Anti-Inflammatory Activity of Populus deltoides Leaf Extract via Modulating NF-κB and p38/JNK Pathways

Abstract

Populus deltoides, known as eastern cottonwood, has been commonly used as a medicinal plant. The aim of the present study was to investigate the mechanism underlying the anti-inflammatory activity of P. deltoides leaf extract (PLE). PLE effectively inhibited the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, but not that of cyclooxygenase-2 (COX-2) and prostaglandin E2. Proinflammatory tumor necrosis factor alpha (TNF-α) levels were also reduced by the extract. PLE inhibited the phosphorylation of nuclear factor-kappa B (NF-κB) and inhibitor of Kappa Bα (IκBα), and blunted LPS-triggered enhanced nuclear translocation of NF-κB p65. In mitogen-activated protein kinase (MAPK) signaling, PLE effectively decreased the phosphorylation of p38 and c-Jun N-terminal protein kinase (JNK), but not of extracellular signal-regulated kinase 1/2 (ERK1/2). Taken together, these results suggest that anti-inflammatory activity of P. deltoides leaf extract might be driven by iNOS and NO inhibition mediated by modulation of the NF-κB and p38/JNK signaling pathways.

Keywords: MAPK; NF-κB; Populus deltoides leaf; anti-inflammation; iNOS.

Figure 1

Effects of P. deltoides leaf extract on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW 264.7 cells. The cells were treated with P. deltoides leaf extract (12.5 and 25 μg/mL) in the presence of 1 μg/mL LPS for 24 h. RAW 264.7 cells were treated with 10 μM of iNOS inhibitor 1400 W simultaneously with LPS treatment. Error bars represent the mean ± SD. *** p < 0.001 compared to LPS alone.

Figure 2

Effect of P. deltoides leaf extract on the production of nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) in LPS-stimulated RAW 264.7 cells. P. deltoides leaf extract (12.5 or 25 μg/mL) suppressed (A) NO production and (B) TNF-α expression in RAW 264.7 cells stimulated by 1 μg/mL LPS. The expressions of TNF-α were also down regulated by treatment of iNOS inhibitor 1400 W (10 μM). Error bars represent the mean ± SD. * p < 0.05 and *** p < 0.001 compared to LPS alone.

Figure 3

Effects of P. deltoides leaf extract on the LPS-induced activation of the nuclear factor-kappa (NF-κB) pathway in RAW 264.7 cells. The cells were treated with P. deltoides leaf extract (12.5 or 25 μg/mL) in the presence of 1 μg/mL LPS for 24 h. Error bars represent the mean ± SD. Values of * p < 0.05 and *** p < 0.001 compared to LPS alone were considered statistically significant.

Figure 4

The inhibitory effect of P. deltoides leaf extract on NF-κB p65 translocation in LPS-stimulated RAW 264.7 cells. Representative photomicrographs of immune-labelled NF-κB (red) and nuclear counterstaining with DAPI (blue) in LPS-stimulated RAW 264.7 cells treated without and with 12.5 or 25 μg/mL of PLE. LPS increased colocalization of NF-κB and DAPI in RAW 264.7 cells. However, PLE reduced the nuclear translocation and accumulation of NF-κB, which was induced by LPS. Scale bar = 10 μm.

Figure 5

Effects of PLE on the phosphorylation of mitogen-activated protein kinase (MAPK) cascade (p-ERK1/2, p-JNK, and p-p38) in LPS-stimulated RAW 264.7 cells. The cells were treated with P. deltoides leaf extract (12.5 or 25 μg/mL) in the presence of 1 μg/mL LPS. Error bars represent the mean ± SD. Values of ** p < 0.01, and *** p < 0.001 compared to LPS alone.

Figure 6

Schematic diagram of the suppressive effects of P. deltoides leaf extract on the inflammatory response via modulating NF-κB and p38/JNK pathway in LPS-stimulated RAW 264.7 cells.