Korean J Physiol Pharmacol.  2016 Jan;20(1):91-99. 10.4196/kjpp.2016.20.1.91.

(E)-3-(3-methoxyphenyl)-1-(2-pyrrolyl)-2-propenone displays suppression of inflammatory responses via inhibition of Src, Syk, and NF-kappaB

• Kim Y
• Jeong EJ
• Han Lee IS
• Kim MY
• Cho JY

• KMID: 2150478
• DOI: http://doi.org/10.4196/kjpp.2016.20.1.91

Abstract

(E)-3-(3-methoxyphenyl)-1-(2-pyrrolyl)-2-propenone (MPP) is an aldol condensation product resulting from pyrrole-2-carbaldehyde and m- and p- substituted acetophenones. However, its biological activity has not yet been evaluated. Since it has been reported that some propenone-type compounds display anti-inflammatory activity, we investigated whether MPP could negatively modulate inflammatory responses. To do this, we employed lipopolysaccharide (LPS)-stimulated macrophage-like RAW264.7 cells and examined the inhibitory levels of nitric oxide (NO) production and transcriptional activation, as well as the target proteins involved in the inflammatory signaling cascade. Interestingly, MPP was found to reduce the production of NO in LPS-treated RAW264.7 cells, without causing cytotoxicity. Moreover, this compound suppressed the mRNA levels of inflammatory genes, such as inducible NO synthase (iNOS) and tumor necrosis factor (TNF)-alpha. Using luciferase reporter gene assays performed in HEK293 cells and immunoblotting analysis with nuclear protein fractions, we determined that MPP reduced the transcriptional activation of nuclear factor (NF)-kappaB. Furthermore, the activation of a series of upstream signals for NF-kappaB activation, composed of Src, Syk, Akt, and IkappaBalpha, were also blocked by this compound. It was confirmed that MPP was able to suppress autophosphorylation of overexpressed Src and Syk in HEK293 cells. Therefore, these results suggest that MPP can function as an anti-inflammatory drug with NF-kappaB inhibitory properties via the suppression of Src and Syk.

Keyword

Anti-inflammatory activity; (E)-3-(3-methoxyphenyl)-1-(2-pyrrolyl)-2-propenone; Macrophages; NF-kappaB

MeSH Terms

Acetophenones
Genes, Reporter
HEK293 Cells
Immunoblotting
Luciferases
Macrophages
NF-kappa B*
Nitric Oxide
Nitric Oxide Synthase
Nuclear Proteins
RNA, Messenger
Transcriptional Activation
Tumor Necrosis Factor-alpha
Acetophenones
Luciferases
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase
Nuclear Proteins
RNA, Messenger
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Chemical structure of (E)-3-(3-methoxyphenyl)-1-(2-pyrrolyl)-2-propenone (MPP).

• 

  Fig. 2 Effect of MPP on the production of NO from LPS-treated RAW 264.7 cells and on the viability of RAW264.7 cells.(A) RAW264.7 cells (1×106 cells/ml) were stimulated with LPS (1 µg/ml) in the presence or absence of MPP (0 to 50 mM) or L-NAME (0 to 1.5 µM) for 24 h. The supernatants were then obtained, and the NO concentration in the supernatants was confirmed using the Griess assay. (B) RAW264.7 cells (1×106 cells/ml) were treated with MPP for 24 h, and cell viability was examined using the MTT assay. All data are expressed as the mean±SD of experiments, which were performed with six samples. *p<0.05 and **p<0.01 compared to normal or control groups.

• 

  Fig. 3 Effects of MPP on transcriptional activation in LPS-stimulated RAW 264.7 cells and MyD88/TRIF-treated HEK293 cells.(A) The mRNA levels of iNOS and TNF-α in LPS (1 µg/ml)-treated RAW264.7 cells in the presence or absence of MPP (10, 25 and 50 µM) were determined by RT-PCR. (B and C) HEK293 cells co-transfected with NF-κB-Luc plasmid construct and β-gals in the presence or absence of MyD88 or TRIF were treated with MPP (25 and 50 µM). Luciferase activity was measured using a luminometer. (D) The nuclear levels of p65 and p50 in LPS (1 µg/ml)-stimulated RAW264.7 cells in the presence or absence of MPP (25 µM) were detected by immunoblotting analysis. Relative intensity was calculated using total levels by the DNR Bio-imaging system. Data (B and C) are expressed as the mean±SD of experiments, which were performed with six samples. *p<0.05 and **p<0.01 compared to normal or control groups.

• 

  Fig. 4 Effects of MPP on the upstream signaling cascade for NF-kB activation in LPS-stimulated RAW 264.7 cells.(A and B) RAW264.7 cells were incubated with LPS (1 µg/ml) in the presence or absence of MPP (50 µM) for 30 min. After preparation of whole cell lysates, the total and phospho-protein levels of Src, Syk, AKT, and IκBα from the lysates were detected by immunoblotting analysis. Relative intensity was calculated using total levels by the DNR Bio-imaging system. *p<0.05 and **p<0.01 compared to normal or control groups.

• 

  Fig. 5 Effects of MPP on autophosphorylation of Src and Syk in Src- or Syk-transfected HEK293 cells.(A and B) Inhibitory activity of MPP (25 and 50 µM) on the autophosphorylation of Src or Syk in Src- or Syk-transfected HEK293 cells was measured by immunoblotting analysis. (C) RAW264.7 cells (1×106 cells/ml) were stimulated with LPS (1 µg/ml) in the presence or absence of PP2 (2.5 and 5 µM) or piceatannol (Picea, 5 and 10 µM) for 24 h. The supernatants were then obtained, and the NO concentration in the supernatants was confirmed using the Griess assay. Relative intensity was calculated using total levels by the DNR Bio-imaging system. *p<0.05 and **p<0.01 compared to normal or control groups.

• 

  Fig. 6 Putative inhibitory pathway of MPP in LPS-treated macrophages.

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