Polyphenols from Lonicera caerulea L. Berry Inhibit LPS-Induced

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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 J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01599 • Publication Date (Web): 02 Jun 2017 Downloaded from http://pubs.acs.org on June 9, 2017

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Journal of Agricultural and Food Chemistry

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Title

Polyphenols from Lonicera caerulea L. Berry Inhibit LPS-induced Inflammation

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through Dual Modulation of Inflammatory and Antioxidant Mediators Running Title

Dual Modulation of Inflammatory and Antioxidant Mediators by Lonicera

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caerulea L. Berry Polyphenols

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Shusong Wua, Satoshi Yanob, Jihua Chenc, Ayami Hisanagab, Kozue Sakaob,d,

Authors

Xi Hea, Jianhua Hea, De-Xing Houa,b,d*

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Affiliations:

a

Core Research Program 1515, Hunan Collaborative Innovation Center for

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Utilization of Botanical Functional Ingredients, Hunan Agricultural University,

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Changsha, Hunan 410128, China b

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The United Graduate School of Agricultural Sciences, Kagoshima University,

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Korimoto 1-21-24, Kagoshima 890-0065, Japan c

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Department of Nutrition Science and Food Hygiene, XiangYa School of Public

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Health, Central South University, Changsha, Hunan 410078, China d

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Department of Food Science and Biotechnology, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan

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

Corresponding author: Professor De-Xing Hou, Department of Food Science and

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Biotechnology, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima

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890-0065, Japan

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E-mail: [email protected] (DX. Hou). Tel/Fax: +81-99-285-8649.

ACS Paragon Plus Environment


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Abstract

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Lonicera caerulea L. berry polyphenols (LCBP) are considered as major components for the

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bioactivities. This study aimed to clarify the molecular mechanisms by monitoring inflammatory

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and antioxidant mediator actions in lipopolysaccharide (LPS)-induced mouse paw edema and

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macrophage cell model. LCBP significantly attenuated LPS-induced paw edema (3.0 ± 0.1 to 2.8

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± 0.1 mm, P < 0.05) and reduced (P < 0.05) serum levels of monocytes chemotactic protein-1

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(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

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pg/ml), macrophage inflammatory protein (MIP)-1α (1761.9 ±208.3 to 1369.1 ±56.4 pg/ml), IL-6

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(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

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±132.0 to 315.2 ±35.1 pg/ml), tumor necrosis factor-α (TNF-α, 2045.5 ±264.9 to 1270.7 ±158.6

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pg/ml). Cell signaling analysis revealed that LCBP inhibited transforming growth factor β

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activated kinase-1 (TAK1)-mediated mitogen-activated protein kinase (MAPK) and nuclear

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factor-κB (NF-κB) pathways, and enhanced the expression of nuclear factor (erythroid-derived

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2)-like 2 (Nrf2) and manganese-dependent superoxide dismutase (MnSOD) in earlier response.

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Moreover, cyanidin 3-glucoside (C3G) and (-)epicatechin (EC), two major components of LCBP,

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directly bound to TAK1. These data demonstrated that LCBP might inhibit LPS-induced

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inflammation by modulating both inflammatory and antioxidant mediators.

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Keywords: Lonicera caerulea L.; Polyphenol; Anti-inflammatory; Antioxidant; Cytokines

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1. Introduction

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Lonicera caerulea L., commonly known as blue honeysuckle or haskap, is a member of the

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Caprifoliaceae family growing natively in Hokkaido of Japan, Siberia of Russia, and northern

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China. Its berry has been reported to possess multiple bioactivities such as anti-microbial1,

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antioxidant2, and anti-inflammatory3. Lonicera caerulea L. berry polyphenols (LCBP) are

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considered to be the major components on these bioactivities. Our recent studies revealed that

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LCBP could inhibit adjuvant-induced arthritis4 and high fat dietary/stress-induced nonalcoholic

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steatohepatitis5 with enhanced expression of antioxidant mediators and decreased expression of

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inflammatory mediators. Thus, we speculated that LCBP possibly inhibit the inflammation

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through dual modulation of inflammatory and antioxidant mediators although the mechanisms that

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how and what kind of mediators LCBP modulate remain unclear.

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Although inflammatory processes are complicated, cytokines such as interleukins,

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chemokines, interferons, and tumor necrosis factors secreted by immune cells are critical

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mediators for inflammatory response6. The release of inflammatory cytokines into circulation can

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induce the recruitment of immune cells such as macrophages to the inflamed tissue to provoke

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inflammation, and further aggravate systemic inflammation7. Among cellular signaling network in

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the inflammatory process, mitogen-activated protein kinase (MAPK)8 and nuclear factor-κB

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(NF-κB)9 cascades are considered as the major pathways that promote the production of

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inflammatory cytokines. Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase

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(JNK), and p38 kinase are the major MAPKs that response to a variety of stimuli in mammalian

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cells8, 10. NF-κB is bound by IκBs in the cytoplasm, and can be translocated into the nucleus to

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function as a transcription factor after the proteolytic degradation of IκBs11. The degradation of



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IκBs is triggered by their phosphorylations12, which are mediated by IκB kinases (IKK)13, 14.

