Scutellarin protects against vascular endothelial dysfunction and prevents atherosclerosis via antioxidation

Accepted Manuscript

Scutellarin protects against vascular endothelial dysfunction and prevents atherosclerosis via antioxidation Jiao Mo , Renhua Yang , Fan Li , Xiaochao Zhang , Bo He , Yue Zhang , Peng Chen , Zhiqiang Shen PII: DOI: Reference:

S0944-7113(18)30056-4 10.1016/j.phymed.2018.03.021 PHYMED 52395

To appear in:

Phytomedicine

Received date: Revised date: Accepted date:

7 August 2017 25 December 2017 12 March 2018

Please cite this article as: Jiao Mo , Renhua Yang , Fan Li , Xiaochao Zhang , Bo He , Yue Zhang , Peng Chen , Zhiqiang Shen , Scutellarin protects against vascular endothelial dysfunction and prevents atherosclerosis via antioxidation, Phytomedicine (2018), doi: 10.1016/j.phymed.2018.03.021

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ACCEPTED MANUSCRIPT

Scutellarin protects against vascular endothelial dysfunction and prevents atherosclerosis via antioxidation 1

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Jiao Mo , Renhua Yang , Fan Li, Xiaochao Zhang, Bo He, Yue Zhang, Peng Chen*, Zhiqiang Shen* School of Pharmaceutical Sciences and Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University, Chun Rong West Street No. 1168, Chenggong, Kunming 650500, PR China *Corresponding authors

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Peng Chen, Zhiqiang Shen, School of Pharmaceutical Sciences and Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University, Chun Rong West Street No. 1168, Chenggong, Kunming 650500, P.R. China Tel.: +86 871 65922781; fax: +86 871 65922780

E-mail addresses: [email protected], [email protected] (P. Chen), [email protected] (Z.-Q. Shen) 1

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These authors contributed equally to this work.

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ABSTRACT Background: Scutellarin is the major constituent responsible for the clinical benefits of Erigeron breviscapus (Vant.) Hand.-Mazz which finds a long history of ethnopharmacological use in Traditional Chinese Medicine. Scutellarin as a pure compound is now under investigation for its protections against various tissue injuries. Purpose: This study aims to examine the effects of scutellarin on oxidative stress-induced vascular endothelial dysfunction and endothelial cell damage, and then to evaluate the therapeutic efficacy of scutellarin in preventing atherosclerosis in rats. endothelium-dependent

relaxation

(EDR)

of

rabbit

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Methods: Radical scavenging ability of scutellarin was determined in vitro. Impact of scutellarin on thoracic

aortic

rings

upon

1,

1-diphenyl-2-picrylhydrazyl (DPPH) challenge was measured. Influences of scutellarin pre-treatment on the levels of reactive oxygen species (ROS), activities of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase and catalase, and the expression of SOD1 and NADPH oxidase 4 (Nox4) in human umbilical vein endothelial cells (HUVECs) injured by H2O2 were examined. Anti-atherosclerotic effect of scutellarin was evaluated in rats fed with high fat diet (HFD).

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Results: Scutellarin showed potent antioxidant activity in vitro. Pretreatment of scutellarin retained the EDR of rabbit thoracic aortic rings damaged by DPPH. In H2O2 injured-HUVECs the deleterious alterations in ROS levels and antioxidant enzymes activity were reversed by scutellarin and the mRNA and protein expression of SOD1 and Nox4 were restored also. Oral administration of scutellarin dose-dependently ameliorated hyperlipidemia in HFD-fed rats and alleviated oxidative stress in rat serum, mimicking the effects of reference drug atorvastatin.

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Conclusion: Scutellarin protects against oxidative stress-induced vascular endothelial dysfunction and endothelial cell damage in vitro and prevents atherosclerosis in vivo through antioxidation. The results

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rationalize further investigation into the clinical use of scutellarin in cardiovascular diseases.

Abbreviations:

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Keywords: Scutellarin, Antioxidation, Oxidative stress, Endothelial dysfunction, Atherosclerosis

Ach, acetylcholine chloride; CAT, catalase; EDCF, endothelium-derived contracting factors; EDRF, relaxing

factor;

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endothelium-derived

EDR,

endothelium-dependent

relaxation;

EIDR,

endothelium-independent relaxation; DPPH, 1,1-dipheyl-2-picrylhydrazyl; eNOS, endothelia nitric oxide synthase; GPx, glutathione peroxidase; HFD, high fat diet; HDL-C, high density lipoprotein

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cholesterol; HUVECs, human umbilical vein endothelial cells; LDL-C, low density lipoprotein cholesterol; •-

MDA, malondialdehyde; NE, noradrenaline; NO, nitric oxide; Nox, NADPH oxidase; O2 , superoxide •

anion; H2O2, hydrogen peroxide; OH , hydroxyl radical; oxLDL, oxidized LDL; SNP, sodium nitroprusside; SOD, superoxide dismutase; TAC, total antioxidant capacity; TC, total cholesterol; TG, triglyceride.

