The fixed structure of Licochalcone A by α, β-unsaturated ketone is necessary for anti-inflammatory activity through the inhibition of NF-κB activation

https://doi.org/10.1016/j.intimp.2010.02.003Get rights and content

Abstract

Glycyrrhiza inflata has been used as a traditional medicine with anti-inflammatory activity. Previously, we reported that a major component, Licochalcone A, potently inhibited TNFα-induced NF-κB activation by inhibiting IKKβ activation. In this study, we investigated whether the fixed structure of Licochalcone A by α, β-unsaturated ketone is required for its inhibitory effect of NF-κB activation. Interestingly, reduced Licochalcone A, which lacks a double bond, failed to inhibit TNFα-induced NF-κB activation. Whereas Licochalcone A potently inhibited TNFα-induced IKK activation, IκBα degradation, nuclear localization of NF-κB and its DNA binding activity, no inhibitory effect was observed by reduced Licochalcone A. In addition, TNFα-induced expression of inflammatory cytokines, CCL2/MCP-1 and CXCL1/KC, was clearly inhibited by Licochalcone A but not reduced Licochalcone A. As a result, culture media pretreated with Licochalcone A but not reduced Licochalcone A following TNFα stimulation significantly inhibited the chemotactic activity of neutrophils. Furthermore, acute carrageenan-induced paw edema in mice was markedly inhibited by administration of Licochalcone A but not reduced Licochalcone A. Taken together, it is suggested that Licochalcone A is a promising anti-inflammatory drug in vivo and its fixed structure is critical for anti-inflammatory activity.

Introduction

In mammals, a transcription factor, the nuclear factor kappa B (NF-κB) family, has 5 members: RelA/p65, RelB, c-Rel, NF-κB1/p50, and NF-κB2/p52 [1]. The p50-RelA/p65 heterodimer is a typical member of the Rel family of transcription factors, which regulate diverse cellular functions, such as immune response, cell growth and development. In unstimulated cells, NF-κB remains inactive in the cytoplasm through the association with inhibitor proteins of the IκB family [2], [3]. In the canonical NF-κB pathway, IκB kinase (IKK) complex, which is composed of two catalytic subunits, IKKα and IKKβ and a regulatory subunit, IKKγ, is rapidly activated by various stimuli, including proinflammatory cytokines. Activated IKKs phosphorylate IκBs, leading to their ubiquitination and proteasomal degradation [4]. These events release NF-κB dimers in the cytosol, allowing them to translocate into the nucleus where they enhance the transcription of target genes [5], [6].

Tumor necrosis factor α (TNFα) is a pleiotropic proinflammatory cytokine with a wide range of biological effects [7]. TNFα participates in the inflammatory effect by inducing various inflammatory cytokines, including CCL2/monocyte chemotactic protein-1(MCP-1) and CXCL1/KC, through the activation of NF-κB [7], [8]. CCL2/MCP-1 stimulates the migration of monocytes and macrophages, and activates these cells to secrete a variety of different cytokines. In turn, CCL2/MCP-1 can exert different biological effects on recipient cells [9]. CXCL1/KC also plays a major role in the recruitment of neutrophils to inflammatory sites. The recruitment of neutrophils is integral to the inflammatory response and is an essential host defense mechanism [10]; therefore, it is easily imagined that abnormality of the TNFα signaling pathway or excess production of TNFα can cause excessive inflammation. In fact, dysregulated TNFα function is implicated in the pathological process of many diseases, including rheumatoid arthritis, Crohn's disease and several neurological diseases [11].

Liquorice root has been used as a traditional medicine in the East and West for the treatment of gastric ulcer, bronchial asthma and inflammation [12]. Licochalcone A is a major and biogenetically characteristic chalcone isolated from the root of Xinjiang liquorice, Glycyrrhiza inflata [13]. Previously, we found that Licochalcone A significantly inhibited TNFα-induced IKK activation [14]. Furthermore, we reported that Licochalcone A inhibited leukemia-derived fusion protein, TEL-JAK2-mediated transformation through the specific inhibition of Stat3 activation [15]. Since the transformation by TEL-JAK2 and TNFα-mediated inflammation are induced through completely different cellular signaling pathways, it is expected that Licochalcone A exhibits anti-transformation activity and anti-inflammatory activity via different mechanisms. In order to further investigate the effect of Licochalcone A, we focused on the relationship of its structure and activity in transformation and inflammation using reduced Licochalcone A. Both Licochalcone A and reduced Licochalcone A exhibited a similar inhibitory effect on STAT3 in transformed cells expressing TEL-JAK2; however, while Licochalcone A had a potent anti-inflammatory effect in vitro and in vivo, reduced Licochalcone A failed to inhibit TNFα-induced IKK activation, NF-κB activation and the expression of CCL2/MCP-1 and CXCL1/KC. In this study, we showed that the planer conformation of Licochalcone A is required for its anti-inflammatory effect.

