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*,† †

Department of Biochemistry and Molecular Biology, Gene Engineering and Biotechnology Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing 100875, People’s Republic of China ‡ Haikou Qili Pharmaceutical Company, Ltd., Haikou 570216, People’s Republic of China § College of Pharmacy, Hubei University of Chinese Medicine, Wuhan 430061, People’s Republic of China S Supporting Information *

ABSTRACT: Isogarcinol (YDIS), a natural compound extracted from Garcinia mangostana L., has a significant immunosuppressive effect on systemic lupus erythematosus and rheumatoid arthritis. This paper reports that it reduced imiquimod-induced psoriasis-like skin lesions in mice. It strongly attenuated the aberrant proliferation and differentiation of keratinocytes. Moreover, the expression of genes involving the interleukin-23 (IL-23)/T-helper 17 (Th17) axis was significantly inhibited in the dorsal skin of the YDIS-treated mice, as was that of the other pro-inflammatory factors TNF-α, IL-2, and even interferon (IFN)-γ. Furthermore, YDIS prevented the abnormal distribution of T cell types and suppressed the differentiation of CD4+ T cells into Th17 cells in the spleens of mice exposed to imiquimod. Interestingly, it elevated numbers of regulatory T cells (Tregs) in the spleen and boosted IL-10 expression in the skin. In agreement with the above, YDIS increased serum IL-10 and reduced serum IL-17. It also caused less damage to the liver and, especially, kidneys of mice than cyclosporine A (CsA). In vitro, YDIS caused more death of HaCaT keratinocytes than CsA. It also strongly inhibited inflammatory factor expression in lipopolysaccharide (LPS)-stimulated HaCaT cells. These findings suggest that YDIS is a promising immunosuppressive agent for treating psoriasis. KEYWORDS: isogarcinol, imiquimod-induced, psoriasis, T-helper 17 cell, regulatory T cell, cyclosporine A, HaCaT

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INTRODUCTION Psoriasis is a common chronic, relapsing immune-mediated inflammatory dermatosis. Histopathologically its lesions are mainly characterized by parakeratosis and acanthosis in the epidermis and blood vessel hyperplasia and inflammatory cell infiltration in the dermis.1 Psoriasis affects approximately 2−3% of the population globally, and most psoriatic sufferers are diagnosed as psoriasis vulgaris or plaque psoriasis.2,3 Although psoriasis does not cause death directly, it leads to massive physiological and psychological burdens. Moreover, it is not limited to skin inflammation but linked to systemic metabolic comorbidities, such as type II diabetes, cardiovascular diseases, atherosclerosis, arthritis, and even melancholia, because of immune system disorders associated with psoriasis.4−6 The underlying cause of psoriasis remains to be identified. Originally it was thought that its onset was due to physical stress or infection, leading to activation of innate immunity, mainly involving plasmacytoid dendritic cells (pDCs), which produce large amounts of IFN-α that trigger adaptive immunity.7,8 T-helper 1 (Th1) cells were considered the key to psoriasis formation in the susceptible population. However, deletion of IFN-α or IFN-γ (master cytokines of Th1 cells) only partially relieved psoriatic symptoms.9,10 Current evidence suggests that a network composed of innate immune cells, proinflammatory factors, T-helper 1 (Th1), and T-helper 17 (Th17) cells underlies the development of human psoriasis. Th17 cells, in particular, may be the key to psoriasis.11,12 As psoriasis progresses, keratinocytes interact with immune cells © 2017 American Chemical Society

via cytokines and/or chemokines, and the innate immune cells such as dendritic cells (DCs) and macrophages in damaged psoriatic areas secrete various cytokines that trigger naı̈ve CD4+ T cells to differentiate into Th1 or Th17 cells, which produce high levels of IFN-γ, IL-17, and IL-22.13,14 These subsequently contribute to further skin inflammation and aberrant proliferation of keratinocytes. Furthermore, abundant proinflammatory factors accelerate the recruitment of inflammatory cells and the differentiation of lymphocytes.15 Regulatory T cells (Tregs) are important in maintaining immune homeostasis. Recent papers suggest that the increased numbers of Th17 cells are accompanied by the accumulation of Tregs, which increase the severity of psoriasis.16 In addition, under inflammatory conditions, Tregs can switch into Th17 cells.17 Hence, a closed pro-inflammatory feedback loop exacerbates the recurrent attack in psoriatic sufferers. To clarify the underlying pathological mechanisms of psoriasis, a convenient and rapid model exhibiting most clinical features of psoriasis is needed. Xenograft models, in which a skin graft from psoriatic tissue is transplanted to an immunodeficient mouse, can produce the main psoriatic symptoms,18 whereas establishing this model is difficult as the required experimental material is hard to obtain. Another Received: Revised: Accepted: Published: 846

November 23, 2016 January 12, 2017 January 13, 2017 January 13, 2017 DOI: 10.1021/acs.jafc.6b05207 J. Agric. Food Chem. 2017, 65, 846−857

