Protocatechuic Acid Attenuates Atherosclerosis by Inhibiting M1 and

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Bioactive Constituents, Metabolites, and Functions

Protocatechuic Acid Attenuates Atherosclerosis by Inhibiting M1 and Promoting M2 Macrophage Polarization Yao Liu, Xu Wang, Juan Pang, Hanyue Zhang, Jing Luo, Xiaoyun Qian, Qian Chen, and Wenhua Ling J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05719 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 2, 2019

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Journal of Agricultural and Food Chemistry

Protocatechuic Acid Attenuates Atherosclerosis by Inhibiting M1 and Promoting M2 Macrophage Polarization Yao Liu

a, b,

Xu Wang

a, b,

Juan Pang

a, b,

Hanyue Zhang

a, b,

Jing Luo

a, b,

Xiaoyun Qian

a, b,

Qian Chen a, b, Wenhua Ling a, b, c *

Author Affiliations: a

Department of Nutrition, School of Public Health, Sun Yat-Sen University (North Campus),

Guangzhou, 510080, P. R. China b

Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Guangzhou, 510080,

P. R. China c

Guangdong Engineering Technology Center of Nutrition Transformation, Guangzhou,

510080, P. R. China

* Corresponding authors and persons to whom reprint requests should be addressed: Wenhua Ling, MD, Ph. D. Department of Nutrition School of Public Health, Sun Yat-Sen University (North Campus) 74 Zhongshan Rd. 2, Guangzhou, Guangdong Province, 510080, P. R. China. Tel: +86-20-87331597; Fax: +86-20-87330446 E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Macrophage polarization has a vital impact on the progression of atherosclerosis (AS).

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Protocatechuic acid (PCA), a flavonol, displays notable atheroprotective effects, but its

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mechanisms have not been clearly defined. We investigated whether PCA attenuated AS by

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regulating macrophage polarization. PCA consumption inhibited HCD-induced plaque

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formation (17.84% and 8.21% in the HCD and HCD+PCA groups, respectively; p < 0.05) and

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inflammatory responses in apolipoprotein E-deficient (ApoE-/-) mice. Moreover, PCA

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suppressed the classically activated macrophage (M1) polarization, which decreased the

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secretion of synthesis of nitric oxide synthase (54.63% and 32.86% in the HCD and HCD+PCA

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group, respectively; p < 0.05) and proinflammatory factors. PCA promoted alternatively

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activated macrophage (M2) activation, which increased the expression of arginine I (6.97% and

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26.19% in the HCD and HCD+PCA group, respectively; p < 0.001) and antiinflammatory

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factors. PCA also regulated M1/M2 polarization in J774 cells and mouse bone marrow-derived

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macrophages. Finally, PCA reduced PI3K/Akt-mediated nuclear factor κB activation, thereby

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suppressing M1 polarization and provoked signal transducers and activators of transcription 6

16

phosphorylation and peroxisome proliferator-activated receptor γ activation, leading to

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enhancing M2 activation. Our data revealed that PCA alleviated AS by regulating M1/M2

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conversion.

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Keywords: Atherosclerosis, Protocatechuic acid, Macrophage polarization, nuclear factor κB,

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Signal transducers and activators of transcription 6

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1. Introduction

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Atherosclerosis (AS), the most important pathological basis of cardiovascular disease (CVD)

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is a multistage and complex process of inflammation.1 Intimal Macrophages exert a vital impact

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on the atherosclerotic inflammatory response, participating in all atherosclerosis-related

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processes.2,3 For one thing, migration, aggregation, proliferation, and apoptosis of macrophages

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influence the stability of the lesions of AS.4 For another thing, macrophages secrete

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proinflammatory and antiinflammatory cytokines, affecting the progression and regression of

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AS.5

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Furthermore, during the progression of atherosclerosis, macrophages faced accumulated factors

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and can mainly polarize into two different functional macrophages: the classically activated

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macrophage (M1) and the alternatively activated macrophage (M2).6,7 M1 is characterized by

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the secretion of proinflammatory cytokines, for example, interleukin 6 (IL-6), tumor necrosis

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factor α (TNF-α), and the synthesis of nitric oxide synthase (iNOS).8 Lee et al. showed that

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TNF-α boosted the progression of AS by promoting endothelial injury and eventually leading

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to aortic stiffness.9 The main features of M2 macrophages are the high level of antiinflammatory

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cytokines such as interleukin 10 (IL-10) and transforming growth factor β (TGF- β) and the

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high secretion of arginine Ⅰ (Arg-1) and the mannose receptor (CD206)

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inhibiting inflammation, these antiinflammatory factors can also reduce protease activity in the

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lesions of AS or reduce the expression of vascular inflammatory adhesion factors, thereby

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increasing the stability of AS plaques.11 The dynamic balance between M1/M2 polarization

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affects the progression and regression of atherosclerosis.6, 12, 13

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Many studies of epidemiology have reported that consumption of polyphenols had a negative 3


10, 11.

