Isogarcinol Extracted from Garcinia mangostana L. Ameliorates

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Isogarcinol Extracted from Garcinia mangostana L. Ameliorates Imiquimod-induced Psoriasis-like Skin Lesions in Mice Shanzao Chen, Kesheng Han, Hu Li, Juren Cen, Yanfang Yang, Hezhen Wu, and Qun Wei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05207 • Publication Date (Web): 13 Jan 2017 Downloaded from http://pubs.acs.org on January 19, 2017

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Journal of Agricultural and Food Chemistry

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Isogarcinol Extracted from Garcinia mangostana L. Ameliorates

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Imiquimod-induced Psoriasis-like Skin Lesions in Mice

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Shanzao Chen†, Kesheng Han ‡, Hu Li†, Juren Cen†, Yanfang Yang§, Hezhen Wu§ &

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Qun Wei†*

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† Department of Biochemistry and Molecular Biology, Gene Engineering and

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Biotechnology Beijing Key Laboratory, College of Life Sciences, Beijing Normal

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University, Beijing, 100875, P. R. China

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‡ Haikou Qili Pharmaceutical Co., Ltd, Haikou, 570216, P. R. China

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§ College of Pharmacy, Hubei University of Chinese Medicine, Wuhan, 430061, P. R.

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China

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* Corresponding author: Qun Wei

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Address: Department of Biochemistry and Molecular biology, Gene Engineering and

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Biotechnology Beijing Key Laboratory, School of Life Sciences, Beijing Normal

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University, Beijing, 100875, P. R. China

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Fax: 86-010-58807365.

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Tel: 86-010-58807365

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E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Isogarcinol (YDIS), a natural compound extracted from Garcinia mangosana L., has a

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significant immunosuppressive effect on systemic lupus erythematosus and

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rheumatoid arthritis. Here we report that it reduced imiquimod-induced psoriasis-like

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skin lesions in mice. It strongly attenuated the aberrant proliferation and

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differentiation of keratinocytes. Moreover, the expression of genes involving the

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interleukin-23(IL-23)/T-helper 17(Th17) axis was significantly inhibited in the dorsal

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skin of the YDIS-treated mice, as was that of the other pro-inflammatory factors

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TNF-α, IL-2 and even interferon(IFN)-γ. Furthermore, YDIS prevented the abnormal

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distribution of T cell types and suppressed the differentiation of CD4+ T cells into

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Th17 cells in the spleens of mice exposed to imiquimod. Interestingly, it elevated

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numbers of regulatory T cells (Tregs) in the spleen and boosted IL-10 expression in

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the skin. In agreement with the above, YDIS increased serum IL-10 and reduced

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serum IL-17. It also caused less damage to the liver and kidneys of mice than

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cyclosporine A (CsA), especially to kidneys. In vitro, YDIS caused more death of

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HaCaT keratinocytes than CsA. It also strongly inhibited inflammatory factor

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expression in lipopolysaccharide (LPS)-stimulated HaCaT cells. Our findings suggest

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that YDIS is a promising immunosuppressive agent for treating psoriasis.

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KEYWORDS: Isogarcinol, imiquimod-induced, psoriasis, T-helper 17 cell,

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regulatory T cell, cyclosporine A, HaCaT

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INTRODUCTION

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Psoriasis is a common chronic, relapsing immune-mediated inflammatory dermatosis.

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Histopathologically its lesions are mainly characterized by parakeratosis and

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acanthosis in the epidermis, and blood vessel hyperplasia and inflammatory cell

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infiltration in the dermis1. Psoriasis affects approximately 2~3% of the population

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globally and most psoriatic sufferers are diagnosed as psoriasis vulgaris or plaque

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psoriasis

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physiological and psychological burdens. Moreover, it is not limited to skin

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inflammation but linked to systemic metabolic comorbidities, such as type- Ⅱ

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diabetes, cardiovascular diseases, atherosclerosis, arthritis and even melancholia,

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because of immune system disorders associated with psoriasis4-6.

