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Mangiferin antagonizes TNF-α-mediated inflammatory reaction and protects against dermatitis in a mice model
International Immunopharmacology 45 (2017) 174–179
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Mangiferin antagonizes TNF-α-mediated inflammatory reaction and protects against dermatitis in a mice model Yunpeng Zhao a,1, Wenhan Wang b,1, Xihai Wu c, Xiaoqian Ma b, Ruize Qu b, Xiaomin Chen b, Chenghao Liu b, Yaoge Liu b, Xiaokai Wang b, Pengcheng Yan b, Hao Zhang b, Jingrui Pan b, Weiwei Li d,⁎ a
Department of Orthopaedics, Qilu Hospital, Shandong University, Jinan, Shandong 250012, PR China School of Medcine, Shandong University, Jinan, Shandong 250012, PR China Department of Gynaecology and Obstetrics, Jinan Central Hospital, Shandong University, Jinan, Shandong, 250012, PR China d Department of Pathology, Qilu Hospital, Shandong University, Jinan, Shandong, 250012, PR China b c
a r t i c l e
i n f o
Article history: Received 28 September 2016 Received in revised form 13 February 2017 Accepted 14 February 2017 Keywords: Dermatitis Inflammation Mangiferin NF-κB signaling pathway RAW264.7 macrophage TNF-α
a b s t r a c t This study aimed to investigate whether mangiferin played a protective role in a well-established dermatitis mouse model and tumor necrosis factor alpha (TNF-α)-induced RAW264.7 macrophages. Contact dermatitis is an inflammatory skin disease in the clinic, while its underlying mechanism still remains to be elucidated. Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-β-D-glucoside (C-glucosyl xanthone), a natural antioxidant that was reported to inhibit inflammatory reactions, has been recently proved to be a potential therapy for inflammation. As a result, the oxazolone-induced dermatitis mice models were established to explore whether mangiferin has an anti-inflammatory role in vivo. The phosphate-buffered saline treatment groups showed emblematic skin inflammation, whereas the administration of mangiferin obviously inhibited dermatitis in the mice models. Furthermore, exogenous mangiferin alleviated the inflammatory reaction in TNF-α-induced macrophages by suppressing the production of inflammation- and oxidative stress–associated molecules. Also, mangiferin treatment repressed the activation of nuclear factor-kappaB signaling pathway. To sum up, mangiferin could provide a new target for the therapy and prevention of skin inflammation. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Contact dermatitis is one of the most common inflammatory skin disorders characterized by immunodysregulation, pruritic skin lesions, and disrupted epidermal barrier function [1]. Contact dermatitis still remains prevalent among populations worldwide, impairing their quality of life. However, much is unknown about the potential mechanism of dermatitis. It is still hard to cure and prevent in the clinic. To date, it is widely accepted that tumor necrosis factor alpha (TNF-α) plays a predominant role in various inflammatory diseases [2,3]. Moreover, antiTNF therapy has been widely used in the clinic for several autoimmune inflammatory diseases, such as rheumatoid arthritis and psoriasis [4,5]. Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-β-D-glucoside (Cglucosyl xanthone), which has a C-glucosyl linkage along multiple –OH
groups, possesses strong free radical–scavenging activity. Mangiferin is a natural antioxidant and is reported to have a positive role in a variety of physiological and disease processes, including tumorigenesis, hypersensitivity, and tissue repair [6,7]. Recent data implied the anti-inflammatory function of mangiferin [8,9]. Mangiferin is reported to antagonize TNF-α expression and function under several conditions [10,11]. Moreover, mangiferin is reported to inhibit rheumatoid arthritis, which is closely associated with TNF-α [9]. The nuclear factor-kappaB (NF-κB) signaling pathway is a key pathway in an inflammatory reaction, which can be significantly induced by TNF-α [12,13]. Mangiferin is known to interfere with the NF-κB pathway [14]. The present study investigated (1) whether mangiferin plays a role in the TNF-α-induced inflammatory reaction in macrophages and (2) the underlying therapeutic function of mangiferin in oxazolone (OXA)-induced contact dermatitis mice models. 2. Materials and methods
Abbreviations: IL-1β, Interleukin-1β; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor-kappaB; OXA, oxazolone; pIκB-α, phosphorylated IκB-α; TNF-α, tumor necrosis factor α; VEH, vehicle; WT, wild type. ⁎ Corresponding author at: Department of Pathology, Qilu Hospital, Shandong University, 107 Wenhuaxi Road, Jinan 250012, PR China. E-mail address: [email protected] (W. Li). 1 These authors contribute equally to this paper.
http://dx.doi.org/10.1016/j.intimp.2017.02.014 1567-5769/© 2017 Elsevier B.V. All rights reserved.
