【作者】 张慧敏；

【导师】 刘坤申；

【作者基本信息】 河北医科大学 ， 内科学， 2009， 博士

【摘要】 充血性心力衰竭(心衰)是发病率、致死率和致残率很高的常见临床综合征,是许多心血管疾病最常见的死亡原因。其病理生理改变主要是神经内分泌-细胞因子系统的激活和心肌重构,过度激活的神经内分泌系统又可导致心肌重构和心力衰竭恶化。神经内分泌系统中以交感-肾上腺素系统为重要,交感神经张力的改变可直接通过肾上腺素受体介导的生物学效应来影响心衰的进程。目前的认识和大型临床试验的结果,已经使心衰的治疗从过去的血流动力学模式发展为以改善心肌生物学行为,提高患者长期生存率和生活质量为目的的循证医学模式,从而使阻断神经激素和心肌重构的恶性循环成为心衰治疗的关键。目前,人类对于心衰的发病机制及治疗的研究已相当深入,在心衰失代偿早期通过药物治疗尚能达到较为满意的疗效,但是对于难治性心衰的患者药物治疗难以奏效,患者往往因顽固性心衰而出现少尿、内环境紊乱、肾功能衰竭而死亡。近几年我们在临床中收治了大量难治性心衰患者,在常规药物治疗基础上加用糖皮质激素,往往能扭转心衰难治的局面,患者尿量明显增多,一般状况及心肾功能好转,并使改善心衰终点事件的β受体阻滞剂和ACEI可以重新启用,并增至靶剂量。肾上腺素能反应系统在调节心血管的稳态上起重要作用。内源性的儿茶酚胺肾上腺素和去甲肾上腺素通过活化9种不同的肾上腺素受体亚型调节着它们的生物作用,研究表明,轻度心衰时β1受体开始下调,严重心衰时β1受体下调至不能对肾上腺素起反应;心衰时β2受体不存在下调。近来发现,心肌还存在β3受体,心衰时β3受体上调,比非衰竭心脏增加2-3倍。β3受体的上调在一定阶段可延缓心肌细胞的进一步损伤,但是在心衰晚期,由于β3受体持续增加导致心肌抑制加重,心功能恶化。心肌的α1-肾上腺素能兴奋可诱发心肌收缩力的适度增强(效应明显小于β1受体),在病理环境中,α1受体在β受体系统下调及G-蛋白和效应器解偶联的情况下,以代偿的方式维持着心肌收缩力。尽管这四种受体亚型在心衰时都有上调或下调的改变,目前还不明确这几种肾上腺素能受体在心衰治疗前后尤其在糖皮质激素治疗后发生何种变化,起多大作用。国内虽然有糖皮质激素治疗心衰的报道,但糖皮质激素对心肌肾上腺素能受体及血流动力学的影响尚未见报道。国外关于上述的研究也很少。目的:1建立大鼠急性心梗后心衰的动物模型,用地塞米松干预治疗心衰的大鼠,观察地塞米松对心衰大鼠的血流动力学的影响;2测定地塞米松治疗前后心衰大鼠心肌的α1、β1、β2、β3受体密度,观察地塞米松对心衰大鼠心肌组织肾上腺素能受体的影响;3选择临床难治性心衰病人35例,在心衰常规治疗无效的基础上应用糖皮质激素强的松,观察能否显著缓解充血性心衰的症状和改善临床状况。方法:第一部分:实验动物为雄性Wistar大鼠40只,水合氯醛腹腔麻醉,小动物呼吸机辅助呼吸,通过结扎左冠状动脉前降支制造大鼠急性心梗模型。12周后大鼠进食活动量明显减少,精神状态差,体重明显增加,口鼻及肢体远端紫绀,建立大鼠心衰模型。随机分成两组,一组肌注地塞米松(1mg /kg),分别于第一、第四天给药,另一组注射相同容量的生理盐水。观察心衰大鼠的症状,4天后对两组大鼠进行血流动力学检测,检测指标包括血压、心率和左室舒张末压;第二部分:取第一部分实验动物大鼠的心肌组织,并取5只正常大鼠的心肌组织作为正常对照,用免疫组化法检测大鼠心衰前后及糖皮质激素治疗前后大鼠心肌组织肾上腺素能受体α1、β1、β2、β3受体的变化,取镜下切片的灰度值反应受体的密度;第三部分:研究对象为临床难治性心衰病人35例,病人的选择标准包括由于急性失代偿的心力衰竭出现的端坐呼吸,难治性水肿,患者病情严重,住院超过1周,包括利尿剂之外药物的静脉治疗。排除标准包括:有感染症状或有糖皮质激素禁忌症的;收缩压低于80 mmHg或高于140 mmHg的心源性休克;血容量不足;机械通气;肥厚型或限制型心肌病;缩窄性心包炎;肺动脉高压;活动性心肌炎。书面征得所有患者同意后,病人开始在心衰常规治疗基础上服用强的松(1mg/kg/day,最大剂量不超过60mg / day至少服用9天),研究的初期终点为每天尿量的变化、病人评价呼吸困难、医生评价总体临床状况及肾功能的变化(血清肌酐和尿素氮)。次级终点为总体安全状况。结果:1在结扎大鼠冠脉建立急性心梗模型中,40只大鼠术中手术成功率为50%,术后24h模型存活率40%,实验期间共死亡6只,研究终点共有10只大鼠(两组各5只)。2地塞米松治疗组的大鼠饮食及精神状态好转,尿量及活动量增加,口鼻及肢体远端紫钳减轻,说明地塞米松干预治疗心衰有效。血流动力学检测结果显示,地塞米松干预治疗组的大鼠与生理盐水对照的大鼠相比,左室舒张末压降低(4.4±0.8mmHg vs 12.1±2.5mmHg, P<0.05),血压和心率没有变化(P>0.05)。3大鼠心衰时,心肌的β1受体密度下调(-5.13±0.097 vs -5.17±0.016, P< 0.01),免疫组化法图像采集显示,心衰大鼠的心肌切片棕黄色颗粒减少,灰度值变大,即β1受体密度变小。地塞米松治疗的心衰大鼠与注射生理盐水的心衰大鼠相比,心肌的β1-AR明显上调(-5.17±0.016 vs -5.14±0.013, P<0.01)。4心衰大鼠的β2受体出现明显上调的趋势(-5.14±0.017 vs -5.10±0.015, P<0.01),心衰大鼠的心肌免疫组化切片棕黄色颗粒明显增多。地塞米松干预治疗的心衰大鼠与注射生理盐水的心衰大鼠相比,心肌的β2-AR有下调趋势,但是没有统计学意义(-5.10±0.015 vs -5.12±0.014, P>0.05)。5大鼠心衰时,心肌的β3受体密度明显上调(-5.18±0.018 vs -5.16±0.004, P<0.05),免疫组化采集的图像显示,心衰大鼠的心肌切片棕黄色颗粒明显增多,灰度值变小,密度变大。地塞米松治疗的心衰大鼠与注射生理盐水的心衰大鼠相比,心肌的β3-AR明显上调(-5.16±0.004 vs -5.13±0.011, P<0.05)。6大鼠心衰时,心肌的α1-AR密度明显上调(-5.16±0.017 vs -5.14±0.003, P<0.01),免疫组化采集的图像显示,心衰大鼠的心肌切片棕黄色颗粒明显增多,灰度值变小,密度变大。地塞米松治疗的心衰大鼠与注射生理盐水的心衰大鼠相比,心肌的α1-AR明显下调(-5.14±0.003 vs -5.17±0.006, P< 0.01)。7对于失代偿的心力衰竭病人,在常规治疗基础上加用强的松治疗后,随着时间的变化,诱导了强大的利尿作用。通过利尿,80%的失代偿心衰病人的充血性心衰症状得到了显著改善(P<0.01),68.6%心衰病人的总体临床状况得到了显著改善(P<0.001)。强的松对血清钠、钾、氯和BUN没有影响,然而强的松能显著降低血清肌酐和尿酸的水平。血清肌酐从基线水平下降了12.21μmol/L (P<0.05) ,血尿酸从基线水平下降了95.22±239.16mmol/L (P< 0.05)。8研究结束时,34个病人体重减轻了,只有1个病人体重没有变化。总体上体重平均减轻了3.17±2.10 kg (P<0.01)。关于安全概况,相对短期的口服强的松是安全的,研究期间没有发现严重副反应(心绞痛恶化、感染和难以控制的高血压等)。