2, 3

. Though psoriasis does not cause death directly, it leads to massive

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The underlying cause of psoriasis remains to be identified. Originally it was

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thought that its onset was due to physical stress or infection, leading to activation of

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innate immunity, mainly involving plasmacytoid dendritic cells (pDCs), which

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produce large amounts of IFN-α that trigger adaptive immunity

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cells were considered the key to psoriasis formation in the susceptible population.

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However, deletion of IFN-α or IFN-γ (master cytokines of Th1 cells) only partially

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relieved psoriatic symptoms

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of innate immune cells, pro-inflammatory factors, T-helper 1 (Th1) and T-helper 17

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(Th17) cells underlies the development of human psoriasis. Th17 cells, in particular,

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may be the key to psoriasis

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immune cells via cytokines or/and chemokines, and the innate immune cells such as

7, 8

. T-helper 1(Th1)

9, 10

. Current evidence suggests that a network composed

11, 12

. As psoriasis progresses, keratinocytes interact with

3



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dendritic cells (DCs) and macrophages in damaged psoriatic areas secrete various

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cytokines that trigger naïve CD4+ T cells to differentiate into Th1 or Th17 cells,

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which produce high levels of IFN-γ, IL-17 and IL-2213,

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contribute to further skin inflammation and aberrant proliferation of keratinocytes.

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Furthermore, abundant pro-inflammatory factors accelerate the recruitment of

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inflammatory cells and the differentiation of lymphocytes15. Regulatory T cells (Tregs)

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are important in maintaining immune homeostasis. Recent reports suggest that the

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increased numbers of Th17 cells are accompanied by the accumulation of Tregs,

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which increase the severity of psoriasis 16. In addition, under inflammatory conditions,

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Tregs can switch into Th17 cells 17. Hence, a closed pro-inflammatory feedback loop

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exacerbates the recurrent attack in psoriatic sufferers.

14

. These subsequently

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In order to clarify the underlying pathological mechanisms of psoriasis, a

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convenient and rapid model exhibiting most clinical features of psoriasis is needed.

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Xenograft models, in which a skin graft from psoriatic tissue is transplanted to an

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immunodeficient mouse, can produce the main psoriatic symptoms

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establishing this model is difficult as the required experimental material is hard to

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obtain. Another approach is to inject IL-23 into the subcutaneous tissue of the ear but

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this only induces a few of the signs of psoriasis, mainly restricted to the skin of the ear.

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Topical application of 5% imiquimod cream (IMQ) on the dorsal skin of mice can

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dramatically activate antigen-presenting cells (APCs) and inflammatory cells in the

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local area, which cause abnormal development of keratinocytes and activate adaptive

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immunity 19. These characteristics resemble most of the features of clinical psoriasis 20, 4


18

, whereas

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, and this model has been widely used for the investigation of psoriasis.

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Nowadays, psoriasis sufferers are treated with a variety of agents, including

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corticosteroids and immunosuppressants, especially the latter. As the intensified

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investigation, drugs affecting specific targets have been developed, such as the human

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monoclonal antibodies etanercept (targeting TNF-α), ustekinumab (targeting

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IL-12p40), ixekizumab and brodalumab (targeting IL-17 receptor A)

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antibody-based immunomodulators can have strong therapeutic effects in early

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treatment, whereas they cannot be used in a long term due to the immune deregulation

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they cause26. The classical immunosuppressants, cyclosporine A (CsA) and tacrolimus

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(FK506), are often used for moderate-to-severe psoriasis

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relieve the psoriasis symptoms in the short term, they also lead to serious

21-25

. These

27, 28

. Although they can

29, 30

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hepatotoxicity and nephrotoxicity

, and, what is worse, once patients discontinue

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to take it, they may experience more serious symptoms 31. Hence, an effective agent

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with low side-effects is urgently needed.

