Int J Clin Exp Med 2017;10(4):6176-6183 www.ijcem.com /ISSN:1940-5901/IJCEM0045887

Original Article Protective effects of quercetin against myocardial /reperfusion injury in rats

Hai-Zhu Zhang1,2, Dao-Xun Wu2, Sai-Li Li2, Xiao-Guang Lu3

1Dali University, Yunnan, China; 2Chengdu University of Traditional Chinese Medicine, China; 3Nanyang City in Henan Province Institute for Food and Drug Control, Henan, China Received December 5, 2016; Accepted February 4, 2017; Epub April 15, 2017; Published April 30, 2017

Abstract: The present study was to investigate the effects of quercetin on myocardial ischemia/reperfusion (MI/R) injury and investigated the underlying mechanisms. The expression of tumor necrosis factor alpha (TNF-α), inter- leukin-6 (IL-6), lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), peroxidase (GSH-Px) and superoxidase dismutase (SOD) was evaluated by enzyme-linked immunosorbent assay (ELISA). Infarct size was measured by /TTC double staining. Pathological changes of heart tissues were observed by HE staining. Levels of nuclear factor-κB (NF-κB) p65, PI3K, Akt, were determined by Western blotting. Pretreatment with quercetin significantly augmented rat cardiac function, as evidenced by increased left ventricular ejection fraction (LVEF) and ± dP/dt. Quercetin reduced myocardial infarct size, markedly decreased LDH, CK, CK-MB, AST and MDA levels, and increased SOD, GSH-Px, CAT in the MI/R group. The protein levels of NF-κB p65 and Bax-2 were signifi- cantly decreased, while the protein levels of PI3K, Akt and Bcl-2 were significantly increased in a dose-dependent manner compared to the model group, respectively. Together, these data demonstrate that quercetin might protect myocardial ischemia via regulating PI3K/Akt/NF-κB p65 signaling.