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Introduction Thirty years ago the historical experiment performed by Robert Furchgott revealed that the local control of vascular tone is dictated by vasoactive substances released by the endothelial cells of arteries (Furchgott, 1988). Subsequent research then identified that the potent endothelium-derived relaxing factor (EDFR) is nitric oxide (NO) and that endothelium-derived contracting factors (EDCF) comprise vasoconstrictor prostanoids and endothelin-1 (De Mey and Vanhoutte, 2014; Vanhoutte and Tang, 2008). Compromised ability of endothelial cells to produce NO coupled with enhanced propensity to release EDCF gives rise to endothelial dysfunction, which is accepted as both the

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hallmark of atherosclerosis and a predictor of cardiovascular diseases (Vanhoutte et al., 2017). NO production of endothelial cells can be downregulated by a number of factors. On cellular basis oxidative stress emerges as a major contributor to curtailing NO release (Montezano and Touyz, 2012). Oxidative stress occurs when the cellular antioxidant defenses are overwhelmed by excessive •-

production of reactive oxygen species (ROS) including superoxide anion (O2 ), hydrogen peroxide •

(H2O2) and hydroxyl radical ( OH) (Victor et al., 2009). Normally ROS are converted to harmless molecules by the action of intracellular superoxide dismutase (SOD), catalase (CAT), and glutathione

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peroxidase (GPx), or neutralized by endogenous nonenzyme antioxidants such as β-carotene and ascorbic acid (Wattanapitayakul and Bauer, 2001). But under conditions of oxidative stress excessive amounts of ROS deplete NO rapidly and eliminate endothelium-dependent relaxation (EDR). Moreover, ROS per se amplify the release of EDCF (Gao and Lee, 2005). Thus oxidative stress is unsuspiciously implicated in endothelial dysfunction and atherosclerosis. A number of therapeutic agents that protect against endothelial dysfunction are evidenced to possess antioxidant activity (Victor et al.,

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2009).

Scutellarin (4’,5,6-trihydroxyflavone-7-glucuronide) (Fig. 1) is a flavone isolated from Erigeron breviscapus (Vant.) Hand.-Mazz which has been used to treat cardiac ischemic and cerebral ischemic

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diseases in Traditional Chinese Medicine with a long history. Modern pharmacological research has identified scutellarin as the major active compound responsible for the clinical actions of Erigeron breviscapus (Liu et al., 2005). Scutellarin now has been drawing increasing attention for its protective

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effects in various tissues. Previous studies find that scutellarin protects against cerebral ischemia in stroke by attenuating microglia-mediated inflammatory responses (Yuan et al., 2016), protects against

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cardiomyocyte ischemia/reperfusion injury by reducing apoptosis and oxidative stress (Wang et al., 2016), and prevents diosbulbin B-induced liver injury by attenuating NF-κB mediated hepatic inflammation and ameliorating liver oxidative stress (Niu et al., 2015). In this study, the protective

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effects of scutellarin against oxidative stress-induced endothelial dysfunction and endothelial cell damage were examined for the first time, and the therapeutic effect of scutellarin in the treatment of atherosclerosis was evaluated in vivo. Materials and methods Reagents Scutellarin (purity > 98.5%) was provided by Professor Zhang Ren-Wei (Yunnan Institute of Materia Medica, China). Vitamin E, 1,1-dipheyl-2-picrylhydrazyl (DPPH), acetylcholine chloride (Ach), and sodium nitroprusside (SNP) were from Sigma-Aldrich (USA). Noradrenaline (NE) bitartrate injection 3 / 23

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was from Shanghai Harvest Pharmaceutical Co., Ltd (China). Atorvastatin was from Pfizer (USA). Total antioxidant capacity (TAC) assay kit (ABTS method) was from Shanghai Baomanbio Co., Ltd. (China). Superoxide assay kit (pyrogallol autoxidation method) and hydroxyl free radical scavenging activity assay kit (Fenton method) were from Beyotime Biotechnology (China). Total superoxide dismutase (SOD) assay kit (hydroxylamine method), malondialdehyde (MDA) assay kit (TBA method), glutathione peroxidase (GPx) assay kit (colorimetric method), catalase (CAT) assay kit (visible light), NADPH oxidase (Nox) test kit, nitric oxide (NO) assay kit (nitrate reductase method), and total protein assay kit (BCA method) were from Nanjing Jiancheng Bioengineering Institute (China). Fura-2/AM was purchased from AnaSpec (USA). Total cholesterol (TC) assay kit (cholesterol oxidase method),

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triglyceride (TG) assay kit (glycerol phosphate oxidase method), high density lipoprotein cholesterol (HDL-C) reagent kit, and low density lipoprotein cholesterol (LDL-C) reagent kit were from Shaoxing Shengkang Bio-tech Co., Ltd. (China). In situ hybridization (ISH) assay kits for Nox4 and SOD1 were from Wuhan Boster Co., Ltd. (China). SOD1 monoclonal primary antibody was from R&D Systems (USA). Nox4 monoclonal primary antibody was from Santa Cruz Biotechnology (USA). Horseradish peroxidase-conjugated secondary antibody was from Tiangen Biotech Co., Ltd. (China). All other

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chemicals were of analytical grade and from commercial sources. In vitro antioxidant assay of scutellarin

The stock solution (2.16 mM) of scutellarin was prepared in 0.02 mM NaOH. Six concentrations of scutellarin were produced by two-fold dilutions. Total antioxidant capacity, superoxide anion scavenging and hydroxyl radical scavenging activities of scutellarin were determined with assay kit by

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following the manufacturer’s instruction. DPPH scavenging activity was determined according to the method of Xu et al. (Xu et al., 2004). The scavenging activity of scutellarin was calculated by the following equation: clearance (%) = (1 − absorbance with scutellarin/absorbance of blank control) ×

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100. Nonlinear regression using a sigmoidal dose–response equation (variable slope) was carried out to calculate the effective concentration of scutellarin required to scavenge radicals by 50% (EC50). 10

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Animals

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μM of Vitamin E was used as the positive control. All tests were carried out in triplicates.

