Studies On Immunoactivities Of Trypsinized Rice Peptides In RAW264.7 Macrophages

Inflammation is a central feature of the host defence against pathogenic mediators such as damaged cells or invading microbes,and it plays a fundamental role in immune modulation and restoration of tissue homeostasis.As essential components of the innate immune response,macrophages are primary immune cells that play a pivotal role in anti-inflammation and host defences against external stimuli such as invading pathogens and bacterial infections.Rice peptides following hydrolysis by proteolytic enzymes have many biological activities,especially for the antioxidant and anti-hypertension properties,but the anti-inflammatory and immune characteristics have not been fully explored.In the previous study,we isolated trypsin-derived hydrolysates from rice protein(RPHs)as a series of multidimensional peptide fractions prepared by different purification processes that promoted proliferation and activity of macrophages.In this study,we decided to measure nitric oxide(NO)concentration to compare inhibition among the different RPHs fractions.We investigated the inhibitory effects of rice peptides on the inflammatory response in LPS-stimulated RAW264.7 macrophages,and probed their underlying molecular mechanisms of action.The LPS-induced RAW264.7 macrophages were used to test and compare the inhibition effects of rice peptides on NO production.The results showed that the NO inhibition rates of RPHs,RPHs-C,RPHs-C-7 and RPHs-C-7-3 were 27.43%,29.51%,27.56% and 61.38%,respectively.Therefore,the fraction RPHs-C-7-3 displayed significant NO suppression activity,and this fraction was chosen for further experiments.Additionally,the study demonstrated the effects of RPHs-C-7-3 on pro-inflammatory mediators expression in LPS-induced RAW264.7 macrophages.The enzyme linked immunosorbent assay(ELISA)and western blot showed that the expression of tumor necrosis factor-?(TNF-?)and inducible nitric oxide synthase(iNOS)was significantly suppressed by RPHs-C-7-3 in the protein level.The reverse transcription PCR assay showed that the transcription of TNF-?,iNOS,interleukin-6(IL-6),and interleukin-1?(IL-1?)were decreased in a dose-dependent manner,indicating the inhibitory effects of RPHs-C-7-3 on pro-inflammatory mediators expression in the mRNA level.Phagocytosis,the main function of macrophages,is critical for the inflammation and immune responses against invading pathogens.The effects of RPHs-C-7-3 on the phagocytosis were examined based on the abilities of cells to internalize fluorescent red latex beads.The flow cytometry showed that stimulation of macrophages with LPS led to a substantial increase in phagocytic rate from 14.3% to 56.87%,but decreased to 36.92%,38.35%,38.90% when pre-treated with 62.50–250.00 ?g/mL RPHs-C-7-3,respectively.These results indicated that RPHs-C-7-3 markedly decreased phagocytosis although no dose-dependence was found.Finally,we investigated the effects of RPHs-C-7-3 on NF-?B and MAPK signaling pathways in LPS-induced RAW264.7 macrophages in the present study.The results showed that LPS facilitated a massive accumulation of p65 in the nucleus,and RPHs-C-7-3 impeded this process in a dose-dependent manner.The phosphorylation of MAPK was found sharply increased after stimulated by LPS,however the p-ERK was sinificantly suppressed by 250.00 ?g/mL RPHs-C-7-3.In summary,our findings demonstrated that RPHs-C-7-3 extracted from rice protein exhibited the highest anti-inflammatory activity by inhibiting the NO production in LPS-induced RAW264.7 macrophages.RPHsC-7-3 inhibited the expression of LPS-induced pro-inflammatory mediators in both protein and mRNA levels,and RPHs-C-7-3 also repressed the phagocytic abilities of the activated macrophages.The anti-inflammatory activity of RPHs-C-7-3 was associated with down-regulation of the NF-?B and MAPK pathway by declining the LPS-induced nuclear translocation of p65 and phosphorylation of ERK.We supported that RPHs-C-7-3 purified fraction is useful anti-inflammatory peptide mixture,and this may assist the development of future anti-inflammatory treatments.