Curcumin shows excellent therapeutic effect on psoriasis in mouse model

Accepted Manuscript Curcumin shows excellent therapeuticeffect on psoriasis in mouse model Di Kang, Bowen Li, Lei Luo, Wenbing Jiang, Qiumin Lu, Mingqing Rong, Ren Lai PII:

S0300-9084(16)00039-0

DOI:

10.1016/j.biochi.2016.01.013

Reference:

BIOCHI 4931

To appear in:

Biochimie

Received Date: 13 August 2015 Accepted Date: 24 January 2016

Please cite this article as: D. Kang, B. Li, L. Luo, W. Jiang, Q. Lu, M. Rong, R. Lai, Curcumin shows excellent therapeuticeffect on psoriasis in mouse model, Biochimie (2016), doi: 10.1016/ j.biochi.2016.01.013. 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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Curcumin shows excellent therapeuticeffect on psoriasis in mouse

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model

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Di Kang1,2¶, Bowen Li1,2¶, Lei Luo1,2¶,Wenbing Jiang3, Qiumin Lu1，Mingqing Rong1*, Ren Lai1*

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Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese

Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming

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650223, Yunnan, China; 2University of Chinese Academy of Sciences, Beijing100009,

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Technology

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China; 3Collegeof Life Science and Technology, Kunming University of Science and

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These authors have the same contribution to this paper

*Authors to whom correspondence should be addressed: 32# Jiaochangdonglu, Kunming 650223, Yunnan, China, Tel: +86 871 6519 6202 Fax: +86 871 6519 9086, E-mail:[email protected] (MR) or [email protected] (RL).

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Abstract Curcumin is an active herbal ingredient possessing surprisingly wide range of

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beneficial properties, including anti-inflammatory, antioxidant, chemopreventive and chemotherapeutic activity. Recently, it has been reported to exhibit inhibitory activity

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on potassium channel subtype Kv1.3. As Kv1.3 channels are mainly expressed in T cells and play a key role in psoriasis, the effects of curcumin were investigated on inflammatory factors secretion in T cells and psoriasis developed in keratin (K) 14-vascular endothelial growth factor (VEGF) transgenic mouse model. Results showed that, 10 µM of curcumin significantly inhibited secretion of inflammatory

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factors including interleukin (IL)-17 ,IL-22, IFN-γ, IL-2, IL-8 and TNF-α in T cells

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by 30-60% in vitro. Notably, more than 50% of T cells proliferation was inhibited by

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application of 100 µM curcumin. Compared with severe psoriatic symptoms observed in the negative control mice, all psoriasis indexes including ear redness, weight,

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thickness and lymph node weight were significantly improved by oral application of curcumin in treatment mouse group. Histological examination indicated that curcumin had anti-inflammatory function in the experimental animals. More than 50% level of inflammatory factors including TNF-α, IFN-γ, IL-2, IL-12, IL-22 and IL-23 in mouse serum was decreased by curcumin treatment as well as cyclosporine. Compared with renal fibrosis observed in the mouse group treated by cyclosporine, no obvious side effect in mouse kidney was found after treated by curcumin. Taken together, curcumin, with high efficacy and safety, has a great potential to treat psoriasis. Keyword: Curcumin, Kv1.3 channel, cytokine, psoriasis 2

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1. Introduction A diversity of physiological and pathophysiological function of potassium channel

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has been well described with some remarkable progress achieved, since the first cloning of Kv channelwas successfully done more than 20 years ago [1]. K+ channels

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are ubiquitous in a variety of cells, neurons, cardiac myocytes, skeletal muscles, smooth muscles, pancreatic β-cells, lymphocytes and tumor cells. Kv1.3 channel has been demonstrated to be expressed predominately in T cells, effector memory T lymphocytes (TEM) and notably up-regulated in activated TEM cells, where in the number of channel is found to be increased from approximately 250 to 1500 channels

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per cell [2, 3]. The activation of Kv1.3, controlling the efflux of K+ out the cells,

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provides persistent electrical signal and a rise in intracellular Ca2+ concentration,

