Dihydromyricetin attenuated Ang II induced cardiac fibroblasts proliferation related to inhibitory of oxidative stress

Author’s Accepted Manuscript Dihydromyricetin attenuated Ang II induced cardiac fibroblasts proliferation related to inhibitory of oxidative stress Qiuyi Song, Lulu Liu, Jin Yu, Jingyao Zhang, Mengting Xu, Linlin Sun, Huiqin Luo, Zhaosong Feng, Guoliang Meng www.elsevier.com/locate/ejphar

PII: DOI: Reference:

S0014-2999(17)30273-X http://dx.doi.org/10.1016/j.ejphar.2017.04.014 EJP71169

To appear in: European Journal of Pharmacology Received date: 23 February 2017 Revised date: 29 March 2017 Accepted date: 12 April 2017 Cite this article as: Qiuyi Song, Lulu Liu, Jin Yu, Jingyao Zhang, Mengting Xu, Linlin Sun, Huiqin Luo, Zhaosong Feng and Guoliang Meng, Dihydromyricetin attenuated Ang II induced cardiac fibroblasts proliferation related to inhibitory of oxidative stress, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2017.04.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Dihydromyricetin attenuated Ang II induced cardiac fibroblasts proliferation related to inhibitory of oxidative stress Qiuyi Song1, Lulu Liu1, Jin Yu, Jingyao Zhang, Mengting Xu, Linlin Sun, Huiqin Luo, Zhaosong Feng, Guoliang Meng* Department of Pharmacology, School of Pharmacy, Nantong University; Key Laboratory of Inflammation and Molecular Drug Target of Jiangsu Province, Nantong, Jiangsu, China *

Corresponding author. Department of Pharmacology, School of Pharmacy, Nantong

University; Key Laboratory of Inflammation and Molecular Drug Target of Jiangsu Province, Nantong 226001, China. Tel: +86 513 8505 1726; fax: +86 513 8505 1728. [email protected]

Abstract Dihydromyricetin (DMY) is one of the most important flavonoids in vine tea, which showed several pharmacological effects. However, information about the potential role of DMY on angiotensin II (Ang II) induced cardiac fibroblasts proliferation remains unknown. In the present study, cardiac fibroblasts isolated from neonatal Sprague-Dawley rats were pretreated with different concentrations of DMY (0- 320 μM) for 4 h, or DMY (80 μM) for different time (0-24 h), followed by Ang II (100 nM) stimulation for 24 h, Then number of cardiac fibroblasts and content of hydroxyproline was measured. The level of cellular reactive oxygen species,

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These authors equally contributed to this work.

malondialdehyde (MDA), activity of superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) were also evaluated. Expression of type I, type III collagen, α-smooth muscle actin (α-SMA), p22phox (one vital subunit of nicotinamide adenine dinuclectide phosphate (NADPH) oxidase), SOD and thioredoxin (Trx) were detected with real time PCR or/and western blot. We found that pre-incubation with DMY (20 μM, 40 μM, 80 μM) for 4 h, 12 h or 24 h attenuated the proliferation of cardiac fibroblasts induced by Ang II. Expression of type I and type III collagen, as well as α-SMA were inhibited by DMY at both mRNA and protein level. DMY also significantly decreased cellular reactive oxygen species production and MDA level, while increased the SOD activity and T-AOC. DMY suppressed p22phox, while enhanced antioxidant SOD and Trx expression in Ang II stimulated cardiac fibroblasts. Thus, dihydromyricetin attenuated Ang II induced cardiac fibroblasts proliferation related to inhibitory of oxidative stress. Keywords: dihydromyricetin; cardiac fibroblasts; proliferation; collagen; reactive oxygen species. Chemical compounds studied in this article DMY (Pubmed CID: 161557)

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1. Introduction Myocardial fibrosis mostly results from long-time overload of the heart and is regarded as an independent risk factor for adverse cardiovascular events (Peng et al., 2016; Zhang et al., 2012). Cardiac fibroblasts are the major source of extracellular matrix in myocardium, and they play an important role in the pathogenesis of cardiac fibrosis (Chen et al., 2012). Previous study suggested that excessive activation of cardiac fibroblasts (including proliferation, collagen synthesis and transformation) contributed to occurrence and development of myocardial fibrosis (Fan and Guan, 2016). However, there are no specific methods to attenuate myocardial fibrosis in the clinical setting. Several factors, such as mechanical stress, electrical signal coupling, inflammatory factors, oxidative stress and so on, are involved in myocardial fibrosis (Kurose and Mangmool, 2016; Patel and Mehta, 2012). These pathological stimulations disorder the sympathetic nervous system and renin angiotensin aldosterone system, then accelerate the proliferation of cardiac fibroblasts and promote collagen synthesis (Lopez et al., 2015; Yong et al., 2015). Moreover, excessive reactive oxygen species due to oxidative stress is one of the most dominant factors to induce myocardial fibrosis (Kong et al., 2014). A major source of reactive oxygen species in myocardium is the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. NADPH oxidase contains multiple subunits and p22phox was the key one (Gray and Jandeleit-Dahm, 2015). There is also a complex antioxidant system to combat oxidative stress, including antioxidant enzymes and substances. The main

