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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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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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

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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.

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. 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

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, 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

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. 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

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. 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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P