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Chicoric Acid ameliorates lipopolysaccharide induced oxidative stress via promoting Keap1/Nrf2 transcriptional signaling pathway in BV-2 microglial cells and mice brain Qian Liu, Yaya Hu, Youfang Cao, Ge Song, Zhigang Liu, and Xuebo Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04873 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 24, 2016

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Journal of Agricultural and Food Chemistry

Chicoric Acid ameliorates lipopolysaccharide induced oxidative stress via promoting Keap1/Nrf2 transcriptional signaling pathway in BV-2 microglial cells and mice brain

Qian Liu, Yaya Hu, Youfang Cao, Ge Song, Zhigang Liu*, Xuebo Liu* Laboratory of Functional Chemistry and Nutrition of Food, College of Food Science and Engineering, Northwest A&F University, Yangling, China

Corresponding author: Dr. Zhigang Liu, College of Food Science and Engineering, Northwest A&F University, 28. Xi-nong Road, Yangling 712100, China. Tel: +86-029-87092817; Fax: +86-029-87092817; E-mail: [email protected] Prof. Xuebo Liu, College of Food Science and Engineering, Northwest A&F University, 28. Xi-nong Road, Yangling 712100, China. Tel: +86-029-87092325; Fax: +86-029-87092325; E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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As a major nutraceutical component of a typical Mediterranean vegetable chicory,

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chicoric acid (CA) has been well-documented due to its excellent antioxidant and

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anti-obesity bioactivities. In current study, the effects of CA on lipopolysaccharide

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(LPS)-stimulated oxidative stress in BV-2 microglia and C57BL/6J mouse and

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underlying molecular mechanisms were investigated. Results demonstrated that CA

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significantly reversed LPS-elicited cell viability decrease, mitochondrial dysfunction,

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activation of NFκB and MAPK stress pathway, and inflammation responses via

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balancing cellular redox-status. Furthermore, molecular modeling study demonstrated

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that CA could insert into the pocket of Keap1 and up-regulated Nrf2 signaling, thus

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transcriptionally regulated downstream expressions of antioxidant enzymes including

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HO-1 and NQO-1 in both microglial cells and i.p. injection of LPS-treated mice brain.

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These results suggested that CA attenuated LPS-induced oxidative stress via

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mediating Keap1/Nrf2 transcriptional pathways and downstream enzymes expressions,

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which indicated that CA has great potential as a nutritional preventive strategy in

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oxidative stress-related neuroinflammation.

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KEYWORDS

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Chicoric acid; lipopolysaccharide; oxidative stress; Keap1/Nrf2; microglia

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INTRODUCTION

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Oxidative stress plays a crucial role in a variety of diseases including diabetes,

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neurodegenerative diseases and cancer. 1, 2 The intercellular production and cleanup of

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free reactive oxygen species (ROS) exists in homeostasis, and excessive ROS will

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cause mitochondrial dysfunction, reduce the levels of ATP and Ca2+, eventually

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leading to cell apoptosis.

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lipopolysaccharide (LPS), a main component of gram-negative bacteria cell walls,

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triggers the phosphorylation of a series protein kinase through specific receptor CD14

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and TLR4 on the cell membrane, releases enormous amounts of ROS, resulting in

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activation of MAPKs and NFκB signaling pathways.

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shown that microglia is the main target of LPS in the brain. Treatment with LPS leads

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to the augment of inflammatory responses and oxidative stress in microglial cells. 6

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Researches indicated that the combination of

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Accumulated studies have

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The intervention of oxidative status in microglia can reduce the risk of some

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neurodegenerative illnesses and developmental disorders. 7 Transcription factor Nrf2,

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a predominant controller of one defense mechanism against oxidative stress damage,

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regulates the induction of various defensive genes encoding detoxifying enzymes such

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as heme oxygenase 1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), and

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antioxidant

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peroxidase (GPx), and catalase (CAT), exerting a variety of cytoprotection effects,

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such as anti-inflammatory response, anti-carcinogenicity, and so forth.

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years, numerous research have found that dietary phytochemicals exerted powerful

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cytoprotection potentials via the activation of Keap1/Nrf2 systems. 9 Previous study

enzymes,

including

super

oxide

dismutase (SOD), glutathione

3


8

In recent


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had demonstrated that apigenin and luteolin markedly activated PI3K/Nrf2/ARE

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signaling pathways, and inhibited expressions of iNOS stimulated by LPS.

