c mice

Journal Pre-proof Artesunate attenuates 2, 4-dinitrochlorobenzene-induced atopic dermatitis by downregulating Th17�cell responses in BALB/c mice Xin-Yu Bai, Ping Liu, Yee-Wen Chai, Yan Wang, Shuang-Hua Ren, Ying-Ying Li, Hong Zhou PII:

S0014-2999(20)30112-6

DOI:

https://doi.org/10.1016/j.ejphar.2020.173020

Reference:

EJP 173020

To appear in:

European Journal of Pharmacology

Received Date: 26 June 2019 Revised Date:

7 February 2020

Accepted Date: 14 February 2020

Please cite this article as: Bai, X.-Y., Liu, P., Chai, Y.-W., Wang, Y., Ren, S.-H., Li, Y.-Y., Zhou, H., Artesunate attenuates 2, 4-dinitrochlorobenzene-induced atopic dermatitis by down-regulating Th17�cell responses in BALB/c mice, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/ j.ejphar.2020.173020. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier B.V.

Contributions: (I)

Conception and design: Xin-Yu Bai;

(II)

Administrative support:; Hong Zhou;

(III)

Provision of study material: Yan Wang and Ying-Ying Li;

(IV)

Collection and assembly of data: Shuang-Hua Ren；

(V)

Data analysis and interpretation: Xin-Yu Bai and Ping Liu;

(VI)

Manuscript writing: Ping Liu and Yee-Wen Chai;

(VII) Final approval of manuscript: All authors.

1

Artesunate attenuates 2, 4-dinitrochlorobenzene-induced atopic dermatitis by

2

down-regulating Th17 cell responses in BALB/c mice

3 4

Xin-Yu Baia†, Ping Liua†, Yee-Wen Chai b, Yan Wang a, Shuang-Hua Ren a, Ying-Ying Li a,

5

Hong Zhou a

6 7

a

8

Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University,

9

Zunyi, Guizhou 563000, China

Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International

10

b

11

Sabah University, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia

Programme of Industrial Chemistry, Faculty of Science and Natural Resources, Malaysia

12 13

†

These authors contributed equally to this work

14 15

* Corresponding author.

16

Email address: [email protected] (H. Zhou)

1

17

ABSTRACT

18

Steroidal agent is a standard clinical treatment of atopic dermatitis; however, have serious side

19

effects. Artesunate is reported to exhibit anti-inflammatory properties although its effect on

20

atopic eczema remains unknown. We investigated the therapeutic effects and possible

21

mechanism of systemic artesunate on DNCB-induced atopic dermatitis in a BALB/c mouse

22

model. To ascertain artesunate (5 and 10 mg/kg) efficacy, skin dermatitis severity and ear, spleen,

23

and lymph node weight were evaluated. Skin tissue mRNA and protein expression and serum

24

cytokine levels were examined. Artesunate significantly improved atopic dermatitis symptoms,

25

decreasing the dermatitis score, ear weight difference, spleen weight, and lymph node weight

26

compared with those following DNCB treatment. Artesunate reduced ear and skin epidermal

27

thickness and mast cell infiltration, as determined using hematoxylin-eosin and toluidine blue

28

staining, respectively. The basal level of IgE (287.67 ± 70.41 ng/ml) and TNF-α (19.94 ± 3.98

29

pg/ml) were Significantly elevated by DNCB (IgE: 1273.23± 176.53 ng/ml; TNF-α: 57.53 ±

30

3.87 pg/ml)，while markedly been suppressed in the treatment group (AS-L: IgE: 1100.25±

31

135.32 ng/ml; TNF-α: 38.47 ± 3.26 pg/ml; AS-H: IgE: 459.46 ± 74.75 ng/ml; TNF-α: 24.38 ±

32

3.85 pg/ml). Among Th17 cell-related factors, DNCB treatment increased mRNA expression of

33

IL-6, IL-17, IL-23, STAT3, and ROR-γt, but reduced TGF-β and SOCS 3; While artesunate

34

reverse these changes. Compared with the model group, artesunate promoted SOCS3 protein and

35

significantly inhibited ROR-γt protein and STAT3 phosphorylation. Thus, artesunate attenuates

36

DNCB-induced atopic dermatitis by inhibiting the release of inflammatory cytokines and

37

downregulating Th17 cell responses in atopic dermatitis mice. 2

38

Keywords: Artesunate, 2,4-Dinitrochlorobenzene, Atopic dermatitis, Cytokine, Th17 cell

39

3

40 41

1. Introduction Atopic dermatitis (AD), also known as atopic eczema, constitutes a prevalent skin disorder

42

that can occur at any age albeit affecting approximately 10–30% of young children (Ku et al.,

43

2017), with increasing prevalence in industrialized and developed countries (Kang et al., 2016).

44

Patients with AD experience significantly reduced quality of life (Ku et al., 2017) along with

45

psychological distress, placing a considerable burden on patients and their families (Serra-

46

Baldrich et al., 2018).

47

AD onset tightly associates with the skin component, skin microbiome, and immune system

48

interaction network (Serra-Baldrich et al., 2018), with complex environmental and genetic factor

49

interaction with skin barrier dysfunction causing inflammation (Hou et al., 2017). AD clinical

50

manifestations include severe edema, pruritus, lichenification, dryness, and erythematous, which

51

are commonly controlled by steroidal agents. However, these exhibit side effects such as skin

52

thinning, atrophy, and immune-suppression upon long-term application (Jiang et al., 2017; Kim

53

et al., 2015). Therefore, safe and effective drugs are required for AD treatment.

54

AD pathogenesis remains incompletely elucidated. Structural and functional skin barrier

55

impairment enhances allergen penetration into the skin and inhibits skin interaction with its

56

microbiome or environmental factors. Inflammation constitutes a hallmark AD pathogenesis.

