Oral Administration of Vanillin Improves Imiquimod-Induced

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Oral Administration of Vanillin Improves ImiquimodInduced Psoriatic Skin Inflammation in Mice Hui-Man Cheng, Feng-Yuan Chen, Chia-Cheng Li, HsinYi Lo, Yi-Fang Liao, Tin-Yun Ho, and Chien-Yun Hsiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04259 • Publication Date (Web): 26 Oct 2017 Downloaded from http://pubs.acs.org on October 27, 2017

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Oral Administration of Vanillin Improves Imiquimod-Induced Psoriatic Skin

2

Inflammation in Mice

3 4

Hui-Man Cheng,†,‡ Feng-Yuan Chen,§ Chia-Cheng Li,§ Hsin-Yi Lo,§ Yi-Fang Liao,†

5

Tin-Yun Ho,*,§,ǁ and Chien-Yun Hsiang*,

6

†

School of Chinese Medicine, China Medical University, Taichung 40402, Taiwan

7

‡

Department of Integration of Traditional Chinese and Western Medicine, China Medical University Hospital, Taichung 40447, Taiwan

8 9

§

ǁ Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan

12 13

Graduate Institute of Chinese Medicine, China Medical University, Taichung 40402, Taiwan

10 11

⊥

⊥

Department of Microbiology, China Medical University, Taichung 40402, Taiwan

14 15

*

16

China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan. Tel.:

17

+886

18

[email protected]

19

*

Corresponding author. Prof. Chien-Yun Hsiang, Department of Microbiology,

4

22053366

x

2163.

Fax:

+886

4

22053764.

E-mail

address:

Corresponding author. Prof. Tin-Yun Ho, Graduate Institute of Chinese Medicine, 1


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20

China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan. Tel.:

21

+886

22

[email protected]

4

22053366

x

3302.

Fax:

+886

4

22032295.

2


E-mail

address:


23

ABSTRACT

24

Vanillin is one of the most widely used flavoring products worldwide. Psoriasis is a

25

chronic inflammatory skin disorder. Interleukin-23 (IL-23)/IL-17 axis plays a critical

26

role in the psoriasis. Here we analyzed the effect of vanillin on imiquimod

27

(IMQ)-induced psoriatic skin inflammation in mice. Mice were treated topically

28

with IMQ on the back skin and orally with various amounts of vanillin for seven

29

consecutive days. Vanillin significantly improved IMQ-induced histopathological

30

changes of skin in a dose-dependent manner. The thickness and the number of cell

31

layer of epidermis were reduced by 29±14.4% and 27.8±11%, respectively, in mice

32

given with 100 mg/kg vanillin. Microarray showed that a total of 9,042

33

IMQ-up-regulated genes was down-regulated by vanillin, and the biological

34

pathways involved in immune system and metabolism were significantly altered by

35

vanillin. The up-regulated expressions of IL-23, IL-17A, and IL-17F genes were

36

suppressed by vanillin, with fold changes of -3.07±0.08, -2.06±0.21, and -1.62±0.21,

37

respectively. Moreover, vanillin significantly decreased both the amounts of IL-17A

38

and IL-23 and the infiltration of immune cells in the skin tissues of IMQ-treated

39

mice. In conclusion, our findings suggested that vanillin was an effective bioactive

40

compound against psoriatic skin inflammation. Moreover, the downregulation of

41

IL-23 and IL-17 expression suggested that vanillin was a novel regulator of 3


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IL-23/IL-17 axis.

43 44

KEYWORDS: vanillin, psoriasis, interleukin-23, interleukin-17, microarray

4



45

INTRODUCTION

46

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a natural phenolic aldehyde

47

extracted from the seed pods of Vanilla planifolia. Vanillin is widely used in food,

48

cosmetic, and pharmaceutical industries. It has been applied as a flavor ingredient in

49

foods, such ice creams, cookies, and chocolates. It has also been used as a fragrance

50

in perfumes and in medicines or cleaning products to mask unpleasant odors or

51

tastes. Moreover, it is one of the most widely used flavoring products worldwide,

52

with an estimated annual volume of 16,000 metric tons.1 In addition to flavoring and

53

fragrance, vanillin exhibits various biological activities. For example, vanillin

54

suppresses cancer cell migration and metastasis in vitro and in vivo through the

55

downregulation of matrix metalloproteinase-9 activity, nuclear factor-κB (NF-κB)

56

signaling pathways, and phosphatidylinositol 3-kinase activity.2-4 Vanillin displays

