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Abstract
Background: This study aims to investigate the anti-inflammatory effects of cinnamaldehyde in atopic dermatitis (AD) in the mouse model. Materials and Methods: Twenty-four mice were divided into four groups: Group A (control), group B [AD with no treatment (AD + NoTre)], group C [AD with corticosteroids (AD + Cort)] and group D [AD with cinnamaldehyde (AD + Cin)]. 2,4-dinitrofluorobenzene was used to form the AD model. Topical corticosteroid was applied to group C, and oral cinnamaldehyde was administered to group D. Dorsal skin biopsies were evaluated immunohistochemically with interleukin (IL)-25, IL-33, thymic stromal lymphopoietin and caspase-3. Results: Epithelial thicknesses were significantly higher in group B–D mice compared to group A (P = 0.002, 0.009, 0.004, respectively). Significantly, higher staining with IL-25 was observed in group B (AD + NoTre) and group D (AD + Cin) than in group A (control) (P = 0.003, 0.002, respectively). However, no significant difference was observed between group D (AD + Cin) and group B (AD + NoTre). All three groups (B–D) had significantly higher staining in terms of diffuseness of IL-33 compared to group A (control) (P = 0.002, 0.002, 0.002, respectively). Caspase-3 staining was significantly lower in group D (AD + Cin) than in group B (AD + NoTre) (P = 0.003, 0.002, respectively). Moreover, caspase-3 staining intensity was significantly lower in group D (AD + Cin) than in group C (AD + Cort) (P = 0.002). Conclusions: Our study demonstrated that IL-33, IL-25 and caspase-3 have a role in the pathogenesis of AD. Furthermore, cinnamaldehyde reduced caspase-3 activity more than topical corticosteroids and anti-inflammatory effects might be investigated in AD therapy with future studies.
Keywords: Atopic dermatitis, caspase-3, cinnamaldehyde, 2,4-dinitrofluorobenzene
Introduction 
Atopic dermatitis (AD) is a chronic and inflammatory skin disorder that presents with itchy, erythematous, exudative and crusted lesions in the acute period; dry, fissured and lichenified lesions in the chronic period.[1] Atopic dermatitis affects the quality of life of patients negatively with the symptoms, the regulation of living conditions for the prevention of attacks, and the possible side effects of the agents used in treatment.[2],[3]
Atopic dermatitis lesions occur based on immunological and environmental factors that result in the development of skin barrier dysfunction.[4] The barrier functions and immunological properties of epidermal keratinocytes play a substantial role in the pathogenesis of AD. Keratinocytes initiate and maintain the inflammation by secreting inflammatory mediators.[5] It has been previously reported that interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP) are released from keratinocytes and precipitate T helper (Th)-2 response resulting in AD pathogenesis.[6–8] Fas/Fas ligand-mediated molecular interactions and caspase activation are demonstrated to play an important role in AD by causing keratinocyte apoptosis.[9] Moreover, it has been shown that increased caspase-3 cleavage, especially in spinous keratinocytes, leads to spongiosis formation in AD. CD4+ and CD8+ lymphocytes play a role in the pathogenesis of AD with increased interferon-gamma activity.[10]
Treatment for AD varies according to age, the severity of disease and accompanying comorbidities. Standard treatment methods are topical anti-inflammatory drugs, phototherapy, systemic steroids and immunosuppressive agents such as cyclosporine and azathioprine.[11],[12] Long-term use of topical corticosteroids, which are the mainstay of mild-moderate AD treatment, are associated with local and systemic side effects such as atrophy, telangiectasia, striae, hypothalamic–pituitary–adrenal axis suppression and growth and development retardation.[11],[13] Immunosuppressive drugs and biological therapies used in patients with severe AD in recent years also have serious side effect profiles.[14] Therefore, the investigation for treatment with high efficacy and low side effect profile continues.
