Asaronic acid attenuates macrophage activation toward M1 phenotype

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Bioactive Constituents, Metabolites, and Functions

Asaronic acid attenuates macrophage activation toward M1 phenotype through inhibition of NF-#B pathway and JAKSTAT signaling in glucose-loaded murine macrophages Hyeongjoo Oh, Sin-Hye Park, Min-Kyung Kang, Yun-Ho Kim, Eun-Jung Lee, Dong Yeon Kim, Soo-Il Kim, Su Yeon Oh, Soon Sung Lim, and Young-Hee Kang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03926 • Publication Date (Web): 17 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

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Journal of Agricultural and Food Chemistry

Asaronic acid attenuates macrophage activation toward M1 phenotype through inhibition of NF-κB pathway and JAK-STAT signaling in glucose-loaded murine macrophages Hyeongjoo Oh, Sin-Hye Park, Min-Kyung Kang, Yun-Ho Kim, Eun-Jung Lee, Dong Yeon Kim, Soo-Il Kim, Su Yeon Oh, Soon Sung Lim, Young-Hee Kang Department of Food science and Nutrition and Korea Nutrition Institute, Hallym University, Chuncheon, 200-702 Korea Hyeongjoo Oh and Sin-Hye Park contributed to this study equally.

Running title: Asaronic acid and macrophage polarization

6041 Words /6 Figures /29 Pages

Funding sources: This study was supported by the Hallym University Research Fund, 2019 (HRF-201902-008). Author Contributions: H. O., S.-H. P. and Y.-H. K. designed research; H.O., S.-H. P., Y.-H. K (Kim), E.-J. L., D. Y. K. and S.-I. K. conducted research; H. O., S.-H. P., M.-K. K. and S. S. L. analyzed data; H. O. and Y.-H. K. wrote the paper. Y.-H. K. had primary responsibility for final content. All authors read and approved the final manuscript. Conflict of interests: The authors declare that they have no conflict of interest. Abbreviations used: AGE, advanced glycation end products; Arg-1, arginase-1; AA, asaronic acid; HIF-1α, hypoxia inducible factor-1α; iNOS, inducible nitric oxide synthase; IGF-1, insulin like growth factor-1; IL-6, interleukin-6; JAK, Janus kinase; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; NF-κB, nuclear factor-kappaB; PGE2, prostaglandin E2; PPARγ, peroxisome proliferator-activated receptor γ; SOCS, suppressor of cytokine signaling; STAT, signal transducers and activators of transcription; TLR4, toll-like receptor 4; VEGF, vascular endothelial growth factor

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

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ABSTRACT

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Macrophage polarization has been implicated in the pathogenesis of obesity and type

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2 diabetes that are recognized as chronic pro-inflammatory diseases. This study investigated

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that high glucose, in common with lipopolysaccharide (LPS), activated macrophages toward

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M1 phenotypes, and that 1-20 μM asaronic acid (AA) counteracted diabetic macrophage

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activation. AA reduced the LPS-promoted secretion of pro-inflammatory interleukin (IL)-6 and

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monocyte chemoattractant protein-1. LPS markedly elevated macrophage induction of the M1

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markers of toll-like receptor 4 (TLR4), CD36 and CD68, which was attenuated by AA. LPS

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significantly enhanced nuclear factor (NF)-κB transactivation, signal transducers and

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activators of transcription 1 (STAT1)/ STAT3 activation and suppressor of cytokine signaling 3

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(SOCS3) induction in macrophages. However, AA highly suppressed the aforementioned

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effects of LPS. Glucose stimulated macrophages to express advanced glycation end products

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(AGE) and receptor for AGE (RAGE). Administration of 20 μM AA to macrophages partly but

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significantly attenuated such effects (1.65±0.12 vs. 0.95±0.25 fold of glucose control for AGE;

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2.33±0.31 vs. 1.40±0.22 fold of glucose control for RAGE). Furthermore, glucose enhanced

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macrophage induction of TLR4 and inducible nitric oxide synthase and IL-6 production, while it

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demoted the production of anti-inflammatory arginase-1 and IL-10. In contrast, AA reversed

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the induction of these markers in glucose-loaded macrophages. AA dose-dependently and

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significantly encumbered NF-κB transactivation, Janus kinase 2 (JAK2), STAT1/STAT3

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activation, and SOCS3 induction upregulated in glucose- supplemented macrophages. These

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results demonstrated for the first time that AA may limit diabetic macrophage activation toward

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the M1 phenotype through inhibition of TLR4-/IL-6-mediated NF-κB/JAK2-STAT signaling

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entailing AGE-RAGE interaction.

