Anti-pruritic and anti-inflammatory effects of oxymatrine in a mouse model of allergic contact dermatitis

Accepted Manuscript Title: Anti-Pruritic and Anti-Inflammatory Effects of Oxymatrine in a Mouse Model of Allergic Contact Dermatitis Authors: Xiaoyun Xu, Wei Xiao, Zhe Zhang, Jianhao Pan, Yixi Yan, Tao Zhu, Dan Tang, Kaihe Ye, Manish Paranjpe, Lintao Qu, Hong Nie PII: DOI: Reference:

S0923-1811(18)30165-8 https://doi.org/10.1016/j.jdermsci.2018.04.009 DESC 3368

To appear in:

Journal of Dermatological Science

Received date: Revised date: Accepted date:

5-11-2017 18-3-2018 16-4-2018

Please cite this article as: Xu Xiaoyun, Xiao Wei, Zhang Zhe, Pan Jianhao, Yan Yixi, Zhu Tao, Tang Dan, Ye Kaihe, Paranjpe Manish, Qu Lintao, Nie Hong.Anti-Pruritic and Anti-Inflammatory Effects of Oxymatrine in a Mouse Model of Allergic Contact Dermatitis.Journal of Dermatological Science (2018), https://doi.org/10.1016/j.jdermsci.2018.04.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Anti-Pruritic and Anti-Inflammatory Effects of Oxymatrine in a Mouse Model of Allergic Contact Dermatitis

Xiaoyun Xu1, Wei Xiao 1, Zhe Zhang1, Jianhao Pan1, Yixi Yan2, Tao Zhu1, Dan Tang1, Kaihe Ye1，

Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs

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1Guangdong

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Manish Paranjpe3, Lintao Qu3, Hong Nie1*

Road No.38, Jinwan district, Zhuhai, Guangdong, China

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2Chuangyebei

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Research, College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong, China

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3 Department of Neurosurgery, Neurosurgery pain research institute, Johns Hopkins University

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School of Medicine, Baltimore, Maryland 21205, U.S.A

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*correspondence ： Hong Nie, Tel: 86-20-85222810, Fax: 86-20-85224476, E-mail: [email protected].

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Highlight

 Identifying oxymatrine has potential bioactive for ACD by Cell Membrane Chromatography.  Creating a mouse model of ACD.

 Using ACD model to evaluate the anti-pruritus effect of OMT and

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its mechanism.

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Abstract Background

Allergic contact dermatitis (ACD) is a highly prevalent inflammatory disease of the

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skin. As a result of the complex etiology in ACD, therapeutic compounds targeting

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refractory pruritus in ACD lack efficacy and lead to numerous side effects.

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Objective

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In this study, we investigated the anti-pruritic effects of oxymatrine (OMT) and explored its mechanism of action in a mouse model of ACD.

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Method

72 male SPF C57BL/6 mice were randomly divided into control group, ACD model

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group, dexamethasone positive control group (0.08 mg.kg-1) and 3 OMT groups (80, 40, 20 mg.kg-1). OMT was administrated by intraperitoneal injection 1 hour before

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video recording on day 10，24hrs after 2nd challenge with SADBE. Cheek skin fold thickness was measured before treatment and after recording. H&E staining was used for pathological observation. RT-qPCR, Immunohistochemistry and LEGENDplexTM

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assay were used to detect cytokines levels. The population of Treg cells in peripheral blood were detected via flow cytometry. Results OMT treatment significantly decreases the skin inflammation and scratching bouts. It rescues defects in epidermal keratinization and inflammatory cell infiltration in ACD mice. Administration of OMT significantly reduced levels of IFN-γ, IL-13, IL-17A,

TNF-α, IL-22 and mRNA expression of TNF-α and IL-1β. Furthermore, it increased the percentage of Treg cells in peripheral blood of ACD mice. Conclusion We have demonstrated that OMT exhibits anti-pruritic and anti-inflammatory effects in ACD mice by regulating inflammatory mediators. OMT might emerge as a potential drug for the treatment of pruritus and skin inflammation in the setting of ACD.