57

The mediators produced in this phase contribute to skin barrier impairment and cell activation

58

including of keratinocytes, thereby enhancing inflammation through proinflammatory cytokine

59

release. In the context of an altered epidermal barrier, antigens encounter epidermal Langerhans

60

cells and inflammatory epidermal dendritic cells bearing trimeric high-affinity Immunoglobulin

61

(Ig)E receptors. Antigen uptake thereby initiates sensitization, leading to T-cell-driven immune

62

responses (Cabanillas & Novak, 2016; Eyerich & Novak, 2013). Antigen presenting cell, 4

63

dendritic cell, and macrophage stimulation during the inflammatory process results in cytokine

64

secretion, promoting naïve T-cell development into four subtypes: (1) Th1, producing e.g. tumor

65

necrosis factor (TNF)-α, interferon-γ, and interleukin (IL)-12; (2) Th2, producing e.g. IL-4, IL-5,

66

and TNF-α; (3) Th17, triggered by IL-6 and transforming growth factor (TGF)-β and producing

67

IL-6, IL-17, and TNF-α; and (4) Treg, triggered in the absence of proinflammatory mediators,

68

which suppress immune responses (Yang et al., 2012). In particular, Th17 cells facilitate

69

autoimmune and inflammatory disease propagation and development and can reflect the

70

underlying immunological mechanisms. As increased Th17 cells promotes inflammation

71

maintenance, Th17 cell upregulation induces inflammatory and autoimmune disease (Noack &

72

Miossec, 2014).

73

The anti-inflammatory agent artesunate, a semi-synthetic derivative of artemisinin isolated

74

from Artemisia annua (Li et al., 2008a), is commonly utilized for severe malaria treatment and

75

antitumor and contragestational applications. For example, artesunate plays a protective role by

76

inhibiting the mRNA expression of TNF-α and decreasing IL-6 secretion (Li et al., 2008b);

77

artesunate also inhibits IL-17, TNF-α, and other inflammatory cytokines secreted by synovial

78

cells (Liu et al., 2017). However, the effect of artesunate on Th17 cell regulation its efficacy in

79

AD treatment still not been elucidated (H. Chen and Maibach, 1994). Therefore, here we

80

investigated whether systemic artesunate was effective for 2, 4-dinitrochlorobenzene (DNCB)-

81

induced AD and the possible molecular mechanisms.

5

82

2. Materials and methods

83

2.1 Animals

84

In this study, we utilized 32 female BALB/c mice (six-week-old) purchased from the

85

Experimental Animal Centre of Army Medical University, formerly known as the Third Military

86

Medical University (Chongqing, China). Each mouse weighed 20 ± 2 g and was maintained

87

under specific pathogen-free conditions. The mice were kept in the animal room at a constant

88

temperature of 22 ± 2 °C and a relative humidity of 75 ± 10% with a 12-h light-dark cycle. They

89

were fed with a laboratory diet and water ad libitum. The experimental procedures were

90

approved by the ethical committee of Zunyi Medical University (license number: ZMUER2017-2-

91

234).

92 93

2.2 Establishment of the mouse model of AD and artesunate treatment

94

Mice were divided into four groups: untreated mice (control group), AD-induced mice

95

(DNCB group), AD-induced mice treated with 5 mg/kg artesunate (AS-L group), and AD-

96

induced mice treated with 10 mg/kg artesunate (AS-H group). Each experimental group

97

consisted of eight mice. To establish the AD model, allergen sensitization and challenge for AD

98

development were performed in accordance with the protocol (Fig. 1A). The dorsal skin of each

99

mouse was shaved over an area of 2 cm × 2 cm. The mice from the DNCB group were sensitized

100

on Day 1, 4, 8, and 11 by applying 100 and 10 µL of 1% DNCB in acetone/olive oil (4:1) to the

101

dorsal skin and right ear respectively. On Day 15, 18, 21, 25, 28, 32, and 35, each mouse within

102

the DNCB group received 100 and 10 µL of 0.5% DNCB applied to the dorsal skin and right ear,

103

respectively. On Day 35, skin scoring was conducted at 30 min following the last challenge with

104

0.5% DNCB. Blood samples were collected from the eye venous plexus of mice. Serum samples 6

105

were obtained by centrifugation and stored at −20 °C until used for analysis. On Day 15, 18, 21,

106

25, 28, 32, and 35, the mice from the AS-L and AS-H groups were treated intraperitoneally with

107

5 and 10 mg/kg of artesunate, respectively, whereas the mice from the control and DNCB groups

108

were administered saline. Drug treatment was conducted twice daily at 08.30 and 20:30.

109 110 111

2.3 Evaluation of skin dermatitis severity The severity of dorsal skin dermatitis was evaluated by scoring clinical symptoms. The skin

112

dermatitis severity score was calculated by summing up the scores for erythema/hemorrhage,

113

edema, excoriation/erosion, and scaling/dryness on the following scale: 0 (none), 1 (mild), 2

114

(moderate), and 3 (severe) (Qiao et al., 2017). Mice were sacrificed by decapitation 24 h

115

following the last 0.5% DNCB challenge and the ear, skin, lymph node, spleen, and blood from

116

the eyes were obtained for analysis.