Keywords: Quercetin, myocardial ischemia/reperfusion, PI3K/Akt/NF-κB signaling

Introduction mechanism underlying Que-mediated cardio- protection is still not completely elucidated. Ischemic heart disease (IHD) is a major health problem and according to the World Health Although the pathogenesis of myocardial isch- Organization, it will be the leading cause of emia is far from clear, the anatomic changes death in the world [1, 2]. In the past few and the characterized biochemical markers decades, extensive studies have been per- have been well illustrated. The overproduction formed to explore effective strategies and of reactive species (ROS) and the acti- drugs to ameliorate or prevent ischemic heart vation of inflammatory cascades are major injury, such as flavonoids, that possess cardio causative factors of cardiomyocyte abnormali- protective effects. Quercetin (Que; 3,3’,4’,5,7- ties. Activation of the PI3K/Akt pathway has pentahydroxyflavone) is one of the major flavo- been reported to prevent neuronal apoptosis noids found in many vegetables and fruits such and protect the brain from cerebral ischemia/ as onion and apple. It is nontoxic and has a reperfusion (I/R) injury [6-8]. Additionally, PI3K/ broad range of pharmacological and biological Akt pathway activation attenuates mitochon- activities including anticarcinogenic, antioxida- dria-mediated apoptosis [9, 10]. /threo- tive, vasoprotective, antidiabetic, and antiplate- nine kinase Akt is a primary mediator of the let effects [3]. Several studies indicated that downstream effects of phosphatidylinositol-3 Que protects the myocardium from ischemic kinase (PI3K), preserving mitochondrial integri- heart injury [4]. Recently, in another study, a ty by phosphorylating molecules such as the significant reduction of the myocardial infarct Bcl-2 family. Nuclear factor-kappaB, a nuclear size in both normal and diabetic animals by Que transcription factor, is a regulator of inflamma- has been reported [5]. However, the molecular tory processes. NF-κB is required for maximal Protective effects of quercetin on myocardial ischemia/reperfusioniujury transcription of numerous cytokines, including treated group and propranolol-treated group TNF-α, IL-1β, and IL-6 [11], which are detrimen- were pretreated with quercetin or propranolol tal to the myocardial functions [12]. every 24 h for 10 days by means of intragastric administration. The present study was to investigate the effects of quercetin on myocardial ischemia/reperfu- Determination of cardiac function sion (MI/R) injury and investigated the underly- ing mechanisms. MI/R-induced cardiac dysfunction was deter- mined by invasive hemodynamic evaluation Materials and methods methods. A micro-catheter was inserted into the left ventricle via the right carotid artery to Materials measure the left ventricular pressure (LVP). ECG and LVP were simultaneously recorded on Male Sprague-Dawley (SD) rats weighing 190- a polygraph. Computer algorithms measured 210 g were provided by Shanghai Slac Labo- left ventricular systolic pressure (LVSP), left ratory Animal Co.LTD (Shanghai, China). Animal ventricular end-diastolic pressure (LVEDP), first experiment was carried out in accordance with derivative of left ventricular pressure (±dP/ the Guide for the Care and Use of Laboratory dtmax) after 3 hours of reperfusion. Animals (US National Research Council, 1996, Jan 12). All animals were allowed free access Determination of myocardial infarct size to food and water, and were maintained at 22-24°C under a 12 hour: 12 hour light-dark After reperfusion conclusion, the coronary ar- cycle. Quercetin with purity of over 99% were tery ligature was retied. 4 mL of 2% Evans blue purchased from National Institutes for Food dye (Shanghai Chemical Reagents, Shanghai, and Drug Control (Beijing, China). Tumor necro- China) was injected into the aorta. Dye was cir- sis factor alpha (TNF-α), interleukin-6 (IL-6), lac- culated and uniformly distributed, except in the tate dehydrogenase (LDH), creatine kinase cardiac region previously perfused by the (CK), malondialdehyde (MDA), glutathione per- occluded coronary artery (defining the ischemic oxidase (GSH-Px) and superoxidase dismutase region or area at risk, AAR). Cardiectomy was (SOD) test kits were all purchased from Nanjing rapidly performed. Hearts were frozen at -20°C Jiancheng Biological Engineering research and sliced into 1-mm sections perpendicular to institute (Nanjing City, China). the base-apex. Slices were incubated in 1% TTC in phosphate buffer at 37°C for 10 minutes (pH Experimental protocol 7.4). Morpho-metric measurements of AAR and infarct area (INF) were performed by image Adult male Sprague-Dawley rats were fasted analysis system. Myocardial infarct size was overnight, and anesthetized via intraperitoneal expressed. (IP) administration of 50 mg/kg . A micro-catheter was inserted into the Determination of LDH, CK, CK-MB, TNF-α, IL-6 left ventricle through the right carotid artery to in the serum measure the left ventricular pressure. Myo- CK, CK-MB and LDH levels were measured by a cardial ischemia was produced after exterioriz- rate assay using anRT-9600 Semi-automatic ing the heart via a left thoracic incision, and Biochemical Analyzer (Shenzhen Lei Du life placing a 6-0 silk slipknot suture around the Science, LLC). TNF-α and IL-6 levels were mea- left anterior descending coronary artery approx- sured by enzyme linked immunosorbent assay imately 2-3 mm from its origin. Ischemia was (ELISA, Wuhan Boshide Biological Technology monitored and confirmed by ST segment eleva- Company, Wuhan, China). All measurements tion upon electrocardiogram (ECG). After 30 were performed according to the kit manufac- minutes ischemia, the slipknot was released, turers’ instructions. and myocardial reperfusion for 3 hours. Rats were randomly divided into the following Determination of SOD and MDA in myocardial groups: (1) sham group; (2) I/R group; (3) I/R+ tissue propranolol (as a positive control, 25 mg/kg); (4) I/R+ Quercetin (200 mg/kg); (5) I/R+ Approximately 0.1 g of myocardial tissue was Quercetin (400 mg/kg). The rats in quercetin- removed from the apical part of the heart,

6177 Int J Clin Exp Med 2017;10(4):6176-6183 Protective effects of quercetin on myocardial ischemia/reperfusioniujury

Figure 1. The effect of quercetin on LVSP, LVEDP, LV+dp/dt, LV-dp/dt of rat subjected to I/R injury. All values pre- sented are mean ± SD. Compared with sham: #P<0.05, ##P<0.01; Compared with I/R: *P<0.05, **P<0.01.

Myocardial tissues histopathology

Immediately after the sacrifice of the rats, the hearts were removed and fixed in 10% formalin solution. The heart tissue was processed for sectioning and staining by standard histological methods. Sections (5 mm, Leica RM 2125, Germany) from the left ventricle were stained with hematoxylin and eosin (H&E) and exam- ined by light microscopy (Nikon, Tokyo, Japan) at 200× magnification.

Western blotting Figure 2. Effects of quercetinon myocardial infarct size in rat subjected to I/R injury. All values present- Myocardium tissues were homogenized in ed are mean ± SD. Compared with sham: #P<0.05, ##P<0.01; Compared with I/R: *P<0.05, **P<0.01. buffe containing phenyl methanesulfonyl fluo- ride. Homogenates were mutated at 4°C for 30 min and centrifuged at 13000× g for 20 min. immersed in ice-cold saline solution (1:9 W/V) Equal amounts of protein were loaded onto and homogenized. The homogenate was centri- each lane and run on SDS-PAGE under reduc- fuged at 3000 rpm for 15 min, and the super- ing conditions. Samples were then electro blot- natant was used for the biochemical assays. ted onto nitrocellulose fiter membrane, blocked SOD and MDA were determined by ELISA kits with 5% nonfat dried milk in Tris-buffered saline according to the instructions recommended by containing 0.1% Triton X-100 (TBST) at room the manufacturers. temperature for 2 h, and incubated overnight at

6178 Int J Clin Exp Med 2017;10(4):6176-6183 Protective effects of quercetin on myocardial ischemia/reperfusioniujury

Figure 3. Effects of quercetin on serum levels of LDH, CK, CK-MB and AST in rat subjected to I/R injury. All values presented are mean ± SD. Compared with sham: #P<0.05, ##P<0.01; Compared with I/R: *P<0.05, **P<0.01.