In addition to


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association with many metabolic diseases including AS.14-16 Reportedly, protocatechuic acid

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(PCA), a main bacterial metabolite of anthocyanins (ACN),17 mediated the protective effect of

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ACN on the cardiovascular system.18,19 PCA was widely found in common foods and has

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attracted widespread attention because of the capacity of inhibiting the oxidation of low-density

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lipoproteins20 and suppressing inflammatory responses.21 PCA significantly diminished the

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secretion of the inflammatory cytokines and mediators driven by lipopolysaccharide (LPS) in

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RAW264.7 cells.21 Our group have demonstrated that PCA exerted an antiatherosclerotic effect

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by enhancing cholesterol efflux,22 and suppressing monocyte adhesion and infiltration in the

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apolipoprotein E-deficient (ApoE-/-) mouse model23,24, as well as blocking vascular smooth

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muscle cell proliferation25 and inhibiting inflammatory responses in vitro.21 However, whether

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PCA might attenuate AS progression by regulating macrophage polarization remains unclear.

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In the present study, we explored the actions of PCA on macrophage polarization in vivo and

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in vitro and investigated the underlying mechanisms.

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2. Materials and Methods

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2.1 Chemicals

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PCA (purity > 97 %) was obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved

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in sterile dimethyl sulfoxide (DMSO) for in vitro experiments. Recombinant mouse interferon

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γ (IFN γ, 200 ng/mL), interleukin 4 (IL-4, 200 ng/mL), insulin-like growth factor (IGF, 100

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ng/mL), LY294002 (25 ng/mL) and A77-1726 (10 μM) were obtained from PeproTech (Rocky

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Hill, NJ, USA). Lipopolysaccharide (LPS, 20 ng/mL) was obtained from Sigma-Aldrich and

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dissolved in sterile PBS. 4


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2.2 Animals model

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Being a well-established animal model for atherosclerosis, male ApoE−/− mice (age, 6 weeks)

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were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing,

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P.R.China). Mice were randomized into three groups (n=10), i.e. normal chow diet (NCD),

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high-cholesterol diet (HCD) and HCD+PCA group. The NCD group was fed with a normal

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chow diet (MD17121, Medicience Ltd. Jiangsu, China) accompanied by a gavage of 0.9 %

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normal saline. The HCD group was fed with a high-cholesterol diet (21 % crude fat, 0.15 %

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cholesterol and 20 % casein, MD12015, Medicience Ltd. Jiangsu, China) accompanied by a

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gavage of 0.9 % normal saline. The HCD+PCA group was fed with a high-cholesterol diet

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accompanied by a gavage of PCA dissolved in 0.9 % normal saline (15 mg/kg bw) for 14 weeks.

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All mice were euthanized and sacrificed after fasting overnight. Blood samples, hearts and

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aortas were harvested according to a previous study.26 Six-week-old male C57BL/6 mice

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(SYSU Animal Center, Guangzhou, China) were fed an AIN-93G diet. All animal related

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experiments were conformed to the Guide of the Animal Care and Use Committee of Sun Yat-

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sen University. (Permit number: SYXK [Yue] 2017-0080).

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2.3 Cell culture

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2.3.1 J774A. 1 mouse macrophage cell line (J774 cells)

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J774 cells were grown in RPMI-1640 medium containing 10 % heat-inactivated fetal bovine

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serum (FBS, Gibco, Grand Island, NY, USA) at 37 C with 5 % CO2 and 95 % humidity.

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2.3.2 Mouse bone marrow-derived macrophages (BMDMs)

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BMDMs were cultured using L929-cells conditioned medium according to a previous study.27

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L929 cells could release numerous of cytokines, which promote bone marrow-derived 5



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monocytes to grow into macrophages. Briefly, L929 cells were maintained in RPMI-1640

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medium with 10 % FBS for 7 days, and then the supernatant of the L929 cells was collected

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and kept at -20 C after filtration through a 0.22-μm filter, which was then referred to as the

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L929-cells conditioned medium. After extraction from C57BL/6 mice, bones were collected

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and suspended in RPMI 1640 medium including 12.5 % FBS and 25 % L929-cells conditioned

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medium. The medium was replaced on day 3 with 10 % FBS and 10 % L929-cells conditioned

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medium. On day 7, immunofluorescence analyses were acquired to determine the purity of

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BMDMs (Supplemental Fig. 1).

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2.4 Quantification of atherosclerotic lesions

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For the lesion at the aortic sinus, hearts from mice cut transversely were imbedded into optimal

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cutting temperature (OCT) compound and stored at −80 C. Cross-sections (10 μm) of the aortic

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sinus were stained using 0.5 % Oil-Red-O (Sigma-Aldrich) for 30 min and hematoxylin

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(Servicebio, Wuhan, China) for 20 s. Then, the cross-sections were rinsed immediately under

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running water for 2 min. Representative micrographs of the lesions were acquired by Leica

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microscope (Leica MD2500, Carl Zeiss, Jena, Germany) and analyzed by Image-Pro Plus

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software (Version: 6.0, Media Cybernetics, Silver Spring, MD, USA).