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Additional research also showed that caffeoylglycolic acid methyl ester up-regulated

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Nrf2 and HO-1 expressions through promoting PI3K and JNK pathways, which

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suppressing the release of pro-inflammatory cytokines such as prostaglandin

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E2 (PGE2) and interleukin-6 (IL-6) in LPS-induced RAW264.7 cells. 11

10

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Chicory (Cichorium intybus L. var. foliosum, Belgian endive), a typical

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Mediterranean vegetable with a bitter taste is gaining increasing interests due to its

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nutritional values and medicinal characteristics. 12 As a major component of chicory,

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chicoric acid (CA), has been regarded as a nutraceutical to have powerful antioxidant,

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and anti-obesity activities, which also existing extensively in Echinacea, lettuce,

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Orthosiphon aristatus and other edible plants and vegetables.

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structure of CA is shown in Fig. 1A. Our previous study demonstrated that CA and its

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metabolites caffeic acid and caftaric acid all exerted remarkable inhibition to DPPH•,

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•OH, ABTS•+ free radicals, and the scavenging activities of CA to these free radicals

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were significantly higher than its metabolites. 15 CA was reported to exert inhibition to

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the production of TNF-α and IL-1β via suppression of NFκB signaling pathways in

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HMC-1 human mast cells. And CA and luteolin synergistically attenuated

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inflammation through the suppression of PI3K/AKT and NFκB signaling pathways in

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LPS-induced RAW264.7 cells.

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CA distributed rapidly and widely in various tissues, and was able to cross the

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blood-brain barrier.

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13, 14

The chemical

Additional in vivo research also demonstrated that

Based on these reports, complementary studies are needed to 4


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determine whether the beneficial effects of CA are applicable to balancing redox

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status in the brain and the intervention of neuroinflammatory response.

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Therefore, this study was intended to evaluate the effects of CA on oxidative

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stress in vivo and in vitro by (a) determining effects of CA on LPS-impaired BV-2

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microglia cell viability and mitochondria function; (b) detecting effects of CA on

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LPS-induced BV-2 cells and mice redox-status imbalance including the production of

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ROS and the levels of antioxidant enzymes; (c) examining of effects of CA on

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redox-sensitive signaling such as PI3K/AKT and MAPKs, and transcriptional

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pathway NFκB; and (d) uncovering the effects of CA on key regulator during stress

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reaction-Nrf2/Keap1 activation. Above all, it provides novel insights into the

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mechanisms of CA on the regulation of LPS-induced redox-status equilibrium and

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neuroinflammation.

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MATERIALS AND METHODS

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Reagents and antibodies

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Chicoric acid (purity ≥ 98%) was purchased from Weikeqi Biological

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Technology

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3-(4,5-dimethylthiazol-2-yl)-2,5-

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2’,7’-dichlorofluorescin diacetate (DCFH-DA) and N-acetyl-L-cysteine (NAC) were

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obtained from Sigma (St. Louis, MO, USA). JC-1 dye and Fura-2 AM were obtained

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from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). ATP, MDA,

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GSH, SOD and CAT assay kits were purchased from Nanjing Jiancheng

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Bioengineering Institute (Nanjing, China).

Co.,

Ltd.

(Sichuan,

China).

Lipopolysaccharide

diphenyltetrazolium

bromide

(LPS), (MTT),

Enzyme-linked immunosorbent assay

5



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(ELISA) kits for PGE2 and cAMP were purchased from Shanghai Xinle

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Biotechnology (Shanghai, China). Amplex Red Hydrogen Peroxide/Peroxidase Assay

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Kit was obtained from Invitrogen (California, USA). All other reagents were made in

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China and were of HPLC-grade or the highest commercially available grade.

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Antibodies against ND1 (SC-20493), COX2 (SC-65239), β-actin (SC-47778),

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Lamin B (SC-6217), COX-2 (SC-1747), HO-1 (SC-1796), NQO-1 (SC-16464), Nrf2

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(SC-722) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

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Antibodies against iNOS (2982), NFκB p65 (8242), IκB (9242), p-IκBα (Ser32/36)

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(5A5), p-p44/42 MAPK (ERK1/2) (9101), p44/42 MAPK (ERK1/2) (9102),

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p-SAPK/JNK (Thr183/Tyr185) (9251), SAPK/JNK (9252), p-p38 MAPK (9211),

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p38MAPK (9212), p-AKT (9271) and AKT (9272) were purchased from Cell

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Signaling Technology Company (Shanghai, China).

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Animals and treatment

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3 month-old C57BL/6J mice were obtained from Xi’an Jiaotong University (Xi’an,

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Shaanxi, China). Mice were housed in the animal facility under standard conditions

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(12/12 light-dark cycle, humidity at 50 ± 15%, temperature 22 ± 2°C) and fed with a

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standard diet (AIN-93M). Mice were assigned into Control, LPS, and CA+LPS group

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(n = 10/group). CA treatment group received 0.05% CA in drinking water for 45 days.

