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A novel chromone derivative with anti-inflammatory property via inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway.

Liu H, Xu R, Feng L, Guo W, Cao N, Qian C, Teng P, Wang L, Wu X, Sun Y, Li J, Shen Y, Xu Q - PLoS ONE (2012)

Bottom Line: In the present study, we show that a novel chromone derivative, DCO-6, significantly reduced lipopolysaccharide (LPS)-induced production of nitric oxide, IL-1β and IL-6, decreased the levels of iNOS, IL-1β and IL-6 mRNA expression in both RAW264.7 cells and mouse primary peritoneal macrophages, and inhibited LPS-induced activation of p38 MAPK but not of JNK, ERK.LPS-induced production of intracellular reactive oxygen species (ROS) was remarkably impaired by DCO-6, which disrupted the formation of the TRAF6-ASK1 complex.Our results indicate that DCO-6 showed anti-inflammatory properties through inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China.

ABSTRACT
The p38 MAPK signaling pathway plays a pivotal role in inflammation. Targeting p38 MAPK may be a potential strategy for the treatment of inflammatory diseases. In the present study, we show that a novel chromone derivative, DCO-6, significantly reduced lipopolysaccharide (LPS)-induced production of nitric oxide, IL-1β and IL-6, decreased the levels of iNOS, IL-1β and IL-6 mRNA expression in both RAW264.7 cells and mouse primary peritoneal macrophages, and inhibited LPS-induced activation of p38 MAPK but not of JNK, ERK. Moreover, DCO-6 specifically inhibited TLR4-dependent p38 activation without directly inhibiting its kinase activity. LPS-induced production of intracellular reactive oxygen species (ROS) was remarkably impaired by DCO-6, which disrupted the formation of the TRAF6-ASK1 complex. Administering DCO-6 significantly protected mice from LPS-induced septic shock in parallel with the inhibition of p38 activation and ROS production. Our results indicate that DCO-6 showed anti-inflammatory properties through inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway. Blockade of the upstream events required for p38 MAPK action by DCO-6 may provide a new therapeutic option in the treatment of inflammatory diseases.

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Effects of DCO-6 on the production of intracellular ROS and the formation of TRAF6-ASK1 complex in RAW264.7 cells.(A) Cells were incubated in the absence or presence of indicated TLR ligands for 6 h. Intracellular ROS production was detected by DCF fluorescence using flow cytometry. (B) Cells were treated with various concentrations of DCO-6 in the absence or presence of LPS. Intracellular ROS production was detected as mentioned above. Data are shown as means ± S.D. of three independent experiments. *P<0.05 vs LPS control. (C) Cells were treated with various concentrations of DCO-6 in the absence or presence of H2O2 for 6 h. Whole cell lysates were prepared for Western blotting analysis. The protein levels of total and phosphorylated p38 were determined at least three times, and representative data are shown. (D) The interaction between TRAF6 and ASK1 was measured by coimmunoprecipitation assay. Representative data are shown.
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pone-0037168-g005: Effects of DCO-6 on the production of intracellular ROS and the formation of TRAF6-ASK1 complex in RAW264.7 cells.(A) Cells were incubated in the absence or presence of indicated TLR ligands for 6 h. Intracellular ROS production was detected by DCF fluorescence using flow cytometry. (B) Cells were treated with various concentrations of DCO-6 in the absence or presence of LPS. Intracellular ROS production was detected as mentioned above. Data are shown as means ± S.D. of three independent experiments. *P<0.05 vs LPS control. (C) Cells were treated with various concentrations of DCO-6 in the absence or presence of H2O2 for 6 h. Whole cell lysates were prepared for Western blotting analysis. The protein levels of total and phosphorylated p38 were determined at least three times, and representative data are shown. (D) The interaction between TRAF6 and ASK1 was measured by coimmunoprecipitation assay. Representative data are shown.

Mentions: Given that the generation of ROS is required for TLR4-dependent activation of p38 but not JNK [18], we examined whether DCO-6 inhibited LPS-induced phosphorylation of p38 by reducing ROS production. After treatment for 6 h, LPS, but not poly (I:C) or CpG, resulted in a large amount of ROS production in RAW264.7 cells (Fig. 5A). DCO-6 inhibited LPS-induced ROS production in a concentration-dependent manner (Fig. 5B) and 30 µM of DCO-6 showed the inhibitory activity comparable to 1 mM of the antioxidant N-acetyl-L-cystein (NAC). In addition, 1 mM of H2O2 in RAW264.7 cells induced p38 phosphorylation and Co-incubation of DCO-6 with H2O2 inhibited the phosphorylation of p38 in a concentration-dependent manner (Fig. 5C).


