Various proteases were employed to hydrolyze walnut protein, and anti-inflammatory effects of the hydrolysates were evaluated in RAW264.7 cells exposed to lipopolysaccharide. The bromelain hydrolysate, which was further separated using ultrafiltration and RP-HPLC, presented the highest anti-inflammatory effect. A total of 100 peptides were detected using UPLC-Q-Orbitrap-MS2, and novel peptide VLRWPR (VR6) was screened. A DSS-induced colitis model in mice was utilized to assess the anti-inflammatory activity of VR6 in vivo. VR6 markedly reduced the disease activity index score by 66%, reversed the colon-length shortening by 28%, regulated the serum inflammatory index, and alleviated histopathological damage within the colon. VR6 suppressed inflammation by modulating the TLR4-NF-κB axis. Concurrently, VR6 maintained intestinal barrier function by elevating zonula occludens-1, occludin, and claudin-1 levels by 3.43, 1.45, and 2.66 times, respectively. Furthermore, 16S rRNA sequencing analysis indicated that VR6 could reverse the gut microbiota imbalance and improve the production of short-chain fatty acids.
Keywords: NO inhibitory activity; anti-inflammatory activity; gut microbiota; peptide; walnut.