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Abbreviations

ACD, allergic contact dermatitis; CHS, contact hypersensitivity OMT, oxymatrine;

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DEM, dexamethasone; SADBE，squaric acid dibutylester； IL-13, interleukin-13; IL2, interleukin-2; IL-6, interleukin-6; TNF-α, tumor necrosis factor α; IL-1β, interleukin1β; IL-17, interleukin-17A; IL-22, interleukin-22; IL-4, interleukin-4, IFN-γ,

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interferon-γ; Th1, T helper 1; Th2, T helper 2

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Keywords

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Oxymatrine; Allergic Contact Dermatitis; Anti-pruritus; Anti-inflammatory

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Introduction

Allergic contact dermatitis (ACD)[1] is a highly prevalent inflammatory disease of the skin, which is mediated by hapten-specific T-cells and caused by repeated exposure of

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the skin to the same hapten [2]. ACD usually presents with inflammatory responses

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including erythema, swelling and immune imbalances. Patients with ACD often also

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exhibit refractory pruritus. Pruritus is an uncomfortable sensation which can trigger the

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urge to scratch [3]. Refractory pruritus, also called chronic pruritus, occurs repeatedly and negatively affects attention and sleep quality. Approximately 25% of individuals

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suffer from serious refractory pruritus in their lifetime [4]. Yet the molecular mechanisms underlying refractory pruritus are largely unexplored [5]. As a result,

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effective therapeutic compounds for refractory pruritus remain elusive. Current therapeutic options for refractory pruritus include, antihistamines,

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anticonvulsants, opioid receptor antagonists, serotonin receptor antagonists and glucocorticoid. However, these compounds have large side effects and are not always effective. A lack of effective therapies poses a significant burden to those suffering from

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refractory pruritus. Therefore, developing safe, effective compounds with minimal side effects for refractory pruritus represents unmet medical need. Traditional Chinese medications (TCMs), like Sophorae Flavesentis Radix, have

been known for thousands of years to have therapeutic effects against pruritic and algetic symptoms with minimal side effects, providing a valuable resource to develop novel compounds for the treatment of refractory pruritus in ACD [6]. Using cell

membrane chromatography to screen components from Sophorae Flavesentis Radix we identified oxymatrine (OMT) as a compound that combines with pruritus nerve conduction dorsal root ganglion (DRG) cells which means it has the potential to act as bioactive compound for curing ACD. This study explored the potential anti-pruritic effects of OMT and the associated mechanism using a mouse model of ACD.

therapeutic compounds for refractory pruritus associated with ACD.

Methods

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Animals

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Fundamental insights gained from this study will guide us to develop the novel

A total of 72 C57BL/6 male mice, aged 6 weeks (20-25 g), were used in the present study (Foshan, China). All animals were housed under a 12 h light/dark cycle. All

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animal welfare and experimental procedures were in strict accordance with the Guide

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for the Care and Use of Laboratory Animals and related ethical regulations of Jinan

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University, according to internationally accepted standards. Mice were given an adlibitum access to a standard diet and water. Mice were divided into a control group

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(also called vehicle group), ACD model group, dexamethasone (DEM) positive control

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group (0.08 mg.kg-1) and 3 OMT groups (80 mg.kg-1, 40 mg.kg-1, 20 mg.kg-1). Establishment of ACD model and evaluation of scratching behavior Contact hypersensitivity (CHS) mouse model of ACD was induced by administering

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squaric acid dibutylester (SADBE) on the cheek as reported previously [7]. Briefly, mice were sensitized by topical application of 25 μL 1% SADBE in acetone onto the

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abdominal skin (0.8 cm in diameter) once daily for three consecutive days. Mice in the control group were treated with an acetone vehicle. Five days later, the right cheek of the ACD mice was challenged once daily for two consecutive days with a topical

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application of 25 μL 1% SADBE in acetone. The right cheek of the control mice was treated with the acetone vehicle. On day 10, 24hrs after 2nd challenge, OMT was administered i.p. 1 h before video recording. The related procedure is schematically summarized in Fig. 1. For behavior measurements, mice were housed in a separate, clear plastic container in 9×9×13 cm3. A small amount of bedding was placed in each container. A video recorder was positioned above the mice to record the behaviors of

two mice simultaneously. Four mirrors per mouse were positioned in each container to offer the video recorder a four-sided view in addition to the top view. The experiment was performed in a sound-proof room. All the mice were trained for adaptability 6 days prior to experimentation, then were habituated to the test chamber for half an hour prior to video recording. The number of bouts of scratching directed to the mouse cheek were counted. All experimenters were blind to experimental group during data collection.

Evaluation of skin fold thickness and pathological observation

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Criterion of itch-like scratching behavior was used as reported previously [8].

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Skin-fold thickness of each mice in each group was measured 3 times with a caliper

before the first challenge of SADBE on day 7 and after treatment with the experimental compound and video recording on day 10. Following testing, the mice were executed and

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serum were collected, the mice skin was promptly excised, washed with pre-cooling PBS, some of

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them were putted into -80 ℃ refrigerator for second day RT-qPCR analysis and the rests were

A part of the fixed cheek skin was

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put into 4% formaldehyde for histopathological analysis.

sent to the Pathology Department of the First Affiliated Hospital of Jinan University

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(Guangzhou, China) for hematoxylin-eosin (HE) staining analysis, and another part was

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sent to the Wuhan Servicebio Technology CO., LTD (Wuhan, China) for immunohistochemistry (IHC) staining analysis. For digital quantitative analysis, pictures taken from the mouse skin were captured in a standard way with a digital

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camera DM6000 (Leica, Germany). The digital images were processed using the software Image Pro Plus to measure the positive area fraction.

