| 1. Background and Objective:Myocardial I/R injury is a clinical problem associated with procedures, such asangioplasty, coronary bypass surgery, transplantation and thrombolysis, which arecommonly used to re-establish blood flow to minimize damage to the heart due to severemyocardial ischemia. In addition, myocardial I/R results in myocardial infarction due tointrinsic cardiomyocyte death. With the application of CABG and thrombolytic therapy,ischemic heart can re-establish blood flow and oxygen supply in a short time to minimizedamage, but reperfusion increases the emergence of arrhythmia, expands infarction area andinduced ventricular systolic dysfunction. Continuous remodeling leads to cardiacdecompensation, ultimately leading to heart failure or even death. How to reverse orameliorate myocardial ischemia reperfusion injury and left ventricular remodeling is criticalfor clinical treatment of ischemic heart disease and significant to the prognosis of patientswith myocardial infarction improvement.Mangiferin is a pharmacologically active phytochemical and a natural polyphenolicantioxidant present in the bark, fruits, roots and leaves of Mangifera indica Linn.Mangiferin has been reported to possess antidiabetic, antioxidant, antiproliferative,immunomodulatory, cardiotonic and diuretic properties. Studies have shown that mangiferincould clear acute injury induced NO and reactive oxygen, reduce CPK and LDH activities,but the cardioprotective mechanism of mangiferin on ventricular remodeling has not beenreported.The aim of this study was to investigate the effects of mangiferin on rat models ofmyocardial I/R and ventricular remodeling, explore its potential mechanisms ofcardioprotection. These findings point to the therapeutic potential of mangiferin could provide a new auxiliary therapy treat patients.2. Methods:2.1Myocardial ischemia-reperfusion model and evaluationMale Sprague-Dawley(SD) rats, ligate the left anterior descending coronary artery (LAD)30min. Myocardial ischemia injury was induced by30min of ischemia followed by120minof reperfusion. The experiment was divided into2groups: sham operation group and ischemiareperfusion (MIRI) group. Rats in Sham group were only wearing a line, not the ligation ofLAD. Apply multichannel physiological recorder to observe the change of ECG and cardiachemodynamics. Records left ventricular systolic pressure (LVSP), left ventricular enddiastolic pressure (LVEDP), maximum Changing Rate of ventricular pressure (±dp/dtmax).Detects myocardial enzymes aspartate amino transferase (AST), creatine kinase (CPK) andlactate dehydrogenase (LDH) activities; determines inflammatory medium of IL-6, IL-10,prostacyclin(PGI2), and thromboxane A2(TXA2); detects myocardial tissue superoxidedismutase (SOD), myeloperoxidase (MPO), malondialdehyde (MDA) and glutathioneperoxidase (GSH-Px) expression levels.2,3,5-three phenyl chloride tetrazolium (TTC)staining calculates the area of myocardial infarction, and myocardial ultrastructure wasobserved by electron microscopy.2.2Myocardial ischemia-reperfusion injury on the ventricular remodelingAccording to the method previously established, MIRI rats fed4w. The experiment wasdivided into2groups: Sham group and MIRI group. The rats in group Sham were wearingonly a line, not the ligation of LAD. ECG detects the left ventricular end diastolic diameter(LVIDd), left ventricular end systolic diameter (LVIDs), ventricular septal end-diastolic wallthickness (IVSTd), ventricular septal systolic thickness (IVSTs), left ventricular posterior walldiastolic thickness (LVPWd), left ventricular posterior wall thickness at end systole (LVPWs);left ventricle systolic blood pressure (LVSP), left ventricular end-diastolic pressure (LVEDP),left ventricular ejection fraction (EF), and left ventricular fractional shortening (FS). Massonstaining determines the myocardial collagen content (CVF); TUNEL staining analyzes themyocardial cell apoptosis to evaluate apoptosis index (AI); fluorescence quantitative PCR andWestern-blot technology analyzes Caspase-3and Bcl-2expression levels.2.3Effect of mangiferin on MIRI and ventricular remodeling2.3.1Different doses of mangiferin on MIRI According to the method previously established, the experiment was divided into4groups: MIRI group, low dose mangiferin group, medium dose mangiferin group, and highdose mangiferin group. During operation, MIRI group were given1ml saline by sublingualvein injection, mangiferin groups were given5,10,20mg/kg mangiferin by sublingual veininjection. Cardiac hemodynamic indices were measured; myocardial AST, CPK, LDHactivities were detected; IL-6, IL-10, PGI2, TXA2contents were