Inhibition of Mast Cell-Mediated Allergic Responses by Arctii Fructus

Subscriber access provided by Technical University of Munich University Library

Article

Inhibition of Mast cell-mediated Allergic Responses by Arctii Fructus Extracts and its Main Compound Arctigenin Ji-Ye Kee, and Seung-Heon Hong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02965 • Publication Date (Web): 04 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 34

Journal of Agricultural and Food Chemistry

1

Inhibition of Mast cell-mediated Allergic Responses by Arctii Fructus Extracts and its Main

2

Compound Arctigenin

3 4

Ji-Ye Kee, Seung-Heon Hong

5

6

Department of Oriental Pharmacy, College of Pharmacy, Wonkwang-Oriental Medicines

7

Research Institute, Wonkwang University, 460 Iksandae-ro, Iksan, Jeonbuk 54538, Republic

8

of Korea

9 10

Correspondence should be addressed to Seung-Heon Hong, [email protected]

11

12

Short title: Anti-allergic effects of Arctii Fructus and arctigenin

1

ACS Paragon Plus Environment


13

ABSTRACT: The Arctium lappa (Arctii Fructus) seed and its major active compound,

14

arctigenin (ARC), are known to have anti-cancer, anti-obesity, anti-osteoporosis, and anti-

15

inflammatory activities. However, the effect of Arctii Fructus and ARC on mast cell-mediated

16

allergic inflammation and the associated mechanism have not been elucidated. Therefore, we

17

attempted to investigate the anti-allergic activity of Arctii Fructus and ARC on mast cells and

18

experimental mouse models. Arctii Fructus water (AFW) or ethanol extract (AFE) and ARC

19

reduced the production of histamine and pro-inflammatory cytokines such as interleukin (IL)-

20

1β, IL-6, IL-8, and TNF-α in mast cells. AFW, AFE, and ARC inhibited phosphorylation of

21

MAPKs and NF-κB in activated mast cells. Moreover, IgE-mediated passive cutaneous

22

anaphylaxis and compound 48/80-induced anaphylactic shock were suppressed by AFW, AFE,

23

and ARC administration. These results suggest that Arctii Fructus and ARC are potential

24

therapeutic agents against allergic inflammatory diseases.

25 26 27

KEYWORDS: Arctii Fructus, Arctigenin, Allergic inflammation, Mast cells, MAPK

2


Page 2 of 34

Page 3 of 34


28

INTRODUCTION

29

Allergic diseases, such as allergic rhinitis, allergic eczema, and atopic dermatitis, are

30

hypersensitivity disorders of the immune system. The number of allergy patients has

31

consistently increased with approximately 10–20% of the world’s population suffering from

32

allergic diseases.1 Thus, allergic diseases are important public health problems that can

33

influence the quality of life and the socioeconomic burden. Antihistamines, steroids, non-

34

steroidal anti-inflammatory drugs, and immunosuppressants are common drugs currently

35

used to treat and prevent allergic diseases. However, these medications can show central side

36

effects, and in certain cases do not result in efficient recovery.2

37

Mast cells play a critical role as regulators of allergic diseases due to their wide

38

distribution in various tissues.3 Allergic reactions result in mast cell degranulation, which

39

leads to the rapid release of histamine and various inflammatory mediators such as

40

leukotrienes, serotonin, chemokines, and proinflammatory cytokines, including interleukin

41

(IL)-1β, IL-6, IL-8, and TNF-α. These mediators aggravate allergic responses through the

42

infiltration, differentiation, and activation of immune cells.4 Therefore, modulation of mast

43

cell degranulation and release of allergic mediators, is an effective treatment strategy for

44

allergic diseases.

45

Mitogen-activated protein kinases (MAPKs) signaling pathway is associated with several

46

pivotal physiological processes such as proliferation, activation, degranulation, and migration.

47

Extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 are

48

major components of the MAPK signaling pathway.5-7 Extracellular stimuli activate MAPKs,

49

which induce the degradation of IκB proteins and the nuclear translocation of nuclear factor-

50

kappaB (NF-κB). Activation of NF-κB causes the transcription of target genes, including

51

inflammatory mediators, which are crucial factors in the development of allergic responses.6, 3



52

8, 9

53

Arctium lappa (Arctii Fructus) seeds have been used as anti-inflammatory, diuretic, and

54

detoxifying drugs in oriental medicine.10 The Arctii Fructus extract improves high

55

fat/cholesterol diet-induced endothelial dysfunction and dermal extracellular matrix

56

metabolism, which reduces wrinkles.11,12 Arctigenin (ARC) is one of the major active

