Asaronic acid attenuates macrophage activation toward M1 phenotype

4 hours ago - Macrophage polarization has been implicated in the pathogenesis of obesity and type 2 diabetes that are recognized as chronic ...
0 downloads 0 Views 2MB Size
Subscriber access provided by Bibliothèque de l'Université Paris-Sud

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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.

Page 1 of 35

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]

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

1

Page 2 of 35

ABSTRACT

2 3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

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

22

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

23

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

24

entailing AGE-RAGE interaction.

25 26

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

27

polarization

2

ACS Paragon Plus Environment

Page 3 of 35

28

Journal of Agricultural and Food Chemistry

INTRODUCTION

29 30

The macrophage polarization is a tightly controlled process by which macrophages

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

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

39

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

40

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

41

polarizing macrophages toward the M1 activation state through regulating signal transducers

42

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

43

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

44

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

45

(PPARγ).12,13,14

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

healing due to dysregulated differentiation of hematopoietic stem cells towards

54

macrophages.18 Collectively, identification of mechanistic molecules responsible for

3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

55

macrophage polarization is crucial for elucidating novel macrophage-mediated therapeutic

56

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

57

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

58

macrophages is often associated with chronic inflammation and metabolic dysfunction,

59

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

60

peptide-1 analogue reduces atherosclerosis associated with insulin resistance by

61

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

62

Thus, reprogramming toward alternatively-activated macrophages could provide new

63

therapeutic strategies for diabetes-associated diseases.

64

Numerous studies have demonstrated that natural compounds can modulate

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

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

79

complications.

Page 4 of 35

80

4

ACS Paragon Plus Environment

Page 5 of 35

81

Journal of Agricultural and Food Chemistry

MATERIALS AND METHODS

82 83 84

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

85

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

86

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

87

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

88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101 102 103

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

104

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

105

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

106

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

107

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

5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

108

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

109

absence and presence of 1-20 μM AA.

110

Page 6 of 35

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

111

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

112

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

113

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

114

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

115

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

116 117 118

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

119

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

120

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

121

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

122 123 124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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

134

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

6

ACS Paragon Plus Environment

Page 7 of 35

Journal of Agricultural and Food Chemistry

135

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

136

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

137

Seoul, Korea).

138 139 140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150 151 152

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

153

experiment. Statistical analyses were performed using Statistical Analysis Systems statistical

154

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

155

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

156

considered significant at P