Article

Journal of Agricultural and Food Chemistry

expression of cytokines associated with psoriasis and on a variety of relevant processes in vivo and in vitro.

approach is to inject IL-23 into the subcutaneous tissue of the ear, but this induces only a few of the signs of psoriasis, mainly restricted to the skin of the ear. Topical application of 5% imiquimod cream (IMQ) on the dorsal skin of mice can dramatically activate antigen-presenting cells (APCs) and inflammatory cells in the local area, which cause abnormal development of keratinocytes and activate adaptive immunity.19 These characteristics resemble most of the features of clinical psoriasis,20,21 and this model has been widely used for the investigation of psoriasis. Nowadays, psoriasis sufferers are treated with a variety of agents, including corticosteroids and immunosuppressants, especially the latter. As the investigations intensified, drugs affecting specific targets have been developed, such as the human monoclonal antibodies etanercept (targeting TNF-α), ustekinumab (targeting IL-12p40), and ixekizumab and brodalumab (targeting IL-17 receptor A).21−25 These antibody-based immunomodulators can have strong therapeutic effects in early treatment, whereas they cannot be used for a long term due to the immune deregulation they cause.26 The classical immunosuppressants cyclosporine A (CsA) and tacrolimus (FK506) are often used for moderate-to-severe psoriasis.27,28 Although they can relieve the psoriasis symptoms in the short term, they also lead to serious hepatotoxicity and nephrotoxicity, 29,30 and, what is worse, once patients discontinue to take it, they may experience more serious symptoms.31 Hence, an effective agent with low side effects is urgently needed. Isogarcinol (YDIS) is a natural bioactive compound extracted from the mangosteen (including its ripe pericarp and bark) by Dr. Cen in our laboratory.32 Mangosteen (Garcinia mangostana L.) is a tropical fruit that, as one of the agricultural cash crops, is popular in Asia. Mangosteen consists of a milky-white tasty pulp and a red-purple pericarp. Mangosteen pericarp as a Thai folk medicine has been widely used against diarrhea, dysentery, and wound infection in Southeast Asia.33,34 More interestingly, some studies recently indicate that mangosteen extract can serve as a dietary antioxidiant and it can reduce high fat diet induced hepatic steatosis.35,36 Besides, the crude extract of mangosteen enriches functional ice cream with the effect of lowering lipid and hepatoprotection.37−39 Recently, mangosteen extract has been used as a dietary supplement in the United States owing to its healthcare function.40 The structure of YDIS has been identified, whereas the research or reports on its function and effect were rare until we did some work. In our previous studies,32,41−43 we were the first to report YDIS as a new potent immunosuppressant selected with calcineurin (CN) as the target enzyme. On the basis of its significant effect on anti-inflammation and immunomodulation,32 we further investigated the effect of YDIS as an immunosuppressive candidate on autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and experimental autoimmune encephalomyelitis (EAE).41−43 Psoriasis is a chronic inflammatory skin disorder, and environmental and genetic factors may trigger or affect the psoriasis event.2,3,11,44 Of course, some researchers also regard psoriasis as an autoimmune disease.3 The definite pathogenesis of psoriasis remains unclear, and to study it from the perspective of immunomodulation is a daring attempt. On these bases, we try to investigate whether YDIS can regulate psoriasis. In this study, we evaluate the effect of YDIS on IMQ-induced psoriasis-like skin lesions and examine its effects on the