Section snippets

Reagents

Licochalcone A was purchased from Calbiochem (San Diego, CA, USA). Reduced Licochalcone A was synthesized as previously described [16]. Murine TNFα was purchased from PEPROTECH (Rocky Hill, NJ, USA). Antibodies recognizing p65, IκBα, IKKγ, iNOS and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-JNK antibody and anti-phospho-JNK (Thr183/Thr185) were from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse

Licochalcone A but not reduced Licochalcone A inhibited TNFα-induced NF-κB activation

A major component of G. inflata, Licochalcone A is 5- (1, 1-dimethy-2-propenyl) -4, 4′-dihydroxy-2-methoxy chalcone, as shown in Fig. 1A [12], [13]. To investigate the relationship between the structure of Licochalcone A and its anti-inflammatory effect, we obtained reduced Licochalcone A by a reductive reaction (Fig. 1A). As shown in Fig. 1B, the 3D structure showed that Licochalcone A has a fixed planer conformation by α, β-unsaturated ketone. On the other hand, it is presumed that reduced

Discussion

In the present study, we clearly demonstrated that Licochalcone A effectively inhibited carrageenan-induced inflammation in a murine model (Fig. 7). The suppressive effect of Licochalcone A on carrageenan-induced paw edema was well correlated with the inhibition of NF-κB activation, expression of chemokines and migration of neutrophils (Fig. 2, Fig. 3, Fig. 4, Fig. 5). Licochalcone A is a major flavonoid isolated from the root of G. inflata, which has been used to treat various disorders,

Acknowledgements

We thank Dr. H. Itoh and Dr. K. Tago (Nara Institute of Science and Technology) for providing KF-8 cells. We also thank Ms. Saeko Tanabe, Ms. Saeko Takei and Mr. Jun-ichi Furusawa for their technical help.

This work was supported in part by grants 16390024 and 19790071 from MEXT and the Hi-Tech Research Center Project for Private Universities in Japan.

References (29)

Cited by (40)

  • Synthetic methods and biological applications of retrochalcones isolated from the root of Glycyrrhiza species: A review

    2021, Results in Chemistry
    Citation Excerpt :

    The anti-inflammatory activity of the G. inflata species is attributed to the effective inhibition of NF-κB by licochalcone A, B and D [28,29,47]. It was observed that the keto-ethylenic bridge of a retrochalcone is critical for its anti-inflammatory activity; for example, reduced licochalcone A failed to inhibit TNFα-induced NF-κB activation, IκB kinase (IKK) activation and the degradation of its catalytic subunit, IκBα [48]. Later on, it was reported [49] that licochalcone C weakens the lipopolysaccharide-interferon-γ (LPS-IFN-γ) induced inflammation by reducing the activity of inducible nitric oxide synthase (iNOS) via nuclear factor kappa-B (NF-κB) by regulating the antioxidant activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT).

  • Screening for bioactive natural products from a 67-compound library of Glycyrrhiza inflata

    2017, Bioorganic and Medicinal Chemistry
    Citation Excerpt :

    The activities of 13 (licochalcone E) and 43 (licoflavone C) were consistent with previous reports.17,18 Moreover, chalcones showed more potent NO inhibitory activities than the other structural types, consistent with previous report.21 Nuclear factor-kappa B (NF-κB), a transcription factor, is considered to contribute to the anti-inflammatory activities of flavonoids.19

  • Synthesis of novel (2E)-1-[4-(2,4-difluorophenyl)phenyl]3-arylprop-2-en-1-ones: Investigation on spectral, antibacterial, molecular docking and theoretical studies

    2015, Journal of Molecular Structure
    Citation Excerpt :

    Some of the naturally occurring chalcones like butein (1) [12], cardamonin (2) [13], isoliquiritigenin (3) [14], licochalcone A (4) and Xanthohumol (5) [15] exhibit anticancer and antimicrobial activities (Fig. 1). The biphenyl moiety is also found in many natural products that exhibits a wide variety of biological activities such as anti-cancer [16–19], anti-angiogenic [16,18], anti-viral [20] and have the ability to display enhanced fluorescence [21]. The emerging application of chalcones, fluorine and biphenyl initiate us to synthesize some novel difluorobiphenyl chalcones.

View all citing articles on Scopus
View full text