibited the production of NO and cytokines in LPS-induced RAW264.7 cells. In particular, hydrolysates prepared by trypsin and alpha-chymotrypsin remarkably decreased the production of NO, cytokines, and PGE(2). Reverse transcription-polymerase chain reaction analysis demonstrated that the decrease in NO and cytokine production was related to the inhibition of the mRNA expression of inducible nitric oxide synthase and cytokine (TNF-alpha, IL-6, and IL-1 beta) genes. Moreover, a reporter gene assay showed that the hydrolysates inhibited NF-kappa B nuclear translocation, and this inhibition was re-confirmed through inhibition of the mitogen-activated protein kinase pathway. The results suggested that the sweetfish-derived protein hydrolysates obtained using trypsin and alpha-chymotrypsin can be used as anti-inflammatory agents.