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MATERIALS AND METHODS

Mice and Reagents. Female C57BL/6 mice (7−8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were individually housed and provided with purified water ad libitum. Before being used in experiments, they were raised under conventional conditions for at least 1 week with ad libitum access to food and water. All studies were approved by the Committee on Animal Experimentation of Beijing Normal University and performed strictly in accordance with institutional guidelines. Five percent imiquimod cream (Aldara) was purchased from iNova Pharmaceuticals Australia Pty Ltd. Petroleum jelly was produced by Kunlun Petrifaction (Baotou, China). Cyclosporine A (CsA) was bought from TCI (Shanghai) Development Co., Ltd. Isogarcinol (purity > 95%, measured by HPLC) was extracted and purified from the mangosteen (including its pericarp and bark) in our laboratory by Dr. Cen.32 Development of IMQ-Induced Psoriasis-like Skin Lesions in a Murine Model. Mice received a daily topical dose of 62.5 mg of commercially available imiquimod cream on the shaved dorsal skin for 6 or 7 consecutive days as described previously, and the equivalent amount of petroleum jelly was applied to the control group. One day before IMQ or vaseline treatment, mice were anesthetized by intraperitoneal injection of 50 mg/kg of sodium pentobarbital; their fur was shaved locally with an electric clipper followed by a depilatory cream (Veet, Reckitt Benckiser, India). To examine the efficacy of isogarcinol (YDIS), mice were randomly divided into four groups (n = 7 per group): a vehicle-treated petroleum jelly-induced control group (abbreviated control), a vehicletreated IMQ-induced model group (model), a YDIS (100 mg/kg)treated IMQ-induced group (YDIS), and a CsA (50 mg/kg)-treated IMQ-induced group (CsA). The appropriate agents formulated in 200 μL of vehicle (consisting of 12% ethanol, 23% normal saline, 5% Tween 80, and 60% peanut oil) were administered by oral gavage with a stomach-filling needle prior to application of 62.5 mg of IMQ cream, whereas the control and model groups received only 200 μL of vehicle. The time we first treated was considered day 0. Scoring the Severity of Psoriasis-like Skin Lesions. A modified clinical psoriasis area and severity index (PASI) was used to score the severity of dorsal skin inflammation, and the affected skin area was not taken into consideration in the overall score. Erythema, scaling, and skin thickness were scored blind and independently on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. A cumulative score of the above three targets served as a measure of the severity of dorsal inflammation (scale 0−12). Quantitative Real-Time Reverse Transcription PCR (RT-PCR). Total RNA was isolated from fresh dorsal biopsies of sacrificed mice using TRIzol reagent (Invitrogen, USA). cDNA was synthesized using reverse transcriptase M-MLV (RNase H−) and oligo (dT)-18 primer. The mRNAs of factors were detected using an ABI5700 real-time PCR system (Thermo Fisher, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was taken as reference gene to normalize and analyze in parallel all of the gene transcriptions using the 2−ΔΔCT method. Primer sequences designed to detect the gene expression in mouse were as follows: IFN-γ, forward primer, 5′-GTT GCT GAT GGC CTG ATT GTC-3′, reverse primer, 5′-CGG CAC AGT CAT TGA AAG CCT A-3′; IL-4, forward primer, 5′-ACG GAG ATG GAT GTG CCA AAC-3′, reverse primer, 5′-AGC ACC TTG GAA GCC CTA CAG A-3′; IL-17A, forward primer, 5′-TCA TGT GGT GGT CCA GCT TTC-3′, reverse primer, 5′-GAA GGC CCT CAG ACT ACC TCA A-3′; IL-22, forward primer, 5′-TTT CCT GAC CAA ACT CAG CA-3′, reverse primer, 5′-CTG GAT GTT CTG GTC GTC AC-3′; IL-23p19, forward primer, 5′-ACA TGC ACC AGC GGG ACA TA-3′, reverse primer, 5′-CTT TGA AGA TGT CAG AGT CAA GCA G-3′; TNF-α, forward primer, 5′-TAT GGC CCA GAC CCT CAC A-3′, reverse primer, 5′-GGA GTA GAC AAG GTA CAA CCC ATC-3′; GAPDH, forward primer, 5′-TGT GTC CGT CGT GGA 847

DOI: 10.1021/acs.jafc.6b05207 J. Agric. Food Chem. 2017, 65, 846−857

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Figure 1. Effect of YDIS on IMQ-induced psoriasis-like skin lesions in mice. (A) Representative photographs of dorsal skin lesions are shown. For treatments see Materials and Methods. The photographs were taken on day 7. Clinical scores for erythema (B), scaling (C), and skin thickness (D) were evaluated daily according to the modified psoriasis area and the severity index (PASI), as described under Materials and Methods. Symbols represent the mean ± SEM (n = 6). (∗∗) p < 0.01 versus control group; (#) p < 0.05 and (##) p < 0.01 versus model group, Student’s t test. TCT GA-3′, reverse primer, 5′-TTG CTG TTG AAG TCG CAG GAG-3′; RoRγt, forward primer, 5′-TCT GCA AGA CTC ATC GAC AAG G-3′, reverse primer, 5′-CAC ATG TTG GCT GCA CAG G-3′; FoxP3, forward primer, 5′-TGC CTT CAG ACG AGA CTT GGA-3′, reverse primer, 5′-GGC ATT GGG TTC TTG TCA GAG-3′. Primers used for detecting the corresponding gene expression in HaCaT cell line: IL-6, forward primer, 5′-AGA GTA GTG AG GAA CAA GCC-3′, reverse primer, 5′-TAC ATT TGC CGA AGA GCC CT-3′; TNF-α, forward primer, 5′-TCC TTC AGA CAC CCT CAA CC-3′, reverse primer, 5′-AGG CCC CAG TTT GAA TTC TT-3′; β-actin, forward primer, 5′-AGG GAA ATC GTG CGT GAC AT-3′, reverse primer, 5′-TCC TGC TTG CTG ATC CAC AT-3′; S100A7, forward primer, 5′-GCA TGA TCG ACA TGT TTC ACA AAT ACA C-3′, reverse primer, 5′-TGG TAG TCT GTG GCT ATG TCT CCC-3′; S100A9, forward primer, 5′-GCT CCT CGG CTT TGA CAG AGT GCA AG3′, reverse primer, 5′-GCA TTT GTG TCC AGG TCC TCC ATG ATG TGT-3′. Cell Preparation and Flow Cytometric Analysis. IMQ application and corresponding agent oral administration lasted for 6 days; 24 h after the final treatment, the mice were sacrificed. Spleens were harvested into a tissue culture dish with 0.9% normal saline and then minced and filtered through a 70 μm nylon mesh in RPMI 1640 medium without fetal bovine serum. Erythrocytes were lysed, and the