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Transforming growth factor β activated kinase-1 (TAK1) is an ubiquitin-dependent kinase of IKK,

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and acts as an upstream regulator of both MAPK and NF-κB pathways15, 16. Lipopolysaccharide

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(LPS) is the endotoxin produced by all gram-negative bacteria that can induce inflammation by

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activating immune cells to produce inflammatory cytokines17, and macrophages are considered as

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the primary cells leading to the production of a variety of inflammatory mediators18.

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On the other hand, antioxidant mediators play a crucial role in cellular defense system that

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counteracts the oxidative stress from pro-inflammatory response such as LPS stimulation19.

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Manganese-dependent superoxide dismutase (MnSOD) is one of the most important antioxidant

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enzymes to reduce mitochondrial oxidative stress20, and has been proven as the most sensitive

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antioxidant enzyme response to LPS-induced inflammation21. Nuclear factor (erythroid-derived

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2)-like 2 (Nrf2) has been identified as a major transcription factor to attenuate LPS-induced

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inflammatory response by regulating the expression of down-stream antioxidant enzymes19, 22.

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Therefore, both inflammatory and antioxidant mediators play critical roles in the initiation

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and progression of inflammation, and have been suggested as potential therapeutic targets of

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inflammatory diseases23. To clarify the regulatory effects of LCBP on inflammatory and

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antioxidant mediators, we used both animal and cultural cell models in the present study. First we

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globally investigated the effects of LCBP on 23 kinds of cytokines in LPS-induced mouse paw

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edema model, and then identified the regulatory effects of LCBP on the cellular antioxidant and

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inflammatory mediators in LPS-activated macrophage cell model. Furthermore, we identified the

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potential target molecule for LCBP bioactive components using ex vivo pull-down assay. Our data

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demonstrated that LCBP inhibited LPS-induced inflammation through dual modulation of


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inflammatory and antioxidant mediators by targeting TAK1, a key kinase located on the

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inflammatory pathway.

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Materials and methods

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2.1 Chemicals and reagents

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LPS (Escherichia coli Serotype 055:B5) was purchased from Sigma-Aldrich (Tokyo, Japan).

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Cyanidin 3-glucoside (C3G, ≥98%) and -(-)epicatechin (EC, ≥98%) from Tokiwa Phytochemical

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Co., Ltd (Chiba, Japan) were dissolved in DMSO (0.2% final concentration in cultural medium).

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CNBr-activated Sepharose 4B was from GE Healthcare (Uppsala, Sweden). Antibodies against

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c-Jun (Ser73), ERK1/2, p38 kinase, JNK1/2, p65, IκB-α, IKKα/β, and TAK1 were purchased from

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Cell Signaling Technology (Beverly, MA, USA). Antibodies against inducible nitric oxide

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synthases (iNOS), MnSOD, Nrf2, 70-kDa heat shock protein (HSP70), α-tubulin (B-7), lamin B,

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and corresponding secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA,

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USA). LCBP was extracted as described in our previous study4. In brief, Lonicera caerulea L.

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berries harvested in Jilin region of China were homogenized in 75% aqueous ethanol (250 g/l) for

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60 min, and then filtered under reduced pressure. The filtrates were applied on a column packed

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with nonionic polystyrene-divinylbenzene resin (D101, Shanghai, China). The column was first

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washed with deionized water, and then eluted by 75% ethanol. The eluates containing phenolic

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compounds were evaporated at 30 ℃ until no alcohol remained, and then freeze-dried into powder

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at -20 ℃. The phenolic fraction obtained was analyzed by HPLC at 280 and 520 nm. According to

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the peak areas in the retention profiles identified by standard samples, C3G (59.5%) and EC

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(25.5%) were identified as the major phenolic components at 280 nm while other minor



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anthocyanins including peonidin 3-glucoside (7.2%), pelargonidin 3-glucoside (2.3%), peonidin

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3-rutinoside (1.9%), cyanidin 3-rutinoside (1.8%), and cyanidin 3,5-diglucoside (1.3%) were also

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detected at 520 nm. The quantitative analysis indicated that each mg of LCBP contains 0.37 mg

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C3G and 0.23 mg EC.