117 118

2.4 Weight measurement of the ears, spleen, and lymph node

119

The weights of the ear, spleen, and lymph node were measured using an electronic balance

120

(Mettler Toledo, Columbus, Ohio, USA). The weight difference between the left and right ears

121

of each mouse was calculated. The sizes and weights of the spleen and lymph node were

122

compared among the groups using the following calculations:

123

Weight index of the spleen = weight of the spleen / body weight × 100%

(1)

124

Weight index of the lymph node = weight of the lymph node / body weight × 100% (2)

125 126

2.5 Histopathological observation

7

127

The dorsal skin and right ear tissues of mice were fixed in formalin, embedded in paraffin,

128

deparaffinized with xylene, and 5-µm-thick sections of skin and ear tissues were cut and

129

mounted on slides. The mounted tissues were stained with either hematoxylin-eosin for counting

130

of inflammatory cells or toluidine blue for counting mast cells. Evaluation of sections was

131

conducted under a light microscope with 100× magnification for quantitative analysis (Lee and

132

Cho, 2017; Wee et al., 2017).

133 134 135

2.6 Enzyme-linked immunosorbent assay (ELISA) The serum levels of TNF-α and IgE were determined using the appropriate ELISA kits

136

(Invitrogen, Vienna, Austria). The IgE and TNF-α concentrations were evaluated by

137

interpolation from a standard curve following the measurement of optical density at 450 nm.

138 139 140

2.7 Quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) RT-qPCR was performed to determine gene expression in the dorsal skin. Isolation of total

141

RNA from the dorsal skin was carried out using RNAiso Plus (TaKaRa, Kusatsu, Shiga, Japan).

142

Total RNA concentration was quantified by measuring the absorbance at 260 nm and RNA

143

purity was assayed using the ratio of absorbance at 260 and 280 nm. Complementary DNA

144

(cDNA) was synthesized using diethylpyrocarbonate water (DEPC-H2O) (Generay Biotech,

145

Shanghai, China) and PrimeScriptTM RT Reagent Kit (Perfect Real Time) (TaKaRa) containing

146

PrimeScript Buffer (for Real Time), PrimeScript RT Enzyme Mix I, Oligo dT Primer, and

147

Random 6 mers. The mRNA expression levels of β-actin, IL-6, IL-17, IL-23, TGF-β, suppressor

148

of cytokine signaling (SOCS)3, signal transducer and activator of transcription (STAT)3,

149

retinoid-related orphan receptor (ROR)-γt, and β-actin were evaluated using RT-qPCR analysis. 8

150

The primers were purchased from Sangon Biotech (Guiyang, China) (Table 1). Amplification of

151

cDNA of IL-6, IL-17, IL-23, TGF-β, SOCS3, STAT3, ROR-γt, and β-actin was performed in

152

Hard Shell® 96-well PCR plates (Bio-Rad, Hercules, CA, USA). The final 15-µL reaction

153

mixture consisted of 3 µL cDNA and 12 µL of a mixture of 7.5 µL iQTM SYBR® Green

154

Supermix (Bio-Rad), 0.5 µL primer, and 4 µL DEPC-H2O. The reaction was performed using the

155

CFX Connect Real-Time System (Bio-Rad). For amplification, the cycling conditions consisted

156

of pre-degeneration at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 45

157

s. The average transcript levels of genes were normalized to β-actin using the following formula:

158

relative mRNA expression = 2−(Ct of target gene –∆ Ct of β-actin), where Ct is the threshold cycle value.

159 160

Table 1

161

Primer sequences for RT-qPCR.

Gene

GenBank

Forward primer (5′–3′)

Reverse primer (5′–3′)

Il4

NM_021283.2

TACCAGGAGCCATATCCACGGATG

TGTGGTGTTCTTCGTTGCTGTGAG

Il5

NM_010558.1

CCTCATCCTCTTCGTTGCATCAGG

TGATCCTCCTGCGTCCATCTGG

Il6

NM_001314054.1

ACTTCCATCCAGTTGCCTTCTTGG

TTAAGCCTCCGACTTGTGAAGTGG

Il17a

NM_010552.3

TCACTCCTGCTGATTCGGGT

CTCAGTGCCACCTCCAGACT

Il23a

NM_031252.2

ACCTGTAGTGGTGGTGGTGGAG

GGACCAGATAACTGTTGGCAGAGC

Tgfb1

NM_011577.2

GCCATGAGCGGTCCATCACG

CAGTCAGCATCCACGCACCAC

Socs3

XM_021176627.1

CTGCTCTTACGACCGCTGTCTCG

ATGTTGGCAGCCGTGAAGTCTAC

Stat3

NM_011486.5

GCATGGAGGCGTGTCTTGGC

TGTACCTCAGCGATCCGGTTAGG

9

RorcNM_011281

TCACTCCTGCTGATTCGGGT

CTCAGTGCCACCTCCAGACT

variant 1a

CATCCGTAAAGACCTCTATGCCAA β-actin

NM_007393.5

ATGGAGCCACCGATCCACA C

162

a

Encodes ROR-γt

163 164 165

2.8 Western blotting analysis Protein was obtained from the skin tissues of mice using Radio Immunoprecipitation Assay

166

lysis buffer (Beyotime Biotechnology, Haimen, Jiangsu, China) and the tissues were lysed by

167

incubating on ice. The mixture was then centrifuged at 13,800 g and 4 °C for 10 min and the

168

supernatant was collected. Concentration of protein in each sample was determined using the

169

enhanced BCA proteins assay kit (Beyotime Biotechnology). Protein samples (30 µg) were

170

separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and

171

electrophoretically transferred onto nitrocellulose blotting membrane (GE Healthcare Life

172

Science, Dassel, Germany). The membranes were blocked with 5% no-fat milk (dissolved in

173

Tris-buffered saline Tween-20), followed by incubation with the appropriate primary antibodies:

174

rabbit anti-ROR-γt (1:2000, Abcam, Cambridge, UK), rabbit anti-STAT3 (1:2000, Cell

175

Signaling Technology, Boston, MA, USA), rabbit anti-p-STAT3 (1:2000, Cell Signaling

176

Technology), rabbit anti-SOCS3 (1:2000, Abcam), and rabbit anti-β-actin (1:2000, Proteintech,

177

Rosemont, IL, USA), at 4 °C overnight. Membranes were then washed thrice with Tris-buffered

178

saline Tween-20 prior to reaction with horseradish peroxidase-conjugated appropriate secondary

179

antibody (1:6000) for 1 h at 20 °C. The membranes were visualized by chemiluminescence using

180

ECL detection reagents (Millipore Corporation, Billerica MA, USA) and exposed to X-ray films.

