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Polyphenols from Lonicera caerulea L. Berry Inhibit LPS-Induced Inflammation through Dual Modulation of Inflammatory and Antioxidant Mediators Shusong Wu,† Satoshi Yano,‡ Jihua Chen,§ Ayami Hisanaga,‡ Kozue Sakao,‡,∥ Xi He,† Jianhua He,† and De-Xing Hou*,†,‡,∥

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Core Research Program 1515, Hunan Collaborative Innovation Center for Utilization of Botanical Functional Ingredients, Hunan Agricultural University, Changsha, Hunan 410128, China ‡ The United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan § Department of Nutrition Science and Food Hygiene, XiangYa School of Public Health, Central South University, Changsha, Hunan 410078, China ∥ Department of Food Science and Biotechnology, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan ABSTRACT: Lonicera caerulea L. berry polyphenols (LCBP) are considered as major components for bioactivity. This study aimed to clarify the molecular mechanisms by monitoring inflammatory and antioxidant mediator actions in lipopolysaccharide (LPS)-induced mouse paw edema and macrophage cell model. LCBP significantly attenuated LPS-induced paw edema (3.0 ± 0.1 to 2.8 ± 0.1 mm, P < 0.05) and reduced (P < 0.05) serum levels of monocyte chemotactic protein-1 (MCP-1, 100.9 ± 2.3 to 58.3 ± 14.5 ng/mL), interleukin (IL)-10 (1596.1 ± 424.3 to 709.7 ± 65.7 pg/mL), macrophage inflammatory protein (MIP)-1α (1761.9 ± 208.3 to 1369.1 ± 56.4 pg/mL), IL-6 (1262.8 ± 71.7 to 499.0 ± 67.1 pg/mL), IL-4 (93.3 ± 25.7 to 50.7 ± 12.5 pg/ mL), IL-12(p-70) (580.4 ± 132.0 to 315.2 ± 35.1 pg/mL), and tumor necrosis factor-α (TNF-α, 2045.5 ± 264.9 to 1270.7 ± 158.6 pg/mL). Cell signaling analysis revealed that LCBP inhibited transforming growth factor β activated kinase-1 (TAK1)mediated mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways, and enhanced the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and manganese-dependent superoxide dismutase (MnSOD) in earlier response. Moreover, cyanidin 3-glucoside (C3G) and (−)-epicatechin (EC), two major components of LCBP, directly bound to TAK1. These data demonstrated that LCBP might inhibit LPS-induced inflammation by modulating both inflammatory and antioxidant mediators. KEYWORDS: Lonicera caerulea L., polyphenol, anti-inflammatory, antioxidant, cytokines

1. INTRODUCTION Lonicera caerulea L., commonly known as blue honeysuckle or haskap, is a member of the Caprifoliaceae family growing natively in Hokkaido of Japan, Siberia of Russia, and northern China. Its berry has been reported to possess multiple bioactivities such as antimicrobial,1 antioxidant,2 and anti-inflammatory.3 Lonicera caerulea L. berry polyphenols (LCBP) are considered to be the major components for these bioactivities. Our recent studies revealed that LCBP could inhibit adjuvant-induced arthritis4 and high fat dietary/stress-induced nonalcoholic steatohepatitis5 with enhanced expression of antioxidant mediators and decreased expression of inflammatory mediators. Thus, we speculated that LCBP possibly inhibit the inflammation through dual modulation of inflammatory and antioxidant mediators although the mechanisms that show how and what kind of mediators LCBP modulate remain unclear. Although inflammatory processes are complicated, cytokines such as interleukins, chemokines, interferons, and tumor necrosis factors secreted by immune cells are critical mediators for inflammatory responses.6 The release of inflammatory cytokines into circulation can induce the recruitment of immune cells such as macrophages to the inflamed tissue to provoke inflammation, and further aggravate systemic inflammation.7 Among the © 2017 American Chemical Society

cellular signaling network in the inflammatory process, mitogen-activated protein kinase (MAPK)8 and nuclear factorκB (NF-κB)9 cascades are considered as the major pathways that promote the production of inflammatory cytokines. Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase are the major MAPKs that response to a variety of stimuli in mammalian cells.8,10 NF-κB is bound by IκBs in the cytoplasm and can be translocated into the nucleus to function as a transcription factor after the proteolytic degradation of IκBs.11 The degradation of IκBs is triggered by their phosphorylations,12 which are mediated by IκB kinases (IKK).13,14 Transforming growth factor β activated kinase-1 (TAK1) is a ubiquitindependent kinase of IKK and acts as an upstream regulator of both MAPK and NF-κB pathways.15,16 Lipopolysaccharide (LPS) is the endotoxin produced by all Gram-negative bacteria that can induce inflammation by activating immune cells to produce inflammatory cytokines,17 and macrophages are Received: Revised: Accepted: Published: 5133

