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A natural dye, Niram improves atopic dermatitis through down-regulation of TSLP
Accepted Manuscript Title: A natural dye, Niram improves atopic dermatitis through down-regulation of TSLP Author: Na-Ra Han Jin-Young Park Jae-Bum Jang Hyun-Ja Jeong Hyung-Min Kim PII: DOI: Reference:
S1382-6689(14)00247-6 http://dx.doi.org/doi:10.1016/j.etap.2014.10.011 ENVTOX 2106
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
15-5-2014 9-10-2014 13-10-2014
Please cite this article as: Han, N.-R., Park, J.-Y., Jang, J.-B., Jeong, H.J., Kim, H.-M.,A natural dye, Niram improves atopic dermatitis through down-regulation of TSLP, Environmental Toxicology and Pharmacology (2014), http://dx.doi.org/10.1016/j.etap.2014.10.011 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.
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Graphical abstract
2 Highlights
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A natural textile dye, Niram inhibited levels of TSLP/caspase-1/RIP 2 in
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Niram ameliorated clinical symptoms in AD mouse.
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Niram inhibited the infiltration of inflammatory cells in lesional skin.
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Niram inhibited the levels of TSLP,
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mast cells.
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caspase-1, IL-4, and IL-6 in lesional skin. Niram inhibited the serum levels of IgE and histamine in AD mouse.
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A natural dye, Niram improves atopic dermatitis through down-regulation of
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TSLP
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Na-Ra Hana, Jin-Young Parkb, Jae-Bum Jangb, Hyun-Ja Jeongc,*, Hyung-Min
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Kim a,*
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a
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Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
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b
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University, 165, Sechul-ri, Baebang-myun, Asan, Chungnam 336-795, Republic of
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Department of Pharmacology, College of Korean Medicine, Kyung Hee University, 1
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Regional Innovation Center and Inflammatory Disease Research Center, Hoseo
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Korea
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c
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Research Center, Hoseo University, 165, Sechul-ri, Baebang-myun, Asan, Chungnam
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336-795, Republic of Korea
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Department of Food Technology, Biochip Research Center, and Inflammatory Disease
Corresponding author. Tel.: +82 2 961 9448; fax: +82 2 967 7707 (H.M. Kim) or Tel.:
+82 41 540 9681; fax: +82 41 542 9681 (H.J. Jeong). E-mail addresses: [email protected] (H.M. Kim), [email protected] (H.J. Jeong).
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ABSTRACT
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Naju Jjok (Polygonum tinctorium Lour.) has been known to treat skin diseases in
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traditional Korean medicine. A natural textile dye, Niram made from Naju Jjok has
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traditionally been used to dye clothes. Thymic stromal lymphopoietin (TSLP) plays an
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important role in the development of atopic dermatitis (AD). Thus, we investigated that
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Niram might ameliorate AD through regulation of TSLP. Niram significantly inhibited
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the levels of TSLP through blockade of caspase-1/receptor-interacting protein 2
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pathway in stimulated mast cells. Further, Niram ameliorated clinical symptoms in AD
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mouse. Niram significantly inhibited the infiltration of inflammatory cells in lesional
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skin. The levels of TSLP, caspase-1, IL-4, and IL-6 were inhibited in lesional skin
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applied topically with Niram. Niram significantly inhibited the serum levels of IgE and
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histamine in AD mouse. Finally, Niram significantly inhibited the levels of TSLP in
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polyriboinosinic polyribocytidylic acid-stimulated human keratinocyte HaCaT cells.
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These results establish Niram as a functional dye embracing the aspects of not only a
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traditional use but also a pharmacological effect.
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Keywords: Niram; TSLP; Caspase-1; Mast cell; Atopic dermatitis; NC/Nga mice
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1. Introduction
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Atopic dermatitis (AD) is an increasingly common inflammatory skin disorder that
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affects at least 15% of children and is characterized by cutaneous hyper-reactivity to
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environmental triggers (Jin et al., 2009). The increase in the incidence of AD in
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developed countries has been related to familiar and environmental factors (Sebõk et al.,
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2006). There are many factors known to worsen the disease, including climatic factors
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and chemical or physical irritants (Ricci et al., 2004). Clothes can be a protective skin
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barrier against persistent scratching, allowing more rapid improvement of the
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eczematous lesions (Ricci et al., 2012). However, lymphomatoid dermatitis resulted
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from contact with textile dyes (Narganes et al., 2013). Contact urticaria resulted from a
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fluorescent dye in clothing (Sugiura et al., 2010).
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Chronic lesions of AD are characterized by diffuse epidermal hyperplasia and
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perivascular infiltration of mast cells (Kawakami et al., 2009). Mast cells have been
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understood as the key effector cell type in IgE-mediated immediate hypersensitivity and
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allergic disorders (Galli, 2000). The activated mast cells secret various cytokines that
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are relevant in chronic skin inflammation (Harvima and Nilsson, 2011). The activated
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mast cells have highly expressed thymic stromal lymphopoietin (TSLP) and triggered
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allergic inflammation (Liu, 2006). Also, TSLP produced from keratinocytes aggravated
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a hyperreactive immune state in AD skin lesions (Kubo et al., 2014). TSLP has been
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linked to the pathogenesis of AD (Soumelis et al., 2002).
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Caspase-1 is considered an important target to control inflammatory diseases
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(Cunha et al., 2010). Receptor interacting protein2 (RIP2) is a caspase recruitment
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domain-containing kinase that interacts with caspase-1 and plays an important role in
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5 NF-κB activation (Sarkar et al., 2006). Transgenic mice over-expressing caspase-1
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displayed high serum levels of IgE and spontaneously develop chronic dermatitis
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(Nakano et al., 2003). The level of TSLP was increased through caspase-1 and RIP2
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signal pathway in mast cells (Song et al., 2012).
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Naju Jjok (Polygonum tinctorium Lour.) has been known to treat skin diseases
6
in traditional Korean medicine. Niram made from Naju Jjok has traditionally been used
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as a natural textile dye in Korea. However, the protective effect of Niram on AD has not
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yet been clarified. Therefore, we investigated the inhibitory effect of Niram on phorbol
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myristate acetate plus calcium ionophore A23187 (PMACI)-stimulated human mast cell
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line (HMC-1) cells in vitro, 2,4-dinitrofluorobenzene (DNFB)-induced AD-like lesional
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dorsal skin of NC/Nga mice in vivo, and polyriboinosinic polyribocytidylic acid
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(poly(I : C))-stimulated human keratinocyte HaCaT cells in vitro.
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2. Materials and Methods
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2.1. Reagents
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We purchased Isocove’s Modified Dulbecco’s Medium (IMDM) and Dulbeco's
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Modified Eagle Medium (DMEM) from Gibco BRL (Grand Island, NY, USA); PMA,
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calcium
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bromide (MTT), DNFB, and poly(I : C) from Sigma Chemical Co (St. Louis, MO,
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USA); TSLP antibody from R&D Systems (Minneapolis, MN, USA); RIP2, caspase-1,
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and actin antibodies from Santa Cruz Biotechnology (Santacruz, CA, USA); IL-4, IL-6,
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and IgE antibodies from BD Pharmingen (Torreyana Road, San Diego, CA, USA).
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2.2. Preparation of Niram
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
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Shin et al. (2012) indicated the crude Niram preparation. Briefly, Naju Jjok was dipped
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in water for 3 days. And then Naju Jjok was taken out of water. Lime was put in the
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water and the water was stirred until the color was changed into indigo blue. After
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indigo dye sank, supernatant which was changed clearly was drained. The indigo dye
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sunk like mud is named Niram. Niram was kindly provided from Naju city, republic of
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Korea. We used liquid produced from this indigo dye. It was lyophilized and reduced to
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powder. It was dissolved in DW and filtered with 0.22 μm syringe filter.