57

neuroprotective effects on potassium bromate-induced neurotoxicity in mice via

58

anti-oxidative and anti-inflammatory activities.5 It also exhibits neuroprotective

59

effects in lipopolysaccharide (LPS)-induced Parkinson's disease models in rats by

60

reducing the expression of inducible nitric oxide, cyclooxygenase-2 (COX-2),

61

interleukin-1β (IL-1β), and IL-6 through regulating NF-κB signaling.6 Vanillin

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displays potent anti-inflammatory effects on carbon tetrachloride-induced acute liver

63

injury in rats via reducing the expression levels of tumor necrosis factor-α (TNF-α), 5


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IL-1β, and IL-6.7 It suppresses LPS-induced NF-κB activation and COX-2 gene

65

expression in mouse macrophages.8 It inhibits the vascular permeability in acetic

66

acid-induced permeability model in mice, a model representing the first stage of

67

inflammatory reactions.9 It also prevents and ameliorates trinitrobenzene sulfonic

68

acid-induced colitis via the downregulation of proinflammatory cytokines and

69

NF-κB signaling pathway in mice.10

70

Psoriasis is a T cell-mediated chronic inflammatory disorder of skin that affects

71

approximately 125 million people worldwide.11 Psoriasis usually presents as scaly

72

red plaques on certain areas, such as elbows, knees, and scalp. The microscopic

73

lesion of psoriasis is characterized by the increased proliferation and thickening of

74

epidermis (acanthosis), the abnormal differentiation of keratinocytes in stratum

75

corneum (parakeratosis), and the accumulation of leukocytes in epidermis and

76

dermis.12 IL-23/IL-17 axis plays a critical role in the pathogenesis of psoriasis. Upon

77

trauma and infections, psoriatic dermal dendritic cells are activated to produce IL-23.

78

IL-23, along with TNF, activates T helper 17 (Th17) cells to produce IL-17A, which

79

in turn stimulates keratinocyte proliferation and dermal inflammation.13-15 IL-17 is a

80

crucial cytokine in the immunopathogenesis of psoriasis.12 IL-17 activates IL-17

81

receptors (IL-17Rs) on the keratinocytes and induces the clinical characteristics of

82

psoriasis. However, blockade of IL-17 abolishes the development of psoriasis. For 6



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example, human monoclonal antibodies, including secukinumab and ixekizumab,

84

are IL-17A antagonists that neutralize IL-17A and improve the clinical symptoms of

85

psoriasis. Human monoclonal antibody brodalumab is an IL-17RA antagonist that

86

blocks IL-17RA and displays significant skin clearance in phase III studies.16 These

87

findings suggest that compounds blocking IL-17 signaling transduction might

88

display beneficial effects on the amelioration of psoriasis.

89

Previous

studies

indicated

that

vanillin

reduces

the

expressions

of

90

proinflammatory cytokines, such as IL-1β, IL-6 and TNF-α, and subsequently

91

displays anti-inflammatory activities in vivo. However, no study reports the effect of

92

vanillin on IL-17-associated chronic inflammatory diseases, such as psoriasis.

93

Imiquimod (IMQ) is a toll-like receptor 7 (TLR7) and TLR8 agonist that has been

94

used for the treatment of papillomas, actinic keratoses, and superficial basal cell

95

carcinomas. Moreover, topical application of IMQ induces the skin inflammation

96

resembling psoriasis, and its induction is through IL-23/IL-17 axis.17 Therefore, we

97

established IMQ-induced psoriasis-like skin inflammation in mice in this study.

98

Vanillin has been applied as a flavor ingredient in foods. Various amounts of vanillin

99

were given orally instead of topically for 7 consecutive days. Effects of vanillin on

100

the psoriasis were assessed by skin lesions and histopathological examinations.

101

Mechanisms of vanillin on the improvement of psoriasis were further analyzed by 7


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gene expression profiles, immunohistochemical (IHC) staining, and cytokine

103

enzyme-linked immunosorbent assay (ELISA). Our data showed that oral

104

administration of vanillin improved the macroscopic and microscopic lesions of

105

IMQ-induced psoriatic skin inflammation. Additionally, its improvement might be

106

associated with the down-regulation of IL-23/IL-17 axis.

