大鼠心肌缺血再灌注损伤心室重构及芒果苷对其作用和机制的研究
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摘要
1．背景与目的
心肌缺血再灌注损伤(myocardial ischemia reperfusion injury，MIRI)是指缺血心肌组织恢复血流灌注时，再灌注区心肌细胞及局部血管网发生显著的病理生理变化，这些变化可导致进一步的心肌损伤。冠状动脉粥样硬化导致的冠脉狭窄或闭塞，特别是心肌梗死是临床引起死亡的主要疾病之一。随着动脉搭桥术、溶栓疗法等手段的应用使得缺血心脏能在短时间内重新恢复血液灌注及氧供应，有效改善缺血心肌损伤，但再灌注也可能引起心肌损伤，出现心律失常、梗死面积扩大、持久性心室收缩功能低下并因此发生相应的心室重构，导致严重的心力衰竭。如何逆转或减轻心肌缺血再灌注损伤及其心室重构，对于临床上提高缺血性心脏病的治疗效果，改善心梗患者预后具有重要的意义。
芒果苷(mangiferin)广泛存在于百合科植物知母，漆树科芒果叶中，是一种四羟基吡酮的碳糖苷，属双苯吡酮类黄酮类化合物。芒果苷除了能止咳、抗氧化、保肝利胆等作用，随着研究的不断深入，其在治疗糖尿病、心肌病、抗肿瘤、抗艾滋病等方面也有明显的作用。现有研究表明芒果苷能清除急性心肌缺血再灌注损伤产生的一氧化氮和活性氧，降低心肌乳酸脱氢酶和磷酸肌酸激酶含量，保护心肌缺血再灌注损伤，但对MIRI心室重构的保护作用及机制的研究尚未见报道。
本实验将研究大鼠心肌MIRI损伤及心室重构的特征，探讨芒果苷对MIRI损伤及心室重构的保护作用，并对其作用机制进行探讨，可望为临床上心肌梗死后患者心脏功能的恢复提供一种新的辅助治疗途径。
2.实验方法
第一章大鼠心肌缺血再灌注模型的建立及评价
选取雄性Sprague-Dawley (SD)大鼠，气管切开，呼吸机支持呼吸，开胸，结扎左冠状动脉前降支(LAD)30min，松解后再灌注120min，建立大鼠心肌MIRI模型。实验分为2组：假手术(Sham)组及缺血再灌注(MIRI)组。Sham组大鼠只穿线，不结扎LAD。观察其心电图改变，记录左心室收缩压(LVSP)，左心室舒张期末压(LVEDP)，左心室内压最大上升和下降速率(±dp/dtmax)。检测心肌酶谱门冬氨酸氨基转换酶(AST)、磷酸肌酸激酶(CPK)及乳酸脱氢酶(LDH)变化；测定炎性介质IL-6、IL-10、前列环素(PGI2)、血栓素A2(TXA2)含量；检测心肌组织中超氧化物歧化酶(SOD)、髓过氧化物酶(MPO)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)的表达水平；通过2，3，5-氯化三苯基四氮唑(TTC)染色，计算心肌梗死面积；电镜观察MIRI心肌超微结构变化。
第二章大鼠心肌缺血再灌注损伤心室重构的研究
按前述方法建大鼠心肌MIRI模型，存活大鼠喂养4w，实验分为2组：Sham组和MIRI组。超声心动图检测左心室舒张末期内径(LVIDd)、左心室收缩末期内径(LVIDs)室间隔舒张末期厚度(IVSTd)、室间隔收缩末期厚度(IVSTs)、左室后壁舒张末期厚度(LVPWd)、左室后壁收缩末期厚度(LVPWs)；左心室收缩压(LVSP)、左室舒张末期压(LVEDP)及左室射血分数(EF)、左室短轴缩短率(FS)；采用Masson染色，测定心肌胶原纤维含量(CVF)；通过Tunel染色对心肌细胞凋亡进行分析，测定细胞凋亡指数(AI)；通过荧光定量PCR及Western-blot技术从基因和蛋白层面分析Caspase-3和Bcl-2的表达水平。
第三章芒果苷对大鼠心肌MIRI损伤及心室重构作用的研究
实验一不同剂量芒果苷对大鼠心肌MIRI损伤作用的研究
按前述方法建大鼠心肌MIRI模型，选取不同剂量的芒果苷对心肌MIRI进行干预。实验分为4组：MIRI组、芒果苷低、中、高剂量组。MIRI组结扎LAD10min给予1ml生理盐水舌下静脉注射，芒果苷组则分别按低(5mg/kg)、中(10mg/kg)、高(20mg/kg)剂量舌下静脉注射干预。分别测量各组心脏血流动力学指标；检测心肌酶谱AST、CPK、LDH变化；测定炎性介质IL-6、IL-10、PGI2、TXA2含量；检测心肌组织中SOD、 MPO、MDA、GSH-Px的表达水平；观察电镜对MIRI心肌超微结构变化。
实验二芒果苷对大鼠心肌MIRI心室重构作用的研究
