Activation of Nrf2 by phloretin attenuates palmitic acid-induced

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Bioactive Constituents, Metabolites, and Functions

Activation of Nrf2 by phloretin attenuates palmitic acid-induced endothelial cell oxidative stress via AMPK-dependent signaling Qing Yang, Lin Han, Jie Li, Han Xu, Xinfeng Liu, Xinyu Wang, Chuanying Pan, Chuzhao Lei, Hong Chen, and Xianyong Lan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05025 • Publication Date (Web): 09 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

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Journal of Agricultural and Food Chemistry

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Activation of Nrf2 by phloretin attenuates palmitic

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acid-induced

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AMPK-dependent signaling

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Qing Yang†, Lin Han#, Jie Li†, Han Xu†, Xinfeng Liu†, Xinyu Wang†,

5

Chuanying Pan†, Chuzhao Lei†, Hong Chen†, Xianyong Lan†*

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†

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Key Laboratory of Molecular Biology for Agriculture, Yangling, 712100, P. R. China

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#

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712100, P. R. China

endothelial

cell

oxidative

stress

via

College of Animal Science and Technology, Northwest A&F University, Shaanxi

College of Food Science and Engineering, Northwest A&F University, Yangling,

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E-mail addresses of authors:

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Qing Yang: [email protected]

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Lin Han: [email protected]

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Jie Li: [email protected]

14

Han Xu: [email protected]

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Xinfeng Liu: [email protected]

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Xinyu Wang: [email protected]

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Chuanying Pan: [email protected]

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Chuzhao Lei: [email protected]

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Hong Chen: [email protected]

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Corresponding author*: Xianyong Lan

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Postal address: College of Animal Science and Technology, Northwest A&F

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University, Yangling, 712100, P. R. China

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E-mail: [email protected]

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Tel: +86-137-7207-1502

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ACS Paragon Plus Environment


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ABSTRACT

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Phloretin, a dihydrochalcone structural flavonoid compound, possesses

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antioxidant activity. In this study, we conducted studies to explore the function of

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phloretin on high palmitic acid-induced oxidative stress in human umbilical vein

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endothelial cells and investigated the potential mechanism using ribonucleic acid

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sequencing (RNA-Seq). Our findings reveal that phloretin significantly decreased the

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levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA),

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increased superoxide dismutase (SOD) and glutathione peroxidase-1 (Gpx-1) activity,

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and restored the loss of mitochondrial membrane potential (MMP). Next, whole

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transcriptome analysis was performed using RNA-Seq. The results indicated more

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than 3,000 differentially expressed genes (DEGs). Gene Ontology analysis revealed

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that the DEGs were categorized functionally mainly by the biological processes, cell

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metabolism, and cellular response to chemical stimulus. The Kyoto Encyclopedia of

39

Genes and Genomes indicated that they were mainly enriched in cAMP, apoptosis,

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and cytoskeletal regulation signaling pathways. Furthermore, based on the results of

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RNA-Seq and western blotting, our study verified that phloretin upregulated the

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expression of p-Nrf2 and HO-1 by promoting the phosphorylation of AMPK at Thr172

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through activation of liver kinase B1. In conclusion, phloretin attenuates PA-induced

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oxidative stress in HUVECs via the AMPK/Nrf2 anti-oxidative pathway.

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KEYWORDS: Phloretin, AMPK, endothelial dysfunction, Nrf2, reactive oxygen

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species (ROS)


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INTRODUCTION

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With the lifestyle changes associated with modern society, various factors, such as

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lifestyle and diet, may increase the risk of developing chronic diseases, especially

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cardiovascular diseases (CVD). For example, published data have suggested that olive

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oil intake is negatively associated with the occurrence of CVD in Italian women and

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in the Spanish general population.1,2 Based on these findings, excessive palmitic acid

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(PA), a saturated fatty acid commonly found in humans, can increase the levels of

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ROS and oxidative stress, even inducing cellular “lipo-toxicity”, which is regarded as

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the main cause of endothelial dysfunction and the sign of CVD.3-6 Therefore, it is

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necessary to identify approaches able to reverse PA-induced oxidative stress,

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particularly in human umbilical vein endothelial cells (HUVECs).