107 108 109

MATERIALS AND METHODS

110

Chemicals and Reagents. All chemicals, unless indicated, were purchased from

111

Sigma-Aldrich (St. Louis, MO). IMQ cream (5%) (Aldara®) was purchased from

112

3M Pharmaceutical (St. Paul, MN). Rabbit polyclonal antibody against IL-17A and

113

mouse monoclonal antibody against CD11c (integrin αX, dendritic cell marker)

114

were purchased from Santa Cruz (Santa Cruz, CA). Rabbit monoclonal antibodies

115

against CD4 (Th cell marker) and CD11b (integrin α-M/β-2, leukocyte marker), rat

116

monoclonal antibody against Gr-1 (Ly6g, granulocyte marker), and rabbit polyclonal

117

antibody against IL-23 were purchased from Abcam (Cambridge, MA).

118 119

Animal Experiment. BALB/cByJ mice (6-week old, female) were purchased

120

from National Laboratory Animal Center (Taipei, Taiwan). Mouse experiments were 8



121

conducted under ethics approval from China Medical University Animal Care and

122

Use Committee (Permit No. 104-75-N). Mice were maintained under a 12:12

123

light-dark cycle with free access to water and food.

124

Psoriasis-like skin inflammation in mice was induced as described previously.17,18

125

Briefly, mice were received a daily topical dose of 62.5 mg IMQ on both the shaved

126

back skin with an area of 2 × 2 cm and the right ear folds. In mock group, mice were

127

received a daily topical dose of 62.5 mg Vaseline cream. A total of 58 mice was

128

randomly divided into 7 groups of 7-10 mice: (1) mock, (2) IMQ, (3) IMQ + 1

129

mg/kg vanillin, (4) IMQ + 5 mg/kg vanillin, (5) IMQ + 10 mg/kg vanillin, (6) IMQ

130

+ 50 mg/kg vanillin, and (7) IMQ + 100 mg/kg vanillin. IMQ was applied topically

131

and vanillin was given orally for 7 consecutive days. Vanillin was dissolved in water

132

at 10 mg/ml. Mice in mock and IMQ groups were orally given with water. The

133

thickness of right ear was measured using a caliper on Day 1, 3, 5, and 7. The

134

severity of inflammation on the dorsal skin was scored according to the clinical

135

psoriasis area and severity index (PASI) on Day 1, 3, 5, and 7. Erythema and scaling

136

were scored blind and independently on a scale from 0 to 4: 0, none; 1, slight; 2,

137

moderate; 3, marked; 4, very marked. On Day 7, mice were sacrificed and skin

138

samples were collected for histopathological examination, IHC staining, and

139

microarray analysis. Blood samples were collected for cytokine measurement. 9


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Histopathological and IHC Analysis. For histopathological analysis, skin

142

samples were fixed, sectioned, and stained with hematoxylin/eosin (H&E).

143

Histopathological changes were assessed by three investigators in a blind fashion.

144

The thickness and the number of cell layer of epidermis were measured by

145

approximately 40-50 random measurements for each group using ImageScope

146

(Leica Biosystems Imaging, Wetzlar, Germany).

147

For IHC staining, skin tissue sections were incubated with antibodies against

148

IL-23, CD11c, IL-17A, CD4, CD11b, and Gr-1 (1:200 dilution) at 40C overnight,

149

biotinylated secondary antibody at room temperature for 10 min, and avidin-biotin

150

complex reagent at room temperature for 10 min. The slides were then stained with

151

3,3'-diaminobenzidine (Histostain®-Plus, Invitrogen, Camarillo, CA). IL-17A- and

152

IL-23-positive areas were measured using ImageJ (Media Cybernetics, Bethesda,

153

MD) and calculated as (area occupied with brown color/area of whole tissue) × 100.

154

The proportions of CD4-, CD11c-, CD11b-, and Gr-1-positive cell (%) were

155

calculated as (the number of brown cells/the total number of cells) × 100. One

156

hundred cells were counted in each view.19

157 158

Cytokine ELISA. The amounts of IL-17A in sera and skin tissues were measured 10



159

on Day 7 after IMQ induction. Skin tissues were lyzed with RIPA buffer (50 mM

160

Tris-HCl, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl

161

sulfate, 150 mM sodium chloride, 2 mM EDTA, 50 mM sodium fluoride) containing

162

protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Boston, MA).

163

The level of IL-17A was quantified using mouse IL-17A ELISA Ready-SET-Go®

164

(Thermo Fisher Scientific, Waltham, MA).