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2.2 Mouse paw edema model

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The animal experimental protocol was drafted according to the guidelines of the Animal Care

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and Use Committee of Kagoshima University (Permission N0. A12005). Male ICR mice (4 weeks

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of age, Japan SLC Inc.) were group-housed under controlled light (12 h light/day) and temperature

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(25℃), and free access to water and feed. After acclimatization for one week, the mice were

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randomly divided into three groups (n=4): control, LPS, and LPS + LCBP. The mice in LPS +

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LCBP group were orally administrated (p.o.) with 300 mg/kg BW of LCBP suspended in 0.1 ml

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normal saline based on our pilot test, while other mice were administrated with 0.1 ml normal

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saline only. After four days of pretreatment, paw edema was induced with LPS (1mg/kg BW) by

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subcutaneous injection (s.c.) as described previously24. Mouse paw edema was determined by

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measuring paw thickness with a digital caliper (model 19975, Shinwa Rules Co. Ltd, Japan)

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before and every hour after LPS injection until 6 h.

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2.3 Cytokine determination by multiplex technology in mouse serum

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Serum levels of cytokines including interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6,

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IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17, eotaxin, granulocyte colony-stimulating factor

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(G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, keratinocyte-


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derived cytokine (KC), monocytes chemotactic protein-1 (MCP-1), macrophage inflammatory

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protein (MIP)-1α, MIP-1β, RANTES, and tumor necrosis factor-α (TNF-α) in mouse sera were

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measured with a Bio-Plex Pro Mouse Cytokine 23-Plex Panel kit (Bio-Rad Laboratories). The

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assay was performed with a Bio-Plex machine (Bio-Plex 200 System, Bio-Rad, USA) according

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to the manufacturer’s instruction, and the data were analyzed with the Bio-Plex manager software.

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2.4 Cell culture and cytokine determination in culture medium

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Mouse macrophage-like RAW264.7 cell line (Cell No. RCB0535, RIKEN Bio-Resource

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Center Cell Bank, Tsukuba, Japan) was cultured in DMEM containing 10% FBS at 37 °C in a 5%

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CO2 atmosphere. The cells were pre-cultured for 24 h and starved in serum-free media for 2.5 h to

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eliminate the influence of FBS before treatment. To measure cytokines in culture media, the cells

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were seeded into 6-well plates (5×105/well), and pretreated with LCBP for 30 min before exposure

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to LPS for 12 h. The levels of IL-1β, IL-6, and TNF-α were measured by their respective ELISA

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kits (Thermo Fisher Scientific Inc., IL, USA) according to the manufacturer’s manual.

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2.5 Cell fractionation

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To determine the expression of cell mediators of inflammation, RAW264.7 cells (1×106)

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were pre-cultured in 6-cm dishes, and then treated with 75, 150, or 300 μg/ml of LCBP for 30 min

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before exposure to LPS (40 ng/ml) for indicated times. Whole cell lysates were obtained by

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ultrasonication in RIPA buffer. Nuclear protein extracts were prepared by a modified method

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based on previous studies10, 25. In brief, the harvested cells were lysed on ice in buffer A [1 mM

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dithiothreitol, 0.1 mM EDTA, 10 mM Hepes-KOH (pH 7.9), 10 mM KCl, 0.5% Nonidet P-40, and



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0.5 mM phenylmethylsulfonyl fluoride] for 30 min. After being centrifuged at 13500 g (4 °C, 15

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min), the nuclear pellets were washed with buffer A for three times and resuspended in buffer B [1

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mM dithiothreitol, 1 mM EDTA, 20 mM Hepes (pH 7.9), 0.5 M KCl, and 1 mM

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phenylmethylsulfonyl fluoride] on a rotating wheel for 30 min at 4 °C. The supernatants contain

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nuclear proteins were obtained by centrifugation (13500 g, 4 °C, 15 min) and then analyzed by

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Western blot assay.

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2.6 Ex vivo pull-down assay

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Ex vivo pull-down assay was performed as described in previous study24. Briefly, EC or C3G

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(5 μM) were coupled to CNBr-activated Sepharose 4B beads (25 mg) overnight at 4 °C in a

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coupling buffer [25% DMSO, 0.5 M NaCl, and 0.1 M NaHCO3 (pH 8.3)] according to the

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manufacturer’s manual. After centrifugation (1000g, 4 °C, 3 min), the beads were washed with

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coupling buffer (5 volumes) and then resuspended in 0.1 M Tris-HCl buffer (pH 8.0, 5 volumes)

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for 2 h rotation. The conjugated beads were then washed with acetate buffer [0.1 M acetic acid

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(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

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cycles. The RAW264.7 cell lysates (500 μg/mL) were then incubated with the EC or

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C3G-conjugated beads (100 μL, 50% slurry), or control beads in a reaction buffer [2 μg/mL BSA,

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1 mM dithiothreitol, 5 mM EDTA, 150 mM NaCl, 0.01% Nonidet P-40, 0.02 mM PMSF, 50 mM

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Tris-HCl (pH 8.5), and 1 μg protease inhibitor cocktail] overnight at 4 °C. The bound lysates were

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washed with the buffer containing 1 mM dithiothreitol, 5 mM EDTA, 200 mM NaCl, 0.02%

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Nonidet P-40, 0.02 mM PMSF, and 50 mM Tris-HCl (pH 7.5) for five times, and the proteins

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bound to the beads were analyzed by Western blot assay.