按前述方法建大鼠心肌MIRI模型，存活大鼠喂养4w，实验分为3组：MIRI组、芒果苷组、雷米普利组。MIRI组术后灌胃植物油，2ml/日；芒果苷组则根据上述实验一结果，按20mg/kg浓度芒果苷灌胃，1次/日；雷米普利组灌胃雷米普利，0.1mg/kg/日。超声心动图检测LVIDd、LVIDs、IVSTd、IVSTs、LVPWd、LVPWs、LVSP、LVEDP、FS、EF。采用Masson染色，测定心肌胶原纤维含量(CVF)；通过Tunel染色对心肌细胞凋亡进行分析，测定细胞凋亡指数(AI)；测定通过荧光定量PCR及Western-blot技术从基因和蛋白层面分析Caspase-3和Bcl-2的表达水平。
第四章芒果苷对大鼠心肌MIRI损伤及心室重构保护作用的机制研究
按前述方法建大鼠心肌MIRI模型，存活大鼠喂养4w，实验分为4组：Sham组、MIRI组、芒果苷组、SB203580组。Sham组、MIRI组灌胃植物油2ml/日，芒果苷组灌胃芒果苷20mg/kg/日，SB203580组灌胃MAPK抑制剂SB2035802mg/kg/日。ELISA法测量血清中肿瘤坏死因子(TNF-α)浓度；蛋白免疫印迹法测量心肌组织中p-P38和t-P38蛋白表达量。
3.结果
第一章大鼠心肌MIRI模型的建立及评价
结扎LAD后成功建立了心肌缺血再灌注模型，结扎后2-5min内MIRI组心电图可见QRS波宽增大，心电图J点或ST段抬高与高耸的T波融合，呈弓背向上单向曲线。与Sham组比较，MIRI组收缩舒张功能下降，表现为LVSP、±dp/dtmax均降低(P<0.05)；LVEDP升高(P<0.05)。 MIRI组血清AST(871.43±69.54)、 CPK(1652.86±101.37)及LDH(2109.86±115.45)活性均较Sham组(555.75±55.74,1078.38±102.14,1328.13±86.99)增加(P<0.05)；心肌组织中MDA含量均较Sham组增加(P<0.05)、而SOD、GSH-Px活性均较Sham组降低(P<0.05)；血清IL-6、TXA2含量均较Sham组增加(P<0.05)，而IL-10及PGI2浓度均较Sham组降低(P<0.05)；MIRI组心肌梗死面积(MIS)为(49.83±7.25%)，Sham组为0%，两组比较统计学差异明显(P<0.05)；MIRI组超微结构损伤严重，肌节结构排列紊乱，肌丝溶解、断裂、消失；线粒体增多、肿胀、嵴断裂；核基质空化，染色质浓缩、形成大小和形状不一的块状及碎片。
第二章大鼠心肌MIRI心室重构的研究
心肌缺血30min，存活大鼠喂养4w后，MIRI组LVIDs明显扩大、LVEDP明显升高(P<0.05)，而IVSTd、IVSTs、LVPWd、LVPWs、EF、FS、LVSP等各指标都明显降低(P<0.05)；MIRI组心肌间质中胶原纤维严重增生，CVF值(9.43±0.9%)较Sham组(3.09±0.45)升高明显(P<0.05)；同样，MIRI组心肌细胞凋亡指数(33.25±4.08%)较Sham组(8.73±1.02%)有明显上升(P<0.05)，其Caspase-3mRNA基因转录水平(7.43±1.05)和蛋白表达水平(55.88±4.45%)均较Sham组明显上升(P<0.05)，而Bcl-2mRNA基因转录水平(0.14±0.03)和蛋白表达水平(13.25±2.38)均较Sham组明显下降(P<0.05)。
第三章芒果苷对大鼠心肌MIRI损伤及心室重构的作用研究
实验一不同剂量芒果苷对大鼠心肌MIRI的作用研究
芒果苷中、高剂量组改善MIRI大鼠心脏功能，表现为芒果苷中、高剂量组LVSP、±dp/dtmax较MIRI组升高(P<0.05)，而LVEDP降低(P<0.05)；而上述两组心肌酶谱分别为AST(729.38±56.43,642.63±47.09)、 CPK(1417.38±89.83,1270.50±86.37)、 LDH(1758.50±115.61,1553.88±91.66)均较MIRI组(871.43±69.54,1652.86±101.37,2109.86±115.45)明显降低(P<0.05)；和MIRI组相比，芒果苷中、高剂量组IL-6、TXA2表达浓度均降低(P<0.05)，IL-10、PGI2浓度均升高(P<0.05)；SOD、GPx增高(P<0.05)，MDA、MPO的表达降低(P<0.05)；芒果苷中、高剂量组心肌梗死面积分别为(29.25±2.38,24.38±2.20)，与MIRI组(38.14±3.02)比较，显著降低(P<0.05)，同时，芒果苷中、高剂量组心肌超微结构的损伤较轻。而芒果苷低剂量组与MIRI组相比，上述指标无统计学差异，心肌超微结构损伤相似。