10

181

Results were normalized to the internal control β-actin and the optical density was quantified

182

using Image J 1.8.0 software (https://imagej.nih.gov/ij/).

183

2.9 CCK-8 assay

184

The effects of SR1001 on the viability of HaCaT cells were analyzed using the CCK-8 kit

185

(L0724; Dojindo, Kumamoto, Japan). HaCaT cells were seeded in 48-well plates at 10,000

186

cells/well. Following various treatments, the serum-free medium was replaced with 20 µl CCK-8

187

working solution and cells were then incubated for 1 h at 37 °C. The absorbance of each well

188

was measured using a Vmax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA,

189

USA) at 450 nm.

190 191 192

2.10

Cell culture and treatment

The HaCaT cell line, purchased from the Cell Lines Service (Cellcook Biotech Co., Ltd.,

193

Guangzhou, China) was cultured in modified Eagle’s medium supplemented with 10% fetal

194

bovine serum (42Q3194K; Gibco, Gaithersburg, MD, USA) at 37 °C in an atmosphere of 5%

195

CO2 and 95% air. HaCaT cells were incubated with lipopolysaccharide (LPS) from Escherichia

196

coli O111:B4 (L1430; Sigma-Aldrich, St. Louis, MO, USA) for 12 h to induce inflammatory

197

injury. The RORα and RORγt inverse agonist SR1001 (#15471; MedChemExpress, Monmouth

198

Junction, NJ, USA) was dissolved in dimethylsulfoxide to obtain a concentration of 10 mM and

199

diluted to 1, 2.5, 5, and 10 µm to treat cells for 12 h. Artesunate (ZA1190208; Gulin

200

Pharmaceutical Co., Ltd., China) was dissolved in sodium bicarbonate ( T6190203; Gulin

201

Pharmaceutical Co., Ltd.) to obtain a concentration of 10 mM and diluted to 20 mg/mL add to

202

medium treat cells for 12 h.

203 11

204 205

2.11

Statistical analysis

The experimental data are expressed as the mean ± standard error of the mean (S.E.M.). One-

206

way analysis of variance (ANOVA) and Tukey’s honest significant difference test were used to

207

compare the results among groups. Two-way ANOVA was used to compare the interaction

208

between artesunate and SR1001. A significance level of P < 0.05 was considered statistically

209

significant. Statistical analysis was performed using SPSS 22.0 software (IBM, Armonk, NY,

210

USA).

12

211

3. Results

212

3.1 Effects of artesunate on skin severity and ear weight difference in the AD mouse model

213

Our results showed that DNCB could induce the development of hemorrhage, edema, erosion,

214

scaling, and dryness in mice. AD symptoms were relieved following treatment with artesunate

215

(Fig. 1B). DNCB sensitization caused a significant increase of the dermatitis severity score in the

216

DNCB group (***P < 0.001 vs. control group), which was markedly decreased by AS-H

217

treatment (†††P < 0.001 vs. the DNCB group) (Fig. 1C).

218

Weight difference between left and right ears was calculated to evaluate the therapeutic

219

effect of artesunate on DNCB-induced AD in the mouse model. The weight difference between

220

the ears was significantly elevated in the DNCB group compared with that in the control group

221

(***P < 0.001 vs. the control group). The ear weight difference in the AS-H group was

222

significantly decreased compared with that of the DNCB group (†††P < 0.001 vs. the DNCB

223

group) (Fig. 1D).

224

3.2 Effects of artesunate on the weight index of the spleen and lymph node

225

Spleen weight in the DNCB group was the heaviest among the four groups, followed by the

226

AS-L, AS-H, and control group (Fig. 2A). The weight of the spleen in the DNCB group was

227

increased as compared to that in the control group (***P < 0.001 vs. the control group). The

228

spleen weight was decreased by low-dose (†P < 0.05 vs. the DNCB group) and high-dose (†††P <

229

0.001 vs. the DNCB group) artesunate compared with that of the DNCB group (Fig. 2A). The

230

spleen index was also calculated. The spleen index in the DNCB group was the largest among

231

the four groups (***P < 0.001 vs. the control group). Low-dose (††P < 0.01 vs. the DNCB group)

232

and high-dose (†††P < 0.001 vs. the DNCB group) artesunate treatment significantly improved

233

the abnormal elevation of the spleen index (Fig. 2B). 13

The weight of the lymph nodes in the DNCB group was markedly increased compared with

234 235

that in the control group (***P < 0.001 vs. the control group). High-dose artesunate (†††P < 0.001

236

vs. DNCB group) significantly reduced the lymph node weight in AD mice sensitized by DNCB.

237

However, the AS-L group did not exhibit any significant difference in comparison with the

238

DNCB group (Fig. 2C). Calculation of the lymph node index (Fig. 2D) revealed a similar trend

239

as that of the lymph node weight.