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LPS and CA+LPS group mice were intraperitoneally injected LPS (0.25 mg/kg body

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weight/day, dissolved in saline) while control group mice were injected normal saline

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for 9 days. Subsequently, mice were sacrificed, and plasma and brain samples were

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collected and stored at -80 °C for further detection. The animal protocol was approved 6


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by the animal ethics committee of Xi’an Jiaotong University. All of the experimental

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procedures were followed by Guide for the Care and Use of Laboratory Animals:

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Eighth Edition, ISBN-10: 0-309-15396-4, and all surgery were performed under

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anesthesia and all efforts were made to minimize animal suffering.

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Cell culture and treatment

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Mouse microglial (BV-2) cells were purchased from Kunming Institute of

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Zoology, Chinese Academy of Sciences (Kunming, China), and cultured in RPMI

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1640 medium (Gibco Co., USA) supplemented with 10% FBS, 100 IU/ml penicillin,

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and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO2. BV-2

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microglial cells were pretreated with CA for 4 h, and then treated with LPS for 12 h

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after washing with PBS. And then the cells were collected for further detected.

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Cell viability assay

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Cell viability was detected by MTT assay according to previous method. 18 BV-2

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cells were seeded in a 96-well plate at a density of 1×106 cells/well at 37 °C with 5%

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(v/v) CO2. After various treatments, the medium was removed, followed by incubation

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with 0.5 mg/ml MTT for 4 h at 37 °C. 100 µl of DMSO was added to each well to

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dissolve the formazan crystals. The optical density (OD) at 490 nm was measured

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with a microplate reader (Bio-Rad Laboratories, China). Cell viability was expressed

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as a percentage of the control group (untreated cells).

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Cell morphology

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BV-2 cells were seeded in a 6-well plate at a density of 1×104 cells/well at 37 °C

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with 5% (v/v) CO2 overnight. Cells were pretreated with 80 µM CA for 4 h, and then 7



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treated with LPS (1 µg/ml) for 12 h. Cell morphology were observed by an inverted

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fluorescence microscope.

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Analysis of mitochondrial membrane potential

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Mitochondrial membrane potential (MMP) was determined using the

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mitochondrion-specific lipophilic cationic fluorescence dye JC-1 as described in

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previous research. 19 Cells were seeded into 96-well plates (7.0 × 104 cells/well) and

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pre-incubated with 80 µM CA for 4 h and incubated with LPS for 12 h. Cells were

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then treated with 5 µg/ml JC-1 for 1.5 h at 37 °C in the dark and were washed twice

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with PBS and fluorescence intensity was measured using a multimode microplate

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reader (Molecular Devices Co., Sunnyvale, CA, USA) at 485 nm excitation, 585 nm

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and 538 nm emission, respectively. The values were expressed as the OD585/OD538

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ratio.

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Detection of intracellular redox status and H2O2 production

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The cell's redox status was detected by fluorescence dye H2DCFDA.

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After

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pre-incubated with 80 µM CA for 4 h and incubated with LPS for 12 h, cells were

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dyed with 10 µM H2DCFDA for 30 min at 37 °C in the dark. The fluorescence were

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observed by an inverted fluorescence microscope and quantified using a multimode

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microplate reader at 485 nm excitation and 538 nm emission. The fluorescence was

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normalized by protein levels and expressed as a percentage of the control group

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(untreated cells).

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The production of H2O2 was determined by Amplex Red Hydrogen

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Peroxide/Peroxidase Assay Kit. Procedures in detail were conducted according to the 8


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manufacturer’s instructions.

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Measurement of pro-inflammatory cytokines and activities of antioxidant enzymes

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BV-2 cells were pretreated with NAC (10 μM) for 30 min with or without CA,

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and then treated with LPS (1 µg/ml) for 12 h. The content of NO in the supernatant

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was measured by Griess method. Samples were reacted with the same volume of the

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Griess reagent [0.1 % (w/v) N-(1-naphathyl)-ethylenediamine and 1 % (w/v)

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sulfanilamide in 5 % (v/v) phosphoric acid] at 37 °C for 10 min. The optical density

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(OD) at 540 nm was measured with a microplate reader (Bio-Rad Laboratories,

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China). In addition, the contents of PGE2 were determined by ELISA kit. Levels of

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MDA and activities of GSH, CAT and SOD in vivo and in vitro were determined using

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commercially available kit from Nanjing Jiancheng Bioengineering Institute (Nanjing,

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China) according to the manufacturer’s protocol.

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RNA Preparation and Quality Control

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Total RNA was extracted from brain tissue using RNA Extraction Kit (TaKaRa

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MiniBEST Universal RNA Extraction Kit, Dalian, China). The purity and integrity of

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RNA were evaluated using the Quawell 5000 UV-Vis Spectrophotometer (Quawell

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Technology, San Jose, CA, USA). RNA was stored at -80 °C prior to further analysis

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by microarray and real-time quantitative PCR (RT-qPCR).