New Zealand rabbits of either gender weighing 2.0–2.5 kg and male Sprague-Dawley rats weighing 180-220 g were purchased from Animal Center of Kunming Medical University [License: SCXK (Yunnan)

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2008-0008]. Animals were housed in individually ventilated cages and maintained in a standard condition with controlled temperature (21-23 ⁰C) and a strict 12-h light/dark cycle. Animals were allowed free access to food and water. All experimental protocols were approved by the Animal Study Committee of Kunming Medical University and were in accordance with the requirements of NIH Guidelines for care and use of laboratory animals. Arterial rings preparation and endothelium-dependent relaxation (EDR) measurement Rabbits were euthanatized and exsanguinated from abdominal aorta. The thoracic aortic rings were prepared by following the method described by Tang et al (Tang et al., 2006) with minor modification. Briefly, the identical position and length of thoracic aorta was dissected from each animal, cut into 4 / 23

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transverse rings of 4 mm long, and placed in Krebs solution (118.3 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM NaH2PO4, 11.1 mM glucose, and 24.0 mM NaHCO3; pH 7.4; 4 ⁰C). Special care was taken to clean the rings of connective tissue without damaging the endothelium. A single ring was then suspended vertically between two stainless steel hooks in a chamber (8 ml) of Krebs solution maintained at 37 ⁰C and aerated with 95% oxygen and 5% carbon dioxide. One hook was fixed to a stand and the other was attached to an isometric force transducer (Hugo Sachs, Germany). The tension was recorded (Linearcorder WR 3320, Japan). Rings were initially stretched with a tension of 4 g and allowed to equilibrate for 120 min. Krebs solution in the chamber was replenished every 20 min. The rings were pre-constricted with NE (1 μM). When the constrictive

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plateau was reached, rings were incubated with or without 0.25 μM DPPH for 30 min, and were exposed to cumulatively increasing concentrations of either Ach (0.01-1 μM) or SNP (0.01-1 μM) in order to measure the endothelium-dependent relaxation (EDR) and endothelium-independent relaxation (EIDR), respectively. To examine the effect of scutellarin alone on quiescent aortic ring, rings were only incubated with various concentrations of scutellarin. To measure the relaxing effect of scutellarin alone, rings were divided into two groups and were pre-constricted with NE. One group was exposed to 1 μM Ach and the other was exposed to cumulatively increasing concentrations of

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scutellarin. To examine the protective effect of scutellarin on DPPH-induced endothelia dysfunction, rings were divided into five groups: control, 0.25 μM DPPH, and 0.25 μM DPPH with three different concentrations of scutellarin pretreatment (25 μM, 50 μM, and 100 μM, respectively). Rings were incubated with or without scutellarin for 20 min according to grouping and were pre-constricted with NE. Then rings were further incubated with or without 0.25 μM DPPH, and then were subjected to Ach relaxation measurement. At the end of this experiment, biopsies of these rings were taken, fixed

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in 3.5% glutaraldehyde then in 1% osmic acid for 2 h, dehydrated in alcohol, embedded in epoxy resin 618, and sectioned (4 μm) using an Olympus microtome. The sections were stained with lead citrate and uranyl acetate. The ultra-micro structure of endothelium was inspected with a Jem-1011

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transmission electron microscope (Nihon Kohden, Japan).

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Cell culture

Human umbilical vein endothelial cells (HUVECs) were purchased from China Center for Type

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Culture Collection (China). HUVECs were cultured in RPMI 1640 (Gibco) medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37 ⁰C in a humidified atmosphere of 5% CO2. The medium was changed every 2 days and cells were maintained in culture

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with passaging.

Cell viability assay 4

HUVECs were seeded into a 96-well plate at a density of 1.0 × 10 cells/well. Cells were allowed to

attach overnight. To determine the cytotoxicity of scutellarin, cells were added with scutellarin of a series of concentrations, incubated for 24 and 48 h, respectively, and determined for viability by MTT assay.

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H2O2-induced HUVECs injury 4

HUVECs were seeded into a 96-well plate at a density of 1.0 × 10 cells/well and were allowed to attach overnight. Cells were incubated with or without scutellarin of various concentrations for 30 min, and then the medium was replaced and cells were further incubated with 1 mM H2O2 for 4 h. 10 mM Vitamin E was included as the reference. Cell viability was determined by MTT assay. The content of •-

O2 , NO, and MDA and the activity of Nox, SOD, GPx and CAT were determined with assay kit by following the manufacturer’s instruction. The cytosolic Ca

2+

was determined using the method

established by Grynkiewicz et al (Grynkiewicz et al., 1985) and described by Lock et al. (Lock et al., 6

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2011) with modifications. Briefly, cells were loaded with 5 μM Fura-2/AM in Tyrode-HEPES buffer (1.0 × 10 cells/ml) for 40 min at 37 ⁰C, washed by PBS for 2 times, and re-suspended in Tyrode-HEPES. Shimadzu RF5000 Fluorescence Spectrophotometer (Japan) was utilized to record the fluorescence of 2+

Fura-2 and Fura-2-Ca . At intervals of 2 seconds, excitation wavelength alternated between 340 and 380 nm, and ratio (R) of emission at the excitation wavelength of 340 nm to emission at the excitation wavelength of 380 nm was recorded at 510 nm using filters appropriate for Fura-2. Then cell membrane was disrupted by adding 0.1% Triton X-100 and the maximal emission ratio (Rmax) was 2+

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recorded. Then 5 mM EGTA was added to chelate Ca and the minimal emission ratio (Rmin) was 2+

2+

recorded. Intracellular Ca concentration [Ca ]i was calculated using Grynkiewicz equation as follows: 2+

[Ca ]i(nM) = Kd*(R-Rmin)/(Rmax-R), where Kd = 224 nM according to the batch information provided by the manufacturer of Fura-2/AM. The mRNA expression of Nox4 and SOD1 was examined by using in situ hybridization histochemistry assay kit, following the manufacturer’s instruction. The protein expression of Nox4 and SOD1 was determined by Western blot. β-actin was used as the internal

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standard. The levels of mRNA and protein were quantified by densitometry using ImageJ software.