240 241

3.3 Effects of artesunate on epidermal thickness and mast cell number of the ear and skin Hematoxylin-eosin staining was conducted to determine the effectiveness of artesunate

242 243

toward moderating ear (Fig. 3A) and skin (Fig. 3B) epidermal thickness. Toluidine blue staining

244

was conducted to determine the effectiveness of artesunate on ear (Fig. 3C) and skin (Fig. 3D)

245

mast cell numbers. Epidermal thickness and mast cell number of the ear and skin were

246

maintained at low levels in the control group. However, administration of DNCB significantly

247

increased the ear and skin epidermal thickness and mast cell number compared with those of the

248

control group (***P < 0.001 vs. the control group). The epidermal thickness of the ear in the AS-L

249

and AS-H groups was markedly decreased as compared to that of the DNCB group (AS-L group,

250

††

251

was observed with regard to the epidermal thickness of the skin. AS-L and AS-H treatment

252

effectively attenuated DNCB-induced AD-like skin inflammation in mice (AS-L group, †P <

253

0.05 vs. the DNCB group; AS-H group, †††P < 0.001 vs. the DNCB group).

P < 0.01 vs. the DNCB group; AS-H group, †††P < 0.001 vs. the DNCB group). A similar effect

254

The DNCB group also exhibited a massive infiltration of mast cells. The numbers of mast

255

cells in the ear and skin were markedly higher in the DNCB group than in the control group (***P

256

< 0.001 vs. the control group). AS-L and AS-H groups exhibited significantly reduced ear mast 14

257

cell numbers compared with that of the DNCB group (AS-L group, †P < 0.05 vs. the DNCB

258

group; AS-H group, †††P < 0.001 vs. the DNCB group). No significant difference was observed

259

between DNCB and AS-L groups although the skin mast cell number in the AS-L group was

260

lower than that in the DNCB group. Conversely, the AS-H group exhibited markedly decreased

261

skin mast cell number as compared to that of the DNCB group (††P < 0.01 vs. the DNCB group).

262 263

3.4 Effects of artesunate treatment on the release of IgE and TNF-α in the serum

264

To further assess the effects of artesunate on the immediate hypersensitivity reaction of AD,

265

we examined the release of IgE and TNF-α in the serum. The concentration of IgE (***P < 0.001

266

vs. the control group) was markedly increased in the DNCB group compared with that in the

267

control group. IgE concentration in the AS-L group was lower than that in the DNCB group,

268

although the difference between these two groups was not statistically significant. The

269

concentration of IgE (†††P < 0.001 vs. the DNCB group) was significantly reduced in the AS-H

270

group compared to that in the DNCB group (Fig. 4A). The concentration of TNF-α (***P < 0.001

271

vs. control group) was significantly increased in the DNCB group compared with that in the

272

control group. AS-L (††P < 0.01 vs. the DNCB group) and AS-H (†††P < 0.001 vs. the DNCB

273

group) treatment markedly decreased the level of TNF-α (Fig. 4B).

274

3.5 Effects of artesunate on Th2-related cytokine expression in mouse skin tissue

275

RT-qPCR was performed to determine whether artesunate could decrease Th2-related

276

cytokine mRNA levels in the skin tissue of DNCB-induced AD mice. Compared with the control

277

group, DNCB markedly increased the levels of IL-4 (Fig. 5A) (*P < 0.05 vs. the control group),

278

IL-5 (Fig. 5B) (***P < 0.001 vs. the control group). Conversely, artesunate exhibited the ability to

279

reverse the DNCB-induced change of Th2-related cytokine expression. AS-L treatment 15

280

significantly suppressed the IL-4 mRNA expression (†P < 0.05 vs. the DNCB group); AS-H

281

treatment significantly suppressed the IL-4 and IL-5 mRNA expression (†P < 0.05 vs. the DNCB

282

group).

283

3.6 Effects of artesunate on Th17-related cytokine expression in mouse skin tissue

284

RT-qPCR was performed to determine whether artesunate could decrease Th17-related

285

cytokine mRNA levels in the skin tissue of DNCB-induced AD mice. Compared with the control

286

group, DNCB markedly increased the levels of IL-6 (Fig. 6A) (***P < 0.001 vs. the control

287

group), IL-17 (Fig. 6B) (***P < 0.001 vs. the control group), and IL-23(Fig. 6C) (***P < 0.001 vs.

288

the control group), albeit significantly decreased the level of TGF-β (Fig. 6D) (*P < 0.05 vs. the

289

control group). Conversely, artesunate exhibited the ability to reverse the DNCB-induced change

290

of Th17-related cytokine expression. AS-H treatment significantly suppressed the mRNA

291

expression of all inflammatory cytokines examined (IL-6, IL-17, and IL-23, ††P < 0.01 vs. the

292

DNCB group) (Fig. 6A-6C). However, TGF-β levels in the skin tissues did not show any

293

significant difference following artesunate treatment (Fig. 6D).

294 295 296

3.7 Effects of artesunate on STAT3, SOCS,3 and ROR-γt expression in AD mice Western blot analysis was conducted to determine the protein expression of STAT3, SOCS3,

297

and ROR-γt, which constitute regulators of Th17 differentiation. DNCB treatment significantly

298

increased the phosphorylation of STAT3 (Fig. 7A1 and 7A2) and the expression of ROR-γt (Fig.

299

7B1 and 7B2) (**P < 0.01 vs. the control group), whereas the expression of SOCS3 was

300

decreased (Fig. 7C1 and 7C2) (*P < 0.05 vs. the control group). The phosphorylation of STAT3

301

in the AS-L and AS-H groups was markedly lowered as compared to that in the DNCB group

302

(AS-L group, †P < 0.05 vs. the DNCB group; AS-H group, ††P < 0.01 vs. the DNCB group) (Fig. 16

303

7A2). Fig. 7B2 illustrates that high-dose artesunate significantly lowered the protein expression

304

of ROR-γt compared with that of the control group (†P < 0.05 vs. the DNCB group). SOCS3

305

protein expression in the AS-H group was significantly increased compared with that in the

306

DNCB group (†P < 0.05 vs. the DNCB group) (Fig. 7C2).