57

constituents present in the Arctii Fructus water (AFW) and ethanol extracts (AFE).13 ARC

58

has anti-hypertension, anti-tumor, anti-osteoclastogenesis, and neuroprotective activities.14-19

59

Specifically, fermented Arctii Fructus extract can suppress IgE-mediated allergic reaction in

60

RBL‑ 2H3 cells.20 Moreover, ARC successfully inhibited ovalbumin (OVA)-induced

61

anaphylaxis, compound 48/80-induced histamine release, and proinflammatory enzyme

62

activities.21 However, the anti-allergic activities of Arctii Fructus and ARC on human mast

63

cells and the underlying mechanisms are unclear. The aim of this study was to determine the

64

effect of Arctii Fructus and ARC on mast cell-mediated allergic inflammation.

65 66

MATERIALS AND METHODS

67

Preparation of extracts and ARC isolation from Arctii Fructus. Dried and ground Arctii

68

Fructus powder were obtained from Omniherb (Uiseong, Republic of Korea). AFW was

69

prepared by extracting the water-soluble components of the Arctii Fructus powder in boiling

70

water (100 g/L of water) at 100°C for 3 h. The solution was filtered using Whatman filter

71

paper and lyophilized. To prepare the ethanol extract of Arctii Fructus (AFE), 100 g of Arctii

72

Fructus was boiled in 70% ethanol (1 L) for 3 h and evaporated using a Rotary evaporator.

73

The extract was freeze-dried at −56 °C and 9 mm Torr. ARC was isolated from A. lappa

74

seeds as reported previously.21 ARC was identified by comparing the 1H-NMR spectrum

75

data.23 AFW was dissolved in distilled water, whereas AFE and ARC were dissolved in 4


Page 4 of 34

Page 5 of 34


76

DMSO (final concentration of DMSO is less than 0.1%).

77 78

Reagents. Anti-phospho-ERK, phospho-JNK, phospho-p38, phosphor-IκBα, NF-κB, and

79

Receptor interacting protein-2 (RIP2) antibodies were purchased from Cell Signaling

80

Technology, Inc. (Danvers, MA, USA). Anti-histone, α-tubulin, ERK, JNK, p38, and caspase-

81

1 antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Thiazolyl Blue

82

Tetrazolium Bromide (MTT), PMA, A23187, DNP-IgE, DNP-BSA, and compound 48/80

83

were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

84 85

Cell culture. The human mast cell line (HMC-1) was provided by Eichi Morri (Osaka

86

University, Japan), and the rat basophilic leukemia mast cell line RBL-2H3 was purchased

87

from Korean Cell Line Bank (Seoul, Republic of Korea). These cell lines were cultured in

88

10% FBS, 100 U/mL of penicillin and streptomycin in Iscove's Modified Dulbecco's Medium

89

and Dulbecco's Modified Eagle's Medium, respectively. Cells were cultured in an incubator

90

with 5% CO2/95% air at 37°C.

91 92

Cell viability. Cells were seeded in 24-well microplates at a density of 5 × 104 cells/well and

93

stabilized overnight. After 24 h of treatment with AFW, AFE, and ARC, cells were treated

94

with MTT reagent (0.5 mg/mL) and incubated for 4 h. Formazan was lysed with DMSO, and

95

colorimetric changes were measured using a VersaMax ELISA Microplate Reader at 540 nm.

96 97

Histamine assay. Histamine production from mast cells was measured using the human

98

histamine ELISA kit (ALPCO Diagnostics, NH, USA) as per the manufacturer’s protocol.

99

HMC-1 cells were pretreated with AFW, AFE, and ARC for 1 h and stimulated with PMA 5



100

plus A23187 (PMACI; PMA 50 nM and A23187 1 µM) for 24 h. RBL-2H3 cells were

101

sensitized with DNP-IgE (100 ng/mL) for 12 h, and the samples were added to cells 1 h

102

before DNP-BSA treatment. The conditioned medium was collected and used as a sample.

103 104

Real-time RT-PCR. Total RNA was isolated from cells, and cDNA was synthesized using

105

RNA-spinTM, Total RNA Extraction Kit, and Power cDNA Synthesis Kit (iNtRon Biotech,

106

Seoul, Republic of Korea). Expression specific genes was determined by step-one plusTM

107

real-time PCR systems (Applied Biosystems, Foster City, CA, USA). All data were

108

normalized to GAPDH mRNA. The primer sequences are summarized in supporting

109

information.

