Liquiritin from Glycyrrhiza uralensis attenuating rheumatoid arthritis via

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Bioactive Constituents, Metabolites, and Functions

Liquiritin from Glycyrrhiza uralensis attenuating rheumatoid arthritis via reducing inflammation, suppressing angiogenesis and inhibiting MAPK signaling pathway Kefeng Zhai, Hong Duan, Cai-yue Cui, Yu-yao Cao, Jia-li Si, Hui-jiao Yang, Yong-chao Wang, Wen-gen Cao, Gui-zhen Gao, and Zhao-Jun Wei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00185 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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Journal of Agricultural and Food Chemistry

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Liquiritin from Glycyrrhiza uralensis attenuating rheumatoid arthritis via reducing

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inflammation, suppressing angiogenesis and inhibiting MAPK signaling pathway

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Ke-feng Zhai†,‡, Hong Duan*,†, Cai-yue Cui†, Yu-yao Cao$, Jia-li Si†, Hui-jiao Yang†,

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Yong-chao Wang†, Wen-gen Cao†, Gui-zhen Gao†, Zhao-Jun Wei*,$

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†Suzhou

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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

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

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‡Department

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Nanjing, 210002, China

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$School

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P.R. China.

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

of Food and Biological Engineering, Hefei University of Technology, Hefei, 230009

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*Corresponding authors.

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Correspondence to Dr. Hong Duan, E-mail: [email protected], Fax & Tel:

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+86-557-2871037

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Correspondence to Dr. Zhao-Jun Wei, E-mail: [email protected], Tel: +86-551-62901539

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ACS Paragon Plus Environment


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Abbreviations used: RA, Rheumatoid arthritis; RA-FLSs, Human rheumatoid arthritis

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fibroblast-like synoviocytes; TNF-α, Tumor necrosis factor alpha; IL-1β, Interleukin-1β;

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MAPKs, mitogen-activated protein kinases; JNKs, the c-Jun N-terminal kinases; ELISA,

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Enzyme-linked immunosorbent assay; CIA, Collagen induced arthritis; VEGF, Vascular

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endothelial growth factor

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Abstract

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Among the various treatments, induction of synoviocytes apoptosis by natural products during

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rheumatoid arthritis (RA) pathological condition can be considered of vast potential. However,

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it is unclear that liquiritin, a kind of natural flavonoid extracted from the roots of Glycyrrhiza

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uralensis, induced the apoptosis of synovial membrane and its molecular mechanism. In this

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study, interleukin-1β (IL-1β)-RA-FLS cells were incubated with different concentration of

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liquiritin. MTT assay, Hoechst 33342 staining, JC-1 staining and western blot were used to

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check the viability, cell apoptosis, mitochondrial membrane potential changes and the

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expression of related proteins, respectively. In vivo, TUNEL assay and HE staining of tissue

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were used for histopathological evaluation. Our results showed that liquiritin significantly

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inhibited proliferation of IL-1β-induced-RA-FLS, promoted nuclear DNA fragmentation and

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changed the mitochondrial membrane potential to accelerate cell apoptosis. Liquiritin

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down-regulated the ratio of Bcl-2/Bax and inhibited the VEGF expression and

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phosphorylation of JNK and P38. Moreover, liquiritin improved the clinical score of

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rheumatism, inflammatory infiltration and angiogenesis, and induced apoptosis of the

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synovial tissue in vivo. Hence, liquiritin ameliorates RA by reducing inflammation, blocking

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MAPK signaling, and restraining angiogenesis.

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Key words: Liquiritin; Rheumatoid arthritis; Inflammation; MAPK signaling pathway;

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Angiogenesis

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Introduction

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Rheumatoid arthritis (RA) is a kind of autoimmune diseases that causes bone joint or cartilage

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damage.1,2 Although the pathogenesis of RA has not been clearly validated, the propagation

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of fibroblast-like synoviocytes (FLSs) in RA synovium is considered to be pathological

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marker.3 RA-FLSs are stimulated by inflammatory cytokines, and display cancer-related

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properties, including abnormal proliferation and apoptosis resistance, which can severely

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damage articular tissue.4,5 Consequently, it is evident that accelerated apoptosis of FLSs may

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be an effective treatment for RA.