结论:1成功的动物模型是实验的首要条件之一,只有通过训练掌握熟练的技术,增加经验积累,才可以提高动物模型的生存率,保证下一步实验的顺利进行。2血流动力学法是目前评价动物模型心功能的方法之一。血流动力学改善是心功能好转的标志之一。在急性心肌梗死后心衰的大鼠模型中,地塞米松治疗组的大鼠与注射生理盐水的大鼠相比,心衰症状好转,血流动力学改善,地塞米松治疗心衰有效。3大鼠心衰时,心肌的β1受体明显下调,α1受体、β2受体和β3受体密度明显上调。4地塞米松治疗的心衰大鼠与注射生理盐水的心衰大鼠相比,心肌的β1受体和β3受体明显上调,α1受体明显下调,β2受体没有变化。从临床效果来看,地塞米松治疗的心衰大鼠在血流动力学及一般状况上,都明显优于注射生理盐水的心衰大鼠,据此推测,β3受体上调和α1受体下调产生的负性肌力之和可能弱于β1受体上调产生的正性肌力。5对于失代偿的充血性心力衰竭病人,在心衰常规治疗基础上加用强的松这种糖皮质激素能显著缓解充血症状,并伴随着强有力的利尿和改善肾功能的作用。研究期间没有发现严重副反应,相对短期的口服强的松是安全的。更多还原

【Abstract】 Congestive heart failure(CHF) is the common clinical syndrome accompanied with a higher morbidity and mortality rate. Its pathophysiologic changes mainly develop in neuroendocrine-cytokine system activation and myocardium remodeling. Excessus activated neuroendocrine system and renin-angiotensin-aldosterone system(RAAS) further lead to myocardium remodeling and CHF aggravation. For patients with CHF the activation of neuroendocrine system is very important. Changes of adrenergic nerve tension influence the CHF proceeding by adrenoceptor mediated biological effect. Recently, the studies of CHF make the CHF therapy change from previous hemodynamics mode to evidence-based medicine mode aimed at improving myocardium biological behaviour and raising patient long term survival rate and quality of life. Accordingly, blocking vicious cycle of neurohormone activation and myocardium remodeling become the key point of CHF treatment. Now, researches on CHF pathogenesis and treatment have evolved deeply. CHF medication can achieve anticipated curative effect at the early stage, however, which is difficult to have the intended effect for decompensate CHF(DCHF). Patients with DCHF often emerge oliguria, internal environment disorders and renal failure and died at the end stages. For a few years, we cured a lot of patients with DCHF. Glucocorticoid adding to usual treatment in patients with DCHF often turn round the refractory situation, making the patients urine volume increase obviously, the general states and heart-renal function improved obviously. Henceβ-blocker and ACEI can be added and improve CHF states and decease end-point events. Adrenergic system plays an important role in regulating cardiovascular system. Endogenic catecholamines(adrenalin and noradrenaline) regulate their biological action by activating nine adrenoceptor(AR) subtypes. Recently, studies on CHF have indicate thatβ1-AR begins down-regulate when CHF is in its light degree.β1-AR is so down-regulate that it can’t react to adrenalin when CHF is in its very severe degree.β2-AR is not down-regulate in the exhausted heart. It was discovered thatβ3-AR existed in myocardium lately.