Inhibitory effects of (−)-α-bisabolol on LPS-induced inflammatory response in RAW264.7 macrophages

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Abstract

Although (−)-α-bisabolol, a natural monocyclic sesquiterpene alcohol, is often used as a cosmetic soothing supplement, little is known about its mechanisms of anti-inflammatory effects. Therefore, this study was designed to investigate anti-inflammatory effects of (−)-α-bisabolol and its mechanisms of action. In this study, we found that (−)-α-bisabolol inhibited lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW264.7 cells. In addition, expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) genes was reduced, as evidenced by Western blot and luciferase reporter assays for COX-2 and iNOS. To assess the mechanism of the anti-inflammatory property of (−)-α-bisabolol, its effects on the activity of AP-1 and NF-κB promoters were examined. LPS-induced activation of AP-1 and NF-κB promoters was significantly reduced by (−)-α-bisabolol. Consistently, (−)-α-bisabolol reduced LPS-induced phosphorylation of IκBα. In addition, while LPS-induced phosphorylation of ERK and p38 was attenuated by (−)-α-bisabolol, significant changes in the level of phosphorylated JNK were not observed. Our results indicate that (−)-α-bisabolol exerts anti-inflammatory effects by downregulating expression of iNOS and COX-2 genes through inhibition of NF-κB and AP-1 (ERK and p38) signaling.

Highlights

► Anti-inflammatory effect of (−)-α-bisabolol in RAW264.7 cells. ► (−)-α-Bisabolol inhibits LPS-induced expression of iNOS and COX-2 genes. ► (−)-α-Bisabolol reduces LPS-induced phosphorylation of IκBα. ► LPS-induced phosphorylation of ERK and p38 is attenuated by (−)-α-bisabolol. ► (−)-α-Bisabolol inhibits NF-κB and AP-1 (ERK and p38) signaling.

Introduction

Inflammation, a physiological response to infection or injury, plays a critical role in health and disease. Among its mediators, iNOS and COX-2 are important enzymes that regulate inflammatory processes. NO is generated in different cell types by at least three isoforms of NO synthase (NOS). iNOS is expressed in all cell types after stimulation with LPS and/or with different cytokines. During inflammation associated with different pathogens, NO production increases significantly and may become cytotoxic (Kharitonov et al., 1994). Moreover, the free radical nature of NO and its high reactivity with oxygen to produce peroxynitrite (ONOO–) makes NO a potent pro-oxidant molecule that is able to induce oxidative damage, and can be potentially harmful towards cellular targets (Epe et al., 1996). COX-1 and COX-2 catalyze the rate-limiting step in the formation of prostaglandins (Ramsay et al., 2003). The expression of COX-2 is inducible and remains undetectable in most mammalian tissues under basal conditions. COX-2 is also regulated at the post-transcriptional and at the enzymatic levels. NO is a modulator of COX-2 activity and directly affects its enzymatic activity (Mitchell et al., 1995).

NF-κB is transcription factor that plays a key role in regulating host immune and inflammatory responses by increasing the level of cytokines, chemokines, growth factors and cell adhesion molecules (Chen et al., 1999). The promoter region of iNOS and COX-2 contains an NF-κB site and activation of NF-κB leads to transcriptional regulation of the expression of these pro-inflammatory genes (Xie et al., 1994). The transcription factor activator protein-1 (AP-1) is also activated by these stimuli, which is also known to be involved in the transcriptional regulation of inflammatory response (Zenz et al., 2008).

(−)-α-Bisabolol is a natural monocyclic sesquiterpene alcohol found in the oils of chamomile (Matricaria chamomilla), Vanillosmopsis erythropappa and other plants (Fig. 1a). It has been used for hundreds of years in cosmetics because of its perceived skin healing properties. It has been reported that (−)-α-bisabolol possesses a variety of biological activities, such as depigmentation (Kim et al., 2008), anti-fungal (Vila et al., 2010), noiception attenuation in mice (Leite Gde et al., 2011), anti-tumor (Cavalieri et al., 2004, Chen et al., 2010, da Silva et al., 2010, Piochon et al., 2009), and gastroprotective activity (Bezerra et al., 2009). Recently, (−)-α-bisabolol was used as a fragrance ingredient in decorative cosmetics, fine fragrances, shampoos, toilet soaps and other toiletries as well as in non-cosmetic products such as household cleaners and detergents (Bhatia et al., 2008). In addition, (−)-α-bisabolol has been introduced as a soothing agent in baby cosmetics. Despite its wide spread use, no studies have examined the effects of (−)-α-bisabolol on inflammation-associated gene expression.

Therefore, in this study, we characterized the inhibitory mechanism of (−)-α-bisabolol against inflammatory activity and demonstrated that (−)-α-bisabolol inhibits LPS-induced inflammatory reaction through inactivation of AP-1 and NF-κB pathway in RAW264.7 cells.

Section snippets

Reagents and cell culture

(−)-α-Bisabolol was obtained from Sigma (St. Louis, MO, USA) and dissolved in DMSO. Lipopolysaccharide (Escherichia coli 0111:B4) and β-actin antibody was purchased from Sigma (St. Louis, MO, USA). iNOS antibody was obtained from Millipore (Bedford, MA ,USA). And COX-2, phospho-p38 MAPK (Thr180/Tyr182) (28B10), phospho-SAPK/JNK (Thr183/Tyr185) (G9), phospho-p42/44 MAPK (Thr202/Tyr204) (E10), p38 MAPK, SAPK/JNK, and p42/44 MAPK antibodies were obtained from Cell Signaling Technology, Inc.

(−)-α-Bisabolol reduced LPS-induced production of NO and PGE2 in RAW264.7 cells

Pro-inflammatory mediators like NO and PGE2 play very important roles in the inflammatory response. To investigate the involvement of (−)-α-bisabolol in the production of these mediators, the levels of secreted NO and PGE2 were measured. As shown in Fig. 1b and c, LPS-induced production of NO and PGE2 was significantly reduced by (−)-α-bisabolol in a dose-dependent manner. To exclude the possibility that this effect was attributed to the cytotoxic effect of (−)-α-bisabolol, the MTT assay was

Discussion

Although (−)-α-bisabolol, which is a sesquiterpene alcohol, has been used as a soothing agent in dermatologic and cosmetic formulations, no studies have systematically examined the effects of (−)-α-bisabolol on inflammation. In this study, the anti-inflammatory effects of (−)-α-bisabolol were demonstrated and its mechanisms of action was characterized. Specifically, (−)-α-bisabolol exerted anti-inflammatory effects by downregulating expression of pro-inflammatory mediators such as iNOS and

Conclusion

(−)-α-Bisabolol inhibited production of NO and PGE2, proinflammatory mediators, in RAW264.7 cells. In addition, (−)-α-bisabolol reduced expression of iNOS and COX-2 genes by inhibiting the NF-κB and AP-1 (ERK and p38) signaling pathway. These findings suggest that (−)-α-bisabolol may be used as both a soothing agent and for the treatment of inflammatory diseases.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgment

This research was supported by a grant from the Korean Ministry of Knowledge and Economy (70011029).

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