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2.3. Cells culture
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7 HMC-1 cells were incubated in IMDM supplemented with 100 units/ml of penicillin,
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100 μg/ml of streptomycin, and 10% fetal bovine serum (FBS) at 37 °C in 5% CO2 with
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95% humidity. HaCaT cells were kindly provided by Prof. Sang-Hyun Kim
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(Kyungpook National University) and were cultured in DMEM supplemented with 100
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units/ml of penicillin, 100 μg/ml of streptomycin, and 10% FBS at 37 °C in 5% CO2
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with 95% humidity.
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2.4. Cytokines assay
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The levels of TSLP, IL-4, IL-6, and IgE were determined using a sandwich ELISA
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method according to the manufacturer’s instructions (R&D Systems; BD Pharmingen).
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2.5. Quantitative real time-polymerase chain reaction (PCR) analysis
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Quantitative real time-PCR was performed using a SYBR Green master mix and the
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detection of mRNA was analyzed using an ABI StepOne real time-PCR System
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(Applied Biosystems, Foster City, CA, USA). Primer sequences for the reference gene
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GAPDH and gene TSLP were as follows: GAPDH (5’ TCG ACA GTC AGC CGC
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ATC TTC TTT 3’; 5’ ACC AAA TCC GTT GAC TCC GAC CTT 3’); TSLP (5’ TAT
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GAG TGG GAC CAA AAG TAC CG 3’; 5’ GGG ATT GAA GGT TAG GCT CTG G
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3’). Typical profile times used were the initial step, 95 °C for 10 min followed by a
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second step at 95 °C for 10 s for 40 cycles with a melting curve analysis. The level of
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target mRNA was normalized to the level of the GAPDH and compared to the control.
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Data were analyzed using the ΔΔCT method.
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2.6. MTT assay
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Cell viability was measured by a MTT assay. HMC-1 cell (4 × 105) was treated with
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Niram for 2 h and stimulated with PMACI for 8 h and then harvested. HaCaT cells were
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(2 × 105) treated with Niram for 2 h and stimulated with poly(I : C) for 24 h and then
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harvested. The cell suspension containing MTT solution (5 mg/ml) was incubated at
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37 °C for an additional 4 h. After washing the supernatant out, the insoluble formazan
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product was dissolved in dimethyl sulfoxide (DMSO). Then, the optical density of 96-
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well culture plate was determined using an ELISA method reader at 540 nm.
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2.7. Western blot analysis
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The stimulated cells were lysed and separated through 10% SDS-PAGE. After
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electrophoresis, the proteins were transferred to nitrocellulose membranes and then the
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membranes were blocked and incubated with primary and secondary antibodies. Finally,
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the protein bands were visualized by an enhanced chemiluminesence assay according to
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the manufacturer’s instructions (Amersham Co. Newark, NJ, USA).
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2.8. Animals
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Six-week-old male NC/Nga mice were obtained from Charles River Laboratories
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International, Inc. (Yokohama, Japan). And the animals were maintained under
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conventional condition and performed under approval from the animal care committee
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of Kyung Hee University [Protocol Number. KHUASP (SE)-11-009]. Mice were
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sacrificed with CO2 inhalation.
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2.9. Sensitization with DNFB
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For active sensitization, 100 l 0.15% DNFB dissolved in acetone was topically applied
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to the shaved abdominal skin of NC/Nga mice. A week later, the shaved dorsal skin of
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NC/Nga mice was applied with 50 l 0.15% DNFB as a control group twice a week for
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4 weeks (Wu et al., 2011). At that time, Niram (100 g/ml) was topically applied to
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shaved dorsal skin of DNFB-sensitized mice twice a week for 4 weeks. The same
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volume of acetone was applied to the shaved abdominal and dorsal skin and saline was
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topically applied as a vehicle group. The lesional dorsal skin and serum were obtained 4
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h after the last DNFB sensitizationon the basis of our previous study (Han et al., 2014b).
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After anesthetization, blood was withdrawn from the heart of mouse into syringes. And
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then, serum was prepared by centrifugation at 3400 rpm at 4 °C for 10 min.
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2.10. Histological analysis
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Formaldehyde (10%)-fixed dorsal skin samples were embedded in paraffin, cut into 4-
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μm-thick sections. After dewaxing and dehydration, sections were stained with
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hematoxylin and eosin (H&E) to count the number of inflammatory cells and visualize
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epidermal thickness.
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2.11. Clinical symptom of AD
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Scratching behavioral test was evaluated by counting the number of scratching at 4 h
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after the last DNFB sensitization for 10 min. After counting, mice were anesthetized
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and taken pictures for observing clinical features.
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2.12. Reverse transcription (RT)-PCR analysis
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Using an easy-BLUE™ RNA extraction kit (iNtRON Biotech, Republic of Korea), we
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isolated the total RNA from dorsal skin samples in accordance with the manufacturer's
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specifications. We performed RT-PCR with the following primers: TSLP (5’ TGC
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AAG TAC TAG TAC GGA TGG GG C 3′; 5′ GGA CTT CTT GTG CCA TTT CCT
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GAG 3’); IL-4 (5’ ACG GAG ATG GAT GTG CCA AA 3′; 5′ CGA GTA ATC CAT
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TTG CAT GA 3’); IL-6 (5’ CGG GAT CCA TGT TCC CTA CTT CAC AA 3′; 5′ CCC
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AAG CTT CTA CGT TTG CC 3’). Reference gene GAPDH (5’ GGC ATG GAC TGT
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GGT CAT GA 3’; 5’ TTC ACC ACC ATG GAG AAG GC 3’) was used to verify that
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equal amounts of RNA. The annealing temperature was 62 °C for TSLP and IL-4; 50 °C
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for IL-6; 60 °C for GAPDH. Products were electrophoresed on a 1.5% agarose gel and
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visualized by staining with ethidium bromide. The levels of each mRNA were
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normalized to the level of the GAPDH.
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2.13. Analysis of confocal laser-scanning microscope
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Dorsal skin samples were immediately fixed with 4% formaldehyde and embedded in
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paraffin. After dewaxing and dehydration, sections were blocked with FBS followed by
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11 1 h of incubation and then stained with the following antibodies. Anti-rat TSLP (R&D
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Systems), FITC-conjugated anti-rat IgG (Sigma Chemical Co), anti-rabbit c-Kit (Santa
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Cruz Biotechnology), and TRITC-conjugated anti-rabbit IgG (Sigma Chemical Co)
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were used to stain mast cells-derived TSLP. Anti-rabbit caspase-1 (Santa Cruz
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Biotechnology), FITC-conjugated anti-rabbit IgG (Santacruz Biotechnology), and PE-
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conjugated anti-mouse c-Kit antibodies (BD Pharmingen) were used to stain caspase-1
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in mast cells. 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame,
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CA, USA) containing mounting medium was used to counterstain the nuclei. The
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images of stained cells and specimens were randomly visualized using a confocal laser-
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scanning microscope.
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2.14. Histamine assay
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Histamine in the serum was measured by the addition of perchloric acid and
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centrifugation at 400 × g for 5 min at 4°C. The histamine content was measured using
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the o-phthalaldehyde spectrofluorometric method described by Shore et al. (1959). The
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fluorescent intensity was measured at 440 nm (excitation at 360 nm) in
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spectrofluorometer.
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2.15. HPLC analysis
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Niram extract and indirubin were passed through a 0.2 μm membrane. 20 μl aliquots of
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the filtrate were injected into the HPLC. Syncronis C18 (150 mm × 2.1 mm; particle
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size 5 μm) was used as an analytical column. The mobile phases was composed of
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12 solvent A (DIwater) and solvent B (Acetonitrile) at the rate of 10:90. The analysis was
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carried out at a flow rate of 0.5 ml/min. The injection volume was 10 μL. HPLC run for
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55 min. The linearity of the peak area (y) vs. concentration (x, μg/ml) curve was used to
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calculate the contents of indirubin in Niram.
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2.16. Statistical analysis
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The results shown in vitro and in vivo experiments are a summary of the data from at
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least-three experiments and are presented as the mean SEM. Statistical evaluation of
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the results was performed by an independent t-test and ANOVA with Tukey post hoc
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test. All statistical analyses were performed using SPSS v12.00 statistical analysis
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software (SPSS Inc.). The results were considered significant at a value of p < 0.05.