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antioxidant enzymes contain superoxide dismutase (SOD) and thioredoxin (Trx) (Ladjouzi et al., 2015; Kikusato et al., 2015). All the antioxidants, coordinating and maintaining a dynamic balance with oxidative injury, scavenge reactive oxygen species, reduce oxidative stress and eliminate oxidative damage in the body. Ampelopsis grossedentata (Hand-Mazz) W.T.wang is a species of plant mainly distributed in southern China. Its tender stems and leaves are widely used as Vine tea. It has been used for herbal tea and traditional Chinese medicine for over hundreds of years, with effect of lowing blood pressure, antibacterial, anti thrombosis, antioxidant, anti-inflammatory and anti alcoholism (He et al., 2003; Huang et al., 2013; Murakami et al., 2004; Ni et al., 2012; Shen et al., 2012; Zeng et al., 2006). Dihydromyricetin (DMY) and myricetin are two most important flavonoids in vine tea. It was reported that DMY significantly decreased reactive oxygen species formation in H2O2-stimulated human umbilical vein endothelial cells and lipopolysaccharide-treated RAW264.7 macrophages (Hou et al., 2015; Wang et al., 2016). One latest research found that DMY abolished reactive oxygen species and glutathione production in HepG2 Cells (Xie et al., 2016). Moreover, DMY seems to be cardioprotective, as it attenuated reactive oxygen species production by rat primary cardiomyocytes in adriamycin-induced cardiotoxicity model (Zhu et al., 2015). Our previous study also suggested that DMY inhibited angiotensin II (Ang II)-induced cardiomyocyte hypertrophy via an anti-oxidative manner (Meng et al., 2015a). However, there is no information about the potential role of DMY in Ang II induced cardiac fibroblasts proliferation until now.

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The aim of the present study was to examine whether DMY attenuated cardiac fibroblasts proliferation and to elucidate the possible role and detailed mechanism of oxidative stress in this protective effect. 2. Materials and methods 2.1 Cell culture and treatment The experiment was conducted according to NIH Guidelines for Care and Use of Laboratory Animals and was approved by the Institutional Animal Ethical Committee of Nantong University (approval no. NTU-20150305). Sprague-Dawley rats of 1- to 3-d-old were provided by Experimental Animal Center of Nantong University (Nantong, China). Hearts were removed from newborn rats immediately after euthanized by decapitation. The ventricles were separated from the atria, trisected and digested with 0.25% trypsin (Beyotime, Shanghai, China) at 37°C for 7-10 cycles until completely digested. After digestion, all supernatants except the first one were collected. Fetal bovine serum (FBS, Hyclone labs, Logan, UT) of 10% was added into dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA) to stop digestion. The cell suspension, re-suspended with DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (Beyotime, Shanghai, China), was incubated at 37°C for 4 h in a humidified 5% CO2 incubator to separate fibroblasts from cardiomyocytes. The cells from 5 rats were planted in a 6-well plate or other culture plate with equal area for the first subculture. Confluent cardiac fibroblasts were treated with trypsin and sub-cultured, and cells of second or third passages were applied in our further experiments. The phenotype of cardiac fibroblasts was

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characterized with α-SMA and vimentin specific immunofluorescence staining (SFig.1). The purity of cardiac fibroblast in the present study was more than 95%. After the culture medium was changed into DMEM supplemented with 0.5% FBS for 24 h, serum-starved cells were pre-incubated with DMY ((2R,3R)-3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-dihydrochromen-4-one, C15H12O8, PubChem CID: 161557, Standard Center of China, Beijing, China, chemical structure in Fig.1) at different concentrations (0, 1 μM,10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM) for different times (0, 1 h, 2 h, 4 h, 12 h or 24 h), (Meng et al., 2015a) followed by Ang II (Sigma-Aldrich, St. Louis, MO; 100 nM) (Zhou et al., 2016) stimulation for an additional 24 h as previous study. The culture medium without DMY was used as a vehicle control.

