【作者】 连榅林；

【导师】 郑兴；

【作者基本信息】 第二军医大学 ， 内科学， 2011， 博士

【摘要】 目前心力衰竭的治疗方案包括拮抗神经内分泌系统的激活,比如β受体阻滞剂、血管紧张素转换酶抑制、血管紧张素受体拮抗剂、醛固酮拮抗剂等药物,对症治疗,比如利尿剂、强心甙等药物,以及机械辅助治疗。虽然心力衰竭的治疗措施已经取得了长足发展,但心力衰竭的患病率和死亡率仍居高不下,晚期心力衰竭患者人数在逐年增多,这些患者需要新的治疗策略来改善他们的预后。正常心脏依靠线粒体氧化磷酸化生成的大量ATP来维持自身舒缩功能,其中约60-90%的ATP来自脂肪酸氧化,10-40%的ATP来自乳酸盐和葡萄糖。既往动物和临床研究证实心力衰竭时心肌能量代谢出现异常,转变心肌底物代谢模式能改善心室功能,减缓心力衰竭患者左心室功能障碍的发生。心肌能量代谢调节有望成为一种新型的心力衰竭治疗手段。Adropin是2008年由Kumar等在研究下丘脑性肥胖小鼠肝脏基因表达时发现的一种由76个氨基酸构成的分泌性蛋白,它由能量动态平衡相关基因（Enho）编码,后者在肝脏、脑部、脐静脉和冠状动脉内皮细胞都有表达。肝脏Enho表达受机体能量代谢状态和摄取食物中的营养成分组成调节,在肥胖状态下发生改变。转基因过表达或重组adropin治疗能改善进食诱导的肥胖模型胰岛素抵抗和葡萄糖耐量受损。Adropin能减弱与肥胖相关的代谢紊乱,而肥胖易与高血压病、胰岛素抵抗、血脂异常等集结出现,是心力衰竭的独立危险因素,据此,我们推测adropin可能参与了心力衰竭的病理生理过程。本研究主要探讨adropin与心力衰竭的关系及其对心肌细胞的作用,希望找到一个以adropin作为靶点的新的心力衰竭能量代谢治疗方法。第一部分:慢性心力衰竭患者外周血adropin水平的变化及意义目的:探讨慢性心力衰竭患者外周血adropin水平的变化及其临床意义。方法:根据最新ACC/AHA慢性心力衰竭诊断和治疗指南,入选56例慢性心力衰竭患者和20例健康对照者;用ELISA法测adropin、TNF-α、IL-6水平;同时采用RIA法检测BNP水平;用全自动生化分析仪测血糖、血脂、肝肾功能;测量患者身高、体重,计算体重指数;超声检测心脏功能。结果:心力衰竭组LVEF随着NYHA心功能分级的加重而降低,BNP浓度随着NYHA心功能分级的加重而明显增高,BNP与LVEF负相关（r=-0.889,p<0.001）。在NYHAⅣ级的心力衰竭组,IL-6和Cr比对照组明显增高（p<0.05,p<0.05）,而TC和LDL-C比对照组降低（p<0.01,p<0.05）。以NYHA心功能分级的3组心力衰竭组及正常对照组间adropin水平差异显著（p<0.001）:血浆adropin水平随着心功能的恶化而升高,对照组:6.0±0.3 ng/mL;NYHAⅡ:7.6±0.4 ng/mL;NYHAⅢ:9.8±0.5 ng/mL; NYHAⅣ:12.4±0.6 ng/mL。慢性心力衰竭患者adropin水平与血浆BNP水平成正比（r=0.723,p<0.001）,与血浆IL-6水平成正比（r=0.326,p<0.01）,与血浆Cr水平成正比（r=0.238,p<0.05）,与BMI成正比（r=0.295,p<0.05）。血浆adropin水平与LVEF负相关（r=-0.710,p<0.001）。多元线性回归分析表明,血浆BNP和BMI是心力衰竭患者血浆adropin水平的主要影响因素。结论:慢性心力衰竭患者血浆adropin水平随着心功能的恶化而增高。多元线性回归分析证实BNP和BMI是血浆adropin水平的主要影响因素。这些发现表明慢性心力衰竭患者释放到血液中的adropin增多可能参与了心力衰竭发展的病理生理过程,但是具体的机制需要进一步的研究。第二部分:重组adropin对缺血性心力衰竭大鼠模型心功能的影响目的:检测缺血性心力衰竭大鼠模型外周血adropin水平的变化;观察重组adropin注射2周后对缺血性心力衰竭大鼠模型心功能的影响。方法:采用50只SD大鼠分为实验组（n=40）和假手术组（n=10）,实验组大鼠通过前降支结扎至心肌梗死的方法构建心力衰竭模型,并经超声证实,术后2周分为早期干预组和晚期干预组,每组再分为生理盐水治疗组（n=6）和adropin治疗组（n=6）,adropin治疗组腹腔注射adropin 100ug/kg/d,生理盐水治疗组腹腔注射同等量生理盐水,治疗2周后进行各项检测。检测早期干预组的生理盐水治疗组和假手术组大鼠术前及术后24h、3d、2w、4w的adropin水平;早期干预组大鼠治疗2周后处死,进行心肌病理组织学检测,用TUNEL试剂盒进行细胞凋亡检测;晚期干预组大鼠治疗2周后进行超声检测大鼠心脏左室舒张末直径（LVEDD）、左室收缩末直径（LVESD）、左室舒张末容积（LVEDV）、左室收缩末容积（LVESV）和左室射血分数（LVEF）。结果:早期干预组与假手术组相比,术后24小时adropin水平显著下降（3.22±0.24ng/mL vs 5.60±0.16ng/mL, p<0.01）,术后3日adropin水平较假手术组升高,但是不明显,直到术后4周较假手术组明显增加（8.06±0.15ng/mL vs 5.96±0.15ng/mL, p<0.01）;在早期干预组中，生理盐水治疗组心肌变性坏死明显，而adropin治疗组虽有心肌变性坏死,但程度明显降低;③在早期干预组中,生理盐水治疗组TUNEL阳性细胞率明显高于adropin治疗组（39.8±8.2% vs 4.67±2.2%, p<0.01, n=6）;③在晚期干预组中,adropin治疗组与生理盐治疗组相比,大鼠心脏LVEF明显改善（48.72±6.58% vs 35.52±2.05, p<0.01, n=6）。结论:心梗后大鼠血清adropin水平早期即出现波动,有短期内先下降而后持续上升特点,进入心力衰竭期后水平进一步升高。重组adropin治疗2周后明显减轻心梗后心肌变性坏死和细胞凋亡,改善缺血性心力衰竭大鼠的心功能。第三部分:Adropin对心肌细胞肥大、凋亡的作用及其机制目的:观察:①adropin对缺氧诱导心肌细胞肥大和凋亡的影响;②adropin对乳鼠原代心肌细胞AMPK信号通路活化的作用。方法:MTT法检测不同浓度adropin的干预对乳鼠原代心肌细胞在缺氧条件下细胞存活率的影响;Bradford法检测不同浓度adropin的干预对心肌细胞蛋白含量和合成速度的影响;Westernblot检测缺氧条件下adropin干预对乳鼠原代心肌细胞0～4小时p-AMPK水平变化的影响;Annexin V-FITC检测adropin对缺氧条件下乳鼠心肌细胞凋亡的影响。结果:与对照组相比,10^-810^-6mol/L的adropin干预后可显著提高心肌细胞在低氧条件下的存活率（p<0.01）,并呈浓度依赖性;与对照组相比，10^-6mol/L adropin干预可明显抑制低氧诱导的心肌细胞总蛋白含量和合成速度增高（p<0.01）;与无血清组相比,adropin组细胞凋亡率无明显改变（p=0.12）,但是adropin+缺氧组凋亡率较缺氧组显著降低（p<0.01）,加入p-AMPK抑制物compound c后adropin+缺氧组凋亡增加,与缺氧组相比无显著差异;④10^-6mol/L的adropin干预可使体外培养的乳鼠心肌细胞p-AMPK水平较对照组增高（p<0.01）。结论:Adropin可增加缺氧条件下心肌细胞的活性,抑制细胞肥大,提高心肌细胞的p-AMPK水平,并通过AMPK通路抑制心肌细胞凋亡。更多还原