β3-AR up-regulate 2 to 3 folds in the exhausted heart than the normal heart. Up-regulation ofβ3-AR at certain phase can delay myocardial cell impairment further, butβ3-AR continuous augment leads to myocardium inhibition aggravation and heart function deterioration. Myocardiumα1-AR stimulation may induce cardiac musculotone moderate enhancement(effect obviously less thanβ1-AR). In pathologic environment,α1-AR sustains myocardial contractility in compensatory form under circumstances ofβ-AR system down-regulation and uncoupling of G-protein and effectors. In spite of the 4 kinds AR existing up-regulation or down-regulation in the exhausted heart, it is not clear how the 4 kinds AR change before and after the treatmentof CHF, especially treated with glucocorticoid.Although there is a few reports on CHF be treated with glucocorticoid at home,it’s very rare abroad,and it isn’t clear if glucocorticoid influence myocardium AR and hemodynamics.Objectives: 1. we establish rat models of CHF after acute myocardial infarction; We treat them with dexamethasone, and observe if and how dexamethasone influence on hemodynamic of rats with CHF; 2. To measure rats myocardialα1、β1、β2、β3-AR density, and to observe if and how dexamethasone influence on myocardial AR of rats with CHF; 3 to enroll the patients with DCHF whom conventional therapy is ineffective, to observe if prednisone added to conventional therapy can significantly relieve hyperemia symptom and improve clinical state.Methods: Part 1: Waster rats (n=40) were anesthetized with chloral hydrate in abdominal cavity, small animal breathing machine assisted respiration, to make acute myocardial infarction model by ligating ramus descendens anterior arteriae coronariae sinistrae of rats. Rats model with CHF formed 12 weeks later when rats showed foodintake and activity amount reduced obviously, mental status dispirited,weight increased obviously, mouth, nose and limbs far-end cyanosis. Rats with CHF were divided randomly into two groups, one group was injected dexamethasone(1mg /kg) intramuscularly at the first and forth day respectively,the other group was injected equi-volume saline likewise, and observing the changes of symptoms of rats with CHF; measuring rats hemodynamics index including blood pressure(BP), heart rate(HR) and left ventricular end-diastolic pressure (LVEDP) 4 days later. Part 2: To measure every group rats myocardialα1、β1、β2、β3-AR density by immunohistochemical method ,and to understand the AR density changes .Taking the first part rats’myocardium tissues for testing myocardialα1、β1、β2、β3-AR density and 5 normal rats for normo-contrast group and taking gray scale of slice as AR density. Part 3: The study enrolled 35 patients with DCHF. Patients were included if they had orthopnea and refractory edema due to acute DCHF that was severe and had taken more 1 week hospitalization and intravenous therapy in addition to diuretics. The exclusion criteria were the following: Patients with an sign of infection or any conditions that would contraindicate glucocorticoids; systolic blood pressure lower than 80 mm Hg or higher than 140 mm Hg; cardiogenic shock; volume depletion; mechanical ventilation; hypertrophic or restrictive cardiomyopathy; constrictive pericarditis, pulmonary arterial hypertension, or active myocarditis. After written confirmed form was obtained, prednisone (1 mg/kg/day with a maximum dose of 60 mg/day was given for at least 9 days) was added. Primary end points were the