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For the lesions in the whole aortas, whole aortas were cut longitudinally after removing excess

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adipose tissue and then stained in Oil-Red-O for 3 h. Afterwards, the stained aortas were placed

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in 75 % alcohol until the artery wall without lesions was cleaned. Representative images of

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lesions were captured by Leica microscope (Leica M205C) and analyzed by Image-Pro Plus

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software.

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2.5 Serum parameters measurements 6


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Serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C)

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and calcium ions were detected by using the TC assay kit, TG assay kit, HDL-C kit and calcium

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ion kit (all from Applygen, Beijing, P. R. China).

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2.6 Flow cytometry analysis

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Cells were gathered and blocked with CD16/CD32 antibody (BD Biosciences, Franklin Lakes,

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NJ, USA) on ice for 15 min. Then, cells were incubated with APC-CD11c, PE-MHC-II (M1

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macrophage markers), FITC-CD206, APC-CD163 (M2 macrophage markers) and F4/80-APC

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(macrophage marker) for 30 min on ice. After antibodies staining and washing with precooled

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PBS with 1 % FBS, cells were suspended in 500 μL of PBS with 1 % bovine serum albumin

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(BSA) and analyzed by CytExpert software (Beckman-Coulter. Miami, FL, USA). All

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antibodies were obtained from BD Biosciences and eBioscience (San Diego, CA, USA).

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2.7 Immunofluorescence

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The sections of the aortic sinus or cells were fixed in 4 % paraformaldehyde (Servicebio, Wuhan,

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China) for 30 min and permeabilized in 0.05 % TritonX-100 (Servicebio, Wuhan, China) for

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10 min. The sections and cells were blocked in 1 % BSA for 1 h and stained with primary

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antibodies against TNF-α (Cell Signaling Technology, Danvers, MA, USA), IL-10 (CST),

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F4/80 (BD Biosciences), iNOS (Abcam, Cambridge, MA, UK), Arg-1 (CST), P-p65 NF-κB

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(CST), and P-STAT (CST) at 4 C overnight, followed by the incubation of FITC anti-mouse

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IgG (Proteintech, Rosemont, IL, USA) and Cy3 anti-rabbit IgG (Proteintech) for 1 h.

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Micrographs were acquired by a laser scanning confocal microscope (Leica TCS SP5, Wetzlar,

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Germany).

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2.8 Nitric oxide (NO) production 7



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NO production in cell supernatant was detected by a total nitric oxide assay kit (Beyotime,

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Shanghai, China) according to the manufacturer’s instructions.

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2.9 Real-time quantitative reverse transcriptase (qRT-PCR)

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Total RNA was extracted from the aorta, J774 cells and BMDMs by Trizol reagent (Invitrogen

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Life Technology, Carlsbad, CA, USA) and was reversed transcribed by a PrimeScript RT

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reagent kit (Takara, Shiga, Japan). Next, qRT-PCR was conducted with a SYBR Premix

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ExTaqII (TliRNaseHPlus) kit (Takara) on a Vii7 system (Applied Biosystems, Waltham, MA,

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USA). All primers (Sangon Biotech, Shanghai, China) are listed in the Supplemental Table.

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2.10 Western blotting

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The western blotting was performed in accordance with a previous study.28 Briefly, the proteins

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of the cytoplasm and nuclei were isolated using a Nuclear and Cytoplasmic Protein Extraction

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Kit (Beyotime, Shanghai, China), and the concentrations of the proteins were quantified by a

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BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The proteins were

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loaded onto sodium dodecyl sulfate-polyacrylamide gels for electrophoresis (SDS-PAGE) for

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80 min, and then separated and transferred to a polyvinylidene fluoride (PVDF) membrane.

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After blocking in 5 % BSA for 1 h, the PVDF membranes were stained with primary antibodies.

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Rabbit anti-PI3k, anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-IκB and mouse anti-

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GAPDH, anti-NF-κB p65, anti-IκB, anti-Arg-1 antibodies were obtained from CST, rabbit anti-

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iNOS antibody was from Abcam, and anti-Lamin B antibody was from Proteintech.

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2.11 Statistical analysis

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The data are presented as the mean ± standard deviation (SD). Differences between two groups

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were analyzed by Student’s t-test and among multiple groups by one-way ANOVA combined 8


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with Tukey’s post hoc test using SPSS 22.0 (IBM Inc. Chicago, IL, USA). p value less than

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0.05 was considered statistically significant.

