Geraniol targets KV1.3 ion channel and exhibits anti-inflammatory activity in vitro and in vivo

Journal Pre-proof Geraniol targets KV1.3 ion channel and exhibits antiinflammatory activity in vitro and in vivo

Chen-Jun Ye, Sheng-An Li, Yun Zhang, Wen-Hui Lee PII:

S0367-326X(19)31153-0

DOI:

https://doi.org/10.1016/j.fitote.2019.104394

Reference:

FITOTE 104394

To appear in:

Fitoterapia

Received date:

31 May 2019

Revised date:

15 October 2019

Accepted date:

20 October 2019

Please cite this article as: C.-J. Ye, S.-A. Li, Y. Zhang, et al., Geraniol targets KV1.3 ion channel and exhibits anti-inflammatory activity in vitro and in vivo, Fitoterapia (2018), https://doi.org/10.1016/j.fitote.2019.104394

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© 2018 Published by Elsevier.

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Geraniol targets KV1.3 ion channel and exhibits anti-inflammatory activity in vitro and in vivo Chen-Jun Yea,b, Sheng-An Lia, Yun Zhanga, * [email protected] and Wen-Hui Leea,* [email protected] a

Key Laboratory of Animal Models and Human Disease Mechanisms of The Chinese

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Academy of Sciences/Key Laboratory of bioactive peptides of Yunnan Province,

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Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming, Yunnan

Kunming College of Life Science, University of Chinese Academy of Sciences,

Kunming, Yunnan 650204, China

Abstract

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Corresponding author.

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*

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b

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650223, China

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Naturally occurring monoterpenes are known for their various pharmacological activities including anti-inflammation. KV1.3 ion channel is a voltage-gated potassium channel and has been validated as a drug target for autoimmune and chronic inflammatory diseases like psoriasis. Here we experimentally test the direct interaction between monoterpenes and KV1.3 ion channel. Our electrophysiological analysis determined that monoterpenes (geraniol, nerol, β-citronellol, citral and linalool) have inhibitory effects on KV1.3 ion channel. Representatively, geraniol reversibly blocked KV1.3 currents in a voltage-dependent manner with an IC50 of 490.50 ± 1.04 μM at +40 1

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mV in HEK293T cells. At the effective concentrations, geraniol also inhibited cytokine secretion of activated human T cells, including IL-2, TNF-α and IFN-γ. In an imiquimod-induced psoriasis-like animal model, geraniol administration significantly reduced psoriasis area and severity index scores, ameliorated the deteriorating histopathology and decreased the degree of splenomegaly. Together, our findings not

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only suggest that monoterpenes may serve as lead molecules for the development of

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KV1.3 inhibitors, but also indicate that geraniol could be considered as a promising

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therapeutic candidate to treat autoimmune diseases.

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Keywords: monoterpenes; geraniol; KV1.3; autoimmune diseases; psoriasis

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1. Introduction

KV1.3, encoded by the KCNA3 gene, is a voltage-gated potassium channel belonging

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to the shaker (KV1) subfamily and mainly expressed in neurons and immune cells [1].

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Owing to its special expression pattern, KV1.3 channel plays a dominant role in effector memory T cells (TEM), and specific inhibitors of KV1.3 suppress function of TEM cells with little effect on naive and central memory T cells (TCM), thus providing an excellent opportunity for selective immunomodulation [2–4]. Autoreactive TEM cells with high KV1.3 channel expression have been identified as major players in many autoimmune diseases and chronic inflammatory diseases, such as multiple sclerosis, rheumatoid arthritis, psoriasis, chronic kidney disease, ulcerative colitis and asthma, and KV1.3 inhibitors have been confirmed effective in different kinds of animal models [1, 5–10]. Therefore, KV1.3 channel has become an attractive drug target for autoimmune and 2

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chronic inflammatory diseases.

Psoriasis is a common chronic autoimmune skin disease bothering about 2.0–2.5% of the world’s population [11]. Despite much progress in etiology study and drug development for this disease are achieved, the current treatments are far from perfect,

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further exploration of new targets and drugs are still needed, of which oral or topical

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available small molecule inhibitors for KV1.3 channel have drawn special attentions

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[11–13]. Natural products from plants are precious deposits for drug development, and

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clinically or experimentally [14].

