Nobiletin and 5‑Hydroxy-6,7,8,3′,4′-pentamethoxyflavone

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Nobiletin and 5‑Hydroxy-6,7,8,3′,4′-pentamethoxyflavone Ameliorate 12‑O‑Tetradecanoylphorbol-13-acetate-Induced Psoriasis-Like Mouse Skin Lesions by Regulating the Expression of Ki-67 and Proliferating Cell Nuclear Antigen and the Differentiation of CD4+ T Cells through Mitogen-Activated Protein Kinase Signaling Pathways Guliang Yang,†,‡ Shiming Li,*,†,‡ Yiwen Yang,† Li Yuan,† Peilei Wang,† Hui Zhao,§ Chi-Tang Ho,∥ and Chi-Chen Lin*,⊥,# †

Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources, Huanggang Normal University, Huanggang, Hubei 438000, People’s Republic of China ‡ Key Laboratory of Cultivation and Protection for Non-wood Forest Trees, Ministry of Education, Central South University of Forestry and Technology, Changsha, Hunan 410004, People’s Republic of China § Tianjin Key Laboratory of Food and Biotechnology, School of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, People’s Republic of China ∥ Department of Food Science, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901, United States ⊥ Institute of Biomedical Science, National Chung-Hsing University, Taichung 402, Taiwan # Department of Medical Research, China Medical University Hospital, Taichung, Taiwan ABSTRACT: Psoriasis is a chronic and benign proliferative skin disease. Flavonoids in chenpi (aged tangerine peel) from tangerine (Citrus reticulate Blanco), such as nobiletin (Nob), tangeretin, and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone (5-HPMF), possess anti-inflammation and regulation of immune activity among others. In this study, psoriasis-like skin lesions were induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and the preventive effect of Nob and 5-HPMF on psoriasis-like skin lesions was evaluated. Results showed that skin lesions were dramatically reduced by Nob and 5-HPMF. Levels of cytokines, including interleukin (IL)-1β, IL-17, IL-4, IL-6, tumor necrosis factor-α, and interferon-γ, were also reduced after Nob and 5-HPMF treatment. The expression levels of p-ERK1/2 and p-p38 mitogen-activated protein kinase (MAPK) in the TPA group were 5.3, 4.8, and 5.7 but downregulated to 2.7, 2.9, and 2.3 in the Nob group and 2.4, 2.7, and 1.2 in the 5-HPMF group, respectively (p ≤ 0.05). The expression of transcription factors Ki-67 and proliferating cell nuclear antigen (PCNA) and the differentiation of CD4+ T cells were reduced by downregulating the expression of the MAPK signaling pathways. The expression levels in TPA, Nob, and 5-HPMF groups were 0.649 ± 0.094, 0.218 ± 0.034, and 0.193 ± 0.042 for Ki-67 and 0.753 ± 0.114, 0.315 ± 0.094, and 0.294 ± 0.035 for PCNA, respectively. Moreover, 5-HPMF showed stronger reduction activity in the prevention of psoriasis than Nob, indicating that the 5-hydroxyl group facilitated the suppression of psoriasis. KEYWORDS: psoriasis, nobiletin, MAPK, transcription factors, CD4+ T cells

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INTRODUCTION Psoriasis is a chronic relapsing inflammatory skin disease with scaly erythema as the typical skin lesion. The prevalence of psoriasis patients among the global population is 2−3%. The occurrence of psoriasis is not affected by gender or age, but there are two incidence peaks in the youth stage and the old age stage.1 Psoriasis has various degrees of symptoms, such as dryness, itching, and pain, leading to a decrease in the quality of life of patients. Pathological examination of psoriatic skin lesions shows that lymphocytes, neutrophils, and dendritic cells among others significantly infiltrate the lesions. Dermal infiltration of T lymphocytes is an important pathological feature of psoriasis, which indicates that the immune system is involved in the pathogenesis of psoriasis.2 Cytokines, such as interleukin (IL)-17, IL-21, IL-22, and tumor necrosis factor-α (TNF-α), are © XXXX American Chemical Society

Figure 1. Chemical structures of (A) Nob and (B) 5-HPMF.