实验二芒果苷对大鼠心肌MIRI心室重构作用的研究
芒果苷及雷米普利组较MIRI组显著降低LVIDs、LVEDP(P<0.05)，而IVSTd、IVSTs、LVPWd、LVPWs、LVSP、EF、FS等指标明显升高(P<0.05)；芒果苷及雷米普利组可显著减少MIRI心肌细胞凋亡，心肌细胞凋亡指数(22.45±3.07%,16.48±2.32%)，较MIRI组(33.25±4.08%)下降(P<0.05)；芒果苷及雷米普利组可显著减轻心肌间质纤维化程度，其CVF值分别为(6.49±0.81%,4.99±0.54%)，较MIRI组(9.43±0.90%)明显降低(P<0.05)；芒果苷及雷米普利组Caspase-3mRNA基因转录水平分别为(3.97±0.53,2.17±0.39)，蛋白表达水平分别为(33.88±3.14%,21.13±2.85%)较MIRI组(7.43±1.05,55.88±4.45%)明显降低(P<0.05)；芒果苷及雷米普利组Bcl-2mRNA基因转录水平分别为(0.27±0.04,0.49±0.09)和蛋白表达水平分别为(24.38±2.56%,35.38±3.07%)较MIRI组(0.14±0.03,13.25±2.38%)明显升高(P<0.05)。与芒果苷组比较，雷米普利组心脏超声LVIDs、LVEDP显著降低(P<0.05),而IVSTd、IVSTs、LVPWd、LVPWs、LVSP、EF、FS等指标明显升高(P<0.05)；雷米普利组心肌细胞凋亡指数及心肌胶原含量较芒果苷组均明显下降(P<0.05); Caspase-3mRNA基因转录水平和蛋白表达水平明显降低(P<0.05)；Bcl-2mRNA基因转录水平和蛋白表达水平较芒果苷组明显升高(P<0.05)。
第四章芒果苷对大鼠心肌MIRI及心室重构保护作用机制的研究
MIRI组、芒果苷组心肌组织中p-P38蛋白磷酸化水平分别为(34.12±4.53%，26.17±3.47%)较Sham组(19.32±2.18%)明显升高(P<0.05)，SB203580组p-P38蛋白表达水平（21.93±2.57%)与Sham组无明显差异（P>0.05）；与MIRI组比较，芒果苷组和SB203580组p-P38蛋白表达水平明显下降(P<0.05)；与芒果苷组比较，SB203580组p-P38蛋白表达水平明显降低(P<0.05)。
MIRI组、芒果苷组、SB203580组血清TNF-α浓度分别为(92.86±9.53ng/ml，73.63±6.65ng/ml，54.38±4.57ng/ml)较Sham组(20.00±浓度2.14ng/ml)明显增加(P<0.05)；与MIRI组比较，芒果苷组和SB203580组血清TNF-α明显下降(P<0.05)；与芒果苷组比较，SB203580组血清TNF-α含量明显降低(P<0.05)。
4.结论
本文通过超声、病理学及分子生物学技术，多层次探讨了大鼠心肌MIRI损伤及心室重构的表现，通过不同剂量芒果苷对大鼠心肌MIRI的保护作用，并通过对P38MAPK信号转导通路的研究，对芒果苷的作用机制进行了探讨，结果提示：
(1)结扎左冠状动脉前降支(LAD)30min，松解后再灌注120min，可成功建立大鼠心肌MIRI模型。气管切开插管人工呼吸支持，可提高该模型成功率，易重复。
(2)大鼠心肌MIRI损伤后心室发生重构，表现为心室腔扩大，梗死壁变薄；心肌间质胶原纤维异常沉积，心肌纤维化明显；心肌细胞凋亡严重，同时，促凋亡基因Caspase-3基因和蛋白表达增高，而抗凋亡基因Bcl-2基因和蛋白表达下降，提示这两种基因失衡在MIRI导致的心室重构进程中扮演重要的角色。
(3)通过不同剂量芒果苷的干预研究，首次证实芒果苷对大鼠心肌MIRI损伤有保护作用，其作用有量效关系。中、高剂量的芒果苷能有效降低血清中心肌酶谱的表达，发挥其抗炎、抗氧化作用，减轻心肌细胞超微结构的损伤。
