DA-9601, a standardized extract of Artemisia asiatica, blocks TNF-alpha-induced IL-8 and CCL20 production by inhibiting p38 kinase and NF-kappaB pathways in human gastric epithelial cells

Abstract

Aim: To investigate whether, or how, DA-9601, which is a new gastroprotective agent, inhibits TNF-alpha-induced inflammatory signals in gastric epithelial AGS cells.

Methods: Cell viability was determined by MTT assay. IL-8 and CCL20 promoter activities were determined by a luciferase reporter gene assay. NF-kappaB-dependent transcriptional activity was determined by I-kappaB alpha degradation, NF-kappaB p65 nuclear translocation and a luciferase activity assay. IL-8 and CCL20 gene expression and protein secretion were determined by RT-PCR and an enzyme-linked immunosorbent assay (ELISA). Total and phosphorylated forms of mitogen-activated protein kinases (MAPKs) were determined by Western blot.

Results: Treatment of AGS cells with DA-9601 reduced TNF-alpha-induced IL-8 and CCL20 promoter activities, as well as their gene expression and protein release. TNF-alpha also induced NF-kappaB-dependent transcriptional activity in AGS cells. In contrast, in cells treated with DA-9601, TNF-alpha-induced NF-kappaB activity was significantly blocked. Although all three MAP kinase family members were phosphorylated in response to TNF-alpha, a selective inhibitor of p38 kinase SB203580 only could inhibit both NF-kappaB-dependent transcriptional activity and IL-8 and CCL20 production, suggesting a potential link between p38 kinase and NF-kappaB-dependent pathways in AGS cells. Interestingly, DA-9601 also selectively inhibited p38 kinase phosphorylation induced by TNF-alpha.

Conclusion: DA-9601 blocked TNF-alpha-mediated inflammatory signals by potentially modulating the p38 kinase pathway and/or a signal leading to NF-kappaB-dependent pathways in gastric epithelial cells.

Figure 1

TNF-α induces IL-8 and CCL20 secretion and mRNA accumulation in AGS cells in a time- and dose-dependent manner. (A and C) AGS cells (5 x 10⁵/well) were treated for 16 h with the indicated concentrations of TNF-α (0-10 ng/mL). After incubation, the supernatants were collected, and the levels of IL-8 (A) and CCL20 (C) were determined by ELISA (top). At the same time, the cells were colleted and the expression of two chemokines was determined by RT-PCR (bottom). (B and D) AGS cells (5 x 10⁵/well) were treated with TNF-α (5 ng/mL) for the indicated time points (0-24 h). IL-8 and CCL20 secretion and mRNA accumulation were determined as described above. For ELISA, results are expressed as means ± SD of three independent experiments.

Figure 2

DA-9601 inhibits the expression and secretion of CCL20 in AGS cells. (A) AGS cells (1 x 10⁵) were treated with various concentrations of DA-9601 (0-100 μg/mL) for 16 h. Quantitative analysis of cell viability was determined by the MTT assay (mean ± SD., n = 3). (B) Cells (5 x 10⁵) were pretreated with various concentrations of DA-9601 (0-100 μg/mL) for 1 h, and then the cells were further incubated for 8 h with TNF-α (5 ng/mL). Levels of IL-8 and CCL20 mRNAs were determined by RT-PCR. (C) Cells (5 x 10⁵) were pretreated with various concentrations of DA-9601 (0-100 μg/mL) for 1 h, and then the cells were further incubated for 16 h with TNF-α (5 ng/mL). IL-8 and CCL20 protein levels were determined by ELISA. These data are representative of three independent experiments.

Figure 3

DA-9601 blocks IL-8 and CCL20 promoter activities in HEK293T and AGS cells. HEK293T (left) and AGS (right) cells were transfected with pGL3-pIL-8 (A) or pGL3-pCCL20 (B) luciferase vectors. After 24 h of incubation, cells (1 x 10⁵) were pre-treated for 1 h with DA-9601 (50 μg/mL) and stimulated for additional 16 h with medium alone or medium containing TNF-α (5 ng/mL). At the end of incubation, cells were lysed, and the relative luciferase activity was measured using Luciferase Assay System. Results are expressed as means ± SD of three independent experiments.