closely related to the disorder of the immune system. It has been confirmed that T helper cell 17 (Th17)-related cytokines play an Received: May 12, 2018 Revised: July 11, 2018 Accepted: July 15, 2018

A

DOI: 10.1021/acs.jafc.8b02524 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. Histopathology of psoriatic-like lesion skin after Nob and 5-HPMF treatment. (A) H&E staining of mouse ear lesion skin following the indicated treatments. H&E-stained paraffin sections, at 200×. Hyperkeratosis is marked by a black small arrow. Munro’s microabscess is marked by a red small arrow. The granular layer is marked by a black big arrow. The spinous layer is marked by a hollow triangle. Trochanterellus is marked by a black solid triangle. Lymphocyte is marked by a purple small arrow, and capillaries are marked by a yellow solid triangle. (B) Data on histology score (n = 6). (C) Ki-67 immunohistochemical staining of paraffin-embedded slides reveals the inhibitory effect of Nob and 5-HPMF on the proliferation of keratinocytes in the mouse epidermis, at 200×. (D) PCNA immunohistochemical staining of paraffin-embedded slides reveals the inhibitory effect of Nob and 5-HPMF on the proliferation of keratinocytes in the mouse epidermis, at 200×. (E) Ki-67 gray value of the epidermis on day 4 based on the analysis of gray value scanning by Image-Pro Plus of 5−6 sites per mouse. Mean ± SD (n = 6). (F) PCNA gray value of the epidermis on day 4 based on the analysis of gray value scanning by Image-Pro Plus of 5−6 sites per mouse. Mean ± SD (n = 3). The scale bar represents 200 μm. Different letters (a−c) represent significant differences of the same indexes of the different groups when p < 0.01.

certain therapeutic effects on some psoriasis patients, they have severe side effects, such as easily relapsing and nephrotoxicity.3−5 In recent years, cytokine antagonists, such as etanercept and monoclonal antibodies against TNF-α, have shown good efficacy in treating psoriasis and ustekinumab, using the monoclonal antibodies against IL-12 and IL-23, has been approved

important role in the pathogenesis of psoriasis. The pathological manifestations of psoriasis include epidermal hyperplasia and inflammation. Traditional therapeutic drugs for the treatment of psoriasis, such as hydroxyurea, methotrexate, halometasone, and betamethasone, mainly aim to fight inflammation and inhibit epidermal hyperplasia. Although these drugs have B

DOI: 10.1021/acs.jafc.8b02524 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry by the United States Food and Drug Administration (FDA) for the treatment of psoriasis.6 However, because of its high price, it is not commonly used in clinical applications. Flavonoids from citrus peels are closely associated with biological activities, such as antioxidation, inhibitory effects of inflammation, antitumor, prevention of rheumatoid arthritis, and regulation of intestinal flora.7−9 In our previous study, we found that nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone, Nob), one of the major polymethoxyflavones in tangerine peel, can inhibit the activity of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)/mitogen-activated protein kinase (MAPK) and prevent the formation of rheumatoid arthritis.10 Nob has very similar anti-inflammatory physiological effects as steroids, such as dexamethasone. These results further support the notion that Nob is a likely a candidate as a novel immunomodulatory and antiinflammatory agent.11 Nob was identified as an inhibitor of both NO and O2− generation in inflammation-associated tumorigenesis.12 In this study, we evaluated the effect of Nob and its monodemethylated derivative at position 5, i.e., 5-hydroxy-6,7,8,3′,4′pentamethoxyflavone (5-HPMF), also existing naturally in tangerine peels, on 12-O-tetradecanoylphorbol-13-acetate (TPA)induced psoriasis-like skin lesions. We examined their effects on the expression of the MAPK signal pathways and the differentiation of CD4+ T cells on a variety of relevant processes to determine the preventive and therapeutic effects and the underlying mechanism on immune regulation and inflammation of psoriasis.