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many kinds of herbs or active products have proven effective in treating psoriasis

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Monoterpenes, the simplest class of terpenes with two isoprene units, are widely

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distributed in aromatic and medicinal plants, especially in essential oils; monoterpenes have a wide range of pharmacological properties, such as antibacterial, antioxidant, anti-nociceptive, anticancer and anti-inflammatory activities [15]. Geraniol, nerol, citronellol, citral and linalool represent five most common monoterpenes. Citronellol, geraniol and nerol are characteristic components of rose oil, which shows analgesic, anti-inflammatory and many other pharmacological activities [16]. Citral is a major constituent of Cymbopogon citratus, a traditional Chinese medicine to treat diseases like rheumatism, headache and pruritus [17]. Linalool is present in many flowers and

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spice plants, particularly lavender and coriander, and has diverse bioactive properties [18].

Recently, there are increasing interests in understanding interactions between monoterpenes and ion channels as reviewed in [19]. However, whether monoterpenes

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direct interact with KV1.3 ion channel is unknown. In this study, we investigated the

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effects of monoterpenes (geraniol, nerol, citronellol, citral and linalool) on KV1.3 ion

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channel, as well as the potential of geraniol as an anti-inflammation drug in isolated

2. Materials and Methods

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human T cells and a psoriasis animal model.

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2.1. Cell culture and transient transfection

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Human embryonic kidney 293T cells (HEK293T) were obtained from the Conservation Genetics Chinese Academy of Sciences Kunming Cell Bank. Cells were grown in DMEM/F12 medium plus 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified incubator (5% CO2, 37°C). Plasmids encoding mKV1.3 ion channels and enhanced green fluorescent protein (GFP) were transfected to HEK293T cells using Lipofectamine 2000 Transfection Reagent (Invitrogen, USA). Cells with GFP fluorescence were selected for further patch clamp recordings after 1–2 days of transfection.

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2.2. Electrophysiology and analysis Whole cell and outside-out patch clamp recordings were performed at room temperature with a Multiclamp700B amplifier and a Digidata 1550A analog-digital converter controlled by a pClamp10 software (Molecular Devices, USA). Pipettes were pulled from borosilicate glass (Sutter Instrument, USA) using a micropipette puller

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(P-97, Sutter Instrument, USA), and fire-polished with a pipette microforge (Narishige,

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Japan) to a resistance of 3–5 MΩ. For measuring KV1.3 currents, the external bath

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solution contained (mM): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 Glucose and 10

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HEPES (pH 7.3 with NaOH), and the internal pipette solution contained (mM): 140

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KCl, 3 Na2ATP, 1 MgCl2, 5 EGTA and 10 HEPES (pH 7.3 with KOH).

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Five monoterpenes (Fig. 1) were tested for their KV1.3 ion channel activities. Geraniol

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(≥ 98%), nerol (≥ 97%), citral (≥ 97%) and linalool (≥ 98%) were purchased from Macklin Reagent (Shanghai, China), and β-citronellol (≥ 99%) was bought from Aladdin Reagent (Shanghai, China). They were prepared as 1 M stock solutions in dimethyl sulfoxide (DMSO), and freshly diluted with bath solution to the desired concentrations and mixed well before patch experiments. A gravity-fed multichannel rapid solution change system (RSC-200, Bio-Logic Science Instrument, France) was used to apply bath solution or drugs directly to the cell.

The pClamp10 and GraphPad Prism 5 software were used for data acquisition and 5

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analysis. The IC50 (drug concentration required for 50% inhibition of currents), Hill coefficient, % inhibition, and membrane conductance (G), half-maximum voltage (V1/2) and slope factor (κ) for activation or inactivation were all obtained by previously reported methods [20].

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2.3. Human T cells isolation, activation and cytokines measurement

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These procedures were mainly followed by the reported methods [21]. First, human

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peripheral blood mononuclear cells (PBMC) were separated from whole blood of

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healthy donors by Ficoll density gradient centrifugation with human lymphocyte

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separation medium (Solabio, Beijing, China). Next, the PBMCs were further purified by the Human Pan T Cells Isolation Kit (Miltenyi Biotec, Germany), yielding CD3+ T

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cells with purity >96% as routinely assessed by flow cytometry. Then, the T cells were

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maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin in a humidified CO2 (5%) incubator at 37°C. The human protocol was approved by the ethics committee of the Institutional Review Board of the Kunming Institute of Zoology, Chinese Academy of Sciences.