Cinnamon is a compound that has been used in various fields such as medicine, fragrance and cosmetics for many years, in addition to its use as a spice.[15] The chemical components of cinnamon are cinnamaldehyde (cinnamic aldehyde, 3-phenyl-2-propenal), mainly found in the peel, and eugenol (2-methoxy-4-phenol) present in the leaves of it. Other ingredients are cinnamyl, alcohol, coumarin, phenolic acids and carbohydrates.[15] Cinnamaldehyde is the organic compound of cinnamon with the highest rate (65–80%), showing anti-inflammatory and immunomodulatory effects.[16],[17]
The aim of this study is to evaluate the effect of cinnamaldehyde on the epidermal thickness of the lesions, keratinocyte apoptosis and cytokines produced from keratinocytes in mice with AD-like lesions formed with dinitrofluorobenzene (DNFB).
Materials and Methods This experimental animal study was conducted between July to September 2017 with the approval of the Experimental Animals Ethics Committee (#571_23.12.2016).
Twenty-four BALB/c type male mice were used in this study. Mice were six-eight weeks old, weighing 30 ± 2 grams, and kept in standard housing cages until the day of the experiment. Each cage included six mice, drinking water was changed daily and routine cage cleaning was done regularly. Mice were kept at a room temperature of 21°C, and twelve-hour light–dark cycles were implemented. The mice were given ad-libitum standard pellet feed and drinking water throughout the experiment.
Mice were divided into four groups:
Group A (control): Control groupGroup B [AD with no treatment (AD + NoTre)]: Only DNFB applied group.Group C [AD with corticosteroids (AD + Cort)]: DNFB and topical corticosteroid applied groupGroup D [AD with cinnamaldehyde (AD + Cin)]: DNFB and cinnamaldehyde 50 mg/kg/day applied group.Induction of dermatitis: The model defined by Sozmen et al. was used to produce AD-like lesions in mice.[18] Dinitrofluorobenzene, which was used in dermatitis formation, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). It was dissolved in acetone and olive oil (4:1) mixture. For sensitisation, 100 μL of 0.5% DNFB was applied to the shaved dorsum of group B (AD + NoTre), group C (AD + Cort) and group D (AD + Cin) mice once a day during the first week. The next four weeks after the first week, 100 μL 0.2% DNFB was administered twice a week. Atopic dermatitis-like skin lesions developed in all mice at the end of the fifth week [Figure 1]. The administration of 100 μL, 0.2% DNFB, once a week was continued for five to eight weeks to maintain inflammation.
Figure 1: (A) Atopic dermatitis-like skin lesions developed in all mice at the end of the fifth week (B) Control GroupAdministration of topical corticosteroids: Mid-potent topical corticosteroid (mometasone furoate) was applied to the dorsum of mice in group C (AD + Cort) as a thin layer once a day between the fifth and eighth weeks.
Administration of cinnamaldehyde: Cinnamaldehyde at 50 mg/kg/day dose was administered to the mice in group D (AD + Cin) by gastric lavage between the fifth and eighth weeks.
At the end of the eighth week, the animals were sacrificed by decapitation following general anesthesia by the CO2 ventilation method. An area of 2 × 2 cm2 from the dorsum, including epidermis and dermis, was excised. Skin samples were put into buffered formol to be evaluated under a light microscope and sent to Istanbul University Cerrahpasa Medical Faculty Department of Pathology for evaluation. After fixation, biopsy specimens were embedded in paraffin blocks, five-μm serial sections were obtained using a Leica SM 2000R model skid microtome. After staining the samples with hematoxylin and eosin, general tissue properties were examined, and epithelial thickness was measured. Photomicrographs were taken with an Olympus DP70 camera (Olympus, Tokyo, Japan) adapted on an Olympus BX51 model microscope (Olympus Optical, Tokyo, Japan). Photomicrographs were taken from five randomly selected areas from each section. All sections were incubated in a 3% H2O2 solution for five minutes to inhibit endogenous peroxidase activity. Sections were incubated for eighteen hours in +4°C humid room at the following concentrations; IL-33 monoclonal antibody (anti-IL-33 monoclonal antibody Millipore MABF204) at 1:100, IL-25 monoclonal antibody (anti-IL-25 monoclonal antibody Millipore 06-1080) at 1:100, TSLP monoclonal antibody (anti-TSLP monoclonal antibody Millipore ABT330) at 1:100 and anti-caspase 3 antibody (anti-caspase 3 monoclonal antibody ThermoScientif RB-1197-P1) at 1:100. Finally, the sections were stained with diaminobenzidine, Mayer's hematoxylin and examined under a light microscope.