2.1. Cell culture RAW 264.7 cells, the immortalized murine macrophage cell line, were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cells were cultured in Dulbecco's modified Eagle's medium (Gibco, USA) with 10% fetal bovine serum
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(Gibco) and 1% penicillin–streptomycin (HyClone, UT, USA). The cells were cultured at 37 °C under 5% CO2. 2.2. Nitric oxide assay The nitric oxide (NO) assay was performed using the Griess reagent (Promega) to detect whether mangiferin could antagonize TNF-α-induced inflammation in RAW264.7 macrophages [15]. In short, the cells were cultured in the presence of 10 ng/mL TNF-α (R&D Systems)with or without mangiferin in the 96-well plates (1 × 105 cells/well) for 24 h. Next, the supernatant was collected, and its absorbance was tested at 540 nm using a commercial kit. 2.3. Detection of the levels of inflammation biomarkers in vitro The cells were cultured in eight 6-well plates (1 × 106 cells/well) and then incubated overnight. They were divided into five groups. Next, group 1 cells were treated with the culture medium for 24 h. Group 2 cells were cultured with 10 ng/mL TNF-α (R&D systems) without mangiferin for 24 h. Group 3 cells were treated with 10 ng/mL TNF-α (R&D systems) and 20 μmol/L or 100 μmol/L mangiferin for 24 h. Group 4 cells were treated with 10 ng/mL TNF-α (R&D Systems) in the presence of 10 μg/mL etanercept for 24 h. Meanwhile, group 5 cells were treated with 20 μmol/L and 100 μmol/L mangiferin for 24 h. Then, total protein was collected from all groups, and Western blotting was performed for inducible nitric oxide synthase (iNOS). Total RNA was extracted from RAW 264.7 cells, and reverse transcription–polymerase chain reaction (RT-PCR) and real-time PCR were performed.
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2.4. Mice All experimental animals and their care conformed to the guidelines set by the Institutional Animal Care and Use Committee of Shandong University. The 12-week-old WT BL6/C57 male mice used in this study were purchased from Jackson Laboratories (Maine, USA). 2.5. Oxazolone-induced dermatitis mice models The method to establish OXA-induced contact dermatitis mice models has been described in a previous study [16]. Briefly, to sensitize mice to oxazolone, 50 μL of OXA (Sigma, USA) dissolved in ethanol (1.5%) was smeared on the dorsal skin every 2 days for 7 days. The right ears of the mice were challenged by daubing 20 μL of 1% OXA dissolved in a mixture of olive oil and acetone (1:4) on both the outer and inner surfaces every 2 days for 2 weeks, while the left ears were daubed with olive oil and acetone (1:4) as vehicle control (VEH). At the same time, PBS or mangiferin (50 mg/kg body weight as previously reported) was administered to the mice orally every day, and the ear thickness was measured every 2 days using a micrometer (Mitutoyo, Kanagawa, Japan). Seven mice from each group were used in the experiments. 2.6. Quantitative real-time PCR RNA was collected from the ear lobes or RAW 264.7 cells of each group using an RNeasy kit (Qiagen, USA), and reverse transcription was performed using an RT-PCR kit (Qiagen, USA). Next, quantitative real-time PCR was performed in 20-μL of SYBR Green PCR Master Mix
Fig. 1. Mangiferin inhibited TNF-α-mediated inflammation in RAW264.7 macrophages. (A–C) The levels of iNOS, IL-1β, and IL-6 in TNF-α stimulated RAW264.7 cells in the presence or absence of mangiferin and etanercept were detected by quantitative real-time RT-PCR. (D) The level of the expression of iNOS in the groups with or without treatment of mangiferin was detected by Western blot. Total proteins were extracted from RAW264.7 macrophages after culturing with TNF-α for 24 h. (E) The nitrite levels in the PBS and mangiferin treatment groups were tested after culturing with TNF-αusing the Griess reagent. The values are mean ± SD. *P b 0.05, **P b 0.01, and ***P b 0.005 versus vehicle control group. #P b 0.05, ## P b 0.01, and ###P b 0.005 versus PBS treatment group.
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in a 96-well optical reaction plate formatted in the 7300 Sequence Detection System (Applied Biosystems, USA). The primers used in the experiment were as follows: 5′-AATCTCACAGCAGCACATCA-3′ and 5′AAGGTGCTCATGTCCTCATC-3′ for interleukin (IL)-1β; 5′-CCTTCCTACC CCAATTTCCAAT-3′ and 5′-GCCACTCCTTCTGTGACTCCAG-3′ for IL-6; 5′ACA GGAGGGGTTAAAGCTGC-3′ and 5′-TTGTCTCCAAGGGACCAGG-3′ for iNOS; 5′ TACAAGCTGGCTGGTGGGGA-3′ and 5′-GTCGCGGGTCTCAG GACCTT-3′ for NF-κB2; and 5′-AGAACATCATCCCTGCATCC-3′ and 5′AGTTGCTGTTGAAGTCGC-3′ for glyceraldehyde-3-phosphate dehydrogenase. The presence of a single specific PCR product was verified by a melting curve analysis, and for each gene, the experiments were repeated three times.
2.7. Western blot Total proteins were collected from the mice ears in each group. Proteins were loaded on a 10% sodium dodecyl sulfate polyacrylamide gel for 4 h running at 80 V and transferred onto polyvinylidene difluoride (PVDF) membranes. Next, PVDF membranes were blocked with 5% milk in Tris-buffered saline + Tween 20 (TBST) at 37 °C and incubated with anti-phosphorylated IκB-α (pIκB-α, diluted 1:1000) or anti-iNOS (diluted 1:1000) antibody for 1 h at 37 °C. After washing with TBST, the secondary antibody (horseradish peroxidase–conjugated antirabbit immunoglobulin; 1:2000 dilution) was added, and the blot was
visualized using an enhanced chemiluminescence system (Amersham Life Science, USA). 2.8. Histological analysis The ear tissues from the mice were collected and fixed in formalin for N 7 days. After dehydration, the ear tissues were cleared with dimethylbenzene and embedded in paraffin. For histological analysis, more than four consecutive 6-μm sections were stained with hematoxylin and eosin (HE) stain. 2.9. Detection of the levels of IL-1β and IL-6 The sera of mice were extracted and collected from blood using a centrifuge, and then the levels of IL-1β and IL-6 were assayed using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's protocol. 2.10. Statistical analysis The data were expressed as mean ± standard deviation (SD). For standard statistical analysis, the Statistical Package for Social Sciences version 17.0 (SPSS, IL, USA) was used for one-way analysis of variance and the Student t-test. Differences were statistically significant when the P value was b 0.05.