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Low ROS levels are known to participate in cell signaling and are beneficial for

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physical health, but high levels of ROS may trigger cell oxidative stress.7 Excessive

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fatty acid in the diet may increase ROS production and disrupt the balance of the

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energy metabolism, inducing mitochondrial dysfunction and eventually triggering the

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initiation and development of diseases.5,8,9 Fortunately, each cell has self-protection

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mechanisms, which scavenge excessive ROS, including the activation of endogenous

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antioxidant enzymes, such as SOD, catalase, Gpx-1, heme oxygenase-1 (HO-1), and

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thioredoxin peroxidase.10 Furthermore, it has been reported that the expression of

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antioxidant enzymes could be regulated by nuclear factor erythroid 2-related factor 2

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(Nrf2).11,12 In addition, AMP-activated protein kinase (AMPK) could activate the

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Nrf2 signaling pathway to participate in the regulation of oxidative stress in cells.13,14



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Therefore, it is essential to elucidate the function of AMPK and Nrf2 in cellular

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oxidative stress.

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Recently, an increasing amount of research has focused on the function of

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polyphenols as factors beneficial to cardiovascular health. Phloretin is a chalcone

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flavonoid found in apple and pear trees, and possesses many beneficial biological

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characteristics, including antioxidant, anticancer, and anti-inflammatory activities.15-19

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The number of reports on the antioxidant activity of phloretin has also recently

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increased. Notably, the antioxidant activity of phloretin is not only dependent on the

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hydroxyl group in its chemical structure, but also initiates the transcription of

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antioxidant genes.20,21 In addition, phloretin can also promote the synthesis of

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non-antioxidant enzymes to exert antioxidant effects. For example, it reduces

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cytoplasmic and glutathione in organelles.22 However, the current literature regarding

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the effect of phloretin on CVD risk is limited.

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High-throughput ribonucleic acid sequencing (RNA-Seq) not only identifies global

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gene expression trends and detects different transcript isoforms, but also determines

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genomic structural variations.23-25 In recent years, based on the results of RNA-Seq,

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many natural compounds have been reported to participate in regulating the

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expression levels of key genes and proteins, including benzo[a]pyrene and

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dexamethasone salvia miltiorrhiza.26,27 Therefore, we conducted a series of

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experiments to explore the function of phloretin in PA-induced oxidative stress in

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HUVECs and elucidate its potential mechanisms.

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MATERIALS AND METHODS

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Materials. Phloretin (from apple wood, ≥98% by HPLC, Yuanye Biotech. Co.,

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Shanghai, China), AICA riboside (AICAR), compound C (Med. Chem. Express.,

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USA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Beyotime Biotechnology,

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Shanghai, China), and STO-609 (Santa Cruz, USA) were solubilized for use in

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dimethyl sulfoxide (DMSO). In this study, 100 mM PA was prepared using ethanol

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diluted to 100 μM using medium with 10% bovine serum albumin. In all of the

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experiments, the concentration of DMSO was diluted to below 0.1% (w/v). The

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antibodies

used

in

this

experiment

were

as

follows:

anti-AMPKα

and

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anti-phospho-AMPKα (Thr172) obtained from Cell Signaling Technology (Shanghai,

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China); anti-phospho-Nrf2, anti-phospho-liver kinase B1 (LKB1) (Ser428), and

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anti-HO-1 purchased from Santa Cruz Biotechnology (USA); anti-GAPDH antibody

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obtained from Bioss (Beijing, China).

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Cell Culture. The HUVECs were gifted from the Fourth Military Medical

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University (Xi’an, Shaanxi, China). HUVECs were cultured using standard protocols

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as previously described.6 In all experiments, cells were incubated at 37°C in a

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humidified incubator with 5% CO2.

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Cell

Viability

Analysis.

Based

on

the

previous

report,

the

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was

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selected and used to detect the cell viability.6 Briefly, cells were seeded and incubated

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in 96-well plates for 24 h, and then treated with different concentrations of phloretin

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(0.1, 1, 10, 50, and 100 μM) for 24 h. Afterwards, the cells in each well were treated



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according to the instructions of the MTT kit (Beyotime Biotechnology, Shanghai,

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China). The survival rate of cells was calculated as a relative percentage of the

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untreated control.6

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Determination

of

ROS

Production

and

MDA

Activity.

The

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2′,7′-dichlorofluorescin diacetate (DCFH-DA) assay was employed to determine the

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intracellular ROS levels. HUVECs were seeded in 96-well plates and pro-protected

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with various concentrations of phloretin (1, 10, and 50 μM) for 30 min, then the cells

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were exposed in PA (100 μM) for 12 h. The HUVECs were then treated according to

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the manufacturer’s protocols of DCFH-DA (Beyotime Institute of Biotechnology,

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Shanghai, China) and the ROS level was determined by the fluorescence intensity

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using a Multi-Mode Microplate Reader (Perkin Elmer, Waltham, MA, USA). The

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results were presented as relative fold-changes compared with the normal controls.8

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For MDA detection, PA-stimulated HUVECs treated with phloretin were harvested