307

The results of RT-qPCR were similar to those of western blotting. Compared with the control

308

group, DNCB markedly increased the mRNA levels of STAT3 (***P < 0.001 vs. the control

309

group) (Fig. 7A3) and ROR-γt (**P < 0.01 vs. the control group) (Fig. 7B3), albeit significantly

310

decreased the level of SOCS3 (*P < 0.05 vs. the control group) (Fig. 7C3). The AS-H group

311

exhibited significant suppression of the mRNA expression of ROR-γt and STAT3 (ROR-γt, †P <

312

0.05 vs. the DNCB group; STAT3, †††P < 0.001 vs. the DNCB group), albeit significant

313

upregulation of the mRNA level of SOCS3 in the skin tissue (†P < 0.05 vs. the DNCB group).

314 315

3.8 Effect of SR1001on artesunate-mediated inhibition of LPS-induced injury to HaCaT cells

316

by regulating TNF-a and IL-17 release

317

In order to verify that artesunate protects against AD of mice by inhibiting Th17

318

differentiation, further in vitro experiments were performed to clarify the mechanism of

319

artesunate action. Pre-experiments found that, 1, 2.5, 5, and 10 µM of SR1001 were

320

administrated for 24 hours had no significant effect on the survival rate of HaCat cells for 24

321

hours（Supplementary materials.6）.Therefore, the dose of 5 µM, selected based on previous

322

literature reports, was used for subsequent experiments (Yang et al., 2019).

323

Artesunate (20 µg/mL), SR1001 (5 µM), and LPS (100 ng/mL) were used to examine the

324

effect of SR1001 on artesunate inhibition of LPS-induced injury of HaCaT cells as revealed by

325

release of TNF-α and IL-17. As shown in Fig. 8A, both artesunate and SR1001 could 17

326

significantly inhibit the release of IL17 caused by LPS (***P < 0.001 for both). No significant

327

difference was detected between simultaneous and single administration. Correlation analysis

328

performed using artesunate and SR1001 as bivariates revealed that SR1001 had significant

329

interaction with artesunate, and that the SR1001 effect significantly correlated with artesunate

330

(*P < 0.05),

331

As shown in Fig. 8B, both artesunate and SR1001 could significantly inhibit the release of

332

TNF-α caused by LPS (***P < 0.001 for both). Concurrent administration of artesunate and

333

SR1001 inhibited the effect of artesunate on LPS-induced TNF-α release.

334

18

335 336

4. Discussion A defective skin barrier increases the allergen and pathogen penetration in AD, which can be

337

effectively modeled by topical DNCB application in mice. In recent studies, numerous Chinese

338

medicinal herbs in addition to their purified components have been shown to exert an

339

ameliorative effect in DNCB-induced AD mice (Alyoussef, 2015; Fan et al., 2019; Wu et al.,

340

2019). To the best of our knowledge, this is the first report demonstrating the protective effects

341

of artesunate in DNCB-induced AD symptoms. The results of our study suggested that artesunate

342

exhibits protective effects against AD-like symptoms including significant decreases in

343

dermatitis score, weight index of the spleen and lymph node, and epidermal thickness of the ear

344

and skin, when compared with those of the DNCB group.

345

AD constitutes a severe skin inflammation disease that increases the infiltration of

346

inflammatory cells (Ku et al., 2017). Skin lesions in patients with AD are characterized by the

347

infiltration and proliferation of inflammatory cells, such as T cells, mast cells, eosinophils, and

348

basophils (Jiang et al., 2017). In particular, mast cells regulate the homeostatic expression of the

349

epidermal differentiation complex (Sehra et al., 2016). In the DNCB-induced AD mouse model,

350

consecutive application of DNCB stimulates the activity of inflammatory cells and disturbs the

351

skin barrier of mice. Conversely, the findings of the present study indicated that artesunate

352

significantly reduced the mast cell number in the ear and skin of DNCB-treated animals. In turn,

353

this suggested that the role of artesunate in suppressing inflammation may be associated with its

354

effects on inflammatory cell infiltration.

355

The secretion of various immune mediators triggers a cytokine cascade and stimulates the

356

accumulation of collagen, increasing tissue damage and serving as a prelude to dermal

357

thickening (Kim et al., 2015). Secretion of IgE constitutes an immediate hypersensitivity reaction 19

358

in AD that is manifested through mast cells (Brandt & Sivaprasad, 2011; Nedoszytko et al.,

359

2014). In addition, TNF-α also functions as a key player in acute and chronic AD (Akram et al.,

360

2016; Pasparakis et al., 2014). In the present study, the level of TNF-α was significantly

361

increased after the application of DNCB, whereas artesunate could significantly reduce the

362

enhanced levels of inflammatory cytokines.

363

Some studies have suggested that AD is characterized by elevated IgE and mixed Th1, Th2 and

364

Th17 cytokine expression, which has been proven by numerous research institutes (Lipozencic et

365

al., 2009). In mice, epicutaneous sensitization of mouse skin with OVA results in local and

366

systemic Th17 as well as Th2 responses (He et al., 2009). IL-4, IL-5, the key cytokines for Th2

367

cells (Brandt et al., 2011). In the present study, high mRNA levels of IL-4, IL-5 were observed

368

after challenging with the DNCB allergen. Conversely, the IL-4 and IL-5 mRNA level was

369

significantly reduced following application of artesunate on the dorsal skin of AD mice.IL-17, a

370

key cytokine for Th17 cells, is involved in inflammatory responses as observed in development

371

of autoimmunity, and allergic reactions (Peiser, 2013). It was also observed that artesunate could

372

inhibit DNCB-induced increase in IL-17 mRNA. These results indicate that artesunate exerts

373

protective effects in AD mice by regulating TH2 and TH17. Filaggrin-deficient mice

374

developspontaneous eczematous inflammation with age and this inflammation is characterized

375

by a local Th17 response, as evidenced by increased skin mRNA levels of IL17A, whereas

376

elevated skin levels of Th2 cytokines were only observed months later (Oyoshi et al., 2009).