suspension was centrifuged and cells were resuspended in PBS (without calcium and magnesium). For staining cell surface antigens, lymphocytes were collected and treated with Fc-block (BD Pharmingen, USA) and then stained with FITC rat anti-mouse CD3 complex (145-2C11, BD Pharmingen), APC rat anti-mouse CD4 (GK1.5, BD Pharmingen), or PE rat anti-mouse CD8α (53-6.7, BD Pharmingen). APC- or FITC-labeled rat anti-mouse IFN-γ (XMG1.2, BD Pharmingen), PE rat anti-mouse IL-4 (11B11, BD Pharmingen), and PE rat anti-mouse IL-17 (TC11-18H10, BD Pharmingen) were used for intracellular staining of cytokines. In these procedures, splenocytes were pre-incubated for at least 24 h at 37 °C and in 5% CO2. In the last 6 h, they were stimulated with phorbol 12-myristate 13-acetate (60 ng/mL) and ionomycin (900 ng/mL). In some cases, they were also activated by plate-coated purified anti-CD3 monoclonal antibody (10 μg/mL) for 48 h. In all cases, Golgistop inhibitor was added for the final 6 h. At the end of incubation, the splenocytes were harvested and blocked with Fc-block before extracellular staining. After cell surface staining, the splenocytes were fixed and permeabilized with a BD Cytofix/Cytoperm Fixation/Permeabilization Kit and then stained for the appropriate cytokines. To stain FoxP3, cells were directly stained without stimulation using the FoxP3 staining buffer set (eBiosciences, USA). 848


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Figure 2. Histopathology of dorsal skin after YDIS treatment. (A) H&E staining of mouse dorsal skin following the indicated treatments. H&Estained paraffin sections, 200×. (B) Ki-67 immunohistochemical staining of paraffin-embedded slides reveals the inhibitory effect of YDIS on the proliferation of keratinocytes in the mouse epidermis. IHC-P, 200×, DAB peroxidase substrate. (C) Data on epidermal thickness, (D) inflammatory cell infiltration in the dermis, and (E) Ki-67-positive cells in the epidermis on day 6 based on analysis of high-power field (HPF) images of five to six sites per mouse per group. Mean ± SEM (n = 5). (∗∗) p < 0.01 versus control group; (#) p < 0.05 and (##) p < 0.01 versus model group. Data were analyzed by ANOVA followed by Bonferroni’s multiple comparison. 849


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Figure 3. YDIS reduces the expression of cytokines associated with psoriasis in dorsal skin. All mice received IMQ once daily (control mice received petroleum jelly), together with corresponding agent (control and model with equal vehicle). RNA was extracted from dorsal skin samples after 6 days of treatment. Levels of transcripts of (A) TNF-α, (B) IL-6, (C) IL-2, (D) IFN-γ, (E) IL-4, (F) IL-10, (G) IL-23p19, (H) IL-17A, and (I) IL-22 were detected by quantitative RT-PCR. Values are presented as the mean ± SEM (n = 6). (∗) p < 0.05 and (∗∗) p < 0.01 versus control group; (#) p < 0.05 and (##) p < 0.01 versus model group. Data were analyzed by ANOVA followed by Bonferroni’s multiple comparison.

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CD4+ T Cell Isolation. Mice in the control group and model group were treated for 6 days as indicated. Spleens were harvested on day 6. Single-cell suspensions were prepared, and CD4+ cells were separated from splenocytes via magnetic microbeads by using a CD4+ T Cell Isolation Kit (Miltenyi Biotec, Germany) and MS columns (Miltenyi Biotec). All of our procedures are in accordance with the manufacturers’ instruction. The purity of CD4+ T cell fractions were always >95%. Histopathology and Immunohistochemistry Analyses. Dorsal skin grafts were collected and immersed in 4% paraformaldehyde solution to fix them for 3−5 days. Briefly, the tissues were embedded in paraffin and sliced into 5 μm. Then deparaffinized sections were stained with hematoxylin and eosin (H&E). For immunohistochemistry, paraformaldehyde-fixed and paraffin-embedded skin specimens were deparaffinized, antoclaved, heat-processed for antigen retrieval in citrate saline buffer, and incubated with purified rabbit anti-mouse Ki67 (abcam, UK) at 1:50 dilution overnight at 4 °C. All procedures were performed according to the manufacturers’ guidelines. Enzyme-Linked Immunosorbent Assays (ELISA) for Cytokines. Cytokines in serum or culture supernatant were assayed with the corresponding ELISA kits (Neobioscience, China). Statistical Analysis. Data from at least three experiments were analyzed using Student’s t test or one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple-comparison test with GraphPad Prism 5.0. Values are presented as the mean ± SEM, and p < 0.05 was considered significant.