【Abstract】 Current heart failure therapeutic options are directed towards disease prevention via neurohormonal antagonism （β-blockers, angiotensin converting enzyme inhibitors and/or angiotensinreceptor blockers and aldosterone antagonists）, symptomatic treatment with diuretics and digitalis and use of biventricular pacing and defibrillators in a special subset of patients. Despite these therapies and device interventions heart failure remains a progressive disease with high mortality and morbidity rates. The number of patients who survive to develop advanced heart failure is increasing. These patients require new therapeutic strategies.Normal cardiac function is dependent on a constant resynthesis of ATP by oxidative phosphorylation in the mitochondria. The healthy heart gets 60–90% of its energy for oxidative phosphorylation from fatty acid oxidation, with the balance from lactate and glucose. The failing heart has been shown to be metabolically abnormal, in both animal models and in patients, and chronic manipulation of myocardial substrate oxidation toward greater carbohydrate oxidation and less fatty acid oxidation may improve ventricular performance and slow the progression of left ventricular dysfunction in heart failure patients. Thus, regulation of cardiac energy metabolism is expected to be a new therapeutic strategy for heart failure.Adropin was initially discovered by Kumar et al. in 2008 during microarray analysis of liver gene expression in mouse models of obesity. Adropin is encoded by Energy Homeostasis Associated gene （Enho） that is expressed in the liver, brain, human umbilical vein and coronary artery endothelial cells. Liver Enho expression is regulated by energy status and dietary nutrient content, and is altered with obesity. Transgenic overexpression or systemic adropin treatment improves diet-induced obesity, insulin resistance, and glucose tolerance. Elevated BMI and obesity have been associated with the cardiovascular disease risk factors of hypertension, insulin resistance and dyslipidemia. Obesity has been linked to the development of HF. Thus, we hypothesized that adropin may also be related to the failing heart. In the present study, we are intent to investigate the relationship between adropin and HF, and to explore the effects of adropin on proliferation, apoptosis and energy metabolism in cardiocytes.PartⅠChanges of circulating adropin and its significance in CHF patients Objective: Adropin is a recently identified protein that has been implicated in the maintenance of energy homeostasis. we investigated plasma adropin levels in patients with CHF and evaluated the relationship between those and the severity of CHF.Methods: According to the present guidelinges for the diagnosis and management of chronic heart failure of the ACC/AHA, 56 patients with CHF and 20 healthy subjects were enrolled in this study. Plasma levels of adropin, TNF-α, IL-6 were measured using a commercial ELISA kit, and plasma BNP levels were measured with a commercial RIA kit. The plasma glucose levels were measured by an automated glucose oxidase method, serum levels of total TC, TG, HDL-C, LDL-C were measured by enzymatic methods using the autoanalyzer. The height and weight of subjects were measured to calculate the BMI. Ultrasonic was used to measure the heart function.Results: The LVEF gradually decreased and, inversely, plasma levels of BNP were exponentially elevated according to NYHA class in the CHF patients. Plasma BNP had a negative correlation with LVEF （r =-0.889, p<0.001,）. Plasma levels of IL–6 and Cr were significantly higher in the classⅣpatients than those in the control group （p<0.05, p<0.05, respectively）. Lipid analysis showed that plasma levels of TC and LDL-C were lower in the classⅣthan those in the control group （p<0.01, p<0.05, respectively）. The plasma level of adropin increased with the deterioration of cardiac function （ control: 6.0±0.3 ng/mL; NYHAⅡ: 7.6±0.4 ng/mL; NYHAⅢ: 9.8±0.5 ng/mL; NYHAⅣ: 12.4±0.6 ng/mL, respectively, p<0.001）. Plasma adropin level had a positive correlation with plasma BNP levels （r=0.723, p<0.001）, plasma IL-6 levels （r=0.326, p<0.05）,plasma Cr （r=0.238, p<0.05） and BMI （r=0.295, p<0.05）. Plasma adropin levels negatively correlated with LVEF （r =-0.710, p<0.001,）. In the multiple regression analysis, plasma BNP and BMI had independent impact on plasma adropin level in patients with CHF.Conclusion: Plasma adropin levels were significantly increased according to the severity of CHF. Multiple regression analysis showed BNP and BMI had independent impact on plasma adropin level. These findings suggest that the augmented release of adropin may be involved in the pathogenesis of CHF, but further study is necessary to explain the exact role of adropin in CHF.PartⅡEffect of synthetic adropin on cardiac function of HF rat Objective: To investigate the changes of circulating adropin levels in ischemic heart failure model of rats and the effects of treatment with adropin for 2 weeks on cardiac function.Methods: The SD rats were divided into experimental group （n = 40） and sham operation group （n=10）, and model of heart failure induced by myocardial infarction was set up by ligating left descending anterior branch. The 40 rat models of heart failure were divided into early intervention group and late intervention group, each group was further divided into saline treatment group （n=6） and adropin treatment group （n=6）. In adropin treatment group, rats were treated with 100ug/kg/d of adropin by intraperitoneal injection, and in saline treatment group, rats were treated with same doses of physiological saline. In early intervention