Figure 4

DA-9601 blocks NF-κB activity in AGS cells. A: AGS cells (1 x 10⁵) were transfected with NF-κB luciferase reporter vector (0.8 μg/well). After 24 h of incubation, cells (1 x 10⁵) were pre-treated for 1 h with DA-9601 (50 μg/mL) and stimulated for additional 16 h with medium alone or medium containing TNF-α (5 ng/mL). At the end of incubation, cells were lysed, and the relative luciferase activity was measured using Luciferase Assay System; B: AGS cells (1 x 10⁵) were transfected with p65-EGFP vector (0.8 μg/well). After 24 h of incubation, cells were pre-treated for 1 h with DA-9601 (50 μg/mL) and stimulated for 1 h with medium alone or TNF-α. Nuclear translocation of p65-EGFP was observed under the fluorescence microscope (original magnification, 200 X); C: AGS cells (5 x 10⁵) were incubated with TNF-α (5 ng/mL) for the indicated time points (0-240 min) (top) or were pretreated with medium alone or with DA-9601 (50 μg/mL) for 1 h, and incubated with TNF-α for 30 min (bottom). The cell lysates were blotted with antibodies specific for the I-κBα and β-actin.

Figure 5

TNF-α induces phosphorylation of MAPKs in AGS cells. AGS cells (5 x 10⁵ cells/well) were incubated for various times (0-240 min) with TNF-α (5 ng/mL). Protein extracts were prepared at the indicated time points, and then the levels of phosphorylated or total MAPKs (ERK-1/2 (top), p38 kinase (middle), and JNK/SAPK (bottom)) were determined by Western blotting using specific antibodies. The arrows indicate the position of specific immunoreactive bands corresponding to distinct MAPKs.

Figure 6

SB203580 blocks NF-κB-dependent transcriptional activity in AGS cells. AGS cells (5 x 10⁵) were transfected with NF-κB luciferase reporter vector (0.8 μg/well). After 24 h of incubation, cells were pre-treated for 1 h with PD098059 (20 μmol/L), SB203580 (10 μmol/L), SP600125 (2 μmol/L), and PDTC (10 μmol/L); the cells were stimulated for additional 16 h with medium alone or medium containing TNF-α (5 ng/mL). At the end of incubation, cells were lysed, and the relative luciferase activity was measured using Luciferase Assay System. Note, ^aP < 0.05, significantly different from control (n = 4).

Figure 7

Effects of MAPK modulators on IL-8 and CCL20 release by TNF-α in AGS cells. AGS cells (1 x 10⁵) were pre-treated for 1 h with or without selective MAPK inhibitors (PD098059, 20 μmol/L; SB203580, 10 μmol/L; SP600125, 2 μmol/L). The cells were then further incubated for 16 h with TNF-α (5 ng/mL). Levels of IL-8 and CCL20 protein were determined by ELISA. These data are representative of three independent experiments.

Figure 8

DA-9601 selectively attenuates TNF-α-mediated phosphorylation of p38 kinase in AGS cells. Cells (5 x 10⁵ cells/well) were pretreated for 1 h with medium alone or medium containing DA-9601 (50 μg/mL). Then, the cells were stimulated for 15 min with or without TNF-α (5 ng/mL). The cell lysates were blotted with antibodies specific for the phosphorylated or total forms of ERK1/2, p38 kinase, and JNK/SAPK, and visualized using a peroxidase-conjugated secondary antibody and the ECL system.

Figure 9

Hypothetical mechanism of action of DA-9601 on TNF-α-induced CCL20 expression in AGS cells. TNF-α induces activation of three MAPKs. Among three MAPKs, however, activation of p38 kinase involves in NF-κB signaling system. DA-9601 may inhibit NF-κB directly or indirectly through the inhibition of p38 kinase pathway. See text for discussion.