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3. Results

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3.1 PCA supplementation reduces atherosclerotic lesion formation and inflammatory

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responses in ApoE−/− mice

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After 14 weeks of the feeding experiment, the HCD group and the HCD+PCA group did not

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differ significantly in weight or serum lipid parameters (Supplemental Fig. 2). As shown in

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Fig. 1A and Fig. 1B, the atherosclerotic lesion area was greater both in the aortic sinus and the

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whole aorta in the HCD-fed mice as compared with the NCD-fed mice, while PCA

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supplementation significantly reduced the lesion area. Since the role of inflammation in

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promoting plaque formation has long been demonstrated,3 we also investigated the effects of

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PCA on inflammation. PCA consumption decreased the mRNA levels of proinflammatory

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enzymes and cytokines, such as iNOS, IL-6, and TNF-α, and elevated the mRNA levels of

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antiinflammatory factors such as CD206, Arg-1 and IL-10 (Fig. 1C). Immunofluorescent

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staining also showed that PCA addition greatly reduced TNF-α and increased IL-10 expression

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(Fig. 1D). Taken together, PCA inhibited the progression of atherosclerosis in ApoE−/− mice,

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which is associated with the effects of PCA on reducing inflammation.

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3.2 PCA shifts M1/ M2 polarization in ApoE-/- mice.

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Macrophages are indispensable in the formation of atherosclerotic lesions, and macrophage

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polarization has been implicated in the inflammation of the lesions.6,7 Therefore, we explored

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whether PCA suppresses inflammation and plaque formation by changing macrophage 9



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polarization. Both iNOS and F4/80 levels elevated and Arg-1 level decreased in HCD mice

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compared with NCD mice (Fig. 2A). In addition, we found that the expression of iNOS was

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positively related to the severity of AS (r = 0.638, p = 0.01). In contrast, Arg-1 level was

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negatively related to the severity of AS (r = -0.877, p < 0.01) (Fig. 2B). Therefore, the severity

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of atherosclerosis was positively correlated with M1 polarization and negatively correlated with

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M2 polarization. Interestingly, PCA obviously increased the level of iNOS but reduced the

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secretion of Arg-1 in the aortic tissues of ApoE−/− mice. Moreover, in Fig. 2C, we observed

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increased expression of major histocompatibility complex II (MHC II) and decreased secretion

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of CD206 in BMDMs of HCD-fed mice compared with those of the NCD-fed mice, but co-

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feeding with PCA reversed these changes. These findings suggested that PCA intervention

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restrained M1 polarization and enhanced M2 activation in ApoE-/- mice.

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3.3 PCA suppresses M1 polarization in vitro

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To further evaluate the effects of PCA on M1 polarization, we used J774 cells and BMDMs for

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in vitro experiments. First, after conducting qRT-PCR and western blotting with multiple

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concentrations (10, 20 50, 100 μM) of PCA, 20 μM was determined as the best intervention

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concentration (Supplemental Fig. 3). Since macrophages that are activated by toll-like receptor

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(TLR) ligands are referred to M1,12 we used IFN γ and LPS to stimulate the conversion of

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resting macrophages into M1. As shown in Fig. 3A, in both J774 cells and BMDMs, mRNA

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levels of M1 markers rose after IFN γ plus LPS stimulation, IL-6, interleukin 1β (IL-1β), iNOS,

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and TNF-α, for example, while the effects mentioned above were inhibited by PCA treatment.

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PCA inhibited the significant increase in iNOS protein expression stimulated by IFN γ plus

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LPS (Fig. 3B), which was consistent with the immunofluorescence results (Fig. 3C). Fig. 3D 10


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showed that IFN γ and LPS treatment led to an increase of NO production (6.7-fold in J774

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cells and 10.6-fold in BMDMs), which was remarkably inhibited by PCA treatment.

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Furthermore, PCA treatment reduced the secretion of MHC II and CD11c stimulated by IFN γ

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plus LPS (Fig. 3D). These results revealed that PCA plays an inhibitory role in M1 polarization.

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3.4 PCA inhibits M1 polarization via the PI3K/Akt-NF-κB-SOCS1 pathway

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Nuclear factor κB (NF-κB) activation modulates M1 polarization that is activated by TLR

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ligands.29 Therefore, we investigated whether NF-κB activation was involved in PCA-mediated

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M1 polarization. As illustrated in Fig. 4A and Fig. 4B, IFN γ and LPS stimulation promoted

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NF-κB nuclear translocation and this effect was suppressed by PCA. Western blotting was

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performed to detect the signaling pathways related to NF-κB activation including

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phosphoinositide 3-kinase (PI3K), Akt and inhibitor of NF-κB (IκB). When J774 cells were

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driven by IFN γ and LPS, Akt, IκB and NF-κB phosphorylation levels were significantly

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increased, whereas PCA pretreatment restrained these effects of IFN γ and LPS (Fig. 4A). In

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addition, the expression of suppressor of cytokine signaling 1 (SOCS1), a well-established

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negative regulator of NF-κB activation, was markedly increased by PCA treatment (Fig. 4A).

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All of the above findings suggested that PCA suppressed M1 activation by the PI3K/Akt-NF-

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κB-SOCS1 signaling pathway.

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To further explore the inhibitory action of PCA on the PI3K/Akt-NF-κB signaling pathway,

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J774 cells were treated with IGF, a well-known agonist of the PI3K/Akt pathway. As the Fig.