saline treatment group and sham operation group, adropin was measured in five points of time, such as preoperative, postoperative 24h, 3d, 2wks, 4wks. In early intervention group, the histopathology of rat heart was observed by Gonori chromotropic acid staining and TUNEL kit after 2 weeks of treatment. In late intervention group, echocardiography was examined after 2 weeks of treatment.Results: Compared with sham operation group, adropin levels of rat in early intervention group were drop significantly in postoperative 24 hour （3.22±0.24ng/mL vs 5.60±0.16ng/mL, p<0.01）, and began to increase in postoperative 3 days, but there was not statistically significant. The adropin levels were significantly increased in postoperative 4 weeks compared with sham operation group （8.06±0.15ng/mL vs 5.96±0.15ng/mL, p<0.01）. In early intervention group, the degeneration and necrosis of rat myocardium in saline treatment group were much more serious than that in adropin treatment group. In early intervention group, TUNEL positive cells rate of rats in saline treatment group was significantly higher than that in adropin treatment group （39.8±8.2% vs 4.67±2.2%, p<0.01）. In the late intervention group, LVEF of rat heart in adropin treatment group was significantly higher than that in saline treatment group （48.72±6.58% vs 35.52±2.05, p<0.01）.Conclusion: After myocardial infarction, adropin levels of rat were transiently drop and then continued to rise. Treatment with adropin attenuated myocardial necrosis and apoptosis, and improved heart function of heart failure rat.PartⅢEffects of adropin on proliferation and apoptosis of cardiomyocyteObjective: Obeserve effects of adropin on AMPK pathways activating, and apoptosis induced by hypoxia in rat neonatal cardiomyocytes.Methods: MTT method was used to detect cell livability; Bradford method was used to measure cell protein level and synthetic speed of protein; Westernblot was used to detect cell p-AMPK levels; Annexin V-FITC was used to detect cell apoptosis in rat neonatal cardiomyocytes.Results: Compared with the control group, adropin significantly increased cell livability in hypoxic conditions （p<0.01）, and reduced the total protein and the protein synthesis speed with a dose-response trend （p<0.01）. Compared with serum-free group, there was no significant changes in cell apoptosis rate in adropin group cell （p=0.12）, but apoptosis rate of adropin+hypoxia group cell was significantly reduced compared with hypoxia group （p < 0.01）,this effect can be reversed by compound c,which is an inhibitor of p-AMPK. Treating with adropin (10^-6 mol/L), the p-AMPK level in adropin+hypoxia group transiently increased in 15min to 30min compared with hypoxia group （p < 0.01）.Conclusion: Adropin increased livability of cell, inhibited cell hypertrophy, elevated p-AMPK level in rat neonatal cardiomyocytes, and it had anti-apoptotic effects through AMPK activities under the condition of hypoxia.更多还原