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Key Words: Asaronic acid, glucose, JAK-STAT signaling, lipopolysaccharide, macrophage

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polarization

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INTRODUCTION

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The macrophage polarization is a tightly controlled process by which macrophages

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formulate disparate functional settings in response to diverse stimuli.1,2 This process is

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imperative for the host defense against pathogens as well as the maintenance of

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homeostasis.2,3 Although macrophages are essential components in the innate immunity, they

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have several functions in manipulating cell proliferation and tissue repair.3,4 Fully-polarized

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macrophages acquire specific phenotype like M1 classically-activated macrophages or M2

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alternatively-activated macrophages.2,5 These specific phenotypes are determined by diverse

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microenvironments where macrophages are placed.6,7 The macrophage activation entails

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signaling pathways, transcriptional interactions and post-transcriptional regulators that skew

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macrophage function towards either the M1 or M2 phenotype.1,3,8,9 The toll-like receptor (TLR)

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pathway triggered by the bacterial endotoxin lipopolysaccharide (LPS) plays a role in

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polarizing macrophages toward the M1 activation state through regulating signal transducers

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and activators of transcription (STAT)-suppressor of cytokine signaling (SOCS).10,11 On the

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contrary, interleukin (IL)-4 and IL-13 promote the induction of M2 macrophage phenotype

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genes such as arginase 1 (Arg-1) and peroxisome proliferator-activated receptor γ

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(PPARγ).12,13,14

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It has been proposed that macrophages are a potential pharmacological target in

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inflammatory or immune response-mediated metabolic diseases. Emerging evidence has

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established a crucial role of macrophage polarization in the development of metabolic

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diseases.3,15,16 Inflammatory monocytes and macrophages may play critical roles in diabetes-

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triggered cardiovascular disease and other complications of diabetes.17 Macrophages derived

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from type 1 diabetes display enhanced inflammatory phenotypes, which may generate wide-

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ranging effects on the immune system.17 One investigation shows that diabetes impairs wound

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healing due to dysregulated differentiation of hematopoietic stem cells towards

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macrophages.18 Collectively, identification of mechanistic molecules responsible for

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macrophage polarization is crucial for elucidating novel macrophage-mediated therapeutic

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strategies.19,20 However, the mechanisms that promote an inflammatory setting evoking

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atherosclerosis in diabetes are poorly understood. An imbalance in the ratio of M1/M2

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macrophages is often associated with chronic inflammation and metabolic dysfunction,

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resulting in various inflammatory diseases.3,5,21 A recent study shows that the glucagon-like

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peptide-1 analogue reduces atherosclerosis associated with insulin resistance by

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reprogramming macrophages towards an M2 phenotype, leading to reduced inflammation.22

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Thus, reprogramming toward alternatively-activated macrophages could provide new

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therapeutic strategies for diabetes-associated diseases.

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Numerous studies have demonstrated that natural compounds can modulate

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macrophage polarization as a promising therapeutic strategy.23,24,25 The present study

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hypothesized that AA (2,4,5-trimethoxybenzoic acid, Figure 1A), newly identified in purple

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perilla extracts, inhibited M1 macrophage phenotype-mediated inflammation in diabetes.

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Purple perilla leaves have been used in Chinese medicine to treat a wide variety of ailments,

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as well as in Asian cooking as a garnish. Although perilla leaf extracts are known to have

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diverse antioxidant, anti-inflammatory, and tumor-preventing properties,26,27 the bioactive

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properties of AA have been rarely reported. To test the hypothesis, this study investigated that

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AA inhibited macrophage activation toward M1 phenotype in endotoxin- or glucose-exposed

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J774A.1 murine macrophages. Furthermore, this study attempted to explore the involvement

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of nuclear factor (NF)-κB pathway/STAT signaling-responsive mechanism(s) in diabetes-

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associated inflammation. It was found that AA inhibited diabetic inflammatory activation of

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macrophages toward the M1 phenotype through inhibition of NF-κB pathway/STAT signaling

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that entailed mediation of TLR4 and advanced glycation end products (AGE). Thus, AA could

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merit exploration as a therapeutic agent in the treatment of diabetes-associated inflammatory

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complications.

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MATERIALS AND METHODS

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Chemicals Dulbecco's Modified Eagle Medium (DMEM) chemicals, fatty acid-bovine serum

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albumin (BSA), lipopolysaccharide (LPS), and D-glucose were purchased from Sigma Aldrich

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Chemical (St. Louis, MO), as were all other reagents, unless specifically stated elsewhere. 3-

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(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Duchefa

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Biochemie (Haarlem, Netherlands). Fetal bovine serum (FBS) and penicillin-streptomycin were

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obtained from Lonza (Basel, Switzerland). Antibodies of TLR4, CD36, CD68, AGE, receptor for

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AGE (RAGE), inducible nitric oxide synthase (iNOS), NF-κB, STAT1, STAT3, Janus kinase

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(JAK)2 and suppressor of cytokine signaling 3 (SOCS3) were purchased from Santa Cruz

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Biotechnology (Santa Cruz, CA, USA). Antibodies of phospho-inhibitory (I)κB, phospho-

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STAT1, phospho-STAT3, hypoxia inducible factor (HIF)-1α, vascular endothelial growth factor

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(VEGF), arginase-1 (Arg-1), and JAK2 were obtained from Cell Signaling Technology (Danvers,

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MA). Advanced glycation end products AGE-BSA antibody was provided by Bioss Antibodies

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(Woburn, MA). The JAK2 inhibitor was purchased from Calbiochem (Darmstadt, Germany). AA

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was purchased from Cayman Chemical (99% purity, Ann Arbor, MI). β-Actin antibody was

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purchased from Sigma Aldrich Chemicals. Horseradish peroxidase (HRP)-conjugated goat

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anti-rabbit IgG, goat anti-mouse IgG, and donkey anti-goat IgG were supplied by Jackson

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Immuno-Research Laboratories (West Grove, PA, USA).