2.2 Cell number and cytotoxicity assay Cell number was measured by Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China) assay to estimate proliferation of cardiac fibroblasts according to the manufacturer’s directions. Briefly, cardiac fibroblasts (1×104) were seeded in a 96-well plate and exposed to Ang II (100 nM, 24 h) after pretreatment with DMY or vehicle (DMEM) for different times, followed by addition of 10 μl 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) mixture, which was similar to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and was reduced by some dehydrogenases to form aurantius formazan. The cells were then

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incubated at 37°C for 1 h in the incubator. The absorbance was measured in a microplate reader (Biotek Instruments, Winooski, VT) at a wavelength of 450 nm (Xie et al., 2016). Cell numbers were represented as optical density (OD) value. Lactate dehydrogenase (LDH) level released from cells was detected by LDH-Cytotoxic Assay Kits (Roche Diagnostics, Mannheim, Germany) to assess cytotoxicity. The detailed protocol was according to the manufacturer’s directions. The medium without cells was served as a blank control. The LDH content in the medium was calculated based on the absorbance which was normalized with the blank control. The average LDH level was normalized by OD value representing cell numbers.

2.3 Determination of hydroxyproline in culture medium After treatment, hydroxyproline content in cell culture medium for evaluating collagen production was measured according to the protocol provide by the manufacturers (Jiancheng Bioengineering Institute, Nanjing, China). The medium without cells was served as a blank control. The hydroxyproline content in the medium was calculated based on the absorbance which was normalized with the blank control. The average hydroxyproline content was normalized by OD value representing cell numbers.

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2.4 Measurement of malondialdehyde (MDA), SOD, and total antioxidant capacity (T-AOC) Cell precipitate of cardiac fibroblasts was re-suspended in lysis buffer containing 20 mmol/l Tris-HCl (pH 7.5), 1 mmol/l EDTA, 150 mmol/l NaCl, 20 mmol/l NaF, 3 mmol/l Na3VO4, 1 mmol/l PMSF, with 1% Triton X-100 and protease inhibitor cocktail and further lysed on ice for 30 min. After centrifugation at 12, 000 g for 5 min, the supernatants were collected. The protein was applied for MDA measurement and western blot. The level of MDA in cardiac fibroblast was measured by Lipid Peroxidation and Assay Kits (Beyotime, Shanghai, China) with thiobarbituric acid (TBA) method and was expressed as μmol/g·protein. Protein, extracted from cells with homogenate in PBS, was applied for SOD activity and T-AOC measurement. SOD activity was assessed by SOD Assay Kits (Beyotime, Shanghai, China) with nitroblue tetrazolium (NBT) method and was expressed as units/mg·protein. T-AOC was detected by T-AOC Assay Kits (Beyotime, Shanghai, China) with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid

(ABTS)

method and was expressed as mmol/g·protein. All the detections were performed according to the manufacturer’s instructions.

2.5 Quantitative real-time polymerase chain reaction (PCR) Total RNA was extracted using Trizol reagent (Takara, Kyoto, Japan) followed by reverse-transcription using a PrimeScriptTM RT Master Mix Kit (Takara, Kyoto, Japan). Quantitative real-time PCR were performed with above cDNAs using SYBR

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Green Fast qPCR mix (Takara, Kyoto, Japan) with ABI 7500 Real Time PCR System (ABI, Carlsbad, CA). 18S was used as a house-keeping gene. Each cDNA from different groups was run and analyzed in triplicate. Primers used to amplify the fragments of α-smooth muscle actin (α-SMA), collagen I, collagen III and 18S were α-SMA-F 5’-CATCAGGAACCTCGAGAAGC-3’, α-SMA-R 5’-TCGGATACTTCAGGGTCAGG-3’; collagen I-F 5’-AGGGTCATCGTGGCTTCTCT-3’, collagen I-R 5’-CAGGCTCTTGAGGGTAGTGT-3’; collagen III-F 5’-AGCGGAGAATACTGGGTTGA-3’, collagen III-R 5’-GATGTAATGTTCTGGGAGGC-3’; 18S-F 5’-AGTCCCTGCCCTTTGTACACA-3’, 18S-R 5’-CGATCCGAGGGCCTCACTA-3’. Experimental cycle threshold values were normalized to 18S, and relative mRNA expression was calculated versus a control sample.

2.6 Western blot analysis The protein samples extracted with lysis buffer (described at part of 2.4) were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred into polyvinylidene fluoride (PVDF) membrane, blocked with 5% non-fat milk at room temperature for 2 h, and immunobloted with primary anti-collagen I, anti-collagen III, anti-α-SMA, anti-p22phox (1:1000, Santa Cruz Biotechnology Inc., San Diego, CA), anti-SOD, anti-Trx (1:1000, Abcam, Cambridge,

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UK), and anti-GAPDH (1:7000, Sigma-Aldrich, St. Louis, MO) at 4°C over nigh

followed by horseradish peroxidase-conjugated secondary antibodies incubation for 2h at room temperature. Protein bands were visualized by enhanced chemiluminescence substrate (ECL, Thermo Fisher Scientific Inc., Rockford, IL, USA).