165 166

RNA Extraction and Microarray. Total RNA was extracted from 30 mg of skin

167

tissues using RNeasy Mini kit (Qiagen, Valencia, CA). The amount of total RNA

168

was quantified using Beckman DU800 spectrophotometer (Beckman Coulter,

169

Fullerton, CA). The integrity of RNA sample was evaluated using Agilent 2100

170

bioanalyzer (Agilent Technologies, Santa Clara, CA). Microarray analysis was

171

performed as described previously.20 Briefly, Cy5 fluorescence-labeled RNA,

172

prepared using MessageAmpTM aRNA kit (Ambion, Austin, TX), was hybridized to

173

Mouse Whole Genome OneArray (Phalanx Biotech Group, Hsinchu, Taiwan). The

174

fluorescent intensity of each spot was scanned by an Axon 4000 scanner (Molecular

175

Devices, Sunnyvale, CA), analyzed by genepix 4.1 software (Molecular Devices,

176

Sunnyvale, CA), and normalized by R program in the limma package using quantile

177

normalization. The number of replicates was three. Fold changes of genes in IMQ 11


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group were calculated by dividing the normalized data of genes in IMQ group by

179

those in mock group. Fold changes of genes in vanillin group were calculated by

180

dividing the normalized data of genes in vanillin group by those in IMQ group.

181

Genes with fold changes ≥ 1.5 or ≤ -1.5 were selected for biological pathway

182

analysis

183

(https://www.advaitabio.com/ipathwayguide.html).

184

pathogenesis

185

(https://www.ncbi.nlm.nih.gov/omim). The hierarchical clustering analysis of

186

inflammation-related genes, including interleukins, chemokines and TNF genes, was

187

performed and displayed using the WebMeV Multiple Experiment Viewer

188

(http://mev.tm4.org). All microarray data are MIAMI compliant database (Gene

189

Expression Omnibus accession number GSE101177).

using

of

psoriasis

iPathwayGuide

were

Genes retrieved

tool involved from

in

the

OMIM®

190 191

Statistical Analysis. Data were presented as mean ± standard error. Data were

192

analyzed by one-way ANOVA and post hoc Bonferroni test using SPSS Statistics

193

version 20 (IBM, Armonk, NY). A p-value < 0.05 was considered as statistically

194

significant.

195 196 12



197

RESULTS

198

Vanillin Improved the IMQ-Induced Psoriasis-Like Skin Lesions in Mice. To

199

investigate whether vanillin was able to improve psoriasis-like skin lesions induced

200

by IMQ, we treated mice with 62.5 mg IMQ topically and various amounts of

201

vanillin orally for 7 consecutive days. The thickness on right ear was measured

202

using a caliper and the severity of inflammation (erythema and scaling) on the back

203

skin was scored according to PASI. As shown in Figure 1A, the mock group,

204

applied with Vaseline cream alone, showed no morphological changes on the back

205

skin. The dorsal skin remained smooth and the thickness of the right ear was

206

consistent during the 7-day trial (Figure 1B). However, there were significant

207

morphological changes in IMQ group. Mice showed psoriasis-like skin

208

inflammation, such as erythema and scaling, from Day 3 onwards. The severity of

209

skin inflammation increased over time and the dorsal skin displayed a rough lesion

210

resembling human plaque psoriasis on Day 7. The thickness of right ear increased

211

from 0.31±0.04 mm on Day 3 to 0.53±0.04 mm on Day 7 after IMQ treatment. By

212

contrast, morphological changes of IMQ-induced skin lesions were improved by

213

vanillin. Less signs of erythema and scaling on the dorsal skin were observed after

214

vanillin treatment. Mice given with 50 and 100 mg/kg vanillin showed significant

215

reduced symptoms on Day 7. Moreover, vanillin significantly reduced the thickness 13


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of right ear in a dose-dependent manner, compared with IMQ group. The thickness

217

of right ear was 044±0.05 mm and 0.41±0.07 mm on Day 7 in mice treated with 50

218

mg/kg and 100 mg/kg vanillin, respectively.

219 220

Vanillin Improved the IMQ-Induced Histopathological Changes of Skin in

221

Mice. H&E staining of skin sections was examined to evaluate the IMQ-induced

222

histopathological changes of skin. In comparison with mock, the application of IMQ

223

induced psoriasiform lesions, including hyperkeratosis, epidermal hyperplasia,

224

acanthosis, and inflammatory cell infiltration (Figure 2A). The thickness of

225

epidermis in IMQ group was 73.3±8.1 µm, compared to 12.7±2.7 µm in mock group

226

(Figure 2B). Moreover, the number of cell layer of epidermis in IMQ group was

227

9.7±2.2 layers, compared to 1.3±0.5 layers in mock group (Supporting information

228

Figure S1). Oral administration of vanillin significantly ameliorated the

229

IMQ-induced histopathological lesions. The thickness and the the number of cell

230

layer of epidermis were significantly reduced by 29.2±14.4% and 27.8±11%,

231

respectively, in mice given with 100 mg/kg vanillin. Moreover, the improvement of

232

vanillin displayed a dose-dependent manner.