(4)芒果苷可促进Bcl-2的基因转录和蛋白表达水平，降低Caspase-3的基因转录和蛋白表达水平的作用进而抑制心肌MIRI细胞凋亡，降低MIRI大鼠心肌间质胶原纤维化，减轻心室重构，改善心肌顺应性，保护心脏收缩舒张功能。
(5)首次研究芒果苷可抑制P38MAPK，降低血清中炎性介质TNF-α的表达，其可能是芒果苷对MIRI损伤及心室重构的保护作用的重要机制之一。
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 evaluation
Male 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 remodeling
According 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 remodeling
2.3.1Different 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 remodeling
According 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 remodeling
According 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 evaluation
MIRI 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 remodeling
After 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 remodeling
3.3.1Different doses of mangiferin on MIRI
The 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 remodeling
LVIDd 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 remodeling
P38MAPK 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.
心肌缺血再灌注损伤(myocardial ischemia reperfusion injury，MIRI)是指缺血心肌组织恢复血流灌注时，再灌注区心肌细胞及局部血管网发生显著的病理生理变化，这些变化可导致进一步的心肌损伤。冠状动脉粥样硬化导致的冠脉狭窄或闭塞，特别是心肌梗死是临床引起死亡的主要疾病之一。随着动脉搭桥术、溶栓疗法等手段的应用使得缺血心脏能在短时间内重新恢复血液灌注及氧供应，有效改善缺血心肌损伤，但再灌注也可能引起心肌损伤，出现心律失常、梗死面积扩大、持久性心室收缩功能低下并因此发生相应的心室重构，导致严重的心力衰竭。如何逆转或减轻心肌缺血再灌注损伤及其心室重构，对于临床上提高缺血性心脏病的治疗效果，改善心梗患者预后具有重要的意义。
芒果苷(mangiferin)广泛存在于百合科植物知母，漆树科芒果叶中，是一种四羟基吡酮的碳糖苷，属双苯吡酮类黄酮类化合物。芒果苷除了能止咳、抗氧化、保肝利胆等作用，随着研究的不断深入，其在治疗糖尿病、心肌病、抗肿瘤、抗艾滋病等方面也有明显的作用。现有研究表明芒果苷能清除急性心肌缺血再灌注损伤产生的一氧化氮和活性氧，降低心肌乳酸脱氢酶和磷酸肌酸激酶含量，保护心肌缺血再灌注损伤，但对MIRI心室重构的保护作用及机制的研究尚未见报道。
本实验将研究大鼠心肌MIRI损伤及心室重构的特征，探讨芒果苷对MIRI损伤及心室重构的保护作用，并对其作用机制进行探讨，可望为临床上心肌梗死后患者心脏功能的恢复提供一种新的辅助治疗途径。
2.实验方法
第一章大鼠心肌缺血再灌注模型的建立及评价
选取雄性Sprague-Dawley (SD)大鼠，气管切开，呼吸机支持呼吸，开胸，结扎左冠状动脉前降支(LAD)30min，松解后再灌注120min，建立大鼠心肌MIRI模型。实验分为2组：假手术(Sham)组及缺血再灌注(MIRI)组。Sham组大鼠只穿线，不结扎LAD。观察其心电图改变，记录左心室收缩压(LVSP)，左心室舒张期末压(LVEDP)，左心室内压最大上升和下降速率(±dp/dtmax)。检测心肌酶谱门冬氨酸氨基转换酶(AST)、磷酸肌酸激酶(CPK)及乳酸脱氢酶(LDH)变化；测定炎性介质IL-6、IL-10、前列环素(PGI2)、血栓素A2(TXA2)含量；检测心肌组织中超氧化物歧化酶(SOD)、髓过氧化物酶(MPO)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)的表达水平；通过2，3，5-氯化三苯基四氮唑(TTC)染色，计算心肌梗死面积；电镜观察MIRI心肌超微结构变化。
