Oleuropein Curtails Pulmonary Inflammation and Tissue Destruction in

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Bioactive Constituents, Metabolites, and Functions

Oleuropein curtails pulmonary inflammation and tissue destruction in models of experimental asthma and emphysema Yun-Ho Kim, Yean-Jung Choi, Min-Kyung Kang, Eun-Jung Lee, Dong Yeon Kim, Hyeongjoo Oh, and Young-Hee Kang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01808 • Publication Date (Web): 27 Jun 2018 Downloaded from http://pubs.acs.org on June 27, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Oleuropein curtails pulmonary inflammation and tissue destruction in models of experimental asthma and emphysema Yun-Ho Kim†, Yean-Jung Choi‡, Min-Kyung Kang†, Eun-Jung Lee† Dong Yeon Kim†, Hyeongjoo Oh†, Young-Hee Kang†* †

Department of Food and Nutrition, Hallym University, Chuncheon, Korea; ‡Department of Bio-Food Science & Technology, Far East University, Eumseong, Korea

Yun-Ho Kim and Yean-Jung Choi contributed equally.

Running Title: Inhibition of pulmonary inflammation by oleuropein Key Words: Asthma, chronic obstructive pulmonary disease, cigarette smoke, oleuropein, ovalbumin inhalation, pulmonary inflammation Total words: 7659 Number of Figures: 8 Page number: 36

Funding sources: This work (grant No. C0501612) was supported by Business for Cooperative R&D between Industry, Academy, and Research Institute funded by Korea Small and Medium Business Administration in 2017.

Abbreviations used: BALF, bronchoalveolar lavage fluid; COPD, chronic obstructive pulmonary disease; CS, cigarette smoke; ICAM-1, intracellular adhesion molecule-1 IL-4, interleukin-4; LPS, lipopolysaccharide; OVA, ovalbumin; PAR-2, proteinase-activated receptor-2; ROS, reactive oxygen species Author Contributions: Y.-H. K. (Kim), Y.-J. C. and Y. -H. K. designed research; Y.-H. K. (Kim), M. K. K., E.-J. L., D. Y. K. and H. O. conducted research; Y.-H. K. (Kim) analyzed data; Y.-H. K. (Kim), Y.-J. C. and Y.-H. K. wrote the paper. Y.-H. K. had primary responsibility for final content. All authors read and approved the final manuscript.

*To whom correspondence should be addressed: Young-Hee Kang, Ph.D Department of Food and Nutrition Hallym University Chuncheon, Kangwon-do, 200-702 Korea Phone: 82-33-248-2132 Fax: 82-33-254-1475 Email: [email protected] 1


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1


ABSTRACT

2 3

Airway inflammation has been implicated in evoking progressive pulmonary disorders

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including chronic obstructive pulmonary disease (COPD) and asthma as a result of exposure to

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inhaled irritants, characterized by airway fibrosis, mucus hypersecretion and loss of alveolar

6

integrity. The current study examined whether oleuropein, a phenylethanoid found in olive leaves,

7

inhibited pulmonary inflammation in experimental models of interleukin (IL)-4-exposed bronchial

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BEAS-2B epithelial cells and ovalbumin (OVA)- or cigarette smoke (CS)-exposed BALB/c mice.

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Nontoxic oleuropein at 1-20 μM diminished eotaxin-1-mediated induction of α-smooth muscle actin

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and mucin 5AC in epithelial cells stimulated by IL-4 at the transcriptional levels. Oral

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supplementation of 10-20 mg/kg oleuropein reduced the airway influx of eosinophils and

12

lymphocytes as well as IL-4 secretion in lung promoted by OVA inhalation or CS. In addition,

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oleuropein suppressed infiltration of macrophages and neutrophils through blocking OVA

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inhalation- and CS-promoted induction of ICAM-1, F4/80, CD68 and CD11b in airways. In OVA-

15

exposed pulmonary fibrosis was detected, while alveolar emphysema was evident in CS-exposed

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mouse lungs. In alveolar epithelial A549 cells exposed to CS extracts, oleuropein attenuated

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apoptotic cell loss. Collectively, oleuropein inhibited pulmonary inflammation leading to asthmatic

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fibrosis and alveolar emphysema driven by influx of inflammatory cells in airways exposed OVA or

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CS. Therefore, oleuropein may be a promising anti-inflammatory agent for treating asthma and

20

COPD.

21

2



22

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INTRODUCTION

23 24

Proinflammatory responses of the airways have been implicated in evoking chronic

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obstructive pulmonary disease (COPD) and asthma.1-3 Airway narrowing, mucus hypersecretion

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and smaller airway stenosis are ascribed to a chronic pulmonary inflammation.2,4 These alterations

27

in airways lead to a loss of alveolar integrity through further activating aberrant inflammatory

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pathways.4,5 Airway inflammation is prompted by diverse mediators including reactive oxygen

29

species (ROS) and cell-derived chemotactic factors owing to the presence of environmental

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toxins.6,7 Although bronchial epithelial cells form the interface between the airway environment and

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the internal milieu, they can induce an airway-specific immune microenvironment and direct

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immune and inflammatory responses against lung tissue damage.8,9 Bronchiolar epithelium-derived

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monocyte chemoattractant protein-1 and interleukin (IL)-8 can be ascribed to the chemotactic

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activity of neutrophils.6 Additionally, pulmonary mesenchymal cells such as airway smooth muscle