Statistical analysis
SPSS 15.0 for Windows program was used for statistical analysis. Descriptive statistics; numbers and percentages for categorical variables, mean, standard deviation and median for numerical variables were given. Comparison of proportions in independent groups was made by Chi-square analysis. Monte Carlo simulation was applied because the conditions were not met. When the numerical variables did not satisfy the normal distribution condition in the groups, independent more than two-group comparisons were made with the Kruskal–Wallis test. Subgroup analyses were performed using the Mann–Whitney U test and interpreted with Bonferroni correction. A statistical significance level of alpha was accepted as P < 0.05.
Results The epithelial thickness of dorsal skin biopsy samples obtained from mice in group A (control), group B (AD + NoTre), group C (AD + Cort) and group D (AD + Cin) was assessed. Epithelial thickness referring to mean keratinocyte count was significantly higher in group B (AD + NoTre), group C (AD + Cort) and group D (AD + Cin) compared to group A (control) (P = 0.002, P = 0.009, P = 0.004, respectively). No significant difference in the epithelial thickness was found between group B (AD + NoTre), group C (AD + Cort) and group D (AD + Cin) [Table 1].
Table 1: Epithelial thickness measurements and comparisons among the groupsSections obtained from dorsal biopsy samples of mice in all groups were incubated with IL-25, IL-33, TSLP and caspase-3 monoclonal antibodies. Diffuseness and density were scored from 0 to 3 as follows: 0 = none, 1 = mild, 2 = moderate and 3 = severe [Figure 2].
Figure 2: Comparison of immunohistochemical analysis between groups. (a-d) Control group; (e-h) AD model with no treatment; (i-l) AD model with corticosteroid; (m-p) AD model with cinnamadehyte. (e and m) Show higher immunostaining for IL-25 compared to a. (f, j and n) Show higher staining for IL-33 compared to b. Caspase-3 staining is lower in p than in H and significantly lower in p than in L. (a-d, h-j, l-p ×40; e-g, k ×200)Group B (AD + NoTre) had significantly greater staining with IL-25 in terms of both diffuseness and density compared to group A (control) (P = 0.003). Likewise, group D (AD + Cin) was found to have significantly higher staining with IL-25 compared to group A (control) (P = 0.002). No significant difference was observed between group B (AD + NoTre) and group D (AD + Cin) for IL-25 staining [Table 2].
Group B (AD + NoTre), group C (AD + Cort) and group D (AD + Cin) were significantly more stained with IL-33 (diffuseness) than group A (control) (P = 0.002). No significant difference was observed among group B (AD + NoTre), group C (AD + Cort) and group D (AD + Cin) in terms of IL-33 staining [Table 3].
Thymic stromal lymphopoietin staining results were demonstrated in [Table 4]. There was no significant difference in TSLP staining among the groups except for the significant difference between group A (control) and group C (AD + Cort) (P = 0.002).
Table 4: Thymic stromal lymphopoietin staining comparisons among the groupsGroup B (AD + NoTre) had significantly greater staining with caspase-3 in terms of diffuseness and density compared to group A (control) (P = 0.003 and P = 0.002). Group B (AD + NoTre) had significantly higher staining with caspase-3 in terms of diffuseness and density than group D (AD + Cin) (P = 0.003 and P = 0.002). Caspase-3 staining was found significantly lower in group D (AD + Cin) compared to group C (AD + Cort) (P = 0.002) [Table 5].
