Protective Effect of RA on Myocardial Infarction-Induced Cardiac

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Protective effect of RA on myocardial infarction induced-cardiac fibrosis via AT1R/p38 MAPK pathway signaling and modulation of the ACE2/ACE ratio qiaofeng liu, Tian Jingwei, Yannan Xu, Chunmei Li, Xiangjing Meng, and Fenghua Fu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03001 • Publication Date (Web): 19 Aug 2016 Downloaded from http://pubs.acs.org on August 22, 2016

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Journal of Agricultural and Food Chemistry

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Protective effect of RA on myocardial infarction induced-cardiac fibrosis via AT1R/p38

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MAPK pathway signaling and modulation of the ACE2/ACE ratio

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Qiaofeng Liu#, Jingwei Tian#,*, Yanan Xu, Chunmei Li *, Xiangjing Meng, and Fenghua Fu

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Abbreviations: HF, heart failure; MI, myocardial infarction; RA, Rosemary acid; ECM, extracellular

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matrix; Hyp, hydroxyproline; RAS, rein-angiotensin system; ACE, angiotensin-converting enzyme; ACE2,

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angiotensin-converting enzymes 2; Ang II, angiotensin II; AT1R, angiotensin type 1 receptor; α-SMA, alpha

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smooth muscle actin; MAPK, mitogen-activated protein kinase; LVSP: left ventricular systolic pressure;

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LVEDP: left ventricular end-diastolic pressure; HR: heart rate; LV, left ventricular; +dp/dtmax, maximum rats

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of left ventricular pressure rise; -dp/dtmax, maximum rats of left ventricular pressure fall; HWI, heart weight

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index; LVWI, left ventricular weight index; CVF, collagen volume fraction; ACEI, ACE inhibitor.

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ABSTRACT: Rosmarinic acid (α-o-caffeoyl-3,4-dihydroxyphenyl lactic acid, RA), a major active

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constituent of Rosmarinus officinalis Linn. (rosemary), having significant anti-inflammatory,

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anti-apoptotic and anti-oxidant effects. However, the cardioprotection of RA is still not understood.

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Present study was designed, for the first time, to investigate the cardioprotection of RA on

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myocardial infarction (MI) induced-cardiac fibrosis and clarify the possible mechanisms. MI was

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induced in adult rats by left anterior descending coronary artery ligation, and animals were then

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administered RA (50, 100, 200 mg/kg) by gavage. Compared with the model group, RA treatment

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ameliorated the changes in left ventricular systolic pressure (LVSP), +dp/dtmax, and −dp/dtmax after 4

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weeks. This was associated with attenuation of infarct size, collagen volume fraction (CVF),

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expression of collagen I, collagen III, alpha smooth muscle actin (α-SMA), and hydroxyproline

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(Hyp) concentrations. RA treatment was also associated with decreased angiotensin-converting

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enzyme (ACE) expression and increased ACE2 expression, as well as decreased expression of

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angiotensin type 1 receptor (AT1R) and phospho-p38 mitogen-activated protein kinase (p38

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MAPK). Thus, RA can protect against cardiac dysfunction and fibrosis following MI, likely due to

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decreasing ACE expression and increasing ACE2 expression via AT1R/ p38 MAPK pathway.

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KEYWORDS: RA; cardiac dysfunction; cardiac fibrosis; ACE, ACE2, AT1R/p38 MAPK

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■ INTRODUCTION

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Myocardial infarction (MI) is the main pathogenic factor underlying heart failure (HF).1 In the

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post-MI phase, the heart undergoes extensive myocardial remodeling in response to the ischemic

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injury, leading to thickening or stiffening of regions of the heart, with progressive deterioration of

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cardiac function, which can progress to HF.2 Cardiac fibrosis plays a major role in cardiac

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remodeling after MI, and is a predisposing factor for HF. The pathological features of cardiac

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fibrosis include phenotypic changes in cardiac fibroblasts, excessive proliferation, and deposition of

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extracellular matrix (ECM) proteins such as collagen types I and III.3

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The rein-angiotensin system (RAS) is important for the induction and development of cardiac

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fibrosis after MI.4 Angiotensin-converting enzyme (ACE) and angiotensin-converting enzymes 2

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(ACE2) control the production of the bioactive angiotensin peptides of the RAS. For example, the

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balance between ACE and ACE2 (ACE/ACE2) regulates angiotensin II (Ang II) level, and

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represents a pivotal process in the activation of MI-induced cardiac fibrosis.5 Indeed, an increased

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ACE/ACE2 ratio is observed in animal models and patients with HF.5 Further, under hypertensive

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conditions, Ang II increases the ratio of ACE/ACE2 via the angiotensin type 1 receptor (AT1R) and