第二章大鼠心肌缺血再灌注损伤心室重构的研究
按前述方法建大鼠心肌MIRI模型，存活大鼠喂养4w，实验分为2组：Sham组和MIRI组。超声心动图检测左心室舒张末期内径(LVIDd)、左心室收缩末期内径(LVIDs)室间隔舒张末期厚度(IVSTd)、室间隔收缩末期厚度(IVSTs)、左室后壁舒张末期厚度(LVPWd)、左室后壁收缩末期厚度(LVPWs)；左心室收缩压(LVSP)、左室舒张末期压(LVEDP)及左室射血分数(EF)、左室短轴缩短率(FS)；采用Masson染色，测定心肌胶原纤维含量(CVF)；通过Tunel染色对心肌细胞凋亡进行分析，测定细胞凋亡指数(AI)；通过荧光定量PCR及Western-blot技术从基因和蛋白层面分析Caspase-3和Bcl-2的表达水平。
第三章芒果苷对大鼠心肌MIRI损伤及心室重构作用的研究
实验一不同剂量芒果苷对大鼠心肌MIRI损伤作用的研究
按前述方法建大鼠心肌MIRI模型，选取不同剂量的芒果苷对心肌MIRI进行干预。实验分为4组：MIRI组、芒果苷低、中、高剂量组。MIRI组结扎LAD10min给予1ml生理盐水舌下静脉注射，芒果苷组则分别按低(5mg/kg)、中(10mg/kg)、高(20mg/kg)剂量舌下静脉注射干预。分别测量各组心脏血流动力学指标；检测心肌酶谱AST、CPK、LDH变化；测定炎性介质IL-6、IL-10、PGI2、TXA2含量；检测心肌组织中SOD、 MPO、MDA、GSH-Px的表达水平；观察电镜对MIRI心肌超微结构变化。
实验二芒果苷对大鼠心肌MIRI心室重构作用的研究
按前述方法建大鼠心肌MIRI模型，存活大鼠喂养4w，实验分为3组：MIRI组、芒果苷组、雷米普利组。MIRI组术后灌胃植物油，2ml/日；芒果苷组则根据上述实验一结果，按20mg/kg浓度芒果苷灌胃，1次/日；雷米普利组灌胃雷米普利，0.1mg/kg/日。超声心动图检测LVIDd、LVIDs、IVSTd、IVSTs、LVPWd、LVPWs、LVSP、LVEDP、FS、EF。采用Masson染色，测定心肌胶原纤维含量(CVF)；通过Tunel染色对心肌细胞凋亡进行分析，测定细胞凋亡指数(AI)；测定通过荧光定量PCR及Western-blot技术从基因和蛋白层面分析Caspase-3和Bcl-2的表达水平。
第四章芒果苷对大鼠心肌MIRI损伤及心室重构保护作用的机制研究
按前述方法建大鼠心肌MIRI模型，存活大鼠喂养4w，实验分为4组：Sham组、MIRI组、芒果苷组、SB203580组。Sham组、MIRI组灌胃植物油2ml/日，芒果苷组灌胃芒果苷20mg/kg/日，SB203580组灌胃MAPK抑制剂SB2035802mg/kg/日。ELISA法测量血清中肿瘤坏死因子(TNF-α)浓度；蛋白免疫印迹法测量心肌组织中p-P38和t-P38蛋白表达量。
3.结果
第一章大鼠心肌MIRI模型的建立及评价
结扎LAD后成功建立了心肌缺血再灌注模型，结扎后2-5min内MIRI组心电图可见QRS波宽增大，心电图J点或ST段抬高与高耸的T波融合，呈弓背向上单向曲线。与Sham组比较，MIRI组收缩舒张功能下降，表现为LVSP、±dp/dtmax均降低(P<0.05)；LVEDP升高(P<0.05)。 MIRI组血清AST(871.43±69.54)、 CPK(1652.86±101.37)及LDH(2109.86±115.45)活性均较Sham组(555.75±55.74,1078.38±102.14,1328.13±86.99)增加(P<0.05)；心肌组织中MDA含量均较Sham组增加(P<0.05)、而SOD、GSH-Px活性均较Sham组降低(P<0.05)；血清IL-6、TXA2含量均较Sham组增加(P<0.05)，而IL-10及PGI2浓度均较Sham组降低(P<0.05)；MIRI组心肌梗死面积(MIS)为(49.83±7.25%)，Sham组为0%，两组比较统计学差异明显(P<0.05)；MIRI组超微结构损伤严重，肌节结构排列紊乱，肌丝溶解、断裂、消失；线粒体增多、肿胀、嵴断裂；核基质空化，染色质浓缩、形成大小和形状不一的块状及碎片。