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4C shows, after treatment with IGF, the suppressive actions of PCA on Akt, IκB and NF-κB

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phosphorylation were attenuated. Consequently, the transposition of NF-κB was enhanced (Fig.

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4D). IGF also diminished the effect of PCA on NO production (Fig. 4E). Moreover, a selective 11



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blocker of the PI3K/Akt pathway, LY294002, was exposed to J774 cells for 2 h. Contrary to

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the effects of IGF, LY294002 further advanced the inhibition of PCA on the PI3K/Akt-NF-κB-

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SOCS1 pathway (Fig. 4F). Therefore, our findings indicated that PCA limited the activation of

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the PI3K/Akt pathway, thereby suppressing NF-κB activation and the subsequent

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proinflammatory response.

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3.5 PCA promotes M2 polarization in vitro.

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Next, we determined whether PCA also regulated M2 polarization in J774 cells and BMDMs.

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IL-4, a classic Th2 cytokine, was used to polarize macrophages toward the M2 phenotype,

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featuring high levels of Arg-1, CD206 and CD163.12 As shown in Fig. 5A, the mRNA levels

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of M2 markers, for example, CD206, Arg-1, CD163, Kruppel-like factor 4 (KLF4) and found

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in inflammatory zone 1 (Fizz1) were increased by PCA treatment. Intriguingly, western blotting

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and immunofluorescence demonstrated that PCA evoked IL-4-induced Arg-1 secretion (Fig.

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5B, Fig. 5C). Flow cytometry further revealed that IL-4 stimulation elevated the secretion of

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CD206 and CD163 whereas macrophages treated with PCA expressed much higher levels of

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these markers (Fig. 5D). In general, we found that PCA could promote macrophage

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differentiation toward the M2 macrophage.

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3.6 PCA enhances M2 polarization via the STAT6-PPAR γ pathway.

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Signal transducers and activators of transcription 6 (STAT6) activation have been demonstrated

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to have a major role in mediating the process of M2 macrophage polarization.30 We thus

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determined the effect of STAT6 signaling on PCA-regulated M2 polarization. IL-4-stimulated

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J774 cells expressed elevated protein levels of STAT6, phosphorylation of STAT6 and

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peroxisome proliferator-activated receptor γ (PPAR γ) compared with those of the control cells 12


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(Fig. 6A). Importantly, these effects could be further increased by PCA intervention (Fig. 6A).

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To validate the stimulating effect of PCA in the STAT6/PPAR γ pathway, J774 cells were

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treated with A77-1726 (10 μM), an antagonist of STAT6. Fig. 6B showed that the effects of

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PCA on STAT6, PPAR γ and the phosphorylation of the STAT6 protein were inhibited by A77-

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1726. The stimulating effect of PCA on CD206 expression was also inhibited by A77-1726

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treatment (Fig. 6C), further indicating that PCA enforced the activation of the M2 phenotype

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by promoting the STAT6/PPAR γ signaling pathway.

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3.7 PCA inhibits the phosphorylation of NF-κB but contributes to phosphorylated STAT6

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expression in ApoE-/- mice.

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According to in vitro data, the phosphorylation of NF-κB (P-NF-κB) and STAT6 (P-STAT6)

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of aortic sinuses from ApoE-/- mice were detected. The results showed that PCA-treated mice

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had lower expression levels of P-NF-κB and higher levels of P-STAT6 than HCD-fed mice,

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indicating that PCA suppressed NF-κB activation and contributed to the expression of STAT6

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phosphorylation in ApoE-/- mice (Fig. 7A).

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4. Discussion

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In this study, we investigated the effects of PCA on macrophage polarization. In the model of

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HCD-induced arteriosclerosis in ApoE-/- mice, we found that PCA inhibited HCD-induced

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lesion formation and inflammatory responses, and the anti-atherosclerotic effect was associated

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with PCA-modulated macrophage polarization. In vitro experiments also demonstrated that

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PCA altered the M1/M2 polarization in J774 cells and BMDMs which were stimulated with

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IFN γ plus LPS or IL-4. These findings demonstrated that PCA possesses the capacity to 13



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regulate macrophage polarization and has the potential to be used as a therapeutic agent for

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atherosclerosis.

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Monocytes/macrophages have been well recognized as the central participants in the

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inflammatory process during the development of atherosclerosis.6,

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proinflammatory M1 macrophages and antiinflammatory M2 macrophages has a vital impact

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on inflammatory responses and lesion formation.33 Kleinbongard et al.34 suggested that M1

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polarization negatively regulated the function of endothelial cells (ECs) and smooth muscle

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cells (SMCs) and motivated the production of reactive oxide species (ROS), resulting in

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endothelial dysfunction. This study indicated that PCA consumption blocked HCD-driven

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inflammation and arteriosclerosis lesion production in ApoE-/- mice, which was related to the

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suppression of M1 polarization and the enhancement of M2 activation of PCA.