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In vivo anti-atherosclerotic study of scutellarin

Sixty rats were randomly divided into six groups of ten animals for each one: control group, high fat diet (HFD) group, HFD + 5 mg/kg scutellarin group, HFD + 10 mg/kg scutellarin group, HFD + 20 mg/kg

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scutellarin group, and HFD + 5 mg/kg atorvastatin group, in which atorvastatin was used as the reference drug. HFD contained regular diet (78.3%), lard (10%), sugar (5%), cholesterol (3%), yolk

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powder (2%), vitamin D (1%), sodium cholate (0.5%), and propylthiouracil (0.2%). Visceral hypersensitivity was induced in all groups of rats except the control group by daily intraperitoneal injection of bovine serum albumin (32 mg/kg) and chicken egg albumin (2.5 mg/kg) for one week. 5

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After a single injection of vitamin E (6 × 10 U/kg), the hypersensitive rats were fed with HFD for eight weeks. Scutellarin or atorvastatin was given to rats once daily by gavage for six weeks from the third week of HFD feeding. At the end of experiment all animals were euthanatized and the blood was collected for determination of serum levels of MDA, SOD, NO, TC, TG, HDL-C, and LDL-C with assay kit following the manufacturer’s instruction. Biopsies of the thoracic aorta were taken, fixed in 10% neutral buffered formaldehyde for 7 days, embedded in paraffin, sectioned (5 μm), stained with hematoxylin and eosin, and inspected under an optical microscope. Statistical analysis Data are presented as mean ± SD. Differences were analyzed with one-way ANOVA followed by 6 / 23

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Bonferroni’s multiple comparison test or by Student’s t test as needed in GraphPad Prism 5.0 software. A significant difference was considered at P < 0.05. Results In vitro antioxidant activity of scutellarin To evaluate the antioxidant capacity of scutellarin, total antioxidant capacity (TAC) and the •-

•

scavenging ability of scutellarin on DPPH, superoxide anion (O2 ), and hydroxyl radical ( OH) were

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assayed in vitro. As listed in Table 1, scutellarin at various concentrations scavenged all types of radicals in a dose-dependent manner. Scutellarin showed a smallest EC50 value in scavenging hydroxyl radical (37.11 μM).

Protective effect of scutellarin against DPPH-induced endothelial dysfunction in vitro

After Incubation with 0.25 μM of DPPH for 30 min the EDR of rabbit aortic rings induced by 0.1 μM

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and 1 μM of Ach decreased significantly (P < 0.01 and P < 0.001, respectively, vs. the control group) while the EIDR induced by SNP did not change, indicating that DPPH treatment injures endothelium function (Fig. 2A). When the aortic rings were treated with various concentrations of scutellarin before the DPPH incubation, the relaxation responses induced by Ach retained. This effect of scutellarin was significant at the concentration of 50 μM (P < 0.05 vs. DPPH group), and was potent at 100 μM (P < 0.001) (Fig. 2B). Scutellarin alone did not elicit relaxation in quiescent aortic ring but relaxed the aortic

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rings pre-constricted with 1 μM NE in a dose dependent manner, mimicking the effect of Ach (Fig. 2C). Electron microscope inspection showed that the ultra-micro structure of normal vascular endothelium was intact. DPPH caused dissection and collapse of endothelia and infiltration of inflammatory cells.

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The disruption of vascular endothelia and inflammation-induced morphological changes in scutellarin 100 μM group were less severe than that in DPPH group (Fig. 2D).

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Cytotoxicity of scutellarin

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Table 2 shows the viability of HUVECs under various concentrations of scutellarin. Scutellarin treatment for 24 h did not affect the growth of HUVECs. But the treatment of 400 μM scutellarin for 48 h inhibited the proliferation of HUVECs significantly (P < 0.05). Thus the highest dose of scutellarin

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that would be used in H2O2-induced oxidative damage experiment was set at 200 μM, a concentration at which scutellarin did not elicit inhibition on HUVECs growth (Table 2). Scutellarin protected HUVECs from H2O2-induced oxidative damage 1 mM H2O2 treatment for 4 h caused HUVECs death by 50%. Pre-incubation with various concentrations of scutellarin for 30 min before H2O2 challenge rescued HUVECs significantly (Table 3). •-

2+

H2O2 challenge increased intracellular levels of O2 , MDA and Ca , while diminished NO production. Scutellarin pre-incubation dose-dependently inhibited the generation of stress factors but increased NO concentration. H2O2 challenge also reduced the activities of physiological antioxidant enzyme SOD, GPx, and CAT, and increased the activity of Nox that catalyzes the generation of radicals. Scutellarin 7 / 23

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pre-treatment restored the activity of antioxidant enzymes while lessened Nox activity obviously (Table 3). Effect of scutellarin on expression levels of Nox4 and SOD1 The mRNA expression of Nox4 and SOD1 were examined by using in situ hybridization histochemistry assay. The results showed that SOD1 mRNA expression was down-regulated upon H2O2 challenge while Nox4 mRNA level was up-regulated. Scutellarin reversed the changes in SOD1 and Nox4 mRNA expression caused by H2O2 in a dose-dependent manner (Fig. 3 and Fig. 4). Alterations in