110 111

Enzyme-linked immunosorbent assay (ELISA). Release of proinflammatory cytokines was

112

measured by using a BD OptEIA ELISA kit (BD Pharmingen, San Diego, CA, USA)

113

according to manufacturer’s protocol. Briefly, 96-well microplates were precoated with

114

capture antibodies at 4°C overnight. After blocking with 5% FBS for 1 h, samples and

115

standards were added and incubated for at least 3 h. The plates were washed with washing

116

buffer and incubated with detection antibodies and streptavidin HRP for 1 h 30 min. This was

117

followed by washing with 0.05% PBST five times, and the plates were incubated with TMB

118

substrate reagent at 37°C for 30 min. Colorimetric development was blocked with a stop

119

solution and measured using a microplate reader at 405 nm.

120 121

Western blot analysis. Collected cells were washed and lysed using pro-prep protein

122

extraction solution (iNtRon Biotech, Seoul, Republic of Korea) to extract total protein. Cell

123

lysates were centrifuged at 13,000 rpm for 5 min, and the supernatants containing the proteins 6


Page 6 of 34

Page 7 of 34


124

were obtained. Nuclear extracts were isolated using NE-PER Nuclear and Cytoplasmic

125

Extraction Reagents (Pierce Thermo Scientific, Rockford, IL, USA). Total protein was

126

quantified using BCA protein assay, mixed with a 5x sample buffer. Following SDS-

127

polyacrylamide gel electrophoresis, proteins were transferred to PVDF membrane. Specific

128

proteins were detected using primary antibodies and Horseradish Peroxidase secondary

129

antibodies. Protein bands were photographed using the FluorChemTM M system

130

(Proteinsimple, San Jose, CA, USA).

131 132

Caspase-1 activity Assay. Caspase-1 colorimetric assay kit (Biovision, CA, USA) was used to

133

determine caspase-1 activity according to the recommended instructions. Briefly, PMACI

134

was added to AFW, AFE, and ARC-pretreated HMC-1 cells for 24 h, and harvested cells were

135

lysed with lysis buffer. Caspase-1-specific peptide YVAD-pNA was used for the

136

measurement of caspase-1 activity. Colorimetric changes were quantified using a microplate

137

reader at 405 nm.

138 139

Animals. ICR mice (five weeks, male) were purchased from Samtaco Korea (Osan, Republic

140

of Korea). Mice were maintained in a laminar airflow room at 22 ± 1°C. An in vivo

141

experiment was performed in accordance with the regulations issued by Wonkwang

142

University guidelines (WKU14-100).

143 144

Compound 48/80-induced anaphylactic shock. AFW, AFE (25–100 mg/kg), and ARC (10–

145

50 mg/kg) were orally administered 1 h prior to injecting compound 48/80. Control mice

146

received the same volume of 0.05% carboxymethyl cellulose. After intraperitoneal injection

147

of compound 48/80 (8 mg/kg), mortality by anaphylactic shock was monitored for 1 h. 7



148 149

Passive cutaneous anaphylaxis (PCA). A day prior to the experiment, hair was completely

150

removed from the back of mice, and anti-dinitrophenyl (DNP)-IgE (0.5 µg) was injected into

151

the dorsal skin. AFW, AFE (100 mg/kg), and ARC (50 mg/kg) were orally administered 1 h

152

before the antigen challenge. DNP-BSA (1 µg) in saline containing 1% Evans blue was

153

intravenously injected into each mouse via the tail vein. After killing, the dorsal skin was

154

resected to evaluate the effect of the compounds on PCA. Blue dye in the dorsal skin was

155

dissolved with formaldehyde for 24 h and colorimetric change was measured using a

156

microplate reader at 620 nm. The inhibition rates of the compounds were calculated using the

157

following equation: Inhibition rate (%) = 100 - (a/b × 100); ‘a’ is the average absorbance of

158

the sample group, and ‘b’ is the average absorbance of the control group.

159 160

Statistical analysis. Results are shown as the mean ± S.D. from at least three experiments. All

161

statistical analyses were performed by using the Student’s t-test, and p < 0.05 indicated

162

statistical significance.

163 164

RESULTS

165

Arctii Fructus extracts and ARC decrease histamine release in mast cells: Mast cell

166

degranulation results in histamine release by antigen stimulation causing allergic reactions.4

167

Therefore; we investigated whether AFW, AFE, and ARC can suppress the release of

168

histamine from activated mast cells. MTT assay was performed to determine the effect of

169

AFW, AFE, and ARC on the viability of HMC-1 cells. As shown in Figure 1A and B, AFW

170

and AFE (2–50 µg/mL) did not result in any cytotoxicity after 24 h of incubation. Doses of

Inhibition of Mast Cell-Mediated Allergic Responses by Arctii Fructus

Recommend Documents