6-7-Dimethoxy-4-methylcoumarin suppresses pro-inflammatory mediator expression through inactivation of the NF-κB and MAPK pathways in LPS-induced RAW 264.7 cells

Abstract

In this study, we investigated the ability of 6,7-dimethoxy-4-methylcoumarin (DMC) to inhibit lipopolysaccharide (LPS)-induced expression of pro-inflammatory mediators in mouse macrophage (RAW 264.7) cells, and the molecular mechanism through which this inhibition occurred. Our results indicated that DMC downregulated LPS-induced nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, thereby reducing the production of NO and prostaglandin E2 (PGE2) in LPS-activated RAW 264.7 cells. Furthermore, DMC suppressed LPS-induced production of pro-inflammatory cytokines such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α. To elucidate the mechanism underlying the anti-inflammatory activity of DMC, we assessed its effects on the mitogen-activated protein kinase (MAPK) pathway and the activity and expression of nuclear transcription factor kappa-B (NF-κB). The experiments demonstrated that DMC inhibited LPS-induced phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38. In addition, it attenuated LPS-induced NF-κB activation via the inhibition of IκB-α phosphorylation. Taken together, these data suggest that DMC exerts its anti-inflammatory effects in RAW 264.7 cells through the inhibition of LPS-stimulated NF-κB and MAPK signaling, thereby downregulating the expression of pro-inflammatory mediators.

Keywords: 6,7-Dimethoxy-4-methylcoumarin (DMC); MAPKs; NF-kappaB; RAW 264.7 cells; anti-inflammatory.

Figure 1. Effect of DMC on RAW 264.7 cell viability. (A) Chemical structure of DMC. (B) RAW 264.7 cells (1.5 × 10⁵ cells/ml) plated on 96-well plates were treated with DMC at 37 °C for 24 h. Cytotoxicity of DMC was assessed by MTT assay. Values are expressed as means ± S.D. of triplicate experiments. *P < 0.05 for the comparison with the LPS-stimulated group

Figure 2. Effect of DMC on LPS-induced expression of iNOS and COX-2 and production of NO and PGE₂ in RAW 264.7 cells. Cells were stimulated with LPS (1 µg/ml) in the presence of DMC (100, 200, and 400 µM) for 24 h at 37 °C. (A) Culture media were collected in order to measure (A) NO and (B) PGE₂ production by the Griess reaction and ELISA, respectively. Values are expressed as means ± S.D. of triplicate experiments. *P < 0.05 for the comparison with the LPS-stimulated group. (C) Cells were stimulated with LPS (1 µg/ml) in the presence of DMC (100, 200, and 400 µM) for 24 h at 37 °C. The levels of iNOS and COX-2 proteins in cell lysates were analyzed by western blot.

Figure 3. Inhibitory effect of DMC on pro-inflammatory cytokine production in RAW 264.7 cells. The production of (A) TNF-α, (B) IL-6, and (C) IL-1ß were assayed in the culture medium of cells stimulated with LPS (1 µg/ml) for 24 h in the presence of DMC (100, 200, and 400 µM). The concentrations of TNF-α, IL-6, and IL-1ß in the supernatants were determined by ELISA. Values are expressed as means ± S.D. of triplicate experiments. *P < 0.05.

Figure 4. Inhibitory effects of DMC on LPS-induced phosphorylation of IκB-α and NF-κB p50. Cells were treated for different times (10, 20, and 30 min) with LPS (1 µg/ml), alone or with 400 µM DMC, as indicated. The levels of p-IκB-α (phosphorylated-IκB-α), IκB-α, p-p50 (phosphorylated p50) and p-p65 (phosphorylated p65) were determined by western blotting.

Figure 5. Inhibitory effects of DMC on ERK, JNK, and p38 protein levels in RAW 264.7 cells. Cells were treated for different times (10, 20, and 30 min) with LPS (1 µg/ml), alone or with 400 µM DMC, as indicated. The levels of p-ERK (phosphorylated-ERK), ERK, p-JNK (phosphorylated-JNK), JNK, p-p38 (phosphorylated-p-38) and p-38 were determined by western blotting.