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MATERIALS AND METHODS

Chemicals. Nob (Figure 1A) and 5-HPMF (Figure 1B) of more than 98% purity were extracted and purified in-house from chenpi (aged tangerine peel) according to the method described previously.13 The purity was measured on high-performance liquid chromatography (HPLC) and 1H nuclear magnetic resonance (NMR) by comparing to two standard compounds: Nob and 5-HPMF. TPA was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Antibodies of p-p38 MAPK, p38 MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, Ki-67, proliferating cell nuclear antigen (PCNA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Abcam (Cambridge, U.K.). Mouse IL-1β enzyme-linked immunosorbent assay (ELISA) kit and mouse IL-17 ELISA kit were purchased from Bio-Swamp (Wuhan, Hubei, China). Anti-mouse CD4 fluorescein isothiocyanate (FITC), anti-mouse CD25 PE CY5, anti-mouse FOXP3 PE, and anti-mouse IL-17 PE were purchased from eBioscience (San Jose, CA, U.S.A.). PE anti-mouse IL-4 and APC anti-mouse interferon-γ (IFN-γ) were purchased from BioLegend (San Diego, CA, U.S.A.). Animals and Experimental Design. A total of 40 female BALB/c mice, aged 4−6 weeks, were obtained from the Experimental Animal Center of China, Three Gorges University [Yichang, Hubei, China; certification SCXK (E) 2011-0012]. The mice were given free access to food and water. All animal operating procedures proceeded according to the Guidelines for the Care and Use of Laboratory Animals, Ministry of Science and Technology of China. The animal experimental protocols used in this study were authorized by the Animal Care and Scientific Committee of the Huanggang Normal University, China. After 7 days of adaption to the laboratory conditions, the mice were randomly divided into four groups (6 mice/group): control group, TPA group, Nob-treated group, and 5-HPMF-treated group. Both ears of the mice were topically treated with 15 μL of acetone (vehicle; control group), 0.8 nmol of TPA in acetone (TPA group), 2 μM of Nob (Nob-treated group), or 5-HPMF (5-HPMF-treated group) in acetone. The treatment was conducted twice a day for 4 days, with the first time at 10 min before TPA application and the second time at 1 h after TPA. The mice were sacrificed 4 h after the last TPA treatment. Histological and Immunohistochemistry Analyses. To observe the pathological symptoms of psoriatic-like lesion skin tissue, the paraffin sections of the lesion skin tissues were stained with

Figure 3. Nob and 5-HPMF reduced the expression of cytokines associated with psoriasis in ear skin. Levels of transcripts of (A) IL-17 and (B) IL-1β were detected by ELISAs, and (C) TNF-α, IFN-γ, IL-4, and IL-6 were detected by quantitative RT-PCR. Values were presented as the mean ± SD (n = 6). The significant difference among each group was analyzed by one-way ANOVA and Duncan’s multiple range tests. Different letters (a−c) represent significant differences of the same indexes of the different groups when p < 0.01. hematoxylin and eosin (H&E, Sigma-Aldrich). The lesion tissue specimens were fixed in 10% (v/v) neutral formalin for 24 h, embedded in paraffin, and then sliced into 5 μm of thick tissue sections. Then, H&E staining was carried out according to standard protocols. Upon the staining, pathological changes were observed at 200× magnification under a BH2 optical microscope (Olympus, Hino, Tokyo, Japan). Paraffinembedded slices were dewaxed and rehydrated. After incubation with anti-Ki-67 antibody and anti-PCNA antibody overnight at 4 °C according to the guidelines of the manufacturers, the staining results were observed under the optical microscope. The severity of psoriatic lesion skin tissue was scored according to the method of Baker et al.,14 where a score of 1.5−2 represents the severity of the formation of Munro’s small abscess in the cuticle, 0.5 indicates the hyperkeratosis in the pathological tissue, 1.0 represents the parakeratosis in the pathological tissue, 0.5−1.5 indicates the severity of the epidermis rod-like downward extending, 0.5 represents the epidermis mastoid upward extending, and 0.5 represents mastoid capillary dilatation. The greater the total score of each index, the higher the lesion degree of the ear skin. Detection of Cytokines. The blood and skin tissue of lesion ears were immediately collected upon mouse sacrifice, and the serum was C