第二章大鼠心肌MIRI心室重构的研究
心肌缺血30min，存活大鼠喂养4w后，MIRI组LVIDs明显扩大、LVEDP明显升高(P<0.05)，而IVSTd、IVSTs、LVPWd、LVPWs、EF、FS、LVSP等各指标都明显降低(P<0.05)；MIRI组心肌间质中胶原纤维严重增生，CVF值(9.43±0.9%)较Sham组(3.09±0.45)升高明显(P<0.05)；同样，MIRI组心肌细胞凋亡指数(33.25±4.08%)较Sham组(8.73±1.02%)有明显上升(P<0.05)，其Caspase-3mRNA基因转录水平(7.43±1.05)和蛋白表达水平(55.88±4.45%)均较Sham组明显上升(P<0.05)，而Bcl-2mRNA基因转录水平(0.14±0.03)和蛋白表达水平(13.25±2.38)均较Sham组明显下降(P<0.05)。
第三章芒果苷对大鼠心肌MIRI损伤及心室重构的作用研究
实验一不同剂量芒果苷对大鼠心肌MIRI的作用研究
芒果苷中、高剂量组改善MIRI大鼠心脏功能，表现为芒果苷中、高剂量组LVSP、±dp/dtmax较MIRI组升高(P<0.05)，而LVEDP降低(P<0.05)；而上述两组心肌酶谱分别为AST(729.38±56.43,642.63±47.09)、 CPK(1417.38±89.83,1270.50±86.37)、 LDH(1758.50±115.61,1553.88±91.66)均较MIRI组(871.43±69.54,1652.86±101.37,2109.86±115.45)明显降低(P<0.05)；和MIRI组相比，芒果苷中、高剂量组IL-6、TXA2表达浓度均降低(P<0.05)，IL-10、PGI2浓度均升高(P<0.05)；SOD、GPx增高(P<0.05)，MDA、MPO的表达降低(P<0.05)；芒果苷中、高剂量组心肌梗死面积分别为(29.25±2.38,24.38±2.20)，与MIRI组(38.14±3.02)比较，显著降低(P<0.05)，同时，芒果苷中、高剂量组心肌超微结构的损伤较轻。而芒果苷低剂量组与MIRI组相比，上述指标无统计学差异，心肌超微结构损伤相似。
实验二芒果苷对大鼠心肌MIRI心室重构作用的研究
芒果苷及雷米普利组较MIRI组显著降低LVIDs、LVEDP(P<0.05)，而IVSTd、IVSTs、LVPWd、LVPWs、LVSP、EF、FS等指标明显升高(P<0.05)；芒果苷及雷米普利组可显著减少MIRI心肌细胞凋亡，心肌细胞凋亡指数(22.45±3.07%,16.48±2.32%)，较MIRI组(33.25±4.08%)下降(P<0.05)；芒果苷及雷米普利组可显著减轻心肌间质纤维化程度，其CVF值分别为(6.49±0.81%,4.99±0.54%)，较MIRI组(9.43±0.90%)明显降低(P<0.05)；芒果苷及雷米普利组Caspase-3mRNA基因转录水平分别为(3.97±0.53,2.17±0.39)，蛋白表达水平分别为(33.88±3.14%,21.13±2.85%)较MIRI组(7.43±1.05,55.88±4.45%)明显降低(P<0.05)；芒果苷及雷米普利组Bcl-2mRNA基因转录水平分别为(0.27±0.04,0.49±0.09)和蛋白表达水平分别为(24.38±2.56%,35.38±3.07%)较MIRI组(0.14±0.03,13.25±2.38%)明显升高(P<0.05)。与芒果苷组比较，雷米普利组心脏超声LVIDs、LVEDP显著降低(P<0.05),而IVSTd、IVSTs、LVPWd、LVPWs、LVSP、EF、FS等指标明显升高(P<0.05)；雷米普利组心肌细胞凋亡指数及心肌胶原含量较芒果苷组均明显下降(P<0.05); Caspase-3mRNA基因转录水平和蛋白表达水平明显降低(P<0.05)；Bcl-2mRNA基因转录水平和蛋白表达水平较芒果苷组明显升高(P<0.05)。
第四章芒果苷对大鼠心肌MIRI及心室重构保护作用机制的研究