2.7 Measurement of reactive oxygen species After treatment, cells were incubated with 2’,7’-dichlorfluorescein-diacetate (DCFH-DA, 10 μM) liposoluble probe (Beyotime, Shanghai, China) at 37°C for 1 h. Intracellular reactive oxygen species, represented as fluorescence, was measured at 488 nm (excitation) and 528 nm (emission) by a fluorescence microscope (Nikon, Tokyo, Japan).

2.8 Immunofluorescence After treatment, the culture medium was removed and the cells were fixed by poly formaldehyde for 10 min, followed by incubation with PBS containing 0.3% Triton X-100 at room temperature for 20 min. Then cells were incubated with rabbit anti-rat α-SMA antibody (1:500) or vimentin (1:500, Boster, Wuhan, China) at 4°C overnight followed by Alexa Fluor 488 conjugated IgG (1:1000) at 37 °C for 1 h. Nuclei were stained by DAPI (5 μg/ml, Beyotime, Shanghai, China) and then visualized with a fluorescence microscope (Nikon, Tokyo, Japan).

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2.9 Statistical Analysis All data were presented as mean ± standard error (S.E.M.) and analyzed using one-way ANOVA with the Student-Newman-Keuls test (Stata 13.0). A value of P less than 0.05 was considered statistically significant.

3. Results 3.1 DMY inhibits Ang II induced cardiac fibroblasts proliferation and collagen deposition To investigate the optimum concentration of DMY on Ang II induced cardiac fibroblasts proliferation and collagen deposition, cardiac fibroblasts were pretreated with different concentrations of DMY (1 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM and 320 μM) for 4 h followed by Ang II (100 nM) stimulation for another 24 h. Ang II significantly increased cardiac fibroblasts numbers, which was attenuated by DMY at 20 μM, 40 μM and 80 μM (Fig.2 A). Meanwhile, the total hydroxyproline content in the medium and average hydroxyproline content normalized by cell numbers representing collagen deposition, was also reduced by DMY (Fig.2 B-C). It is noting that DMY at 80 μM produced the most profound effect, and this concentration was chosen for further experiments. To confirm the optimum pretreatment time of DMY on Ang II induced cardiac fibroblasts proliferation and collagen deposition, cardiac fibroblasts was administrated with DMY (80 μM) for different time (1 h, 2 h, 4 h, 12 h and 24 h), followed by Ang II (100 nM) for another 24h. DMY at 80 μM pretreated for 4h, 12h and 24h

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significantly decreased cardiac fibroblasts numbers, total and average hydroxyproline content, and the attenuating effect peaked at DMY pretreatment for 4 h (Fig.2 D-F). Moreover, no significant alteration of cardiac fibroblasts numbers and hydroxyproline content was found after DMY administration of 80 μM alone (the right column, Fig. 2 A-F). 3.2 DMY has no toxicity on cardiac fibroblasts In order to verify the above inhibition of DMY in Ang II-induced myocardial fibroblasts proliferation was not due to the toxicity of DMY itself, LDH activity was detected to assess the degree of the cell damage. DMY (1 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM and 320 μM) pretreated for 4 h, or DMY (80 μM) pretreated for different time (1 h, 2 h, 4 h, 12 h and 24 h), did not show any significant difference on total LDH content in the medium or average LDH level normalized by cell numbers (Fig.3 A-D). It suggested that DMY had no cytotoxicity on cardiac fibroblasts in the present study.

3.3 DMY decreases expression of collagen I, collagen III and α-SMA in Ang II-induced cardiac fibroblasts To detect whether DMY inhibited collagen synthesis of Ang II-induced cardiac fibroblasts, expression of collagen I and III at level of both mRNA and protein was firstly measured. Ang II significantly increased collagen I and III in cardiac fibroblasts, which was attenuated by DMY pretreatment (Fig.4 A, B). α-SMA is a robust and sensitive markers of cardiac fibroblasts differentiation (Bai et al., 2013),

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which was measured by real time PCR, western blot and immunofluorescence. It was found that level of α-SMA increased significantly after Ang II stimulation in cardiac fibroblasts, which was also reduced by DMY (80 μM, 4 h) pre-treatment (Fig.5 A-C). Taken together, DMY inhibited collagen synthesis in Ang II-induced cardiac fibroblasts.

