【作者】 杨莹；

【导师】 胡申江； Michael Fu；

【作者基本信息】 浙江大学 ， 临床医学， 2009， 博士

【摘要】 第一部分瑞舒伐他汀对心肌梗死大鼠心功能的影响及其机制研究背景:心力衰竭终末期细胞内Ca²⁺稳态的调节及其病理生理变化主要涉及收缩期Ca²⁺从肌质网中的释放和肌质网对Ca²⁺的重新摄取等机制。肌浆网Ca²⁺-ATP酶(sarcoplasmic reticulum Ca²⁺-ATPase,SERCA)和磷酸受钙蛋白（Phospholamban,PLB）被认为是主要的心肌收缩/舒张的动态调节蛋白。通过干预使心力衰竭时异常的SERCA和PLB表达恢复的治疗方式有望应用于临床治疗心力衰竭患者。近年来的一些临床研究显示3-羟基3-甲基戊二酰辅酶A（HMG-CoA）还原酶抑制剂即他汀类药物在心肌梗死后病人中应用可以减少心室重塑,改善心功能。但对其具体机制的研究中关于他汀类是否可以直接影响钙调蛋白的研究很少。目的:本课题观察心肌梗死后瑞舒伐他汀长期应用对左室重构和心功能的影响。并在此基础上,研究瑞舒伐他汀是否通过影响心脏的肌浆网钙调蛋白SERCA和PLB的表达而产生对心功能的影响,同时检测心肌白细胞介素-6（interleukin-6,IL-6）和白细胞介素-10（interleukin-10,IL-10）的表达。方法:以开胸结扎冠状动脉左前降支的方法建立心肌梗死模型。冠脉结扎一天后,以灌胃方式每天一次给予给药组大鼠10 mg·kg^-1的瑞舒伐他汀。对照组和假手术组大鼠以同样方式给予等量蒸馏水。结扎冠脉后第2、4、6、8、10周以超声心动图方法检测左室大小（左室舒张末期内径和左室收缩末期内径,即LVIDd和LVIDs）、左室短轴缩短率（FS）、左室射血分数（EF）和舒张末期左室后壁厚度（LVPWd）。结扎冠脉第2和第10周超声心动图检测大鼠心肌梗死面积,结扎冠脉10周后测定左室血流动力学指标。大鼠处死后,测量左室、右室重量与体重比值、血脂水平、肌浆网SERCA活性、大鼠心肌组织SERCA2a、PLB和16位丝氨酸磷酸化-PLB（pSer16-PLB）蛋白表达水平以及心肌梗死区、梗死周围区和远离梗死区IL-6和IL-10的表达水平。结果:10周的瑞舒伐他汀治疗未影响实验各组大鼠的血脂水平和心梗面积。但超声心动图和血流动力学检测结果显示,心肌梗死大鼠予以瑞舒伐他汀10mg·kg^-1灌胃给药10周后,左室重构减轻,心功能明显改善。同时,左室心肌降低的SERCA活性、SERCA2a蛋白表达增高,升高的PLB蛋白表达不变,pSer16-PLB蛋白表达升高,降低的pSer16-PLB/PLB比值增加。衰竭心脏左室梗死周围区和远离梗死区升高的IL-6水平降低,升高的IL-10水平进一步升高。结论:心肌梗死大鼠予以瑞舒伐他汀10 mg·kg^-1灌胃给药10周后,显著改善了左室重构和左室功能。该有益作用并非通过其降脂效应或是改变心梗面积而产生。可能与瑞舒伐他汀治疗可以部分恢复钙调节蛋白的异常表达及对炎症因子的致损伤机制有一定的减轻作用有关。第二部分藻酸双酯钠对心肌缺血再灌注后心功能的影响及机制探讨研究背景:藻酸双酯钠（Polysaccharide sulfate,PSS）是以海藻提取物为基础原料,用化学方法引入有效基团合成的一种新型的类肝素药物。基础和临床研究显示PSS具有抗凝血、降低血粘度、降低TC和TG、升高HDL-C、降低血小板释放、明显的刺激合成PGI₂及改善微循环的功能。临床上有很多种情况会出现心脏暂时性缺血再灌注后心功能下降。基础研究显示PSS对大鼠脑缺血再灌注损伤有保护作用。目前关于PSS对心肌缺血再灌注后心功能影响的研究未见报道。目的:本实验应用大鼠心肌缺血再灌注后（Langendorff）模型,评价急性PSS应用能否对缺血再灌注损伤后心脏的心功能产生影响,并在此基础上,研究PSS是否是通过影响心脏丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）信号转导通路及肿瘤坏死因子-α（TNF-α）的表达而产生对心功能的影响。方法:成年雄性Sprague-Dawley（SD）大鼠,随机分成8组:A1组（正常对照组,normalcontrol）:给予改良的Krebs-Henseleit（K-H）液灌注60 min;A2组（缺血再灌注对照组,ischemia control）:缺血20 min,复灌时给予改良的K-H液灌注60 min;A3-A8组（缺血再灌注PSS给药组,ischemia control plus PSS 0.3,1,3,10,30 or 100）:缺血20 min,复灌时给予含0.3,1,3,10,30,100 mg/L PSS的改良K-H液灌注60min。记录心脏收缩舒张功能指标,包括左心室发展峰压（left ventricular灌注developed pressure,LVDP）、左心室舒张末压（left ventricular end diastolic pressure,LVEDP）、左室内压最大上升及下降速率（maximum rate of intraventricular pressuredevelopment and relaxation;+dp/dt max,-dp/dt max）和心率（heart rate）。记录冠脉流量（coronary flow）。收集冠脉流出液,测定其中肌酸激酶（creatine kinase,CK）和乳酸脱氢酶（lactate dehydrogenase,LDH）含量。裂解心脏组织,测定心肌组织TNF-α蛋白含量及MAPK信号转导通路的关键性调控蛋白包括细胞外信号调节激酶（extracellular signal-regulated kinases,ERKs）,c-Jun氨基末端激酶（c-junamino-terminal kinase,JNKs）和p38丝裂原活化蛋白激酶（p38 mitogen-activatedprotein kinase,p38 MAPK）的活性。结果:1～30 mg/L PSS可以提高缺血再灌注后心脏的LVDP%、+dp/dt max%、-dp/dtmax%和冠脉流量百分比,降低LVEDP%,对心率无明显影响。同时,1-30 mg/L PSS明显降低缺血再灌注后心脏释放的CK和LDH量,降低心肌组织升高的TNF-α水平及升高的p-p38MAPK和p-p54/p46-JNK蛋白表达水平。0.3 mg/L PSS对心脏舒缩功能和冠脉流量、心率、CK和LDH释放、TNF-α水平及p-p38MAPK和p-p54/p46-JNK水平均未产生影响。100 mg/L PSS则使本已受损的心功能进一步恶化,导致心脏舒缩功能、冠脉流量和心率的明显下降。0.3～100 mg/L PSS对P-p44/p42-ERK蛋白表达均为产生明显影响。结论:一定浓度的PSS（1～30mg/L）对于缺血再灌注损伤后的心脏,具有提高心脏舒缩功能,减轻心肌细胞损伤,提高冠脉流量而不改变心率等药理效应。该效应可能与减少缺血再灌注损伤所致的TNF-α表达水平的升高,逆转缺血再灌注损伤引起的MAPK通路改变相关。更多还原