April 6, 2017 June 1, 2017 June 2, 2017 June 2, 2017 DOI: 10.1021/acs.jafc.7b01599 J. Agric. Food Chem. 2017, 65, 5133−5141

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Journal of Agricultural and Food Chemistry

randomly divided into three groups (n = 4): control, LPS, and LPS + LCBP. The mice in the LPS + LCBP group were orally administrated (po) with 300 mg/kg BW of LCBP suspended in 0.1 mL of normal saline based on our pilot test, while other mice were administrated with 0.1 mL of normal saline only. After 4 days of pretreatment, paw edema was induced with LPS (1 mg/kg BW) by subcutaneous injection (sc) as described previously.24 Mouse paw edema was determined by measuring paw thickness with a digital caliper (model 19975, Shinwa Rules Co. Ltd., Japan) before and every hour after LPS injection until 6 h. 2.3. Cytokine Determination by Multiplex Technology in Mouse Serum. Serum levels of cytokines including interleukin (IL)1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL12(p70), IL-13, IL-17, eotaxin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GMCSF), IFN-γ, keratinocyte- derived cytokine (KC), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, RANTES, and tumor necrosis factor-α (TNF-α) in mouse sera were measured with a Bio-Plex Pro Mouse Cytokine 23-Plex Panel kit (Bio-Rad Laboratories). The assay was performed with a Bio-Plex machine (Bio-Plex 200 System, Bio-Rad, USA) according to the manufacturer’s instruction, and the data were analyzed with the Bio-Plex manager software. 2.4. Cell Culture and Cytokine Determination in Culture Medium. Mouse macrophage-like RAW264.7 cell line (Cell No. RCB0535, RIKEN Bio-Resource Center Cell Bank, Tsukuba, Japan) was cultured in DMEM containing 10% FBS at 37 °C in a 5% CO2 atmosphere. The cells were precultured for 24 h and starved in serumfree media for 2.5 h to eliminate the influence of FBS before treatment. To measure cytokines in culture media, the cells were seeded into 6-well plates (5 × 105/well), and pretreated with LCBP for 30 min before exposure to LPS for 12 h. The levels of IL-1β, IL-6, and TNF-α were measured by their respective ELISA kits (Thermo Fisher Scientific Inc., IL, USA) according to the manufacturer’s manual. 2.5. Cell Fractionation. To determine the expression of cell mediators of inflammation, RAW264.7 cells (1 × 106) were precultured in 6 cm dishes, and then treated with 75, 150, or 300 μg/mL LCBP for 30 min before exposure to LPS (40 ng/mL) for the indicated times. Whole cell lysates were obtained by ultrasonication in RIPA buffer. Nuclear protein extracts were prepared by a modified method based on previous studies.10,25 In brief, the harvested cells were lysed on ice in buffer A [1 mM dithiothreitol, 0.1 mM EDTA, 10 mM Hepes−KOH (pH 7.9), 10 mM KCl, 0.5% Nonidet P-40, and 0.5 mM phenylmethylsulfonyl fluoride] for 30 min. After being centrifuged at 13500g (4 °C, 15 min), the nuclear pellets were washed with buffer A three times and resuspended in buffer B [1 mM dithiothreitol, 1 mM EDTA, 20 mM Hepes (pH 7.9), 0.5 M KCl, and 1 mM phenylmethylsulfonyl fluoride] on a rotating wheel for 30 min at 4 °C. The supernatants containing nuclear proteins were obtained by centrifugation (13500g, 4 °C, 15 min) and then analyzed by Western blot assay. 2.6. Ex Vivo Pull-down Assay. Ex vivo pull-down assay was performed as described in a previous study.24 Briefly, EC or C3G (5 μM) was coupled to CNBr-activated Sepharose 4B beads (25 mg) overnight at 4 °C in a coupling buffer [25% DMSO, 0.5 M NaCl, and 0.1 M NaHCO3 (pH 8.3)] according to the manufacturer’s manual. After centrifugation (1000g, 4 °C, 3 min), the beads were washed with coupling buffer (5 volumes) and then resuspended in 0.1 M Tris-HCl buffer (pH 8.0, 5 volumes) for 2 h rotation. The conjugated beads were then washed with acetate buffer [0.1 M acetic acid (pH 4.0) and 0.5 M NaCl] and wash buffer [0.5 M NaCl and 0.1 M Tris-HCl (pH 8.0)] for three cycles. The RAW264.7 cell lysates (500 μg/mL) were then incubated with the EC or C3G-conjugated beads (100 μL, 50% slurry), or control beads in a reaction buffer [2 μg/mL BSA, 1 mM dithiothreitol, 5 mM EDTA, 150 mM NaCl, 0.01% Nonidet P-40, 0.02 mM PMSF, 50 mM Tris-HCl (pH 8.5), and 1 μg of protease inhibitor cocktail] overnight at 4 °C. The bound lysates were washed with the buffer containing 1 mM dithiothreitol, 5 mM EDTA, 200 mM NaCl, 0.02% Nonidet P-40, 0.02 mM PMSF, and 50 mM Tris-HCl (pH 7.5) five times, and the proteins bound to the beads were analyzed by Western blot assay.