DOI: 10.1021/acs.jafc.8b02524 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 4. Nob and 5-HPMF reduced the serum levels of (A) SCr, (B) BUN, (C) ALT, (D) AST, and (E) TB. Values were presented as the mean ± SD (n = 6). The significant difference among each group was analyzed by one-way ANOVA and Duncan’s multiple range tests. Different letters (a−c) represent significant differences of the same indices of the different groups when p < 0.01. obtained after centrifugation at 1500 rpm for 10 min for serum cytokine detection. The levels of pro-inflammatory cytokines (IL-1β and IL-17) were evaluated using ELISA (Bio-Swamp, Wuhan, Hubei, China) according to the instructions. The optical density (OD) value of each well at a wavelength of 450 nm was measured immediately using a microtiter plate reader (LabSystems, Helsinki, Finland). Quantitative Real-Time Polymerase Chain Reaction (RT-PCR). Total RNA was purified from lesion ears using TRIzol reagent (Ambion, Austin, TX, U.S.A.) and then transcribed to cDNA using an Advantage RT-for-PCR kit (Clontech, Palo Alto, CA, U.S.A.) according to the protocol of the manufacturer. Quantitative RT-PCR was performed in the Bio-Rad RT-PCR system (Bio-Rad, Hercules, CA, U.S.A.) using SYBR Green Master Mix (Kapa Biosystems, Wilmington, MA, U.S.A.) with primers listed as follows: IL-4, F, 5′-CTGCTCTTCTTTCTCG-3′ and R, 5′-GTGGTGTTCTTCGTTG-3′; IL-6, F, 5′-ACGGCCTTCCCTACTT-3′ and R, 5′-TTCCACGATTTCCCAG-3′; TNF-α, F, 5′-TCACCCACACCGTCAG-3′ and R, 5′-GAGTTGGTCCCCCTTC-3′; IFN-γ, F, 5′-GAACGCTACACACTG-3′ and R, 5′-CTTTTCTTCCACATC3′; and β-actin, F, 5′-CCCATCTACGAGGGCTAT-3′ and R, 5′-TGTCACGCACGATTTCC-3′. The mRNA expression level in each sample was normalized against β-actin, and the expression level was calculated using the delta delta threshold cycle (2−ΔΔCt) method. Determination of Serum Creatinine (SCr), Blood Urea Nitrogen (BUN), Alanine Aminotransferase (ALT), Aspartate Transaminase (AST), and Total Bilirubin (TB) Levels in Serurm. Blood samples obtained from mice naturally coagulated for 20 min after collection

and then centrifuged at 1000 rpm for 10 min. The supernatant was collected and stored at −20 °C until use. The levels of SCr, BUN, ALT, AST, and TB were determined using the detection kit according to the instructions of the manufacturer. Above-mentioned SCr, BUN, ALT, AST, and TB were from Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China. Absorbance was measured on a MK3 microplate reader (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Protein Extraction and Western Blotting. The relative expression value of p-p38 MAPK, p38 MAPK, p-ERK1/2, and ERK1/2 was detected using GAPDH as the internal control. The tissue of lesion ears separated from each group was homogenized in iced radioimmunoprecipitation assay (RIPA) buffer containing 0.1% phenylmethylsulfonyl fluoride (Bio-Swamp). The bicinchoninic acid (BCA) assay (Bio-Swamp) was employed for protein quantification. Protein extracts were loaded with a 15% sodium dodecyl sulfate (SDS)− polyacrylamide gel for electrophoresis and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, U.S.A.). The PVDF membranes were blocked with 5% skim milk in Tris buffer saline and then incubated at 4 °C overnight with respective primary antibodies. After washing with Tris-buffered saline with Tween 20 (TBST) 3 times, the PVDF membranes were incubated with a horseradish-peroxidase-conjugated secondary antibody (1:10 000) at 4 °C for 12 h. The visualized antibody-reactive bands were examined using enhanced chemiluminescence (ECL) and quantified with the Image-Pro Plus software (Media Cybernetics, Rockville, MD, U.S.A.). D

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Journal of Agricultural and Food Chemistry Flow Antibody Labeling and Detection. Spleen tissues of each experimental mouse were fragmented into small pieces and extracted via mechanical disruption. A single cell suspension was obtained using 200-mesh screens for filtration. The operational processes of dyeing, breaking, and fixing were carried out according to the operation description from the kit. Final detection was performed on a Beckman Coulter FC500 cytometer (Beckman Coulter, Brea, CA, U.S.A.), and analysis was with FC500 flow cytometry analysis software CXP. Statistical Analysis. Data statistics and analysis were performed by running the one-way analysis of variance (ANOVA) or one-way Student’s t test and Duncan’s multiple range test. Data were presented as the mean ± standard deviation (SD) for the indicated numbers of independently performed experiments. A probability value of p < 0.05 or p < 0.01 was considered to be statistically significant.