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2.7 Western blot assay

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Western blotting was performed as described previously10. Briefly, obtained protein extracts

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were quantitated by protein assay and then boiled in SDS sample buffer for 5 min, and equal

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amounts of protein (40 μg) were run on 10% SDS-PAGE followed by transfer to PVDF membrane.

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After blocking, the membrane was incubated with specific primary antibody (4 °C, overnight) and

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corresponding HRP-conjugated secondary antibody (room temperature, 1 h). The bound

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antibodies were then detected by the ECL system with a Lumi Vision PRO machine (TAITEC Co.,

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Saitama, Japan), and the relative amount of proteins was quantified by Lumi Vision Imager

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software (TAITEC Co., Saitama, Japan).

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2.8 Statistical analysis

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Results are expressed as means ±SD. Significant differences were determined using one-way

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ANOVA followed by Duncan’s Multiple Range test (SPSS19, IBM Corp., Armonk, NY, USA).

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Protein expression was expressed as induction folds to that of control by densitometry. A

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probability of P < 0.05 was considered significant.

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3

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3.1 The inhibitory effect of LCBP on LPS-induced paw edema

Results

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Mouse paw edema experimental scheme was shown in Figure 1A, and the change in paw

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thickness was shown in Figure 1B. The initial paw thicknesses of mice in each group were not

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different, but injection of LPS (1 mg/kg BW) increased average paw thickness (n=4) from 2.7 to



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3.3 mm at 1 h, and the paw swelling remained till the end (paw thickness=3.0 mm) in 6 h. Oral

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administration of LCBP (300 mg/kg BW daily) for 4 days inhibited LPS-induced paw edema

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significantly (P < 0.05). Particularly, the paw thickness was reduced from 3.3 to 3.1 mm at 1 h and

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2.8 mm at 6 h. In comparing with LPS treatment alone, LCBP decreased the edema by 36.9% at 1

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h, and 63.0% at 6 h, although the paw swelling was not completely disappeared within 6 h (Figure

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1B). As a control, normal saline showed no effects on paw edema.

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3.2 Modulation of serum cytokine levels by LCBP

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To understand the regulatory effects of LCBP on the production of inflammatory cytokines,

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we examined 23 kinds of cytokines in mouse serum simultaneously by multiplex assay as

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described in Section 2. The results showed that injection of LPS induced more than fivefold

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increase in serum levels of G-CSF, RANTES, MCP-1, KC, IL-10, MIP-1α, Eotaxin, IL-6, MIP-1β,

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IL-1β, and IL-12(p40); more than twofold increase in the levels of IL-4, IL-9, IFN-γ, IL-1α, IL-13,

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IL-12(p70), IL-2, and TNF-α; but less than twofold in the levels of GM-CSF, IL-3, IL-5, and

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IL-17; comparing with that of mice without LPS injection (Figure 2). Oral administration of LCBP

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at 300 mg/kg body weight significantly decreased (P < 0.05) the levels of RANTES, MCP-1, KC,

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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

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effect (P > 0.05) on the levels of G-CSF, Eotaxin, MIP-1β, IL-12(p40), IL-9, IFN-γ, IL-1α, IL-13,

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GM-CSF, IL-5, and IL-17. These results suggested that LCBP attenuated LPS-induced mouse paw

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edema by reducing the production of multiple inflammatory cytokines.

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3.3 Modulation of the MAPK pathway by LCBP


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To further understand the regulatory effects of LCBP on cellular inflammatory pathways, we

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next used a LPS-activated microphage (RAW264.7) cell model, which can mimic a situation of

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infection. The production of representative IL-1β, IL-6, and TNF-α was first measured in the

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culture media to confirm the anti-inflammatory effect of LCBP in the model. As shown in Figure

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3A, the levels of IL-1β, IL-6, and TNF-α were significantly increased (P < 0.05) by LPS

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stimulation, and concentration-dependently reduced by pretreatment with 75-300 μg/ml of LCBP.

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Moreover, the expression of iNOS, another marker of LPS-induced inflammation, was also

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decreased by pretreatment with LCBP (75-300 μg/ml) in the same manner (Figure 3B).

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Accumulated data indicate that LPS can induce the activation of MAPKs including JNK,

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ERK and p38 kinase, and subsequently activates transcription factors such as AP-1 to promote the

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production of cytokines10. To investigate the cellular signaling pathways involved in the inhibition

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of inflammatory mediators by LCBP, we first checked the phosphorylation of c-Jun, which is a

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major component of AP-1 in c-Jun/c-Fos heterodimer form. The results showed that pretreatment

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with 75-300 μg/ml of LCBP concentration-dependently inhibited LPS-induced c-jun

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phosphorylation in RAW264.7 cells (Figure 4A). Correspondingly, pretreatment with LCBP also

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suppressed the phosphorylation of JNK1/2, ERK1/2 and p38 kinase in the same manner (Figure

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4B). These results suggested that down-regulation of MAPK pathways was involved in the

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inhibition of inflammatory cytokines production by LCBP.