MIRI组、芒果苷组心肌组织中p-P38蛋白磷酸化水平分别为(34.12±4.53%，26.17±3.47%)较Sham组(19.32±2.18%)明显升高(P<0.05)，SB203580组p-P38蛋白表达水平（21.93±2.57%)与Sham组无明显差异（P>0.05）；与MIRI组比较，芒果苷组和SB203580组p-P38蛋白表达水平明显下降(P<0.05)；与芒果苷组比较，SB203580组p-P38蛋白表达水平明显降低(P<0.05)。
MIRI组、芒果苷组、SB203580组血清TNF-α浓度分别为(92.86±9.53ng/ml，73.63±6.65ng/ml，54.38±4.57ng/ml)较Sham组(20.00±浓度2.14ng/ml)明显增加(P<0.05)；与MIRI组比较，芒果苷组和SB203580组血清TNF-α明显下降(P<0.05)；与芒果苷组比较，SB203580组血清TNF-α含量明显降低(P<0.05)。
4.结论
本文通过超声、病理学及分子生物学技术，多层次探讨了大鼠心肌MIRI损伤及心室重构的表现，通过不同剂量芒果苷对大鼠心肌MIRI的保护作用，并通过对P38MAPK信号转导通路的研究，对芒果苷的作用机制进行了探讨，结果提示：
(1)结扎左冠状动脉前降支(LAD)30min，松解后再灌注120min，可成功建立大鼠心肌MIRI模型。气管切开插管人工呼吸支持，可提高该模型成功率，易重复。
(2)大鼠心肌MIRI损伤后心室发生重构，表现为心室腔扩大，梗死壁变薄；心肌间质胶原纤维异常沉积，心肌纤维化明显；心肌细胞凋亡严重，同时，促凋亡基因Caspase-3基因和蛋白表达增高，而抗凋亡基因Bcl-2基因和蛋白表达下降，提示这两种基因失衡在MIRI导致的心室重构进程中扮演重要的角色。
(3)通过不同剂量芒果苷的干预研究，首次证实芒果苷对大鼠心肌MIRI损伤有保护作用，其作用有量效关系。中、高剂量的芒果苷能有效降低血清中心肌酶谱的表达，发挥其抗炎、抗氧化作用，减轻心肌细胞超微结构的损伤。
(4)芒果苷可促进Bcl-2的基因转录和蛋白表达水平，降低Caspase-3的基因转录和蛋白表达水平的作用进而抑制心肌MIRI细胞凋亡，降低MIRI大鼠心肌间质胶原纤维化，减轻心室重构，改善心肌顺应性，保护心脏收缩舒张功能。
(5)首次研究芒果苷可抑制P38MAPK，降低血清中炎性介质TNF-α的表达，其可能是芒果苷对MIRI损伤及心室重构的保护作用的重要机制之一。
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 evaluation
Male 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 remodeling
According 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 remodeling
2.3.1Different 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 remodeling
According 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 remodeling
According 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 evaluation
MIRI 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 remodeling
After 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 remodeling
3.3.1Different doses of mangiferin on MIRI
The 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 remodeling
LVIDd 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 remodeling
P38MAPK 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.
引文
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