considered as the primary cells leading to the production of a variety of inflammatory mediators.18 On the other hand, antioxidant mediators play a crucial role in cellular defense system that counteracts the oxidative stress from proinflammatory responses such as LPS stimulation.19 Manganese-dependent superoxide dismutase (MnSOD) is one of the most important antioxidant enzymes to reduce mitochondrial oxidative stress20 and has been proven to be the most sensitive antioxidant enzyme response to LPS-induced inflammation.21 Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has been identified as a major transcription factor to attenuate LPSinduced inflammatory response by regulating the expression of downstream antioxidant enzymes.19,22 Therefore, both inflammatory and antioxidant mediators play critical roles in the initiation and progression of inflammation and have been suggested as potential therapeutic targets of inflammatory diseases.23 To clarify the regulatory effects of LCBP on inflammatory and antioxidant mediators, we used both animal and cultural cell models in the present study. First we globally investigated the effects of LCBP on 23 kinds of cytokines in LPS-induced mouse paw edema model, and then we identified the regulatory effects of LCBP on the cellular antioxidant and inflammatory mediators in LPS-activated macrophage cell model. Furthermore, we identified the potential target molecule for LCBP bioactive components using ex vivo pull-down assay. Our data demonstrated that LCBP inhibited LPS-induced inflammation through dual modulation of inflammatory and antioxidant mediators by targeting TAK1, a key kinase located on the inflammatory pathway.

2. MATERIALS AND METHODS 2.1. Chemicals and Reagents. LPS (Escherichia coli Serotype 055:B5) was purchased from Sigma-Aldrich (Tokyo, Japan). Cyanidin 3-glucoside (C3G, ≥98%) and (−)-epicatechin (EC, ≥98%) from Tokiwa Phytochemical Co., Ltd. (Chiba, Japan) were dissolved in DMSO (0.2% final concentration in cultural medium). CNBr-activated Sepharose 4B was from GE Healthcare (Uppsala, Sweden). Antibodies against c-Jun (Ser73), ERK1/2, p38 kinase, JNK1/2, p65, IκB-α, IKKα/ β, and TAK1 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against inducible nitric oxide synthases (iNOS), MnSOD, Nrf2, 70-kDa heat shock protein (HSP70), α-tubulin (B-7), lamin B, and corresponding secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). LCBP was extracted as described in our previous study.4 In brief, Lonicera caerulea L. berries harvested in the Jilin region of China were homogenized in 75% aqueous ethanol (250 g/L) for 60 min, and then filtered under reduced pressure. The filtrates were applied on a column packed with nonionic polystyrene−divinylbenzene resin (D101, Shanghai, China). The column was first washed with deionized water and then eluted by 75% ethanol. The eluates containing phenolic compounds were evaporated at 30 °C until no alcohol remained and then freeze-dried into powder at −20 °C. The phenolic fraction obtained was analyzed by HPLC at 280 and 520 nm. According to the peak areas in the retention profiles identified by standard samples, C3G (59.5%) and EC (25.5%) were identified as the major phenolic components at 280 nm while other minor anthocyanins including peonidin 3-glucoside (7.2%), pelargonidin 3-glucoside (2.3%), peonidin 3-rutinoside (1.9%), cyanidin 3rutinoside (1.8%), and cyanidin 3,5-diglucoside (1.3%) were also detected at 520 nm. The quantitative analysis indicated that each mg of LCBP contains 0.37 mg of C3G and 0.23 mg of EC. 2.2. Mouse Paw Edema Model. The animal experimental protocol was drafted according to the guidelines of the Animal Care and Use Committee of Kagoshima University (Permission No. A12005). Male ICR mice (4 weeks of age, Japan SLC Inc.) were group-housed under controlled light (12 h light/day) and temperature (25 °C), and free access to water and feed. After acclimatization for 1 week, the mice were 5134