RESULTS YDIS Ameliorates IMQ-Induced Psoriasis-like Skin Lesions in Mice. To investigate whether YDIS could alleviate psoriasis, an IMQ-induced psoriasis-like skin lesions mouse model was established as described.19 YDIS (100 mg/kg) or CsA (50 mg/kg) was orally administered once daily followed by application of 62.5 mg of IMQ to the bare skin on the backs of mice for 7 consecutive days. Compared with the mice in the control group, the body weight of those in the model group decreased rapidly after IMQ application, whereas oral administration of YDIS or CsA arrested this decline after the first 3 days (data not shown). However, the mice receiving CsA were weak and their fur lost color (Figure 1A). The mice receiving YDIS had lower mean scores for erythema, scales, and skin thickness (Figure 1A−D). Even though CsA was more effective than YDIS in its effect on epidermal scales and thickness early on, the situation was reversed later (Figure 1C,D). In terms of cumulative scores, the effects of YDIS and CsA were similar (Figure 1E). Histopathological analysis of hematoxylin and eosin (H&E) staining of the dorsal skin suggested that IMQ application induced epidermal hyperplasia, parakeratosis (arrowheads indicated in Figure 2A), and acanthosis (Figure 2A). In addition, IMQ application led to loss of the granular layer, 850


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Figure 4. Oral administration of YDIS inhibits the formation of Th17 cells in the spleen and promotes the formation of Tregs. Representative flow cytometric results for Th1, Th2, Th17, and Treg cell numbers in splenocytes are from the mice treated for 6 consecutive days. Mice were treated as described under Materials and Methods. Intracellular levels of cytokines IL-4 (A) IL-17, and IFN-γ (B) were detected by flow cytometry, and the cells were gated for CD4+. (C−E) Statistical analysis of the above results. (F, G) Splenocytes were stained and gated for CD4+CD25+ with a Foxp3 Staining Buffer Set without stimulation. (H) Graphs depict CD3+ cell populations in splenocytes and the changes of CD3+CD4+/CD3+CD8+ proportions. Values in the graphs are shown as the mean ± SEM (n = 5−6) of three independent experiments. (∗) p < 0.05 &and (∗∗) p < 0.01 versus control group; (#) p < 0.05 and (##) p < 0.01 versus model group. 851


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Figure 5. Influence of YDIS on the expression of transcription factors RORγt and FoxP3 in isolated CD4+ T cells. Splenocytes were harvested and isolated from the control and model groups with a CD4+ T Cell Isolation Kit. CD4+ T cells were stimulated with plate-coated anti-CD3 (10 μg/mL) monoclonal antibody for 48 h in the presence or absence of 5 μM YDIS. The relative expression levels of transcription factors (A) RORγt and (B) FoxP3 were detected by RT-PCR. Data are shown as the mean ± SEM (n = 5). (∗) p < 0.05 and (∗∗) p < 0.01 versus medium.

Figure 6. YDIS administration alters the levels of IL-17 and IL-10 in serum. Oral administration of YDIS attenuated the production of (A) IL-17 in serum, whereas it boosted (B) IL-10. Mice were treated as indicated for 6 consecutive days (n = 5−7 mice per group). On day 6, sera were obtained, and the concentrations of IL-17 and IL-10 were measured by ELISA. Bars above show the mean ± SEM; each symbol represents an individual mouse. ANOVA was followed by Bonferroni’s multiple-comparison tests. (∗∗) p < 0.05 versus control group; (#) p < 0.05 and (##) p < 0.01 versus model group.

inflammatory environment induced by IMQ application in mouse skin. Immunoregulatory Effects of YDIS on CD4+ T Cells in the IMQ-Induced Psoriasis-like Murine Model. To explore the influence of YDIS on Th1, Th2, and Th17 cells in the mouse spleen, single splenocyte suspensions were prepared after 6 days of agent administration and stimulated ex vivo with phorbol 12-mysistate 13-acetate (PMA) and ionomycin. Thereafter, the intracellular cytokines IL-4, IFN-γ, and IL-17 as well as surface CD4 expression were determined by flow cytometric analysis. IMQ treatment increased IL-17+CD4+ T cells (Th17) on day 6 (Figure 4B,E), but it hardly affected the proportions of IFN-γ+CD4+ T cells (Th1) and IL-4+CD4+ T cells (Th2) (Figure 4A−D). YDIS treatment significantly dampened the Th17 cell response (Figure 4B,E), but Th1 and Th2 cell numbers were not affected (Figure 4A−E). Surprisingly, the proportion of CD4+CD25+FoxP3+ T cells (Tregs) was dramatically increased by YDIS treatment but not by CsA, even though, like YDIS, it diminished the Th17 response (Figure 4F,G). It is widely accepted that T cells play an important role in the pathogenesis of psoriasis2,14 and that IMQ application can affect the distribution of T cell types.19 Therefore, we next examined if YDIS could reverse the abnormal splenic proportions caused by IMQ. Flow cytometric analysis showed that total numbers of CD3+ T cells were not affected significantly by IMQ (Figure 4H, left panel), whereas the