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There are growing studies focusing on the antiinflammatory effects of polyphenols from plant

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foods.14, 35, 36 PCA is widespread in common foods, especially vegetables and foods, such as

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mushrooms (34.27 mg/100g dry weight), green chicory (30.18 mg/100g fresh weight), black

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olives (21.00 mg/100g dry weight).19 There are a few studies investigation on the human dietary

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intake of PCA. One study by Manach et al. reported that dietary PCA intake was 6-10 mg/d.37

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PCA has extensive biological effects.17,37 Min et al. indicated that the inhibition of PCA on the

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LPS-stimulated expression of the inflammatory mediators NO and prostaglandin E2 (PGE2)

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was dose-dependent in RAW264.7 cells.21 An in vitro experiment showed that PCA resisted

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lipid aggregation in THP-1-derived macrophages.38 In our previous studies, PCA exerted an

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antiatherosclerotic role by enhancing cholesterol efflux via inhibiting ATP-binding cassette

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transporter A1 (ABCA1) and G1 (ABCG1) secretion,22 suppressing monocyte adhesion23 and 14


32

The balance between

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infiltration24 associated with downregulation of vascular cell adhesion molecule 1 (VCAM-1),

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intercellular adhesion molecule 1 (ICAM-1) and CC chemokine receptor 2 (CCR2) in ApoE-/-

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mice. Moreover, our recent study reported that chicory, rich in PCA enhanced e-NOS-

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modulated endothelium-dependent vasodilation.39 However, no study has explored whether

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PCA exerts antiinflammatory and antiatherosclerotic properties by promoting the conversion

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of M1/M2 phenotype. To our understanding, this is the first time that PCA has been found to

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suppress the establishment of AS in ApoE-/- mice, which was at least partly mediated by M1/M2

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polarization.

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Wei et al.31 suggested that feeding mice an HCD for 12 weeks resulted in a proinflammatory

294

activation of macrophages in ApoE-/- mice; furthermore, the increased secretion of the M1

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phenotype was regulated by miR-342-5p. In our ApoE-/- mouse model, we found that feeding

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mice a high cholesterol diet for 14 weeks promoted the progression of plaque formation and the

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inflammatory response. Importantly, compared with the control group, we observed increased

298

expression of M1 markers and decreased secretion of M2 markers in the HCD group, but co-

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feeding with PCA reversed these changes. The concentration of PCA used in our previous

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animal studies related to anti-atherosclerosis was between 5-30 mg/kg body weight.22-24 In this

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study, we chose 15 mg/kg body weight as the consumption concentration of PCA. Despite the

302

different concentrations, PCA has the same anti-inflammatory and anti-atherosclerosis effects,

303

which leads us to speculate whether PCA has an optimal intervention concentration. In addition,

304

our group has previously measured the part of pharmacokinetic parameters in the plasma of

305

ApoE-/- mice orally administrated PCA with 25 mg/kg body weight. The results demonstrated

306

that plasma PCA reached the maximum levels (4380.8440.6 nmol/L) at 0.5 h and was 15



307

undetectable at 8 h after the treatment.24 It has been well known that the cellular responses to

308

phytochemicals varied greatly between in vivo and in vitro system. Therefore, in in vitro

309

experiments, we explored different concentration of PCA (at 10, 20 50, 100 μM) to examine

310

their influence on macrophage transform to M1 and M2 phenotypes and chose 20 μM as the

311

intervention concentration of PCA.

312

A few investigations have demonstrated that other polyphenols can also inhibit inflammatory

313

responses by modulating M1/M2 polarization. Aharoni et al.40 reported that pomegranate juice

314

and its polyphenols notably increased IL-10 expression and promoted antiinflammatory M2

315

macrophage activation. A recent study indicated that curcumin could be considered a novel

316

antiinflammatory agent due to its capacity of suppressing M1 polarization and enhancing M2

317

activation.41 In addition, Dugo et al.42 suggested that polyphenols extracted from cocoa also had

318

a similar effect on M1/M2 polarization. Nevertheless, detailed molecular mechanisms have not

319

been investigated in these studies.

320

Some studies found that a few cytokines are able to stimulate M1/M2 polarization, such as LPS,

321

IFN γ, IL-4 and revealed possible pathways involved in the regulation of M1/M2 phenotype

322

polarization.12,

323

macrophages by activating the NF-κB signaling pathway, characterized by increased activity

324

and transposition into the nucleus. PCA reduced NF-κB (p65) secretion and activation in mouse

325

aortic endothelial cells and RAW 264.7 cells.21 Notably, Lin et al.45 indicated that PCA blocked

326

the nuclear translocation of NF-κB, rather than blocking the activation of other nuclear

327

transcription factors such as activator protein 1 (AP-1). In this study, PCA counteracted NF-κB

328

activation and then promoted the expression of SOCS1, a classic negative feedback regulatory

13, 43

Arranz et al.44 reported that LPS polarized macrophages into M1

16


Page 16 of 35

Page 17 of 35


329

factor of the JAK-STAT pathway, which plays an inhibitory role in the process of

330

inflammation.46, 47 The STAT6 signaling pathway is a classic pathway that regulates the IL-4-

331

induced M2 polarization.30 IL-4 activates the JAK pathway by increasing the phosphorylation

332

of JAK, which accumulates STAT6 and promotes the phosphorylation of STAT6.11

333

Phosphorylated STAT6 can directly bind to KLF4 and PPAR γ, thereby contributing to M2

334

polarization. Similarly, our findings demonstrated that PCA could further activate the STAT6

335

signaling pathway and promote M2 polarization on the basis of IL-4 stimulation.