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3. Results
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3.1. Effect of Niram on the level of TSLP in stimulated HMC-1 cells
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First, we investigated whether Niram could regulate the production and mRNA
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expression of TSLP in mast cells. Niram (100 g/ml) significantly inhibited the
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production and mRNA expression of TSLP in PMACI-stimulated HMC-1 cells (p <
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0.05; Fig. 1A and B). Niram (100 g/ml) did not have influence on the production of
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TSLP in unstimulated HMC-1 cells. As shown in Fig.1C, the cytotoxicity did not show
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at doses of 1, 10, and 100 g/ml.
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3.2. Effect of Niram on the expressions of caspase-1 and RIP2 in stimulated HMC-1
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cells
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Next, we investigated how Niram could regulate the level of TSLP mechanistically.
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Because Moon and Kim (2011) have reported that TSLP was regulated through caspase-
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1 signal pathway in mast cells, we analyzed the expressions of caspase-1 and RIP2 (an
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upstream activator of caspase-1) by Niram with Western blot analysis. Niram inhibited
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the activation of caspase-1 in PMACI-stimulated HMC-1 cells (Fig. 2). Niram also
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reduced the expression of RIP2 (Fig. 2).
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3.3. Effect of Niram on clinical symptoms in DNFB-induced AD mouse model
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14 We mimicked the traditional use of Niram as a textile dye and applied Niram (100
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g/ml) topically to DNFB-sensitized lesional dorsal skin. The conspicuous erythema
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and hemorrhage were present in the lesional dorsal skin; whereas the application with
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Nirma (100 g/ml) noticeably ameliorated these phenotypes in the lesional dorsal skin
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(Fig. 3A). In addition, Niram significantly inhibited the infiltration of inflammatory
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cells and epidermis thickness in the lesional dorsal skin (p < 0.05; Fig. 3B). Niram
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significantly inhibited scratching behavior (Fig. 3C).
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3.4. Effect of Niram on the level of TSLP and caspase-1 in DNFB-induced AD mouse
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model
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Based on the regulatory effect of TSLP and caspase-1 by Niram in vitro, we
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investigated whether Niram also could regulate the level of TSLP and caspase-1 in the
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AD-like lesional dorsal skin. As shown in Fig.4A and B, the application with Niram
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inhibited the protein level and mRNA expression of TSLP in the lesional dorsal skin.
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Niram inhibited the expression of TSLP in mast cells from the lesional dorsal skin
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(Fig .4C). Furthermore, Niram inhibited the mast cells-derived caspase-1 expression in
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the lesional dorsal skin (Fig. 4D).
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3.5. Effect of Niram on the levels of AD-related factors in DNFB-induced AD mouse
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model
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Finally, we investigated whether Niram could regulate AD-related factors in DNFB-
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induced AD mouse model. The mRNA expressions of IL-4 and IL-6 were inhibited in
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15 the lesional dorsal skin applied with Niram (Fig. 5A). Also, the protein levels of IL-4
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and IL-6 were significantly inhibited by Niram (p < 0.05, Fig. 5B). In addition, Niram
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significantly inhibited the levels of serum IgE and histamine (p < 0.05, Fig. 5C).
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3.6. Effect of Niram on the level of TSLP in stimulated HaCaT cells
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Finally, we investigated whether Niram also could regulate the production and mRNA
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expression of TSLP in HaCaT cells. Niram (100 g/ml) significantly inhibited the
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production of TSLP in poly(I : C)-stimulated HaCaT cells (p < 0.05; Fig. 6A). The
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mRNA level of TSLP was elevated, peaking 2 h following exposure to poly(I:C) in
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HaCaT cells (p < 0.05; Fig. 6B). And Niram (100 g/ml) significantly inhibited the
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mRNA expression of TSLP 2h after exposure to poly(I : C) (p < 0.05; Fig. 6C). The
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cytotoxicity did not show at all doses of Niram in stimulated HaCaT cells (Fig. 6D).
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3.7. HPLC analysis of indirubin from Niram
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Finally, the indication of potential single compund was determined from Niram. We
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analyzed the content of indirubin from Niram through HPLC. Indirubin has reported to
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be a main compound from Niram (Shin et al., 2012). The retention time of the indirubin
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was approximately 4 min (Fig. 7). Indirubin from Niram was included about 42.3%.
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4. Discussion
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We determined an inhibitory effect of Naju Jjok which is the main ingredient of Niram
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on AD in previous study (Han et al., 2014a). And Chu and Kim (2014) reported that the
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number of mast cells was decreased in 2,4-dinitrochlorobenzene-induced AD-like
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lesional dorsal skin of BALB/c mice. Furthermore, the present study provides the
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additional and specific evidence that Niram has an inhibitory effect on AD by regulating
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the levels of TSLP in mast cells. Niram inhibited the production and mRNA expression
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of TSLP through the blockade of caspase-1 and RIP2signal pathway in stimulated
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human mast cells. In addition, Niram inhibited the levels of TSLP, IL-4, IL-6, and
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caspase-1 in the lesional dorsal skin of AD mouse model. Niram inhibited the levels of
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serum IgE and histamine in AD mouse model. Also, Niram inhibited the levels of TSLP
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in stimulated keratinocytes.
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The defect of the skin barrier has played an important role in the pathogenesis
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of AD causing the development of eczematous lesions after exposure to repeated
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irritants (Ricci et al., 2004). Mast cells play an important role in triggering the skin
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inflammation (Harvima and Nilsson, 2011). Disrupted skin barrier of patients with
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atopic eczema facilitated contact between mast cells in the skin and environmental
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triggers of the disease (Ribbing et al., 2011). Mast cells generated from patients with
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atopic eczema have enhanced levels of granule mediators such as histamine and IL-6
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(Ribbing et al., 2011). Also, Keratinocytes are involved in skin inflammation through
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the regulation of expression of chemokines and cytokines (Giustizieri et al., 2001).
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TSLP is a general biomarker for skin-barrier defects and the expression of TSLP is
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sustained as long as barrier defects persist (Demehri et al., 2008). TSLP was
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17 overproduced in keratinocytes of human AD skin lesions (Soumelis et al., 2002).
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Furthermore, Demehri et al. (2009) have reported that the skin-derived TSLP was
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sufficient to trigger the atopic march, sensitizing the lung airways to inhaled allergens.
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Blockade of signal pathway of TSLP inhibited TSLP-driven Th2 inflammatory cell
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infiltration, cytokine secretion, and IgE production (Seshasayee et al., 2007). In this
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study, Niram inhibited the production and mRNA expression of TSLP in stimulated
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human mast cells. Niram inhibited the mRNA expression and protein expression of
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TSLP in the lesional dorsal skin of AD. Also, Niram decreased the expression of mast
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cells-derived TSLP in the lesional dorsal skin. In addition, Niram inhibited the
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production and mRNA expression of TSLP in stimulated human keratinocytes. Thus,
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we imply that Niram might prevent AD aggravated by increased TSLP.
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TSLP was regulated through caspase-1 signal pathway in mast cells (Moon and
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Kim, 2011). Caspase-1 transgenic mice overexpressing caspase-1 spontaneously
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suffered from chronic dermatitis and had a significant increase in the serum levels of
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histamine and IgE (Murakami et al., 2006; Yamanaka et al., 2000). And infiltration of
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mast cells was markedly elevated in lesions of caspase-1 transgenic mice (Murakami et
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al., 2006). The level of RIP2 protein was up-regulated in allergic airway inflammation.
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The deficiency of RIP2 reduced the levels of IL-4 and serum IgE, infiltration of
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inflammatory cells, and mRNA expression of TSLP in mouse asthma model (Goh et al.,
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2013). In this study, Niram inhibited the expressions of caspase-1 and RIP2 in
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stimulated human mast cells. Niram inhibited the expression of mast cells-derived
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caspase-1 in the lesional dorsal skin. Thus, we imply that Niram has an anti-AD effect
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by regulating TSLP through the blockade of RIP2 and caspase-1 in mast cells.