For induction of cytokines secretion, isolated T cells were seeded to 96 well plates at a density of 1 × 105 cells/well in 200 μL of RPMI 1640 medium and activated using anti-CD3/CD28 dynabeads (Invitrogen, USA) at a cell: bead ration of 1：1. Geraniol and control medium were added 1 h before bead stimulation. After 16 h of activation, 6

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the supernatants were collected and measured for IL-2, TNF-α and IFN-γ respectively by ELISA following the manufacturer’s instructions (NeoBioscience, Shenzhen, China).

2.4. Cell viability and cell proliferation assay

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Isolated naïve T cells were seeded into 96 well plates and incubated with geraniol

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with various concentrations for 24 h. Cell viability was measured using the MTS

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assay kit (Promega) according to the manufacturer’s instructions.

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To test geraniol’s effect on T cell proliferation, isolated T cells were seeded into 96 well plates and stimulated for 24 h, and geraniol were added 1 h before bead

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stimulation. Cell proliferation was detected using the MTS assay kit.

2.5. Western Blot

To measure geraniol’s effect on KV1.3 protein expression, Jurkat T cell line were used. Jurkat cell were seeded to 6 well plate and treated with or without 500 μM geraniol for 24 h, then cell lysates were prepared and used for western blot analysis as detailed in [22]. Primary antibodies against KV1.3 (Immunoway) and GAPDH (Proteintech) were used.

2.6. Animals 7

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Male BALB/c mice from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China) were housed in a standard condition (22 ± 2°C, 50–60% humidity) under a 12 h light/dark cycle with free access to standard laboratory food and water. Mice at 8 to 11 week that had undergone acclimatization for at least one week were used for experiments, and all procedures of this study were approved by the Animal Care and

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Use Committee at Kunming Institute of Zoology, Chinese Academy of Sciences.

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2.7. Imiquimod (IMQ)-induced psoriasis-like model

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Psoriasis-like mice model was induced according to the previous method with slight

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modifications [23]. The back hair of mice were shaved two days before experiments, and mice were randomly divided into five groups (five mice per group): control group,

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IMQ + Vehicle group, IMQ + geraniol (Ger) low group, IMQ + Ger high group, and

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IMQ + Dexamethasone (DXM) group. The four IMQ treated groups of mice all received 40 mg of 5% IMQ cream (Sichuan Mingxin Pharmaceuticals, China) topically applied to a 3 cm × 2 cm shaved area once a day for 6 consecutive days. Vaseline cream (Macklin, China) was applied to the control group mice similarly.

2.8. Drug administration To test the immunomodulatory effect of geraniol, mice of IMQ + Ger low group and IMQ + Ger high group were orally administrated with 50 mg/kg and 200 mg/kg of geraniol daily for 6 days respectively. Pure geraniol was dissolved in corn oil with a 8

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volume of 0.25 mL/100g body weight as described in [24]. Mice of IMQ + Vehicle group received only corn oil. DXM sodium phosphate (Macklin, China) was dissolved in distilled water and was given as a positive control at a dose of 10 mg/kg by gavage for mice of IMQ + DXM group [25]. All the above drug treatments were conducted 1h

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before IMQ challenge.

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2.9. Scoring severity of skin inflammation

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From day 0 to day 6, severity of back skin inflammation were evaluated by the clinical

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Psoriasis Area and Severity Index (PASI) scores before any treatment was done every

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day [23]. Three parameters (erythema, thickness and scales) were assessed independently, scoring from 0 to 4 (0, none; 1, slight; 2, moderate; 3, marked; 4, very

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marked). The total score (0–12) is the sum of the three values. On the day 6, mice were

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photographed, and then sacrificed, samples of skin lesions were collected for further histological analysis and immumohistochemical staining, and spleens were weighed for spleen index analysis.

2.10. Histological analysis Skin samples were fixed in 10% buffered formalin and embedded in paraffin. Sections of 5 μm thickness were made and stained with hematoxylin and eosin (H&E). Microscopic images of histological slides were digitalized and visualized with CaseViewer software (3DHistech, Hungary). For each section, thickness of the 9

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interfollicular epidermis (epidermal thickness) were measured at 10 representative sites [26].

