N-(p-Coumaryol)-Tryptamine Suppresses the Activation of JNK/c-Jun Signaling Pathway in LPS-Challenged RAW264.7 Cells

Abstract

N-(p-Coumaryol) tryptamine (CT), a phenolic amide, has been reported to exhibit anti-oxidant and anti-inflammatory activities. However, the underlying mechanism by which CT exerts its pharmacological properties has not been clearly demonstrated. The objective of this study is to elucidate the anti-inflammatory mechanism of CT in lipopolysaccharide (LPS)-challenged RAW264.7 macrophage cells. CT significantly inhibited LPS-induced extracellular secretion of pro-inflammatory mediators such as nitric oxide (NO) and PGE2, and protein expressions of iNOS and COX-2. In addition, CT significantly suppressed LPS-induced secretion of pro-inflammatory cytokines such as TNF-α and IL-1β. To elucidate the underlying anti-inflammatory mechanism of CT, involvement of MAPK and Akt signaling pathways was examined. CT significantly attenuated LPS-induced activation of JNK/c-Jun, but not ERK and p38, in a concentration-dependent manner. Interestingly, CT appeared to suppress LPS-induced Akt phosphorylation. However, JNK inhibition, but not Akt inhibition, resulted in the suppression of LPS-induced responses, suggesting that JNK/c-Jun signaling pathway significantly contributes to LPS-induced inflammatory responses and that LPS-induced Akt phosphorylation might be a compensatory response to a stress condition. Taken together, the present study clearly demonstrates CT exerts anti-inflammatory activity through the suppression of JNK/c-Jun signaling pathway in LPS-challenged RAW264.7 macrophage cells.

Keywords: COX-2; JNK; Lipopolysaccharide; N-(p-Coumaroyl) tryptamine; RAW 264.7 cells; c-Jun; iNOS.

Fig. 1.

Chemical structure of N-(p-coumaryol) tryptamine.

Fig. 2.

Effects of N-(p-coumaryol) tryptamine on LPS-induced extracellular release of NO (A) and PGE₂ (B) in RAW264.7 macrophage cells. RAW264.7 cells were pretreated with indicated concentrations of N-(p-coumaryol) tryptamine for 1 hr before incubation with LPS (200 ng/ml) for 24 hr. The level of nitrite and PGE₂ were measured using Griess reagent and ELISA assay, respectively. N-(p-Coumaryol) tryptamine significantly suppressed LPS-induced extracellular release of NO and PGE₂. (C) Effect of N-(p-coumaryol) tryptamine on the viability of RAW264.7 cells. No significant cell death was observed with N-(p-coumaryol) tryptamine concentrations used in the present study. The data were obtained from three independent experiments and expressed as mean ± S.D. (n=3). ^*p<0.05 and ^**p<0.01 indicate statistically significant differences from treatment with LPS alone. CT stands for N-(p-coumaryol) tryptamine.

Fig. 3.

Effects of N-(p-coumaryol) tryptamine on LPS-induced expression of iNOS and COX-2 in RAW264.7cells. (A) The cell lysates were subjected to SDS-PAGE, and then protein levels of iNOS and COX-2 were determined by Western blot analysis. N-(p-Coumaryol) tryptamine significantly attenuated LPS-induced over expression of iNOS and COX-2. Images are representative of three independent experiments that shows reproducible results. (B) Quantitative analysis of immunoblots of iNOS and COX-2. N-(p-Coumaryol) tryptamine significantly suppressed LPS-induced iNOS and COX-2 expression. The data were obtained from three independent experiments and expressed as mean ± S.D. (n=3). ^*p<0.05 and ^**p<0.01 indicate statistically significant differences from treatment with LPS alone. CT stands for N-(p-coumaryol) tryptamine.

Fig. 4.

Effect of N-(p-coumaryol) tryptamine on LPS-induced extracellular secretion of IL-1β and in RAW264.7 macrophage cells. RAW264.7 cells were pretreated with indicated concentrations of N-(p-coumaryol) tryptamine for 1 hr, then incubated with LPS (200 ng/ml) for 24 hrs. The concentrations of IL-1β (A) and TNF-α (B) in collected cell culture media were measured by ELISA assay as described in the methods. N-(p-Coumaryol) tryptamine meaningfully reduced LPS-stimulated IL-1β and TNF-α cytokines in aconcentration-dependent manner. The values are expressed as mean ± SD for three independent experiments. ^*p<0.05 and ^**p<0.01 indicate statistically significant differences from treatments with LPSalone.

Fig. 5.

Effect of N-(p-coumaryol) tryptamine on LPS-induced activation of MAPK signaling pathway in RAW264.7 macrophage cells. (A) representative immunoblots, (B) quantitative analysis of immunoblots. Cellswere challenged with 200 ng/ml LPS in the absence or presence of N-(p-coumaryol) tryptamine. LPS-induced increased phosphorylation of JNK and c-jun was significantly attenuated with N-(p-coumaryol) tryptamine treatment. However, phosphorylation of ERK and p38 was not affected with N-(p-coumaryol) tryptamine treatment, suggesting that JNK/c-jun signaling might play a key role in the LPS-induced activation of RAW264.7 cells. Images are representative of three independent experiments that shows reproducible results. The values are expressed as mean ± SD for three independent experiments. ^*p<0.05 and ^**p<0.01 indicate statistically significant differences from treatments with LPS alone. CT stands for N-(p-coumaryol) tryptamine.

Fig. 6.

Effect of N-(p-coumaryol) tryptamine on LPS-induced activation of Akt signaling pathway in RAW264.7 macrophage cells. (A) representative immunoblots, (B) quantitative analysis of immunoblots. N-(p-coumaryol) tryptamine appeared to significantly attenuate Akt phosphorylation in RAW264.7 macrophage cells. The images shown are representative of three independent experiments and the values are expressed as mean ± SD for three experiments. ^*p<0.05 and ^**p<0.01 indicate statistically significant differences from treatments with control alone. CT stands for N-(pcoumaryol) tryptamine.

Fig. 7.

Role of JNK and Akt signaling pathways in N-(p-coumaryol) tryptamine-mediated suppression of LPS-induced RAW264.7 cell activation. RAW264.7 cells were pretreated with CT (N-(p-coumaryol) tryptamine), SP (SP600125, JNK inhibitor), or W (wortmannin, Akt inhibitor), and then exposed to LPS (200 ng/ml) for 1 hr. The cell lysates were prepared and subjected to Western blotting analysis. (A) Phosphorylation levels of Akt and c-jun were examined in the presence of CT, SP, or W. CT and SP significantly suppressed LPS-induced phosphorylation of Akt and c-jun. However, W did not inhibit LPS-induced c-jun phosphorylation. (B) The protein levels of iNOS and COX-2 were examined. Suppression of LPS-induced iNOS and COX-2 expressions were observed with N-(p-coumaryol) tryptamine (CT) and also with JNK inhibitor (SP) but not with Akt inhibitor (W). The images on top are representatives of three independent experiments. The data for quantitative analyses on bottom were obtained from three independent experiments and expressed as mean ± SD (n=3). ^*p<0.05 and ^**p<0.01 indicate statistically significant differences from treatments with control alone.