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Cytokine imbalance could initiate inflammation in patients with AD, thus
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18 emphasizing the important role of cytokines in AD (Gharagozlo et al., 2013). The
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overproduction of cytokines such as IL-4 and IL-6 could activate B cell to produce IgE
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and cause atopic manifestations (Novak et al., 2002). Overproduction of IL-4 has been
4
reported in lesional from patients with AD (Jung et al., 2003). Peripheral blood T cells
5
derived from patients with AD spontaneously produced more IL-6 than that from T cells
6
from normal subjects (Toshitani et al., 1993). The level of IgE is associated with
7
severity of AD and contributed by abnormality of skin barrier, a key feature of AD (Liu
8
et al., 2011). TSLP induced the histamine release from mast cells through signaling
9
cascade (Ito et al., 2012). All effects of histamine were decreased by caspase-1 inhibitor,
10
indicating that histamine action is dependent on cytokines mediated by caspase-1
11
(Nishibori et al., 2001). In this study, Niram inhibited the levels of IL-4 and IL-6 in the
12
lesional dorsal skin and serum IgE and histamine. Thus, we postulate that Niram might
13
inhibit the clinical symptoms through down-regulating the levels of predominant
14
mediators of AD, such as IL-4, IL-6, IgE, and histamine.
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Naju Jjok has been reported to have effects of anti-oxidant, anti-cancer, anti-
16
inflammation, etc. (Jang et al., 2012; Kim et al., 2010). Indirubin is an active compound
17
of Naju Jjok (Kim et al., 2010). Indirubin inhibited inflammatory reactions in delayed-
18
type hypersensitivity (Kunikata et al., 2000). Indirubin also inhibited allergic contact
19
dermatitis through regulating Th cell-mediated immune responses (Kim et al., 2013).
20
Also, we indicated indirubin as a potential compound of Niram made from Naju Jjok
21
through HPLC chromatographic analysis. Thus, we imply that indirubin, an active
22
compound of Niram, might regulate the level of TSLP and inhibit AD in this study.
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23
Taken together, Niram inhibited the levels of TSLP in stimulated mast cells,
24
keratinocytes, and AD mouse model. Senti et al. (2006) have reported that antimicrobial
Page 18 of 38
19 silk clothing showed potential to become an effective treatment of AD. Similarly, we
2
propose that clothes dyed with Niram could also be functional clothes to prevent skin
3
disorders in every life. Furthermore, Niram might provide the beneficial effect as
4
clothes for AD patients.
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20 1
Conflict of interest
2 3
The authors declare that there are no conflicts of interest.
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Acknowledgments
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This research was supported by Basic Science Research Program through the National
8
Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and
9
Technology (2012R1A1A2A10044645) and partially supported by Naju city (2012).
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Yamanaka, K., Tanaka, M., Tsutsui, H., Kupper, T.S., Asahi, K., Okamura, H.,
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Figure legends
2
Fig. 1. Niram inhibits the level of TSLP in stimulated HMC-1 cells. (A) HMC-1 cells (4
3
× 105) were pretreated with Niram (1, 10, and 100 g/ml) for 2 h and stimulated with
4
PMACI for 7 h. The production of TSLP was analyzed with the ELISA. (B) HMC-1
5
cells (1 × 106) were pretreated with Niram (1, 10, and 100 g/ml) for 2 h and stimulated
6
with PMACI for 5 h. The mRNA expression of TSLP was analyzed with the real time-
7
PCR analysis. (C) Cell viability was analyzed with the MTT assay. Each datum
8
represents the mean ± SEM of three independent experiments. #p < 0.05; significantly
9
different from unstimulated cells. *p < 0.05; significantly different from PMACI-
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stimulated cells.
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Fig. 2. Niram regulates the expressions of caspase-1 and RIP2 in stimulated HMC-1
13
cells. HMC-1 cells (5 × 106) were pretreated with Niram (1, 10, and 100 g/ml) for 2 h
14
and stimulated with PMACI for 1 h. The expressions of caspase-1 and RIP2 were
15
analyzed with Western blot analysis.
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Fig. 3. Niram ameliorates clinical symptoms in DNFB-induced AD mouse model. (A)
18
The clinical features were observed 4 h after the last DNFB sensitization. (B)
19
Histological analysis of the lesional dorsal skin was examined by H&E staining for
20
inflammatory cells and epidermis thickness. The inflammatory cells were counted from
21
five tissue sections (1.5 × 103 μm2) selected randomly. The inflammatory cells were
22
indicated by arrows. Representative photomicrographs were examined at 400×
23
magnification. (C) The time of scratching behavior was measured at 4 h after the last
24
DNFB sensitization for 10 min. *p < 0.05; significantly different from DNFB-sensitized
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29 1
group (control). n = 3.
2
Fig. 4. Niram regulates the levels of TSLP and caspase-1 in AD mouse model. (A) The
4
protein expression of TSLP from the lesional dorsal skin homogenates was analyzed
5
with ELISA. (B) The mRNA expression of TSLP was analyzed with RT-PCR analysis.
6
(C) The TSLP+ (FITC) and c-Kit+ (TRITC) cells in the DNFB-sensitized skin lesions
7
were examined with a confocal laser-scanning microscope. Mast cells are identified as
8
c-Kit+ cells. The arrows in merged images show the colocalization of mast cells and
9
TSLP. (D) The caspase-1+ (FITC) and c-Kit+ (PE) cells in the DNFB-sensitized skin
10
lesions were examined with a confocal laser-scanning microscope. The arrows in
11
merged images show the colocalization of mast cells and caspase-1. #p < 0.05;
12
significantly different from vehicle group. *p < 0.05; significantly different from
13
DNFB-sensitized group (control). n = 3.
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Fig. 5. Inhibitory effect of Niram on the levels of AD-related factors in AD mouse
16
model. (A) The mRNA expressions of IL-4 and IL-6 were analyzed with RT-PCR
17
analysis. (B) The protein expressions of IL-4 and IL-6 from the lesional dorsal skin
18
homogenates were analyzed with ELISA. (C) The level of serum IgE was analyzed with
19
ELISA. The intensity of serum histamine was assayed as described in materials and
20
methods section. #p < 0.05; significantly different from vehicle group. *p < 0.05;
21
significantly different from DNFB-sensitized group (control). n = 3.
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Fig. 6. Niram inhibits the level of TSLP in stimulated HaCaT cells. (A) HaCaT cells (2
24
× 105) were pretreated with Niram (1, 10, and 100 g/ml) for 2 h and stimulated with
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30 poly(I : C) 10 g/ml for 24 h. The production of TSLP was analyzed with the ELISA.
2
(B) HaCaT cells (1 × 106) were stimulated with poly(I : C) 10 g/ml for 10 h. The
3
mRNA expression of TSLP was analyzed with the real time-PCR analysis. (C) HaCaT
4
cells (1 × 106) were pretreated with Niram (1, 10, and 100 g/ml) for 2 h and stimulated
5
with poly(I : C) for 2 h. The mRNA expression of TSLP was analyzed with the real
6
time-PCR analysis. (D) Cell viability was analyzed with the MTT assay. Each datum
7
represents the mean ± SEM of three independent experiments. #p < 0.05; significantly
8
different from unstimulated cells. *p < 0.05; significantly different from poly(I : C)-
9
stimulated cells.
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Fig. 7. Representative HPLC chromatogram of Niram. The HPLC fraction was
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monitored by recording the UV absorbance at 230 nm.
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S1382-6689(14)00247-6 http://dx.doi.org/doi:10.1016/j.etap.2014.10.011 ENVTOX 2106
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
15-5-2014 9-10-2014 13-10-2014
Please cite this article as: Han, N.-R., Park, J.-Y., Jang, J.-B., Jeong, H.J., Kim, H.-M.,A natural dye, Niram improves atopic dermatitis through down-regulation of TSLP, Environmental Toxicology and Pharmacology (2014), http://dx.doi.org/10.1016/j.etap.2014.10.011 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.
