Salicin from Alangium chinense Ameliorates Rheumatoid Arthritis by

Jun 1, 2018 - Department of Clinical Laboratory, Jinling Hospital, School of Medicine, ... of Pharmacy, University of Central Punjab , Lahore 54000 , ...
1 downloads 0 Views 2MB Size
Subscriber access provided by University of Winnipeg Library

Bioactive Constituents, Metabolites, and Functions

Salicin from Alangium chinense ameliorates rheumatoid arthritis by modulating the Nrf2-HO-1-ROS pathways Kefeng Zhai, Hong Duan, Ghulam Jilany Khan, Hui Xu, Fang-kai Han, Wen-gen Cao, Gui-zhen Gao, Lingling Shan, and Zhao-Jun Wei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02241 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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 28

Journal of Agricultural and Food Chemistry

1

Salicin from Alangium chinense ameliorates rheumatoid arthritis by

2

modulating the Nrf2-HO-1-ROS pathways

3 4

Short title: Salicin attenuates rheumatoid arthritis by reducing oxidative stress

5 6

Ke-feng Zhai*,†,‡, Hong Duan†, Ghulam Jilany Khan#, ††, Hui Xu†, Fang-kai Han†, Wen-gen

7

Cao†, Gui-zhen Gao†, Ling-ling Shan†, §, Zhao-Jun Wei*,$

8 9



Engineering Research Center of Natural medicine and Functional Food, Institute of

10

Pharmaceutical Biotechnology, School of Biological and Food Engineering, Suzhou

11

University, 49, Bianhe Road, Suzhou, 234000, P.R. China.

12



13

Nanjing, 210002, P.R. China.

14

††

15

Engineering, Southeast University, Nanjing, 210096, P.R. China.

16

#

17

Punjab, Lahore, 54000 Pakistan.

18

$

19

China.

20

§

21

Imaging and Bioengineering, National Institutes of Health, Bethesda 20892, USA.

Department of Clinical Laboratory, Jinling Hospital, School of Medicine, Nanjing University,

State Key Laboratory of Bioelectronics, School of Biological Science and Medical

Department of Pharmacology and Therapeutics, Faculty of Pharmacy, University of Central

School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009 P.R.

Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical

22 23

*Corresponding authors.

24 25

Correspondence to Dr. Ke-feng Zhai at Engineering Research Center of Natural medicine 1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 28

26

and Functional Food, Institute of Pharmaceutical Biotechnology, School of Biological and

27

Food Engineering, Suzhou University, 49, Bianhe Road, Suzhou, 234000, P.R. China. E-mail:

28

[email protected] , Fax & Tel: +86-557-2871037

29

Correspondence to Dr. Zhao-Jun Wei at School of Food Science and Engineering, Hefei

30

University of Technology, Hefei, P.R. China, E-mail: [email protected] , Tel:

31

+86-551-62901539

32 33

Abbreviations used: RA, Rheumatoid arthritis; ROS, Reactive oxygen species; RA-FLSs,

34

Human rheumatoid arthritis fibroblast-like synoviocytes; CAT, Catalase; MDA,

35

Malonyldialdehyde; GSH, Glutathione; SOD, Superoxide dismutase; MPO, Myeloperoxidase;

36

LPO, Lipid peroxidase; IL-1β, Interleukin-1β; Nrf2, Nuclear-related factor 2; HO-1, Heme

37

oxygenase-1; MMP-1, Matrix metalloproteinases-1; MMP-3, Matrix metalloproteinases-3;

38

CIA, Collagen induced arthritis; ELISA, Enzyme-linked immunosorbent assay

39

2

ACS Paragon Plus Environment

Page 3 of 28

Journal of Agricultural and Food Chemistry

40

Abstract

41

Rheumatoid arthritis (RA) is a chronic inflammatory disorder linked to oxidative stress of rheumatoid

42

arthritis fibroblast like-synoviocytes (RA-FLSs). The effects and potential mechanism of salicin on

43

inflammation and oxidative stress of RA-FLSs were examined by MTT, ELISA, and western blot methods.