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protein expression of Nox4 and SOD1 were in consistence with that in mRNA expression. H 2O2 elevated Nox4 protein level and lowered SOD1 protein level, while scutellarin restored the protein expression levels of these two enzymes (Fig. 5). Anti-atherosclerotic activity of scutellarin in vivo

High fat diet (HFD) feeding for consecutive eight weeks elevated rat serum levels of TC, TG, and

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LDL-C by 289.4%, 951.6%, and 1445.2%, respectively, and reduced the HDL-C to 72.2%. In the same time the rat serum levels of SOD and NO decreased markedly while MDA level increased, indicating that the rats developed hyperlipidemia and were highly susceptible to atherosclerosis. Six weeks of scutellarin oral administration reduced serum TC, TG and LDL-C, and increased serum HDL-C, mimicking the effect of atorvastatin in alleviating hyperlipidemia. In addition, scutellarin dose-dependently raised serum levels of SOD and NO and lowered MDA level (Table 4).

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Histopathological inspection revealed that rats fed with HFD had obvious atherosclerotic plaque in thoracic aorta, accompanied by macrophages infiltration and intima inflammation. Scutellarin administration dramatically diminished the size and number of atherosclerotic plaque and subdued

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inflammatory cells infiltration, showing the same effect as the positive control atorvastatin (Fig 6).

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Discussion

In common with most plant derived flavonoids, scutellarin possesses in vitro antioxidant property

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which was validated in the present study. As the correlation between oxidative stress and the pathogenesis of atherosclerosis has long been established, we ask the question whether the antioxidant activity may endow scutellarin with benefit in attenuating and/or preventing

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cardiovascular diseases. Thus in this study we for the first time investigated the effect of scutellarin in DPPH-induced endothelia dysfunction of rabbit aorta. The stable radical DPPH undermined EDR but not EIDR, indicating that oxidative stress injures endothelium but not vascular enervation (Tang et al., 2006). Scutellarin was able to lessen EDR impairment caused by DPPH and ameliorate the inflammatory morphologic changes in vascular endothelium. This result suggests that scutellarin acts on endothelium and the mechanism is associated with scavenging radicals. Intriguingly, scutellarin alone relaxed NE-pre-restricted aortic rings, mimicking the effect of Ach. This suggests a possible impact of scutellarin on NO release in endothelium. In a study performed with the rat cerebral ischemia/reperfusion model, scutellarin was found to up-regulate endothelia nitric oxide synthase (eNOS) (Hu et al., 2005), the isoform of NOS that produces NO with beneficial effects such as vasodilation and inhibition on pro-inflammatory events (Sugimoto and Iadecola, 2002). The 8 / 23

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modulation on eNOS expression thus may underlie the effect of scutellarin on NO release. Although the endothelium-dependent relaxing effect of scutellarin is evidenced, the present study did not include the test of scutellarin’s effect on EIDR. The influence of scutellarin on SNP-induced relaxation of endothelium-deprived aortic rings was not determined. Thus the endothelium-independent vasodilatory actions of scutellarin cannot be excluded and need further exploration. In order to identify the molecules involved in the mechanism of action of scutellarin, we analyzed the changes of endothelial ROS levels in HUVECs upon oxidative challenge. Incubation with 1 mM •-

H2O2 for 4 h affected the viability of HUVECs. In survived cells NO was depleted and O2 , MDA, and Ca

2+

•-

were up-regulated. Low NO production leads to compromised EDR, while excessive O2 reacts -

-

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with NO to form ONOO or nitrogen dioxide (NO2 ) that exacerbate oxidative stress (Ellinsworth, 2015; Hu et al., 2005). MDA generated by ROS degrading polyunsaturated lipids damages DNA, and then massive mitochondrial DNA damage accelerates atherosclerosis (Andreassi and Botto, 2003). Intracellular Ca

2+

overload initiates inflammatory process and mitochondrial dysfunction, and then

directs cell into apoptotic pathway (Madamanchi and Runge, 2007). Pretreatment of scutellarin checked the deleterious alterations occurring in HUVECs and thus increased cell viability. Cellular antioxidant enzymatic systems in HUVECs were definitely impaired upon oxidative stress. Activity of •-

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SOD, GPx, and CAT decreased but Nox increased. SOD converses O2 into H2O2 which is then detoxified by GPx and CAT (Victor et al., 2009). Over-expression of SOD retards the development of -/-

atherosclerosis in apolipoprotein E mice (Mueller et al., 2005; Santilli et al., 2015). On the contrary, Nox is a major source of ROS (Touyz et al., 2011). Nox4 overexpression worsened cardiac impairment •-

and Nox4 knockout decreased mouse cardiac O2

level (Panth et al., 2016). Scutellarin

dose-dependently increased the activity of SOD, GPx, and CAT, meanwhile inhibited Nox to generate

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excess ROS. This result demonstrated that scutellarin spares HUVECs from oxidative damage through boosting cellular antioxidant defenses. Furthermore, scutellarin recovered the mRNA and protein •-

expression of SOD1 (the cytosolic Cu/Zn-SOD that mainly detoxifies cellular O2 ) and suppressed Nox4

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(the most abundant Nox isoform in endothelium) expression in HUVECs, thus the regulation of SOD1 and Nox4 expression by scutellarin also contribute to the protection of scutellarin on endothelial cells. Although the etiology of atherosclerosis involves the interaction of a number of genes and

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environmental factors (Victor et al., 2009), an important pathologic aspect has been identified as lipid peroxidation, one of the detrimental consequences of oxidative stress (Libby et al., 2011). Human