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the changes in TPA-stimulated inflammation. These results revealed that Nob and 5-HPMF were effective in preventing TPA-induced psoriasis. Nob and 5-HPMF Corrected Abnormal SCr, BUN, ALT, AST, and TB in TPA-Induced Mice. Concentrations of SCr (Figure 4A), BUN (Figure 4B), ALT (Figure 4C), AST (Figure 4D), and TB (Figure 4E) in mouse serum were measured with an automatic biochemical analyzer. There is an apparent pattern that the values of serum SCr, BUN, ALT, AST, and TB in the TPA-treated group of mice were significantly higher than that of the control group. However, treatment with Nob or 5-HPMF significantly lowered the serum levels of SCr, BUN, ALT, AST, and TB (panels A−E of Figure 4). These experimental results suggested that Nob and 5-HPMF prevented damage to the liver and kidney. Effects of Nob and 5-HPMF on the Expression of MAPK-Mediated Inflammatory Signaling in Ear Skin Lesions. To investigate the preventive effect of Nob and 5HPMF on the degree of inflammation in the pathological tissues of TPA-induced psoriasis-like mice, we extracted protein from the ear lesions of mice in each group and detected the relative expression levels of p-ERK1/2, ERK1/2, p-p38 MAPK, and p38 MAPK by the western blotting method (Figure 5). In comparison

RESULTS

Histological Assessment. The histopathological H&E staining in Figure 2A indicated that the ear lesions in the TPA group showed hyperkeratosis and Munro’s microabscess in some keratinized areas. The granular layer became thinner or even disappeared. The thickness of the spinous layer increased. Trochanterellus lengthened and widened. Dermal papilla extended upward, and capillary proliferation and congestion emerged. A large number of lymphocytic infiltrates arose surrounding the blood vessels. In comparison to the TPA group, the ear lesion tissues of Nob and 5-HPMF topically applied to mice ears showed mild hyperkeratosis. The spinous layer was much thinner but was still thicker than that of the control group. Telangiectasia and perivascular lymphocytic infiltration were alleviated. Histopathological scores of the lesion tissues revealed that Nob and 5-HPMF could significantly inhibit the symptoms of psoriasis induced by TPA. The pathological score (Figure 2B) of the 5-HPMF treatment group was 1.93, whereas the Nob-treated group had a value of 2.15. Hence, 5-HPMF showed stronger activity in prevention of TPA-induced psoriasis-like skin lesions compared to Nob. Effects of Nob and 5-HPMF on Expression of Ki-67 and PCNA in Ear Lesion Tissue. Immunohistochemistry results (panels C−F of Figure 2) showed that there were low levels of expression of Ki-67 (Figure 2C) and PCNA (Figure 2D) in the basal layer and the spinous layer of the control group. After TPA induction, these two proteins showed strong positive expression in the basal, acanthosis, and granular layers of the skin lesions (Figure 2F). However, after Nob or 5-HPMF treatment, expression of these two proteins was mildly positive or close to that of normal skin in the control group and was significantly different from that of the TPA group (panels C−F of Figure 2). Effects of Nob and 5-HPMF on the Expression of Proinflammatory Cytokines. Serum pro-inflammatory cytokine levels of IL-17 (Figure 3A) and IL-1β (Figure 3B) were determined by ELISA. The results in Figure 3 demonstrated that the serum pro-inflammatory cytokine levels of IL-1β and IL-17 in the TPA group increased significantly compared to the control group. Levels of these two serum pro-inflammatory cytokines in Nob- and 5-HPMF-treated groups were significantly reduced (panels A and B of Figure 3). The effects of Nob and 5-HPMF on expression levels of mRNA associated with inflammation in mouse skin were detected (Figure 3C). The results showed that TPA application increased the transcription of TNF-α, IL-6, and IFN-γ but IL-4 was not significantly affected (Figure 3C), indicating that Nob and 5-HPMF have no weakening effect of the antipsoriatic function of IL-4, while, at the same time, counteracting