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Isogarcinol (YDIS) is a natural bioactive compound extracted from the mangosteen

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(including its ripe pericarp and bark) by Dr. Cen in our lab32. Mangosteen (Garcinia

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mangostana L.) is a tropical fruit which as one of the agricultural cash crops is

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popular with Asian. Mangosteen consists of milky-white tasty pulp and red-purple

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pericarp. Mangosteen pericarp as a Tai folk medicine has been widely used against

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diarrhea, dysentery and wound infection in Southeast Asia33,

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interestingly, some reports recently indicate that mangosteen extract can serve as a

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dietary antioxidiant and it can reduce high fat-diet induced hepatic steatosis35, 36. 5


34

. And more


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Beside, crude extract of mangosteen enriches functional ice-cream with the effect on

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lowering lipid and hepatoprotection

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used as a dietary supplement in America owing to its healthcare function40.

37-39

. And recently, mangosteen extract has been

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The structure of YDIS has been identified, whereas the researches or reports on its

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function and effect are rare until we do some works. In our previous studies32, 41-43, we

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were the first to report YDIS as a new potent immunosuppressant selected with

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calcineurin (CN) as the target enzyme. Based on its significant effect on

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anti-inflammation and immunomodulation32, we further investigated the effect of

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YDIS as an immunosuppressive candidate on autoimmune diseases, such as

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rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and experimental

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autoimmune encephalomyelitis (EAE)

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disorder, and environmental and genetic factors may trigger or impact the psoriasis

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event

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disease 3. The definite pathogenesis of psoriasis remains unclear, and to study it from

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the perspective of immunomodulation is a daring attempt. Based on these, we try to

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investigate whether YDIS can regulate psoriasis.

41-43

. Psoriasis is a chronic inflammatory skin

2, 3, 11, 44

. Of course, some researchers also regard psoriasis as an autoimmune

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In this study, we evaluate the effect of YDIS on IMQ-induced psoriasis-like skin

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lesions and examine its effects on the expression of cytokines associated with

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psoriasis and on a variety of relevant processes in vivo and in vitro.

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MATERIALS AND METHODS

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Mice and Reagents. 7-to-8-week-old female C57BL/6 mice were purchased from

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Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were individually 6


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housed and provided with purified water ad libitum. Before being used in experiments,

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they were raised under conventional conditions for at least 1 week with ad libitum

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access to food and water. All studies were approved by the Committee on Animal

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Experimentation of Beijing Normal University and performed strictly in accordance

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with institutional guidelines.

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5% Imiquimod cream (Aldara) was purchased from iNova Pharmaceuticals

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Australia Pty Ltd. Vaseline was produced by Kunlun Petrifaction (Baotou, China).

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Cyclosporine A (CsA) was bought from TCI (Shanghai) Development Co., Ltd.

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Isogarcinol (Purity>95%, measured by HPLC) was extracted and purified from the

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mangosteen (including its pericarp and bark) in our laboratory by Dr. Cen32.

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Development of IMQ-induced Psoriasis-Like Skin Lesions in a Murine

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Model.Mice received a daily topical dose of 62.5 mg of commercially available

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imiquimod cream on the shaved dorsal skin for 6 or 7 consecutive days as described

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previously, and the equivalent amount of vaseline was applied to the control group.

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One day before IMQ or vaseline treatment, mice were anesthetized by intraperitoneal

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injection of 50mg/kg of sodium pentobarbital; their fur was shaved locally with an

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electric clipper followed by a depilatory cream (Veet, Reckitt Benckiser, India).

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To examine the efficacy of Isogarcinol (YDIS), mice were randomly divided into 4

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groups (n=7 per group): a vehicle-treated vaselin-induced control group (abbreviated

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to Control), a vehicle-treated IMQ-induced model group (Model), a YDIS

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(100mg/kg)-treated IMQ-induced group (YDIS) and a CsA (50mg/kg)-treated

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IMQ-induced group (CsA). The appropriate agents formulated in 200ul vehicle 7



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(consisting of 12% ethanol, 23% normal saline, 5% Tween 80 and 60% peanut oil)

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were administered by oral gavage with a stomach-filling needle prior to application of

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62.5mg IMQ cream, while the control and model groups received only 200ul vehicle.