Fig. 2. Mangiferin inhibited inflammatory reaction in the oxazolone-induced dermatitis mice models. (A) Mangiferin treatment attenuated the increase in ear thickness in the mouse model, detected using a micrometer. (B) Histological analysis of inflammation in the skin tissue of dermatitis mice models, as detected by HE staining. (C) Mangiferin treatment attenuated thickening of epithelium in oxazolone-induced dermatitis, as measured by histological analysis. (D) Histological analysis of macrophages in the skin tissue of dermatitis mice models, as assayed by immunohistochemistry of CD68. Every experiment was repeated three times. The values are the mean ± SD. *P b 0.05, **P b 0.01, and ***P b 0.005 versus vehicle control group. #P b 0.05, ##P b 0.01 and ###P b 0.005 versus PBS treatment group. Scale bar, 50 μm.
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3. Results 3.1. Mangiferin protected against TNF-α-mediated inflammation in macrophages Macrophages play a crucial role in skin inflammation [17]. RAW264.7 macrophages were cultured with TNF-α(10 ng/mL), a critical inflammatory cytokine that plays a role in inflammatory reactions such as dermatitis, with or without 100 μmol/L mangiferin at specific time points to assess the anti-inflammatory function of mangiferin in macrophages. Mangiferin greatly reduced the levels of IL-1β, IL-6, and iNOS, which are inflammatory biomarkers, in the presence of TNF-α. As previously reported, iNOS acts as an inflammatory mediator in various forms of dermatitis (Fig. 1A–C). Western blot was performed to study the expression of iNOS induced by TNF-α with or without treatment with mangiferin. Mangiferin markedly decreased the levels of iNOS expression in RAW264.7 cells (Fig. 1D). Moreover, the amount of nitrite was assessed using the Griess test. The elevation of nitrite levels by TNF-α was inhibited by mangiferin dose-dependently (Fig. 1E). 3.2. Mangiferin protected against skin inflammation in the chemically induced contact dermatitis mice models A previous study showed that mangiferin protected against inflammatory arthritis in mice models [9]. OXA-induced dermatitis models were established in the WT BL6/C57 male mice to evaluate whether mangiferin had the same function in inflammatory dermatitis. The mice were treated with PBS or mangiferin orally, as described earlier. The ear lobes were collected from each group at indicated time points, followed by histological analysis. The ear thickness was measured using a micrometer. The mangiferin therapy group showed an obviously less increase in thickness compared with the PBS group (Fig. 2A). The histological analysis of the ears revealed that the thickening of epithelium and the infiltration of inflammatory cells were repressed in the mangiferin treatment group (Fig. 2B and C). It is well accepted that CD68 is a critical biomarker of macrophages [18]. The expression of CD68 was detected in ear tissues of each group using
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immunohistochemistry. Mangiferin remarkably suppressed the expression of CD68 in dermatitis (Fig. 2D). Furthermore, total RNA was extracted from the ear lobes of each group. Quantitative real-time PCR results showed that the levels of inflammation-associated molecules, including iNOS, IL-1β, andIL-6, were significantly lower in the mangiferin treatment group (Fig. 3A–C). Sera were collected from all groups, and ELISA was used to test the circulating levels of IL-1β and IL-6. The levels of inflammatory biomarkers produced in the OXA-induced dermatitis mice models significantly decreased on mangiferin administration (Fig. 3D and E). Moreover, total protein was extracted from the ear tissues of each group (n = 3), followed by the Western blot analysis for the level of iNOS. Mangiferin treatment significantly diminished the protein level of iNOS in the mice models (Fig. 3F). 3.3. Mangiferin repressed the activation of NF-κB signaling pathway in vivo Mangiferin repressed the activation of NF-κB pathway, the pathway that plays an important role in a variety of skin inflammations. Next, whether mangiferin directly inhibited the NF-κB pathway in skin inflammation was explored. RNA was isolated from the ear lobes of both the mangiferin and PBS treatment groups, and real-time PCR was performed for determining the levels of NF-κB2. The elevation of the levels of NF-κB2 in dermatitis models was diminished by additional treatment of mangiferin (Fig. 4A). Furthermore, total proteins were isolated and collected from the mice ear samples. Mangiferin inhibited the phosphorylation of IκB in OXA-induced skin inflammation (Fig. 4B). Additionally, p-IκBα was detected through immunohistochemistry. Mangiferin greatly abolished the activation of NF-κB pathway in dermatitis (Fig. 4C). Taken together, it implied that the NF-κB signaling pathway might be a potential target for the therapeutic role of mangiferin in skin inflammation. 4. Discussion To date, mangiferin is reported to play a therapeutic role in several inflammatory diseases [19,20]. Moreover, mangiferin was reported to
Fig. 3. Mangiferin reduced the level of inflammatory cytokines in serum and skin from the oxazolone-induced dermatitis mice models. (A–C) Levels of iNOS, IL-1β, and IL-6 in the ear lobes of dermatitis mice models with or without PBS and mangiferin treatment were detected by quantitative real-time PCR. (D and E) Level of IL-1β and IL-6 in the serum of dermatitis mice models with or without PBS and mangiferin treatment was detected by ELISA. Sera were extracted from each experimental mice group, and ELISA was used to test the level of IL-1β and IL6. (F) Level of iNOS expression in total protein collected from the ear tissue of PBS- or mangiferin-treated mice was detected using Western blot. Every experiment was repeated three times. The values are the mean ± SD. *P b 0.05, **P b 0.01, and ***P b 0.005 versus vehicle control group. #P b 0.05, ##P b 0.01, and ###P b 0.005 versus PBS treatment group.