Figure 5. Nob and 5-HPMF inhibited the expression of MAPK. Protein expression levels were determined relative to actin in three independent experiments. Data were expressed as the mean (n = 6) and statistically analyzed using a one-way ANOVA and Duncan’s multiple range test. The significant difference among each group was analyzed by one-way ANOVA and Duncan’s multiple range tests. Different letters (a−c) represent significant differences of the same indices of the different groups when p < 0.01.

to the control group, the relative expression values of p-ERK1/2 and p-p38 MAPK in the TPA group were 5.3, 4.8, and 5.7, respectively. The expression levels of ERK1/2 and p38 MAPK were basically the same in each group. In the Nob- and 5-HPMF-treated groups, the expression values of the phosphorylated proteins, p-ERK1/2 and p-p38 MAPK, were 2.7, 2.9, and E

DOI: 10.1021/acs.jafc.8b02524 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 6. Nob and 5-HPMF regulated the differentiation of CD4+ T cells. The differentiation of CD4+ T cells in spleen tissues was detected by flow cytometry in three dependent experiments: (A) Th1, (B) Th17, (C) Th2, and (D) Treg. The significant difference among each group was analyzed by one-way ANOVA and Duncan’s multiple range tests. Different letters (a−c) represent significant differences of the same indices of the different groups when p < 0.01.

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DISCUSSION Pathological changes in psoriasis are mainly epidermal keratinocyte hyperproliferation, abnormal differentiation, dermal papillary capillary proliferation, and inflammatory cell infiltration.15−17 In this study, we found that the expression levels of p-ERK1/2 and p-p38 in skin lesions of the TPA group were significantly enhanced. Subsequently, under the effect of p-ERK1/2 and p-p38, the differentiation level of Th1 cells was significantly increased, whereas the differentiation degree of Th2 and Treg cells was significantly reduced. In comparison to the TPA-treated group, the expression levels of p-ERK and p-p38 in the lesion tissue of Nob- and 5-HPMF-treated mice were significantly reduced, the differentiation degree of Th1 cells was decreased, and Th2 and Treg cell differentiation significantly increased. Hence, Nob and 5-HPMF can regulate the differentiation of CD4+ T cells by regulating the activity of the MAPK signaling pathways to prevent the formation of TPA-induced psoriasis-like lesions. MAPK is a protein kinase that can be activated by stimulation of various mitogens and plays a critical role in the regulation of cell proliferation and

2.3 for the Nob treatment and 2.4, 2.7, and 1.2 for the 5-HPMF treatment, respectively. The significant reduction indicated that Nob and 5-HPMF reduced inflammation by inhibiting ERK1/2 and p38 phosphorylation. Immunoregulatory Effects of Nob and 5-HPMF on CD4+ T Cells in the TPA-Induced Psoriasis-Like Mouse Model. To investigate the effects of Nob and 5-HPMF on the differentiation of Th1, Th2, Th17, and Treg cells in the spleen of TPA-treated mice, the expressions of intracellular cytokines IL-4, IFN-γ, and IL-17 as well as the cell surface of CD4 receptor proteins were determined by flow cytometric analysis. Results in Figure 6 showed that TPA treatment increased CD4+ IFN-γ+ (Th1; Figure 6A) and IL17+ CD4+ (Th17; Figure 6B) cells but diminished numbers of CD4+ IL4+ (Th2; Figure 6C) and CD4+ CD25+ FoxP3+ (Treg; Figure 6D) cells significantly on day 4. Surprisingly, the proportion of Th2 and Tregs dramatically increased, and the proportion of Th2 cells was reduced by Nob and 5-HPMF treatment. This finding indicates that Nob and 5-HPMF have immunomodulatory effects on the differentiation of CD4+ T cells. F

DOI: 10.1021/acs.jafc.8b02524 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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(201615803) and the National Natural Science Foundation of China (31571832).