determined; myocardialtissue SOD, MPO, MDA and GSH-Px expressions were characterized, myocardialultrastructure was observed by electron microscopy.2.3.2Different doses of mangiferin on ventricular remodelingAccording to the method previously established, MIRI rats fed4w. The experiment wasdivided into3groups: MIRI group, mangiferin group and ramipril group. After MIRI, ratswere garaged vegetable oil,2ml/day. Mangiferin group was selected the20mg/kg by gavage,1time/day; ramipril group was garaged by administration of ramipril,0.1mg/kg/day. ECGdetects the LVIDd, LVIDs, IVSTd, IVSTs, LVPWd, LVPWs, LVSP, LVEDP, FS and EF.Masson staining determines the myocardial collagen content (CVF); TUNEL staininganalyzes the myocardial cell apoptosis to evaluate apoptosis index (AI); fluorescencequantitative PCR and Western-blot technology analyzes Caspase-3and Bcl-2expressionlevels.2.4The mechanism of mangiferin on MIRI and ventricular remodelingAccording to the method previously established, MIRI rats fed4w. The experiment wasdivided into4groups: Sham group, MIRI group, mangiferin group, and MAPK inhibitorSB203580group. Sham and MIRI group were gavaged by vegetable oil,2ml/day, mangiferingroup was gavaged by mangiferin20mg/kg/day, and inhibitor group was gavaged bySB2035802mg/kg/day. Serum tumor necrosis factor alpha (TNF-α) concentrations weremeasured by ELISA; Western blot detects myocardial tissue p-P38and t-P38proteinexpression.3. Results:3.1Myocardial ischemia-reperfusion model and evaluationMIRI model was successfully established. After the ligation of LAD in2-5min,electrocardiographic was changed significantly, ST was elevated and QRS was widened, STand QRS were the indicators of successful coronary occlusion. LVSP and±dp/dtmaxin MIRI group were decreased (P<0.05), LVEDP was increased (P<0.05) indicated that systolic anddiastolic function were decreased. Serum AST (71.43±69.54), CPK (1652.86±101.37) andLDH (2109.86±115.45) activities in MIRI group were higher than those in Sham group(555.75±55.74,1078.38±102.14,1328.13±86.99, respectively)(P<0.05); oxide content ofMDA in myocardial tissue were higher than that in Sham group (P<0.05), activity ofantioxidant enzymes SOD, GSH-Px were lower than those in group Sham (P<0.05). Serumlevels of pro-inflammatory mediators (IL-6, TXA2) were higher than those in Sham group(P<0.05), and anti-inflammatory mediators of IL-10and PGI2were lower than those in Shamgroup (P<0.05); myocardial infarction area (MIS) increased significantly (49.83±7.25%) thanthat in Sham group (0%, P<0.05). Electron microscope indicated that there was no damage inthe sham-operated rats. While in MIRI group, mitochondria appeared to be swollen withdisorganized cristae and wrinkled bodies. Loss of normal striations and disorganization ofsarcomeres were also found, and the shape of the nuclei was abnormal.3.2Myocardial ischemia-reperfusion injury on the ventricular remodelingAfter establishing MIRI model, rats reperfusion4w. LVIDs, LVEDP were increasedsignificantly (P<0.05), while IVSTd, IVSTs, LVPWd, LVPWs, EF, FS, LVSP and otherindicators were significantly reduced (P<0.05) in MIRI group. During ventricularremodeling, myocardial interstitial collagen fibers underwent severe hyperplasia, the CVF(9.43±0.9%) was higher than that in Sham group (3.09±0.45, P<0.05); myocardial cellapoptosis index (33.25±4.08%) was increased significantly compared to Sham group(8.73±1.02%, P<0.05); Caspase-3gene transcriptional level (7.43±1.05) and proteinexpression level (55.88±4.45%) were increased significantly compared to Sham group;Bcl-2gene transcriptional level(0.14±0.03) and protein expression level (13.25±2.38)significantly were decreased significantly compared to Sham group.3.3Effect of mangiferin on MIRI and ventricular remodeling3.3.1Different doses of mangiferin on MIRIThe medium and high dose of mangiferin groups significantly improved MIRI heartfunction, elevated LVEDP,±dp/dtmax (P<0.05), reduced LVSP (P<0.05); myocardialenzymes AST(729.38±56.43,642.63±47.09, respectively), CPK(1417.38±89.83,1270.50±86.37, respectively), LDH (1758.50±115.61,1553.88±91.66, respectively)expression weresignificantly lower compared to model group(871.43±69.54,1652.86±101.37,2109.86± 115.45, respectively, P<0.05);proinflammatory cytokine IL-6, TXA2expressions werelower compared to model group (P<0.05); IL-10and PGI2concentrations were elevated(P<0.05); the expression of antioxidant enzymes SOD and GPx were increased (P<0.05),the expression of MDA and MPO were decreased (P<0.05); mangiferin could significantlyreduce the myocardial ultrastructural injury of MIRI.3.3.2Different doses of mangiferin on ventricular remodelingLVIDd was significantly