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p38 mitogen-activated protein kinase (MAPK) both in vivo and in vitro.6

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Rosmarinus officinalis Linn. (rosemary), belonging to the family Lamiaceae, is an evergreen

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aromatic plant with upright stems, dark green leaves, and whitish-blue flowers distributed in the

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Mediterranean region. 7 Extract of rosemary leave was shown to be a popular herbal products that

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has been consumed as an anti-oxidant agent and flavoring in cosmetics and food conservation. 8,9 In

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some countries, rosemary is usually used as a medicinal plant in the modern and traditional

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medicines for hypertension and diabetes complications treatment. 10-12

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Rosmarinic acid (Figure 1, α-o-caffeoyl-3,4-dihydroxyphenyl lactic acid, RA), a major active

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constituent of rosemary, has marked anti-inflammatory, anti-apoptotic, and anti-oxidant effects.13-15

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Recently, Kim et al. and our previous study reported that RA has antifibrogenic effects in kidney


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and liver fibrosis.16-17 The protective effects of RA against cardiac fibrosis have not been reported,

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although beneficial effects of RA against cardiac cell injury induced by anti-hypoxia and

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re-oxygenation

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MAPK expression in vitro and in vivo.20 Thus, in the present study, we examined the hypothesis that

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long-term oral RA treatment would prevent cardiac dysfunction and cardiac fibrosis following MI

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in rats, through down-regulating ACE expression and up-regulating ACE2 expression via the

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AT1R/p38 MAPK pathway.

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■ MATERIALS AND METHODS

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and acute ischemia in rats19 were reported. Interestingly, RA also inhibits p38

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Plant material. RA (Formula: C18H16O8, Figure 1) was supplied by Shandong Engineering

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Research Center for Nature Drug (Yantai, China) with a purity of > 99.5% as determined by HPLC

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(Figure S1).

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Animals. Sprague-Dawley male rats, weighing 260 ± 20 g, were obtained from Shandong

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Luye Pharmaceutical Co. Ltd. (SCXK Lu 2014 0002), and housed in a specific-pathogen-free grade

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laboratory at 23 ± 3°C, 40–65% humidity, and on a 12-h light/dark cycle. Before the trial, animals

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were adapt to the environment for 1 week with free access to food and drinking water. All animals

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showed normal drinking, eating, and activity, with no adverse reactions. Experimental protocols

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were approved by the Animal Ethics Committee of Yantai University (IACUU-201500513).

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Coronary artery ligation surgery. Animals were anesthetized (sodium pentobarbital, 35

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mg/kg) by intraperitoneal injection (i.p). A left thoracotomy was performed and the heart was

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ligated around the left anterior descending coronary artery (LAD). The wound was stitched by a 6-0

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prolene suture after the air was completely vented out. Successful MI was confirmed by apparent

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ST-segment elevation in electrocardiogram. Sham-operated animals underwent the same surgical

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procedure without the coronary ligature. All animals received routine intramuscular injections of

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buprenorphine-HCl (0.2 mg/kg) for analgesia and penicillin (1000 U) to prevent infection.

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Experimental design. Rats were randomly divided into five groups: (1) sham rats treated



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with 0.9% physiologic saline (n = 15), (2) MI rats treated with 0.9% physiologic saline (n = 15), (3)

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MI rats treated with 50 mg/kg RA (n = 15), (4) MI rats treated with 100 mg/kg RA (n = 15), and (5)

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MI rats treated with 200 mg/kg RA (n = 15). All rats were treated daily with RA by gastric gavage

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once a day for 4 weeks from the 1st day after surgery.

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Assessment of hemodynamic parameters. After 4 weeks, all rats were anesthetized

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with sodium pentobarbital (35 mg/kg, i.p.) for hemodynamic assessment. A micromanometer-tipped

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catheter (Model SPR-838; Millar instruments) was introduced via the right carotid artery into the

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left ventricle to measure left ventricular systolic pressure (LVSP), left ventricular end-diastolic

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pressure (LVEDP), +dp/dtmax, and −dp/dtmax of the left ventricle.

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Assessment of heart weight index and left ventricular weight index. After

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hemodynamic measurements, blood samples were taking from the abdominal aorta, centrifuged,

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and the supernatant was stored at −20°C. The heart and the left ventricle (LV) were isolated, washed,

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and weighed. Relative values (organ weight/body weight) of the heart weight index (HWI, mg/g)

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and the left ventricular weight index (LVWI, mg/g) were determined.