2.11. Immumohistochemical staining Deparaffinized and hydrated sections were microwaved in sodium citrate buffer to

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unmask antigens. After blocking with 5% goat serum, sections were incubated with

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primary antibodies specific for CD3 (Bioss) overnight at 4˚C. After quenching

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endogenous peroxidase, sections were treated with 2-step plus polymer HRP detection

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system (Beijing Zhongshan Jinqiao), and then stained with Enhanced HRP-DAB

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2.12. Statistical analysis

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Chromogenic Kit (TIANGEN BIOTECH).

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Data were expressed as the mean ± SEM and analyzed using GraphPad Prism 5. Unpaired Student's t test was used for two group comparison and values of P < 0.05 were considered significant.

3. Results 3.1. Monoterpenes inhibit KV1.3 currents In an attempt to find new KV1.3 inhibitors from natural compounds, five monoterpenes were tested using whole-cell recordings on HEK293T cells overexpressing KV1.3. As shown in Figure 1, they all exhibited inhibitory effects on KV1.3. In the presence of 1 10

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mM compounds, the ratios of currents inhibition were: 88.48 ± 2.83% for geraniol, 79.53 ± 3.96% for nerol, 78.46 ± 1.05% for β-citronellol, 50.71 ± 4.82% for citral and

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49.53 ± 1.64% for linalool, respectively (Fig. 1A–E; mean ± SEM; n = 4).

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Fig. 1. Structures and KV1.3 inhibitory effects of five monoterpenes. Inhibition of 1

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mM geraniol (A), nerol (B), β-citronellol (C), citral (D) and linalool (E) on KV1.3 whole cell currents expressed in HEK293T cells. Currents were evoked by a 200 ms depolarization to +40 mV from a holding potential of −80 mV every 15 s. Representative current traces in the absence and presence of indicated drugs are shown. 3.2. Effects of geraniol on KV1.3 channel We used geraniol as the representative chemical for further study. As shown in Figure 2A, different concentrations (from 100 to 2000 μM) of geraniol were tested by whole-cell recordings. The current amplitudes were decreased following higher concentrations application, indicating that the inhibitory effect of geraniol was specific 11

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and concentration-dependent. The dose-response data of the currents at the end of pulse were analyzed and fitted with a Hill equation, produced the concentration-response curve in Figure 2B. After applying 500 μM geraniol, the steady-state block of KV1.3 current was reached within 90 s, and the time-course of wash-out was rather quick and complete within 15 seconds (Fig. 2C). Therefore, geraniol possesses a fast and

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reversible interaction with KV1.3 ion channel. To rule out the possibility of indirect

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reactions, we performed the outside-out patch recordings. In the presence of 500 μM

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geraniol, we observed the similar effect as that of whole-cell configuration (Fig. 2A and

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D).

Next, we observed the voltage-dependent inhibition of KV1.3 by geraniol.

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Current-voltage relationships indicated that geraniol inhibited KV1.3 currents

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apparently at potentials above −20 mV (Fig. 2E). The inhibition ratio of geraniol on KV1.3 was plotted as a function of the tested membrane potential. The inhibition percentage increased steeply from 16.80% to 50.48% between −20 mV and +20 mV, consistent with the range for channel opening, while the inhibition reached a plateau from +30 mV to +60 mV, when the channels were fully activated (Fig. 2F). This blocking character suggests that geraniol exerts an open channel block on KV1.3. The effect of geraniol on KV1.3 channel inactivation rate was also analyzed. The inactivation time constant at +40 mV was generated by fitting the current inactivation phase to a mono-exponential equation. Geraniol accelerated the current inactivation 12

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rate dose-dependently (Fig. 2G), which is another indication for open channel block.

Further kinetic studies were performed to investigate the effects of geraniol on activation and inactivation of KV1.3. The steady-state activation curves were generated by plotting the normalized conductance against step potentials from −70 mV to +60 mV

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and fitted with Boltzman equation. In the control, the value of V1/2 for activation was

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−10.30 ± 0.84 mV and slope factor κ was 15.00 ± 0.82; after geraniol application, the

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V1/2 value was −27.07 ± 1.53 mV and κ was 13.24 ± 1.33 (Fig. 2H). Thus, geraniol

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shifted the activation curve in the negative direction. The steady-state inactivation

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curves were determined using a standard double-pulse protocol as described in [20]. In the control, the value of V1/2 for inactivation was −38.08 ± 0.62 mV and slope factor κ

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was 4.24 ± 0.58; after geraniol application, the V1/2 value was −49.89 ± 1.02 mV and κ

curve.