A novel chromone derivative with anti-inflammatory property via inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway.

Liu H, Xu R, Feng L, Guo W, Cao N, Qian C, Teng P, Wang L, Wu X, Sun Y, Li J, Shen Y, Xu Q - PLoS ONE (2012)

Effects of DCO-6 on the production of intracellular ROS and the formation of TRAF6-ASK1 complex in RAW264.7 cells.(A) Cells were incubated in the absence or presence of indicated TLR ligands for 6 h. Intracellular ROS production was detected by DCF fluorescence using flow cytometry. (B) Cells were treated with various concentrations of DCO-6 in the absence or presence of LPS. Intracellular ROS production was detected as mentioned above. Data are shown as means ± S.D. of three independent experiments. *P<0.05 vs LPS control. (C) Cells were treated with various concentrations of DCO-6 in the absence or presence of H2O2 for 6 h. Whole cell lysates were prepared for Western blotting analysis. The protein levels of total and phosphorylated p38 were determined at least three times, and representative data are shown. (D) The interaction between TRAF6 and ASK1 was measured by coimmunoprecipitation assay. Representative data are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376149&req=5

pone-0037168-g005: Effects of DCO-6 on the production of intracellular ROS and the formation of TRAF6-ASK1 complex in RAW264.7 cells.(A) Cells were incubated in the absence or presence of indicated TLR ligands for 6 h. Intracellular ROS production was detected by DCF fluorescence using flow cytometry. (B) Cells were treated with various concentrations of DCO-6 in the absence or presence of LPS. Intracellular ROS production was detected as mentioned above. Data are shown as means ± S.D. of three independent experiments. *P<0.05 vs LPS control. (C) Cells were treated with various concentrations of DCO-6 in the absence or presence of H2O2 for 6 h. Whole cell lysates were prepared for Western blotting analysis. The protein levels of total and phosphorylated p38 were determined at least three times, and representative data are shown. (D) The interaction between TRAF6 and ASK1 was measured by coimmunoprecipitation assay. Representative data are shown.
Mentions: Given that the generation of ROS is required for TLR4-dependent activation of p38 but not JNK [18], we examined whether DCO-6 inhibited LPS-induced phosphorylation of p38 by reducing ROS production. After treatment for 6 h, LPS, but not poly (I:C) or CpG, resulted in a large amount of ROS production in RAW264.7 cells (Fig. 5A). DCO-6 inhibited LPS-induced ROS production in a concentration-dependent manner (Fig. 5B) and 30 µM of DCO-6 showed the inhibitory activity comparable to 1 mM of the antioxidant N-acetyl-L-cystein (NAC). In addition, 1 mM of H2O2 in RAW264.7 cells induced p38 phosphorylation and Co-incubation of DCO-6 with H2O2 inhibited the phosphorylation of p38 in a concentration-dependent manner (Fig. 5C).

Bottom Line: In the present study, we show that a novel chromone derivative, DCO-6, significantly reduced lipopolysaccharide (LPS)-induced production of nitric oxide, IL-1β and IL-6, decreased the levels of iNOS, IL-1β and IL-6 mRNA expression in both RAW264.7 cells and mouse primary peritoneal macrophages, and inhibited LPS-induced activation of p38 MAPK but not of JNK, ERK.LPS-induced production of intracellular reactive oxygen species (ROS) was remarkably impaired by DCO-6, which disrupted the formation of the TRAF6-ASK1 complex.Our results indicate that DCO-6 showed anti-inflammatory properties through inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China.

ABSTRACT
The p38 MAPK signaling pathway plays a pivotal role in inflammation. Targeting p38 MAPK may be a potential strategy for the treatment of inflammatory diseases. In the present study, we show that a novel chromone derivative, DCO-6, significantly reduced lipopolysaccharide (LPS)-induced production of nitric oxide, IL-1β and IL-6, decreased the levels of iNOS, IL-1β and IL-6 mRNA expression in both RAW264.7 cells and mouse primary peritoneal macrophages, and inhibited LPS-induced activation of p38 MAPK but not of JNK, ERK. Moreover, DCO-6 specifically inhibited TLR4-dependent p38 activation without directly inhibiting its kinase activity. LPS-induced production of intracellular reactive oxygen species (ROS) was remarkably impaired by DCO-6, which disrupted the formation of the TRAF6-ASK1 complex. Administering DCO-6 significantly protected mice from LPS-induced septic shock in parallel with the inhibition of p38 activation and ROS production. Our results indicate that DCO-6 showed anti-inflammatory properties through inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway. Blockade of the upstream events required for p38 MAPK action by DCO-6 may provide a new therapeutic option in the treatment of inflammatory diseases.

Show MeSH
Related in: MedlinePlus