Abstract:

The study aimed to explore the anti-inflammatory peptides in Clinacanthus nutans (Burm. f.) Lindau and elucidate their potential molecular mechanisms. Proteins were extracted using alkaline extraction and isoelectric precipitation followed by ultrafiltration techniques. After screening out two fractions with anti-inflammatory activities, a total of 116 polypeptide sequences were identified by using liquid chromatography-mass spectrometry. Subsequent screening using an activity scoring platform and polypeptide toxicity prediction identified 25 non-toxic polypeptide sequences with scores >0.8. Molecular docking with the mitogen-activated protein kinase (MAPK) target identified three polypeptide sequences with superior efficacy compared to the MAPK inhibitor cetirizine. These sequences were synthesized using solid-phase synthesis, and their inhibitory effects on lipopolysaccharide- induced inflammation in RAW 264.7 cells were evaluated. Ultimately, a polypeptide sequence, WDDC, demonstrating significant anti-inflammatory potential, was identified. To minimize redundancy with human proteins and enhance stability, modifications involving glycine and acetyl moieties were implemented, resulting in the designation of the modified polypeptide as Ac-WDDCG. The findings indicated that Ac-WDDCG significantly inhibited the mRNA expression of genes associated with the Nod-like receptor family (NLRP3) signaling pathway, including toll-like receptor 4 (TLR4),NLRP3,and nuclear factor-kappa B(NF-κB) in human umbilical vein endothelial cells (HUVEC cells) (P <0.05).Moreover,it markedly reduced the secretion levels of downstream inflammatory mediators,including tumor necrosis factor-alpha (TNF-α),interleukin-6 (IL-6) and NO,in the LPS-induced the RAW 264.7 cell model (P <0.01),and inhibited the migration of neutrophils in zebrafish induced by CuSO₄ (P <0.05).These results suggest that Ac-WDDCG exhibits potent anti-inflammatory activity,potentially through the inhibition of the NLRP3 signaling pathway.

Key words: Clinacanthus nutans (Burm.f.) Lindau, peptides, mitogen-activated protein kinase, Nod-like receptor family, inflammatory cytokines