233 234

Gene Expression Profiles Affected by Vanillin in IMQ-Induced Skin Tissues. 14



235

To elucidate the mechanism of vanillin on the improvement of IMQ-induced skin

236

inflammation, we extracted RNA samples from mock, IMQ, and IMQ+100 mg/kg

237

vanillin groups, and the gene expression profiles were analyzed by microarray. In

238

comparison with mock, the transcripts of 14,915 genes and 1,030 genes were

239

up-regulated and down-regulated, respectively, by 1.5-fold in IMQ group. However,

240

a total of 9,042 IMQ-upregulated genes was down-regulated by 100 mg/kg vanillin.

241

Genes with fold changes ≥ 1.5 or ≤ -1.5 in vanillin group were selected for

242

biological pathway analysis. Table 1 shows that 20 pathways were significantly

243

altered by vanillin, and half of the altered pathways were associated with

244

metabolism and immune system. Vanillin significantly affected the immune system

245

pathways, including chemokine signaling pathway, T cell receptor signaling pathway,

246

NF-κB signaling pathway, Toll-like receptor signaling pathway, and NOD-like

247

receptor signaling pathway. Therefore, we further evaluated the effects of vanillin on

248

inflammatory gene expression. In a total of 89 interleukin-associated genes, 59

249

genes were up-regulated by IMQ, while half of the IMQ-upregulated gene

250

expressions were down-regulated by vanillin (Figure 3). Additionally, the majority

251

of chemokine- and TNF-associated genes were up-regulated by IMQ and

252

down-regulated by vanillin. Among 58 chemokine-associated genes, the expressions

253

of 41 genes were induced by IMQ, while 16 IMQ-upregulated genes were 15


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down-regulated by vanillin. About 23 TNF-associated genes were up-regulated by

255

IMQ, while 13 IMQ-induced genes were down-regulated by vanillin.

256

We further evaluated the effects of vanillin on psoriasis-related gene expression.

257

Psoriasis-associated genes were retrieved from OMIM® and genes with fold changes

258

≥ 1.5 or ≤ -1.5 in IMQ group are listed in Table 2. Among 58 genes, only two genes,

259

including IL-17D and CDK5 regulatory subunit associated protein 1-like 1 genes,

260

were down-regulated by IMQ. Twenty IMQ-upregulated genes were down-regulated

261

by vanillin, with fold changes ≤ -1.5. These genes included chemokine (C-X3-C)

262

receptor 1, TNF, chemokine (C-C motif) ligand 20, IL-21, IL-17A, IL-17C, and

263

IL-17F genes. IL-23/IL-17 axis has been known to play a crucial role in the

264

pathogenesis of psoriasis. IL-23 is a heterodimeric cytokine composed of IL-23A

265

(IL-23 alpha subunit p19) and IL-12B (IL-12 subunit p40). IMQ up-regulated the

266

expression of IL-17 family genes, except IL-17D gene (data not shown). However,

267

IMQ-induced IL-17 gene expressions were down-regulated by vanillin. IMQ and

268

vanillin decreased the expression of IL-12B gene by 1.43±0.07 fold and 1.08±0.11

269

fold, respectively (data not shown). However, IMQ up-regulated the expression of

270

IL-23A gene by 6.01±1.01 fold, while IMQ-induced IL-23A gene expression was

271

decreased by vanillin by 3.07±0.78 fold. These findings suggested that IMQ induced

272

the expression of inflammatory genes, such as interleukins, chemokines and TNFs, 16



273

which in turn contributed to the pathogenesis of psoriasis. However, vanillin might

274

ameliorate psoriasis by the downregulation of IMQ-induced inflammatory genes,

275

especially IL-23 and IL-17 genes, in skin tissues.

276 277

Vanillin Reduced the Levels of IL-17A and IL-23 and the Infiltration of

278

Inflammatory Cells in Skin Tissues. To verify the microarray data, we measured

279

the amounts of IL-17A in sera and skin tissues by ELISA. As shown in Figure 4A,

280

the amounts of IL-17A in sera and skin tissues were increased by IMQ. However,

281

vanillin treatment significantly decreased the amount of IL-17A to a basal level.

