Salicin from Alangium chinense Ameliorates Rheumatoid Arthritis by

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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

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Journal of Agricultural and Food Chemistry

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Salicin from Alangium chinense ameliorates rheumatoid arthritis by

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modulating the Nrf2-HO-1-ROS pathways

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Short title: Salicin attenuates rheumatoid arthritis by reducing oxidative stress

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

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

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†

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

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Pharmaceutical Biotechnology, School of Biological and Food Engineering, Suzhou

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

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‡

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Nanjing, 210002, P.R. China.

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††

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

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#

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Punjab, Lahore, 54000 Pakistan.

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$

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China.

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§

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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

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*Corresponding authors.

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

ACS Paragon Plus Environment


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

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

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

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

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

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+86-551-62901539

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

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

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

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

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

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

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Abstract

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

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

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

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

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

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

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

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

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

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

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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

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

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

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pathways in RA-FLSs

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

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Introduction

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

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

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

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

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

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

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

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

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

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

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

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against RA.

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

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

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

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

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

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

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

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

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

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

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

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inhibition of ROS 13.

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

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

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

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


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

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

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

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

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

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Materials and methods

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Chemicals

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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Primary RA-FLSs culture

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

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

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

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

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

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within two to eight passages.

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Cell viability assays

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

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

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

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

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

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

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

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

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

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to optical density of control cell group.

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Measurement of cytokines by ELISA

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

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

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Western blot analysis

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


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

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

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

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

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

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

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

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Measurement of ROS

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

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

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

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

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

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

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

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Immunofluorescence staining

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

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

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

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

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Nrf2 siRNA transfection

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

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

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

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

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

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treated cells.

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Animals and experimental design

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Histochemical analysis

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

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

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

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Eosin (H and E).

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SOD, MDA, GSH, and CAT measurements

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

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were performed.

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Statistical analysis

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

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


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ANOVA followed by Dunnett post hoc test. p-value

Salicin from Alangium chinense Ameliorates Rheumatoid Arthritis by

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