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Fig. 4. Mangiferin attenuated TNF-α-induced augmentation of NF-κB signaling in the mice. (A) Total RNA was extracted from the dermatitis model and control groups. The level of NF-κB2 was detected by quantitative real-time PCR. (B) Phosphorylation of IκB in skin tissue was sharply reduced by mangiferin treatment, as detected by Western blot. (C) Mangiferin treatment greatly impaired oxazolone-mediated p-IκB expression in skin tissue, as assayed by immunohistochemistry. *P b 0.05, **P b 0.01, and ***P b 0.005 versus vehicle control group. #P b 0.05, ##P b 0.01, and ###P b 0.005 versus PBS treatment group. Scale bar, 50 μm.
antagonize TNF function [21]. In recent years, mangiferin has been found to have a protective effect on inflammatory arthritis in the mice models, suggesting that mangiferin might play a role in dermatitis. Macrophages play an important role in skin inflammation; it is a major target for treating dermatitis [22]. TNF-α is a well-known inflammatory mediator [23]. Moreover, it is reported that the inhibition of TNF-α function could attenuate skin inflammation. Anti-TNF agents have been widely used for the therapy of several diseases such as psoriasis. Moreover, iNOS, IL-1β, andIL-6, which can be induced by TNF-α [24,25], are various important biomarkers for inflammation. The most commonly used macrophage cell line, RAW264.7 cells, was employed in the present study. The level of inflammatory biomarkers such as iNOS, IL-1β, and IL-6 greatly decreased in the presence of mangiferin, suggesting that TNF-α-induced skin inflammation was greatly inhibited by mangiferin. The OXA-induced dermatitis model is the most acceptable model for investigating dermatitis [26]. This model was used in the present study, and mangiferin therapy markedly attenuated the severity of skin inflammation. Macrophages are widely explored in the therapy of dermatitis and have become the most commonly used target for various inflammatory diseases [27,28]. Moreover, CD68 is well accepted as a characteristic biomarker of macrophages [29]. The expression of CD68 in skin tissue was measured using immunohistochemistry in this study. Mangiferin greatly reduced the expression level of CD68, suggesting that the role of mangiferin in dermatitis might depend on the antagonization of macrophages. The NF-κB pathway is a key signaling pathway for the TNF-αinduced inflammatory reaction; it plays an important role in skin inflammation [30]. Furthermore, as previously reported, mangiferin inhibited TNF-α-mediated inflammation in several disease models via the NF-κB signaling pathway [31,32]. The present study showed that mangiferin markedly lowered the expression and inhibited the activity of NF-κB signaling pathway. It also affected the levels of NF-κB downstream molecules in vivo, suggesting that mangiferin might play a role in skin inflammation through repressing the activation of NF-κB pathway. To sum up, mangiferin plays a positive role in inhibiting dermatitis
and may serve as a potential therapeutic agent in the treatment of skin inflammation. The OXA-induced skin inflammation mice model is the most frequently used animal model to study the effect of new therapeutic agents for dermatitis. However, human dermatitis still remains to be elucidated. The present study confirmed that mangiferin inhibited dermatitis in the mice. However, it could not confirm whether mangiferin could also inhibit dermatitis in humans. The study indicated the immense potential of mangiferin in inflammatory diseases. However, the question arises whether the anti-inflammatory effect would be affected on changing the structure of mangiferin. Future studies should focus on exploring a new structure or some new conditions to improve the effect and safety of mangiferin in treating dermatitis in humans. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 81501880), the Natural Science Foundation of Shandong Province (Grant No. BS2014YY048), the Natural Science Foundation of Shandong Province (Grant No. BS2015SW028), Major Project of Science and Technology of Shandong Province (No. 2015ZDJS04001), and Key Research and Development Projects of Shandong Province (No. 2015GSF118115). References [1] S.C. Han, D.H. Koo, N.J. Kang, W.J. Yoon, G.J. Kang, H.K. Kang, E.S. Yoo, Docosahexaenoic acid alleviates atopic dermatitis by generating Tregs and IL-10/ TGF-beta-modified macrophages via a TGF-beta-dependent mechanism, J. Invest. Dermatol. 135 (6) (2015) 1556–1564. [2] W. Tang, Y. Lu, Q.Y. Tian, Y. Zhang, F.J. Guo, G.Y. Liu, N.M. Syed, Y. Lai, E.A. Lin, L. Kong, J. Su, F. Yin, A.H. Ding, A. Zanin-Zhorov, M.L. Dustin, J. Tao, J. Craft, Z. Yin, J.Q. Feng, S.B. Abramson, X.P. Yu, C.J. Liu, The growth factor progranulin binds to TNF
