Protocatechuic acid ameliorated palmitic acid-induced oxidative

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Bioactive Constituents, Metabolites, and Functions

Protocatechuic acid ameliorated palmitic acid-induced oxidative damage in endothelial cells through activating endogenous antioxidant enzymes via an AMPK-dependent pathway Lin Han, Qing Yang, Wenfang Ma, Jia Li, Liu-Zhu Qu, and Min Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03414 • Publication Date (Web): 16 Sep 2018 Downloaded from http://pubs.acs.org on September 16, 2018

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Protocatechuic acid ameliorated palmitic acid-induced

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oxidative damage in endothelial cells through activating

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endogenous antioxidant enzymes via an AMPK-dependent

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pathway

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Lin Han†,# , Qing Yang§, Wenfang Ma†, Jia Li†, Liuzhu Qu#, Min Wang*,†

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†

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712100, P. R. China

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#

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of the Three Gorges Reservoir Area’s Medicinal Herbs, College of Biology and Food

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

The Chongqing Engineering Laboratory for Green Cultivation and Deep Processing

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Engineering, Chongqing Three Gorges University, Chongqing 404100, P. R. China

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§

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712100, P. R. China

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E-mail addresses of authors:

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Lin Han: [email protected]

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Qing Yang: [email protected]

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Wenfang Ma: [email protected]

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Jia Li: [email protected]

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Liuzhu Qu: [email protected]

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Corresponding author*: Min-Wang

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Postal address: College of Food Science and Engineering, Northwest A&F University,

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712100 Yangling, PR China

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E-mail address: [email protected]

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


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Tel:+86-130-3293-8796

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ABSTRACT

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Protocatechuic acid (PCA, 3, 4-dihydroxybenzoic acid), the main metabolite of anthocyanins, is

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widely distributed in fruits and vegetables, and has been reported to possess a strong antioxidant

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activity. Herein, we aimed to investigate the protective effect of PCA against high palmitic

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acid-induced oxidative damage and the underling molecular mechanisms in human umbilical vein

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endothelial cells (HUVECs). PCA reduced the levels of intracellular reactive oxygen species (ROS)

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and malondialdehyde (MDA), and increased the activities of endogenous antioxidant enzymes

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including superoxide dismutases (SOD), glutathione peroxidases-1 (Gpx-1), and heme

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oxygenase-1 (HO-1). Metabolomic analysis showed that PCA affected numerous metabolites,

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especially some of which were related with energy metabolism. PCA also up-regulated the

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phosphorylation of AMPK at Thr172 through activating LKB1, then promoted the expression of

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p-Nrf2 and HO-1. Moreover, PCA reversed the decreased expression of PGC-1α and significantly

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increased the mitochondrial density. Collectively, these results demonstrated that PCA attenuated

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PA-induced oxidative damage in HUVECs via an AMPK-dependent pathway.

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KEYWORDS: PCA, HUVECs, oxidative damage, AMPK, Nrf2

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INTRODUCTION

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Reactive oxygen species (ROS) are chemically reactive chemical species mainly containing

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peroxides, superoxide, hydroxyl radical, and singlet oxygen.1 In mammalian cells, ROS are

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produced in many ways, however, the most accepted two ways are mitochondrial respiratory chain

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complexes and NADPH-oxidase (NOX) catalysed reactions.2 Additionally, mitochondrial energy

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metabolism is recognized as the most quantitatively important source of ROS in the different types

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of eukaryotic cells.3 O2▪-, the primary ROS, is generated by electrons leaking to oxygen from

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different sites of mitochondrial electron transport chain, including 6 sites at complex I

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(NADH-CoQ reductase) and 5 sites at complex III (CoQH2-cytochrome c reductase).4, 5 O2▪- is

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mainly distributed in the inner membrane and can be converted rapidly to hydrogen peroxide

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(H2O2) by superoxide dismutase (SOD). H2O2 readily diffuses across the mitochondrial

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membranes and is catalyzed to non-toxic H2O and O2 by several antioxidant enzymes including

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catalase (Cat), peroxiredoxins, and glutathione peroxidases-1 (Gpx-1) that have differential

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sub-cellular distributions.4 Palmitic acid (PA), the most common saturated fatty acid found in

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human, was reported to induce a cellular toxic response known as “lipotoxicity”.6 As reported, the

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PA-induced lipotoxicity was mediated by an increased level of ROS, or oxidative stress, and

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several mechanisms were involved, including enhancing Complex I-associated O2▪- generation by

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β-oxidation, interfering with various enzymatic processes, and interacting with components of the

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respiratory chain, etc.7 Then, the excessive ROS can “attack” the components of cell such as DNA,

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protein, lipids, and result in cumulative cellular oxidative damages, which plays a pivotal role in

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development and progression of diseases like diabetes, neurodegenerative diseases, cancer and

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cardiovascular diseases (CVDs).8,9



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PA overloading induced oxidative stress is a crucial event that in the initial development of

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endothelial dysfunction.4 Fortunately, each cell is endowed with elaborate and powerful protective

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mechanisms to neutralize superoxide radicals and peroxides. Among them, the endogenous