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Evaluation of mRNA levels of IL-1β and TNF-α in the cheek skin The mRNA levels of IL-1β and TNF-α in the cheek skin were measured by real-time PCR (RT-qPCR). Total RNA was extracted by using Trizol reagent according to the

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manufacturer’s instructions. The cDNA was synthesized from 1000 ng of total RNA by PrimeScript™RT reagent Kit with gDNA Eraser (Perfect Real Time). Each cDNA sample was amplified for the gene of interest and β-actin in a 25 μL reaction volume using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus). All primers used are listed in Table 1. The real-time PCR conditions were 95 °C for 30 s followed by 40 cycles of

95 °C for 5 s, 55 °C for 30 s and 72 °C for 60 s. The mRNA levels of all genes were normalized to GAPDH. Evaluation of Treg cells percentages in peripheral blood Peripheral blood was collected after recording on day 10. Expression of cell surface markers was assessed on fresh, whole heparin sodium-anticoagulated peripheral blood. Lymphocytes were stained with monoclonal antibodies according to manufacturer’s

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recommendation (PE/Cy7-labeled anti-mouse CD4, PE-labeled anti-mouse CD25, APC-labeled anti-mouse CD127). Appropriate κ isotype control (Rat lgG2a-APC,

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BioLegend; Rat lgG2b-PE) were used to evaluate nonspecific staining and create a positive threshold. Red blood cells were lysed using RBC Lysis Buffer. All samples were analyzed on a BD Accuri C6 flow cytometer. Results are expressed as percentages

Evaluation of cytokine levels in serum and spleen

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of CD4+CD25+CD127-/low cells out of the total CD4+ lymphocytes.

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It was reported that SADBE had a whole body reaction[9], the peripheral blood and

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spleen tissue was collected on day 10 after recording. Peripheral blood was incubated

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on ice and the serum layer was removed from the top. Spleen lysate solution was extracted by IP cell lysate and PMSF. Levels of cytokines (IFN-γ, IL-2, IL-4, IL-6, IL-

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13, IL-17A, TNF-α, IL-22) were measured using a LEGENDplexTM Mouse Th

Statistics

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Cytokine Panel by BD FACS VERSE flow cytometer.

All data were presented as mean ± SEM. Data were analyzed via one-way ANOVA with

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Dunnett t3 test. P < 0.05 was considered as statistically significant. Reagents

Squaric acid dibutylester (SADBE) (Tokyo Chemicals Inc., Japan, lot #XZS2F-IP),

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acetone (Sigma Inc, America, lot# P3761), normal saline (Changzhou Lanling Pharmaceutical Co., Ltd, China, lot #H52020069), oxymatrine (Chengdu Push Biotechnology Co., ltd, Chengdu, China, lot #PS0187-0020), dexamethasone (Hubei Tianyao Pharmaceutical Co., Ltd, China, lot # H42020019) pentobarbital sodium (Sigma Inc., America, lot# P3761), TRIZOL (Invitrogen Corp, Carlsbad, CA, USA, lot #103106), PrimeScript™RT Reagent Kit with gDNA Eraser (Perfect Real Time)

(Takara, Dalian, Japan, lot #AK3501), SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, Japan, lot #AK7806), RBC Lysis Buffer (10X) (BioLegend Inc., San Diego, CA, USA, lot #420301), PE/Cy7 anti-mouse CD4 (Biolegend, San Diego, CA, USA,lot#100528), PE anti-mouse CD25 (Biolegend, San Diego, CA, USA, lot#101904), PE Rat IgG2b, κIsotype Ctrl (Biolegend, San Diego, CA, USA, lot#400607), APC anti-mouse CD127 (Biolegend, San Diego, CA, USA, lot#135012),

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APC Rat IgG2a, κIsotype Ctrl (Biolegend, San Diego, CA, USA, lot#400511),

LEGENDplexTM Kits, Th Cytokine Panel (Biolegend, San Diego, CA, USA,

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lot#740005) were used in this study.

Results

Administration of oxymatrine reduced scratching behavior in ACD mice

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As demonstrated in Fig 2, the ACD model group experienced significantly more

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scratching bouts compared to the control group (p < 0.01). ACD mice administered

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DEM or OMT (80 mg.kg-1, 40 mg.kg-1 and 20 mg.kg-1) groups experienced significantly less scratching bouts compared to the model group (p < 0.01).