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with ice-cold RIPA containing 1 mM PMSF, and the BCA protein assay kit

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(Beyotime Biotechnology, Shanghai, China) was employed to determine the protein

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concentration. The MDA levels were then measured using the corresponding

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detection kit (Jiancheng, Nanjing, China) according to the manufacturer’s protocols.6

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Detection of SOD and Gpx-1 Activities. PA-stimulated cells with different

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concentrations of phloretin were collected and centrifuged before harvesting the

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supernatant and detecting the SOD and Gpx1 activity using the corresponding

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detection kits (Beyotime Biotechnology, Shanghai, China). In addition, the protein

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was determined as previously described. The results were presented as relative


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fold-changes compared with the normal controls. 8

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Detection of Mitochondrial Membrane Potential. In this study, HUVECs were

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plated in a 48-well plate and incubated with various concentrations of phloretin (0.1, 1,

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and 10 μM) for 30 min. Subsequently, 100 μM PA solution was used to stimulate

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cells for another 24 h. Then, after removing the growth medium and washing the

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wells with PBS, TMRE solution (Abcam, London, UK) at a final concentration of 500

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nM was used to treat the cells for 20 min at 37oC. Then, the mitochondrial membrane

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potential (MMP) was determined using a TMRE-Mitochondrial Membrane Potential

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Assay Kit (Abcam, London, UK). 6

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Sample Collection, RNA Isolation, and Reverse Transcription PCR. Three

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groups (control group, PA-treated group, and 50 μM phloretin and PA co-treated

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group) were selected for this study. In this experiment, the total RNA of each sample

148

was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). After

149

quantification, 1 μg total RNA were reverse transcribed using a cDNA synthesis kit

150

(Life Technologies, Shanghai, China).28

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Library Construction and Sequencing. The RNA purity was determined via the

152

OD260/OD280 and OD260/OD230 ratios. Then, the RNA was treated with DNase I to

153

eliminate any remaining DNA. Afterwards, beads with oligo (dT) were used to enrich

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and purify the mRNA. Nine libraries were constructed using the TruSeq SBS v3-HS

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kit, before sequencing to investigate the global transcriptome of the control group

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using Illumina HiSeq 2500 (Illumina, Inc.). The quality controls of all reads were

157

checked using FastQC (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/).



158

Alignment, Assembly, and Functional Annotation of Differentially Expressed

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Genes. All the reads were filtered using Trimmomatic29, then mapped onto the human

160

genome GRCh38.p12 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.38)

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by hisat2 2.1.0 using the paired-end mapping method.30 Transcript assembly was

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carried out using Stringtie.31 Then, the fragments per kilobase of exon per million

163

reads mapped (FPKM) was calculated using the number of reads to quantify the gene

164

expression levels. Genes with adjusted p-values ≤0.05 and a fold change (FC) ≥ 2

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were considered significant.32

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The differentially expressed genes (DEGs) were subjected to Gene Ontology (GO)

167

(http://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genomes

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(KEGG) (http://www.kegg.jp) analysis.33-35 Functional annotations were evaluated

169

using the KOBAS website (http://kobas.cbi.pku.edu.cn/anno_iden.php).36

170

Validation of RNA Sequencing Data by Quantitative Real-time PCR.

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Twenty-two significant DEGs from our transcriptome data were randomly selected to

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detect expression levels using the quantitative real-time PCR (qRT-PCR) method. The

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specific primers for the genes are shown in Table 1. The composition and program of

174

qRT-PCR used was the same as previous reports. 37 The 2-∆∆CT method was selected to

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calculate the relative gene expression, and the data were normalized using GAPDH

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relative expression (Table S1).38

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Western Blot Analysis. Cells were harvested, and the protein was quantified as

178

previously described.39 Then, SDS/PAGE was used to separate the samples before

179

transferring the samples onto PVDF membranes (Millipore, Germany). After blocking


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and washing, the membranes were incubated with primary antibodies and then probed

181

with secondary antibodies as previously reported.8 The antibody-antigen complexes

182

were determined using a chemiluminescence assay system. ImageJ software was

183

employed to calculate the density of protein bands. GAPDH was selected as the

184

internal standard.

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Statistical Analysis. All data are showed as the mean ± SD from no fewer than

186

three independent experiments. Statistical differences were evaluated by two-tailed

187

t-test or ANOVA followed by Student–Newman–Keuls test. p0.05). Similarly, the MDA test results showed that,

217

compared with the control group, PA stimulation caused cells to produce a large

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amount of MDA (p

Activation of Nrf2 by phloretin attenuates palmitic acid-induced

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