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which alters the proliferation and activation of T cells [4,5]. Many scientists believe that blockers of the Kv1.3 channel could be potential therapeutic interests to treat

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autoimmune diseases. A sea anemone toxin ShK, with 35 amino acid residues, is reported to show a high affinity to Kv1.3 with an IC50 value of 10 pM [6]. A synthetic

analog of ShK, ShK-186, is reportedly supposed to be a therapeutic for autoimmune diseases and has already been scheduled to phase-1 clinical in 2011 [7]. Millions of people have been suffering from psoriasis, a kind of serious autoimmune inflammatory disease usually occurred in the skin and joints. Intraregional T-lymphocytes trigger primed basal stem keratinocytes to proliferate and perpetuate the disease process of psoriasis [8-11]. Transgenic, spontaneously mutated or chemically induced mouse models were used for understanding the 3

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pathogenesis of psoriasis and subsequently screening for anti-psoriatic compounds. Those ideal mouse models are supposed to show the same symptom to human, such

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as histological, cellular and molecular features of psoriatic skin. A mouse cDNA encoding VEGF164 was used to generate K14-VEGF transgenic mice imitate the

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psoriatic of human [12]. These transgenic mice resulting inflammatory skin condition with profound cellular and molecular features of psoriasis, including the characteristic vascular changes and epidermal alterations.

Curcumin (diferuloylmethane), isolated from a rhizomatous herbaceous perennial plant Curcumalonga (Linn) (turmeric), has been used as a traditional Chinese

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Medicine for a very long period to treat abdominal pain, as well as forliver disorders,

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diabetic wounds, rheumatism, anorexia, and menstrual difficulties [13,14]. In recent

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years, curcumin has been well studied and some of its pharmacological properties have been described. For instance, it is reported to interact with multiple targets and

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possess activities on decreasing cell proliferation and enhancing apoptosis of tumor cells. Importantly, curcumin shows potential effects against the development of pancreatic cancer, skin cancer, breast cancer and colorectal cancerin animal models [15]. It has been regarded as a complementary therapy to the treatment of psoriasis in clinical, but the mechanism is still unknown [16-19]. Besides those functions stated above, curcumin reduces the Kv currents in rabbit coronary arterial smooth muscle cells where it acts as a potent blocker of Kv channels [20]. In addition, curcumin also inhibits Kv1.3 currents in Jurkat Tcells and TEM [21,

22]. In this study, curcumin was found to inhibit currents of Kv1.3 channel and the 4

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proliferation of T cells as well. Moreover, curcumin showed a potenteffect on a

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psoriasis model of 14-VEGF transgenic mice.

2. Methods

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2.1Reagents

Curcumin, purchased from Sigma-Aldrich(St Louix, MO), was prepared as a 10 mM stock solution in dimethyl sulfoxide (DMSO). Antibodies to human CD3 and CD28 were from BD Biosciences (San Jose, CA). All other chemicals used were

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purchased from Sigma-Aldrich.

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2.2 Transient transfection and cell culture

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Two plasmids encoding Kv1.3 and green fluorescent protein were transfected to human embryonic kidney 293 (HEK293) cells [23]. Cells were grown under standard

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tissue culture conditions (5% CO2, 37℃) in DMEM supplemented with 10% FBS. After 3 hours, HEK293 cells were washed with fresh medium.

2.3 Patch-clamp recording on human Kv1.3 channel HEK293 cells with GFP fluorescence were selected for patch-clamp recordings after 36-72 h of transfection. K+ currents were recorded using an internal solution containing the followings (in mM): KCl 140, MgCl2 2.5, HEPES 10, and EGTA 11 (pH 7.2). The external bathing solution contained the followings (in mM): NaCl 150, KCl 5, CaCl2 25, MgCl2 12, HEPES 10, and D-glucose 10 (pH 7.2). Whole-cell 5

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patch-clamp recordings were carried out at room temperature (20-25℃) using an

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EPC-10 amplifier (HEKA, Germany) [24].