differentiation. MAPK has three major family members, i.e., ERK1/2, c-Jun N-terminal kinase (JNK), and p38 MAPK. Under normal conditions, ERK1/2 and p38 are present in an inactive form. Both ERK1/2 and p38 can be activated by phosphorylation. Phosphorylated ERK1/2 can promote the differentiation of Treg cells,18 and the activated p38 MAPK will induce Th1 cell differentiation, leading to a disproportional ratio of Th1/Th2.19 In the current study, we found that TPA-induced expression of Ki-67 and PCNA in the ear lesions of the psoriatic-like mouse was significantly higher than that of normal mice in the control group but Nob and 5-HPMF effectively inhibited the expression of Ki-67 and PCNA through inhibiting the activation of MAPK signaling pathways and reducing abnormal cell proliferation and parakeratosis in the TPA-induced lesion tissue. The transcription factors Ki-67 and PCNA were involved in cell proliferation and differentiation. The expression of p-p38 MAPK positively correlated with the expression level of Ki-67, indicating that p-p38 MAPK could regulate Ki-67 expression.20 At the same time, PCNA expression was also affected by the MAPK signaling pathways.21−23 In psoriatic lesions, the overexpression of Ki-67 and PCNA promoted the sustained proliferation of keratinocytes, resulting in abnormal proliferation and parakeratosis of keratinocytes in the non-proliferative stratum spinosum and granular layers.24,25 We discovered that the effective inhibition of the expression of Ki-67 and PCNA by Nob and 5-HPMF was through inhibiting the activation of MAPK signaling pathways and reducing abnormal cell proliferation and parakeratosis in the TPA-induced lesions. In this study, we also found that 5-HPMF possessed a stronger inhibitory activity in TPA-induced psoriasis-like skin lesions than Nob, although it was not statistically significant in this study, whereas it has been reported that 5-HPMF has a much better anti-inflammatory property than Nob.26 Nob has six methoxy groups, while 5-HPMF has a hydroxyl group at position 5 and five methoxy groups. Both the position and number of methoxyl groups in the flavonoid skeleton have influence on the biological property of polymethoxyflavones, such as Nob and 5-HPMF. The hydroxyl group on the flavonoid ring, particularly at position 5, was found to have higher activity than the parent permethoxylated flavones.26 In summary, Nob and 5-HPMF inhibited the activation of the MAPK signaling pathways and reduced the synthesis of inflammatory factors, which affected the differentiation of CD4+ T cells and the expression of transcription factors, namely, Ki-67 and PCNA, thereby reducing the biological effects of psoriatic skin lesions. In addition, we also found that the hydroxyl group at position 5 of 5-HPMF is likely the reason for better activity of polymethoxyflavones in prevention of psoriasislike skin lesions.

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Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ALT, alanine aminotransferase;; AST, aspartate transaminase; BCA, bicinchoninic acid assay; BUN, blood urea nitrogen; ECL, enhanced chemiluminescence; ELISA, enzyme-linked immunosorbent assay; ERK1/2, extracellular signal-regulated kinase 1/2; H&E, hematoxylin and eosin; IFN-γ, interferon-γ; IL, interleukin; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; Nob, nobiletin; OD, optical density; PCNA, proliferating cell nuclear antigen; PVDF, polyvinylidene difluoride; RT-PCR, real-time polymerase chain reaction; SCr, serum creatinine; TB, total bilirubin; TBST, Tris-buffered saline with Tween 20; Th17, T helper cell 17; TNF-α, tumor necrosis factor-α; TPA, 12-O-tetradecanoylphorbol 13-acetate; 5-HPMF, 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone; ΔΔCt, delta delta threshold cycle

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: 01-973-919-3702. Fax: 01-732-932-6776. E-mail: [email protected]. *Fax: 886-4-23592705. E-mail: [email protected]. ORCID

Shiming Li: 0000-0002-6167-0660 Hui Zhao: 0000-0002-7517-5054 Funding

This study received support from the High Level Scientific Research Cultivation Project of Huanggang Normal University G

DOI: 10.1021/acs.jafc.8b02524 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.jafc.8b02524 J. Agric. Food Chem. XXXX, XXX, XXX−XXX