3.4 DMY reduces oxidative stress in Ang II-induced cardiac fibroblasts As well as we know, oxidative stress plays an important role in the proliferation of cardiac fibroblasts. Effect of DMY on oxidative stress in Ang II-stimulated cardiac fibroblasts was further evaluated. It was showed that DMY significantly decreased the fluorescence intensity of DCFH-DA in cardiac fibroblasts, which suggested that higher level of reactive oxygen species induced by Ang II was significantly reduced by DMY (80 μM, 4 h) pretreatment (Fig.6 A). MDA is the metabolites of lipid peroxidation which was a main index for reactive oxygen species. The MDA level was decreased after DMY (80 μM, 4 h) pretreatment followed by Ang II stimulation (Fig.6 B). To investigate the possible anti-oxidative mechanisms of DMY, SOD activity and T-AOC levels was further checked. Ang II decreased SOD activity and T-AOC in cardiac fibroblasts, which was enhanced significantly by DMY (Fig.6 C, D).

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3.5 DMY suppresses p22phox but increases SOD and Trx expression in Ang II stimulated cardiac fibroblasts Above results showed that DMY effectively inhibited oxidative stress during Ang II-induced cardiac fibroblasts proliferation. To explore the detailed mechanism, expression of p22phox, SOD and Trx was assessed by western blot. Ang II significantly increased expression of p22phox, while decreased the expression of antioxidant enzymes of SOD and Trx. DMY (80 μM) pretreatment restored the expression of p22phox protein, SOD and Trx (Fig.7 A-C).

3.6 DMY alone increased SOD activity and T-AOC in cardiac fibroblasts Above data suggested the protective effect on DMY pre-treated cardiac fibroblast followed by Ang II stimulation for another 24 h. However, whether DMY induced some other effects on the cells (although not affecting proliferation) in the absence of Ang II was unknown. Next, reactive oxygen species, MDA level, SOD activity, T-AOC, expression of SOD, Trx and p22phox were evaluated after DMY administration alone for 24h. We found that DMY alone increased SOD activity and T-AOC. However, there is no significant difference on reactive oxygen species production, MDA level, as well as expression of SOD, Trx and p22phox (SFig.2 A-E).

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4. Discussion Our present study found that DMY attenuated Ang II induced cardiac fibroblasts proliferation related to inhibitory of oxidative stress. DMY, belonging to flavonoid compounds, shows potential inhibition on proliferation of breast cancer MCF-7 and MDA-MB-231 cells, nasopharyngeal carcinoma HK-1 cells, human hepotoma HepG2 cells, leukemia HL-60, K-562 cells, and lung cancer H1299 cells (Ni et al., 2012; Jeon et al., 2008; Wu et al., 2013). DMY also suppressed platelet aggregation, attenuated release of 5-serotonin and increased the concentration of intracellular calcium with platelet activating factor stimulation, which is beneficial to cardiovascular system (Wang et al., 2014). Recent studies found that DMY reduced the levels of alanine aminotransferase, LDH and MB isoenzyme of creatine kinase in adriamycin treated mice, suggesting a protective effect on adriamycin induced cardiac toxicity (Zhu et al., 2015). The latest research revealed that DMY pretreatment provided significant protection against myocardial ischemia-reperfusion injury via antioxidant capacity enhancement and apoptosis inhibition (Liu et al., 2016). Our previous studies revealed that DMY inhibited cardiomyocyte hypertrophy induced by Ang II, which was possibly related to the antioxidative effect via a NO-dependent manner (Meng et al., 2015a). However, the effect of DMY on cardiac fibroblasts proliferation remains unknown. Cardiac fibroblasts proliferation is one of the most vital characteristics in myocardial fibrosis, which mainly manifest as increased cell numbers and collagen accumulation in the extracellular matrix. These events could result in an irreversible

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damage in a variety of cardiovascular diseases including heart failure and hypertension (Liao et al., 2014). Many studies have shown that Ang II increased cardiac fibroblasts numbers and collagen synthesis via type 1 receptor of Ang II and G protein coupled receptors activation (Etelvino et al., 2014). In this study, cardiac fibroblasts isolated from newborn Sprague-Dawley rat were stimulated with Ang II to induce proliferation. It was found that DMY concentration-dependently and time-dependently inhibited Ang II induced cardiac fibroblast proliferation. More importantly, high concentration of DMY up to 320 μM showed no damage on activity or survival quality of cardiac fibroblasts. That is to say that DMY has potential ability to inhibit cardiac fibroblasts proliferation, which has nothing to do with cytotoxicity. It is noting that DMY also suppressed proliferation in hepatocellular carcinoma cells via promoting apoptosis (Liu et al., 2014) and inducing G2/M phase cell cycle arrest through the Chk1/Chk2/Cdc25C pathway (Huang et al., 2013), while DMY attenuated apoptosis in myocardial ischemia-reperfusion injury (Liu et al., 2016). Meanwhile, DMY has pro-apoptotic effects on AGS human gastric cancer cells (Ji et al., 2015), human non-small cell lung cancer (Kao et al., 2016) and human hepatoma HepG2 cells (Wu et al., 2013). Hydroxyproline accounted for about 13.4% in collagen, while very few in elastin and was absent in other proteins, and hydroxyproline concentration was a good index to reflect collagen secretion (Meng et al., 2015b). Collagen type I and III are two major structural proteins for myocardial collagen matrix. We found that DMY inhibited both collagen expression and collagen secretion in Ang II stimulated cardiac fibroblasts.