2.4 Human T cell isolation.

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T cells from peripheral blood mononuclear cells (PBMCs) of healthy volunteers were isolated by negative magnetic depletion using biotin-conjugated CD14, CD15, CD16, CD19, CD34, CD56, CD123 and CD235a (Human Pan T Cells Isolation Kit; Miltenyi Biotec). The PBMCs were separated using Ficoll-Hypaque density gradient centrifugation for 15 min at 3,000 rpm following the manufacturer’s protocol. The

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purity of TEM cells was assessed by flow cytometry to be more than 95%. Then the T

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cells were maintained in RPMI 1640 medium (Thermo) supplemented with 10% FBS

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(Thermo), penicillin (100 U/ml) and streptomycin (100 µg/ml) in 5% CO2 at 37℃ on 96-well plates (105 cells/well) for 1 h. Approval to conduct these studies was obtained

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from the ethics committee of the Institutional Review Board of the Kunming Institute of Zoology, Chinese Academy of Sciences (2013-116). All participants were provided written informed consent for the use of blood samples. 2.5 Cytokine secretion by human CD3+ T cells. Curcumin and Cyclosporin A (Cs A) were diluted in PBS and then added 1 h prior

to bead stimulation. The isolated CD3+ T cells were activated using anti-CD3+/ CD28+ dynabeads (Invitrogen) at a T cell: bead ratio of 1:1 in 200 µL of RPMI medium in 96-well plates, in triplicate. After a 16-h activation, cells were counted and supernatant were analyzed for determining the concentration of cytokines (hTNF-α, 6

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hIL-2, hIL-8, hIL-17, hIL-22 and hIFN-γ ) using ELISA (R&D, USA) following the

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manufacturer’s instructions.

2.6 Effects of curcumin in a mouse model of psoriatic.

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Three months old keratin 14-VEGF transgenic mice with a psoriatic form phenotype were selected for further experiment (♀18, ♂18) [12]. Mice were divided into three group, then were treated with saline, curcumin or Cs A. Curcumin (40 mg/kg) or Cs A (40 mmol/kg) were administered orally to mice every 24 hours and a psoriatic-form phenotype of ears was photographed every 5 days during the treatment.

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Four parameters of psoriasis including ear redness, ear thickness, ear weight, and

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lymph node weight were used to estimate the severity of psoriasis. The concentration

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of cytokines (mTNF-α, mIL-2, mIL-8, mIL-17, mIL-22 and mIFN-γ) in serum sampled was determined by ELISA.

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All mice in this study were fed in housed in standard cages (391 x 199 x 160 mm)

with enough food and water. Each group of this study contained five mice and then were sacrificed by use of analgesia. This study with all the experimental protocols was approved and the methods were carried out in accordance within the guidelines of the Animal Care and Use Committee at Kunming Institute of Zoology, Chinese Academy of Sciences (2013-115).

2.7 Histology and immunohistochemistry The biopsy specimens of the mice skin in the central part of psoriasis (each 5 days 7

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after treatment) were observed under mice for light microscopy. Skin specimens were fixed in 10% formalin, dehydrated through a graded series of ethanol, cleared in

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xylene, and embedded in paraffin wax. Thick sections (5 µm) were prepared and stained with hematoxylin and eosin (H&E) for histological analysis. Renal fibrosis

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was evaluated by Masson trichrome staining [25]. For immunohistochemical analysis, ear sections were stained with antibodies to CD3 or E-selectin (CD62E) (diluted 1:100; Abcam) [26].

2.8 Statistical analysis

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Data was analysis by using the GraphPad Prism 4 and SigmaPlot. All data points

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are shown as mean ± SE. Dose-response curves were fitted using the Hill logistic

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equation: y=1-(1-fmax)/[1+([Tx]/IC50)n] where fmax is the fraction of current resistant to inhibition at high toxin (Tx) concentration and n is an empirical Hill coefficient.

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Statistical differences were determined using the 1-way analysis of variance test or Student’s t test. Values of P < 0.05 were considered significant.

3 Results 3.1 Effect of curcumin on hKv1.3 currents Curcumin was previously identified as an inhibitor of Kv1.3 channel [27]. In order to confirm this effect, hKv1.3 channels were expressed in HEK293 cells and electrophysiological experiment was performed. Currents were elicited by a 300 ms depolarization of +10 mV from a holding potential of -80 mV. About 45% of Kv1.3 8

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currents were depressed by curcumin at the concentration of 10 µM (Fig. 1A). Fitting the Hill equation to the data points (Fig. 1B) yielded an IC50 of 12.5±3.2 µM.