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Administration of oxymatrine reduced elevations in skin fold thickness in ACD mice

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As shown in Fig 3, changes in cheek skin fold thickness in ACD model group were significantly greater compared to the control group (p < 0.01). Treatment with DEM or

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OMT (80 mg.kg-1, 40 mg.kg-1 and 20 mg.kg-1) significantly decreased cheek skin fold thickness of ACD mice (p < 0.01).

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Defects in epidermal keratinization and inflammatory cell infiltration in ACD mice cheek skin H&E staining was used to characterize pathological abnormalities in the experimental groups (Fig 4). In the control group, epidermal cells were arranged regularly without

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large gaps or morphological abnormalities (Fig 4A). Compared with the control group, severe hyperkeratosis was observed in the cheek skin of ACD model group as a result of increased scratching behavior. Increased lymphocyte infiltration was observed in the ACD model group and the boundary between the dermis and epidermis (Fig 4B). Administration of DEM and OMT (80 mg.kg-1, 40 mg.kg-1 and 20 mg.kg-1) rescued these defects in epidermal keratinization and inflammatory cell

infiltration in ACD mice, however, the immune infiltration was still higher than in healthy control mice, the administration time may be too short, and the mice in administration group could still be in the recovery period.(Fig 4C, 4D, 4E, 4F). Administration of oxymatrine partially rescued elevations in TNF-α and IL-1β mRNA expression in 1% SADBE challenge cheek skin As shown in Fig 5, mean mRNA expression levels of TNF-α and IL-1β measured in the

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cheek skin, were significantly elevated in the ACD model group (p < 0.01) compared

to control. mRNA expression levels of TNF-α in the cheek skin were decreased (p <

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0.01) in the DEM and OMT 80 mg.kg-1, 40 mg.kg-1 and 20 mg.kg-1 groups compared to the ACD model group.

Administration of oxymatrine rescued reductions in Treg cells percentages of

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CD4+ T cells in the peripheral blood of ACD mice

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As shown in Fig 6, the Treg cell percentages of CD4+ T cells in the peripheral blood

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were decreased in the ACD model group (p < 0.05). The OMT 80 mg.kg-1 group showed a significant increase in the Treg cells percentage of CD4+ T cells in the peripheral

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blood (p < 0.01) compared to the model group. The DEM group and OMT 40 mg.kg-1 groups also showed significant increases in Treg cells percentages of CD4+ T cells in

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the peripheral blood (p < 0.05) compared to the model group. The OMT 20 mg.kg-1 group had no statistically significances compared to the model group.

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Administration of oxymatrine rescued elevations of serum concentrations of cytokines in ACD mice

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As shown in Fig 7, serum concentrations of IFN-γ, IL-13, IL-17A, TNF-α and IL-22 were increased in the ACD model group (p < 0.01 or p < 0.05) compared to control. Administration of DEM significantly reduced serum concentrations of IFN-γ, IL-17A,

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TNF-α and IL-22 in ACD mice (p < 0.01). OMT 80 mg.kg-1 reduced serum concentrations of IFN-γ, IL-13, TNF-α and IL-22 (p < 0.01 or p < 0.05). Administration of OMT 40 mg.kg-1 significantly reduced serum concentrations of IFN-γ, IL-17A, TNFα and IL-22 in ACD mice (p < 0.01). Administration of OMT 20 mg.kg-1 significantly reduced serum concentrations of IFN-γ and IL-22 of ACD mice (p < 0.01 or p < 0.05).

Concentrations of IL-2 and IL-4 did not show statistically significant changes between groups. The effect of oxymatrine on cytokine concentrations in spleen As shown in Fig 8, concentrations of IFN-γ, IL-2, IL-6 and IL-17A in spleen were increased in the ACD model group (p < 0.01 or p < 0.05) compared to control. Mice administered DEM showed reduced concentrations of IFN-γ, IL-2, IL-6 and IL-17A in

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the spleen compared to ACD mice (p < 0.01 or p < 0.05). OMT 80 mg.kg-1 mice showed

reduced concentrations of IFN-γ and IL-2 in the spleen compared to ACD mice (p <

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0.01 or p < 0.05). OMT 40 mg.kg-1 mice showed reduced concentrations of IFN-γ, IL-

2, IL-6 and IL-17A in the spleen compared to ACD mice (p < 0.01 or p < 0.05). ACD mice showed decreased levels of IL-4 compared to control (p < 0.05), however, there

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were no statistically significant changes in IL-4 levels in mice administrated DEM or

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OMT compared to the ACD model group.