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RT-qPCR

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Total RNA (1 mg) was reverse transcribed into cDNA using the PrimeScriptTM

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RT Master Mix reverse transcription kit (TaKaRa PrimeScript RT Master Mix, Dalian,

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China), and the mRNA expression was quantified by RT-qPCR using SYBR green 9



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PCR kit (TaKaRa SYBR® Premix Ex TaqTM II, Dalian, China) and CFX96TM

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real-time system (Bio-Rad, Hercules, CA). Gene-specific mouse primers were used as

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mentioned in Table 1. Ct values were normalized to GAPDH, and the relative gene

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expression was calculated with the 2-△△Ct method.

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Western blotting

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The treated BV-2 cells were harvested and lysed with cell lysis buffer

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(Beyotime Institute of Biotechnology, Jiangsu, China) and brain tissue were

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homogenated with normal saline. Nuclear extraction reagent (Xianfeng Biotechnology,

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Xi’an, China) was used for the separation of cytosolic extract (cytosol) and nuclear

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extract (nucleus). The total protein concentration was determined using the BCA

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Protein Kit (Thermo Fisher, Shanghai, China). Cell lysate was solubilized in SDS

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sample buffer and then immediately heated at 95 °C for 10 min. The proteins were

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separated by SDS/PAGE, and transferred onto PVDF membranes. After using

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appropriate antibodies, the immunoreactive bands were visualized with an enhanced

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chemiluminescence reagent.

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Molecular modeling

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To investigate the possible binding mode of chicory acid to Keap1 as the

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potential inhibitor, molecular docking analysis was carried out by using Autodock

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Vina 1.0.2.

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4L7B)

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http://www.rcsb.org/pdb/home/home.do) and the coordinates of chain B was extracted

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as receptor input file. The three-domination conformer of CA (PubChem CID:

21

was

The cocrystalized structure of Keap1-inhibitor complex (PDB code derived

from

RCSB

Protein

10


Data

Bank

(PDB,

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5281764)

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(https://pubchem.ncbi.nlm.nih.gov/) as ligand input file. The receptor and ligand input

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files were prepared using Graphical User Interface program AutoDock Tools 1.5.6. 22

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The search space was included in a box of 21.528 × 22.079 × 22.267 Å, centered on

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the binding site of the ligand in cocrystalized structure. The num_modes and

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exhaustiveness parameters were set to 100 and 16, respectively, and default values

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were used for other parameters. After docking the top scored pose was selected for the

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detail analysis.

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Statistical analysis

205

was

downloaded

from

PubChem

database

Data in vivo are performed as means ± SEM at least three independent

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experiments and in vitro were presented as the means ± SD. Student's t-test was used

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for determination of significant differences. Means were considered to be statistically

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distinct if p < 0.05.

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RESULTS

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Effects of CA on cell viability and mitochondria function in LPS-treated BV-2

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microglial cells

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Numerous studies had demonstrated that LPS could induce inflammation and

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even cell death in BV-2 microglial cells. 23 Morphological changes of BV-2 cells were

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observed in Fig. 1B. LPS-treated BV-2 cells showed fewer branches that were shorter

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and even resorbed into the cell body, while control group had small soma with distal

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arborization, which was consistent with previous studies. 24 Surprisingly, CA reversed

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these morphological changes induced by LPS. Moreover, as illustrated in Fig. 1D, 11



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incubation with LPS for 12 h successfully reduced BV-2 microglia cell viability, while

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CA treatment alone had no effects on it (p < 0.05). To assess the effect of CA on BV-2

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microglial cell viability, cells were pretreated with various concentration (20, 40, 80

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µM) of CA for 4 h before treated with LPS. The results showed that CA remarkably

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inhibited LPS-induced cell viability decrease and this suppression was correlated with

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the CA dosage treated (p < 0.05). 80 µM CA further inhibited LPS-induced cell

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viability decrease from 82.73% to 90.43%.

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Mitochondria are not only cellular majority “energy factory”, but also play a

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central role in regulating cellular redox status, by relasing free radicals such as H2O2,

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a second messager that mediating different cell signaling pathways. As shown in Fig.

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1C&E, LPS elicited mitochondrial membrane potential (MMP) loss, which is a

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marker of mitochondrial dysfunction, while CA significantly restored defective MMP

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(p < 0.01).

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Effect of CA on the LPS-elicited cellular redox status unbalance in BV-2 microglia

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cells

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Since intracellular ROS generated by mitochondrial under various exogenous

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stimuli have been reported participated in mediating many cell signaling pathways, 25

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the levels of intracellular ROS and H2O2 had been measured. As demonstrated in Fig.

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2A&B, LPS stimulated the generation of ROS by 36.65% compared with the control

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group, while CA significantly quenched intracellular ROS to the normal level (p

Chicoric Acid Ameliorates Lipopolysaccharide ... - ACS Publications

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