336

In summary, our data concluded that PCA alleviated AS by inhibiting M1 polarization and

337

promoting M2 activation. Previous studies also reported that PCA could inhibit AS progression

338

by reducing inflammatory cell infiltration, promoting cholesterol efflux, and suppressing

339

smooth muscle cell proliferation and endothelial cell dysfunction.22,

340

results of previous studies with those of our present study, we might conclude that PCA could

341

serve as a therapeutic agent for atherosclerosis. The potential clinical application of PCA in AS

342

need to be explored in further studies.

24, 25, 48

Combining the

343 344

Abbreviations used

345

ACN, anthocyanins; ApoE-/-, apo lipoprotein E-deficient; Arg-1, arginine I; AS,

346

atherosclerosis; BMDMs, bone marrow-derived macrophages; BSA, bovine serum albumin;

347

CD206, mannose receptor; COX-2, cyclooxygenase-2; CVD, cardiovascular disease; ECs,

348

endothelial cells; ERK, extracellular regulated protein kinase; FBS, fetal bovine serum; Fizz1,

349

found in inflammatory zone 1; HDL-C, high-density lipoprotein cholesterol; IFN γ, interferon

350

γ; IGF, insulin-like growth factor; IκB, inhibitor of NF-κB; IL-1β, interleukin 1β; IL-4, 17



351

interleukin 4; IL-6, interleukin 6; IL-10, interleukin 10; iNOS, synthesis of nitric oxide

352

synthase; LPS, lipopolysaccharide; JNK, c-JunN-terminlkinase; KLF4, kruppel-like factor 4;

353

MAPK, mitogen-activated protein kinase; MHC II, Major histocompatibility complex II; NF-

354

κB, nuclear factor κB; NO, nitric oxide; OCT, optimal cutting temperature; PCA,

355

protocatechuic acid; PG E2, prostaglandin E2; PI3K, phosphoinositide 3-kinase; PVDF,

356

polyvinylidene fluoride; PPAR γ, peroxisome proliferators-activated receptor γ; ROS, reactive

357

oxide species; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide

358

gels for electrophoresis; SMCs, smooth muscle cells; SOCS3, suppressor of cytokine

359

signaling 3; SRs, scavenger receptors; STAT6, signal transducers and activators of

360

transcription 6; TC, total cholesterol; TG, triglyceride; TGF-β, transforming growth factor β;

361

TNF-α, tumor necrosis factor α.

362 363

Funding

364

This work was received funding from the Major Projects of Guangzhou Health Collaborative

365

Innovation [grant number 201604020002], the State Key Program of National Natural Science

366

Foundation of China [grant number 81730090] and the Guangdong Science and Technology

367

Project [grant number 2016A050502013].

368 369

Supporting Information Description

370

Information of primer Sequences used for qRT-PCR; purity determination of BMDMs; the

371

effects of PCA on weight, serum TC, TG, HDL-C and calcium ions in ApoE-/- mice;

372

determination of the best intervention concentration of PCA. 18


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(48) Chan, K.; Chui, S. H.; Wong, D. Y.; Ha, W. Y.; Chan, C. L.; Wong, R. N. Protective effects

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516

induced endothelial dysfunction. Life Sci. 2004, 75, 3157-3171.

25



517

Figure captions

518

Figure 1. PCA inhibits the progression of atherosclerosis. Male 6-week-old ApoE-/- mice

519

were divided into three groups: NCD (normal chow diet, gavage of 0.9% normal saline); HCD

520

(high-cholesterol diet, gavage of 0.9% normal saline); HCD+PCA (high-cholesterol diet,

521

gavage of 15 mg/kg bw PCA) and were fed for 14 weeks. (A) Quantification of the lesion area

522

in Oil-Red-O stained aortic sinuses of ApoE−/−mice (n = 8) (B) Representative images and

523

quantification of Oil-Red-O stained atherosclerotic lesions in whole aorta. (n = 6) (C) mRNA

524

levels of M1 markers and M2 markers in the aorta. (D) Representative images of the expression

525

of TNF-α and IL-10 in the aortic sinus. Data are shown as the means ± SD. *p < 0.05, NCD vs.

526

HCD; #p < 0.05, HCD vs. HCD+PCA.