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CyclosporineA (Cs A), an immunosuppressive agent of T lymphocytes, which has been using to treat psoriasis since 1979. After added 100 µM Cs A, no effects were

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detected on KV1.3 channel (data not shown).

3.2 Effect of curcumin on T cells

Kv1.3 channels were expressed on T cells involved in proliferation and cytokine secretion. Now, it has been regarded as an important therapeutic target for diverse

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autoimmune diseases. Curcumin and Cs A were incubated with T cells for 1 h before

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T cells were activated by anti-CD3+/CD28+ dynabeads. ELISA was carried out to

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detect the concentration of cytokines (TNF-α, IL-2, IL-8, IL-17, IL-22 and IFN-γ) after activation for 16 h. It was found that 10 µM curcumin significantly reduced

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production of IL-17, IL-22, IFN-γ, IL-2, IL-8 and TNF-α by about 36%, 24%,

50%,69%, 49% and 29%, respectively（Fig. 2A-F). Moreover, 100 µM curcumin almost completely inhibited the production of TNF-α, IL-2 and IFN-γ (Fig. 2C, D, F).

Similar to curcumin, 10 µM Cs A reduced the production of IL-17, IL-22, IFN-γ, IL-2, IL-8 and TNF-α by about 36%, 33%, 77%, 75%, 48% and 56%, respectively (Fig. 2A-F). Meanwhile, 100 µM of both curcumin and Cs A depressed T cells proliferation by more than 50% (Fig. 2G).

3.3 Therapeutic effects of curcumin on transgenic mouse model of psoriasis 9

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As Kv1.3 is a critical target of autoimmune diseases, a transgenic mouse model was used to test the effect of curcumin on psoriasis. In 3 months old K14-VEGF

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transgenic mice, occasional focal skin lesions begin to occur on the ear, and then expand to the dorsal as well as lateral skin with age increased [10]. Mice with visibly

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red vascular and little signs of thickening on the ears were selected to investigate the function of curcumin and Cs A (Fig. 3). As shown in Fig 3, red psoriatic forms of phenotype (thickening and erythema) were visible and continuously worsened with the days increased. A little signs of thickening and erythema phenotypes were detected on ears under the treatment of curcumin and Cs A at the first 5 days (Fig. 3).

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After treatment for 15-20 days, the psoriatic forms of phenotypes gradually

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disappeared, while the ear skins of the control group were visibly redder and became

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worse than that observed the beginning of the treatment (Fig. 3). The significance of therapeutic benefits was evaluated by four parameters such as

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ear redness, ear thickness, ear weight and lymph node weight. Initially, ear redness of male and female was 8.1 and 6.2, respectively, while it increased to 11.8 in control group of both male and female after 20 days. Ear thickness of male and female in control group, after 20 days, increased about to 133 and 138 µM, respectively. Ear

thickness of male and female after curcumin treatment for 20 days was reduced to 241 and 227 µM, respectively, while in Cs A treated group it was reduced to 276 µM in both male and female. Ear weight of male and female in control group was about 28 and 26 mg, respectively. After 20 days, ear weight of male and female increased to 32 and 30 mg, respectively. Ear weight of male and female after curcumin treatment for 10

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20 days was reduced to 25.4 and 23.6 mg, respectively. Meanwhile, ear weight of male and female after treated by Cs A was 25.8 and 25.4 mg, respectively. At the

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beginning of treatment, lymph node weight of male and female in control group was 8.3 and 7.9 mg, respectively, while after 20 days later, lymph node weight of male

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and female increased to 9.5 and 9.1 mg, respectively. In contrast to the control group, the lymph node weight of both male and female in curcumin treated group decreased to 6.0 mg after treatment for 20 days. The lymph node weight in Cs A treated group for male and female was 7.4 and 7.6 mg, respectively.