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cells and lung fibroblasts react with various mediators and produce autocrine responses. In COPD,

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neutrophils, macrophages, and lymphocytes are prominent, while eosinophils are salient cells in

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asthmatic airways.6,11,12 However, the underlying mechanisms for airway inflammation of asthma

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and COPD remain unsolved. Underlying mechanisms of small airway destruction and structural

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changes may reveal potential therapeutic targets in pulmonary diseases.13

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Several studies are ongoing to pinpoint diverse molecular targets in the airway process to

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develop novel therapeutic agents.14-16 The primary therapeutic option for chronic airway diseases is

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to combat airway inflammation.14,15 In addition, the immunomodulatory therapies in airways may

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effectively alleviate pulmonary diseases.15 Inhaled corticosteroids and β-adrenergic receptor

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agonists, phosphodiesterase antagonists and statins in clinical applications can influence COPD-

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associated mechanisms underlying inflammation, mucin overproduction and tissue destruction.16-18

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However, the long-term use of these drugs results in undesirable consequences such as risk of

47

pneumonia in patients.19 Macrolides with anti-microbial, immunomodulatory, and anti-viral activities,

48

stand for a potential therapeutic remedy for asthma characterized by airway 3


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hyperresponsiveness.15 Currently, the use of macrolides in chronic asthma or acute exacerbations

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is not yet validated. Therefore, new therapeutic strategies with natural agents that have mostly

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proven to be safe need to be ripened for pulmonary inflammation to relieve exacerbating rates and

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all-cause mortality.14 Numerous studies reveal potential roles for several prevailing natural

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compounds for the treatment of inflammatory lung diseases.20-22 Anti-inflammatory resveratrol,

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quercetin, mangiferin rich in mango leaf extract, and dihydroquercetin rich in grape leaf extract

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suppress bleomycin-induced lung damage and pulmonary fibrosis.22 Our new studies reveal that

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astragalin combated epithelial apoptosis, allergic Inflammation and airway thickening in ovalbumin

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(OVA)-challenged mice.23,24

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It has been well documented that anti-inflammatory resveratrol protects lung diseases

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such as acute lung injury, asthma, COPD and lung fibrosis.25 Oleuropein (Figure 1A) is one of

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phenolic compounds rich in olives, and has antioxidant, anti-microbial, and anti-obesity

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properties.26-28 However, the beneficial effects of oleuropein on asthma and smoking-induced

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emphysema have not been reported. Based on the evidence that polyphenols abundant in various

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fruits and vegetables may inhibit inflammatory diseases in airways,22,29 this study investigated that

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oleuropein had the protective properties in asthma model characteristic of eosinophilic

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inflammation caused by IL-4 and OVA administration. Since In contrast to asthma, inflammation in

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COPD by cigarette smoke (CS) leads to progressive remodeling of lung tissues, 30 this study

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explored the beneficial effects of oleuropein on neutrophilic inflammation in CS-challenged mouse

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models. The current study demonstrated that oleuropein suppressed OVA- or CS-induced

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recruitment of eosinophils, macrophages, neutrophils and lymphocytes, and that this compound

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inhibited alveolar destruction causing emphysema in CS-challenged mice.

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MATERIALS AND METHODS

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Chemicals

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M199, human epidermal growth factor (EGF), hydrocortisone, gelatin, human insulin, apo-

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transferrin, oleuropein, chicken egg white albumin and thrombin were provided from the Sigma-

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Aldrich Chemical (St. Louis, MO), unless specifically described elsewhere. Fetal bovine serum

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(FBS), penicillin-streptomycin and trypsin-EDTA were supplied by the Lonza (Walkersville, MD).

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Human proteins of IL-4 and eotaxin-1 (CCL11), and goat polyclonal eotaxin-1 antibody were

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supplied from R&D systems (Minneapolis, MN). Rabbit polyclonal antibodies of intracellular

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adhesion molecule (ICAM)-1 and CD68 were provided from the Santa Cruz Biotechnology (Dallas,

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TX). Rabbit polyclonal antibodies of α-smooth muscle actin (α-SMA) and CCR3, mouse

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monoclonal mucin 5AC (MUC5AC) antibody, rat monoclonal F4/80 antibody (clone A3-1, IgG2b),

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goat polyclonal CD11b antibody, and rabbit monoclonal proteinase-activated receptor (PAR)-2

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(clone EPR13675, IgG) were purchased from Abcam (Cambridge, UK). Rabbit monoclonal

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phospho-STAT6 (Tyr641) antibody (Clone 46H1L12, IgG) was obtained from Thermo Fisher

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Scientific (Rockford, IL). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, donkey

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anti-goat IgG and goat anti-mouse IgG were obtained from Jackson Immuno-Research

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Laboratories (West Grove, PA). Mouse monoclonal β-actin antibody (clone AC-74, IgG2a) was

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obtained from Sigma-Aldrich Chemicals. Essential fatty acid free bovine serum albumin (BSA) and

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skim milk were supplied by Becton Dickinson Company (Sparks, MD). 4',6-Diamidino-2-

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phenylindole (DAPI) was obtained from Santa Cruz Biotechnology. Oleuropein was dissolved in

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dimethyl sulfoxide (DMSO) for cell culture and animal gavage; a final concentration of DMSO was

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Oleuropein Curtails Pulmonary Inflammation and Tissue Destruction in

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