527

Figure 2. PCA modulates macrophage polarization in the progression of atherosclerosis

528

in ApoE-/- mice. (A) Representative images of co-expression of F4/80 (green) and iNOS (red)

529

or Arg-1 (red) in aortic sinuses of ApoE−/−mice. (B) Correlation between the number of F4/80

530

macrophages and iNOS-positive macrophages (M1 type) (r = 0.638, p = 0.01) or Arg-1-positive

531

macrophages (M2 type) (r = -0.877, p < 0.01). (C) Cytokine concentrations of F4/80

532

(macrophage) and MHC II (M1) or CD206 (M2) in BMDMs extracted from ApoE-/- mice. Data

533

are shown as the means ± SD. *p < 0.05, NCD vs. HCD; #p < 0.05, HCD vs. HCD+PCA.

534

Figure 3. PCA inhibits IFN γ plus LPS-induced M1 polarization in vitro. J774 cells (left)

535

and BMDMs (right) were pretreated with or without PCA (20 μM) for 24 h prior to the

536

stimulation of IFN γ (200 ng/ml) plus LPS (20 ng/ml). (A) After 12 h, mRNA levels of M1

537

markers were determined. (B) After 24 h, NO production in the cell supernatant. (C and D)

538

Protein expression of iNOS by western blotting and immunofluorescence. (E) Cytokine 26


Page 26 of 35

Page 27 of 35


539

concentrations of CD11c and MHCII (M1). Data are shown as the means ± SD. *p < 0.05,

540

Control vs. IFN γ + LPS; #p < 0.05, IFN γ + LPS vs. IFN γ + LPS + PCA.

541

Figure 4. PCA inhibits IFN γ plus LPS-induced M1 polarization through the Pi3k/AKT-

542

NF-κB-SOCS1 signaling pathway. J774 cells were incubated with PCA (20 μM) for 24 h and

543

then treated with IFN γ (200 ng/ml) and LPS (20 ng/ml) for another 24 h. (A) Respective

544

western blots showing phosphorylated AKT, IκB and NF-κB proteins, nuclear NF-κB proteins

545

and total SOCS1 proteins. (B) Respective image depicting NF-κB nuclear translocation. (C, D

546

and E) J774 cells were incubated with IGF (100 ng/ml) and PCA (20 μM) for 24 h then treated

547

with IFN γ (200 ng/ml) and LPS (20 ng/ml) for another 24 h. (C) Respective western blots

548

showing phosphorylated AKT, IκB and NF-κB proteins, nuclear NF-κB proteins and total

549

SOCS1 proteins. (D) Representative images depicting NF-κB nuclear translocation. (E) NO

550

production in the cell supernatant. (F) J774 cells were incubated with PCA (20 μM) for 24 h,

551

and then treated with LY294002 (20 μM) for 2 h prior to the stimulation with IFN γ (200 ng/ml)

552

and LPS (20 ng/ml). Respective western blots showing phosphorylated AKT, IκB and NF-κB

553

proteins, nuclear NF-κB proteins and total SOCS1 proteins. Data are shown as the means ± SD.

554

*p < 0.05, Control vs. IFN γ + LPS; #p < 0.05, IFN γ + LPS vs. IFN γ + LPS + PCA.

555

Figure 5. PCA advances IL-4-induced M2 activation in vitro. J774 cells (left) and BMDMs

556

(right) were pretreated with or without PCA (20 μM) for 24 h prior to stimulation with IL-4

557

(200 ng/ml). (A) After 12 h, mRNA levels of M2 markers were analyzed. (B and C) After 24

558

h, Arg-1 protein expression was analyzed by western blotting and immunofluorescence. (D)

559

Cytokine concentrations of CD206 and CD163. Data are shown as the means ± SD. *p < 0.05,

560

Control vs. IL-4; #p < 0.05, IL-4 vs. IL-4 + PCA. 27



561

Figure 6. PCA advances IL-4-induced M2 activation by promoting the STAT6-PPAR γ

562

signaling pathway J774 cells were pretreated with or without PCA (20 μM) for 24 h prior to

563

the stimulation with IL-4 (200 ng/ml). (A) Respective western blots showing total STAT and

564

PPAR γ proteins and phosphorylated STAT proteins. (B and C) J774 cells were treated with

565

A77-1726 (10 μM) and PCA (20 μM) for 24 h and then stimulated with IL-4 (200 ng/ml), for

566

another 24 h. (F) Respective western blots showing total STAT and PPAR γ proteins and

567

phosphorylated STAT protein (G) Cytokine concentrations of CD206. Data are shown as the

568

means ± SD. *p < 0.05, Control vs. IL-4; #p < 0.05, IL-4 vs. IL-4 + PCA.

569

Figure 7. The expression of phosphorylated NF-κB and STAT6 in ApoE-/- mice. (A)

570

Representative images of NF-κB (P-NF-κB) and STAT6 (P-STAT6) phosphorylation. Data are

571

shown as the means ± SD. *p < 0.05 HCD vs. HCD+PCA.

28


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Figure graphics Figure 1

29



Figure 2

30


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Figure 3

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Figure 4

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Figure 5

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Figure 6

34


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Figure 7

Graphic for table of contents

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Protocatechuic Acid Attenuates Atherosclerosis by Inhibiting M1 and

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