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The time we first treated was considered as day 0.

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Scoring the Severity of Psoriasis-like Skin Lesions. A modified clinical Psoriasis

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Area and Severity Index (PASI) was used to score the severity of dorsal skin

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inflammation, and the affected skin area was not taken into consideration in the

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overall score. Erythema, scaling, and skin thickness were scored blind and

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independently on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4,

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very marked. The cumulative score of the above 3 targets served as a measure of the

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severity of dorsal inflammation (scale 0-12).

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Quantitative Real-time Reverse Transcription PCR. Total RNA was isolated

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from fresh dorsal biopsies of sacrificed mice using TRIzol reagent (Invitrogen, USA).

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cDNA was synthesized using Reverse Transcriptase M-MLV (Rnase H-) and Oligo

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(dT)-18 primer. The mRNAs of factors were detected using an ABI5700 Real-time

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PCR system (Thermo Fisher, USA). Glyceraldehyde-3-phosphate dehydrogenase

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(GAPDH) was taken as reference gene to normalize and analyze in parallel all the

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gene transcription using the 2-∆∆CT method. Primer sequences designed to detect the

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gene expression in mouse were as follows: IFN-γ, forward primer, 5’- GTT GCT GAT

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GGC CTG ATT GTC-3’, reverse primer, 5’-CGG CAC AGT CAT TGA AAG CCT

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A-3’; IL-4, forward primer, 5’-ACG GAG ATG GAT GTG CCA AAC-3’; reverse

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primer, 5’- AGC ACC TTG GAA GCC CTA CAG A-3’; IL-17A, forward primer, 8


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5’-TCA TGT GGT GGT CCA GCT TTC-3’, reverse primer, 5’-GAA GGC CCT CAG

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ACT ACC TCA A-3’; IL-22, forward primer, 5’-TTT CCT GAC CAA ACT CAG

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CA-3’, reverse primer, 5’- CTG GAT GTT CTG GTC GTC AC-3’; IL-23p19, forward

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primer, 5’-ACA TGC ACC AGC GGG ACA TA-3’, Reverse primer, 5’-CTT TGA

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AGA TGT CAG AGT CAA GCA G-3’ ; TNF-α, forward primer, 5’-TAT GGC CCA

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GAC CCT CAC A-3’, reverse primer, 5’-GGA GTA GAC AAG GTA CAA CCC

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ATC-3’ ; GAPDH, forward primer, 5’- TGT GTC CGT CGT GGA TCT GA-3’,

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reverse primer, 5’-TTG CTG TTG AAG TCG CAG GAG-3’; RoRγt, forward primer,

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5’-TCT GCA AGA CTC ATC GAC AAG G-3’,reverse primer, 5’-CAC ATG TTG

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GCT GCA CAG G-3’; FoxP3, forward primer, 5’-TGC CTT CAG ACG AGA CTT

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GGA-3’, reverse primer, 5’-GGC ATT GGG TTC TTG TCA GAG-3’. Primers used

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for detecting the corresponding gene expression in HaCaT cell line: IL-6, forward

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primer, 5’-AGA GTA GTG AG GAA CAA GCC-3’, reverse primer, 5’-TAC ATT

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TGC CGA AGA GCC CT-3’; TNF-α, forward primer, 5’-TCC TTC AGA CAC CCT

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CAA CC-3’, reverse primer, 5’-AGG CCC CAG TTT GAA TTC TT-3’; β-actin,

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forward primer, 5’-AGG GAA ATC GTG CGT GAC AT-3’, reverse primer, 5’-TCC

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TGC TTG CTG ATC CAC AT-3’; S100A7, forward primer, 5’-GCA TGA TCG ACA

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TGT TTC ACA AAT ACA C-3’, reverse primer, 5’- TGG TAG TCT GTG GCT ATG

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TCT CCC-3’; S100A9, forward primer, 5’-GCT CCT CGG CTT TGA CAG AGT

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GCA AG-3’, reverse primer, 5’-GCA TTT GTG TCC AGG TCC TCC ATG ATG

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TGT-3’.