【Abstract】 Part 1 Beneficial effect of rosuvastatin on heart failure is associated with alterations of calcium-regulatory proteinsBackground:The release of Ca²⁺ from sarcoplasmic reticulum（SR） and sarcoplasmic Ca²⁺ reuptake mainly involve in the regulation of intracellular Ca²⁺ homeostasis and its pathophysiological change.Sarcoplasmic reticulum Ca²⁺ ATPase（SERCA） and SERCA regulatory protein phospholamban（PLB） are the key proteins responsible for maintaining Ca²⁺ homeostasis throughout the excitation-contraction coupling cycle in cardiomyocyte.Interfering with the abnormal expression of SERCA and PLB may be a promising method in the clinical treatment of HF patients.Recent clinical and experimental studies indicated that statins（3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors） can attenuate ventricular remodeling and improve ventricular function after myocardial infarction（MI）.But there are few data exploring whether statins exhibit special effect in improving left ventricular（LV） remodeling and function through influence on calcium-regulatory proteins. Aim:We undertook our research to investigate effect of rosuvastatin on LV remodeling and cardiac function in a rat model of HF induced by MI.In addition,tissue levels of SERCA2a,SERCA activity,PLB and pSerl6-PLB as well as interleukin（IL）-6 and IL-10 in myocardium of rats with MI were detected to explore the molecular mechanism.Methods:Adult male Sprague-Dawley rats were anesthetized and the left coronary artery was ligated 2 to 3 mm from its origin to induce MI.For sham-operated animals,the suture was placed but not ligated（S group）.MI rats were randomized to receive gavage with either 1 ml·day-1 distilled water（MI group） or 10 mg·kg-1·day-1 rosuvastatin dissolved in 1 ml distilled water for 10 weeks（MI+R group）.Normal control（NC group） and normal control plus rosuvastatin treatment rats（NC+R group） were randomly selected.Rats were evaluated by echocardiography 2,4,6,8 and 10 weeks after operation.Left ventricle（LV） internal diameter at end-diastolic phase（LVIDd）,LV internal diameter at end-systolic phase（LVIDs）,LV posterior wall thickness at diastolic phase（LVPWd）,LV shortening fraction（FS） and ejection fraction（EF） were measured. Infarct size was also detected by echocardiography 2 and 10 weeks after MI.10 weeks after MI,rats were anesthetized,and hemodynamic variables were measured.After rats were sacrificed,LV relative weight（LVRW） and right ventricular relative weight （RVRW） were obtained,when the LV actual weight（LVAW） and right ventricular actual weight（RVAW） were corrected for body weight.Serum total cholesterol,triglycerides, and low- and high-density lipoprotein cholesterol were measured using enzymatic colorimetric assays.SR was prepared from LV tissue for Determination of SERCA activity.The protein expression of SERCA2a,PLB and phospho-PLB at serine-16 （pSer16-PLB） were detected by western blot.IL-6 and IL-10 levels were assessed by enzyme-linked immunosorbent assay.Results:After rosuvastatin treatment,LV remodeling and dysfunction were attenuated. Rosuvastatin partially restored the low expression of SERCA2a and pSerl6-PLB, increased SERCA activity,but showed no effect on PLB expression.Furthermore, rosuvastatin reduced the increased IL-6 level and further elevated IL-10 level in the peri-infarct and remote zones of MI.Serum lipid levels and infarct size remained unchanged.Conlusions:Rosuvastatin is effective in reducing LV remodeling and dysfunction in the failing heart.The molecular mechanism may be related to normalization of SERCA2a expression,SERCA activity,and