Flaxseed orbitides, linusorbs, inhibit LPS-induced THP-1 macrophage inflammation

Abstract

Linusorbs (flaxseed orbitides) are a family of naturally-occurring cyclic peptides. Previously, we reported that their anticancer effects were associated with their structures. In this study, we investigated the anti-inflammatory activities of 2 different linusorbs ([1-9-NαC]-linusorb B2 and [1-9-NαC]-linusorb B3) in lipopolysaccharide (LPS)-induced THP-1 macrophage activation as well as the underlying mechanism of this inflammatory response. Both molecules suppressed pro-inflammatory mediators (TNF-α, IL-1β, IL-6, NO and COX-2) and were involved in downregulating the NF-κB signaling pathway. The suppressive effects on pro-inflammatory mediators were comparable and the concentration range of action was similar (1-4 μM). However, the concentration of compound that induced downregulation of the NF-κB pathway was different for each compound. While [1-9-NαC]-linusorb B3 could inhibit the activation of the NF-κB pathway at concentrations of 1 and 2 μM, [1-9-NαC]-linusorb B2 induced a comparable inhibitory effect at a concentration of 4 μM.

Fig. 1. Chemical structures of LOB2 and LOB3.

Fig. 2. Effects of LOB3 (A) and LOB2 (B) on the viability of THP-1 macrophages determined by MTT assay. THP-1 macrophages were pretreated with various concentrations (0, 0.5, 5, 10, 20, 50, 100 and 200 μM) of LOB3 or LOB2 for 1 h and then the cells were stimulated with LPS (1 μg mL⁻¹) for 3 h. Significant differences compared with control group are indicated as *p < 0.05.

Fig. 3. LOB3 and LOB2 prevented the NO production of LPS-induced THP-1 macrophage inflammation model. THP-1 macrophages were pretreated with various concentrations (0, 0.5, 1, 2, 4 and 8 μM) of LOB3 or LOB2 for 1 h and then the cells were stimulated with LPS (1 μg mL⁻¹) for 3 h. After treatment, NO production was detected by the Griess reagent. Significant differences among LPS treatments are indicated as *p < 0.05, **p < 0.01. ^##p < 0.01 versus the control group.

Fig. 4. LOB3 and LOB2 inhibited the LPS-stimulated release of TNF-α (A), IL-1β (B) and IL-6 (C) in THP-1 macrophages. THP-1 macrophages were pretreated with different concentrations (0, 0.5, 1, 2, 4 and 8 μM) of LOB3 or LOB2 for 1 h and then the cells were stimulated with LPS (1 μg mL⁻¹) for 3 h. After treatment, the production of cytokines was evaluated by corresponding human ELISA kits. Significant differences among LPS treatments are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ^###p < 0.001 versus the control group.

Fig. 5. LOB3 and LOB2 suppressed the protein expression of COX-2 in the LPS-stimulated THP-1 cells analyzed by western blot analysis. THP-1 macrophages were pretreated with different concentrations (0, 1, 2 and 4 μM) of LOB3 or LOB2 for 1 h and then the cells were stimulated with LPS (1 μg mL⁻¹) for 3 h. COX-2 protein level was corrected to β-actin and was expressed relative to the LPS group (set as 1). ^###p < 0.001 versus the control group. Significant differences compared with LPS group are indicated as **p < 0.01, ***p < 0.001.

Fig. 6. LOB3 and LOB2 inhibited the phosphorylation of IKKα/β (A), IκBα (B) and p65-NF-κB (C) in the LPS-stimulated THP-1 cells evaluated by western blot analysis. THP-1 macrophages were pretreated with different concentrations (0, 1, 2 and 4 μM) of LOB3 or LOB2 for 1 h and then the cells were stimulated with LPS (1 μg mL⁻¹) for 3 h. Protein levels of p-IKKα/β, p-IκBα and p-p65-NF-κB were corrected to β-actin and expressed relative to the LPS group (set as 1). ^###p < 0.001 versus the control group. Significant differences compared with LPS group are indicated as *p < 0.05, **p < 0.01, ***p < 0.001.