Korean J Physiol Pharmacol.  2017 May;21(3):345-352. 10.4196/kjpp.2017.21.3.345.

JS-III-49, a hydroquinone derivative, exerts anti-inflammatory activity by targeting Akt and p38

• Yi YS
• Kim MY
• Cho JY

• KMID: 2376963
• DOI: http://doi.org/10.4196/kjpp.2017.21.3.345

Abstract

Since previous studies have reported that hydroquinone (HQ) exerted immunosuppressive and anti-inflammatory activity, various HQ derivatives have been synthesized and their biological activities investigated. In this study, we explored the anti-inflammatory activity of JS-III-49, a novel HQ derivative, in macrophage-mediated inflammatory responses. JS-III-49 suppressed the production of the inflammatory mediators nitric oxide (NO) and prostaglandin E2 (PGE2) and down-regulated the mRNA expression of the inflammatory enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as well as the expression of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-1b without cytotoxicity in LPS-stimulated RAW264.7 cells. JS-III-49 inhibited nuclear translocation of the NF-kB transcription factors p65 and p50 by directly targeting Akt, an upstream kinase of the NF-kB pathway, in LPS-stimulated RAW264.7 cells. However, JS-III-49 did not directly inhibit the kinase activities of Src and Syk, which are upstream kinases of Akt, in LPS-stimulated RAW264.7 cells. Moreover, JS-III-49 suppressed the nuclear translocation of c-Fos, one of the components of AP-1, by specifically targeting p38, an upstream mitogen-activated protein kinase (MAPK) in the AP-1 pathway in LPS-stimulated RAW264.7 cells. These results suggest that JS-III-49 plays an anti-inflammatory role in LPS-stimulated macrophages by targeting Akt and p38 in the NF-kB and AP-1 pathways, respectively.

Keyword

Akt; Anti-inflammatory activity; HQ derivative; JS-III-49; Macrophages; p38

MeSH Terms

Cyclooxygenase 2
Cytokines
Dinoprostone
Interleukin-6
Macrophages
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Phosphotransferases
Protein Kinases
RNA, Messenger
Transcription Factor AP-1
Transcription Factors
Cyclooxygenase 2
Cytokines
Dinoprostone
Interleukin-6
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Phosphotransferases
Protein Kinases
RNA, Messenger
Transcription Factor AP-1
Transcription Factors

Figure

• 

  Fig. 1 Suppressive effect of JS-III-49 on the production of inflammatory mediators.(A) Chemical structure of JS-III-49. (B) RAW264.7 cells pretreated with the indicated doses of (left panel) JS-III-49 or (right panel) L-NAME for 30 min were treated with LPS (1 µg/ml) for 24 h, and NO levels in the cell culture media were determined. (C) RAW264.7 cells pretreated with the indicated dose of (left panel) JS-III-49 or (right panel) indomethacin (Indo) for 30 min were treated with LPS (1 µg/ml) for 24 h, and PGE2 levels in the cell culture medium were determined by EIA. (D) RAW264.7 cells were treated with the indicated dose of JS-III-49 for 24 h, and the cell viability was measured by MTT assay. *p<0.05, **p<0.01 compared to control.

• 

  Fig. 2 Suppressive effect of JS-III-49 on the mRNA expression of pro-inflammatory genes.(A) RAW264.7 cells pretreated with the indicated doses of JS-III-49 for 30 min were treated with LPS (1 µg/ml) for 6 h, and mRNA expression levels of COX-2, iNOS, IL-6, IL-1β, and GAPDH were determined by semi-quantitative RT-PCR. (B, C) RAW264.7 cells pretreated with the indicated doses of JS-III-49 for 30 min were treated with LPS (1 µg/ml) for 6 h, and mRNA expression levels of (B) iNOS and (C) COX-2 were determined by quantitative real-time PCR. GAPDH was used as an internal control to normalize the mRNA expression levels of target genes. **p<0.01 compared with control.

• 

  Fig. 3 The suppressive effect of JS-III-49 on the NF-κB pathway is mediated by direct targeting of Akt.(A) RAW264.7 cells pretreated with JS-III-49 (30 µM) for 30 min were treated with LPS (1 µg/ml) for the indicated time, and protein levels of p65 and p50 in the nuclear fraction of the cells were determined by Western blot analysis. (B) RAW264.7 cells pretreated with JS-III-49 (30 µM) for 30 min were treated with LPS (1 µg/ml) for the indicated time, and phosphorylated and total levels of IκBα in the total cell lysate were determined by Western blot analysis. (C) RAW264.7 cells pretreated with JS-III-49 (30 µM) for 30 min were treated with LPS (1 µg/ml) for the indicated time, and phosphorylated and total levels of Src, Syk, and Akt in the total cell lysate were determined by Western blot analysis. (D) Effects of JS-III-49 on the kinase activities of Src and Syk were determined by a conventional in vitro kinase assay using purified Src and Syk as described in the Materials and Methods. (E) RAW264.7 cells pretreated with the indicated doses of JS-III-49 for 30 min were treated with LPS (1 µg/ml) for 5 min, and phosphorylated and total levels of Akt in the total cell lysate were determined by Western blot analysis. Lamin A/C and β-actin were used as internal controls for the nuclear fraction and total cell lysate, respectively.

• 

  Fig. 4 The suppressive effect of JS-III-49 on the AP-1 pathway is mediated by targeting of c-Fos and p38.(A) RAW264.7 cells pretreated with JS-III-49 (30 µM) for 30 min were treated with LPS (1 µg/ml) for the indicated time, and protein levels of c-Jun and c-Fos in the nuclear fraction of cells were determined by Western blot analysis. (B) RAW264.7 cells pretreated with JS-III-49 (30 µM) for 30 min were treated with LPS (1 µg/ml) for the indicated time, and phosphorylated and total levels of ERK, JNK, and p38 in the total cell lysate were determined by Western blot analysis. Lamin A/C and β-actin were used as internal controls for the nuclear fraction and total cell lysate, respectively.

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  Fig. 5 Proposed model to illustrate the inhibitory pathway of JS-III-49-mediated anti-inflammatory responses in macrophages.

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