which was reversed by YDIS or CsA (arrows in Figure 2A). Moreover, mice treated with either agent had less epidermal thickness and infiltration of inflammatory cells into the dermis (Figure 2A−D). Ki-67, a nuclear antigen that is used to label the latest proliferated cell, was immunohistochemically stained in paraffin sections, which provided further evidence that YDIS could reduce the proliferation of keratinocytes (Figure 2B,E). Overall, oral administration of 100 mg/kg YDIS and 50 mg/kg CsA had largely similar effects on the histopathological changes induced by IMQ. Effect of YDIS on Expression of Psoriasis-Associated Cytokines in Mouse Skin. We examined the effects of YDIS on the mRNA levels of genes associated with psoriasis. IMQ application increased the transcription of TNF-α, IL-2, IL-6, and other cytokines involved in Th1 and Th17 responses, such as IFN-γ, IL-23p19, IL-17, and IL-22 (Figure 3A−D,H,I). It also elevated the anti-inflammatory factor IL-10, a key cytokine of Tregs (Figure 3F), but IL-4 was not significantly affected (Figure 3E). YDIS administration dramatically decreased the expression of genes encoding the key cytokines implicated in psoriasis, including TNF-α, IL-6, IL-2, and IFN-γ (Figure 3A− D) and especially factors of the IL-23/Th17 axis (Figure 3G− I). Furthermore, IL-10 was further increased by YDIS (Figure 3F), but not by CsA. Thus, YDIS reduced the expression of pro-inflammatory factors including the IL-23/Th17 inflammatory axis and also increased the secretion of IL-10. Overall, oral administration of YDIS to some extent improved the 852


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Figure 7. Damage to liver and kidneys by YDIS and CsA. Mice were treated as indicated for 7 consecutive days (n = 5 or 6 mice per group). Sera were collected and analyzed on day 7. Liver function indices: alanine aminotransferase (A), aspartate transaminase (B), and total bilirubin (C). Kidney function indices: blood urea nitrogen (D) and serum creatinine (E). Values in the graphs are the mean ± SEM (n = 5−6). ANOVA followed by Bonferroni’s multiple comparison tests was used. (∗) p < 0.05 and (∗∗) P < 0.01 versus control group treated with vehicle; (#) p < 0.05 and (##) p < 0.01 versus CsA group.

Figure 8. Impact of YDIS versus CsA on HaCaT keratinocytes. The effects of YDIS and CsA on the viability of (A) HaCaT, (B) Jurkat, and (C) RAW264.7 cells were measured. After pre-incubation overnight, the corresponding concentration of YDIS or CsA was added to the cell culture system, and then the cells were incubated at 37 °C in 5% CO2 for another 24 h. Cell viability was measured with a Cell Counting Kit-8 (CCK-8). Data are the mean ± SD (n = 6) of triplicate experiments.

ratio of CD3+CD4+/CD3+CD8+ T cells decreased markedly (Figure 4H, right panel). YDIS treatments reversed this effect, whereas CsA had a less marked effect. These data suggest that YDIS ameliorates IMQ-induced psoriasis-type lesions by modulating Th17 cells and Tregs. YDIS Inhibits Th17 Cells but Increases the Number of Tregs. On the basis of the above, we determined if YDIS affected the differentiation of CD4+ T cells. We isolated CD4+ T cells and stimulated them with plate-coated anti-CD3 (10 μg/mL) for 48 h in the presence or absence of YDIS (5 μM). According to a report of Cen et al., YDIS has little toxic effect on splenic lymphocytes at this concentration. 32 After incubation, RNA was extracted and analyzed. YDIS significantly inhibited RORγt (master transcription factor of Th17) expression in CD4+ T cells from control and IMQ-induced mice (Figure 5A). However, it merely increased transcription of FoxP3 (master transcription factor of Tregs) in the CD4+ T cells from IMQ-treated mice (Figure 5B), but had no clear effect on those from controls. Furthermore, we confirmed these results by assaying the production of key cytokines of Th17 cells and Tregs in serum by ELISA. In agreement with the effect of YDIS on splenocytes of IMQ-induced mice, IL-17 (Th17) was markedly reduced in serum (Figure 6A), whereas IL-10 was increased (Figure 6B). Collectively, we considered that YDIS

could efficiently inhibit Th17 and that its elevated response of Tregs was relevant to its suppression on Th17 and improvement of inflammatory conditions. YDIS Causes Less Damage to the Liver and Kidneys of Mice than CsA. CsA, as a powerful classical immunosuppressant, is widely used to prevent organ transplant rejection and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus.45,46 CsA treatment can also relieve diabetes and psoriasis to some extent.29,47 However, its severe hepatotoxicity and nephrotoxicity result in a narrow therapeutic index, and large individual differences in both pharmacokinetics and pharmacodynamics further affect its application.29,48 We compared the effects of CsA with YDIS on liver and kidneys. Neither IMQ application nor YDIS administration affected significantly liver and kidney function indices (Figure 7), whereas CsA increased total bilirubin, blood urea nitrogen, and serum creatinine (Figure 7C−E). These findings indicate that YDIS does less damage to the liver and kidneys than CsA, especially the kidneys. YDIS Has a Specific Lethal Effect on HaCaT Keratinocytes and Reduces the Expression of Proinflammatory Factors in LPS-Stimulated HaCaT Cells. We also assessed the influence of YDIS on HaCaT keratinocytes (an immortal cell line derived from human 853