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Assessment of infarct size and cardiac fibrosis. The left ventricles of the tissue below

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the ligature line (3–5 mm thick) were then fixed with 4% paraformaldehyde, dehydrated, and

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embedded in paraffin. To assay the degree of cardiac fibrosis, the collagenous fibrotic area fraction

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was assessed using the Masson’s trichrome staining kit (Maixin Bio-Technology Co., Ltd, Fuzhou,

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China) in paraffin sections (4 µm thick). The sections were digitally imaged using a microscope

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(Olympus DP25) and the collagen volume fraction (CVF) in the peri-infarcted areas of LV was

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evaluated as the percentage of fibrosis area (blue staining) to the total LV area in an average of five

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sections in each heart (NIH Image software). The infarct size was calculated as the ratio of total

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infarct circumference to total LV circumference (NIH Image software).

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Assessment of hydroxyproline concentrations. The hydroxyproline (Hyp) content in

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blood serum was assayed using a commercial kit (Nanjing Jiancheng Bioengineering Institute,


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Nanjing, China) according to the manufacturers’ instructions. The concentrations of Hyp were

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expressed as µg/ml.

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Western blotting. Myocardial tissue was ground in radio-immunoprecipitation assay lysis

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buffer, and the homogenate was stored on ice for 30 min, centrifuged at 14,000 ×g for 15 min at

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−4°C, and the supernatant was collected. A BCA kit (Beyotime Institute of Biotechnology, Nanjing,

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China) was used to determine the protein concentration. The supernatant was diluted in SDS-PAGE

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loading buffer (5X) and then boiled for 5 min. Protein samples were subjected to SDS-PAGE and

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transferred to polyvinylidene fluoride membranes. The membranes were blocked in TBST

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containing 5% skim milk for 2 h at room temperature, and subsequently incubated with the

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following antibodies: rabbit polyclonal anti-α-SMA antibody (abcam), rabbit polyclonal

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anti-collagen I antibody (abcam) and anti-collagen III antibody (abcam), anti-Ang II antibody

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(abcam), anti-p38 antibody (abcam) and anti-phospho-p38 antibody (abcam), anti-AT1R antibody

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(abcam), rabbit polyclonal anti-ACE antibody (Santa Cruz) and anti-ACE2 antibody (Santa Cruz),

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and mouse monoclonal anti-GADPH antibody (Beyotime) for overnight at -4℃. The membranes

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were then washed three times with TBST, probed with appropriate secondary antibodies for 1.5 h at

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room temperature, and proteins detected by chemiluminescence.

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Statistical analysis. All data were reported as mean ± SD. Inter-group differences were

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analyzed with one-way analysis of variance (ANOVA) followed by Dunnett's test. The analyses

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were performed using Graph Pad Prism Software, V5.0. Differences were considered statistically

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significant at a value of P < 0.05.

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■ RESULTS

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Effects on cardiac function. Hemodynamic outcomes obtained at 4 weeks after MI are

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shown in Table 1. After MI, there was a decrease in diastolic function, as shown by a significant

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increase in LVEDP (P < 0.05) and decrease in −dp/dtmax (P < 0.01). MI also impaired systolic 6 ACS Paragon Plus Environment


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cardiac function, with a significant decrease in LVSP (P < 0.01) and +dp/dtmax (P < 0.01). Treatment

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with RA markedly improved diastolic and systolic cardiac function, as shown by a significant

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increase in LVSP with 100 mg/kg RA (P < 0.05), and a significant increase in LVSP, +dp/dtmax and

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−dp/dtmax with 200 mg/kg RA (P < 0.01, P < 0.05), compared with the model group. There were no

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significant differences in HR between any of the groups.

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Effects on infarct size, heart weight index and left ventricular weight index. The

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infarct sizes in the model group and RA groups are shown in Figure 2. There was a significant

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increase in infarct size in MI rats compared to sham rats, which was obviously decreased with RA

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treatment compared with the model group (P < 0.05, P < 0.01). As shown in Table 1, HWI and

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LVWI were markedly higher in the model group compared with the sham group (P < 0.01).

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However, compared with the model group, there was a trend towards a decrease in HWI and LVWI

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in the RA groups.

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Effects on cardiac fibrosis. Also, we assessed the effects of RA on cardiac fibrosis. By

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Masson’s trichrome stained sections (collagen stains blue), there was a significant increase in the

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CVF in the model group compared with the sham group (P < 0.01). By contrast, CVF was

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attenuated in the RA treatment groups (P < 0.01) (Figure 3).

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Effects on type I and III collagen content, hydroxyproline concentration and

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α-smooth muscle actin expression. Next, collagen I and collagen III protein content were

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assessed by Western blotting. There was a significant increase in collagen I and collagen III protein

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expression in the model group compared with the sham group (P < 0.01 for both), which was

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markedly reduced with RA treatment (P < 0.01) (Figure 4 A). On the other hand, there was a

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significant increase in Hyp concentration in the model group compared with the sham group (P

Protective Effect of RA on Myocardial Infarction-Induced Cardiac

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