282

IL-23-producing dendritic cells, Th17 cells, and granulocytes are considered as key

283

factors for the pathogenesis of psoriasis. Therefore, we analyzed whether vanillin

284

inhibited the infiltration and the cytokine secretion of these cell subsets in psoriatic

285

skin by IHC staining (Figure 4B). In comparison with mock, IMQ increased the

286

proportions of CD11c- and CD4-positive cells, which were localized in the dermis

287

and in the margin between dermis and epidermis. IMQ also increased the

288

proportions of CD11b- and Gr-1-positive cells, which were mainly localized in the

289

dermis. Additionally, IMQ increased the levels of IL-23 and IL-17A, which were

290

localized in both dermis and epidermis. However, vanillin significantly decreased

291

the proportions of stained cells and the levels of IL-23 and IL-17A in IMQ-induced 17


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psoriatic skin tissues. These findings suggested that vanillin inhibited the infiltration

293

and the cytokine (IL-17A and IL-23) secretion of dendritic cell, Th17 cell, and

294

granulocyte subsets in psoriatic skin tissues.

295 296 297

DISCUSSION

298

Here we induced psoriasis-like skin inflammation by IMQ in BALB/c mice. There

299

are several mouse models of psoriasis. For example, spontaneous mutated mouse

300

strains, such as flaky skin mice (Ttc7fsn/Ttc7fsn), chronic proliferative dermatitis

301

mutant mice (cpdm/cpdm) and homozygous asebia mutant mice (Scd1ab/Scd1ab),

302

display psoriasiform phenotypes.21 Transgenic mice (K5.Stat3) carrying the signal

303

transducers and activators of transcription 3 gene driven by bovine keratin 5

304

promoter develop skin lesions resembling human psoriasis.21 In addition to

305

transgenic mouse models, IMQ induces psoriasis in patients during topical treatment

306

of actinic keratoses and superficial basal cell carcinomas, suggesting its application

307

on the mouse model of psoriasis.22 IMQ induces a psoriasis-like dermatitis with

308

characteristics of scaling and skin thickening in BALB/c mice. Moreover, IMQ

309

induction is mediated via the IL-23/IL-17 axis, resembling the pathogenesis of

310

human psoriasis.17 Luo et al (2016) compared the psoriatic mouse models induced 18



311

by two different brands of IMQ and found that IMQ cream (Aldara®) induces more

312

characteristic psoriasiform lesions than another brand of IMQ cream.23 Therefore,

313

we established psoriatic mouse model in BALB/c mice by topical application of

314

IMQ (Aldara®) on the dorsal skin. Histopathological examination showed psoriatic

315

characteristics, such as hyperkeratosis, epidermal hyperplasia, acanthosis, and

316

infiltration of inflammatory cells. Moreover, IMQ induced the expression of

317

IL-23/IL-17 genes in skin tissues was also consistent with the findings in other

318

reports.

319

The development of psoriasis is mediated via IL-23/IL-17 axis.12-15 IL-23 is a

320

heterodimeric cytokine composed of IL-23A (IL-23 alpha subunit p19) and IL-12B

321

(IL-12 subunit p40). It is released from dendritic cells and mediates the terminal

322

differentiation and the activation of Th17 cells. IL-17 is a pro-inflammatory

323

cytokine produced by activated Th17 cells. Members of the IL-17 family include

324

IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. IL-17A, together with

325

IL-17F, forms the homodimer or heterodimer, interacts with IL-17R on the

326

keratinocytes, and then mediates the hyper-proliferation and differentiation of

327

keratinocytes. It also acts in synergy with TNF-α to induce proinflammatory

328

cytokine production by keratinocytes.12 IL-17C is mainly expressed by epithelial

329

cells following the stimulation with TNF-α. It induces a similar pattern of gene 19


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330

expression of IL-17A. IL-17E is expressed by innate immune cells and tissue

331

stromal cells. Overexpression of IL-17E in transgenic mice induces common

332

features of IL-17A-driven inflammation. IL-17B and IL-17D are expressed in many

333

cell types. However, their roles on the development of psoriasis are still unclear.24 In

334

this study, we found that the expressions of IL-23, IL-17A, IL-17B, IL-17C, IL17E,

335

IL-17F, as well as TNF-α genes in the skin were up-regulated by IMQ. However,

336

IMQ-upregulated

337

down-regulation of IL-23/IL-17-related genes and TNF-α gene might explain the

338

amelioration of psoriatic lesion and the reduction of inflammation in the skin by

339

vanillin. In addition to IL-17, we found that IMQ up-regulated the expressions of

340

IL-4 and IL-10 genes, while vanillin down-regulated the IMQ-induced expressions

341

of IL-4 and IL-10 genes. IL-4 and IL-10 are anti-inflammatory cytokines that inhibit