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Mangiferin antagonizes TNF-α-mediated inflammatory reaction and protects against dermatitis in a mice model Yunpeng Zhao a,1, Wenhan Wang b,1, Xihai Wu c, Xiaoqian Ma b, Ruize Qu b, Xiaomin Chen b, Chenghao Liu b, Yaoge Liu b, Xiaokai Wang b, Pengcheng Yan b, Hao Zhang b, Jingrui Pan b, Weiwei Li d,⁎ a
Department of Orthopaedics, Qilu Hospital, Shandong University, Jinan, Shandong 250012, PR China School of Medcine, Shandong University, Jinan, Shandong 250012, PR China Department of Gynaecology and Obstetrics, Jinan Central Hospital, Shandong University, Jinan, Shandong, 250012, PR China d Department of Pathology, Qilu Hospital, Shandong University, Jinan, Shandong, 250012, PR China b c
a r t i c l e
i n f o
Article history: Received 28 September 2016 Received in revised form 13 February 2017 Accepted 14 February 2017 Keywords: Dermatitis Inflammation Mangiferin NF-κB signaling pathway RAW264.7 macrophage TNF-α
a b s t r a c t This study aimed to investigate whether mangiferin played a protective role in a well-established dermatitis mouse model and tumor necrosis factor alpha (TNF-α)-induced RAW264.7 macrophages. Contact dermatitis is an inflammatory skin disease in the clinic, while its underlying mechanism still remains to be elucidated. Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-β-D-glucoside (C-glucosyl xanthone), a natural antioxidant that was reported to inhibit inflammatory reactions, has been recently proved to be a potential therapy for inflammation. As a result, the oxazolone-induced dermatitis mice models were established to explore whether mangiferin has an anti-inflammatory role in vivo. The phosphate-buffered saline treatment groups showed emblematic skin inflammation, whereas the administration of mangiferin obviously inhibited dermatitis in the mice models. Furthermore, exogenous mangiferin alleviated the inflammatory reaction in TNF-α-induced macrophages by suppressing the production of inflammation- and oxidative stress–associated molecules. Also, mangiferin treatment repressed the activation of nuclear factor-kappaB signaling pathway. To sum up, mangiferin could provide a new target for the therapy and prevention of skin inflammation. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Contact dermatitis is one of the most common inflammatory skin disorders characterized by immunodysregulation, pruritic skin lesions, and disrupted epidermal barrier function [1]. Contact dermatitis still remains prevalent among populations worldwide, impairing their quality of life. However, much is unknown about the potential mechanism of dermatitis. It is still hard to cure and prevent in the clinic. To date, it is widely accepted that tumor necrosis factor alpha (TNF-α) plays a predominant role in various inflammatory diseases [2,3]. Moreover, antiTNF therapy has been widely used in the clinic for several autoimmune inflammatory diseases, such as rheumatoid arthritis and psoriasis [4,5]. Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-β-D-glucoside (Cglucosyl xanthone), which has a C-glucosyl linkage along multiple –OH
groups, possesses strong free radical–scavenging activity. Mangiferin is a natural antioxidant and is reported to have a positive role in a variety of physiological and disease processes, including tumorigenesis, hypersensitivity, and tissue repair [6,7]. Recent data implied the anti-inflammatory function of mangiferin [8,9]. Mangiferin is reported to antagonize TNF-α expression and function under several conditions [10,11]. Moreover, mangiferin is reported to inhibit rheumatoid arthritis, which is closely associated with TNF-α [9]. The nuclear factor-kappaB (NF-κB) signaling pathway is a key pathway in an inflammatory reaction, which can be significantly induced by TNF-α [12,13]. Mangiferin is known to interfere with the NF-κB pathway [14]. The present study investigated (1) whether mangiferin plays a role in the TNF-α-induced inflammatory reaction in macrophages and (2) the underlying therapeutic function of mangiferin in oxazolone (OXA)-induced contact dermatitis mice models. 2. Materials and methods
Abbreviations: IL-1β, Interleukin-1β; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor-kappaB; OXA, oxazolone; pIκB-α, phosphorylated IκB-α; TNF-α, tumor necrosis factor α; VEH, vehicle; WT, wild type. ⁎ Corresponding author at: Department of Pathology, Qilu Hospital, Shandong University, 107 Wenhuaxi Road, Jinan 250012, PR China. E-mail address: [email protected] (W. Li). 1 These authors contribute equally to this paper.
http://dx.doi.org/10.1016/j.intimp.2017.02.014 1567-5769/© 2017 Elsevier B.V. All rights reserved.