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Cell Culture Mouse macrophage-like cell line J774A.1 (American Type Culture Collection,

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Manassas, VA) were grown in DMEM supplemented with 10% FBS at 37C in a humidified

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atmosphere of 5% CO2 in air. However, in culture experiments J774A.1 macrophages were

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incubated in DMEM supplemented with 0.4% BSA. The macrophages were pretreated with 1-

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20 μM AA and exposed to 2 μg/ml LPS for up to 48 h. In another set of experiments, J774A.1

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macrophages were incubated in media containing 33 mM glucose for up to 72 h in the

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absence and presence of 1-20 μM AA.

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The cytotoxicity of AA was determined from cell growth by using MTT assay. Cells

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were treated with AA for 24 h were incubated with 1 mg/ml MTT solution at 37C for 3 h,

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resulting in the formation of insoluble purple formazan product dissolved in 250 μl isopropanol.

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Optical density was measured using a microplate reader (Bio-Rad Model 550, Hercules, CA)

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at λ = 570 nm. This study found that AA had no cytotoxicity within the doses of 1-20 μM

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(Figure 1B). The current experiments employed AA in the range of 1-20 μM.

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Enzyme-Linked Immunosorbent Assay (ELISA) Cell media were collected from culture of J774A.1 macrophages and stored at -20C.

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The secretion of IL-6, insulin like-growth factor (IGF)-1, monocyte chemoattractant protein

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(MCP)-1, and IL-10 and was examined in culture media by using ELISA kits (R&D System,

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Minneapolis, MN), according to a manufacturer’s instruction.

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Western Blot Analysis Following the culture protocols, cells were lysed in a lysis buffer. Equal protein

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amounts of cell lysates were electrophoresed on 8-12% sodium dodecyl sulfated-

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polyacrylamide gel (SDS-PAGE) and transferred onto a nitrocellulose membrane. After

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blocking with 5% skim milk or 3% BSA for 3 h at room temperature, the membranes were

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incubated with polyclonal or monoclonal antibodies of TLR4, CD36, CD68, phospho-IκBα,

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phospho-STAT1, phospho-STAT3, SOCS3, AGE, receptor for AGE (RAGE), HIF-1α, VEGF,

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iNOS, Arg-1 and phospho-JAK2 for overnight at 4C. After three times of washing with Tris-

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buffered saline-tween 20 buffer, the membranes were incubated with anti-rabbit or anti-mouse

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IgG conjugated to HRP for 1 h. The individual protein level was detected by Immobilon

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Western Chemiluminescent HRP substrate (Millipore, Billerica, MA). For the internal control,

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the membranes were incubated with β-actin antibody (Sigma Aldrich Chemicals). After the

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performing immunoblot analyses, the blot bands were visualized on Agfa X-ray film (Agfa

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HealthCare NV, Mortsel, Belguim), developing signals with X-ray developer and fixer (Duksan,

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Seoul, Korea).

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Immunocytochemical Analysis After J774A.1 macrophages were exposed to 2 μg/ml LPS or 33 mM glucose in the

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absence and presence of 1-20 μM AA for 48-72 h, cells were fixed with 4% formaldehyde for

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15 min permeated with 0.1% Triton X-100 and 0.1% sodium citrated for 1 min on the ice. Cells

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were treated with a 5% BSA for 1 h. For the Immunofluorescent cytochemical staining, cells

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was incubated with a specific primary antibody against NF-κB overnight and further with Cy3-

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or FITC-conjugated IgG (Rockland, Pottstown, PA) for 1 h, and washed with phosphate-

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buffered saline-tween 20. For the nuclear staining, cells were incubated with 4’,6-diamidino- 2-

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phenylindole (DAPI, Santa Cruz Biotechnology) for 10 min. Each slide was mounted in

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VectaMount mounting medium (Vector Laboratories, Burlingame, CA). Images were taken

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using an optical Axiomager microscope system (Zeiss, Oberkochen, Germany).

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Data Analysis The results were expressed as mean ± SEM for each treatment group in each

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experiment. Statistical analyses were performed using Statistical Analysis Systems statistical

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software package (SAS Institute, Cary, NC). Significance was determined by one-way analysis

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of variance, followed by Duncan range test for multiple comparisons. Differences were

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considered significant at P

Asaronic acid attenuates macrophage activation toward M1 phenotype

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