Abstract: This study evaluated the anti-inflammatory effect of a fibrinolytic enzyme from Lyophyllum ulmarium (LUFE) on lipopolysaccharide-induced J774A.1 macrophages. The cells were divided into four groups: normal control, LPS, LPS + low-dose LUFE (LUFE-L), and LPS + high-dose (LUFE-H). The LPS + LUFE-L and LPS + LUFE-H groups were first incubated with 10 and 20 μg/mL of LUFE for 24 h, respectively, and then all groups except the normal control group were treated with 1 μg/mL of LPS for 24 h. Enzyme-linked immunosorbent assay (ELISA) was conducted to determine interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and monocyte chemotactic protein-1 (MCP-1) levels in cell culture supernatants; the Transwell assay was used to observe cell chemotaxis ability; cell adhesion function was detected by the cell-extracellular matrix assay and cell phagocytosis by neutral red phagocytosis assay; immunofluorescence staining was performed to observe the level of cellular reactive oxygen species (ROS); colorimetric assays were used to detect cellular malondialdehyde (MDA) and catalase (CAT) levels and superoxide dismutase (SOD) activity. Western blotting was performed to determine the expression of Toll-like receptor 4 (TLR4) and nuclear factor erythroid 2-related factor (Nrf2) pathway-related proteins. The results showed that LUFE intervention reduced the levels of IL-6, IL-1β, MCP-1, ROS, and MDA, inhibited LPS-induced macrophage chemotaxis, adhesion, and phagocytosis, and elevated cellular SOD and CAT levels. Meanwhile, it down-regulated the expression and activation of TLR4, myeloid differentiation primary response protein 88 (MyD88), Kelch-like ECH-associated protein 1 (Keap-1) and nuclear factor kappa-B (NF-κB) proteins and up-regulated the levels of Nrf2, heme oxygenase 1 (HO-1) and NAD(P)H-quinone oxidoreductase 1 (NQO1). In summary, LUFE could inhibit LPS-induced inflammatory responses in macrophages by modulating the Nrf2 and TLR4/MyD88 signaling pathways.

Key words: Lyophyllum ulmarium; thrombolytic enzyme; inflammation; oxidative stress; macrophage