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3.4 Modulation of the NF-κB pathway by LCBP

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Since NF-κB is one of the major transcription factors to mediate the production of

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inflammatory cytokines26, we next investigated the effects of LCBP on the translocation of NF-κB.



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The results showed that LPS induced a fourfold increase in nuclear NF-κB p65, and pretreatment

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with LCBP (300 μg/ml) reduced the nuclear translocation of p65 to 1.4 fold (Figure 5A). We

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further confirmed that the phosphorylation of p65, IκB-α were also suppressed by pretreatment

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with 75-300 μg/ml of LCBP in a concentration-dependent manner (Figure 5B). Correspondingly,

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the total protein of IκB-α was decreased by LPS, and restored by LCBP. Moreover, the activation

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of IKKα/β, an upstream kinase, was also suppressed by LCBP. Thus, LCBP potentially reduced

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the production of inflammatory cytokines also by down-regulating NF-κB pathway.

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3.5 Modulation of cellular antioxidant mediators by LCBP

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Recent studies have shown that antioxidant mediators also play critical roles in counteracting

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inflammatory response19. Thus, we investigated the effects of LPS and LCBP on the expression of

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Nrf2 and MnSOD, two important antioxidant mediators, with a time-course experiment in

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RAW264.7 cells. Our results showed that LPS induced more than threefold expression of Nrf2 and

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MnSOD after 12 and 24h treatment (Figure 6A), and LCBP induced more than threefold

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expression of MnSOD and Nrf2 from 6-24h (Figure 6B). However, the oxidative stress-response

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proteins such as HSP70 and iNOS were induced only by LPS, not by LCBP. These results

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suggested that LCBP might activate antioxidant response pathways early to counteract the

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oxidative stress induced by LPS.

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The major phenolic components of LCBP were identified as C3G and EC in our previous

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study4. To know whether C3G and EC are the major biological modulators of LCBP, the

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regulatory effects of C3G and EC on the inflammatory and antioxidant mediators were

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investigated in LPS-activated RAW264.7 cells. As shown in Figure 7A, both EC (69 μg/ml) and


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C3G (111 μg/ml) enhanced the expression of MnSOD and Nrf2, but decreased LPS-induced

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expression of HSP70 and iNOS. Especially, the combination of EC and C3G, which is equal to the

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amount in LCBP, showed similar effect as LCBP. Thus, C3G and EC of LCBP might be the major

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modulators for these actions.

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Since TAK1 is a key regulator of both MAPK and NF-κB pathways, we speculated that C3G

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and EC of LCBP may target TAK1 to modulate the inflammatory mediators. Thus, we first

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examined whether these components attenuate the TAK1 activation. As shown in Figure 7B, the

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combination of C3G and EC effectively suppressed LPS-induced phosphorylation of TAK1 as

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well as LCBP. Next, we examined whether C3G and EC directly bind to TAK1 to suppress TAK1

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activation, using ex vivo pull-down assay as described in Section 2. As shown in Figure 7C, TAK1

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was detected in the beads coupled with EC (51.9% binding rate) or C3G (38.6% binding rate), but

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not in the beads alone (Figure 7C). These data demonstrated that C3G and EC might directly bind

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to TAK1 to attenuate the activation.

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4

Discussion The present study demonstrated that LCBP inhibited LPS-induced inflammation by dually

modulating inflammatory and antioxidant mediators.

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Although inflammatory and antioxidant mediators are complicated, cytokines and

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Nrf2-activated antioxidant mediators are critical mediators in inflammatory response19, 27. In this

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study, we first used mouse paw edema model induced by LPS, which is a strong inducer for

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cytokine production and significantly increased serum levels of 21 kinds of cytokines except IL-5

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and IL-17. Oral administration of LCBP significantly reduced serum levels of 12 kinds of which



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including RANTES, MCP-1, KC, IL-10, MIP-1α, IL-6, IL-1β, IL-4, IL-12(p-70), IL-2, TNF-α,

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and IL-3. Among these cytokines, IL-10 and IL-4 are considered as anti-inflammatory cytokines

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against excessive inflammation and self-immunity28. IL-6, IL-1β, IL-12(p-70), IL-2, and TNF-α

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are the initially modulated cytokines, since RANTES can be regulated by TNF-α29, MCP-1 is one

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of the downstream targets of IL-630, and IL-1β can induce the expression of KC, MIP-1α, and

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MIP-1β31, 32. On the other hand, LPS also can activate oxidative stress response pathways in

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macrophages19. For example, LPS can activate NADPH oxidase through TLR4 leading to the

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excessive production of superoxide radicals (O2·-) and subsequent hydrogen peroxide (H2O2)33.