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antioxidant enzymes including SOD, Cat, Gpx, heme oxygenase-1 (HO-1), and thioredoxin

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peroxidase establish the most important antioxidant defenses.2 Nuclear factor erythroid-derived

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factor 2-related factor 2 (Nrf2) is a transcription factor that regulates the expression of antioxidant

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and detoxification genes in response to oxidative stress.10 Perxisome proliferator activated

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receptor (PPAR) γ coactivator 1-α (PGC-1α) is a transcriptional coactivator that regulates

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biosynthesis of mitochondria and reduces the accumulation of ROS.11 Both Nrf2 and PGC-1α are

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response to the oxidative stress, as well as energy metabolism that can be activated by

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AMP-activated protein kinase (AMPK).12 As a sensor of cellular energy, AMPK can be directly

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activated by AMP on the γ subunit and salicylate on the β subunit.13 But most natural compounds

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increase the expression of p-AMPK through an indirect ways. Our previous researches showed

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that both D-chiro inosital and D-fagomine could up-regulate the expression of p-AMPK via

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activating the upstream protein, p-LKB1, and then increase the activity of Nrf2 and PGC-1α,

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respectively.14,15

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Protocatechuic acid (PCA, 3, 4-dihydroxybenzoic acid), a catechol-type o-diphenol phenolic

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acid, is widely distributed in fruit and vegetables such as tea, grape, chicory, raspberry, cauliflower,

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and buckwheat, but with low concentrations.16,17 However, as the main metabolite of anthocyanins,

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the concentration of PCA in vivo may be higher than the simple quantity ingested, and the daily

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intake probably reaches up to 180-250 mg/d, which is much higher than that of the other

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polyphenols (estimated at 20-30 mg/d).16 Therefore, PCA has elicited the interest of researchers in


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the recent years on its health functions, such as antioxidant, anti-inflammatory, anti-diabetic, and

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anti-apoptotic activities, as well as anti-cardioascular diseases.18-21 Previous studies indicated that

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PCA had strong activity to scavenge free radicals in vitro and reduce the oxidative stress via

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activating the endogenous antioxidant enzymes in vivo.22,23 It has been reported that PCA activated

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AMPK and suppressed its downstream kinase mTOR/S6K in murine hepatic cell line and in the

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liver of C57BL/6 mice.24 Meanwhile, several lines of evidence suggested that PCA protected

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against oxidative damage and induced the expression of endogenous antioxidant enzymes via

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activating Nrf2 in different cell lines.23,25 However, the detailed mechanisms and signal pathways

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of PCA that protecting against oxidative stress were fewer reported. In the current study, we aimed

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to investigate the effect of PCA on the high fat-induced oxidative stress in HUVECs and elucidate

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the potential mechanisms underlying the activation of AMPK.

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MATERIALS AND METHODS

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Chemicals and Antibodies. Protocatechuic acid (purity ≥ 98%; Yuanye Biotech. Co., China),

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AICAR and Compound C (purity > 99%, MedChemExpress, USA), and STO-609 (Santa Cruz,

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USA) were dissolved in DMSO, however, the final concentration of DMSO in culture medium

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was controlled to below 0.1% (w/v). Palmitic acid was dissolved in ethanol and diluted with

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medium containing 10% BSA to the 100 µM. Antibodies: anti-AMPKα and anti-phospho-AMPKα

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(Thr172) were purchased from Cell Signaling Technology (Shanghai, China); anti-phospho-Nrf2,

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anti-phospho-LKB1 (Ser428), anti-PGC-1α, and anti-HO-1 antibodies were purchased from Santa

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Cruz Biotechnology (USA); anti-GAPDH antibody was purchased from Bioss (Beijing, China).

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Cell Culture. The human umbilical vein endothelial cells (HUVECs) were purchased from

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American Type Culture Collection (San Diego, CA, USA) and gifted from The Fourth Military



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Medical University (Xi’an). HUVECs were grown in Dulbecco’s modified Eagle medium

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(DMEM; HyCloneTM, Logan Utah, USA) with 10% fetal bovine serum (FBS; Biological

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Industries, Kibbutz Beit Haemek, Israel), 100 µg/mL streptomycin and 100 U/ml

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penicillin–streptomycin (HyCloneTM, Logan Utah, USA), and incubated at 37 ℃ in a humidified

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5% CO2 atmosphere. Cells at passages 3-15 were used in the current study.

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Cell Viability Assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

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(MTT) assay was employed to determine cell viability according to the previously described.15

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Briefly, HUVECs were plated in 96-well plates and incubated for 24 h, then treated with different

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concentrations of PCA for 12 h. Following incubation, 0.5 mg/mL solution of MTT was added to

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each well. After 4 h at 37 ℃, 100 µL Formanzan solution was added and incubated for another 4 h.

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The absorbance was recorded at a wavelength of 570 nm, and the cell viability was expressed as a

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relative percentage to the non-treated control group.

