Elsevier

Life Sciences

Volume 88, Issues 25–26, 20 June 2011, Pages 1077-1087
Life Sciences

The involvement of TRPA1 channel activation in the inflammatory response evoked by topical application of cinnamaldehyde to mice

https://doi.org/10.1016/j.lfs.2011.03.017Get rights and content

Abstract

Aims

In the present work, we characterize the inflammatory process induced by the topical application of cinnamaldehyde on the skin of mice and verify the participation of transient receptor potential A1 TRPA1 receptors in this process.

Main methods

We measured mouse ear edema and sensitization/desensitization after topical application of cinnamaldehyde or/and capsaicin. We also quantified cellular infiltration through myeloperoxidase (MPO) activity and histological and immunohistochemical analyses and evaluated the expression of TRPV1 and TRPA1 by western blot.

Key findings

Cinnamaldehyde induced ear edema in mice (1–6 μg/ear) with a maximum effect of 4 μg/ear. Cinnamaldehyde promoted leukocyte infiltration as detected by increasing MPO activity and confirmed by histological analyses. The edema and cellular infiltration evoked by the application of 4 μg/ear of cinnamaldehyde were prevented by topical application of ruthenium red, a non-selective TRP antagonist as well as camphor and HC030031, two TRPA1 receptor antagonists. Cinnamaldehyde-induced edema, but not cellular infiltration, was prevented by topical application of the tachykinin NK1 antagonist, aprepitant, indicating a neuropeptide release phenomenon in this process. Additionally, we observed that repeated topical applications of cinnamaldehyde did not induce changes in sensitization or desensitization with respect to the edema response. Interestingly, repeated treatment with the TRPV1 agonist, capsaicin, abrogated it edematogenic response, confirming the desensitization process and partially decreasing the cinnamaldehyde-induced edema, suggesting the involvement of capsaicin-sensitive fibers.

Significance

Our data demonstrate that the topical application of cinnamaldehyde produces an inflammatory response that is dependent on TRPA1 receptor stimulation.

Introduction

Various natural compounds can induce contact irritant dermatitis upon skin exposure. Commonly used products such as cosmetics may contain these ingredients. Animal and human studies have demonstrated the irritant properties of several compounds, such as cinnamaldehyde, which is found in cinnamon oil and used in body lotions, hair sprays, shampoos and face creams (Cocchiara et al., 2005). Cinnamaldehyde is considered a moderately potent sensitizing hapten that is capable of penetrating the skin, and the use of up to 3% cinnamaldehyde can induce dermal irritation in human volunteers. In spite of this, 10% cinnamaldehyde can be found in some cosmetic products. However, the mechanism by which this irritant compound produces skin reactions is still unclear (Johansen et al., 1996, Bickers et al., 2005, Thorne et al., 1991).

Other natural skin irritants, such as allyl isothiocyanate from mustard oil and capsaicin from red pepper, produce dermatitis by stimulating members of the transient receptor potential (TRP) family of ion channels. TRP channels are widely expressed in mammalian tissues and play diverse roles in sensorial detection. These channels involve six related protein subfamilies that comprise the TRPV1 (vanilloid subfamily) and the TRPA1 (ankyrin subfamily) receptors, which are gated by diverse stimuli, such as chemical and thermal activation (Caterina et al., 1997, Story et al., 2003, Bandell et al., 2004; for review see: Calixto et al., 2005).

TRPV1, a heat-activated channel (≥ 43 °C), is stimulated by a broad array of natural, synthetic and endogenous substances, such as capsaicin, resiniferatoxin and anandamide. It has been hypothesized that TRPA1 could be activated by mechanical and cold stimuli, but this is still under debate (Corey et al., 2004, Bandell et al., 2004, Kwan et al., 2006, Jordt et al., 2004, Caspani and Heppenstall, 2008). Also, TRPA1 is chemically activated by a wide range of natural and synthetic compounds, including endogenous ligands (4-hydroxynonenal), and several irritant substances (allyl isothiocyanate and cinnamaldehyde) (Trevisani et al., 2007, Bautista et al., 2005; for review see: Calixto et al., 2005, Zurborg et al., 2007). Allyl isothiocyanate and capsaicin have been historically used as chemical irritants in models of ear edema in rodents. Their application results in characteristic neurogenic inflammation, which is observed as increased blood flow, vascular permeability, leukocyte infiltration and release of neuropeptides into the rodent skin, a phenomenon that is also observed in some cases of dermatitis (Jancsó et al., 1977, Gamse et al., 1980, Gábor and Ra'zga, 1992, Inoue et al., 1997, Huang et al., 2003).

Cinnamaldehyde may induce ear edema in mice (Thorne et al., 1991), however, the effect of cinnamaldehyde on other inflammatory parameters, such as leukocyte infiltration, is unclear. Despite knowing that cinnamaldehyde acts as an agonist of TRPA1, the role of TRPA1 in skin irritation produced by cinnamaldehyde is still unknown. Therefore, the aim of this study was to better characterize the inflammatory process induced by cinnamaldehyde in the skin and to verify TRPA1's participation in this process.

Section snippets

Animals

Experiments were performed on adult male Swiss mice (weight 25–35 g) bred in our animal house. The animals were kept in a temperature-controlled room (22 ± 2 °C) under a 12-h light–dark cycle. Food and water were freely available. The animals (4 to 6 per group) were acclimatized to the laboratory for at least 24 h before testing and were used only once. All protocols are in accordance with the US guidelines for the care and use of laboratory animals (Zimmermann, 1983) and all procedures were

Histology

To verify histological changes in the mouse ear after capsaicin, allyl isothiocyanate or cinnamaldehyde application, samples were collected 0.5, 6, 12 and 24 h after the induction of inflammation. Mice were euthanized and ear tissue was removed and fixed in alfac solution (a 16:2:1 mixture of ethanol 80%, formaldehyde 40% and acetic acid). Each sample was embedded in paraffin wax, sectioned at 5 μm and stained with hematoxylin-eosin. A representative area was selected for qualitative light

Characterization of edema induced by cinnamaldehyde and other TRP receptor agonists

The cinnamaldehyde-induced edema (4 μg/ear) became detectable 15 min after application and reached its maximal effect at 30 min and lasted for 1 h (Fig. 1A). The edematogenic effects induced by allyl isothiocyanate (4 μg/ear) or capsaicin (200 μg/ear) also peaked 30 min after application. However, the effects were longer than for cinnamaldehyde, lasting up to 6 and 2 h after their applications, respectively (Fig. 1C and E). Based on these results, dose–response edema curves were made 30 min after the

Discussion

The present study shows that the topical application of cinnamaldehyde on mouse ears causes acute inflammatory responses with edema formation and leukocyte infiltration, which are dependent on TRPA1 activation in sensory neurons and other cellular types.

Irritant contact dermatitis is one of the most common skin diseases with a lifetime prevalence of over 30% (Kadyk et al., 2003, Slodownik et al., 2008). At concentrations between 3% and 8%, cinnamaldehyde produces local harm and sever

Conclusion

In conclusion, we demonstrated that the topical application of cinnamaldehyde to the mouse ear produces an acute skin inflammatory response dependent on TRPA1 receptor stimulation. Our findings suggest a possible role for TRPA1 in inflammatory skin reactions.

Conflict of interest

No competing interest.

Acknowledgments

This study was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Brazil. The fellowships from CNPq and CAPES are also acknowledged.

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