1
1
Graphical abstract
2 Highlights
4
A natural textile dye, Niram inhibited levels of TSLP/caspase-1/RIP 2 in
5
Niram ameliorated clinical symptoms in AD mouse.
6
Niram inhibited the infiltration of inflammatory cells in lesional skin.
7
Niram inhibited the levels of TSLP,
8
mast cells.
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caspase-1, IL-4, and IL-6 in lesional skin. Niram inhibited the serum levels of IgE and histamine in AD mouse.
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Page 1 of 38
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A natural dye, Niram improves atopic dermatitis through down-regulation of
2
TSLP
3
Na-Ra Hana, Jin-Young Parkb, Jae-Bum Jangb, Hyun-Ja Jeongc,*, Hyung-Min
4
Kim a,*
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a
7
Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
8
b
9
University, 165, Sechul-ri, Baebang-myun, Asan, Chungnam 336-795, Republic of
us
cr
Department of Pharmacology, College of Korean Medicine, Kyung Hee University, 1
an
Regional Innovation Center and Inflammatory Disease Research Center, Hoseo
10
Korea
11
c
12
Research Center, Hoseo University, 165, Sechul-ri, Baebang-myun, Asan, Chungnam
13
336-795, Republic of Korea
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Department of Food Technology, Biochip Research Center, and Inflammatory Disease
Corresponding author. Tel.: +82 2 961 9448; fax: +82 2 967 7707 (H.M. Kim) or Tel.:
+82 41 540 9681; fax: +82 41 542 9681 (H.J. Jeong). E-mail addresses: [email protected] (H.M. Kim), [email protected] (H.J. Jeong).
Page 2 of 38
3
ABSTRACT
2
Naju Jjok (Polygonum tinctorium Lour.) has been known to treat skin diseases in
3
traditional Korean medicine. A natural textile dye, Niram made from Naju Jjok has
4
traditionally been used to dye clothes. Thymic stromal lymphopoietin (TSLP) plays an
5
important role in the development of atopic dermatitis (AD). Thus, we investigated that
6
Niram might ameliorate AD through regulation of TSLP. Niram significantly inhibited
7
the levels of TSLP through blockade of caspase-1/receptor-interacting protein 2
8
pathway in stimulated mast cells. Further, Niram ameliorated clinical symptoms in AD
9
mouse. Niram significantly inhibited the infiltration of inflammatory cells in lesional
10
skin. The levels of TSLP, caspase-1, IL-4, and IL-6 were inhibited in lesional skin
11
applied topically with Niram. Niram significantly inhibited the serum levels of IgE and
12
histamine in AD mouse. Finally, Niram significantly inhibited the levels of TSLP in
13
polyriboinosinic polyribocytidylic acid-stimulated human keratinocyte HaCaT cells.
14
These results establish Niram as a functional dye embracing the aspects of not only a
15
traditional use but also a pharmacological effect.
17 18 19
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Keywords: Niram; TSLP; Caspase-1; Mast cell; Atopic dermatitis; NC/Nga mice
Page 3 of 38
4 1
1. Introduction
2
Atopic dermatitis (AD) is an increasingly common inflammatory skin disorder that
4
affects at least 15% of children and is characterized by cutaneous hyper-reactivity to
5
environmental triggers (Jin et al., 2009). The increase in the incidence of AD in
6
developed countries has been related to familiar and environmental factors (Sebõk et al.,
7
2006). There are many factors known to worsen the disease, including climatic factors
8
and chemical or physical irritants (Ricci et al., 2004). Clothes can be a protective skin
9
barrier against persistent scratching, allowing more rapid improvement of the
10
eczematous lesions (Ricci et al., 2012). However, lymphomatoid dermatitis resulted
11
from contact with textile dyes (Narganes et al., 2013). Contact urticaria resulted from a
12
fluorescent dye in clothing (Sugiura et al., 2010).
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Chronic lesions of AD are characterized by diffuse epidermal hyperplasia and
14
perivascular infiltration of mast cells (Kawakami et al., 2009). Mast cells have been
15
understood as the key effector cell type in IgE-mediated immediate hypersensitivity and
16
allergic disorders (Galli, 2000). The activated mast cells secret various cytokines that
17
are relevant in chronic skin inflammation (Harvima and Nilsson, 2011). The activated
18
mast cells have highly expressed thymic stromal lymphopoietin (TSLP) and triggered
19
allergic inflammation (Liu, 2006). Also, TSLP produced from keratinocytes aggravated
20
a hyperreactive immune state in AD skin lesions (Kubo et al., 2014). TSLP has been
21
linked to the pathogenesis of AD (Soumelis et al., 2002).
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22
Caspase-1 is considered an important target to control inflammatory diseases
23
(Cunha et al., 2010). Receptor interacting protein2 (RIP2) is a caspase recruitment
24
domain-containing kinase that interacts with caspase-1 and plays an important role in
Page 4 of 38
5 NF-κB activation (Sarkar et al., 2006). Transgenic mice over-expressing caspase-1
2
displayed high serum levels of IgE and spontaneously develop chronic dermatitis
3
(Nakano et al., 2003). The level of TSLP was increased through caspase-1 and RIP2
4
signal pathway in mast cells (Song et al., 2012).
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Naju Jjok (Polygonum tinctorium Lour.) has been known to treat skin diseases
6
in traditional Korean medicine. Niram made from Naju Jjok has traditionally been used
7
as a natural textile dye in Korea. However, the protective effect of Niram on AD has not
8
yet been clarified. Therefore, we investigated the inhibitory effect of Niram on phorbol
9
myristate acetate plus calcium ionophore A23187 (PMACI)-stimulated human mast cell
10
line (HMC-1) cells in vitro, 2,4-dinitrofluorobenzene (DNFB)-induced AD-like lesional
11
dorsal skin of NC/Nga mice in vivo, and polyriboinosinic polyribocytidylic acid
12
(poly(I : C))-stimulated human keratinocyte HaCaT cells in vitro.
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Page 5 of 38
6 1
2. Materials and Methods
2 3
2.1. Reagents
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We purchased Isocove’s Modified Dulbecco’s Medium (IMDM) and Dulbeco's
6
Modified Eagle Medium (DMEM) from Gibco BRL (Grand Island, NY, USA); PMA,
7
calcium
8
bromide (MTT), DNFB, and poly(I : C) from Sigma Chemical Co (St. Louis, MO,
9
USA); TSLP antibody from R&D Systems (Minneapolis, MN, USA); RIP2, caspase-1,
10
and actin antibodies from Santa Cruz Biotechnology (Santacruz, CA, USA); IL-4, IL-6,
11
and IgE antibodies from BD Pharmingen (Torreyana Road, San Diego, CA, USA).
A23187,
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2.2. Preparation of Niram
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
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ionophore
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Shin et al. (2012) indicated the crude Niram preparation. Briefly, Naju Jjok was dipped
16
in water for 3 days. And then Naju Jjok was taken out of water. Lime was put in the
17
water and the water was stirred until the color was changed into indigo blue. After
18
indigo dye sank, supernatant which was changed clearly was drained. The indigo dye
19
sunk like mud is named Niram. Niram was kindly provided from Naju city, republic of
20
Korea. We used liquid produced from this indigo dye. It was lyophilized and reduced to
21
powder. It was dissolved in DW and filtered with 0.22 μm syringe filter.
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2.3. Cells culture
24
Page 6 of 38
7 HMC-1 cells were incubated in IMDM supplemented with 100 units/ml of penicillin,
2
100 μg/ml of streptomycin, and 10% fetal bovine serum (FBS) at 37 °C in 5% CO2 with
3
95% humidity. HaCaT cells were kindly provided by Prof. Sang-Hyun Kim
4
(Kyungpook National University) and were cultured in DMEM supplemented with 100
5
units/ml of penicillin, 100 μg/ml of streptomycin, and 10% FBS at 37 °C in 5% CO2
6
with 95% humidity.