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The systematic cell death is known as apoptosis and its regulated by receptor mediated and

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mitochondrial-mediated pathways to retain homeostasis.6-8 Apoptosis affects the cytochrome

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c,9,10 mitogen-activated protein kinases (MAPKs).11,12 The signaling pathway (p38 and c-Jun

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N-terminal kinases (JNKs) induces apoptosis by activating caspases.13,14

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Liquiritin is a kind of flavonoid compound from the root of Glycyrrhiza uralensis.15 Various

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pharmacological activities, such as neuroprotective,16 antioxidant17, 18 and anti-myocardial

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fibrosis effects of liquiritin were demonstrated.19 In addition, liquiritin has recently been

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reported to induce apoptosis. Previously, liquiritin has induced apoptosis of lung cancer cells

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through the up regulation of p53.20 Liquiritin combined with cisplatin could seize the cell at

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G0/G1, through down regulation of cyclin A, cyclin D1, CDK4 and upregulation of p53 and

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p21.21

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Since, the effect of liquiritin on the apoptosis of RA-FLSs in RA remains unexplored.

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Therefore, we examined the outcome of liquiritin exposure on the apoptosis of RA-FLS and

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its molecular mechanism.

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Materials and methods

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Chemicals

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Liquiritin (PubChem CID: 503737) (Figure 1A) extracted and separated from the Glycyrrhiza

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uralensis which was obtained from Yanchi, Ningxia Province, China. For the extraction, ratio

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of solid to liquid was set at 1:40 which was followed by purification by macroporous resin

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adsorption. The resulting extraction was additionally separated with preparative liquid

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chromatograph column which was further treated with sephadex LH-20 and polyamide

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column. Purity of liquiritin was examined by HPLC-UV (Supplementary Figure 1A) and

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identification was completed using NMR (Supplementary Figure 1B). Liquiritin was

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dissolved into DMEM for cell processing and saline solution for animal level experiments,

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respectively.

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Cell culture media, antibiotics and reagents were obtained from Life Technologies (Carlsbad,

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CA, USA). HE staining kit, hoechst 332342 staining kit, DAPI, trypsin, BCA protein

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concentration determination kit and JC-1 mitochondrial membrane potential assay kit

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(Nantong, China).

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Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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bromide (MTT) were procured from Sigma (St. Louis, MO, USA). ELISA kits for IL-1β,

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

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antibodies against Bax, Bcl-2, COX-IV, cytochrome C, and pro-caspase-3 were purchased

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from Cell Signalling Technology (Beverly, MA, USA); antibodies against β-actin, JNK, p38,

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P-JNK, P-p38, and GAPDH were purchased from EnoGene BIO Inc. (Nanjing, China);

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antibody to Vascular endothelial growth factor (VEGF) detection kit was purchased from

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Boster Biological Technology (Wuhan, China). Secondary antibodies were obtained from

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Bioworld Technology Inc. (St. Louis Park, MN, USA). TUNEL brightgreen apoptosis

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detection kit was obtained from Vazyme Biotech (Nanjing, China).

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Cell culture

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Primary RA-FLSs (ATCC, Manassas, VA, USA) were cultured, digested and passaged until

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confluence of 90% was achieved.22 RA-FLSs at four to ten generation are selected for

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experiment.

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Cell viability assays

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For the viability evaluation, RA-FLSs (8 × 104 cells/well) were incubated with range of

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liquiritin (0, 0.345, 3.45, 34.5 and 345 μmol/L) for 48 h followed by adding of 100 μL of

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MTT (5 mg/mL) and then further incubation at 37 °C for 4 h. After centrifugation,

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supernatant was discarded and DMSO (150 μL) was added followed by oscillation for 10 min

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to make crystal dissolution prior to absorbance measurement at 570 nm (Thermo, Waltham,

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MA, USA). MTT assay was also used to detect the apoptosis effect of liquiritin on

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IL-1β-induced RA-FLSs.22 The cells were pretreated with increasing concentrations (0, 0.345,

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3.45, 34.5 and 345 μmol/L) of liquiritin for 48 h with or without IL-1β (10 ng/mL).