4°C with different first antibodies. The mem- tionally, quercetin markedly decreased LVEDP brane was washed with TBST for three times compared to MI/R group. Hemodynamic data per 15 min. The HRP-linked secondary antibody support quercetin improved rat cardiac systolic was incubated with the membrane for 1 h at and diastolic function after MI/R. room temperature. Excess antibody was washed off with TBST for three times per 15 Quercetin decreased infarct size and reduced min before incubation ECL for 1 min. biochemical marker enzymes

Statistical analysis Myocardial infarct size and LDH, CK, CK-MB and AST were measured to assess myocardial All values were expressed as the mean ± S.D. injury. Myocardial INF expressed as percentage and analyzed by one-way analysis of variance of AAR (Figure 2). No was (ANOVA) followed by Duncan’s Multiple Range observed in sham-group hearts. Quercetin Test using SPSS version 13.0 software; a (400 mg/kg) treatment significantly decreased P-value of less than 0.05 was considered sig- infarct size. Significant increases in the myocar- nificant and P<0.01 was considered to be sta- dial injury biomarker enzymes, CK, AST, CK-MB tistically very significant. and LDH were observed in I/R group compared Results with the sham-group. Pretreatment with quer- cetin decreased CK, AST, CK-MB and LDH lev- Quercetin improved rat cardiac systolic and els compared with rats in I/R group in a dose- diastolic function after MI/R dependent manner (Figure 3).

Quercetin had no effects on blood glucose, Quercetin increased SOD activity and de- blood pressure and cardiac function in the creased MDA, TNF-α and IL-6 levels absence of MI/R. Pretreatment with quercetin enhanced ± LVdP/dt max after 3 hours reperfu- To determine whether quercetin attenuated sion compared to MI/R group (Figure 1). Addi- MI/R-induced oxidation index, plasma SOD,

6179 Int J Clin Exp Med 2017;10(4):6176-6183 Protective effects of quercetin on myocardial ischemia/reperfusioniujury

Figure 4. Effects of quercetin on serum levels of SOD, MDA, TNF-α and IL-6 in rat subjected to I/R injury. All values presented are mean ± SD. Compared with sham: #P<0.05, ##P<0.01; Compared with I/R: *P<0.05, **P<0.01.

Figure 5. Effects of quercetin onmorphological changes of rats’ myocardial tissues. A: Sham; B: I/R, C: Propranolol; D: Quercetin (200 mg/kg); E: Quercetin (400 mg/kg). Scar bar: 200 mM (upper panel), Scar bar: 50 mM (lower panel).

MDA, TNF-α and IL-6 levels were measured dependent manner compared with the I/R after reperfusion conclusion. Compared with group. Compared with the sham-group, the the sham-group, SOD levels in the I/R group MDA, TNF-α and IL-6 levels in the I/R group decreased significantly. Pretreatment with increased significantly. Pretreatment with quer- quercetin increased SOD activity in a dose- cetin decreased MDA, TNF-α and IL-6 levels in a

6180 Int J Clin Exp Med 2017;10(4):6176-6183 Protective effects of quercetin on myocardial ischemia/reperfusioniujury

Figure 6. Effect of quercetin on PI3K/Akt/NF- kB signaling. Compared with sham: #P<0.05, ##P<0.01; Compared with I/R: *P<0.05, **P<0.01.

dose-dependent manner compared with the Quercetin increased PI3K, Akt and Bcl-2 levels I/R group rats (Figure 4). and decreased NF-κB p65, Bax-2 levels

Quercetin showed normal, well preserved of The expression of proteins of PI3K, NF-κB p65, cardiac muscle cell histology Akt, Bcl-2 and Bax-2 were changed in heart. As Histological evaluation of the myocardial tis- shown in Figure 6, compared with the sham- sues (Figure 5) by light microscopy demonstrat- group, the protein levels of NF-κB p65 and ed myocardium cell in I/R group rats arranged Bax-2 in I/R group were significantly increased. in disorder and swell significantly. Obvious myo- In quercetin treated groups, the protein levels cardial cell swelling, degeneration, loss of of NF-κB p65 and Bax-2 were significantly transverse striations, and large numbers of decreased in a dose-dependent manner com- infiltrating inflammatory cells also observed in pared to the model group, respectively. I/R group. Pretreatment with quercetin showed Compared with the sham-group, the protein lev- normal, well preserved of cardiac muscle cell els of PI3K, Akt and Bcl-2 in I/R group were sig- histology. The results indicated that quercetin nificantly decreased. In quercetin treated can reduce the degree of pathological changes groups, the protein levels of PI3K, Akt and Bcl-2 in myocardial tissues in MI/R. were significantly increased in a dose-depen-