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atherosclerotic lesions are abundant with oxidation products of linoleic acid and arachidonic acid (Waddington et al., 2003). As most of linoleic acid and arachidonic acid are esterified with cholesterol to form cholesteryl linoleate and cholesteryl arachidonate which primarily comprise LDL, LDL is easily

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oxidizable and oxidized LDL (oxLDL) is the major atherogenic lipoprotein in arterial wall (Ellinsworth, 2015). Cholesterol itself can also be oxidized to oxysterols, which are present plentifully in atherosclerotic lesions and human macrophage foam cells. Oxysterols level is directly proportional to the severity of atherosclerosis (Brown and Jessup, 1999). As a result, manipulating plasma lipoprotein and cholesterol metabolisms and protecting endothelium from oxidative damage are unsurprisingly accepted as approaches to preventing atherosclerosis (Rader and Daugherty, 2008). This rationale is substantiated by the anti-atherosclerotic effect of probucol, an anti-hyperlipidemic drug which lows cholesterol and displays antioxidant activity (Mashima et al., 2001). In the present study, HFD leaded to high serum levels of TG, TC, and LDL, marking the development of hyperlipidemia, meanwhile SOD activity decreased and MDA level increased, outlining oxidative stress. Scutellarin regimen lowered TG, TC, and LDL and alleviated oxidative stress, exhibiting beneficial effects similar to that of atorvastatin. 9 / 23

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Taking into account the protection of scutellarin against DPPH-induced endothelium dysfunction and H2O2-induced oxidative cell damage, it is safe to at least partly attribute the in vivo anti-atherosclerotic effect of scutellarin to its antioxidant activity. However, whether scutellarin directly acts on enzymes that involved in lipid metabolism, as statins do, is beyond the mechanisms that this study would shed light upon. Conclusion This study for the first time reports the protection of scutellarin against oxidative stress-induced

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endothelial dysfunction and endothelial cells damage. In rat model of atherosclerosis, oral administration of scutellarin alleviated hyperlipidemia and retarded atherosclerosis development. The underlying mechanism mainly involves antioxidation. In addition, our results suggest impacts that scutellarin might have on NO release and lipid metabolism, the mechanisms of which await illustration. Due to many disappointing results of large-scale clinical trials of systemic and unspecific antioxidant therapy exemplified by vitamin E supplementation (Steven et al., 2015), future antioxidant strategies will shift from focusing on the classical antioxidant vitamins to the activators of intrinsic antioxidant

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enzyme system, inhibitors of critical ROS sources, and cell organelle-specific antioxidants. This study shows that scutellarin may meet the criteria of a promising category of antioxidants and is worth of in depth investigation as a drug candidate in the prevention and treatment of atherosclerosis. Conflict of interest

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The authors, Jiao Mo, Renhua Yang, Fan Li, Xiaochao Zhang, Bo He, Yue Zhang, Peng Chen, and Zhiqiang Shen, declare that they have no conflict of interest and that they have no financial

Acknowledgments

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relationship with the organization that sponsored the research.

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This work was supported by the National Natural Science Foundation of China (grant No.: 81660613,

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81260493) and the Natural Science Foundation of Yunnan Province, P.R. China (grant No.: 2015FA021).

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References Andreassi, M.G., Botto, N., 2003. DNA damage as a new emerging risk factor in atherosclerosis. Trends in Cardiovascular Medicine 13, 270-275. Brown, A.J., Jessup, W., 1999. Oxysterols and atherosclerosis. Atherosclerosis 142, 1-28. De Mey, J.G.R., Vanhoutte, P.M., 2014. End O' the line revisited: moving on from nitric oxide to CGRP. Life Sciences 118, 120-128. Ellinsworth, D.C., 2015. Arsenic, reactive oxygen, and endothelial dysfunction. The Journal of Pharmacology and Experimental Therapeutics 353, 458-464.

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Furchgott, R.F., 1988. Studies on relaxation of rabbit aorta by sodium nitrite: the basis for the proposal that acid-activatable inhibitory factor from bovine retractor penis is inorganic nitrite and the endothelium-derived relaxing factor is nitric oxide. In: Vanhoutte, P.M. (Ed.), Vasodilatation: Vascular Smooth Muscle Peptides, Autonomic Nerves, and Endothelium. Raven Press, New York, NY., pp. 401-414.

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Fig. 1. Chemical structure of scutellarin

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A

C

100

Control

80

***

60 40

**

20

80

a,b,c

***

60

a,b a

40

*** 20

***

0 1

0 0.01

0.1

1

0.01

Ach

B

0.1

1

(M)

Ach

SNP

D

Control DPPH

100

DPPH+100 M scutellarin DPPH+50 M scutellarin DPPH+25 M scutellarin

*** *

60

** *

a) Control

-1.0

-0.5

100 (M)

b) 0.25 μM DPPH

0.0

c) 0.25 μM DPPH + 100 μM scutellarin

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lg[Ach] (M)

50

M

20

-1.5

25

Scutellarin

*** *

40

0 -2.0

12.5

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*** 80

Relaxation (%)

***

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Relaxation (%)

100

Relaxation (%)

0.25 M DPPH

Fig. 2. Effects of scutellarin on EDR of rabbit aortic rings. (A) EDR of rabbit aortic rings was impaired by 0.25 μM DPPH. (B) Pre-incubation of scutellarin retained EDR of aortic rings treated by 0.25 μM DPPH.

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(C) Scutellarin alone relaxed aortic rings pre-constricted with 1 μM NE. (D) Morphological appearances of vascular endothelia under electron microscope (25000 ×). Data are expressed as mean ± SD, n = 6. * a

P < 0.05, ** P < 0.01, and *** P < 0.001 vs. the control group or 1μM Ach group in (C); P < 0.05 vs. b

c

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12.5 μM scutellarin group, P < 0.05 vs. 25 μM scutellarin group, and P < 0.05 vs. 50 μM scutellarin group, one-way ANOVA followed by Bonferroni’s posttest.