Discussion In this study, we found that the keratinocyte-derived cytokines (IL-25 and IL-33) and caspase-3 were increased in the AD model, supporting their role in pathogenesis. It was also found that among the other substances examined in this study, only caspase-3 was found to decrease more with cinnamaldehyde than with corticosteroids.
Anti-inflammatory effects of cinnamaldehyde were demonstrated in various studies.[16],[19],[20] In a study in which Wistar albino rats were used, nasal administration of cinnamaldehyde reduced inflammatory cell infiltration and vascular occlusion in plasma cells, eosinophils and lamina propria in an allergic rhinitis rat model, thereby reducing allergic symptoms.[21] Another study reported that cinnamaldehyde had been shown to reduce inflammation by lowering nuclear translocation of toll-like receptor 4, tumour necrosis receptor-associated factor 6 and nuclear factor kappa light chain-enhancing B cells in the cerebral ischemia.[20] Although all these studies showed the various anti-inflammatory effects of cinnamaldehyde, no study investigated the impact of keratinocyte-derived cytokines and keratinocyte apoptosis in experimental AD-like lesions to the best of our knowledge.
Atopic dermatitis arises from the interaction between various cell types including T cells, Langerhans cells, basophils, eosinophils and keratinocytes. Keratinocytes are involved in the persistence of inflammation and immune response through the secretion of proinflammatory mediators.[22] In chronic skin lesions of AD, clinically lichenified plaques show histopathologically psoriasiform acanthosis and epidermal thickening.[23] In our study, the epithelial thickness in group D (AD + Cin) was significantly lower than those in group B (AD + NoTre), though there was no statistical significance.
Keratinocytes are thought to have a critical role in the pathogenesis of AD because of the mediator they secrete and their interactions with other cell types. It was demonstrated in recent studies that the epidermal cytokines IL-25, IL-33 and TSLP play an essential role in the T-helper 2 (Th2) response and the innate immune system with the effect of various environmental factors.[24],[25] Th2 cells and Th2-related cytokines are involved in the development of AD. It has been shown that there is an increase in the levels of Th2-dependent cytokines (IL-4, IL-5 and IL-13), especially in the acute phase of the disease. Recently, studies have shown that mutations in the filaggrin (FLG) gene are associated with impaired skin barrier function in a significant proportion of patients with AD.[25] IL-25 is a newly identified cytokine belonging to IL-17 family and has been shown in mouse models to be involved in the generation and maintenance of the Th2 immune response.[26],[27] It was found that IL-25 and its receptor levels increased in human AD lesions. Moreover, the production of IL-4, IL-5 and IL-13, eosinophilia and IgE levels were found to increase with IL-25 in the asthma mouse model.[28],[29] In our study, Group B (AD + NoTre) stained with IL-25 was significantly higher in diffuseness and density than group A (control). This result supports the role of IL-25 in the formation of AD. IL-25 staining density in group D (AD + Cin) was lower than in group B but it was not statistically significant.
IL-33 is a cytokine belonging to the IL-1 family, which is secreted by stimulation from keratinocytes and endothelial cells. Proinflammatory cytokines induced by IL-33 are known to aggravate atopic diseases such as AD and asthma.[30] In a study, IL-33 was found to have an essential role in the development of AD in mice with ovalbumin.[31] Another study investigated the effects of various IL-33 concentrations in human keratinocytes on FLG expression.[32] IL-33 has been shown to act in AD pathogenesis not only by inducing proinflammatory cytokines but also by contributing to the disruption of the epidermal barrier by reducing FLG.