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was 4.53 ± 1.01 (Fig. 2I). Thus, geraniol also caused a negative shift of the inactivation

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Fig. 2. Inhibition of KV1.3 channels by geraniol (Ger). (A) Representative current

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traces were recorded in bath solution and in presence of 100, 200, 500, 600, 1000 or 2000 μM geraniol. (B) The concentration-response curve for the current at the end of pulse. Hill equation fitting gives an IC50 and Hill coefficient of 490.50 ± 1.04 μM and 2.64 ± 0.40 (mean ± SEM; n = 4). (C) Representative trace of time-course for inhibition of 500 μM geraniol on KV1.3 current at the end of pulse. (D) Representative traces of 500 μM geraniol on KV1.3 currents recorded from outside-out patches. (E) Current-voltage relationship curves before and after applying 500 μM geraniol. Whole cell current were elicited by 200 ms steps from −50 mV to +60 mV in 10 mV increments every 15 s from the holding potential of −80 mV. (F) Inhibition ratios at 14

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different test voltages. (G) Time constant of current inactivation at different concentrations of geraniol. (mean ± SEM; n = 3; *, P < 0.05; **, P < 0.01; vs. control). (H, I) The steady-state activation (H) and inactivation (I) curves before and after applying 500 μM geraniol were fitted with Boltzman equation.

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3.3. Geraniol inhibits T cell proliferation and cytokine secretion

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As well documented, KV1.3 inhibitors depolarize the membrane potential, thus

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inhibiting Ca2+ influx required for T cell activation and finally suppressing cytokine

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production and cell proliferation of T cells [2, 27, 28].

To test whether geraniol also has such effects, human CD3+ T cells was isolated. Patch

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clamp experiments showed that geraniol also inhibited Kv1.3 currents in native T cells

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(Fig. 3A). Viability assay and proliferation assay revealed that geraniol potently inhibited T cell proliferation upon stimulation, while had no obvious cytotoxic effect on naïve T cells (Fig. 3B and C).

To test whether the inhibitory effect of geraniol on KV1.3 channel could cause functional immunosuppression, cytokine levels of activated human CD3+ T cells were detected by ELISA. Consistent with our expectations, geraniol concentration-dependently decreased cytokine secretion of T cells upon stimulation. As shown in Figure 3D–E, 200 μM geraniol reduced production of IL-2, TNF-α and IFN-γ 15

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by 32.82 ± 4.19%, 23.55 ± 2.98% and 56.05 ± 7.16%, respectively; while the reductions were more significantly for 500 μM geraniol by 47.12 ± 3.99%, 48.59 ± 1.97% and 78.22 ± 1.07%; moreover, 1000 μM geraniol almost blocked the three kinds of cytokine release completely. The potencies of these effects are consistent with KV1.3 ion channel blockage (Fig. 2A and B). Thus, geraniol demonstrates strong

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immunosuppression function on human CD3+ T cells.

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Besides direct and acute inhibition of KV1.3 channel, Kv1.3 blockers could serve

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anti-inflammatory function by chronic down-regulation of channel expression [20]. To

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determine the effect of geraniol on KV1.3 expression, Western blot analysis were performed, and no obvious change in protein level was observed (Fig. 3G and H).

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Therefore, geraniol suppresses T cell functions without affecting KV1.3 expression.

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Fig. 3. Geraniol (Ger) depressed T cell proliferation and cytokine secretion without affecting KV1.3 expression. (A) Representative traces of 500 μM geraniol on KV1.3 currents recorded from whole cell patches of Isolated human CD3+ T cells. (B) Unstimulated T cells were incubated with geraniol for 24 h, and cell viability was detected by MTS assay. (C) T cells were activated by anti-CD3/CD28 dynabeads for 24 h, and geraniol were applied 1h before stimulation. Cell proliferation were measured by MTS assay. (D–F) T cells were activated by anti-CD3/CD28 dynabeads for 16 h, and geraniol were applied 1h before stimulation. Supernatants were assessed for levels of 17

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IL-2 (D), TNF-α (E) and IFN-γ (F) by ELISA, respectively. Data are shown as mean ± SEM (n=3). Statistically significant differences compared to the control (no geraniol) groups are indicated (**P < 0.01, ***P < 0.001). (G) Representative Western blot detection of KV1.3 expression with or without 500 μM geraniol for 24 h. (H) Relative

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KV1.3 expressions were normalized to GAPDH (n = 3).