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Administration of oxymatrine rescued elevations of protein expression of cytokines in ACD mice

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As shown in Fig 9, the protein expression of IFN-γ, TNF-α, IL-2, IL-6 and IL-17A in

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mice skin were significantly increased in the ACD model group compared to control group (p < 0.001 or p < 0.01). Mice administered with DEM showed reduced protein expression of IFN-γ, TNF-α, IL-2 and IL-6 compared to ACD mice (p < 0.001 or p <

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0.01). OMT 80 mg.kg-1 mice showed reduced protein expression of IFN-γ, TNF-α, IL2, IL-6 and IL-17A compared to ACD mice (p < 0.001 or p < 0.01 or p < 0.05). OMT

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40 mg.kg-1 mice showed reduced protein expression of IFN-γ, TNF-α and IL-6 compared to ACD mice (p < 0.001 or p < 0.01). OMT 20 mg.kg-1 mice showed reduced protein expression of TNF-α and IL-6

compared to ACD mice (p < 0.001 or p < 0.01).

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ACD mice showed increased levels of IL-1β compared to control, however, there were no statistically significant changes in IL-1β levels in mice administrated DEM or OMT compared to the ACD model group.

Discussion Chronic pruritus, also called refractory pruritus, is often associated with

repeated episodes has been met with limited therapeutic response. The mechanism underlying refractory remains elusive. Refractory pruritus damages both the physical and psychological health of its patients and, as a result, negatively impacts their quality of life. ACD-associated pruritic symptoms often present with repeated episodes for long periods of time. Therefore, ACD not only involves refractory pruritus but also represents an excellent model to study refractory pruritus.

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SADBE was used in this study to establish a model of ACD. While SADBE is

most commonly used for the treatment of pelade, SABDE can also induce ACD with

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severe itching after repeated usage [10]. In the present study, SADBE was used as an antigen to induce ACD and establish a contact hypersensitivity model for the study of ACD [11, 12]. LEGENDplexTM Mouse Th Cytokine Panel is a fluorescence-encoded-

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bead-based multiplex assay panel suitable for use on various flow cytometers. It

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provides a higher detection sensitivity and broader dynamic range than the traditional

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ELISA.

Itch is a subjective sensation which cannot be accurately represented in an animal

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model. But scratching, a response to itch, can be studied by using the cheek model of

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mice [13]. Therefore, we measured scratching with the hindlimb as an indicator of itch [14]. The itch sensation can be quantified by using a video camera to record the scratching behavior of mice over a period of time and analyzing the number of bouts of

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scratching. In this study, after the usage of SADBE to establish the ACD model, scratching bouts were significantly increased in the model group compared to control,

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which indicates that mice felt a strong itch sensation after the challenge with SADBE. This further supports that the ACD model was successfully established in this study. When compared with model group, the OMT groups showed significant decreases in

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their number of scratching bouts, which indicates that OMT can partially rescue itch sensation and reduce the number of scratching bouts on the cheek in ACD mice. Th17 is a newly identified lineage of effector T cells involved in autoimmunity and immune responses to pathogens[15]. Previous findings have reported an increase in Th17 cell

infiltration and IL-17A expression in the skin of ACD patients[16]. Th17 cells can secrete IL-17A, IL-17F, TNF-α and IL-6 , modulate keratinocyte immune responses and amplify a

nonspecific cytotoxic cascade which further results in a severe and sustained cutaneous inflammatory reaction[17, 18]. In this study, we found the protein expression of IL-17A,

TNF-α, IL-6 in skin was significantly increased in the ACD model group compared to control group. After treatment with OMT, the protein expression of IL-17A, TNF-α and IL-6 in the skin was decreased compare to ACD group., which might explain the antipruritic effects of OMT. IL-13 is an inflammatory cytokine which can induce pruritus

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and is secreted by Th2 cells. Overexpression of IL-13 in the skin can lead to serious

dermatitis with pruritus [19, 20]. Our result shown that serum levels IL-13 were

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significantly increased in the ACD model group compared to control group. Administration of OMT (80 mg.kg-1) decreased the serum concentration of IL-13, which might account for the anti- pruritic effects of OMT.

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We also assessed the anti-inflammatory effect of OMT in ACD mice. Mice in the

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ACD model group showed a significant increase in cheek skin fold thickness compared

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to control. Administration of OMT significantly decreased cheek skin fold compared to ACD mice. H&E staining showed severe hyperkeratosis and increased infiltration of

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lymphocytes in the ACD model group compared to control. Administration of OMT

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partially rescued the cheek skin from epidermal keratinization and inflammatory cell infiltration in ACD mice. Therefore, in this study, we showed that administration of OMT can rescue defects in the skin fold thickness and inflammatory cell infiltration in

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ACD mice. IL-1β is one of the initial markers of Langerhans cell migration in many allergic diseases [21, 22]. The concentration of IL-1β can reflect the severity of

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inflammation. TNF-α is secreted by activated monocytes and macrophages. Secretion of TNF- α can induce secretion of platelet activating factor, IL-6 and IL-1. Further, TNF-α, as a central mediator of inflammation, plays an important role in