In a study comparing skin biopsies of AD patients and healthy controls, it was found that IL-33 and ST2L (a transmembrane receptor), which is the receptor for this cytokine, were higher in AD patients. Moreover, IL-33 was found to participate in the pathogenesis of inflammatory responses by increasing IL-6, CXCL-8/IL-8 (C-X-C motif chemokine 8), CCL-20 (CC chemokine ligand-20), CCL-17 (CC chemokine ligand-17) and CCL-5 (CC chemokine ligand-5) levels.[6] In our study, the diffuseness of IL-33 in staining was significantly higher in group B (AD + NoTre) than in group A (control). Our results support the findings in the literature. Moreover, IL-33 staining was lower in group D (AD + Cin) than in group B (AD + NoTre) in our study; however, no statistical significance was found.
It has been shown that TSLP is highly expressed in keratinocytes in AD patients and the severity of AD might be correlated with TSLP levels according to human studies and animal models.[33] In a study conducted to determine the role of TSLP in AD, transgenic mice that express keratinocyte-specific, tetracycline-inducible TSLP transgene were produced.[34] Skin-specific overexpression of TSLP showed the development of eczematous lesions containing dermal cellular infiltrates. A dramatic increase in Th2 cells and the formation of AD-like phenotype with increased serum IgE levels were observed. These transgenic mice demonstrate that TSLP can induce a series of allergic inflammations in the skin and may provide a valuable animal model for the future study of this common disease.[34] In a study that included 71 adults, 61 pediatric patients and 31 adult controls using standard enzyme-linked immunoabsorbent testing techniques, serum TSLP and IL-33 levels were measured, and these values were found to be significantly higher in AD patients compared to the control group.[35] In our study, there was no significant difference between group B (AD + NoTre) and group A (control). This result might be caused by the application of DNFB to the restricted area or an inadequate dose administration.
The aspartate-specific cysteine protease (caspase) cascade is thought to be the main pathway of apoptosis. Caspase-3 is one of the most common proteases in cells and is majorly involved in cellular death pathways.[36] Keratinocyte apoptosis mediated by Fas/Fas ligand molecular interactions and subsequent caspase activation is thought to have an essential role in AD pathogenesis, particularly in the formation of spongiosis.[33] In a study, caspase-3 cleavage was analysed by immunohistology to calculate epidermal caspase activation in normal and AD skin. In the acute phase lesion of AD, increased expression of degraded caspase-3 in keratinocytes in the basal layer and caspase-3 cleavage in one or more layers of the spongiotic area was detected.[10] In another study, it was shown that caspase-3/7, caspase-8, and caspase-9 were increased in biopsy samples from mice with an AD model.[37] In our study, the diffuseness and density of caspase-3 were significantly higher in group B (AD + NoTre) than in group A (control). There was a significantly lower caspase-3 staining in group D (AD AD + Cin) than in group B (AD + NoTre). Furthermore, group D (AD + Cin) significantly reduced caspase-3 more than group C (AD + Cort), in which the topical corticosteroids were used. These results show that cinnamaldehyde might have an anti-inflammatory effect by reducing caspase-3.
In this study, IL-25, IL-33 and TSLP staining were reduced in mice when cinnamaldehyde was administered. However, the results were not statistically significant compared to the control group. We think that the insufficient number of animals or the inadequate concentration of cinnamaldehyde administered orally might have caused it. This is the limitation of our study. However, we believe that our study provides an important finding that caspase-3 is reduced to a greater extent with cinnemaldehyde than with steroid. Caspase-3, together with IL-25 and IL-33, might be valuable markers in the treatment research of AD. Thus, we think that our research will guide future studies.
Acknowledgement
The authors acknowledge the Turkish Society of Dermatology and Istanbul Haseki Training and Research Hospital for the grand that enable the work to be carried out.
Financial support and sponsorship
This study was supported financially by the Turkish Society of Dermatology and Istanbul Haseki Training and Research Hospital.
Conflicts of interest
There are no conflicts of interest.
References 
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