DOI: 10.1021/acs.jafc.7b01599 J. Agric. Food Chem. 2017, 65, 5133−5141

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Journal of Agricultural and Food Chemistry 2.7. Western Blot Assay. Western blotting was performed as described previously.10 Briefly, obtained protein extracts were quantitated by protein assay and then boiled in SDS sample buffer for 5 min, and equal amounts of protein (40 μg) were run on 10% SDS− PAGE followed by transfer to a PVDF membrane. After blocking, the membrane was incubated with specific primary antibody (4 °C, overnight) and corresponding HRP-conjugated secondary antibody (room temperature, 1 h). The bound antibodies were then detected by the ECL system with a Lumi Vision PRO machine (TAITEC Co., Saitama, Japan), and the relative amount of proteins was quantified by Lumi Vision Imager software (TAITEC Co., Saitama, Japan). 2.8. Statistical Analysis. Results are expressed as means ± SD. Significant differences were determined using one-way ANOVA followed by Duncan’s multiple range test (SPSS19, IBM Corp., Armonk, NY, USA). Protein expression was expressed as induction folds to that of control by densitometry. A probability of P < 0.05 was considered significant.

3.2. Modulation of Serum Cytokine Levels by LCBP. To understand the regulatory effects of LCBP on the production of inflammatory cytokines, we examined 23 kinds of cytokines in mouse serum simultaneously by multiplex assay as described in section 2. The results showed that injection of LPS induced more than 5-fold increase in serum levels of G-CSF, RANTES, MCP-1, KC, IL-10, MIP-1α, eotaxin, IL-6, MIP-1β, IL-1β, and IL12(p40); more than 2-fold increase in the levels of IL-4, IL-9, IFN-γ, IL-1α, IL-13, IL-12(p70), IL-2, and TNF-α; but less than 2-fold in the levels of GM-CSF, IL-3, IL-5, and IL-17; compared with that of mice without LPS injection (Figure 2). Oral administration of LCBP at 300 mg/kg body weight significantly decreased (P < 0.05) the levels of RANTES, MCP-1, KC, IL-10, MIP-1α, IL-6, IL-1β, IL-4, IL-12(p-70), IL-2, TNF-α, and IL-3 but showed no significant effect (P > 0.05) on the levels of GCSF, eotaxin, MIP-1β, IL-12(p40), IL-9, IFN-γ, IL-1α, IL-13, GM-CSF, IL-5, and IL-17. These results suggested that LCBP attenuated LPS-induced mouse paw edema by reducing the production of multiple inflammatory cytokines. 3.3. Modulation of the MAPK Pathway by LCBP. To further understand the regulatory effects of LCBP on cellular inflammatory pathways, we next used a LPS-activated microphage (RAW264.7) cell model, which can mimic a situation of infection. The production of representative IL-1β, IL-6, and TNF-α was first measured in the culture media to confirm the anti-inflammatory effect of LCBP in the model. As shown in Figure 3A, the levels of IL-1β, IL-6, and TNF-α were significantly increased (P < 0.05) by LPS stimulation, and concentrationdependently reduced by pretreatment with 75−300 μg/mL LCBP. Moreover, the expression of iNOS, another marker of LPS-induced inflammation, was also decreased by pretreatment with LCBP (75−300 μg/mL) in the same manner (Figure 3B). Accumulated data indicate that LPS can induce the activation of MAPKs including JNK, ERK, and p38 kinase, and subsequently activates transcription factors such as AP-1 to promote the production of cytokines.10 To investigate the cellular signaling pathways involved in the inhibition of inflammatory mediators by LCBP, we first checked the phosphorylation of c-Jun, which is a major component of AP-1 in the c-Jun/c-Fos heterodimer form. The results showed that pretreatment with 75−300 μg/mL LCBP concentrationdependently inhibited LPS-induced c-jun phosphorylation in RAW264.7 cells (Figure 4A). Correspondingly, pretreatment with LCBP also suppressed the phosphorylation of JNK1/2, ERK1/2, and p38 kinase in the same manner (Figure 4B). These results suggested that downregulation of MAPK pathways was involved in the inhibition of inflammatory cytokines production by LCBP. 3.4. Modulation of the NF-κB Pathway by LCBP. Since NF-κB is one of the major transcription factors to mediate the production of inflammatory cytokines,26 we next investigated the effects of LCBP on the translocation of NF-κB. The results showed that LPS induced a 4-fold increase in nuclear NF-κB p65, and pretreatment with LCBP (300 μg/mL) reduced the nuclear translocation of p65 to 1.4-fold (Figure 5A). We further confirmed that the phosphorylation of p65 and IκB-α was also suppressed by pretreatment with 75−300 μg/mL LCBP in a concentration-dependent manner (Figure 5B). Correspondingly, the total protein of IκB-α was decreased by LPS and restored by LCBP. Moreover, the activation of IKKα/β, an upstream kinase, was also suppressed by LCBP. Thus, LCBP potentially reduced the production of inflammatory cytokines also by downregulating NF-κB pathway.