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Measurement of Intracellular ROS and MDA Levels. Intracellular ROS in HUVECs was

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investigated by the conversion of 2’, 7’-dichlorofluorescin diacetate (DCFH-DA) to highly

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fluorescent dichlorofluorescein (DCF). Cells were seeded in a black 96-well plate and incubated

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for 24 h at 37 ℃, the growth medium was removed and the 96-wells were washed with PBS (100

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µL)，then different concentrations of PCA were added to pre-protect for 30 min, followed by 12 h

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of treatment with PA (100 µM). After that, cells were incubated with DCFH-DA (Beyotime

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Institute of Biotechnology, Shanghai, China) at a final concentration of 10 µM for 30 min at 37°C.

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Fluorescence was measured using a multi-mode Microplate Reader (Perkin Elmer, Waltham, MA,

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USA). Results were expressed as fold changes in fluorescence intensity vs control.

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For MDA measurement, cells were lysed with ice-cold RIPA containing 1 mM PMSF and


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collected to determine the protein concentration by using BCA protein assay kit (Beyotime

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Biotechnology, Shanghai, China). The levels of MDA were determined by corresponding

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detection kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the

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manufacturer’s protocols.

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Measurement of SOD and Gpx-1. At the end of incubating with or without different

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concentrations of PCA for 12 h, the cells lysates were collected and centrifuged at 10000 rpm for

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10 min at 4 ℃. The supernatant was collected to investigated the SOD and Gpx1 activity by using

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commercial assay kits (Beyotime Biotechnology, Shanghai, China), according to the

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manufacturer’s instructions. Meanwhile, the protein of the supernatant was measured by using

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BCA protein assay kit (Beyotime Biotechnology, Shanghai, China).

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mtDNA/DNA Assay. HUVECs mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) were

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determined by Qrt-PCR as previously described.26 Total DNA from HUVECs was extracted by

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using TIANamp Genomic DNA extraction kit (Tiangen Biotech, Beijing, China). To determine the

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content of mtDNA relative to nDNA, the amount of tfam and β-actin gene were selected to

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represent mtDNA and nDNA, respectively. The primers used for tfam and β-actin amplification

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were:

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5’-AATCGTGCGTGACATTAAG-3’; reverse primer: 5’-CAGAACACCGTGGCTTCTAC-3’ and

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5’-GAAGGAAGGCTGGAAGAG-3’, respectively, and the ratio of mitochondrial tfam and

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β-actin (mtDNA/nDNA) was calculated as the mitochondrial density.27

forward

primer:

5’-GGCACAGGAAACCAGTTAGG-3’

and

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NMR Analysis. The NMR analysis for the metabolites of cell was performed according to

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previous reports.28,29 After being treated with or without PCA (100 µM) under 100 µM PA for 6 h,

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cells were collected and the polar metabolites were extracted by adding a solvent composed of



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distilled water, methanol, and chloroform (1:1:1). The extracts were freeze dried and resolved in

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D2O buffer. 1H-NMR spectra were detected using a 500-MHz NMR instrument (Bruker,

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Switzerland) with a room temperature probe. The residual water signal was suppressed using a

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NOESYPRESAT pulse sequence, 128 transients, 11600 spectral width, relaxation delay 6 s, an

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acquisition time 4s. All NMR spectra were baseline-corrected using MestReNova software.

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Western Blot Analysis. Cells were lysed with ice-cold RIPA containing 1 mM PMSF as

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previous method.15 After protein quantification, samples were separated by SDS/PAGE and

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transferred onto PVDF membranes (Millipore, Germany). Membranes were blocked with 5% BSA,

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and incubated with primary antibodies: p-AMPK and AMPK antibodies (CST, Shanghai, China),

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p-Nrf2, p-LKB1, LKB1, PGC-1α, and HO-1 antibodies (Santa Cruz, Texas, USA), GAPDH

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antibody (Bioss,

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Chemiluminescence assay system was employed to assay antibody-antigen complexes and the

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density of protein bands was quantified using ImageJ software (Bio-Rad Company, California,

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USA).

Beijing,

China),

followed

by probing

with

secondary antibodies.

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Statistical Analysis. Data were expressed as the means ± SD from no fewer than three

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independent experiments. Statistical differences between groups were evaluated by using a

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one-way ANOVA followed with multiple tests. p < 0.05 was considered to be significant.

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RESULTS

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PCA Reduces ROS and MDA Generation in HUVECs. The result of MTT assay (Fig. 1A)

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showed that PCA revealed no toxic effect at concentrations up to 200 µM on the viability of

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HUVECs. Considering the concentrations achievable in the plasma under normal circumstance,16

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we chose PCA at concentration up to 100 µM in the following experiments.


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ROS are mainly generated as a result of the escaping of electrons from electron transport chains

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and reacting with oxygen to form superoxide.30 Physiological levels of ROS are important to cell

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signaling and host defense, however, excess ROS can “attack” the biological macromolecules

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including protein, DNA, lipid and results in oxidative stress and cellular damage.31 As shown in

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Fig. 1B, the intracellular ROS level in PA treated group was 1.368-times higher than that of

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control group. PCA significantly reduced ROS production to 1.063 ± 0.03 (fold to control, p

Protocatechuic acid ameliorated palmitic acid-induced oxidative

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