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immunoregulation. In this study, we show that administration of OMT can significantly down regulate protein expression level of INF-γ, TNF-α, IL-2, IL-6, IL-17A, also reduce the mRNA expression levels of IL-1β and TNF-α in ACD mice. Further, we show that OMT can significantly reduce serum concentrations of TNF-α in ACD mice. However, due to individual differences in mice, drug absorption capacity inconsistency

may lead to poor dose-dependence, but taken all into consideration, these results demonstrate the anti-inflammatory effects of OMT in ACD mice. Anti-inflammatory effects are often mediated through the regulation of immune responses. In response to hapten, CD4+ T cells differentiate into functionally distinct Th1 or Th2 subsets of cells that can be characterized by the presence of IFN-γ and IL4 signal cytokines, respectively [23, 24]. Th1 cells have been regarded as critical for

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immunity against intracellular microorganisms. ACD associated inflammation is largely mediated by Th1-specific adaptive immunity [25]. Therefore, a Th1/Th2

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imbalance may underlie the onset of ACD. In this study, we demonstrate the ability of

OMT to significantly reduce serum and blood levels of IFN-γ and spleen levels of IL2 in ACD mice. Though we found no statistically significant differences in IL-4 levels

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between control, ACD and OMT mice, reductions in IFN-γ and IL-2 in mice

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administered OMT demonstrate the ability of OMT to down regulate Th1 cell

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expression and rescue Th1/Th2 imbalances in ACD mice. Further, regulating the number of Th1/Th2 cells might be an immunoregulatory effect of OMT.

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The regulation of balance in Th1/Th2 is closely related to Treg cells. Treg cells,

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formerly known as suppressor T cells, are a subpopulation of T cells which modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. The number of Treg cells can influence the development of ACD [22, 26]. A

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2009 study found that anti-inflammatory agents reduce inflammatory response in ACD mice by increasing the number of Treg cells [27]. Th17 cells secrete mainly IL-17A,

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IL-17F, IL-22 and IL-21 cytokines. IL-6 and TGF- β regulate the Treg/Th17 cell balance. In the presence of IL-6, TGF-β can promote Th17 cells to secrete IL-17A, which promotes the development of inflammation [28-30]. Therefore, Th17 /Treg cell

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balance is important for the presence and development of ACD. In this study, we observed that mice administered OMT had decreased cheek skin and serum, spleen levels of IL-17A, decreased cheek skin and serum levels of IL-2, decreased cheek skin and spleen levels of IL-6. In order to detect Treg cells, we used flow cytometry to isolate CD4+CD25+ CD127low/- cells. When compared to the ACD model group, mice administered OMT show an increased Treg cell percentage of CD4+ T cells in the

peripheral blood. These results demonstrate that mice with ACD have more Th17 cells and fewer Tregs. Our results further demonstrate that OMT can rescue this Th17/Treg cell imbalance in ACD mice, suggesting a possible immunoregulatory effect of OMT. In conclusion, by establishing a SADBE-induced ACD cheek model in mice and performing behavioral, histologic, proteomic and transcriptomic analysis, we demonstrate the anti-pruritic effect of OMT on refractory pruritus in ACD mice. OMT-

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mediated rescue of increased IL-13 in ACD mice further confirm the anti-pruritic effect

of OMT. We show that the anti-inflammatory effects of OMT are mediated through a

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decrease in cheek skin fold thickness and downregulation of IFN-γ, TNF-α, IL-2, IL-6,

IL-17A and IL-1β in the cheek skin. The immunomodulatory effects of OMT are rescued Th1/Th2 and Th17/Treg cell imbalances in ACD mice. This study has

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established the anti-pruritic effects of OMT in ACD OMT might emerge as a potential

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drug for the treatment of pruritus and skin inflammation in the setting of ACD and

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potentially other chronic pruritus associated conditions.

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Conflict of interest

The authors declare no conflicts of interest.

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Author contributions

Xiaoyun Xu, Wei Xiao, Zhe Zhang, Jianhao Pan, Yixi Yan, Tao Zhu, and Dan Tang and

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Kaihe Ye designed and carried out the experiment, analyzed the behavioral data,

measured the Treg cells and concentrations of the cytokines in serum and spleen and

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wrote the draft of manuscript. Manish Paranjpe and Lintao Qu revised the manuscript. Hong Nie was in charge of the experiment design, supervision of the project, and final approval of the manuscript.

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Acknowledgements This work was supported by grants from the Chinese National Natural Science

Foundation to H.N. (No. 81373993 and 81673634), Innovation and entrepreneurship training of Jinan University to H.N. (No. 201610559016), Climbing project of Guangzhou province to H.N. (No. PDJH2016B0062).