3. RESULTS 3.1. The Inhibitory Effect of LCBP on LPS-Induced Paw Edema. The mouse paw edema experimental scheme is shown in Figure 1A, and the change in paw thickness is shown in Figure

Figure 1. The inhibitory effect of LCBP on LPS-induced mouse paw edema. (A) The experimental procedure of the mouse paw edema model. ICR mice were randomly divided into three groups (n = 4): control (CTL), LPS, and LPS + LCBP. Mice were orally administrated (po) with LCBP (300 mg/kg BW daily) for 4 days, and LPS (1 mg/kg) was then injected subcutaneously (sc) into the mouse paw. Paw thickness was measured before and every hour after LPS injection until 6 h. (B) The change in paw thickness. The data represent mean ± SD, and values labeled with different letters differ significantly (P < 0.05): the label b represents significant difference with the CTL group (a), while c represents significant difference with the LPS group (b).

1B. The initial paw thicknesses of mice in each group were not different, but injection of LPS (1 mg/kg BW) increased average paw thickness (n = 4) from 2.7 to 3.3 mm at 1 h, and the paw swelling remained until the end (paw thickness = 3.0 mm) in 6 h. Oral administration of LCBP (300 mg/kg BW daily) for 4 days inhibited LPS-induced paw edema significantly (P < 0.05). Particularly, the paw thickness was reduced from 3.3 to 3.1 mm at 1 h and 2.8 mm at 6 h. In comparing with LPS treatment alone, LCBP decreased the edema by 36.9% at 1 h, and 63.0% at 6 h, although the paw swelling was not completely disappeared within 6 h (Figure 1B). As a control, normal saline showed no effects on paw edema. 5135

DOI: 10.1021/acs.jafc.7b01599 J. Agric. Food Chem. 2017, 65, 5133−5141

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Journal of Agricultural and Food Chemistry

Figure 2. Modulation of serum cytokine levels by LCBP. The blood sera of mice were obtained from the mice 6 h after LPS injection. Serum levels of 23 kinds of cytokines were measured by multiplex technology, and arranged in an order from high to low change in LPS-induced mice. The data represent mean ± SD, and values labeled with different letters differ significantly (P < 0.05): the label b represents significant difference with the CTL group (a), while c represents significant difference with the LPS group (b).