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Cell Preparation and Flow Cytometric Analysis. IMQ application and 9



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corresponding agent oral administration lasted for 6 days. Namely, 24 hours after the

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final treatment the mice were sacrificed. Spleens were harvested into a tissue culture

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dish with 0.9% normal saline, and then minced and filtered through a 70µm nylon

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mesh in RPMI 1640 medium without fetal bovine serum. Erythrocytes were lysed,

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and the suspension was centrifuged and cells resuspended in PBS (without calcium

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and magnesium). For staining cell surface antigens, lymphocytes were collected and

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treated with Fc-block (BD Pharmingen, USA), and then stained with FITC rat

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anti-mouse CD3 complex (145-2C11, BD Pharmingen), APC rat anti-mouse CD4

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(GK1.5, BD Pharmingen), or PE rat anti-mouse CD8α (53-6.7, BD Pharmingen).

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APC- or FITC-labeled rat anti-mouse IFN-γ (XMG1.2, BD Pharmingen), PE rat

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anti-mouse IL-4 (11B11, BD Pharmingen), and PE rat anti-mouse IL-17

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(TC11-18H10, BD Pharmingen) were used for intracellular staining of cytokines. In

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these procedures, splenocytes were pre-incubated for at least 24 hours at 37︒C and in

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5% CO2. In the last 6 hours, they were stimulated with phorbol 12-myristate

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13-acetate (60ng/ml) and ionomycin (900ng/ml). In some cases, they were also

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activated by plate-coated purified anti-CD3 monoclonal antibody (10µg/ml) for 48

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hours. In all cases, Golgistop inhibitor was added for the final 6 hours. At the end of

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incubation, the splenocytes were harvested and blocked with Fc-block before

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extracellular staining. After cell surface staining, the splenocytes were fixed and

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permeabilized with a BD Cytofix/CytopermTM Fixation/Permeabilization Kit, then

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stained for the appropriate cytokines. To stain FoxP3, cells were directly stained

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without stimulation using the FoxP3 staining Buffer Set (eBiosciences, USA). 10


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CD4+ T Cell Isolation. Mice in control group and model group were treated for 6

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days as indicated. Namely, spleens were harvested on day 6. Single-cell suspensions

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were prepared, and CD4+ cells were separated from splenocytes via magnetic

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microbeads by CD4+ T Cell Isolation Kit (Miltenyi Biotec, Germany) and MS

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Columns (Miltenyi Biotec, Germany). All our procedures are in accordance with the

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manufacturers’ instruction. The purity of CD4+ T cell fractions were always > 95%.

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Histopathology and Immunohistochemistry Analyses. Dorsal skin grafts were

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collected, and immersed in 4% paraformaldehyde solution to fix them for 3-5 days.

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Briefly, the tissues were embedded in paraffin and sliced into 5µm. Then

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deparaffinized sections were stained with haematoxylin and eosin (H&E). For

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immunohistochemistry,

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specimens were deparaffinized, antoclaved, heat-processed for antigen retrieval in

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citrate saline buffer, and incubated with purified rabbit anti-mouse Ki-67 (abcam, UK)

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at 1:50 dilution overnight at 4︒C. All procedures were performed according to the

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manufacturers’ guidelines.

paraformaldehyde-fixed

and

paraffin-embedded

skin

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Enzyme-linked Immunosorbent Assays (ELISA) for Cytokines. Cytokines in

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serum or culture supernatant were assayed with the corresponding ELISA kits

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(Neobioscience, China).

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Statistical Analysis. Data from at least three experiments were analyzed using

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Student’s t-test or one-way analysis of variance followed by Bonferroni’s Multiple

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Comparison test with GraphPad Prism 5.0. Values are presented as mean±SEM, and

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Isogarcinol Extracted from Garcinia mangostana L. Ameliorates

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