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b)

c)

d)

e)

f)

a ###

80

a ###

**

##

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Control 1 mM H2O2

100

*** ***

40

***

M

60

20 0 Control

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Integrated optical density of SOD1 mRNA

g)

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a)

12.5

50

200

Scutellarin

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a) Control

10 (M)

b) 1 mM H2O2

c) 1 mM H2O2 + 12.5 μM scutellarin

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d) 1 mM H2O2 + 50 μM scutellarin e) 1 mM H2O2 + 200 μM scutellarin f) 1 mM H2O2 + 10 μM VE

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g) Quantification of SOD1 mRNA level

Fig. 3. Effect of scutellarin on SOD1 mRNA expression in HUVECs. Treatment of 1 mM H2O2 significantly decreased SOD1 mRNA expression in HUVECs as visualized by in situ hybridization. Scutellarin dose-dependently raised SOD1 mRNA expression. SOD1 mRNA levels were quantified by ImageJ 1.49v Software. The Data are expressed as mean ± SD of five independent experiments. ** P < 0.01 and *** P < 0.001 vs. the control group;

##

P < 0.01 and

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a

P < 0.001 vs. 1 mM H2O2 group; P <

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b)

c)

d)

e)

f)

***

80

###

***

a ###

***

60

a, b ###

***

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**

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40

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Control 1 mM H2O2

100

20 0 Control

12.5

50

200

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Integrated optical density of Nox4 mRNA

g)

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10 (M)

b) 1 mM H2O2

c) 1 mM H2O2 + 12.5 μM scutellarin

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d) 1 mM H2O2 + 50 μM scutellarin e) 1 mM H2O2 + 200 μM scutellarin f) 1 mM H2O2 + 10 μM VE

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g) Quantification of Nox4 mRNA level

Fig. 4. Effect of scutellarin on Nox4 mRNA expression in HUVECs. Treatment of 1 mM H2O2 significantly increased Nox4 mRNA expression as visualized by in situ hybridization. Scutellarin dose-dependently decreased Nox4 mRNA expression. Nox4 mRNA levels were quantified by ImageJ 1.49v Software. Data are expressed as mean ± SD of five independent experiments. ** P < 0.01 and *** P < 0.001 vs. the control group;

###

a

b

P < 0.001 vs. 1 mM H2O2 group; P < 0.05 vs. 12.5 μM scutellarin group and P <

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a)

b)

c)

d)

e)

f)

SOD1 32 kd Nox4 65 kd β-actin 43 kd

1.5

###

***

1.0

###

***

a ###

***

***

50

200 (M)

***

0.0 Control

10

12.5

h)

Scutellarin

Control 1 mM H2O2 1.5

*** ***

###

##

***

***

0.5

0.0

10

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VE

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***

200 (M)

Scutellarin

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Normalized integrated optical density of Nox4 protein

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Normalized integrated optical density of SOD1 protein

g)

b) 1 mM H2O2

c) 1 mM H2O2 + 10 μM VE

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d) 1 mM H2O2 + 12.5 μM scutellarin e) 1 mM H2O2 + 50 μM scutellarin f) 1 mM H2O2 + 200 μM scutellarin g) Quantification of SOD1 protein level h) Quantification of Nox4 protein level Fig. 5. Effects of scutellarin on protein expression of SOD and Nox4 in HUVECs. The protein levels of SOD1 and Nox4 were normalized to β-actin, respectively, and were quantified by ImageJ 1.49v Software. Data are expressed as mean ± SD of five independent experiments. *** P < 0.001 vs. the control group;

##

P < 0.01 and

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a

P < 0.001 vs. 1 mM H2O2 group; P < 0.05 vs. 12.5 μM scutellarin

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c)

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e)

f)

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a) Control b) HFD

c) HFD + 5 mg/kg scutellarin

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d) HFD + 10 mg/kg scutellarin e) HFD + 20 mg/kg scutellarin

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f) HFD + 5 mg/kg atorvastatin

Fig. 6. Histological appearances of the rat thoracic aorta after eight weeks of high fat diet (HFD)

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Table 1 Antioxidant capacity of scutellarin in vitro Substance

Clearance (%)

(μM)

TAC

DPPH

O2

•

Control

0.06 ± 0.05

0.18 ± 0.10

0.08 ± 0.04

0.11 ± 0.07

VE 10

71.22 ± 4.88*

65.33 ± 10.05*

84.10 ± 7.95*

76.94 ± 10.23*

Sclr 400

73.43 ± 9.13*

70.82 ± 11.24*

82.51 ± 6.74*

75.75 ± 13.31*

Sclr 200

72.65 ± 5.16*

67.57 ± 7.98*

70.35 ± 9.57*

76.25 ± 10.03*

Sclr 100

62.70 ± 9.12*

53.08 ± 12.89*

57.29 ± 5.90*

71.02 ± 8.40*

Sclr 50

47.81 ± 7.54*

36.55 ± 7.23*

44.42 ± 5.67*

56.81 ± 12.46*

Sclr 25

40.23 ± 5.38*

30.46 ± 9.67*

36.82 ± 11.33*

42.59 ± 7.15*

Sclr 12.5

19.94 ± 4.53*

25.82 ± 6.19*

27.31 ± 10.68*

30.03 ± 6.55*

EC50

56.87

85.84

58.48

37.11

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(μM)

•-

Data are expressed as mean ± SD, n = 3. * P < 0.05 vs. the control group, one-way ANOVA followed by

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Bonferroni’s posttest. VE: Vitamin E, Sclr: scutellarin.