377

Therefore, we continue to study with TH17 as the focus.

378

In turn, TGF-β constitutes a multifunctional cytokine that promotes immunosuppression

379

through the direct induction of Tregs, which are marked by expression of the transcription factor

380

forkhead box P3 (Foxp3) (Worthington et al., 2012). Moreover, TGF-β promotes expression of 20

381

the master transcription factor ROR-γt in combination with the inflammatory cytokines IL-6 or

382

IL-23 (Korn et al., 2009). TGF-β levels represent an important determinant regarding whether T-

383

cell responses are inhibited or promoted during an immune response. At low concentrations,

384

Th17 cells are differentiated when TGF-β synergizes with proinflammatory cytokines through

385

induction of ROR-γt expression. In contrast, Foxp3 induction and Treg cell formation are

386

promoted by TGF-β at higher concentrations (Zhou et al., 2008). In the present study, the mRNA

387

expression level of TGF-β in the DNCB group was lower than those in control and artesunate-

388

treated groups. This indicated that the lower concentration of TGF-β synergizing with ROR-γt in

389

AD mice promoted the generation of Th17 cells, in turn leading to severe skin inflammation,

390

whereas artesunate increased TGF-β concentration, thereby inhibiting Th17 cell formation to

391

suppress inflammation.

392

Previous studies indicated that IL-23 promotes Th17 differentiation as characterized by

393

related proinflammatory cytokines, such as IL-17. IL-23 alone is not sufficient to differentiate

394

naïve T cells to Th17 cells as they do not express the IL-23 receptor (Zhou et al., 2008). Rather,

395

TGF-β drives Th17 differentiation from naïve CD4+ T cells to Th17 cells together with IL-6, IL-

396

21, and subsequently IL-23 (Bettelli et al., 2006; Korn et al., 2007; Mangan et al., 2006;

397

Veldhoen et al., 2006). The differentiation of naïve CD4+ T cells to Th17 and Treg cells occurs

398

under distinct cytokine milieus, among which IL-6 plays an important role in determining the

399

production of Th17 or Treg cells in concert with TGF-β (Bettelli et al., 2006).

400

STAT3, an important player in the IL-23 signaling pathway, mediates signals that lead to

401

lineage commitment for Th17 differentiation, in addition to the feedback control that leads to the

402

production of immunosuppressive cytokines, such as IL-10. Thus, it could serve to maintain the

403

balance of Th17/Treg cells (Commins et al., 2010; Wei et al., 2008). Our results showed that the 21

404

increased mRNA expression levels of STAT3 and ROR-γt in the DNCB group were lowered by

405

consecutive application of artesunate, thereby attenuating the AD symptoms.

406

Notably, SOCS3 functions as a critical regulator of Th17 generation regardless of the

407

presence of TGF-β, IL-6, or IL-23. In the absence of SOCS3, the STAT3 phosphorylation

408

induced by IL-23 is enhanced. STAT3 is able to bind to the gene promoters of IL-17A and IL-

409

17F (Chen et al., 2006). SOCS3 deficiency in T cells leads to higher concentrations of Th17 cells.

410

Alternatively, STAT3 phosphorylation is reduced consequent to SOCS3 overexpression T cells,

411

thereby suppressing the development of Th17 cells (Qin et al., 2009). This finding is consistent

412

with the present results in which the mRNA expression level of SOCS3 in the DNCB group was

413

lower than that in the artesunate groups, whereas that of STAT3 was higher.

414

In order to verify that artesunate protected against AD in mice by inhibiting Th17 cell

415

differentiation, further in vitro experiments were utilized to clarify the mechanism of artesunate

416

action. These findings suggested that the administration of SR1001, a specific inhibitor of ROR-

417

γt, weakened the effect of artesunate. This further indicated that artesunate may also act on ROR-

418

γt, in turn inhibiting the release of TNF-α to ultimately inhibit AD.

419

In summary, application of artesunate significantly decreased the severity of DNCB-induced

420

AD, as evaluated by histopathological symptoms, IgE production, and expression of Th17-

421

related cytokines and proteins. These results suggested that artesunate exerts therapeutic effects

422

on skin inflammation by inhibiting the numerous DNCB-stimulated inflammatory responses via

423

the regulation of Th17 cell responses. Nevertheless, although our findings suggested that the

424

expression of inflammatory cytokines was suppressed by application of artesunate, further

425

studies with larger sample sizes and research on human samples should be carried out in order to

426

confirm the effectiveness of artesunate in AD. 22

427

Acknowledgements

428

This work was supported by the fourth batch of “Thousand People Innovation and

429

Entrepreneurship Talents Fund” in Guizhou Province; National Natural Science Foundation of

430

China-Guizhou Provincial People's Government Joint Fund Project, sub-project [NSFC-

431

81673495, NSFC-U1812403-4-1].

432 433 434

Declarations of interest: none.

435 436

Appendix A. Supplementary material.

437

Supplementary data associated with this article can be found in the online version at:

438 439

23

440

Figure Legends

441

Fig. 1. Experimental schedule and observation indicators. (A) Schematic diagram of the

442

experimental protocol in mice. Mice were divided into four groups (n = 8 per group). DNCB was

443

applied to the dorsal skin and ear for sensitization. (B) Effects of artesunate on AD-like skin

444

severity induced by DNCB. Representative images for the control group, DNCB group, 5 mg/kg

445

artesunate group (AS-L group), and 10 mg/kg artesunate group (AS-H group). (C) Skin

446

dermatitis scores and (D) ear weight difference between control, DNCB, AS-L, and AS-H groups.