2.1. Cell culture RAW 264.7 cells, the immortalized murine macrophage cell line, were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cells were cultured in Dulbecco's modified Eagle's medium (Gibco, USA) with 10% fetal bovine serum
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(Gibco) and 1% penicillin–streptomycin (HyClone, UT, USA). The cells were cultured at 37 °C under 5% CO2. 2.2. Nitric oxide assay The nitric oxide (NO) assay was performed using the Griess reagent (Promega) to detect whether mangiferin could antagonize TNF-α-induced inflammation in RAW264.7 macrophages [15]. In short, the cells were cultured in the presence of 10 ng/mL TNF-α (R&D Systems)with or without mangiferin in the 96-well plates (1 × 105 cells/well) for 24 h. Next, the supernatant was collected, and its absorbance was tested at 540 nm using a commercial kit. 2.3. Detection of the levels of inflammation biomarkers in vitro The cells were cultured in eight 6-well plates (1 × 106 cells/well) and then incubated overnight. They were divided into five groups. Next, group 1 cells were treated with the culture medium for 24 h. Group 2 cells were cultured with 10 ng/mL TNF-α (R&D systems) without mangiferin for 24 h. Group 3 cells were treated with 10 ng/mL TNF-α (R&D systems) and 20 μmol/L or 100 μmol/L mangiferin for 24 h. Group 4 cells were treated with 10 ng/mL TNF-α (R&D Systems) in the presence of 10 μg/mL etanercept for 24 h. Meanwhile, group 5 cells were treated with 20 μmol/L and 100 μmol/L mangiferin for 24 h. Then, total protein was collected from all groups, and Western blotting was performed for inducible nitric oxide synthase (iNOS). Total RNA was extracted from RAW 264.7 cells, and reverse transcription–polymerase chain reaction (RT-PCR) and real-time PCR were performed.
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2.4. Mice All experimental animals and their care conformed to the guidelines set by the Institutional Animal Care and Use Committee of Shandong University. The 12-week-old WT BL6/C57 male mice used in this study were purchased from Jackson Laboratories (Maine, USA). 2.5. Oxazolone-induced dermatitis mice models The method to establish OXA-induced contact dermatitis mice models has been described in a previous study [16]. Briefly, to sensitize mice to oxazolone, 50 μL of OXA (Sigma, USA) dissolved in ethanol (1.5%) was smeared on the dorsal skin every 2 days for 7 days. The right ears of the mice were challenged by daubing 20 μL of 1% OXA dissolved in a mixture of olive oil and acetone (1:4) on both the outer and inner surfaces every 2 days for 2 weeks, while the left ears were daubed with olive oil and acetone (1:4) as vehicle control (VEH). At the same time, PBS or mangiferin (50 mg/kg body weight as previously reported) was administered to the mice orally every day, and the ear thickness was measured every 2 days using a micrometer (Mitutoyo, Kanagawa, Japan). Seven mice from each group were used in the experiments. 2.6. Quantitative real-time PCR RNA was collected from the ear lobes or RAW 264.7 cells of each group using an RNeasy kit (Qiagen, USA), and reverse transcription was performed using an RT-PCR kit (Qiagen, USA). Next, quantitative real-time PCR was performed in 20-μL of SYBR Green PCR Master Mix
Fig. 1. Mangiferin inhibited TNF-α-mediated inflammation in RAW264.7 macrophages. (A–C) The levels of iNOS, IL-1β, and IL-6 in TNF-α stimulated RAW264.7 cells in the presence or absence of mangiferin and etanercept were detected by quantitative real-time RT-PCR. (D) The level of the expression of iNOS in the groups with or without treatment of mangiferin was detected by Western blot. Total proteins were extracted from RAW264.7 macrophages after culturing with TNF-α for 24 h. (E) The nitrite levels in the PBS and mangiferin treatment groups were tested after culturing with TNF-αusing the Griess reagent. The values are mean ± SD. *P b 0.05, **P b 0.01, and ***P b 0.005 versus vehicle control group. #P b 0.05, ## P b 0.01, and ###P b 0.005 versus PBS treatment group.
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in a 96-well optical reaction plate formatted in the 7300 Sequence Detection System (Applied Biosystems, USA). The primers used in the experiment were as follows: 5′-AATCTCACAGCAGCACATCA-3′ and 5′AAGGTGCTCATGTCCTCATC-3′ for interleukin (IL)-1β; 5′-CCTTCCTACC CCAATTTCCAAT-3′ and 5′-GCCACTCCTTCTGTGACTCCAG-3′ for IL-6; 5′ACA GGAGGGGTTAAAGCTGC-3′ and 5′-TTGTCTCCAAGGGACCAGG-3′ for iNOS; 5′ TACAAGCTGGCTGGTGGGGA-3′ and 5′-GTCGCGGGTCTCAG GACCTT-3′ for NF-κB2; and 5′-AGAACATCATCCCTGCATCC-3′ and 5′AGTTGCTGTTGAAGTCGC-3′ for glyceraldehyde-3-phosphate dehydrogenase. The presence of a single specific PCR product was verified by a melting curve analysis, and for each gene, the experiments were repeated three times.
2.7. Western blot Total proteins were collected from the mice ears in each group. Proteins were loaded on a 10% sodium dodecyl sulfate polyacrylamide gel for 4 h running at 80 V and transferred onto polyvinylidene difluoride (PVDF) membranes. Next, PVDF membranes were blocked with 5% milk in Tris-buffered saline + Tween 20 (TBST) at 37 °C and incubated with anti-phosphorylated IκB-α (pIκB-α, diluted 1:1000) or anti-iNOS (diluted 1:1000) antibody for 1 h at 37 °C. After washing with TBST, the secondary antibody (horseradish peroxidase–conjugated antirabbit immunoglobulin; 1:2000 dilution) was added, and the blot was
visualized using an enhanced chemiluminescence system (Amersham Life Science, USA). 2.8. Histological analysis The ear tissues from the mice were collected and fixed in formalin for N 7 days. After dehydration, the ear tissues were cleared with dimethylbenzene and embedded in paraffin. For histological analysis, more than four consecutive 6-μm sections were stained with hematoxylin and eosin (HE) stain. 2.9. Detection of the levels of IL-1β and IL-6 The sera of mice were extracted and collected from blood using a centrifuge, and then the levels of IL-1β and IL-6 were assayed using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's protocol. 2.10. Statistical analysis The data were expressed as mean ± standard deviation (SD). For standard statistical analysis, the Statistical Package for Social Sciences version 17.0 (SPSS, IL, USA) was used for one-way analysis of variance and the Student t-test. Differences were statistically significant when the P value was b 0.05.