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3.4 Effect of curcumin on cytokine levels of transgenic mouse model

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Cytokine levels (TNF-α, IFN-γ, IL-2, IL-12, IL-22, IL-23) of psoriasis mice were

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also tested to confirm the therapeutic effect. As showed in Fig 5, the cytokine levels of IFN-γ, TNF-α, IL-2, IL-12, IL-22 and IL-23 of both male and female mice

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chronically increased in control mice after 20 days (Fig. 5). At the beginning of the treatment, cytokine (IFN-γ,TNF-α, IL-2, IL-12, IL-22, IL-23) of male and female in

the three groups was almost at the same levels. After treatment with saline for 20 days, TNF-α, IFN-γ, IL-2, IL-12, IL-22 and IL-23 of male increased to 105, 114,118, 102, 104 and 110 pg/ml, respectively (Fig. 5), while the cytokine level of IFN-γ, IL-2,

IL-12, IL-22, IL-23 in female was 119, 117, 122, 112, 109 and 120 pg/ml, respectively (Fig. 5). In contrast to control group, IFN-γ,TNF-α, IL-2, IL-12, IL-22 and IL-23 in male mice under 20 days treatment of curcumin were reduced to 80, 85, 81, 76, 73 and 89, respectively, while the level in female were 78, 83, 84, 69, 73 and 11

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92, respectively (Fig. 5). IFN-γ, IL-2, IL-12, IL-22 and IL-23 of male in Cs A treatment for 20 days were 65, 82, 66, 64, 63 and 67, respectively, while the level in

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female were 64, 79, 63, 60, 59 and 60, respectively (Fig. 5).

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3.5 Histology and immunohistochemistry results after treatment

H&E-staining results of the three groups revealed a mild and potentially pre-psoriatic phenotypein mouse of 3 months old. After treatment for 20 days, an increase of epidermal hyperplasia with acanthosis, absence of the granular layer of the epidermis, focal parakeratosis and intralesional mononuclear cells were found in

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control male and female mice, while no psoriatic form of phenotype appeared in the

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ear of curcumin and Cs A treatment group (Fig. 6A). No difference was observed in

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histologic examinaion of both male and female in the three groups. Compared with the beginning of the treatment, baker scores showed a remarkable reduction after

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curcum in and Cs A treatment for 10 or 20 days, while those of the control group were notably increased (Fig. 6B). CD3+ is a sign of active T cells and stands for the distribution of T cells. As shown in Fig. 7B, T cells were significantly up-regulated and detected widely in the ear skin in control group, whereas only few were found in curcumin and Cs A treated group after 20 days later (Fig. 6C). Histological results showed a big difference among the three groups (Fig. 6D). In addition, E-selectin, distributed in basal keratinocytes and vasculature, was a hallmark of the hyperplastic and inflamed vessels. Immunochemistry and histologic evaluation revealed that only few inflammatory vessels or cells were found in those mice treated with curcumin and 12

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cyclosporine. In contrast to treatment groups, a larger number of inflammatory vessels or cells were detected in control group after 20 days (Fig. 6E). The results

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fromhistological examination of both curcumin and Cs A treated group were almost

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same, but exhibited a great difference compared with control group (Fig. 6F).

3.6 Side effect of curcumin on kidney

It is well known that Cs A has indisputable efficacy in the treatment of severe plaque psoriasisin clinical trials. However, Cs A exhibits serious side effects in administration of therapy such as increase of vascular resistance and may result in

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renal fibrosis [27, 28]. The masson-trichrome staining results showed an obvious

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renal fibrosis area in kidney after treatment with cyclosporine for 20 days in

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K14-VEGF transgenic mice (Fig. 7). Similar to the control group, the stained kidney from curcumin treated mice showed the same results to the control group, indicating

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no side effects on kidney.

4. Discussion

Curcumin is a traditional drug possessing diverse activities such as antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal, and anticancer activity. According to recent discoveries, curcumin influences a variety of ion channels and transporters including potassium channel, calcium channel, chloride channel. Thus, curcumin has potential therapeutic effects against a wide range of diseases. In this study, we demonstrated that curcumin inhibited hKv1.3 channel with IC50 value of 12 13

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µM, which possibly interacted with the pore blocker binding site of potassium channel [29].