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3.4. Geraniol attenuates psoriasis-like inflammation

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To further evaluate whether geraniol has immunosuppressive function in vivo, we next

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examined the therapeutic effects of geraniol on psoriasis-like inflammation in a

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psoriasis animal model, which is induced by topically applying IMQ cream to shaved mice back skin. As an activator of TLR7 and TLR8, IMQ has potent immune activation

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effect and is clinically used to treat papilloma virus infection of skin. However, this

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impact induces inflammatory skin lesions in mice that resembling human plaque type psoriasis in many aspects [23].

After 6 days of treatments, the back skin (IMQ + Vehicle group) showed typical psoriasis-like inflammation, such as erythema, thickening and scaling, compared with control mice (Fig. 4A). Mice treated with geraniol and DXM exhibited significant improvements of morphological feathers and reductions of PASI scores from day 2 to day 6 (Fig. 4B). These results are consistent with the histological analysis. Skin lesions (IMQ + Vehicle group) showed apparently increased hyperkeratosis, acanthosis, and 18

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elongation of rete-like ridge, resembling human psoriasis [29], but H&E staining from geraniol and DXM treated mice exhibited significantly diminished hyperkeratosis, reduced epidermal thickness and few elongation of rete-like ridge (Fig. 5A and B). The therapeutic effects of higher dose of geraniol (200 mg/kg) were comparable with DXM, a corticosteroid used for psoriasis treatment clinically (Fig. 4 and Fig. 5A–B).

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Splenomegaly is another feather of psoriasis-like inflammation induced by IMQ [23,

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30]. The spleen index (spleen weight/body weight) of geraniol and DXM treated groups

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was significantly decreased (Fig. 5C). T lymphocytes infiltration in the skin lesions was

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detected by immumohistochemical staining of CD3, a T cell marker. IMQ treatment

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increased CD3 expression, while geraniol (200 mg/kg) administration significantly reversed this effect (Fig. 5D and E). Taken together, these results demonstrated that

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geraniol effectively alleviated psoriasis-like skin inflammation in IMQ-induced mice.

Fig. 4. Geraniol (Ger) improved the morphological features of imiquimod (IMQ)-induced psoriasis-like skin lesions in mice. To induce psoriasis-like skin inflammation, IMQ cream were topically applied every day from day 0 to day 5. 19

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Vaseline cream was used in control group. Geraniol were orally administrated at a dose of 50 mg/kg (Ger low) or 200 mg/kg (Ger high). Dexamethasone (DXM, 10 mg/kg) was used as a positive control. (A) Representative images of back skin of mice after 6 days of indicated treatments. (B) PASI scoring of skin erythema, thickness and scaling

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was performed daily (mean ± SEM; n = 5).

Fig. 5. Geraniol (Ger) improved the structural features and reduced splenomegaly of IMQ-induced psoriasis in mice. (A) Representative H&E staining images of back skin from each group (Scale bar = 100 μm). Pound sign, asterisk, and arrow indicate 20

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hyperkeratosis, acanthosis, and elongation of rete-like ridge respectively. (B) Epidermal thickness of each group was analyzed (mean ± SEM; n = 5; ***P < 0.001 vs. the IMQ + Vehicle group). (C) Spleen index of each group was analyzed (mean ± SEM; n = 4–5; *P < 0.05, **P < 0.01, ***P < 0.001 vs. the IMQ + Vehicle group). (D) Representative CD3 immumohistochemical staining images of back skin from each

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group (Scale bar = 50 μm). (E) CD3 expression of each group was qualified by average

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optical density (AOD). (mean ± SEM; n = 5; ***P < 0.001 vs. the IMQ + Vehicle

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group).