3.5. Modulation of Cellular Antioxidant Mediators by LCBP. Recent studies have shown that antioxidant mediators also play critical roles in counteracting inflammatory response.19 Thus, we investigated the effects of LPS and LCBP on the

expression of Nrf2 and MnSOD, two important antioxidant mediators, with a time-course experiment in RAW264.7 cells. Our results showed that LPS induced more than 3-fold expression of Nrf2 and MnSOD after 12 and 24 h treatment 5136

DOI: 10.1021/acs.jafc.7b01599 J. Agric. Food Chem. 2017, 65, 5133−5141

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Journal of Agricultural and Food Chemistry

Figure 4. Modulation of the MAPK pathway by LCBP. RAW264.7 cells were treated with 75, 150, or 300 μg/mL LCBP for 30 min and then exposed to LPS (40 ng/mL) for another 30 min. (A) The total and phosphorylated proteins of c-Jun and (B) MAPKs including JNK1/2, ERK1/2, and p38 kinase in whole cell lysates. The induction folds of the phosphorylated proteins were calculated as the intensity of the treatment relative to that of control normalized to total proteins by densitometry. The blots shown are the examples of three independent experiments.

Figure 3. The inhibitory effect of LCBP on the production of IL-1β, IL6, TNF-α, and iNOS in LPS-activated RAW264.7cells. The cells were treated with 75, 150, or 300 μg/mL LCBP for 30 min and then exposed to LPS (40 ng/mL) for 12 h. (A) The levels of IL-1β, IL-6, and TNF-α in culture media. The data represent mean ± SD of six repeats, and values labeled with different letters differ significantly (P < 0.05): the label b represents significant difference with normal cells (a), while c or d represents significant difference with LPS-activated cells (b). (B) The total protein of iNOS in whole cell lysates by Western blotting. The induction fold of iNOS protein was calculated as the intensity of the treatment relative to that of control normalized to α-tubulin by densitometry. The blots shown are the examples of three independent experiments.

(Figure 6A), and LCBP induced more than 3-fold expression of MnSOD and Nrf2 from 6 to 24 h (Figure 6B). However, the oxidative stress-response proteins such as HSP70 and iNOS were induced only by LPS, not by LCBP. These results suggested that LCBP might activate antioxidant response pathways early to counteract the oxidative stress induced by LPS. The major phenolic components of LCBP were identified as C3G and EC in our previous study.4 To know whether C3G and EC are the major biological modulators of LCBP, the regulatory effects of C3G and EC on the inflammatory and antioxidant mediators were investigated in LPS-activated RAW264.7 cells. As shown in Figure 7A, both EC (69 μg/mL) and C3G (111 μg/ mL) enhanced the expression of MnSOD and Nrf2, but decreased LPS-induced expression of HSP70 and iNOS. Especially, the combination of EC and C3G, which is equal to the amount in LCBP, showed a similar effect as LCBP. Thus, C3G and EC of LCBP might be the major modulators for these actions.

Figure 5. Modulation of the NF-κB pathway by LCBP. Cell treatment was same as in Figure 4. (A) The total protein of p65 in nuclear fraction. (B) The total and phosphorylated proteins of p65, IκB-α, and IKKα/β in whole cell lysates. The induction folds were calculated as the intensity of the treatment relative to that of control normalized to lamin B (for nuclear p65), α-tubulin (for IκB-α and p-IκB-α), or respective total proteins (for p-p65 and p-IKKα/β) by densitometry. The blots shown are the examples of three independent experiments.

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Figure 6. Time-course experiments of LPS- and LCBP-induced expression of Nrf2, MnSOD, HSP70, and iNOS. RAW264.7 cells were treated with 40 ng/mL LPS (A) or 300 μg/mL LCBP (B) for 0.5− 24 h. The total proteins of Nrf2, MnSOD, HSP70, and iNOS in whole cell lysates were detected by Western blotting. Induction folds of the proteins were calculated as the intensity of the treatment relative to that of control normalized to α-tubulin by densitometry. The blots shown are the examples of three independent experiments.

Figure 7. Modulation of inflammatory and antioxidant mediators by LCBP, EC, and C3G. (A) The total proteins of Nrf2, MnSOD, HSP70, and iNOS in whole cell lysates. RAW264.7 cells were treated with EC (69 μg/mL), C3G (111 μg/mL), or LCBP (300 μg/mL) for 30 min, and then exposed to LPS (40 ng/mL) for 12 h. (B) The phosphorylated protein of TAK1 in whole cell lysates. Cells were treated with EC, C3G, or LCBP for 30 min, and then exposed to LPS for another 30 min. (C) The binding abilities of EC and C3G to TAK1 protein. The ex vivo pulldown assay was performed as described in section 2.6. The binding efficiency of EC or C3G to TAK1 protein was presented as the ratio of input control (lane 1). The blots shown are the examples of three independent experiments.