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Table 2 Cytotoxicity of scutellarin to HUVECs

24 h

48 h

0

0.80 ± 0.05

1.30 ± 0.10

12.5

0.81 ± 0.07

1.33 ± 0.14

25

0.80 ± 0.08

1.29 ± 0.12

50

0.81 ± 0.20

1.33 ± 0.10

100

0.80 ± 0.10

1.31 ± 0.07

200

0.84 ± 0.06

1.32 ± 0.07

400

0.82 ± 0.13

1.02 ± 0.09*

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Cell Viability (A570)

Scutellarin (μM)

Cell viability was indicated by absorbance at 570 nm as measured by using MTT method. Data are

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expressed as mean ± SD, n = 16.* P < 0.05 vs. 0 μM scutellarin group, one-way ANOVA followed by

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Bonferroni’s posttest.

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Table 3 Influences of scutellarin upon HUVECs injured by H2O2 Measurement

Control

H2O2 (1 mM) Scutellarin (μM) 12.5

50

200

#

0.50 ± 0.03*

0.53 ± 0.04*

#

0.34 ± 0.08*

#

0.27 ± 0.14*

#

#

6.45 ± 0.92*

0.34 ± 0.03*

0.48 ± 0.03*

O2 (A560)

0.09 ± 0.03

0.94 ± 0.15*

0.39 ± 0.12*

MDA (nM)

2.14 ± 1.71

12.23 ± 2.87*

9.01 ± 3.42*

7.23 ± 1.04*

[Ca ]i (nM)

2+

99.12 ± 4.55

201.38 ± 8.07*

198.11 ± 5.64*

171.25 ± 3.86*

NO (μM)

874.5 ± 36.9

509.3 ± 25.7*

557.1 ± 22.5*

SOD (U/ml)

131.36 ± 34.40

47.15 ± 16.08*

87.83 ± 18.55*

GPx (U/ml)

157.20 ± 6.20

42.70 ± 5.60*

71.80 ± 5.60*

93.41 ± 2.80*

CAT (U/ml)

10.05 ± 1.49

4.27 ± 1.28*

5.32 ± 1.50*

5.89 ± 1.12*

Nox (U/ml)

17.34 ± 2.57

119.70 ± 10.13*

76.80 ± 11.42*

#

#a

#a

0.25 ± 0.09

#

#a

6.29 ± 1.70

#

#ab

652.8 ± 34.1*

#

115.83 ± 47.48

#a

121.60 ± 3.40*

89.57 ± 25.93* #

#

#

#ab

#a

#

0.52 ± 0.03

155.49 ± 9.91*

604.2 ± 26.6* #

#a

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0.67 ± 0.05

•-

10

#

#ab

125.70 ± 3.70*

#ab

31.60 ± 8.29*

#

665.0 ± 30.4*

112.54 ± 13.21

#

#a

# #

#

6.95 ± 1.03*

#

85.42 ± 8.83*

#

Data are expressed as mean ± SD, n = 16.* P < 0.05 vs. the control group, P < 0.05 vs. 1 mM H2O2 a

#

#

6.33 ± 1.05*

53.06 ± 9.14*

100.27 ± 3.73

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Viability (A570)

VE (μM)

b

group, P < 0.05 vs. 12.5 μM scutellarin group, and P < 0.05 vs. 50 μM scutellarin group, one-way

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Table 4 Effect of oral administration of scutellarin on HFD-fed rats Control

Measurement

HFD

0.31 ± 0.06

2.95 ± 0.22*

Atorvastatin

(mg/kg)

(mg/kg)

10

5 TG (mmol/L)

Scutellarin 20 #a

2.89 ± 0.42*

2.52 ± 0.17*

10 #ab

0.78 ± 0.10*

#a

5.12 ± 0.49*

#a

1.31 ± 0.14

#ab

2.90 ± 0.52*

2.54 ± 0.22

7.35 ± 0.57*

7.30 ± 0.28*

6.32 ± 0.51*

6.01 ± 0.28*

HDL-C (mmol/L)

1.51 ± 0.18

1.09 ± 0.10*

1.04 ± 0.15*

1.24 ± 0.18*

1.29 ± 0.08*

LDL-C (mmol/L)

0.42 ± 0.09

6.07 ± 0.28*

5.93 ± 0.37*

5.26 ± 0.43*

SOD (U/ml)

151.22 ± 7.91

86.34 ± 10.27*

112.54 ± 9.19*

125.71 ± 8.53*

MDA (mmol/L)

2.09 ± 0.62

8.13 ± 0.80*

7.96 ± 0.77*

5.52 ± 0.51*

NO (μmol/L)

40.25 ± 2.94

25.50 ± 3.76*

26.36 ± 3.28*

31.02 ± 2.59*

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TC (mmol/L)

#a

4.35 ± 0.40*

#a

#a

#a

134.02 ± 11.40*

# #

129.22 ± 8.35* 3.41 ± 0.69*

#a

37.08 ± 4.11

34.38 ± 2.01*

#

#

Data are expressed as mean ± SD, n = 10. * P < 0.05 vs. the control group, P < 0.05 vs. HFD-fed group, a

b

P < 0.05 vs. 5 mg/kg scutellarin group, and P < 0.05 vs. 10 mg/kg scutellarin group, one-way ANOVA

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followed by Bonferroni’s posttest.

#

#ab

3.79 ± 0.65*

#a

#

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#

#

1.76 ± 0.32*

#a

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Graphical Abstract

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