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2.4. Cytokines assay
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The levels of TSLP, IL-4, IL-6, and IgE were determined using a sandwich ELISA
11
method according to the manufacturer’s instructions (R&D Systems; BD Pharmingen).
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2.5. Quantitative real time-polymerase chain reaction (PCR) analysis
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Quantitative real time-PCR was performed using a SYBR Green master mix and the
16
detection of mRNA was analyzed using an ABI StepOne real time-PCR System
17
(Applied Biosystems, Foster City, CA, USA). Primer sequences for the reference gene
18
GAPDH and gene TSLP were as follows: GAPDH (5’ TCG ACA GTC AGC CGC
19
ATC TTC TTT 3’; 5’ ACC AAA TCC GTT GAC TCC GAC CTT 3’); TSLP (5’ TAT
20
GAG TGG GAC CAA AAG TAC CG 3’; 5’ GGG ATT GAA GGT TAG GCT CTG G
21
3’). Typical profile times used were the initial step, 95 °C for 10 min followed by a
22
second step at 95 °C for 10 s for 40 cycles with a melting curve analysis. The level of
23
target mRNA was normalized to the level of the GAPDH and compared to the control.
24
Data were analyzed using the ΔΔCT method.
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Page 7 of 38
8 1 2
2.6. MTT assay
3
Cell viability was measured by a MTT assay. HMC-1 cell (4 × 105) was treated with
5
Niram for 2 h and stimulated with PMACI for 8 h and then harvested. HaCaT cells were
6
(2 × 105) treated with Niram for 2 h and stimulated with poly(I : C) for 24 h and then
7
harvested. The cell suspension containing MTT solution (5 mg/ml) was incubated at
8
37 °C for an additional 4 h. After washing the supernatant out, the insoluble formazan
9
product was dissolved in dimethyl sulfoxide (DMSO). Then, the optical density of 96-
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well culture plate was determined using an ELISA method reader at 540 nm.
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2.7. Western blot analysis
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The stimulated cells were lysed and separated through 10% SDS-PAGE. After
15
electrophoresis, the proteins were transferred to nitrocellulose membranes and then the
16
membranes were blocked and incubated with primary and secondary antibodies. Finally,
17
the protein bands were visualized by an enhanced chemiluminesence assay according to
18
the manufacturer’s instructions (Amersham Co. Newark, NJ, USA).
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2.8. Animals
21 22
Six-week-old male NC/Nga mice were obtained from Charles River Laboratories
23
International, Inc. (Yokohama, Japan). And the animals were maintained under
24
conventional condition and performed under approval from the animal care committee
Page 8 of 38
9 1
of Kyung Hee University [Protocol Number. KHUASP (SE)-11-009]. Mice were
2
sacrificed with CO2 inhalation.
3
2.9. Sensitization with DNFB
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For active sensitization, 100 l 0.15% DNFB dissolved in acetone was topically applied
7
to the shaved abdominal skin of NC/Nga mice. A week later, the shaved dorsal skin of
8
NC/Nga mice was applied with 50 l 0.15% DNFB as a control group twice a week for
9
4 weeks (Wu et al., 2011). At that time, Niram (100 g/ml) was topically applied to
10
shaved dorsal skin of DNFB-sensitized mice twice a week for 4 weeks. The same
11
volume of acetone was applied to the shaved abdominal and dorsal skin and saline was
12
topically applied as a vehicle group. The lesional dorsal skin and serum were obtained 4
13
h after the last DNFB sensitizationon the basis of our previous study (Han et al., 2014b).
14
After anesthetization, blood was withdrawn from the heart of mouse into syringes. And
15
then, serum was prepared by centrifugation at 3400 rpm at 4 °C for 10 min.
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2.10. Histological analysis
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Formaldehyde (10%)-fixed dorsal skin samples were embedded in paraffin, cut into 4-
20
μm-thick sections. After dewaxing and dehydration, sections were stained with
21
hematoxylin and eosin (H&E) to count the number of inflammatory cells and visualize
22
epidermal thickness.
23 24
2.11. Clinical symptom of AD
Page 9 of 38
10 1
Scratching behavioral test was evaluated by counting the number of scratching at 4 h
3
after the last DNFB sensitization for 10 min. After counting, mice were anesthetized
4
and taken pictures for observing clinical features.
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2.12. Reverse transcription (RT)-PCR analysis
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Using an easy-BLUE™ RNA extraction kit (iNtRON Biotech, Republic of Korea), we
9
isolated the total RNA from dorsal skin samples in accordance with the manufacturer's
10
specifications. We performed RT-PCR with the following primers: TSLP (5’ TGC
11
AAG TAC TAG TAC GGA TGG GG C 3′; 5′ GGA CTT CTT GTG CCA TTT CCT
12
GAG 3’); IL-4 (5’ ACG GAG ATG GAT GTG CCA AA 3′; 5′ CGA GTA ATC CAT
13
TTG CAT GA 3’); IL-6 (5’ CGG GAT CCA TGT TCC CTA CTT CAC AA 3′; 5′ CCC
14
AAG CTT CTA CGT TTG CC 3’). Reference gene GAPDH (5’ GGC ATG GAC TGT
15
GGT CAT GA 3’; 5’ TTC ACC ACC ATG GAG AAG GC 3’) was used to verify that
16
equal amounts of RNA. The annealing temperature was 62 °C for TSLP and IL-4; 50 °C
17
for IL-6; 60 °C for GAPDH. Products were electrophoresed on a 1.5% agarose gel and
18
visualized by staining with ethidium bromide. The levels of each mRNA were
19
normalized to the level of the GAPDH.
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2.13. Analysis of confocal laser-scanning microscope
22 23
Dorsal skin samples were immediately fixed with 4% formaldehyde and embedded in
24
paraffin. After dewaxing and dehydration, sections were blocked with FBS followed by
Page 10 of 38
11 1 h of incubation and then stained with the following antibodies. Anti-rat TSLP (R&D
2
Systems), FITC-conjugated anti-rat IgG (Sigma Chemical Co), anti-rabbit c-Kit (Santa
3
Cruz Biotechnology), and TRITC-conjugated anti-rabbit IgG (Sigma Chemical Co)
4
were used to stain mast cells-derived TSLP. Anti-rabbit caspase-1 (Santa Cruz
5
Biotechnology), FITC-conjugated anti-rabbit IgG (Santacruz Biotechnology), and PE-
6
conjugated anti-mouse c-Kit antibodies (BD Pharmingen) were used to stain caspase-1
7
in mast cells. 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame,
8
CA, USA) containing mounting medium was used to counterstain the nuclei. The
9
images of stained cells and specimens were randomly visualized using a confocal laser-
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scanning microscope.
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2.14. Histamine assay
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Histamine in the serum was measured by the addition of perchloric acid and
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centrifugation at 400 × g for 5 min at 4°C. The histamine content was measured using
16
the o-phthalaldehyde spectrofluorometric method described by Shore et al. (1959). The
17
fluorescent intensity was measured at 440 nm (excitation at 360 nm) in
18
spectrofluorometer.
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2.15. HPLC analysis
21 22
Niram extract and indirubin were passed through a 0.2 μm membrane. 20 μl aliquots of
23
the filtrate were injected into the HPLC. Syncronis C18 (150 mm × 2.1 mm; particle
24
size 5 μm) was used as an analytical column. The mobile phases was composed of
Page 11 of 38
12 solvent A (DIwater) and solvent B (Acetonitrile) at the rate of 10:90. The analysis was
2
carried out at a flow rate of 0.5 ml/min. The injection volume was 10 μL. HPLC run for
3
55 min. The linearity of the peak area (y) vs. concentration (x, μg/ml) curve was used to
4
calculate the contents of indirubin in Niram.
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2.16. Statistical analysis
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The results shown in vitro and in vivo experiments are a summary of the data from at
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least-three experiments and are presented as the mean SEM. Statistical evaluation of
10
the results was performed by an independent t-test and ANOVA with Tukey post hoc
11
test. All statistical analyses were performed using SPSS v12.00 statistical analysis
12
software (SPSS Inc.). The results were considered significant at a value of p < 0.05.