44

Salicin significantly reduced cell viability (82.03 ± 7.06, P < 0.01), cytokines (47.70 ± 1.48 ng/L for TNF-α,

45

30.03 ± 3.49 ng/L for IL-6) (P < 0.01), matrix metalloproteinases-1/-3 expression (P < 0.01) in

46

IL-1β-induced RA-FLSs, inhibited ROS generation and p65 phosphorylation (P < 0.01) as compared to

47

IL-1β-induced treatment. Moreover, salicin promoted Nrf2 nuclear translocation (2.15 ± 0.21) and HO-1

48

expression (1.12 ± 0.05), and reduced ROS production in IL-1β-induced RA-FLSs (P < 0.01). Salicin not

49

only reduced the collagen induced arthritis by reducing the clinical score (P < 0.01), inflammatory

50

infiltration and synovial hyperplasia in vivo, but also suppressed the oxidative damage indexes (SOD

51

155.40 ± 6.53 U/mg tissue, MDA 152.80 ± 5.89 nmol/g tissue, GSH 50.98 ± 3.45 nmol/g tissue, and CAT

52

0.92 ± 0.10 U/g protein) (P < 0.01) of ankle joint cells. Conclusively, our findings indicate that salicin

53

ameliorates rheumatoid arthritis which may be associated with oxidative stress and Nrf2-HO-1-ROS

54

pathways in RA-FLSs

55

Keywords: Rheumatoid arthritis; Salicin; Fibroblast-like synoviocytes; Oxidative stress; ROS

56

3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 28

57

Introduction

58

Rheumatoid arthritis (RA) is characterized by inflammation, bone and cartilage destruction 1-2 which may

59

markedly lower quality of life. Among the various causes of RA, oxidative stress is an important risk factor

60

known to play an imperative pathophysiological role in initiation and development of RA 3-4. The studies

61

have indicated that inflammation, oxidative stress, lipid peroxidation and increased reactive oxygen species

62

(ROS) can affect progression and severity of RA 5-6. Pro-inflammatory mediators such as interleukin-1β

63

(IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α), are crucial

64

element to regulate inflammation and joint damage during the course of RA 7. Additionally, ROS 7 and

65

related enzymes associated with oxidative stress including catalase (CAT), malonyldialdehyde (MDA),

66

glutathione (GSH), superoxide dismutase (SOD), myeloperoxidase (MPO), lipid peroxidase (LPO) 6, 8 are

67

significant inflammatory modulators in RA . As a consequence, the suppression of oxidative stress induced

68

by pro-inflammatory mediators in the synoviocytes has been proposed as a potential therapeutic strategy

69

against RA.

70

Alangium chinense (Lour.) Harms. (Alangiaceae) is widely distributed throughout the tropical and

71

subtropical regions of the Eastern Hemisphere, particularly in South China 9. It is a deciduous shrub and

72

cultivated for both forestal and medicinal uses specifically in traditional Chinese medicine (TCM) to treat

73

rheumatic arthritis, acroanesthesia, and fractures 10. Salicin (structure shown in Fig. 1A), a prodrug form of

74

acetyl salicylic acid (Aspirin) 11, is a major natural compound found in the stems and roots of Alangium

75

chinense. Previous studies have reported that salicin from Alangium chinense is one of the major active

76

ingredients in Fengshiding capsule as TCM for its therapeutic potential in rheumatic disease 12. Recent

77

studies have shown that salicin has vast range of pharmacological effects including antioxidant 13,

78

antinociceptive 11, anti-inflammation 14, antitumor 15, and neurite outgrowth 16. In addition, it has also been

79

reported that the extracts, enriched with salicin as a constituent or salicin alone deteriorate carcinogenicity

80

as well as reduce angiogenesis in mice by reducing oxidative stress 17 through upregulation of Nrf2 and

81

inhibition of ROS 13.

82

Earlier studies have reported that in human body, salicin is metabolized into salicylic acid and an

83

administration of 838 micromol (240 mg) of salicin may lead to a peak serum concentration of 1.2 mg/L 18.

84

In another double blind clinical trial studies, it has been reported that oral administration of 240 mg of

85

salicin is therapeutically effective to produce analgesic effect 19. However, this analgesic effect may not be 4

ACS Paragon Plus Environment

Page 5 of 28

Journal of Agricultural and Food Chemistry

86

solely due to active metabolite of salicin (salicylic acid) and require further exploration of effects of salicin

87

as an analgesic agent itself and its effects against rheumatoid arthritis.

88

So far, no extensive studies have been reported on the antirheumatic activity of salicin on RA-FLSs.

89

Herein, we examined the suppressive effects of salicin against IL-1β-induced oxidative stress responses in

90

RA-FLSs and elucidated the possible antioxidative mechanisms of salicin.

91 92

Materials and methods

93

Chemicals

94

Extracted and purified salicin (PubChem CID: 439503) from the Alangium chinense was obtained from

95

Wuwei County, Anhui Province, China. Before the extraction and purification, plant species was identified

96

by Prof. Jian-li Zhou, Anhui University of Traditional Chinese Medical University (He-fei, China) and

97

sample was deposited in the herbarium of the university (voucher number 20170605). The extraction

98

technique for salicin was as follows: the ratio of solid to liquid was 1∶30; the extraction was done 3 times

99

and the duration for each time was 1.5 hours. The purification of salicin from the Alangium chinense was

100

carried out by macroporous resin adsorption. Overall, HPD-826 showed good performance for purification

101

of salicin, and the proposed procedure is stable as well as feasible for industrial applications. The purified

102

extraction was further treated with n-butanol and silicagel column. Finally, salicin of more than 98% purity

103

was determined by HPLC-UV and identified by LC-MS and NMR (Supplementary Fig. 1A-C).