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4. Discussion KV1.3 inhibitors are promising to treat autoimmune diseases. Our research identified geraniol, nerol, β-citronellol, citral and linalool as new KV1.3 inhibitors. They are naturally occurring monoterpenes extensively used in perfumes, food, cosmetic and human health care products [15]. They all possess anti-inflammatory properties with

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other mechanisms as previously reported. Geraniol and citronellol were reported to

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exhibit anti-inflammatory effects by inhibiting the production of nitric oxide and

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prostaglandin E2 in macrophages [31]; nerol showed therapeutic potential in ulcerative

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colitis verified by the oxazolone-induced colitis model [32]; citral was found to

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decrease cytokines production by murine macrophages [17]; linalool was reported to reduce cigarette smoke-induced inflammatory cells infiltration and cytokines release

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by inhibiting NF-κB activation [33]. Our work indicates that KV1.3 ion channel is a

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novel pharmacological target of monoterpenes, which should play at least part of their anti-inflammatory effects. And these findings suggest that monoterpenes are likely the templates for KV1.3 modulator development, given their simple structures.

Naïve and TCM cells require antigen priming in lymph nodes before migrating to inflammation sites, while TEM cells rapidly enter the local inflamed tissues and exhibit immediate effector functions by secreting massive cytokines, thus playing important roles in autoimmune diseases [1, 3]. KV1.3 inhibitors have been demonstrated to inhibit T cell activation and suppress cytokine secretion. Our results showed that geraniol 22

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potently inhibited the production of cytokines in activated T cells, at concentration ranges consistent with KV1.3 inhibition. D-limonene, another monoterpene, was reported to have a similar effect, but with lower potency (IC50 >2 mM for IL-2, TNF-α and IFN-γ) compared with geraniol (IC50 about 0.5 mM, Fig. 3A–C) [34]. Pharmacokinetic and bioavailability studies reveal that geraniol has a high

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bioavailability of 92% and could reach a peak plasma concentration of 270 μg/mL

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(about 1.75 mM) after oral administration of geraniol (50 mg/kg) to rat [35].

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Accordingly, the effective concentration of geraniol for KV1.3 blockade could have a

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good clinical relevance.

Geraniol was reported to treat inflammatory diseases such as asthma and colitis

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effectively in animal models [24, 36], but whether geraniol has therapeutic potential in

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psoriasis is unknown. In the present study, oral application of geraniol exhibited excellent therapeutic effects on psoriasis-like inflammation in IMQ-induced mice model (Fig. 4), and no obvious changes in motility and vitality of mice were observed during the experiments. Geraniol is classified as Generally Recognized As Safe (GRAS) and it was reported that geraniol could be considered safe even at high doses and for long periods of administration, as results showed that treatment with 120 mg/kg of geraniol on mice for 4 weeks increased anti-oxidative defenses with no signs of liver toxicity but slightly affected cytochrome P450 enzyme activities [35]. Derived No Effect Level (DNEL) of geraniol is 13.5 mg/kg for humans, corresponding to 100–120 23

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mg/kg in mice [36]. In our experiments, 50 mg/kg geraniol is sufficient to alleviate the inflammation of skin lesions, showing potentials to translate geraniol for human treatment.

Essential oils have been widely used in skincare and many of them contain geraniol. It

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will be ideal to use geraniol externally to treat skin inflammation, however, our

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preliminary experiments showed that mice had no obvious improvement after topical

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application of geraniol (data not shown). We suspect that geraniol promotes IMQ

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absorption as a skin penetration enhancer [37], thus counteracting its anti-inflammatory

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effects. Whether topical administration of geraniol has beneficial effect on inflamed

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5. Conclusion

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skin remains to be validated by using other animal models.

In summary, our study identifies KV1.3 ion channel as a novel pharmacological target of monoterpenes, thus providing a new explanation for their anti-inflammatory activities and addressing the possibility of serving monoterpenes as templates of KV1.3 inhibitors. Further in vitro and in vivo studies confirmed the excellent immunosuppressive function of geraniol in both human T cells and psoriasis-like animal model, suggesting that geraniol has great potentials to treat psoriasis and other autoimmune diseases.

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Conflicts of interest None.

Acknowledgments We thank Dr. Shilong Yang (Kunming institute of zoology, CAS) for valuable

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suggestions.

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This study was supported by National Natural Science Foundation of China (31572268)

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and the Yunling Scholar Program (U1602225).

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Graphical abstract:

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Drug Discovery Today. 12 (2007) 1061–1067. doi:10.1016/j.drudis.2007.09.001.

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