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Hoechst 33342 staining

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In this study, the effect of liquiritin on RA-FLSs apoptosis was noticed by hoechst 33342

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staining. RA-FLSs (3.5 × 105 cells/well) were exposed to various concentrations of liquiritin

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(0, 0.345, 3.45 and 34.5 μmol/L) for 48 h with or without IL-1β (10 ng/mL). The RA-FLS

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cells in 24-well plates were flushed thrice with cold PBS and fixed with 4%

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paraformaldehyde for 30 min after 48 h, and then washed with PBS again and stained with

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Hoechst 33342 for 30 min in the dark. Finally, they were observed by fluorescence

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microscopy (model number: CKX31; Olympus, Japan).

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JC-1 staining

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RA-FLSs (3.5 × 105 cells/well) were exposed to various concentrations of liquiritin (0, 0.345,

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3.45 and 34.5 μmol/L) for 48 h with or without IL-1β (10 ng/mL). The RA-FLS cells were

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washed with cold PBS, mixed with 1 mL of DMEM and 1 mL of JC-1 staining solution, and

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then incubated at 37 °C. After incubation, it was flushed with JC-1 Buffer (1×) and 2 mL of

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cell culture medium was added prior to fluorescence microscopy (model number: CKX31;

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Olympus, Japan).

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Western blot Analysis

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RA-FLSs cultured in 60 mm dishes were consequently cleaned three times in PBS, incubated

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with 60 μL of lysis buffer, and stored at -80 °C for 10 min. The RA-FLS cells were scraped

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and transferred into the doff tube, nurtured with lysis buffer, and centrifuged at 12,000×g for

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5 min. After centrifugation, the upper layer was transferred into another doff tube. 4 μL of

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each sample was for taken for quantitative, and the remaining samples were added to loading

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buffer at 100 °C for 10 min. Equal amounts (30 μg) of protein samples were resolved by

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SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene

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difloride (PVDF) membranes. The primary and secondary antibodies were incubated

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overnight and washed.23 Signals were visualized using ECL reagents, and images were

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acquired using the FluorChem HD2 Imaging system (Protein Simple, CA, USA).

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In vivo experiment design

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Male wistar rats (200 to 240 g) were obtained from Yangzhou University, Yangzhou, China.

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The air flow in the animal room was unobstructed with 12 h light/12 h dark cycle with

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relative humidity 55% - 65% and temperature (22 ± 2) °C. All rats were fed with conventional

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methods, monitored and recorded in general. All animal procedures comply with the

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application of the guidelines for experimental animal care at Suzhou University. Wistar rats

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divided into three categories after normal feeding for 10 days with 8 rats in each group. The

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three groups were named as: vehicle control; the CIA group; the CIA + liquiritin group (8 mg

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kg-1 liquiritin was administered to the rats by intragastric route from day 11 to day 48). The

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rats were treated with a mixed solution of type II collagen and an equal amount of Freund's

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adjuvant (collagen induced arthritis (CIA) model).1,2,24 The clinical symptoms and scores of

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the extremities were confirmed by two independent blinded operators after the second

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immunization. Each limb of clinical arthritis was assessed using a range of 0-3.24

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Histochemical analysis

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For this, the hind limbs and overlying skin were fixed in 10% neutral buffered formalin.

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Tissue was fixed in 10% ethylene diamine tetraacetic acid (EDTA) paraffin and sectioned (5

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μm thick). Then after, staining was completed with HE and TUNEL, respectively.

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Measurement of cytokines by ELISA

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Serum samples were used cytokines determination (i.e. IL-1β, TNF-α and IL-6) by using

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ELISA kits.

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Statistical analysis

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The data was analyzed using one-way analysis of variance (ANOVA) followed by Dunnett

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post hoc test. p-value

Liquiritin from Glycyrrhiza uralensis attenuating rheumatoid arthritis via

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