Elsevier

Cellular Signalling

Volume 21, Issue 5, May 2009, Pages 778-785
Cellular Signalling

Licochalcone A significantly suppresses LPS signaling pathway through the inhibition of NF-κB p65 phosphorylation at serine 276

https://doi.org/10.1016/j.cellsig.2009.01.021Get rights and content

Abstract

Licorice root, Glycyrrhiza inflata, has been used as a traditional medicine for the treatment of bronchial asthma and inflammation; however, the mechanism of its anti-inflammatory activity has not been clarified. Here, we investigated the effect of Licochalcone A, a major component of G. inflata, on the LPS signaling pathway. We found that Licochalcone A remarkably inhibited LPS-induced NO production, and TNFα expression and MCP-1 expression in both RAW264.7 cells and primary macrophages. Furthermore, when injected with Licochalcone A prior to injection of LPS, the serum level of TNFα and MCP-1 in C57BL/6 mice was clearly decreased, indicating that Licochalcone A has a potent anti-inflammatory effect both in vitro and in vivo. Strikingly, Licochalcone A significantly inhibited LPS-induced NF-κB transcriptional activation; however, it had no effect on not only the phosphorylation and degradation of IκBα but also nuclear translocation and DNA binding activity of NF-κB p65. Interestingly, Licochalcone A markedly inhibited the phosphorylation of p65 at serine 276. As a result, it reduced NF-κB transactivation by preventing the interaction of p65 with p300. Taken together, Licochalcone A might contribute to the potent anti-inflammatory effect of G. inflata through the unique mechanism of NF-κB inhibition.

Introduction

Innate immunity is an ancient form of host defense that is activated rapidly to enable, through a multiplicity of effector mechanisms, defense against bacterial or viral infections and stresses; however, excessive innate immunity responses may cause conditions such as sepsis and chronic inflammation [1], [2]. Innate immunity responses are mediated through host factors, including proinflammatory cytokines and glycerophospholipids induced by bacterial cell wall components, such as lipopolysaccharide (LPS) [3]. Among the host factors, nitric oxide (NO) is the foremost key mediator responsible for sepsis syndrome. In fact, it has been reported that high amounts of NO are generated during sepsis [4], [5], [6]. Additionally, previous reports have shown that decreased responses to bacterial infections and endotoxic shock appeared in mice lacking inducible NO synthase (iNOS), suggesting that NO in an important inflammatory mediator in vivo [7]. Moreover, it is well known that activated macrophages and monocytes produce various proinflammatory cytokines and chemokines, such as interleukin 1 (IL-1), tumor necrosis factor α (TNFα), IL-8 and MCP-1 [8], [9], [10]. As a result, various biological responses, including tissue injury, shock and apoptosis, were rapidly induced in vivo [1], [2], [3]. Therefore, novel compounds which suppress the LPS-induced production of NO or proinflammatory cytokine are expected for the development of effective anti-inflammatory drugs [11].

The cellular pathways involved in the production of NO and proinflammatory cytokines due to Gram-negative bacterial infections have been clearly described. In this model, LPS stimulates Toll-like receptor 4 (TLR4) and causes the recruitment of a set of adaptor proteins, such as TNF receptor-associated factor 6 (TRAF6) to TLR4 [12], [13], [14], [15]. These interactions result in the triggering of downstream signaling cascades leading to the activation of major transcription factors, nuclear factor kappaB (NF-κB) and activating protein-1 (AP-1) through IκB kinases (IKKs) and MAP kinase families, respectively [12], [13], [14], [15], [16]. These transcription factors positively control the gene expression of iNOS and proinflammatory cytokines during bacterial infection [17], [18], [19].

Licorice root has been used as a traditional medicine in the East and West for the treatment of gastric ulcer, bronchial asthma and inflammation [20]. Licochalcone A is a major and biogenetically characteristic chalcone isolated from the root of Xinjiang licorice, Glycyrrhiza inflata Batalin [21]; therefore, it is easily imagined that Licochalcone A could have a potent anti-inflammatory effect, however; its detailed anti-inflammatory mechanism has not been clarified. It is important to gain insight into the mechanisms responsible for the gene expression of iNOS and proinflammatory cytokines and to control their production during bacterial infection. In an effort to determine the mechanisms underlying the anti-inflammatory effects of Licochalcone A, we investigated its inhibitory mechanism of the LPS signaling pathway.

Section snippets

Antibodies and reagents

Licochalcone A was a gift from Minophagen Pharmaceutical Co., Ltd (Tokyo, Japan) [22]. LPS (Escherichia coli 055:B5;) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies recognizing p65, lamin, IκBα, iNOS, p300 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies recognizing phosphorylated p65 at Ser276, phosphorylated p65 at Ser536, SAPK/JNK, phosphorylated SAPK/JNK at Thr183/Tyr185, ERK, phosphorylated ERK at Thr202/Try204, p38, and

Licochalcone A reduced LPS-induced NO production through the inhibition of iNOS expression

NO is generated by immune-activated macrophages in inflammatory regions and acts as an important mediator in inflammation [4], [5], [6], [7]. In order to determine the effects of Licochalcone A on NO production, a murine macrophage cell line, RAW264.7 cells were treated with various concentrations of Licochalcone A for 1 h following LPS stimulation. Upon LPS stimulation, the NO level rose to 40 μM after 24 h. Interestingly, Licochalcone A potently inhibited the LPS-induced production of NO in a

Discussion

For many years, licorice root, G. inflata, has been used to treat various disorders, including gastric ulcer, bronchial asthma and inflammation [20], [21]; however, the detailed mechanism of its effect has not been clarified. So far, Licochalcone A, which is a major chalcone isolated from G. inflata, has been reported to possess anti-malarial and anti-microbial activity [36], [37], [38]; however, only the phenomena have been reported and the detailed mechanisms of the various effects of

Acknowledgements

We thank Ms. Kayoko Tani and Mr. Yusuke Sumiyoshi for the gift of Licochalcone A. This work was supported in part by grants (16390024, 9590075, 19790071) from MEXT, Open Research Project and the Hi-Tech Research Center Project for Private Universities in Japan.

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