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We also found that DMY alone was not able to modify the baseline proliferation and collagen synthesis. Associated with the inhibitory effect of DMY on Ang II induced cardiac fibroblasts proliferation and synthesis of collagen, it is suggested that DMY has potential to attenuate cardiac fibroblasts proliferation in pathological but not physiological condition. It is noting that DMY of 160 μM or 320 μM did not attenuate the effect elicited by Ang II. Although there is no cytotoxicity on cardiac fibroblasts of DMY, too high concentration of DMY might be potentially adverse to cardiac fibroblasts to attenuate the protective effect of DMY itself. It is suggested that appropriate dosage was beneficial for scientific usage of DMY. Myofibroblast is a special cell exhibiting typical characteristics of both smooth muscle cells and fibroblasts (Xu et al., 2015). Compared with normal fibroblasts, myofibroblast has a stronger ability to proliferate and secrete collagen. The expression of α-SMA in cardiac fibroblasts is a marker protein of myofibroblasts, which is a robust and sensitive sign of proliferation (Costa-Almeida et al., 2015). Our study found that DMY reduced the expression of α-SMA in Ang II-induced cardiac fibroblasts, suggesting the potential effect of DMY on inhibiting activation of fibroblast and transformation of fibroblast to myofibroblast. Oxidative stress is one of the important mechanisms to initiate myocardial fibrosis (Ilkun and Boudina, 2013). In normal physiological conditions, about 90% of the oxygen in the cell is consumed by mitochondria, and was then reduced into water. However, in many pathological processes, both expression and activity of NADPH oxidase increases, and then enhances oxidation production, induces cardiac fibroblast

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cell damage, results in extracellular matrix accumulation, and finally leads to myocardial fibrosis (Li et al., 2015). Studies have confirmed that Ang II promoted the production and release of reactive oxygen species in cardiac fibroblasts (Yu et al., 2012), which was consistent with the increased fluorescence intensity of DCFH-DA, enhanced level of MDA, but decreased SOD activity and T-AOC levels after Ang II stimulation in cardiac fibroblasts of our present study. DMY exhibited powerful antioxidant ability represented as decreased reactive oxygen species production and MDA level but enhanced SOD activity and T-AOC. It might be one key mechanism of DMY for protective effect on Ang II-induced cardiac fibroblasts proliferation and collagen expression or secretion. NAPDH oxidase inhibitor GKT137831 inhibited the level of reactive oxygen species and reduced the degree of myocardial fibrosis (Zhao et al., 2015). Previous research found that DMY concentration-dependently attenuated production of oxidative stress and promoted Hepal-6 cells apoptosis, which was related to the prohibitive effect of DMY on NADPH oxidase 4 (NOX4) expression (Liu et al., 2015). Our present study found that DMY down regulated the expression of NADPH oxidase subunit p22phox, which might be one of the most important mechanisms to attenuate oxidative stress in Ang II-stimulated cardiac fibroblasts. Meanwhile, there is also a complex system to fight against the reactive oxygen species damage. In patients suffering from myocardial remodeling, there was a higher level of oxidative stress but lower expression of SOD in myocardium (Frustaci et al., 2015). Myocardial fibrosis and cardiac dysfunction in diabetic cardiomyopathy symptoms was significantly attenuated in skeletal muscle-specific SOD transgenic