6181 Int J Clin Exp Med 2017;10(4):6176-6183 Protective effects of quercetin on myocardial ischemia/reperfusioniujury dent manner compared to the I/R group, apoptosis. Bcl-2 regulates mPTP opening in respectively. opposition to Bax, blocking cytochrome c re- lease, inhibiting caspase activity, and decreas- Discussion ing cell apoptosis. Western blot revealed I/R significantly decreased the Bcl-2 and decreased In this study, quercetin improved rat cardiac Bax-2, an effect reversed by quercetin adminis- systolic and diastolic function after MI/R, tration, suggesting quercetin-mediated cardio- decreased infarct size and reduced biochemi- protection against I/R injury may occur partially cal marker enzymes (CK, AST, CK-MB and LDH). via modulating Bcl-2/Bax expression. The ser- Pretreatment with quercetin also increased ine survival kinase Akt is activated downstream SOD activity and decreased MDA, TNF-α and of phosphatidylinositol 3-kinase (PI3K). Activ- IL-6 levels. And pretreatment with quercetin ation of PI3K and Aktis cardioprotective against showed normal, well preserved of cardiac mus- I/R injury, and prevents cardiomyocyte apopto- cle cell histology. These results suggested that sis. The activation of NF-κB pathway triggers quercetin at the doses used in this study had the overproduction of pro-inflammatory cyto- cardioprotective effects in myocardial isch- kines, such as IL-1β, IL-6 and TNF-α, which are emia/reperfusion that could be attributed to detrimental to the myocardial functions [17]. their anti-oxidative and anti-inflammatory proper- AS shown in Figure 6, The expression of pro- ties. teins of PI3K, Akt, NF-κB p65, Bcl-2 and Bax-2 were changed in heart. Compared with the con- Excessive oxidative stress induced the overpro- trol group, the protein levels of NF-κB p65 and duction of pro-inflammatory cytokines, includ- Bax-2 in I/R group were significantly increased, ing IL-6, IL-1β and TNF-α. Some studies have In quercetin pretreatment group, the protein documented that the elevated levels of inflam- levels of NF-κB p65 and Bax-2 were significant- matory markers were related to ischemia. ly decreased in a dose-dependent manner Quercetin preconditioning decreased the con- compared to the I/R group, respectively. Com- tents of pro-inflammatory cytokines, which sug- pared with the sham-group, the protein levels gested that the cardioprotective effects of of PI3K, Akt and Bcl-2 in I/R group were signifi- quercetin were partially attributed to its anti- cantly decreased. In quercetin, the protein lev- inflammatory property. These findings were els of PI3K, Akt and Bcl-2 were significantly consistent with the histological evaluation of increased in a dose-dependent manner com- inflammatory celluar infiltration in myocardial pared to the I/R group, respectively. tissues.

SOD (a key antioxidant enzyme) and MDA (a In conclusion, quercetin exerted anti-inflamma- reactive oxygen species) are important indexes tion and anti-oxidation effects against myocar- of myocardial oxidative damages [13]. It is well dial ischemia/reperfusion injury in rats, which known that SOD activity reflects the cellular might be associated with regulating PI3K/Akt/ capability of scavenging/quenching free radi- NF-κB p65 signaling cals [14]. Under physiological conditions, low Disclosure of conflict of interest levels of reactive oxygen species play impor- tant roles in signal transduction and metabolic None. pathways [15, 16]. However, under pathologic conditions, excessive reactive oxygen species Address correspondence to: Dr. Xiao-Guang Lu, Nan- resulted in the imbalance of antioxidant sys- yang City in Henan Province Institute for Food and tem. MDA, the end product of lipid peroxida- Drug Control, Henan, China. E-mail: luxiaoguangh- tion, was overproduced in the I/R rats. [email protected] According to the obtained data, the level of MDA was down-regulated with quercetin pre- References treatment and the activities of SOD was recov- ered versus those in I/R rats. [1] Booth EA, Lucchesi BR. Estrogen-mediated protection in myocardialischemia reperfusion The Bcl-2 protein family, compromised of both injury. Cardiovasc Toxicol 2008; 8: 101-113. anti-and pro-apoptotic members, are important [2] Chen F, Fu, YL, Zou LT, Lu XY, Wang CY. Cin- mitochondrial regulators during cardiomyocyte namic acid on new ischemia and reperfusion

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