Snake natriuretic peptide (NP) Lebetin 2 (L2) has been shown to improve cardiac function and reduce fibrosis as well as inflammation by promoting M2-type macrophages in a reperfused myocardial infarction (MI) model. However, the inflammatory mechanism of L2 remains unclear. Therefore, we investigated the effect of L2 on macrophage polarization in lipopolysaccharide (LPS)-activated RAW264.7 cells in vitro and explored the associated underlying mechanisms. TNF-α, IL-6 and IL-10 levels were assessed using an ELISA assay, and M2 macrophage polarization was determined by flow cytometry. L2 was used at non-cytotoxic concentrations determined by a preliminary MTT cell viability assay, and compared to B-type natriuretic peptide (BNP). In LPS-activated cells, both peptides reduced TNF-α and IL-6 release compared to controls. However, only L2 increased IL-10 release in a sustained manner and promoted downstream M2 macrophage polarization. Pretreatment of LPS-activated RAW264.7 cells with the selective NP receptor (NPR) antagonist isatin abolished both IL-10 and M2-like macrophage potentiation provided by L2. In addition, cell pretreatment with the IL-10 inhibitor suppressed L2-induced M2 macrophage polarization. We conclude that L2 exerts an anti-inflammatory response to LPS by regulating the release of inflammatory cytokines via stimulating of NP receptors and promoting M2 macrophage polarization through activation of IL-10 signaling.

Ulcerative colitis is characterized by inflammation, oxidative stress, and excessive free radical production. This study investigated the antioxidant, anti-inflammatory, and protective effects of N-acetylcysteine (NAC), Tiron, and their fixed-ratio combination in acetic acid-induced colitis. An integrated approach was used, combining ligand–ligand docking, acellular antioxidant and protein-denaturation assays, and an in vivo model in male Wistar rats. Colitis was induced by intrarectal administration of 3% acetic acid after 14 days of intraperitoneal pretreatment with NAC, Tiron, or NAC–Tiron. Docking analysis suggested physicochemical compatibility through non-covalent interactions. In vitro, NAC, Tiron, and their combination showed antioxidant and anti-denaturation activities. NAC–Tiron displayed greater activity than the individual compounds in selected endpoints, particularly 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, but did not consistently outperform them across the other assays. In vivo, NAC, Tiron, and NAC–Tiron attenuated macroscopic and histological colonic damage, reduced inflammatory cell infiltration and edema, decreased lipid peroxidation and protein carbonylation, and helped preserve superoxide dismutase (SOD) activity, reduced glutathione (GSH), and total thiols. The treatments also attenuated increases in C-reactive protein (CRP) and free iron without evident worsening of the measured systemic biochemical parameters. Overall, these findings provide an exploratory proof of concept for fixed-ratio NAC–Tiron co-administration but do not establish pharmacological synergy or superiority over standard therapies.