References [1] Ikoma A, Steinhoff M, Ständer S, Yosipovitch G, Schmelz M: The neurobiology of itch. Nature

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Reviews Neuroscience 7: 535, 2006.

[2] Peng C, Hu JH, Zhu QG, Liu JY, Qin Z, Gao HL, et al.: Studies of effective constituent and compatibility mechanism of prescription of Sophorae Flavesentis Radix. Chinese herbal medicine 11:

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1631-1635, 2008.

[3] Ikoma A, Steinhoff M, Stander S, Yosipovitch G, Schmelz M: The neurobiology of itch. Nature Reviews Neuroscience 7: 535-547, 2006.

[4] Matterne U, Apfelbacher CJ, Vogelgsang L, Loerbroks A, Weisshaar E: Incidence and determinants of chronic pruritus: a population-based cohort study. Acta dermato-venereologica 93: 532-537, 2013.

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[5] Duque MI, Vogel CA, Fleischer AB, Jr., Yosipovitch G: Over-the-counter topical antipruritic agents are commonly recommended by office-based physicians: an analysis of US practice patterns. The Journal

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of dermatological treatment 15: 185-188, 2004.

[6] YZ Z: Sophora lotion of Sophora lotion in the treatment of allergic contact dermatitis Guide of China

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Medicine 10: 308, 2012.

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[7] Fu K, Qu L, Shimada SG, Nie H, Lamotte RH: Enhanced scratching elicited by a pruritogen and an algogen in a mouse model of contact hypersensitivity. Neuroscience Letters 579: 190-194, 2014. [8] Lamotte RH, Shimada SG, Sikand P: Mouse models of acute, chemical itch and pain in humans.

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Experimental Dermatology 20: 778-782, 2011.

[9] Marhaba R, Vitacolonna M, Hildebrand D, Baniyash M, Freyschmidt-Paul P, Zöller M: The importance of myeloid-derived suppressor cells in the regulation of autoimmune effector cells by a

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chronic contact eczema. Journal of Immunology 179: 5071, 2007. [10] Happle R, Kalveram KJ, Buchner U, Echternacht-Happle K, Goggelmann W, Summer KH: Contact allergy as a therapeutic tool for alopecia areata: application of squaric acid dibutylester. Dermatologica

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161: 289-297, 1980.

[11] Scott AE, Kashon ML, Yucesoy B, Luster MI, Tinkle SS: Insights into the quantitative relationship between sensitization and challenge for allergic contact dermatitis reactions. Toxicol Appl Pharmacol 183: 66-70, 2002.

[12] Camouse MM, Swick AR, Ryan CA, Hulette B, Gerberick F, Tinkle SS, et al.: Determination of in

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vivo dose response and allergen-specific T cells in subjects contact-sensitized to squaric acid dibutyl ester. Dermatitis 19: 95-99, 2008. [13] Qu L, Fan N, Ma C, Wang T, Han L, Fu K, et al.: Enhanced excitability of MRGPRA3- and MRGPRD-positive nociceptors in a model of inflammatory itch and pain. Brain : a journal of neurology 137: 1039-1050, 2014. [14] Shimada SG, LaMotte RH: Behavioral differentiation between itch and pain in mouse. Pain 139: 681-687, 2008. [15] Eyerich K, Foerster S, S, Seidl H, Behrendt H, Hofmann H, Ring J, et al.: Patients with chronic

mucocutaneous candidiasis exhibit reduced production of Th17-associated cytokines IL-17 and IL-22. Journal of Investigative Dermatology 128: 2640, 2008. [16] Louten J, Boniface K, de Waal Malefyt R: Development and function of TH17 cells in health and disease. J Allergy Clin Immun 123: 1004-1011, 2009. [17] Milner JD, Brenchley JM, Laurence A, Freeman AF, Hill BJ, Elias KM, et al.: Impaired T(H)17 cell differentiation in subjects with autosomal dominant hyper-IgE syndrome. Nature 452: 773-776, 2008. [18] Zelante T, Luca AD, D'Angelo C, Moretti S, Romani L: IL-17/Th17 in anti-fungal immunity: what's new? European Journal of Immunology 39: 645-648, 2009. [19] Zheng T, Oh MH, Oh SY, Schroeder JT, Glick AB, Zhu Z: Transgenic expression of interleukin-13

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in the skin induces a pruritic dermatitis and skin remodeling. The Journal of investigative dermatology 129: 742-751, 2009.

[20] Dillon SR, Sprecher C, Hammond A, Bilsborough J, Rosenfeld-Franklin M, Presnell SR, et al.:

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Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice. Nature immunology 5: 752-760, 2004.

[21] Schwarz GT: Immunoregulatory Mechanisms Involved in Elicitation of Allergic Contact Hypersensitivity. Immunology Today 7: 37-44, 1996.