increased, and interventricular septum thickness in diastole andsystole and left ventricle posterior wall thickness in diastole and systole were significantlydecreased in the MIRI group compared with those in the sham-operated group. The indices ofsystolic function (ejection fraction and fractional shortening) were significantly reducedcompared with the sham-operated as well as the rate of LV contraction in the MIRI group.Interventricular septum thickness in diastole and systole and left ventricle posterior wallthickness in diastole and systole were increased in mangiferin and ramipril treatment groupscompared with those in the MIRI group. Improvement of systolic function induced bymangiferin and ramipril was also accompanied by increased percent ejection fraction andfractional shortening (P<0.05for mangiferin and ramipril). Damaged myocardium stains blueand viable myocardium stains red. The induction of fibrosis in the MIRI group correspondedwith extracellular fibrosis, while this fibrosis disappeared in MIRI rats treated withmangiferin. Marked collagen deposition was found in the MIRI hearts, which was confirmedby increased collagen volume fraction (9.43±0.90%in the MI group vs.3.09±0.45%in thesham-operated group, P<0.05). Mangiferin and ramipril inhibited collagen aggregation anddecreased collagen volume fraction (6.49±0.81,4.99±0.54%, respectively, P<0.05vs. MIRIrats).3.4The mechanism of mangiferin on MIRI and ventricular remodelingP38MAPK activity was transiently increased soon after MIRI. Phosphorylation of p38was decreased in the MIRI+mangiferin (26.17±3.47%) and MIRI+SB groups (21.93±2.57%)compared with that in the MIRI group (34.12±4.53%). TUNEL results were detected througha light microscope; a dark brown diaminobenzidine signal indicated positive staining, whileshades of blue-green to greenish tan signified a nonreactive cell. Results showed that theapoptosis index was increased after MIRI. After mangiferin treatment, the apoptosis indexwas significantly decreased (P<0.05). A few of the brown nuclei are found in the sham group hearts, while the number of brown nuclei increased significantly in model groups. Themangiferin and ramipril treatment could attenuate the number of apoptotic cells. In the MIRIgroup, TNF-α was significantly elevated (92.86±9.53) compared with that in the sham-operated group (20.00±2.14, P<0.05). Both mangiferin and the p38MAPK inhibitorSB203580reduced the accumulation of TNF-α after MIRI (73.63±6.65,54.38±4.57,respectively).4. Conclusions:Our study was investigated the cardioprotective role of mangiferin on MIRI andventricular remodeling from multiple layers in rats. By varying the dose of mangiferinintervention and P38MAPK signal transduction pathway studies, we explored furthermechanism of mangiferin, the results were indicated as follows:4.1A tracheotomy was performed to insert a catheter into the trachea, especially tofacilitate breathing with the ventilator support, ligation LAD30min followed by120minreperfusion, we successfully established the MIRI model. The model has a good repeatabilityand a high success rate.4.2After MIRI, ventricular remodeling was happened, which represents enlargedchamber, thinned infarction wall, abnormal deposited myocardial interstitial collagen fiber,and pronounced myocardial fibrosis. Myocardial compliance was decreased and systolic anddiastolic function was severely damaged. Pro-apoptotic gene expression of Caspase-3mRNAand protein were increased and anti-apoptosis gene expression of Bcl-2were decreased,suggested that the two factors played an important role in MIRI induced ventricularremodeling.4.3Through the different dosage of mangiferin intervention studies, we confirmed thecardioprotect effect of mangiferin exist dose-effect relationship. In the medium and highdose, mangiferin can effectively decrease the serum myocardial enzyme spectrumexpression, exerts its anti-inflammatory, antioxidant,attenuates myocardial ultrastructuralinjury, and protects cardiac function.4.4Mangiferin promotes Bcl-2gene transcription and protein expression levels,decreased Caspase-3gene transcription and protein expression levels, inhibits myocardialapoptosis in MIRI cells, reduces myocardial interstitial collagen fibrosis, improvesmyocardial compliance and protects cardiac systolic and diastolic function. 4.5To the best of our knowledge, this is the first study on the cardioprotective effect ofmangiferin in LV remodeling. We confirmed that mangiferin inhibited P38MAPK activation,reduced serum TNF-α expression, which may be the important mechanism of mangiferin onMIRI induced remodeling. |
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