104

Alexa Fluor®488 Donkey Anti-Goat IgG (H+L) antibody, Fetal bovine serum (FBS), Dulbecco’s modified

105

Eagle’s medium (DMEM), Penicillin and Streptomycin were purchased from Life Technologies (Carlsbad,

106

CA, USA). IL-1β was purchased from PeproTech Inc. (NJ, USA). Enzyme linked immunosorbent assay

107

(ELISA) kits for TNF-α and IL-6 were purchased from YIFEIXUE BIO TECH (Nanjing, China).

108

Superoxide dismutase (SOD) kit, Malondialdehyde (MDA) kit, Glutathione (GSH) kit, Catalase (CAT) kit,

109

Reactive oxygen species (ROS) kit, Hematoxylin and Eosin (HE) staining kit, and DAPI were purchased

110

from Beyotime Institute of Biotechnology (Nantong, China). CelLytic™ NuCLEAR™ extraction kit,

111

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO)

112

were purchased from Sigma (St. Louis, MO, USA). Primary antibodies used for immunoblotting analyses

113

were including antibodies against pp65, p65, HO-1, Nrf2, and Histone H3 were purchased from Cell

114

Signalling Technology (Beverly, MA, USA); antibodies against MMP-3, GAPDH and β-actin were 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 28

115

purchased from EnoGene BIO Inc. (Nanjing, China). An antibody for MMP-1 detection was purchased

116

from Boster Biological Technology (Wuhan, China). Horseradish peroxidase (HRP)-conjugated secondary

117

antibodies were purchased from Bioworld Technology Inc. (St. Louis Park, MN, USA). RIPA Lysis Buffer,

118

protease inhibitor and enhanced chemiluminescene (ECL) reagents were purchased from Vazyme Biotech

119

(Nanjing, China). Nrf2 siRNA (h), non-overlapping sequence of human Nrf2, were purchased from Santa

120

Cruz Biotech (sc-37030, Dallas, TX, USA).

121

Primary RA-FLSs culture

122

After obtaining RA-FLSs from American Type Culture Collection (Manassas, VA, USA) we cultivated

123

them in DMEM with 10% FBS. To avoid bacterial contamination, the media was enriched with antibiotics

124

including Streptomycin (100 µg/mL) and Penicillin (100 units/mL). The cells were allowed to grow at a

125

uniformly humidified atmosphere at 37°C temperature with 95% and 5% O2 and CO2 supply respectively.

126

The media was replaced after 72 hours of incubation and RA-FLSs were utilized for experimentation

127

within two to eight passages.

128

Cell viability assays

129

We evaluated the cell viability using MTT assay for which RA-FLSs were seeded into 96 well plate. Upon

130

a mass of 8 × 104 cells per well in 100 µL medium, the cells were exposed to different concentrations of

131

salicin ranging from 0 to 1200 µmol/L for 48 h with or without 10 ng/mL of IL-1β. After the treatment with

132

or without salicin along-with or without IL-1β for 48 h, the cells were further treated MTT solution (25

133

µL/well) and allowed to stand for additional 4 h at 37 °C as per manufacturer’s recommended standard

134

protocols. After the incubation for 4 h, DMSO (150 µL/well) was added and the 96 well plate was

135

subjected to observe the optical density by microplate reader (Multiskan FC, Thermo Fisher Scientific, MA,

136

USA) against 540 nm of wavelength. Final viability for each experiment was calculated by using the

137

standard formula [(ODE/ODC) × 100%] where ODE refers to optical density of treated cells; and ODC refers

138

to optical density of control cell group.

139

Measurement of cytokines by ELISA

140

The obtained supernatants of cell cultures were investigated for quantitative measurement of cytokines such

141

as TNF-α and IL-6 by Enzyme-linked immunosorbent assay (ELISA) kits.

142

Western blot analysis

143

The cell lysates preparation and the quantification of obtained protein dilutions were done following the 6

ACS Paragon Plus Environment

Page 7 of 28

Journal of Agricultural and Food Chemistry

144

earlier described protocols 20. Briefly, proteins with equal amounts of 25 µg were determined by

145

SDS-PAGE and moved to PVDF membranes accordingly and further subjected for blockage by 5%

146

skimmed milk at room temperature for 2 h. The membranes were allowed to stand with primary antibodies

147

for 12 h at 4 °C followed by incubation with secondary antibodies for another 2 h at room temperature. The

148

final immunoreactive bands of proteins on the membranes were observed with the aid of ECL reagents and

149

images were taken by FluorChem HD2 (Protein Simple, CA, USA). Densitometrical analyses of the

150

intensities from obtained protein bands, were done by AlphaView SA (Protein Simple, CA, USA).