pSerl6-PLB levels,as well as through cytokine alterations independent of its effect of lipid-lowering or MI size reduction. Part 2 Cardioprotection and mechanisms of polysaccharide sulfate against ischemia/reperfusion injury in isolated rat heartsBackground:Polysaccharide sulfate（PSS） is a new type heparinoid extracted from Phylum Phaeophyta.By chemically introducing sulfuryl and propylene glycol residues in the hydroxyl and carboxyl group of alginic acid sodium,PSS obtained is a diester sodium. Basic and clinical research showed PSS have the effect of anticoagulation,lowering blood viscosity,decreasing TC and TG,increasing HDL-C,depressing platelet release, stimulating PGI2 synthesis and improving microcirculation.Clinically,there is a sort of myocardial injury,which occurs with transient ischemia followed by re-establishment of flow,and results in prolonged postischemic contractile dysfunction of myocardium. Although PSS is successfully applied in ischemic cardio-cerebrovascular disease,its effect on cardiac function after ischemia/reperfusion（I/R） injury has not been investigated previously.Aim:The aim of the present study was to investigate whether acute application of PSS can protect the heart from I/R injury in a model of myocardial I/R of the isolated rat hearts.In addition,the present study is designed to explore whether the cardioprotective effect of PSS is based on the alterations of tumor necrosis factor-a（TNF-α） expression and mitogen-activated protein kinase（MAPK） signal transduction pathway.Methods:Isolated rat hearts were perfused（Langendorff） and subjected to 20 min global ischemia followed by 60 min reperfusion with Kreb’s Henseleit solution or different concentrations of PSS（0.3～100 mg/L）.Myocardial contractile and diastolic function （including left ventricular developed pressure,LVDP;left ventricular end diastolic pressure,LVEDP;maximum rate of intraventricular pressure development and relaxation,+dp/dt max and -dp/dt max） was continuously recorded.Heart rate and the coronary flow were measured.Creatinine kinase（CK） and lactate dehydrogenase（LDH） leakage were measured.Tumor necrosis factor-α（TNF-α） expression by cardiomyocytes was investigated.Western blot analysis for extracellular regulated kinases（ERKs）,c-jun amino-terminal kinase（JNKs） and p38 mitogen-activated protein kinase（MAPK） activity were performed.Results:PSS administration at concentrations of 1-30 mg/L improved cardiac contractility, reduced CK and LDH releasing and inhibited TNF-αproduction.Phosphorylated-p38MAPK（p-p38MAPK） and p-p54/p46-JNK increased in I/R rat hearts but diminished in PSS（1～30 mg/L） treated hearts.0.3 mg/L PSS had no effect on cardiac contractility,CK and LDH releasing,TNF-αproduction as well as p-p38MAPK and p-p54/p46-JNK expression.In contrast,high concentration of PSS（100 mg/L） had adverse effects that caused worsening of heart function.P-p44/p42-ERK level was unchanged after 0.3～100 mg/L PSS treatment.Conlusions:PSS has cardioprotective effect against I/R injury on the rat heart dose-dependently. The beneficial pharmacological effect including improving cardiac performance, increasing coronary flow but not affecting heart rate and lowering cardiocyte injury may be mediated through normalization of p38 MAPK and JNK pathways as well as TNF-αexpression.更多还原