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Kv1.3 blockers, such as clofazimine, canspecifically suppress the disease-causing TEM cells and reduce the production of the inflammatory factors without significantly

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affecting TCM cells cytokines. Furthermore, clofazimine inhibits IL-2 production in Jurkat T cells [30]. In this study, curcumin reduced the expression of inflammatory cytokines such as TNF-α, IL-2, IL-8, IL-17, IL-22 and IFN-γ in T cells and depressed the proliferation of T cells. These results show similarity to a previous report, although the target of curcumin was not revealed previously [31]. Thus, we

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demonstrated that curcumin influenced activity as well as proliferation of T cells by

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inhibiting currents of Kv1.3 channel.

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Effector memory (TEM) cells can migrate to inflamed sites of autoimmune diseases and perform immediate effectory functions by secreting massive cytokines

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[27]. Pathologic cytokines comprise of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interferon-α (IFN-α), T-helper-1 (Th1) cytokines (for

instance, IFN-γ), Th17 cytokines such as interleukin-17 (IL-17) and IL-22, and

antigen presenting cell (APC)-derived cytokines (e.g.IL-12 and IL-23) [32].Cytokines and chemokines especially IL-2 and IL-23 of T cells have clearly been a central role in the pathogenesis of psoriasis [33, 34]. IL-2 and gamma interferon (IFN-γ) predominantly promoted cell-mediated immunity [35, 36]. Curcumin showed a great therapeutic value to the mouse model of psoriasis Psoriaticform phenotypes including ear redness, ear thickness, ear weight, and lymph 14

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node weight were significantly reduced after treatment with 40 mg/kg curcumin for 20 day. Imaging results further confirmed that ear skin of treated mice turned back to

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normal condition. Furthermore, cytokine levels of TNF-α, IFN-γ, IL-2, IL-12, IL-22 and IL-23 were reduced to normal level after 20 days of curcumin treatment. T cells

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were activated and up-regulated in K14-VEGF transgenic mice, but significantly decreased in curcumin treated group. It implies that inhibition of Kv1.3 channel by curcumin induced a regulation of inflammatory cytokines and prevented the proliferation of T cells. In addition, to reduce the level of cytokines, curcumin may influence the signaling pathways including mitogen-activated protein kinases

mice [37, 38].

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(MAPKs), activator protein (AP)-1 and nuclear factor (NF)-κB to cure psoriasis of

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Cs A inhibited the activation of T-cells and reduced the production of IL-2 and IFN-γ [39]. Cyclosporine at doses of 2.5 to 5.0 mg kg−1 d−1 over 12 to 16 weeks

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exhibits significant improvement in psoriasis, in some cases it shows complete clearance of the disease [28]. Besides the significant therapeutic effect, the main side effects of cyclosporine used for the treatment of psoriasis are hypertension, renal toxicity, and cancer [27]. Interestingly, in this study, curcumin showed no toxicity to kidney after treatment for 20 days. Thus, curcumin shows high safety for further use. Curcumin was demonstrated to inhibit Kv1.3 channel and depress the expression of

cytokines and proliferation of T cells. It was also found to show a great therapeutic effect on psoriasis in mouse model without obvious side effects. Conclusively, our study suggests that curcumin may be a potential drug candidate to treat psoriasis. 15

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Conflict of interest

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The authors declare no competing financial interests.

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Acknowledgements

This work was supported by MOST (2013CB911300, 2010CB529800), NSFC (31025025, 31025025, U1132601, 31200590), the West light Doctoral program and Youth Innovation Promotion Association of CAS and Yunan Province (2013FB072).

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inhibiting IL-1beta and IL-6 production in mice.PLoS One. 8（2013）:e67078

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NestleFO. Psoriatics. Curr. Dir. Autoimmun. 10(2008) 65-75.