151

Measurement of ROS

152

Previously described 21 DCFH-DA fluorescence assay was used to detect the cellular ROS generation,

153

mainly H2O2. Initially, cells (3×104 per well) were incubated overnight in 24-well plates. The cells were

154

exposed to suitable concentrations of salicin followed by incubation with a serum-free medium containing

155

25 µM DCFH-DA at 37 °C for 30 min. Thereafter, cells were washed twice by PBS, and the fluorescence

156

intensity of dichlorofluorescein (DCF) fluorescence was observed by inverted fluorescence microscope

157

(Olympus CKX31, Tokyo, Japan) after fixation by 4% (w/v) paraformaldehyde solution. Image-Pro Plus

158

6.0 software was used to analyze of the obtained ROS fluorescence intensity.

159

Immunofluorescence staining

160

For the staining procedure, eight-well chamber slides were used for seeding the active cell cultures using

161

previously described procedure 22. The fixed cells were allowed to stand overnight with primary antibody at

162

4°C followed by incubation with Alexa Fluor-conjugated secondary antibody for another 1 h at room

163

temperature. The cells were visualized using a confocal microscope (Olympus, Tokyo, Japan).

164

Nrf2 siRNA transfection

165

FLS-RA were quickly transfected with small interfering RNA (siRNA) targeting to Nrf2 by Lipofectamine

166

2000 TM (Invitrogen) according to the manufacturer’s instructions. Nontargeting siRNA was used as a

167

negative control. After 48 h of transfection, cells were exposed to salicin for 1 h, followed by treatment

168

with IL-1β for the indicated times. The western blot analysis (BIO-RAD, Hercules, California,USA) and

169

intracellular ROS level measurements (Olympus CKX31, Tokyo, Japan) were performed by using the

170

treated cells.

171

Animals and experimental design

172

We obtained normal healthy 10 weeks old male Wistar rats weighing between 200 to 240 g from Model 7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 28

173

Animal Research Centre of Yangzhou University, Yangzhou, China. The animals were acclimatized for 10

174

days before the start of formal experimentation. During the whole period of experimental study, the animals

175

were kept at standard environmental conditions including temperature, humidity and light/dark cycle

176

(22±2 °C, 60±10%, and 12/12 h respectively); and were fed on regular rat chow and water ad libitum. All

177

the animal handling and experimental procedure were approved by the guideline of experiment animals,

178

Suzhou University and “Institutional Animal Use and Care Committee of Suzhou University”.

179

A total of 24 animals were categorized into 3 groups each comprising 8 rats (n=8). In order to develop

180

arthritis in Wistar rats, other than vehicle control group, the animals were treated with collagen type II

181

(collagen induced arthritis (CIA) model) associated with Freund’s adjuvants (day 0 to 10). The 16 CIA

182

animals were then again casually divided into two groups for further, thus a total of 3 groups were made.

183

Group one (G1) was taken as vehicle control group, group two (G2) was collagen induced arthritis group

184

without any treatment, while group three (G3) was collagen induced arthritis along with a treatment of

185

salicin 15 mg/kg. The treatment was given to Wistar rats for 38 days after the induction of arthritis, through

186

intragastric route with a dose gap of 48 h. Two independently blinded operators were employed for the

187

assessment of clinical symptoms and limb score. The arthritis scores (ranging from 0 - 3) against each limb

188

were assessed following the protocols as described earlier 2. At the termination (48th day after the first

189

post-immunization), the animals were euthanized with 60 mg/kg pentobarbital (administered through

190

intra-peritonial route) and appropriate samples were taken for further analyses.

191

Histochemical analysis

192

As per the previous study 23, excised mice hind limbs were fixed in 10% neutral buffered formalin followed

193

by decalcification of the tissues in 10% EDTA and then after embedding in paraffin. The sections (5µm

194

thick) were made by paraffin slicing machine (Leica, Wetzlar, Germany) and stained with Hematoxylin and

195

Eosin (H and E).

196

SOD, MDA, GSH, and CAT measurements

197

Tests were performed calorimetrically using rat’s joint tissue homogenate. SOD, MDA, GSH, and CAT kits

198

were performed.

199

Statistical analysis

200

All the experiments were performed in triplicates and the data were presented as means ± standard error of

201

mean (SEM). While the significance of the difference was calculated using Student t test or one way 8

ACS Paragon Plus Environment

Page 9 of 28

Journal of Agricultural and Food Chemistry

202

ANOVA followed by Dunnett post hoc test. p-value