Fig. 2. Mangiferin inhibited inflammatory reaction in the oxazolone-induced dermatitis mice models. (A) Mangiferin treatment attenuated the increase in ear thickness in the mouse model, detected using a micrometer. (B) Histological analysis of inflammation in the skin tissue of dermatitis mice models, as detected by HE staining. (C) Mangiferin treatment attenuated thickening of epithelium in oxazolone-induced dermatitis, as measured by histological analysis. (D) Histological analysis of macrophages in the skin tissue of dermatitis mice models, as assayed by immunohistochemistry of CD68. Every experiment was repeated three times. The values are the mean ± SD. *P b 0.05, **P b 0.01, and ***P b 0.005 versus vehicle control group. #P b 0.05, ##P b 0.01 and ###P b 0.005 versus PBS treatment group. Scale bar, 50 μm.
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3. Results 3.1. Mangiferin protected against TNF-α-mediated inflammation in macrophages Macrophages play a crucial role in skin inflammation [17]. RAW264.7 macrophages were cultured with TNF-α(10 ng/mL), a critical inflammatory cytokine that plays a role in inflammatory reactions such as dermatitis, with or without 100 μmol/L mangiferin at specific time points to assess the anti-inflammatory function of mangiferin in macrophages. Mangiferin greatly reduced the levels of IL-1β, IL-6, and iNOS, which are inflammatory biomarkers, in the presence of TNF-α. As previously reported, iNOS acts as an inflammatory mediator in various forms of dermatitis (Fig. 1A–C). Western blot was performed to study the expression of iNOS induced by TNF-α with or without treatment with mangiferin. Mangiferin markedly decreased the levels of iNOS expression in RAW264.7 cells (Fig. 1D). Moreover, the amount of nitrite was assessed using the Griess test. The elevation of nitrite levels by TNF-α was inhibited by mangiferin dose-dependently (Fig. 1E). 3.2. Mangiferin protected against skin inflammation in the chemically induced contact dermatitis mice models A previous study showed that mangiferin protected against inflammatory arthritis in mice models [9]. OXA-induced dermatitis models were established in the WT BL6/C57 male mice to evaluate whether mangiferin had the same function in inflammatory dermatitis. The mice were treated with PBS or mangiferin orally, as described earlier. The ear lobes were collected from each group at indicated time points, followed by histological analysis. The ear thickness was measured using a micrometer. The mangiferin therapy group showed an obviously less increase in thickness compared with the PBS group (Fig. 2A). The histological analysis of the ears revealed that the thickening of epithelium and the infiltration of inflammatory cells were repressed in the mangiferin treatment group (Fig. 2B and C). It is well accepted that CD68 is a critical biomarker of macrophages [18]. The expression of CD68 was detected in ear tissues of each group using
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immunohistochemistry. Mangiferin remarkably suppressed the expression of CD68 in dermatitis (Fig. 2D). Furthermore, total RNA was extracted from the ear lobes of each group. Quantitative real-time PCR results showed that the levels of inflammation-associated molecules, including iNOS, IL-1β, andIL-6, were significantly lower in the mangiferin treatment group (Fig. 3A–C). Sera were collected from all groups, and ELISA was used to test the circulating levels of IL-1β and IL-6. The levels of inflammatory biomarkers produced in the OXA-induced dermatitis mice models significantly decreased on mangiferin administration (Fig. 3D and E). Moreover, total protein was extracted from the ear tissues of each group (n = 3), followed by the Western blot analysis for the level of iNOS. Mangiferin treatment significantly diminished the protein level of iNOS in the mice models (Fig. 3F). 3.3. Mangiferin repressed the activation of NF-κB signaling pathway in vivo Mangiferin repressed the activation of NF-κB pathway, the pathway that plays an important role in a variety of skin inflammations. Next, whether mangiferin directly inhibited the NF-κB pathway in skin inflammation was explored. RNA was isolated from the ear lobes of both the mangiferin and PBS treatment groups, and real-time PCR was performed for determining the levels of NF-κB2. The elevation of the levels of NF-κB2 in dermatitis models was diminished by additional treatment of mangiferin (Fig. 4A). Furthermore, total proteins were isolated and collected from the mice ear samples. Mangiferin inhibited the phosphorylation of IκB in OXA-induced skin inflammation (Fig. 4B). Additionally, p-IκBα was detected through immunohistochemistry. Mangiferin greatly abolished the activation of NF-κB pathway in dermatitis (Fig. 4C). Taken together, it implied that the NF-κB signaling pathway might be a potential target for the therapeutic role of mangiferin in skin inflammation. 4. Discussion To date, mangiferin is reported to play a therapeutic role in several inflammatory diseases [19,20]. Moreover, mangiferin was reported to
Fig. 3. Mangiferin reduced the level of inflammatory cytokines in serum and skin from the oxazolone-induced dermatitis mice models. (A–C) Levels of iNOS, IL-1β, and IL-6 in the ear lobes of dermatitis mice models with or without PBS and mangiferin treatment were detected by quantitative real-time PCR. (D and E) Level of IL-1β and IL-6 in the serum of dermatitis mice models with or without PBS and mangiferin treatment was detected by ELISA. Sera were extracted from each experimental mice group, and ELISA was used to test the level of IL-1β and IL6. (F) Level of iNOS expression in total protein collected from the ear tissue of PBS- or mangiferin-treated mice was detected using Western blot. Every experiment was repeated three times. The values are the mean ± SD. *P b 0.05, **P b 0.01, and ***P b 0.005 versus vehicle control group. #P b 0.05, ##P b 0.01, and ###P b 0.005 versus PBS treatment group.