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Figure 9. Inhibition by YDIS of pro-inflammatory factor expression in LPS-stimulated HaCaT cells. Cells were cultured overnight and then treated with LPS (10 μg/mL) and agents as indicated above for 48 h. At the end of incubation, total RNA was extracted, and gene expression was measured by RT-PCR. YDIS attenuated the expression of pro-inflammatory cytokines TNF-α (A) and IL-6 (B).The production of psoriasins S100A7 (C) and S100A9 (D) was also decreased by YDIS; CsA acted as the positive control. Data were analyzed by ANOVA followed by Bonferroni’s multiplecomparison tests. Results are the mean ± SEM (n = 5) of triplicate experiments. (∗) P < 0.05 and (∗∗) p < 0.01 versus control group; (#) p < 0.05 and (##) p < 0.01 versus LPS group.

mg/kg) followed by IMQ application to the dorsal skin once daily for 7 consecutive days significantly reduced the psoriasis symptoms, including erythema, scaling, and epidermal thickness, compared with vehicle-treated IMQ-induced model mice. In addition, YDIS reduced the retention of nuclei in the stratum corneum due to the excessive proliferation and differentiation of keratinocytes induced by IMQ, and Ki-67 immunohistochemical staining confirmed the striking inhibitory effect of YDIS on keratinocyte development. It is known that excess production of pro-inflammatory cytokines and chemokines produced by activated antigenpresenting cells accounts for the migration of inflammatory cells, including T lymphocytes, dendritic cells, macrophages, and neutrophil granulocytes, into psoriatic sites.1 The recruited inflammatory cells in turn secrete more cytokines and stimulate the proliferation of immune cells and keratinocytes. This selfamplifying cycle constantly reinforces the psoriasis.2 Th1 and Th17, two distinct subsets of CD4+ T cells, have been thought to play important roles in human psoriasis.20 Recently, attention has focused on Th17 cells. The IMQ-induced psoriasis-like lesions in mice also involve a pivotal role for Th17.19 In our study, IMQ stimulation significantly elevated pro-inflammatory factors such as TNF-α, IL-2, IL-6, and IL-23 and activated expression of IL-17 and IL-22, two cytokines produced mainly by Th17 cells. IL-23 can exacerbate psoriasis, and it is a key upstream factor in Th17 cell differentiation.12 IMQ application promoted the differentiation of CD4+ T cells into the Th17 subset and decreased the ratio of CD4+/CD8+ T cells in the spleen. Oral administration of YDIS dropped the

keratinocytes), which are often used to test the effects of candidates on psoriasis. The effects of 24 h of exposure to YDIS and CsA on the viability of HaCaT cells were measured with CCK-8. YDIS inhibited HaCaT cell growth at 2 μM and caused nearly 50% death at 8 μM; it had a significantly more lethal effect than CsA on the HaCaT keratinocytes (Figure 8A). On the other hand, it was less toxic than CsA at the same concentrations to Jurkat (Figure 8B) and RAW264.7 cells (Figure 8C). In further experiments, HaCaT cells were exposed to LPS (10 μg/mL) with or without YDIS as indicated for 48 h. CsA was the positive control. As shown in Figure 9, LPS significantly up-regulated TNF-α, IL-6, S100A7, and S100A9, and this effect was attenuated by YDIS at 2 and 4 μM, in a dose-dependent manner. Furthermore, YDIS and CsA had almost the same impact on TNF-α, S100A7, and S100A9 levels (Figure 9A,C,D), whereas IL-6 was more effectively reduced by YDIS than by CsA (Figure 9B). In summary, YDIS but not CsA has a specific lethal effect on HaCaT keratinocytes and also reduces the production of inflammatory factors in HaCaT keratinocytes.

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DISCUSSION Agricultural products are regarded as a rich resource library to develop natural bioactive constituents against oxidation, cardiovascular diseases, and inflammation. YDIS is a natural immunosuppressant extracted from mangosteen (G. mangostana L.).32 Our aim was to investigate the effect of YDIS as an immunosuppressant on IMQ-induced psoriasis-like skin lesions in a murine model. We found that oral gavage of YDIS (100 854