Since TAK1 is a key regulator of both MAPK and NF-κB pathways, we speculated that C3G and EC of LCBP may target TAK1 to modulate the inflammatory mediators. Thus, we first examined whether these components attenuate the TAK1 activation. As shown in Figure 7B, the combination of C3G and EC effectively suppressed LPS-induced phosphorylation of TAK1 as well as LCBP. Next, we examined whether C3G and EC directly bind to TAK1 to suppress TAK1 activation, using ex vivo pull-down assay as described in section 2. As shown in Figure 7C, TAK1 was detected in the beads coupled with EC (51.9% binding rate) or C3G (38.6% binding rate), but not in the beads alone (Figure 7C). These data demonstrated that C3G and EC might directly bind to TAK1 to attenuate the activation.

12(p-70), IL-2, and TNF-α are the initially modulated cytokines, since RANTES can be regulated by TNF-α,29 MCP-1 is one of the downstream targets of IL-6,30 and IL-1β can induce the expression of KC, MIP-1α, and MIP-1β.31,32 On the other hand, LPS also can activate oxidative stress response pathways in macrophages.19 For example, LPS can activate NADPH oxidase through TLR4 leading to the excessive production of superoxide radicals (O2•−) and subsequent hydrogen peroxide (H2O2).33 These excessive products cause NF-κB-mediated inflammatory response,34 and simultaneously activate Nrf2-mediated oxidative stress response pathway to counteract the reactive oxygen species.35 In the present study, increased levels of both antioxidant response proteins (Nrf2 and MnSOD) and oxidative stress markers (HSP70 and iNOS) were observed in LPSactivated RAW264.7 cells. LCBP also increased the expression of Nrf2 and MnSOD, but not HSP70 and iNOS. The time-course experiments revealed that the induction time of Nrf2 and MnSOD expression by LCBP was from 6 h, which was earlier than from 12 h by LPS. These data suggested that LCBP might activate antioxidant response pathways early to counteract the oxidative stress induced by LPS. Several lines of studies have demonstrated that the expression level of Nrf2 is correlated with

4. DISCUSSION The present study demonstrated that LCBP inhibited LPSinduced inflammation by dually modulating inflammatory and antioxidant mediators. Although inflammatory and antioxidant mediators are complicated, cytokines and Nrf2-activated antioxidant mediators are critical mediators in inflammatory responses.19,27 In this study, we first used the mouse paw edema model induced by LPS, which is a strong inducer for cytokine production and significantly increased serum levels of 21 kinds of cytokines except IL-5 and IL-17. Oral administration of LCBP significantly reduced serum levels of 12 kinds of cytokines including RANTES, MCP-1, KC, IL-10, MIP-1α, IL-6, IL-1β, IL-4, IL12(p-70), IL-2, TNF-α, and IL-3. Among these cytokines, IL-10 and IL-4 are considered as anti-inflammatory cytokines against excessive inflammation and self-immunity.28 IL-6, IL-1β, IL5138

DOI: 10.1021/acs.jafc.7b01599 J. Agric. Food Chem. 2017, 65, 5133−5141

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Journal of Agricultural and Food Chemistry

glucoside, pelargonidin 3-glucoside, peonidin 3-rutinoside, cyanidin 3-rutinoside, and cyanidin 3,5-diglucoside were also contained in LCBP according to HPLC analysis, peonidin and pelargonidin showed limited effects on LPS-induced inflammation in our previous study.10 Cyanidin 3-rutinoside and cyanidin 3,5-diglucoside might have potential bioactive effects against inflammatory factors and oxidative stress,51,52 but their contents in LCBP were far below the effective concentration. Thus, C3G and EC are considered as the major biological modulators of LCBP for antioxidant and inflammatory activities. Taken together, LCBP, rich in C3G and EC, inhibited LPSinduced inflammation through the dual modulation of inflammatory and antioxidant mediators, reducing the production of multiple inflammatory cytokines and enhancing the expression of Nrf2 and MnSOD. These findings provided a comprehensive understanding of the anti-inflammatory property of LCBP at the molecular level.

