The Cardioprotective Effect Of Naringenin Against Ischemia/reperfusion Injury And Underlying Mechanism In Rat

Object: Naringenin (Nari) is a representative of the flavanone derivingfrom immature or nearly mature dry outer layer of Rutaceae grapefruit peel.Naringenin has been proved to have broad spectrum of biologicalfunctions,such as hypolipidemic，anti-oxidative, anti-bacterial, anti-inflamma-tory, anti-tumor, and anti-atherosclerosis pharmacological effects. In recentyears, researches demonstrated that naringenin can inhibit TNF-α-inducedproliferation and migration in vascular smooth muscle cell，and produceanti-tumor effect. It was also reported that Nari improves insulin signaling andsensitivity, enhances the biological activity of insulin. Naringenin has beenfound to have a protective effect on rat cranial nerves against ischemic injury,which might be related to its antioxidation, inhibition of apoptosis andanti-inflammation. However, the effect of naringenin on ischemia/reperfusionheart has not been reported.The aim of present study was to investigate the protective effect ofnaringenin on heart against ischemia/reperfusion injury in rat and theunderlying mechanism in aspects of cardiac function, antioxidant, andmyocardial infarct size etc. through using Langendorff isolated heart perfusiontechnique and Nari pretreatment on ischemia/reperfusion rat heart.Methods: Adult male Sprague-Dawley (SD) rats weighting280-320gwere randomly divided into four groups: the control group (CON), Naringeningroup (NARI), Naringenin+glibenclamide (NARI-GLI) group andNaringenin+5-hydroxy decanoic acid group (NARI-5-HD). The CON ratheart was treated with30min global ischemia followed by60or120minreperfusion; the NARI rat heart was pretreated with1,2.5,5,10,20, or40μmol/L Nari before ischemia and reperfusion; the NARI-GLI rat heart wasgiven10μmol/L glibenclamide before Nari pretreatment; the NARI-5-HD heart was given100umol/L5-HD before Nari pretreatment.After anesthetized, rats were fixed on backlying position, thoracotomy, andthe hearts were quickly excised and mounted on a Langendorff isolated heartperfusion apparatus. After stabilization of30min perfusion with K-H solution,the hearts were subjected to30min no-flow global ischemia followed by60min of reperfusion. The heart was soaked in37℃perfusate during the entireischemia to maintain the heart constant temperature. For the hearts used tomeasure infarct size, reperfusion time extended to120min. A water-filledlatex balloon connecting to a pressure transducer was introduced into the leftventricle through the atria to record isovolumic left ventricular pressure. Thefunctional parameters of isolated rat heart, including left ventricular developedpressure (LVDP), left ventricular end diastolic pressure (LVEDP), maximalpositive and negative velocity of left ventricular pressure (±dP/dtmax),coronary flow (CF) and heart rate (heart rate, HR), were recorded under thebasical condition and during ischemia/reperfusion continously.During the experiment, coronary effluent was collected at each time pointof reperfusion, and the content of lactate dehydrogenase (LDH) was measuredwith a spectrophotometer in accordance with instruction of the lactatedehydrogenase kit. At the end of the experiment, the heart was taken downand one part of the heart was sliced to determine the infarct size by TTCreagent. Another part of the heart was placed in liquid nitrogen forpreservation. The activity of myocardial superoxide dismutase (SOD) wasassessed by xanthine oxidase method, and malondialdehyde (MDA) weremeasured by the thiobarbituric acid method.Results:1Under the basical condition, every parameter of caridc function in ratwas not different significantly among CON, NARI, NARI-GLY andNARI-5-HD groups (P>0.05).2At60min of ischemia/reperfusion, LVDP of hearts at low concentration(1μmol/L) Nari was17.2±3.7mmHg (recovery rate14.3±2.3%), and wasnot significant difference compared with15.6±4.8mmHg (15.4±2.4%) in control hearts (P>0.05); LVDP at2.5,5,10,20and40μmol/L Nari was29.7±9.6mmHg (23.8±6.1%),29.2±7.6mmHg (24.1±5.3%),41.0±10.1mmHg(33.6±8.5%),43.7±6.9mmHg (35.2±6.1%) and41.5±2.9mmHg (35.4±1.7%), respectively, and significantly higher than that in control hearts(P<0.05). LVEDP at1μmol/L Nari was78.9±7.2mmHg，and was notsignificant different compared with80.5±4.7mmHg in control group(P>0.05). LVEDP at2.5,5,10,20and40μmol/L Nari was72.9±1.7,71.6±4.7,62.3±5.0,59.3±6.6and57.7±6.8mmHg, respectively andsignificantly lower than that in CON hearts (P<0.05).+LVdp/dtmax at1μmol/L Nari was422.7±19.3mmHg/s，and was no significant differencecompared with368.8±24.3mmHg/s in control hearts (P>0.05);+LVdp/dtmax at2.5,5,10,20and40μmol/L Nari was542.0±64.1,613.4±80.4,848.5±78.3,917.8±73.5and945.0±177.7mmHg/s, respectively, andsignificantly higher than that in control hearts (P<0.05).-LVdP/dtmax at1μmol/L Nari was-400.8±30.3mmHg/s，was no significant differentcompared with-306.8±18.31mmHg/s in control hearts (P>0.05);-LVdP/dtmax at at2.5,5,10,20and40μmol/L Nari was-537.2±94.7,-583.3±71.9,-731.9±87.5,-787.4±38.0and-825.3±61.5mmHg/s，respectively,and significantly higher than that in control hearts (P<0.05).3At60min of ischemia/reperfusion, CF at1μmol/L Nari was3.2±0.8ml，was not significant different compared with2.9±0.5ml in control hearts(P>0.05); CF at2.5,5,10,20and40μmol/L Nari was3.6±0.5、3.8±0.6,4.3±0.4,4.6±0.5and4.8±1.2ml, respectively, and significantly higher thanthat in control hearts (P<0.05).4At60min of ischemia/reperfusion, LDH in coronary effluent at1μmol/L Nari was809.8±32.0U/L, and was not significant differentcompared with957.3±30.5U/L in control hearts (P>0.05); LDH at2.5,5,10,20,40μmol/L Nari was634.6±88.4,530.6±83.1,518.6±41.7,473.5±44.9and468.1±59.8U/L, respectively, and significantly lower than that in controlgroup (P <0.05).5At60min of ischemia/reperfusion, myocardial infarct size in the control and1μmol/L Nari treated rats was49.3±3.6%and43.6±4.8%, respectively，and there was not significant different between them (P>0.05); myocardialinfarct size at2.5,5,10,20,40μmol/L Nari was35.7±4.2,33.4±3.1,29.0±1.2,25.4±2.3and24.3±4.3%, respectively, and significantly lower than thatin control hearts (P <0.05).6At ischemia/reperfusion, the activity of myocardial SOD at1μmol/LNari was269.31±40.49U/mgprot， and was not significant differentcompared with248.53±17.96U/mgpro in control hearts (P>0.05); theactivity of SOD at2.5,5,10,20,40μmol/L Nari was307.72±23.48,316.62±25.27,328.88±47.43,333.62±31.29and337.08±45.53U/mgprot，respectively, and significantly higher than that in control hearts (P<0.05). MDA at1μmol/L Nari was0.74±0.08nmol/mgprot, and was notsignificant different compared with0.83±0.05nmol/mgprot in control hearts(P>0.05); MDA at2.5,5,10,20,40μmol/L Nari was0.68±0.06,0.67±0.07,0.64±0.04,0.63±0.01and0.60±0.02nmol/mgprot, respectively, andsignificantly lower than that in control hearts (P <0.05).7In NARI-GLI and NARI-5-HD hearts, the recovery of left ventricularfunction decreased, LDH in the coronary effluent elevated, myocardial infarctsize increased, SOD increased and MDA decreased after30-min ischemia and60-min reperfusion. There were no significant difference of cardiac function,LDH, infarct size, SOD and MDA among NARI-GLI, NARI-5-HD andcontrol hearts (P <0.05).Conclusion: Naringenin has effective protection on heart againstischemia/reperfusion injury, promoting recovery of cardiac function afterischemia/reperfusion， lowering LDH in the coronary effluent，increasingcoronary flow, and reducing myocardial infarct area. The cardioprotectiveeffect of Naringenin may be carried out through enhancing myocardialantioxidant capacity and opening ATP-sensitive potassium channels located inthe cell membrane and mitochondrial membrane.