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mice (Call et al., 2015). More exercises enhanced the expression of SOD in elderly heart, while overexpression of SOD significantly reduced oxidative stress, and inhibited cardiac fibrosis (Kwak et al., 2015). Trx was an antioxidant to maintain the balance of the thiol-related redox status, and it also played vital roles in redox signal regulation (Koharyova and Kollarova, 2015; Lu et al., 2010; Tan et al., 2015; Zhu et al., 2013). Cardiac specific Trx knockout disrupted mitochondrial function, increased oxygen free radicals and impaired cardiac function (Huang et al., 2015). Over-expression of Trx in bone marrow mesenchymal stem cell increased pro-angiogenic factor production in myocardium, and ameliorated myocardial fibrosis, improve cardiac function after myocardial infarction (Suresh et al., 2015). Previous research suggested that DMY attenuated H2O2-induced apoptosis in human umbilical vein epithelial cells and inhibited intracellular reactive oxygen species production (Hou et al., 2015). DMY remarkably improved 3-nitropropionic acid-induced striatal metabolic abnormality via decreasing MDA and increasing SOD activity (Mu et al., 2016). DMY also increased SOD to reduced lipid accumulation in oleic acid-induced hepatic steatosis in L02 and HepG2 cells (Xie et al., 2016). The present study found that DMY enhanced the expression of antioxidant enzymes SOD and Trx, which might be one of the important mechanisms of DMY to prevent oxidative stress and proliferation of myocardial fibroblasts. However, Trx is mainly divided into two types, including Trx1 in cytoplasm and nucleus, and Trx2 in mitochondria (Whayne et al., 2015). How DMY regulated different subtypes of Trx to eliminate reactive oxygen species remains further research.

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Although DMY alone increased SOD activity and T-AOC in cardiac fibroblasts, which was beneficial to resist pathological stimulus. It is noting that DMY alone did not alter the level of oxidative stress and the expression of SOD, Trx or p22phox, which suggested that DMY might have slight effect on the physiological oxidative stress and anti-oxidative system. In other words, DMY specifically exerted antioxidant ability in pathological state. In conclusion, our research provides critical evidence that DMY pretreatment inhibited the proliferation and collagen synthesis, which might be related the attenuated effect on oxidative stress in cardiac fibroblasts induced by Ang II. It is beneficial to offer new therapeutic strategy for myocardial fibrosis prevention. Conflict of interest statement The authors declare no conflict of interest.

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Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (grant nos. 81400203), the Natural Science Foundation of Nantong City (grant no. MS12015015) and a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions. We would like to thank Dr. Muhammad Arslan Ishfaq in School of Medicine, Nantong University for the English writing revision. References Bai, J., Zhang, N., Hua, Y., Wang, B., Ling, L., Ferro, A., Xu, B., 2013. Metformin inhibits angiotensin II-induced differentiation of cardiac fibroblasts into myofibroblasts. PLoS One 8, e72120. Call, J.A., Chain, K.H., Martin, K.S., Lira, V.A., Okutsu, M., Zhang, M., Yan, Z., 2015. Enhanced skeletal muscle expression of extracellular superoxide dismutase mitigates streptozotocin-induced diabetic cardiomyopathy by reducing oxidative stress and aberrant cell signaling. Circ. Heart Fail. 8, 188-197. Chen, Q.Q., Zhang, W., Chen, X.F., Bao, Y.J., Wang, J., Zhu, W.Z., 2012. Electrical field stimulation induces cardiac fibroblast proliferation through the calcineurin-Nfat pathway. Can. J. Physiol. Pharmacol. 90, 1611-1622. Costa-Almeida, R., Gomez-Lazaro, M., Ramalho, C., Granja, P.L., Soares, R., Guerreiro, S.G., 2015. Fibroblast-endothelial partners for vascularization strategies in tissue engineering. Tissue Eng. Part A 21, 1055-1065. Etelvino, G.M., Peluso, A.A., Santos, R.A., 2014. New components of the renin-angiotensin

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Figure Legends Fig. 1. Chemical structure of dihydromyricetin (DMY). Fig. 2. Effect of DMY on cell numbers and of hydroxyproline content in Ang II induced cardiac fibroblasts proliferation. (A) Neonatal rat cardiac fibroblasts were pretreated with different concentrations of DMY (0, 1 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM) for 4 h followed by Ang II (100 nM) stimulation for a further 24 h. The number of cells was represented as an OD value using a cell counting assay. (B) Content of total hydroxyproline in cell culture medium was determined. (C) Average hydroxyproline level was normalized by OD value