inflammatory pathways, in which, knockdown of Nrf2 promoted the expression of NF-κB-mediated inflammatory pathways.34,36 Thus, our data suggested that LCBP primarily activated Nrf2mediated oxidative stress response pathways to counteract LPSinduced reactive oxygen species, and further prevent against LPSinduced inflammation. In this study, we found that LCBP inhibited TLR4-mediated inflammation by downregulating both MAPK and NF-κB pathways. Thus, we speculated that TAK1 might be a target molecule for LCBP-inhibited inflammatory signaling since TAK1 is a common upstream kinase that activated by TLR4. The ex vivo binding data revealed that C3G and EC, two major active compounds of LCBP, could directly bind to TAK1, and inhibit its phosphorylation. These data suggested that TAK1 might be the primary target for suppressing inflammatory mediator activation by LCBP. On the other hand, we previously identified MEK1 as a binding target to delphinidin or its glycoside Dp-3-sam,37 analogues of cyanidin or C3G. Thus, we cannot exclude the possibility that C3G and/or EC may target other cellular kinases to exert their biological activity, which is valuable to be investigated in further work. The effective concentrations of C3G and EC in cell experiments in this study were over 50 μM, which are similar to the effective concentrations of other polyphenols such as other anthocyanidins10 and tea proanthocyanidins38 as reported previously. Accumulated data have shown that the effective concentrations of polyphenols in culture cells are, in general, quite higher than those measured in animal tissue or plasma. Although it is still hard to fill the gap between the two, some lines of studies have indicated that cells cultured under laboratory conditions of 95% air/5% CO2 are in a state of hyperoxia, experiencing about 150 mmHg of O2, whereas most cells in the human body are exposed to O2 concentrations in the range of 1− 10 mmHg.39 Polyphenols in higher O2 concentration tend to be broken down.40 Another reason is that most polyphenols can bind to bovine serum albumin (BSA) in cultural medium and form a polyphenol−BSA complex,41 which reduced the amount of free polyphenols for action. Therefore, the effective concentrations of C3G and EC to reach the targeting molecules might be quite lower than that at addition in cultural cells. These facts can partially explain why most polyphenols show the effects in culture cells with higher concentrations than in animal serum, and suggest that the concentration of polyphenols in cell culture could not be simply considered as the concentration in animal serum or tissues. Most animal and human studies have found that anthocyanins present in natural food source were absorbed mainly in their intact glycosidic form42,43 from the stomach after ingestion by a process that may involve bilitranslocase,44 while EC could be absorbed as its original form.45 Therefore, the major active compounds, C3G and EC, might exert the anti-inflammatory function partially as the structures at absorption. It is also reported that C3G could be degraded into phenolic metabolites such as protocatechuic acid, phloroglucinaldehyde, and ferulic acid,46 which may act as bioactive molecules. The biological activities of these metabolites after absorption will be investigated in our coming work. The phenolic components of LCBP were analyzed by HPLC in our previous study, and the major components were identified as C3G and EC.4 Other studies also suggested that C3G is the major anthocyanin in Lonicera caerulea L. berry,1,47−49 while the concentration of EC is diverse in different cultivars.1,48,50 Although other minor anthocyanins including peonidin 3-

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AUTHOR INFORMATION

Corresponding Author

*Department of Food Science and Biotechnology, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan. E-mail: [email protected]. Tel/fax: +81-99-285-8649. ORCID

De-Xing Hou: 0000-0002-0449-8331 Funding

This work was partially supported by the funds from Core Research Program 1515 and Hunan Collaborative Innovation Center for Utilization of Botanical Functional Ingredients of Hunan Agricultural University, China, and by the funds from the Scholar Research of Kagoshima University, Japan (D.-X.H.). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED C3G, cyanidin 3-glucoside; EC, (−)-epicatechin; ERK, extracellular signal-regulated kinase; HSP70, 70 kDa heat shock protein; IKK, IκB kinases; IL, interleukin; iNOS, inducible nitric oxide synthases; JNK, c-Jun N-terminal kinase; KC, keratinocytederived cytokine; LPS, lipopolysaccharide; LCBP, Lonicera caerulea L. berry polyphenols; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemotactic protein-1; MIP, macrophage inflammatory protein; MnSOD, manganese-dependent superoxide dismutase; NF-κB, nuclear factor-κB; Nrf2, nuclear factor (erythroid-derived 2)-like 2; TAK1, transforming growth factor β activated kinase-1; TNF-α, tumor necrosis factor-α

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REFERENCES

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