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had similar effects on Jurkat and RAW264.7 cells. Furthermore, YDIS significantly decreased the overexpression of TNF-α, IL6, S100A7, and S100A9 in LPS (10 μg/mL) induced HaCaT cells. IL-6 and TNF-α play important roles in psoriasis, possibly via the MAPK pathway,18 and S100A7 and S100A9 are psoriasis-associated molecules that are highly expressed in psoriatic skin lesions.51 On the basis of the above observations, it appears that YDIS does less damage to liver and kidneys than CsA both in vivo and in vitro. Hence, it could be a promising candidate for psoriasis. In summary, YDIS interacts with antigen-presenting cells to inhibit the expression of pro-inflammatory cytokines; it reduces the differentiation of CD4+ T cells into Th17 cells and decreases IL-17 and IL-22 expression subsequently. This combined with reductions in other pro-inflammatory factors limits the shift of Tregs into Th17 cells. Meanwhile, low levels of IL-17 and IL-22 reduced the proliferation and differentiation of keratinocytes, and the accumulated Tregs favor the maintenance of homeostasis. Thus, the psoriatic symptoms are gradually mitigated. In conclusion, the experimental data demonstrate that YDIS is an effective bioactive compound against psoriasis with low toxicity, and its therapeutic effect is associated with down-regulation of pro-inflammatory cytokines and Th17 cells. YDIS may be a useful agent for treating psoriasis.

production of pro-inflammatory factors to improve inflammatory condition in skin lesions induced by IMQ. Results of flow cytometry analysis also suggested that YDIS attenuated the aberrant splenic differentiation. IFN-γ, a principal cytokine associated with Th1 cells, was only slightly increased in mRNA level in skin, whereas we did not find a marked rise about the proportion of Th1 cells in spleen from the model mice, and the response of Th2 cells was also little affected. These findings are in accordance with previous studies.19,21 YDIS depressed IFN-γ levels only in the skin and not the spleen and did not affect Th2 cell numbers. Recently, IL-17+γδ T cells were reported to play an important role in psoriasis;49 IL-17-producing dermal γδ T cells in psoriatic patients were elevated when compared with healthy controls, and IL-23 induced γδ T cells to produce IL-17 in the skin. IMQ treatment of IL-17 receptor A-deficient mice also led to a rise in IL-17+γδ T cells.44 Additionally, expression of IL-23 and IL-17 was markedly increased in mouse skin in response to IMQ. These findings suggest that it would be interesting to examine if YDIS affects γδ T cells. We intend to do this in future work. Spontaneously occurring regulatory T cells (Tregs), components of the CD4+CD25+ T cell population, as well as the transcription factor FoxP3 are important in immune homeostasis.50 Dysfunctional Tregs exacerbate psoriatic symptoms, and both Th17 and Tregs influence the development of psoriasis.16 Moreover, it has been reported that Tregs can differentiate into Th17 cells under pro-inflammatory conditions, which would have serious consequences for the progression of human disease.17 Furthermore, IL-23 boosts this differentiation process. Even though Tregs increase in psoriatic patients, this hardly attenuates the psoriatic symptoms due to the abundant inflammatory factors such as IL-22- and IL-17producing Th17 cells in the lesions. Instead, a closed feedback loop may enhance the loss of FoxP3 and promote the accumulation of RoRγt, which would aggravate the psoriasis.16,17 Our experiments showed that YDIS administration for 6 days significantly elevated Tregs in the spleen. Furthermore, IL-10 expression was also up-regulated in skin and serum. A reasonably logical explanation of the effect of YDIS can be proposed on the basis of the above findings, as follows. Oral gavage of YDIS first decreases the expression of proinflammatory factors, especially IL-23, IL-17, and IL-22, which reduces inflammatory conditions. The low levels of IL23 and IL-6 then restrict the transcription of RoRγt, which decreases the differentiation of Th17. Moreover, the weakened inflammatory environment limits the shift from Tregs to Th17 cells and promotes the accumulation of FoxP3, so increasing the numbers of Tregs. This increases the suppressive action of Tregs on the skin inflammation and reduces psoriatic symptoms. CsA, which was used as a type of positive control, reduced only Th17 responses but had little effect on Tregs. When we have evaluated the immunosuppressive effect of YDIS as a bioactive constituent on psoriasis, whether it has significant side effect will be taken into consideration. In our study, we compared CsA and YDIS in terms of liver and kidney function indices. CsA treatment for 7 days significantly elevated total bilirubin, blood urea nitrogen, and serum creatinine, but not alanine aminotransferase and aspartate transaminase. On the other hand, oral gavage of YDIS hardly had any effect on these indices. Excessive proliferation of keratinocytes (KCs) is the most striking feature of psoriasis, and we showed that YDIS had a much more pronounced lethal effect on HaCaT cells than cyclosporine A (CsA) at the same concentrations, whereas they

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05207. Proportion of CD3+CD4+ cells and CD3+CD8+ cells in spleen of mouse after relative administration for 6 days (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(Q.W.) Fax: 86-010-58807365. Phone: 86-010-58807365. Email: [email protected]. ORCID

Qun Wei: 0000-0001-6241-0679 Funding

The work was supported by the National Natural Science Foundation of China (No. 30970636). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED YDIS, isogarcinol; IMQ, imiquimod; Th1, T-helper 1 cell; Th17, T-helper 17 cell; Treg, regulatory T cell; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon gamma; CsA, cyclosporine A; CCK-8, cell counting kit-8; LPS, lipopolysaccharide

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REFERENCES

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