[22] Pichowki JS, Cumberbatch M, Dearman RJ, Basketter DA: Investigation of induced changes in sensitization testing. Toxicol In Vitro 14: 351-360, 2000.

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interleukin 1 beta mRNA expression by cultured human dendritic cells as an in vitro approach to skin

N

[23] Matsui K, Mori A, Ikeda R: Langerhans cell‐like dendritic cells stimulated with an adjuvant direct the development of Th1 and Th2 cells in vivo. Clinical & Experimental Immunology 182: 101-107, 2015.

A

[24] Zhu J: T helper 2 (Th2) cell differentiation, type 2 innate lymphoid cell (ILC2) development and

M

regulation of interleukin-4 (IL-4) and IL-13 production. Cytokine 75: 14-24, 2015. [25] Slodownik D, Lee A, Nixon R: Irritant contact dermatitis: a review. Australasian Journal of Dermatology 49: 10-11, 2008.

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[26] Wagner AH, Wittjen I, Stojanovic T, Middel P, Meingassner JG, Hecker M: Signal transducer and activator of transcription 1 decoy oligodeoxynucleotide suppression of contact hypersensitivity. J Allergy Clin Immun 121: 158-165, 2008.

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[27] Lucas JL, Mirshahpanah P, Haas-Stapleton E, Asadullah K, Zollner TM, Numerof RP: Induction of Foxp3+ regulatory T cells with histone deacetylase inhibitors. Cellular immunology 257: 97-104, 2009. [28] Bettelli E, Carrier Y, Gao W: Reciprocal developmental pathways for the generation of pathogenic

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effector TH17 and regulatory T cells. Nature 441: 235-238, 2006. [29] Mangan PR, Harrington LE, O’Quinn DB: Transforming growth factor-β induces development of the TH17 lineage. Nature: 231-234, 2006. [30] Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, B ， S: TGF- β in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 24:

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179-189, 2006.

Figures

Figure 1. The schematic experimental schedule Figure 2. Scratching bouts of mice 30 min after administration of the compound. * p < 0.05, ** p < 0.01, compared with Model Group by one-way ANOVA with Dunnett t3 test. (n =12) Figure 3. Change in skin fold thickness. * p < 0.05, ** p < 0.01, compared with Model Group by

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one-way ANOVA with Dunnett t3 test. (n =12) Figure 4. HE staining of the challenged right cheek skin (×200). (A) Control Group; (B) Model

Group; (C) DEM Group; (D) OMT 80 mg.kg-1 Group; (E) OMT 40 mg.kg-1 Group; (F) OMT 20

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mg.kg-1 Group. The arrows indicate regions of inflammatory cell infiltration.

Figure 5. Normalized relative mRNA expression in the challenged right cheek skin. (A) TNF-α; (B) IL-1β. * p < 0.05, ** p < 0.01, compared with Model Group by one-way ANOVA with Dunnett t3

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test. (n =3)

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Figure 6. Treg cells percentage of CD4+ T cells in peripheral blood. * p < 0.05, ** p < 0.01,

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compared with Model Group by one-way ANOVA with Dunnett t3 test. (n =3) Figure 7. Serum levels of cytokines. (A) IFN-γ; (B) IL-2; (C) IL-4; (D) IL-13; (E) IL-17A; (F) TNF-

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α; (G) IL-22. * p < 0.05, ** p < 0.01, compared with Model Group by one-way ANOVA with

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Dunnett t3 test. (n =3)

Figure 8. Concentration of cytokines in the spleen. (A) IFN-γ; (B) IL-2; (C) IL-4; (D) IL-6; (E) IL-

test. (n =3)

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17A. * p < 0.05, ** p < 0.01, compared with Model Group by one-way ANOVA with Dunnett t3

Figure 9. Normalized relative protein expression in the challenged right cheek skin (×200). (B) A:

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IFN-γ; B: TNF-α; C: IL-2; D: IL-6; E: IL-17A; F: IL-1β. * p < 0.05, ** p < 0.01, *** p < 0.001,

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compared with Model Group by one-way ANOVA with Dunnett t3 test. (n =4-5)

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A

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N

A

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N

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Primer

IL-1β

forward primer

5′- CCTTGTGCAAGTGTCTGAAGCAGC-3′

IL-1β

reverse primer

5′- GCCACAGCTTCTCCACAGCCA -3′

TNF-α

forward primer

5′ TCTACTGAACTTCGGGGTGATCG 3′

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Gene

Sequence 5′-3′

reverse primer

5′ ACGTGGGCTACAGGCTTGTCA 3′

GAPDH

forward primer

5′- TTCAGTGATGTGGACTTGGAC -3′

reverse primer

5′- CTGAGAGGGAAATCGTGCGT -3′

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TNF-α

GAPDH

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Table 1. Primers used for RT-qPCR