342

the production of pro-inflammatory cytokines and subsequently diminish the

343

pathological inflammation in tissues.25 Did the down-regulation of IL-4 and IL-10

344

genes by vanillin suggest the unfavorable effects of vanillin as an anti-psoriatic

345

medicine? Immune homeostasis is a complex process and the balancing of the

346

immune response is achieved partly through the actions of cytokines. Physiological

347

counterbalances have been observed between pro-inflammatory cytokines and

348

anti-inflammatory cytokines.26-27 Therefore, although vanillin down-regulated the

gene

expressions

were

suppressed

20


by

vanillin.

The


349

expressions of both pro-inflammatory cytokine genes and anti-inflammatory

350

cytokine genes, counterbalances between cytokines might contribute to the

351

amelioration of psoriatic skin inflammation by vanillin. Whether vanillin alone

352

reduced the expressions of IL-4 and IL-10 gene in mock-treated skin without IMQ

353

and increased the risk of inflammation remained to be further studied.

354

Our data showed that oral administration of vanillin improved IMQ-induced skin

355

inflammation in mice. In human, vanillin is broken down to vanillic acid in the liver

356

and subsequently excreted in the urine. When vanillin is administered orally at a

357

dose of 100 mg/kg to rats, most metabolites are excreted in urine within 24 h. In 48

358

h, 94% of the dose is accounted for as follows: vanillin (7%), vanillic acid (47%),

359

vanillyl alcohol (19%), vanilloylglycine (10%), catechol (8%), 4-methylcatechol

360

(2%), guaiacol (0.5%), and 4-methylguaiacol (0.6%).28 Vanillic acid, the major

361

metabolite of vanillin after ingestion, displays anti-inflammatory activities via the

362

inhibition of oxidative stress, pro-inflammatory cytokine (IL-1β, TNF-α, and IL-33)

363

production, and NF-κB activation in the carrageenan-induced paw edema model in

364

mice.29 Oral administration of vanillic acid significantly reduces the clinical signs of

365

ulcerative colitis and the levels of inflammatory mediators in the dextran sulfate

366

sodium-induced colitis model in mice.30 Treatment of vanillic acid on

367

non-stimulated Jurkat CD4+ T cells reduces the release of IL-12, also suggesting 21


Page 22 of 44

Page 23 of 44


368

that vanillic acid might be an anti-inflammatory metabolite of dietary polyphenols.31

369

Although vanillic acid is one of the metabolites of vanillin after ingestion, vanillin is

370

a natural phenolic aldehyde present in Vanilla planifolia and has been widely used

371

in food, cosmetic, and pharmaceutical industries. Therefore, we evaluated the effects

372

of vanillin instead of vanillic acid on the IMQ-induced psoriatic skin inflammation

373

in mice.

374

We found that oral administration of vanillin down-regulated the expression of

375

IL-23, IL-17A, and IL-17F genes, which were released from dendritic cells and

376

Th17 cells. These findings suggested that vanillin might interact with dendritic cells

377

or Th17 cells and inhibit the expression of these proinflammatory cytokine genes.

378

How did vanillin influence the functions of dendritic cells or Th17 cells in the skin?

379

Transient receptor potential caption channel subfamily V member 1 (TRPV1), also

380

called vallinoid receptor, is a nociceptor present in sensory neurons, skeletal muscles,

381

lymph nodes, and digestive tracts.32 Imaging data showed that TRPV1 closely

382

contacts with dendritic cells and regulates the IL-23/IL-17 pathway, which in turn

383

controls the psoriasiform skin inflammation. Ablation of TRPV1 by resiniferatoxin

384

attenuates skin inflammation and draining lymph node hypertrophy in the IMQ

385

model, also suggesting that TRPV1 promotes local immune responses directly in the

386

skin.33 In this study, we found that IMQ up-regulated the expression of TRPV1 gene, 22



387

while vanillin down-regulated the IMQ-induced expression of TRPV1 gene, with a

388

fold change of -1.47±0.67. Some natural substances, such as capsaicin, zingerone

389

and eugenol, have shown agonistic or antagonistic activities on TRPV1.34 Because

390

the chemical structural similarity of vanillin, we speculated that oral administration

391

of vanillin might display antagonistic effects on TRPV1 in digestive tracts or

392

down-regulate the expression of TRPV1 gene, and subsequently control the skin

393

inflammation in IMQ-induced psoriasis model (Figure 5). Moreover, because the

394

attenuated inflammatory response by TRPV1 is not limited to the skin, it might also

395

explain why vanillin improves other IL-23/IL-17-mediated chronic inflammatory

396

diseases, such as inflammatory bowel disease, in our previous study.10 The affinity

397

of vanillin with TRPV1 receptor and the tissue distribution of vanillin remained to

398

be further studied.