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3. Results
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3.1. Effect of Niram on the level of TSLP in stimulated HMC-1 cells
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First, we investigated whether Niram could regulate the production and mRNA
6
expression of TSLP in mast cells. Niram (100 g/ml) significantly inhibited the
7
production and mRNA expression of TSLP in PMACI-stimulated HMC-1 cells (p <
8
0.05; Fig. 1A and B). Niram (100 g/ml) did not have influence on the production of
9
TSLP in unstimulated HMC-1 cells. As shown in Fig.1C, the cytotoxicity did not show
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at doses of 1, 10, and 100 g/ml.
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3.2. Effect of Niram on the expressions of caspase-1 and RIP2 in stimulated HMC-1
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Next, we investigated how Niram could regulate the level of TSLP mechanistically.
16
Because Moon and Kim (2011) have reported that TSLP was regulated through caspase-
17
1 signal pathway in mast cells, we analyzed the expressions of caspase-1 and RIP2 (an
18
upstream activator of caspase-1) by Niram with Western blot analysis. Niram inhibited
19
the activation of caspase-1 in PMACI-stimulated HMC-1 cells (Fig. 2). Niram also
20
reduced the expression of RIP2 (Fig. 2).
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3.3. Effect of Niram on clinical symptoms in DNFB-induced AD mouse model
23
Page 13 of 38
14 We mimicked the traditional use of Niram as a textile dye and applied Niram (100
2
g/ml) topically to DNFB-sensitized lesional dorsal skin. The conspicuous erythema
3
and hemorrhage were present in the lesional dorsal skin; whereas the application with
4
Nirma (100 g/ml) noticeably ameliorated these phenotypes in the lesional dorsal skin
5
(Fig. 3A). In addition, Niram significantly inhibited the infiltration of inflammatory
6
cells and epidermis thickness in the lesional dorsal skin (p < 0.05; Fig. 3B). Niram
7
significantly inhibited scratching behavior (Fig. 3C).
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3.4. Effect of Niram on the level of TSLP and caspase-1 in DNFB-induced AD mouse
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model
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Based on the regulatory effect of TSLP and caspase-1 by Niram in vitro, we
13
investigated whether Niram also could regulate the level of TSLP and caspase-1 in the
14
AD-like lesional dorsal skin. As shown in Fig.4A and B, the application with Niram
15
inhibited the protein level and mRNA expression of TSLP in the lesional dorsal skin.
16
Niram inhibited the expression of TSLP in mast cells from the lesional dorsal skin
17
(Fig .4C). Furthermore, Niram inhibited the mast cells-derived caspase-1 expression in
18
the lesional dorsal skin (Fig. 4D).
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3.5. Effect of Niram on the levels of AD-related factors in DNFB-induced AD mouse
21
model
22 23
Finally, we investigated whether Niram could regulate AD-related factors in DNFB-
24
induced AD mouse model. The mRNA expressions of IL-4 and IL-6 were inhibited in
Page 14 of 38
15 the lesional dorsal skin applied with Niram (Fig. 5A). Also, the protein levels of IL-4
2
and IL-6 were significantly inhibited by Niram (p < 0.05, Fig. 5B). In addition, Niram
3
significantly inhibited the levels of serum IgE and histamine (p < 0.05, Fig. 5C).
4 5
3.6. Effect of Niram on the level of TSLP in stimulated HaCaT cells
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Finally, we investigated whether Niram also could regulate the production and mRNA
8
expression of TSLP in HaCaT cells. Niram (100 g/ml) significantly inhibited the
9
production of TSLP in poly(I : C)-stimulated HaCaT cells (p < 0.05; Fig. 6A). The
10
mRNA level of TSLP was elevated, peaking 2 h following exposure to poly(I:C) in
11
HaCaT cells (p < 0.05; Fig. 6B). And Niram (100 g/ml) significantly inhibited the
12
mRNA expression of TSLP 2h after exposure to poly(I : C) (p < 0.05; Fig. 6C). The
13
cytotoxicity did not show at all doses of Niram in stimulated HaCaT cells (Fig. 6D).
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3.7. HPLC analysis of indirubin from Niram
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Finally, the indication of potential single compund was determined from Niram. We
18
analyzed the content of indirubin from Niram through HPLC. Indirubin has reported to
19
be a main compound from Niram (Shin et al., 2012). The retention time of the indirubin
20
was approximately 4 min (Fig. 7). Indirubin from Niram was included about 42.3%.
Page 15 of 38
16 1
4. Discussion
2
We determined an inhibitory effect of Naju Jjok which is the main ingredient of Niram
4
on AD in previous study (Han et al., 2014a). And Chu and Kim (2014) reported that the
5
number of mast cells was decreased in 2,4-dinitrochlorobenzene-induced AD-like
6
lesional dorsal skin of BALB/c mice. Furthermore, the present study provides the
7
additional and specific evidence that Niram has an inhibitory effect on AD by regulating
8
the levels of TSLP in mast cells. Niram inhibited the production and mRNA expression
9
of TSLP through the blockade of caspase-1 and RIP2signal pathway in stimulated
10
human mast cells. In addition, Niram inhibited the levels of TSLP, IL-4, IL-6, and
11
caspase-1 in the lesional dorsal skin of AD mouse model. Niram inhibited the levels of
12
serum IgE and histamine in AD mouse model. Also, Niram inhibited the levels of TSLP
13
in stimulated keratinocytes.
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The defect of the skin barrier has played an important role in the pathogenesis
15
of AD causing the development of eczematous lesions after exposure to repeated
16
irritants (Ricci et al., 2004). Mast cells play an important role in triggering the skin
17
inflammation (Harvima and Nilsson, 2011). Disrupted skin barrier of patients with
18
atopic eczema facilitated contact between mast cells in the skin and environmental
19
triggers of the disease (Ribbing et al., 2011). Mast cells generated from patients with
20
atopic eczema have enhanced levels of granule mediators such as histamine and IL-6
21
(Ribbing et al., 2011). Also, Keratinocytes are involved in skin inflammation through
22
the regulation of expression of chemokines and cytokines (Giustizieri et al., 2001).
23
TSLP is a general biomarker for skin-barrier defects and the expression of TSLP is
24
sustained as long as barrier defects persist (Demehri et al., 2008). TSLP was
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Page 16 of 38
17 overproduced in keratinocytes of human AD skin lesions (Soumelis et al., 2002).
2
Furthermore, Demehri et al. (2009) have reported that the skin-derived TSLP was
3
sufficient to trigger the atopic march, sensitizing the lung airways to inhaled allergens.
4
Blockade of signal pathway of TSLP inhibited TSLP-driven Th2 inflammatory cell
5
infiltration, cytokine secretion, and IgE production (Seshasayee et al., 2007). In this
6
study, Niram inhibited the production and mRNA expression of TSLP in stimulated
7
human mast cells. Niram inhibited the mRNA expression and protein expression of
8
TSLP in the lesional dorsal skin of AD. Also, Niram decreased the expression of mast
9
cells-derived TSLP in the lesional dorsal skin. In addition, Niram inhibited the
10
production and mRNA expression of TSLP in stimulated human keratinocytes. Thus,
11
we imply that Niram might prevent AD aggravated by increased TSLP.
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TSLP was regulated through caspase-1 signal pathway in mast cells (Moon and
13
Kim, 2011). Caspase-1 transgenic mice overexpressing caspase-1 spontaneously
14
suffered from chronic dermatitis and had a significant increase in the serum levels of
15
histamine and IgE (Murakami et al., 2006; Yamanaka et al., 2000). And infiltration of
16
mast cells was markedly elevated in lesions of caspase-1 transgenic mice (Murakami et
17
al., 2006). The level of RIP2 protein was up-regulated in allergic airway inflammation.