33. Nickoloff BJ. The cytokine network in psoriatics. Arch. Dermatol. 127 (1991) 871-84. 34. Schön MP, et al., Psoriatics: the plot thickens. Nat. Immunol. 2(2001) 91. 35. Capon F, et al., Sequencevariants in the genes for the interleukin-23 receptor (IL23R) and its ligand(IL12B) confer protection against psoriatics. Hum. Genet. 122 (2007) 201–206. 36. Cargill M, et al., A largescalegenetic association study confirms IL12B and leads 19

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to the identification ofIL23R as psoriatics-risk genes. Am. J. Hum. Genet. 80 (2007) 273–290.

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37. Liacini A, et al., Induction of matrix metalloproteinase-13 gene expression by TNF-alpha is mediated by MAP kinases, AP-1, and NF-kappaB transcription

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factors in articular chondrocytes. Exp. Cell. Res.288 (2003) 208-17.

38. Ju JH, et al., IL-23 induces receptor activator of NF-kappaB ligand expression on CD4+ T cells and promotes osteoclastogenesis in an autoimmune arthritis model. J. Immunol. 181 (2008)1507-18.

39. Clipstone NA, et al., Identification of calcineurin as a key signalling enzyme in

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T-lymphocyte activation.Nature. 357 (1992)695-7.

Figure 1. Effect of curcumin on Kv1.3 channel expressed in HEK293 cells. (A)

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Currents were elicited by a 300 ms depolarization of +10 mV from a holding potential of -80 mV. The red line was the currents elicited after added 10 µM curcumin. (B) Concentration-response curve for curcumin inhibition of Kv1.3 currents in HEK293T cells (n=5).

Figure 2. Curcumin depressed cytokines levels and cell proliferation. Curcumin and Cs A inhibited secretion of IL-17 (A), IL-22 (B), IFN-γ (C), IL-2 (D), IL-8 (E) and TNF-α (F) by T lymphocytes. (G) The effect of different concentrations of curcumin and Cs A on human T cell proliferation.All data points are shown as mean ±S.E (n = 20

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indicated by * (p﹤0.05) and ** (p﹤0.01).

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5). Statistically significant differences compared to the saline control group are

Figure 3. Developmentof psoriaticform phenotype on the ear skin of male mice

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(A)and female mice (B) treated with either saline (control group), cyclosporine A or curcumin. Curcumin (40 mg/kg) or Cs A (40 mmol/kg) were administered orally to mice every 24 hours for 20 days. Images were photographed each 5 daysduring the treatment.

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Figure 4.Analysis of psoriaticform phenotype in keratin 14-VEGF mice treated with

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saline, curcumin (40 mg/kg) or Cs A (40 mmol/kg). The phenotypesof

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psoriasisinclude ear redness (A), ear thickness (B), ear weight (C), and lymph node

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weight (D). All data points are shown as mean ±S.E.

Figure 5. Cytokine levels in male and female mice treated with saline, cyclosporine and curcumin for 20-day. Levels of IFN-γ (A), TNF-α (B), IL-2 (C), IL-12 (D), IL-22

(E) and IL-23 (F) were measured by Elisa methods. All data points are shown as mean ±S.E.

Figure 6. Histological evaluation of the different treatment groups (saline, cyclosporine A or curcumin). Photomicrographs of ear sections stained with hematoxylin and eosin stain (A), CD3 (C), and CD62E (E) and each of their baker 21

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score of hematoxylin and eosin stain (B), CD3 (D), CD62E (F). Photomicrographs of each group contained three male mice and three female mice and observed every 10

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days.Scale bars =50 µm. All data points are shown as mean ±S.E (n = 5). Statistically significant differences compared to the saline control groupare indicated by ** (p﹤

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0.01).

Figure 7. Side effects of curcumin and CsA on the kidney of keratin 14-VEGF transgenic mice. Histological evaluation of the different treatment groups (saline, CsA or curcumin). The kidney sections were stained by masson trichrome. Blue area

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wasstand for fibrous tissue. Photomicrographs of each group were observed every 10

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days. Scale bars =100 µm.

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ACCEPTED MANUSCRIPT 1. T cells proliferation was inhibited by curcumin though Kv1.3 channel.

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2. Oral application of curcumin showed an obvious effects on psoriasis. 3. No obvious side effects of curcumin was found in mouse kidney.

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4. curcumin has a great potential to treat psoriasis.