399

Oral administration of 300 mg/kg vanillin displays no toxicity in rats.35 The oral

400

lethal dosage at 50% (LD50) is 1.58 g/kg in rats, equivalent to 3.16 g/kg in mice.36

401

Pharmacokinetic study in rats shows that oral administration of vanillin has a

402

relatively good bioavailability (7.6%).37 Our findings showed that vanillin improved

403

the IMQ-induced skin inflammation at 100 mg/kg, a dosage below LD50. Moreover,

404

the lipophilic property of vanillin suggested that vanillin might be stored in fat

405

tissues in the skin and released over time. In conclusion, these findings suggested 23


Page 24 of 44

Page 25 of 44


406

that vanillin was a bioactive compound against psoriatic skin inflammation with a

407

low toxicity. Moreover, the downregulation of IL-23 and IL-17 expression

408

suggested that vanillin was a novel regulator of IL-23/IL-17 axis.

409 410

FUNDING SOURCES

411

This work was supported by grants from Ministry of Science and Technology

412

(MOST104-2320-B-039-018-MY3,

413

MOST105-2320-B-039-017-MY3), China Medical University (CMU104-H-01 and

414

CMU104-H-02), and CMU under the Aim for Top University Plan of the Ministry of

415

Education, Taiwan.

MOST105-2811-B-039-009,

and

416 417

NOTES

418

The authors declare no competing financial interest.

419 420

ABBREVIATIONS USED

421

COX-2, cyclooxygenase-2; ELISA, enzyme-linked immunosorbent assay; H&E,

422

hematoxylin/eosin; IMQ, imiquimod; IHC, immunohistochemical staining; IL,

423

interleukin, IL-17R, IL-17 receptor; LD50, lethal dosage at 50%; LPS,

424

lipopolysaccharide; NF-κB, nuclear factor-κB; PASI, psoriasis area and severity 24



425

index; Th17, T helper 17; TLR, toll-like receptor, TRPV1, transient receptor

426

potential caption channel subfamily V member 1; TNF-α, tumor necrosis factor-α

25


Page 26 of 44

Page 27 of 44


427

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Casagrande, R.; Verri, W. A. Jr. Vanillic acid inhibits inflammatory pain by

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Alvarez, D.; Paust, S.; Wood, J. N.; von Andrian, U. H. Nociceptive sensory

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(37) Beaudry, F.; Ross, A.; Lema, P. P.; Vachon, P. Pharmacokinetics of vanillin

544

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545

Phytother. Res. 2010, 24, 525-530.

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Page 34 of 44

546

FIGURE CAPTIONS

547

Figure 1. Effect of vanillin on the IMQ-induced psoriasis-like skin lesions in mice.

548

Mice were treated daily with Vaseline cream (mock) or IMQ on the shaved back

549

skin and the right ear for 7 consecutive days. In vanillin groups, mice were applied

550

topically with IMQ and given orally with various amounts (1, 5, 10, 50, or 100

551

mg/kg) of vanillin. (A) Photos are representative images of back skin lesions, taken

552

on Day 7 (n=7-10/group) after IMQ induction. (B) Erythema and scaling of the

553

dorsal skin were scored using a scale from 0 to 4. The thickness of right ear was

554

measured using a caliper. Values are mean ± standard error. *p < 0.05,

555

***

**

p < 0.01,

p < 0.001, compared with IMQ group.

556 557

Figure 2. Effect of vanillin on the IMQ-induced histopathological changes of skin in

558

mice. Mice were treated daily with Vaseline cream (mock) or IMQ on the shaved

559

dorsal skin for 7 consecutive days. In vanillin groups, mice were applied topically

560

with IMQ and given orally with various amounts (1, 5, 10, 50, or 100 mg/kg) of

561

vanillin. (A) H&E stain of skin samples. Skin samples were fixed, and sections were

562

stained with H&E (original magnification 200×). Photos are representative images

563

(n=7-10/group). Scale bar = 100 µm. (B) Quantification of the thickness and the

564

number of cell layer of epidermis. Data were measured from 4-5 sites per mouse per 33


Page 35 of 44


###

p < 0.001, compared with mock. *p

Oral Administration of Vanillin Improves Imiquimod-Induced

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