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Fig. 4. Mangiferin attenuated TNF-α-induced augmentation of NF-κB signaling in the mice. (A) Total RNA was extracted from the dermatitis model and control groups. The level of NF-κB2 was detected by quantitative real-time PCR. (B) Phosphorylation of IκB in skin tissue was sharply reduced by mangiferin treatment, as detected by Western blot. (C) Mangiferin treatment greatly impaired oxazolone-mediated p-IκB expression in skin tissue, as assayed by immunohistochemistry. *P b 0.05, **P b 0.01, and ***P b 0.005 versus vehicle control group. #P b 0.05, ##P b 0.01, and ###P b 0.005 versus PBS treatment group. Scale bar, 50 μm.
antagonize TNF function [21]. In recent years, mangiferin has been found to have a protective effect on inflammatory arthritis in the mice models, suggesting that mangiferin might play a role in dermatitis. Macrophages play an important role in skin inflammation; it is a major target for treating dermatitis [22]. TNF-α is a well-known inflammatory mediator [23]. Moreover, it is reported that the inhibition of TNF-α function could attenuate skin inflammation. Anti-TNF agents have been widely used for the therapy of several diseases such as psoriasis. Moreover, iNOS, IL-1β, andIL-6, which can be induced by TNF-α [24,25], are various important biomarkers for inflammation. The most commonly used macrophage cell line, RAW264.7 cells, was employed in the present study. The level of inflammatory biomarkers such as iNOS, IL-1β, and IL-6 greatly decreased in the presence of mangiferin, suggesting that TNF-α-induced skin inflammation was greatly inhibited by mangiferin. The OXA-induced dermatitis model is the most acceptable model for investigating dermatitis [26]. This model was used in the present study, and mangiferin therapy markedly attenuated the severity of skin inflammation. Macrophages are widely explored in the therapy of dermatitis and have become the most commonly used target for various inflammatory diseases [27,28]. Moreover, CD68 is well accepted as a characteristic biomarker of macrophages [29]. The expression of CD68 in skin tissue was measured using immunohistochemistry in this study. Mangiferin greatly reduced the expression level of CD68, suggesting that the role of mangiferin in dermatitis might depend on the antagonization of macrophages. The NF-κB pathway is a key signaling pathway for the TNF-αinduced inflammatory reaction; it plays an important role in skin inflammation [30]. Furthermore, as previously reported, mangiferin inhibited TNF-α-mediated inflammation in several disease models via the NF-κB signaling pathway [31,32]. The present study showed that mangiferin markedly lowered the expression and inhibited the activity of NF-κB signaling pathway. It also affected the levels of NF-κB downstream molecules in vivo, suggesting that mangiferin might play a role in skin inflammation through repressing the activation of NF-κB pathway. To sum up, mangiferin plays a positive role in inhibiting dermatitis
and may serve as a potential therapeutic agent in the treatment of skin inflammation. The OXA-induced skin inflammation mice model is the most frequently used animal model to study the effect of new therapeutic agents for dermatitis. However, human dermatitis still remains to be elucidated. The present study confirmed that mangiferin inhibited dermatitis in the mice. However, it could not confirm whether mangiferin could also inhibit dermatitis in humans. The study indicated the immense potential of mangiferin in inflammatory diseases. However, the question arises whether the anti-inflammatory effect would be affected on changing the structure of mangiferin. Future studies should focus on exploring a new structure or some new conditions to improve the effect and safety of mangiferin in treating dermatitis in humans. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 81501880), the Natural Science Foundation of Shandong Province (Grant No. BS2014YY048), the Natural Science Foundation of Shandong Province (Grant No. BS2015SW028), Major Project of Science and Technology of Shandong Province (No. 2015ZDJS04001), and Key Research and Development Projects of Shandong Province (No. 2015GSF118115). References [1] S.C. Han, D.H. Koo, N.J. Kang, W.J. Yoon, G.J. Kang, H.K. Kang, E.S. Yoo, Docosahexaenoic acid alleviates atopic dermatitis by generating Tregs and IL-10/ TGF-beta-modified macrophages via a TGF-beta-dependent mechanism, J. Invest. Dermatol. 135 (6) (2015) 1556–1564. [2] W. Tang, Y. Lu, Q.Y. Tian, Y. Zhang, F.J. Guo, G.Y. Liu, N.M. Syed, Y. Lai, E.A. Lin, L. Kong, J. Su, F. Yin, A.H. Ding, A. Zanin-Zhorov, M.L. Dustin, J. Tao, J. Craft, Z. Yin, J.Q. Feng, S.B. Abramson, X.P. Yu, C.J. Liu, The growth factor progranulin binds to TNF
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