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These excessive products cause NF-κB-mediated inflammatory response34, and simultaneously

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activate Nrf2-mediated oxidative stress response pathway to counteract the reactive oxygen

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species35. In the present study, increased levels of both antioxidant response proteins (Nrf2 and

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MnSOD) and oxidative stress markers (HSP70 and iNOS) were observed in LPS-activated

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RAW264.7 cells. LCBP also increased the expression of Nrf2 and MnSOD, but not HSP70 and

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iNOS. The time-course experiments revealed that the induction time of Nrf2 and MnSOD

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expression by LCBP was from 6 h, which was earlier than from 12 h by LPS. These data

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suggested that LCBP might activate antioxidant response pathways early to counteract the

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oxidative stress induced by LPS. Several lines of studies have demonstrated the expression level

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of Nrf2 is correlated with inflammatory pathways, in which, knockdown of Nrf2 promoted the

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expression of NF-κB-mediated inflammatory pathways34, 36. Thus, our data suggested that LCBP

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primarily activated Nrf2-mediated oxidative stress response pathways to counteract LPS-induced

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reactive oxygen species, and further prevent against LPS-induced inflammation.

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In this study, we found that LCBP inhibited TLR4-mediated inflammation by down-


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regulating both MAPK and NF-κB pathways. Thus, we speculated that TAK1 might be a target

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molecule for LCBP-inhibited inflammatory signaling since TAK1 is a common upstream kinase

311

that activated by TLR4. The ex vivo binding data revealed that C3G and EC, two major active

312

compounds of LCBP, could directly bind to TAK1, and inhibit its phosphorylation. These data

313

suggested that TAK1 might be the primary target for suppressing inflammatory mediator

314

activation by LCBP. On the other hand, we previously identified MEK1 as a binding target to

315

delphinidin or its glycoside Dp-3-sam37, analogues of cyanidin or C3G. Thus cannot exclude the

316

possibility that C3G and/or EC may target other cellular kinases to exert their biological activity,

317

which is valuable to be investigated in further work.

318

The effective concentrations of C3G and EC in cell experiments in this study were over 50

319

μM, which are similar to the effective concentrations of other polyphenols such as other

320

anthocyanidins10 and tea proanthocyanidins38 as reported previously. Accumulated data have

321

shown that the effective concentrations of polyphenols in culture cells are, in general, quite higher

322

than those measured in animal tissue or plasma. Although it is still hard to fill the gap between the

323

two, some lines of studies have indicated that cells cultured under laboratory conditions of 95%

324

air/5% CO2 are in a state of hyperoxia, experiencing about 150 mmHg of O2, whereas most cells

325

in the human body are exposed to O2 concentrations in the range of 1-10 mmHg 39. Polyphenols in

326

higher O2 concentration tend to be broken down 40. Another reason is that most polyphenols can

327

bind to bovine serum albumin (BSA) in cultural medium and form polyphenols-BSA complex41,

328

which reduced free polyphenols amount for action. Therefore, the effective concentrations of C3G

329

and EC to reach the targeting molecules might be quite lower than that at addition in cultural cells.

330

These facts can partially explain why most polyphenols show the effects in culture cells with



Page 16 of 44

331

higher concentrations than in animal serum, and suggest that the concentration of polyphenols in

332

cell culture could not be simply considered as the concentration in animal serum or tissues.

333

Most animal and human studies have found that anthocyanins present in natural food source

334

were absorbed mainly in their intact glycosidic form42, 43 from the stomach after ingestion by a

335

process that may involve bilitranslocase44, while EC could be absorbed as its original form45.

336

Therefore, the major active compounds, C3G and EC might exert the anti-inflammatory function

337

partially as the structures at absorption. It is also reported that C3G could be degraded into

338

phenolic metabolites such as protocatechuic acid, phloroglucinaldehyde, and ferulic acid46, which

339

may act as bioactive molecules. The biological activities of these metabolites after absorption will

340

be investigated in our coming work.

341

The phenolic components of LCBP were analyzed by HPLC in our previous study, and the

342

major components were identified as C3G and EC4. Other studies also suggested that C3G is the

343

major anthocyanin in Lonicera caerulea L. berry 1, 47-49, while the concentration of EC is diverse

344

in different cultivars1, 48, 50. Although other minor anthocyanins including peonidin 3-glucoside,

345

pelargonidin

346

3,5-diglucoside were also contained in LCBP according to HPLC analysis, peonidin and

347

pelargonidin showed limited effects on LPS-induced inflammation in our previous study

348

Cyanidin 3-rutinoside and cyanidin 3,5-diglucoside might have potential bioactive effects against

349

inflammatory factors and oxidative stress

350

effective concentration. Thus, C3G and EC are considered as the major biological modulators of

351

LCBP for antioxidant and inflammatory activities.

352

3-glucoside,

peonidin

3-rutinoside,

cyanidin

3-rutinoside,

and

cyanidin

10

.