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representing cell numbers. (D) Neonatal rat cardiac fibroblasts were pretreated with DMY (80 μM) for different times (0, 1 h, 2 h, 4 h,12 h or 24 h) followed by Ang II (100 nM) stimulation for a further 24 h. The number of cells was represented as an OD value using a cell counting assay. (E) Content of total hydroxyproline in cell culture medium was determined. (F) Average hydroxyproline level was normalized by OD value representing cell numbers. Cells treated with culture medium were served as a vehicle control. Data are expressed as mean ± S.E.M. of values from eight independent experiments. **P<0.01 versus control; #P<0.05, ##P<0.01 versus only Ang II stimulated group. Fig. 3. Effect of DMY on cell viability and cytotoxicity in cardiac fibroblasts proliferation. (A) Neonatal rat cardiac fibroblasts were pretreated with different concentrations of DMY (1 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM) for 4 h followed by Ang II (100 nM) stimulation for a further 24 h. Total LDH content in the medium was evaluated. (B) Average LDH was normalized by OD value representing cell numbers. (C) Neonatal rat cardiac fibroblasts DMY (80 μM) was pretreated in for different time (1 h, 2 h, 4 h, 12 h and 24 h) followed by Ang II (100 nM) stimulation for a further 24 h. Total LDH content in the medium was evaluated. (D) Average LDH was normalized by OD value representing cell numbers. Data are expressed as mean ± S.E.M. of values from four independent experiments. Fig. 4. Effect of DMY on Ang II induced collagen I and collagen III expression in cardiac fibroblasts. (A) Neonatal rat cardiac fibroblasts were pretreated with DMY (80 μM) for 4 h followed by Ang II (100 nM) stimulation for a further 24 h. Collagen

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I and collagen III mRNA expressions were assessed by real time PCR. 18S was used as a house-keeping gene. (B) Collagen I and collagen III protein expression in cell lysates were measured by western blot. GAPDH was used as a loading control. Cells treated with culture medium were served as a vehicle control. Data are expressed as mean ± S.E.M. of values from four independent experiments. **P<0.01 versus control, ##

P<0.01 versus only Ang II stimulated group.

Fig. 5. Effect of DMY on Ang II induced α-SMA expression in cardiac fibroblasts. (A) Neonatal rat cardiac fibroblasts were pretreated with DMY (80 μM) for 4 h followed by Ang II (100 nM) stimulation for a further 24 h. Expressions of α-SMA mRNA were assessed by real time PCR. 18S was used as a house-keeping gene. (B) Expressions of α-SMA protein expression in cell lysates were measured by western blot. GAPDH was used as a loading control. (C) Cellular α-SMA was immunofluorescence stained using Alexa Fluor 488 conjugated IgG (Green). The nuclei were counter-stained with DAPI (Blue). Bar =100 μm. Cells treated with culture medium were served as a vehicle control. Data are expressed as mean ± S.E.M. of values from four independent experiments. **P<0.01 versus control; #P<0.05, ##

P<0.01 versus only Ang II stimulated group.

Fig. 6. Effect of DMY on oxidative stress in Ang II-stimulated cardiac fibroblasts. (A) Neonatal rat cardiac fibroblasts were pretreated with DMY (80 μM) for 4 h followed by Ang II (100 nM) stimulation for a further 24 h. Cellular reactive oxygen species production was staining with DCFH-DA (10 μM) fluorescent probe under a fluorescence microscope. Bar=100 μm. (B-D) Levels of MDA, activity of SOD, and

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T-AOC in cardiac fibroblasts were measured. Cells treated with culture medium were served as a vehicle control. Data are expressed as mean ± S.E.M. of values from six independent experiments. **P<0.01 versus control, #P<0.05 versus only Ang II stimulated group. Fig. 7. Effect of DMY on p22phox, SOD and Trx expression in Ang II stimulated cardiac fibroblasts. Neonatal rat cardiac fibroblasts were pretreated with DMY (80 μM) for 4 h followed by Ang II (100 nM) stimulation for a further 24 h. Expressions of p22phox (A), SOD (B) and Trx (C) protein expression in cell lysates were measured by western blot. GAPDH was used as a loading control. Cells treated with culture medium were served as a vehicle control. Data are expressed as mean ± S.E.M. of values from four independent experiments. **P<0.01 versus control; #P<0.05, ##

P<0.01 versus only Ang II stimulated group.

Supplementary Figure legends SFig. 1. Characterization of the cardiac fibroblast phenotype. The cardiac fibroblast was characterized by α-SMA or vimetin specific immunofluorescence staining using Alexa Fluor 488 conjugated IgG (Green). The nuclei were counter-stained with DAPI (Blue). Bar =50 μm.

SFig. 2. Effect of DMY alone on ROS, MDA, SOD, T-AOC, Trx and p22phox in cardiac fibroblasts. Neonatal rat cardiac fibroblasts were administrated with DMY (80 μM) for 24 h. (A) Cellular ROS production was staining with DCFH-DA (10 μM)

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fluorescent probe under a fluorescence microscope. Bar=100 μm. (B-D) Levels of MDA, activity of SOD, and T-AOC in cardiac fibroblasts were measured. (E) Expressions of p22phox, SOD and Trx protein expression in cell lysates were measured by western blot. GAPDH was used as a loading control. Cells treated with culture medium were served as a vehicle control. Data are expressed as mean ± S.E.M. of values from four independent experiments. *P<0.05, **P<0.01 versus control.

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