18
The deficiency of RIP2 reduced the levels of IL-4 and serum IgE, infiltration of
19
inflammatory cells, and mRNA expression of TSLP in mouse asthma model (Goh et al.,
20
2013). In this study, Niram inhibited the expressions of caspase-1 and RIP2 in
21
stimulated human mast cells. Niram inhibited the expression of mast cells-derived
22
caspase-1 in the lesional dorsal skin. Thus, we imply that Niram has an anti-AD effect
23
by regulating TSLP through the blockade of RIP2 and caspase-1 in mast cells.
24
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Cytokine imbalance could initiate inflammation in patients with AD, thus
Page 17 of 38
18 emphasizing the important role of cytokines in AD (Gharagozlo et al., 2013). The
2
overproduction of cytokines such as IL-4 and IL-6 could activate B cell to produce IgE
3
and cause atopic manifestations (Novak et al., 2002). Overproduction of IL-4 has been
4
reported in lesional from patients with AD (Jung et al., 2003). Peripheral blood T cells
5
derived from patients with AD spontaneously produced more IL-6 than that from T cells
6
from normal subjects (Toshitani et al., 1993). The level of IgE is associated with
7
severity of AD and contributed by abnormality of skin barrier, a key feature of AD (Liu
8
et al., 2011). TSLP induced the histamine release from mast cells through signaling
9
cascade (Ito et al., 2012). All effects of histamine were decreased by caspase-1 inhibitor,
10
indicating that histamine action is dependent on cytokines mediated by caspase-1
11
(Nishibori et al., 2001). In this study, Niram inhibited the levels of IL-4 and IL-6 in the
12
lesional dorsal skin and serum IgE and histamine. Thus, we postulate that Niram might
13
inhibit the clinical symptoms through down-regulating the levels of predominant
14
mediators of AD, such as IL-4, IL-6, IgE, and histamine.
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Naju Jjok has been reported to have effects of anti-oxidant, anti-cancer, anti-
16
inflammation, etc. (Jang et al., 2012; Kim et al., 2010). Indirubin is an active compound
17
of Naju Jjok (Kim et al., 2010). Indirubin inhibited inflammatory reactions in delayed-
18
type hypersensitivity (Kunikata et al., 2000). Indirubin also inhibited allergic contact
19
dermatitis through regulating Th cell-mediated immune responses (Kim et al., 2013).
20
Also, we indicated indirubin as a potential compound of Niram made from Naju Jjok
21
through HPLC chromatographic analysis. Thus, we imply that indirubin, an active
22
compound of Niram, might regulate the level of TSLP and inhibit AD in this study.
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Taken together, Niram inhibited the levels of TSLP in stimulated mast cells,
24
keratinocytes, and AD mouse model. Senti et al. (2006) have reported that antimicrobial
Page 18 of 38
19 silk clothing showed potential to become an effective treatment of AD. Similarly, we
2
propose that clothes dyed with Niram could also be functional clothes to prevent skin
3
disorders in every life. Furthermore, Niram might provide the beneficial effect as
4
clothes for AD patients.
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Page 19 of 38
20 1
Conflict of interest
2 3
The authors declare that there are no conflicts of interest.
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Acknowledgments
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This research was supported by Basic Science Research Program through the National
8
Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and
9
Technology (2012R1A1A2A10044645) and partially supported by Naju city (2012).
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BALB/c
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Cunha, T.M., Talbot, J., Pinto, L.G., Vieira, S.M., Souza, G.R., Guerrero, A.T., Sonego,
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Figure legends
2
Fig. 1. Niram inhibits the level of TSLP in stimulated HMC-1 cells. (A) HMC-1 cells (4
3
× 105) were pretreated with Niram (1, 10, and 100 g/ml) for 2 h and stimulated with
4
PMACI for 7 h. The production of TSLP was analyzed with the ELISA. (B) HMC-1
5
cells (1 × 106) were pretreated with Niram (1, 10, and 100 g/ml) for 2 h and stimulated
6
with PMACI for 5 h. The mRNA expression of TSLP was analyzed with the real time-
7
PCR analysis. (C) Cell viability was analyzed with the MTT assay. Each datum
8
represents the mean ± SEM of three independent experiments. #p < 0.05; significantly
9
different from unstimulated cells. *p < 0.05; significantly different from PMACI-
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stimulated cells.
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Fig. 2. Niram regulates the expressions of caspase-1 and RIP2 in stimulated HMC-1
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cells. HMC-1 cells (5 × 106) were pretreated with Niram (1, 10, and 100 g/ml) for 2 h
14
and stimulated with PMACI for 1 h. The expressions of caspase-1 and RIP2 were
15
analyzed with Western blot analysis.
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Fig. 3. Niram ameliorates clinical symptoms in DNFB-induced AD mouse model. (A)
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The clinical features were observed 4 h after the last DNFB sensitization. (B)
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Histological analysis of the lesional dorsal skin was examined by H&E staining for
20
inflammatory cells and epidermis thickness. The inflammatory cells were counted from
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five tissue sections (1.5 × 103 μm2) selected randomly. The inflammatory cells were
22
indicated by arrows. Representative photomicrographs were examined at 400×
23
magnification. (C) The time of scratching behavior was measured at 4 h after the last
24
DNFB sensitization for 10 min. *p < 0.05; significantly different from DNFB-sensitized
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group (control). n = 3.
2
Fig. 4. Niram regulates the levels of TSLP and caspase-1 in AD mouse model. (A) The
4
protein expression of TSLP from the lesional dorsal skin homogenates was analyzed
5
with ELISA. (B) The mRNA expression of TSLP was analyzed with RT-PCR analysis.
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(C) The TSLP+ (FITC) and c-Kit+ (TRITC) cells in the DNFB-sensitized skin lesions
7
were examined with a confocal laser-scanning microscope. Mast cells are identified as
8
c-Kit+ cells. The arrows in merged images show the colocalization of mast cells and
9
TSLP. (D) The caspase-1+ (FITC) and c-Kit+ (PE) cells in the DNFB-sensitized skin
10
lesions were examined with a confocal laser-scanning microscope. The arrows in
11
merged images show the colocalization of mast cells and caspase-1. #p < 0.05;
12
significantly different from vehicle group. *p < 0.05; significantly different from
13
DNFB-sensitized group (control). n = 3.
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Fig. 5. Inhibitory effect of Niram on the levels of AD-related factors in AD mouse
16
model. (A) The mRNA expressions of IL-4 and IL-6 were analyzed with RT-PCR
17
analysis. (B) The protein expressions of IL-4 and IL-6 from the lesional dorsal skin
18
homogenates were analyzed with ELISA. (C) The level of serum IgE was analyzed with
19
ELISA. The intensity of serum histamine was assayed as described in materials and
20
methods section. #p < 0.05; significantly different from vehicle group. *p < 0.05;
21
significantly different from DNFB-sensitized group (control). n = 3.
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Fig. 6. Niram inhibits the level of TSLP in stimulated HaCaT cells. (A) HaCaT cells (2
24
× 105) were pretreated with Niram (1, 10, and 100 g/ml) for 2 h and stimulated with
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30 poly(I : C) 10 g/ml for 24 h. The production of TSLP was analyzed with the ELISA.
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(B) HaCaT cells (1 × 106) were stimulated with poly(I : C) 10 g/ml for 10 h. The
3
mRNA expression of TSLP was analyzed with the real time-PCR analysis. (C) HaCaT
4
cells (1 × 106) were pretreated with Niram (1, 10, and 100 g/ml) for 2 h and stimulated
5
with poly(I : C) for 2 h. The mRNA expression of TSLP was analyzed with the real
6
time-PCR analysis. (D) Cell viability was analyzed with the MTT assay. Each datum
7
represents the mean ± SEM of three independent experiments. #p < 0.05; significantly
8
different from unstimulated cells. *p < 0.05; significantly different from poly(I : C)-
9
stimulated cells.
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Fig. 7. Representative HPLC chromatogram of Niram. The HPLC fraction was
12
monitored by recording the UV absorbance at 230 nm.
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