51, 52

, but their contents in LCBP were far below the

Taken together, LCBP, rich in C3G and EC, inhibited LPS-induced inflammation through the


Page 17 of 44


353

dual modulation of inflammatory and antioxidant mediators, reducing the production of multiple

354

inflammatory cytokines and enhancing the expression of Nrf2 and MnSOD. These findings

355

provided a comprehensive understanding of the anti-inflammatory property of LCBP at the

356

molecular level.

357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374



375

Abbreviations used: C3G, cyanidin 3-glucoside; EC, -(-)epicatechin; ERK, extracellular

376

signal-regulated kinase; HSP70, 70-kDa heat shock protein; IKK, IκB kinases; IL, interleukin;

377

iNOS, inducible nitric oxide synthases; JNK, c-Jun N-terminal kinase; KC, keratinocyte-derived

378

cytokine; LPS, lipopolysaccharide; LCBP, Lonicera caerulea L. berry polyphenols; MAPK,

379

mitogen-activated protein kinase; MCP-1, monocytes chemotactic protein-1; MIP, macrophage

380

inflammatory protein; MnSOD, manganese-dependent superoxide dismutase; NF-κB, nuclear

381

factor-κB; Nrf2, nuclear factor (erythroid-derived 2)-like 2; TAK1, transforming growth factor β

382

activated kinase-1; TNF-α, tumor necrosis factor-α

383 384

Conflict of interest

385

The authors have declared no conflict of interest.

386 387

Funding sources

388

This work was partially supported by the funds from Core Research Program 1515 and Hunan

389

Collaborative Innovation Center for Utilization of Botanical Functional Ingredients of Hunan

390

Agricultural University, China, and by the funds from the Scholar Research of Kagoshima

391

University, Japan (D.X. Hou).

392 393 394 395 396


Page 18 of 44

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397

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46. Amin, H. P.; Czank, C.; Raheem, S.; Zhang, Q.; Botting, N. P.; Cassidy, A.; Kay, C. D.,

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Anthocyanins and their physiologically relevant metabolites alter the expression of IL-6 and

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VCAM-1 in CD40L and oxidized LDL challenged vascular endothelial cells. Mol. Nutr. Food Res.

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47. Chaovanalikit, A.; Thompson, M. M.; Wrolstad, R. E., Characterization and quantification of


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anthocyanins and polyphenolics in bluehHoneysuckle (Lonicera caerulea L.). J. Agric. Food Chem.

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2004, 52, 848-52.

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48. Oszmiański, J.; Wojdyło, A.; Lachowicz, S., Effect of dried powder preparation process on

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polyphenolic content and antioxidant activity of blue honeysuckle berries (Lonicera caerulea L.

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var. kamtschatica). LWT - Food Science and Technology 2016, 67, 214-222.

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49. Kusznierewicz, B.; Piekarska, A.; Mrugalska, B.; Konieczka, P.; Namiesnik, J.; Bartoszek, A.,

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Phenolic composition and antioxidant properties of Polish blue-berried honeysuckle genotypes by

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HPLC-DAD-MS, HPLC postcolumn derivatization with ABTS or FC, and TLC with DPPH

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visualization. J. Agric. Food Chem. 2012, 60, 1755-63.

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50. Rupasinghe, H. P.; Boehm, M. M.; Sekhon-Loodu, S.; Parmar, I.; Bors, B.; Jamieson, A. R.,

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Anti-Inflammatory Activity of Haskap Cultivars is Polyphenols-Dependent. Biomolecules 2015, 5,

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51. Youdim, K. A.; Martin, A.; Joseph, J. A., Incorporation of the elderberry anthocyanins by

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52. Chen, P. N.; Chu, S. C.; Chiou, H. L.; Kuo, W. H.; Chiang, C. L.; Hsieh, Y. S., Mulberry

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anthocyanins, cyanidin 3-rutinoside and cyanidin 3-glucoside, exhibited an inhibitory effect on the

546

migration and invasion of a human lung cancer cell line. Cancer Lett. 2006, 235, 248-59.

547 548 549 550



551

Figure captions

552 553

Figure 1

554

experimental procedure of mouse paw edema model. ICR mice were randomly divided into three

555

groups (n=4): control (CTL), LPS, and LPS+LCBP. Mice were orally administrated (p.o.) with

556

LCBP (300 mg/kg BW daily) for 4 days, and LPS (1mg/kg) was then injected subcutaneously (s.c.)

557

into mouse paw. Paw thickness was measured before and every hour after LPS injection until 6 h.

558

(B) The change in paw thickness. The data represent mean ± SD, and values with different

559

superscript letters differ significantly (P < 0.05), the superscript b represents significant difference

560

with CTL group (a), while c represents significant difference with LPS group (b).

The inhibitory effect of LCBP on LPS-induced mouse paw edema. (A) The

561 562

Figure 2

563

obtained from the mice 6 h after LPS injection. Serum levels of 23 kinds of cytokines were

564

measured by multiplex technology, and arranged in an order from high to low change in

565

LPS-induced mice. The data represent mean ± SD, and values with different superscript letters

566

differ significantly (P < 0.05), the superscript b represents significant difference with CTL group

567

(a), while c represents significant difference with LPS group (b).