447

Data are presented as the means ± standard error of the mean (S.E.M.). ***P < 0.001 vs. the

448

control group, †††P < 0.001 vs. the DNCB group. DNCB, 2,4-dinitrochlorobenzene; AD, atopic

449

dermatitis.

450 451

Fig. 2. Effects of artesunate on weights of the spleen and lymph node indices in mice (n = 8 per

452

group). Comparison of the spleen weight (A)/index (B) and lymph node weight (C)/index (D)

453

between the control, DNCB, AS-L, and AS-H groups. Data are presented as the means ±

454

standard error of the mean (S.E.M.). ***P < 0.001 vs. the control group; †P < 0.05, ††P < 0.01, †††P

455

< 0.001 vs. the DNCB group. DNCB, 2,4-dinitrochlorobenzene; AS-H, 10 mg/kg artesunate; AS-

456

L, 5 mg/kg artesunate.

457 458

Fig. 3. Histopathologic and serum biochemistry evaluation following DNCB treatment on the ear

459

and dorsal skin of mice (n = 4 per group). (A1) Representative images of hematoxylin-eosin

460

staining for ear epidermal thickness; scale bar, 200 µm. (A2) Comparison of skin epidermal

461

thickness between control, DNCB, AS-L, and AS-H groups. (B1) Representative images of

462

hematoxylin-eosin staining for the skin epidermal thickness; scale bar, 200 µm. (B2) Comparison 24

463

of skin epidermal thickness between the control, DNCB, AS-L, and AS-H groups. (C1)

464

Representative images of toluidine staining for ear mast cell number; scale bar, 200 µm. (C2)

465

Comparison of ear mast cell number between the control, DNCB, AS-L, and AS-H groups. (D1)

466

Representative images of toluidine staining for skin mast cell number; scale bar, 200 µm. (D2)

467

Comparison of skin mast cell number between control, DNCB, AS-L, and AS-H groups. Data

468

are presented as the means ± standard error of the mean (S.E.M.). ***P < 0.001 vs. the control

469

group; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. the DNCB group. DNCB, 2,4-dinitrochlorobenzene;

470

AS-H, 10 mg/kg artesunate; AS-L, 5 mg/kg artesunate.

471 472

Fig. 4. Serum biochemistry evaluation, IgE (A) and TNFα (B) following DNCB treatment of

473

mice (n = 8 per group). Data are presented as the means ± standard error of the mean (S.E.M.).

474

***

475

dinitrochlorobenzene.

P < 0.001 vs. the control group; ††P < 0.01, †††P < 0.001 vs. the DNCB group. DNCB, 2,4-

476 477

Fig. 5. Expression of IL-4 (A) and IL-5 (B) in the skin tissue by quantitative real time reverse

478

transcription-polymerase chain reaction (n = 6 per group). Data are presented as the means ±

479

standard error of the mean (S.E.M.). *P < 0.05, ***P < 0.001 vs. the control group, †P < 0.05 vs.

480

the DNCB group. DNCB, 2, 4-dinitrochlorobenzene.

481 482

Fig. 6. Expression of IL-6, IL-17, IL-23, and TGF-β in the skin tissue by quantitative real time

483

reverse transcription-polymerase chain reaction (n = 6 per group). Data are presented as the

484

means ± standard error of the mean (S.E.M.). *P < 0.05, ***P < 0.001 vs. the control group, †P <

485

0.05, ††P < 0.01 vs. the DNCB group. DNCB, 2, 4-dinitrochlorobenzene. 25

486 487

Fig. 7. Effects of artesunate on p-STAT3, SOCS3, and ROR-γt expression in AD mice. Relative

488

protein expression of p-STAT3 and STAT3 (A1, A2), ROR-γt (B1, B2), and SOCS3 (C1,

489

C2),was determined by western blot and the data were normalized with β-actin (n = 3 per group).

490

mRNA expression of STAT3 (A3), ROR-γt (B3), andSOCS3 (C3) in skin tissue as determined

491

by quantitative real time reverse transcription-polymerase chain reaction (RT-qPCR) (n = 6 per

492

group). Data are presented as the means ± standard error of the mean (S.E.M.). *P < 0.05, **P <

493

0.01, ***P < 0.001 vs. the control group, †P < 0.05, ††P < 0.01, †††P < 0.001 vs. the DNCB group.

494

AD, atopic dermatitis; DNCB, 2,4-dinitrochlorobenzene.

495 496

Fig. 8. The effect of AS with SR1001 in IL-17 (A) and TNFα (B) release induced by LPS on

497

HaCaT cell. A, HaCaT cell were treatment with LPS, SR1001 or AS at the same time incubate

498

for 12h. n=3. Data were presented as mean ± standard error mean (S.E.M.). One-way ANOVA

499

and Tukey’s HSD test: ***P<0.001 vs. Medium, †††P<0.001 vs. LPS. Interaction Analysis：

500

$

501

incubate for 12h. n=3. ***P<0.001 vs. Medium, †††P<0.001 vs. LPS. ###P<0.001 vs. LPS+AS.

502

ANOVA and Tukey’s HSD test: ***P<0.001 vs. Medium, †††P<0.001 vs. LPS, ###P<0.001 vs.

503

LPS+AS.

P<0.05 vs.LPS+AS. B, HaCaT cell were treatment with LPS, SR1001 or AS at the same time

26

504

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Authorship contribution statement Xin-Yu Bai: Conceptualization, Methodology, Data curation. Ping Liu: Methodology, Writing-original draft. Yee-Wen Chai: Data curation. Yan Wang: Data curation. Shuang-Hua Ren: Methodology. Ying-Ying Li: Methodology. Hong Zhou: Conceptualization, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.