Modulation of serum cytokine levels by LCBP. The blood sera of mice were

568 569

Figure 3 The inhibitory effect of LCBP on the production of IL-1β, IL-6, TNF-α, and iNOS

570

in LPS-activated RAW264.7cells. The cells were treated with 75, 150, or 300 μg/ml of LCBP for

571

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

572

culture media. The data represent mean ± SD of six repeats, and values with different superscript


Page 26 of 44

Page 27 of 44


573

letters differ significantly (P < 0.05), the superscript b represents significant difference with

574

normal cells (a), while c or d represents significant difference with LPS-activated cells (b). (B)

575

The total protein of iNOS in whole cell lysates by Western blotting. The induction fold of iNOS

576

protein was calculated as the intensity of the treatment relative to that of control normalized to

577

-tubulin by densitometry. The blots shown are the examples of three independent experiments.

578 579

Figure 4

580

150, or 300 μg/ml of LCBP for 30 min and then exposed to LPS (40 ng/ml) for another 30 min. (A)

581

The total and phosphorylated proteins of c-Jun, and (B) MAPKs including JNK1/2, ERK1/2, and

582

p38 kinase in whole cell lysates. The induction folds of the phosphorylated proteins were

583

calculated as the intensity of the treatment relative to that of control normalized to total proteins

584

by densitometry. The blots shown are the examples of three independent experiments.

Modulation of the MAPK pathway by LCBP. RAW264.7 cells were treated with 75,

585 586

Figure 5 Modulation of the NF-κB pathway by LCBP. Cell treatment was same as in Figure 4.

587

(A) The total protein of p65 in nuclear fraction. (B) The total and phosphorylated proteins of p65,

588

IκB-α, and IKKα/β in whole cell lysates. The induction folds were calculated as the intensity of

589

the treatment relative to that of control normalized to Lamin B (for nuclear p65), α-tubulin (for

590

IκB-α and p-IκB-α), or respective total proteins (for p-p65 and p-IKKα/β) by densitometry. The

591

blots shown are the examples of three independent experiments.

592 593

Figure 6

594

HSP70, and iNOS. RAW264.7 cells were treated with 40 ng/ml of LPS (A) or 300 μg/ml of

Time-course experiments of LPS and LCBP-induced expression of Nrf2, MnSOD,



595

LCBP (B) for 0.5-24 h. The total proteins of Nrf2, MnSOD, HSP70, and iNOS in whole cell

596

lysates were detected by Western blotting. Induction folds of the proteins were calculated as the

597

intensity of the treatment relative to that of control normalized to α-tubulin by densitometry. The

598

blots shown are the examples of three independent experiments.

599 600

Figure 7 Modulation of inflammatory and antioxidant mediators by LCBP, EC and C3G.

601

(A) The total proteins of Nrf2, MnSOD, HSP70, and iNOS in whole cell lysates. RAW264.7 cells

602

were treated with EC (69 μg/ml), C3G (111 μg/ml) or LCBP (300 μg/ml) for 30 min, and then

603

exposed to LPS (40 ng/ml) for 12 h. (B) The phosphorylated protein of TAK1 in whole cell lysates.

604

Cells were treated with EC, C3G or LCBP for 30 min, and then exposed to LPS for another 30

605

min. (C) The binding abilities of EC and C3G to TAK1 protein. The ex vivo pull-down assay was

606

performed as described in Section 2.6. The binding efficiency of EC or C3G to TAK1 protein was

607

presented as the ratio of input control (lane 1). The blots shown are the examples of three

608

independent experiments.

609 610 611 612 613 614 615 616


Page 28 of 44

Page 29 of 44

617


Figure 1

618 619 620 621 622 623 624 625 626 627



628

Figure 2

629


Page 30 of 44

Page 31 of 44

630


Figure 3

631 632



633

Figure 4

634 635 636 637 638 639


Page 32 of 44

Page 33 of 44

640


Figure 5

641 642 643 644



645

Figure 6

646 647 648 649


Page 34 of 44

Page 35 of 44

650


Figure 7

651 652 653 654 655 656



657

Table of Contents Graphic

658

659


Page 36 of 44

Page 37 of 44


218x201mm (300 x 300 DPI)



285x484mm (300 x 300 DPI)


Page 38 of 44

Page 39 of 44


177x293mm (300 x 300 DPI)



178x218mm (300 x 300 DPI)


Page 40 of 44

Page 41 of 44


176x242mm (300 x 300 DPI)



179x243mm (300 x 300 DPI)


Page 42 of 44

Page 43 of 44


221x273mm (300 x 300 DPI)



249x153mm (96 x 96 DPI)


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Polyphenols from Lonicera caerulea L. Berry Inhibit LPS-Induced

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