effects on daily urine volume; patient assessed dyspnea and physician assessed global clinical status on day 1, day 2, day 3, day 4 and day 9; changes in renal function (serum creatinine, blood uric acid). Secondary end point was overall safety profile.Results:1. Operation achievement ratio is 50% during ligating rat coronary artery to make acute myocardial infarction model. Survival rate of model is 40% postoperative 12 hours.6 rats died during experimental session, there are 10 rats in the study end point (each group has 5 rats,respectively) . 2 .Compared with rats treated with saline,rats treated with dexamethasone had better mental status and appetite,urine volume and activity amount increased,mouth, nose and limbs far-end cyanosis lessened. LVEDP cut down(4.4±0.8 mmHg vs 12.1±2.5mmHg, P<0.05), BP and HR unchanged(P>0.05). 3. Myocardialβ1-AR density down- regulation(-5.13±0.097 vs -5.17±0.016, P< 0.01) in rats with CHF compared with normal rats, Buffy particle diminished in myocardium slice, gray scale value greatened, and i.e.β1-AR density grew down. Compared with rats treated with saline,myocardialβ1-AR density up-regulation obviously(-5.17±0.016 vs -5.14±0.013, P<0.01)in rats treated with dexamethasone.4 Myocardialβ2-AR density up-regulation obviously(-5.14±0.017 vs -5.10±0.015, P<0.01)in rats with CHF compared with normal rats, Buffy particle increased in myocardium slice. Myocardialβ2-AR density had down-regulation tendency in rats treated with dexamethasone compared with rats treated with saline, but statistical analysis is no difference(-5.10±0.015 vs -5.12±0.014, P>0.05). 5. Myocardialβ3-AR density up-regulation obviously(-5.18±0.018 vs -5.16±0.004, P<0.05)in rats with CHF compared with normal rats, buffy particle increased in myocardium slice. Myocardialβ3-AR density up-regulation obviously(-5.16±0.004 vs -5.13±0.011, P<0.05)in rats treated with dexamethasone compared with rats treated with saline. 6. Myocardialα1-AR density up-regulation obviously(-5.16±0.017 vs -5.14±0.003, P<0.01)in rats with CHF compared with normal rats, buffy particle increased in myocardium slice. Gray scale value grew down, and i.e.α1-AR density greatened. Compared with rats treated with saline, Myocardialα1-AR density down-regulation obviously(-5.14±0.003 vs -5.17±0.006, P< 0.01)in rats treated with dexamethasone compared with rats treated with saline. 7. For patients with DCHF, adding prednisone to the usual care resulted in significant relief of the congestive symptoms, which was accompanied with a potent diuresis with time and an improvement in renal function. At the end of study, dyspnea patient-assessed was markedly improved in 80% DCHF patients (P< 0.01), while global clinical status was markedly improved in 68.6 % DCHF patients(P<0.001)due to diuresis. Prednisone had no impact on serum sodium, potassium, chloride, BUN. However, it could significantly lower creatinine and uric acid. Changes from baseline in serum creatinine and uric acid was -12.21μmol/L (P<0.05) and -95.22±239.16 mmol/L (P<0.05), respectively. 8. At the end of study, 34 patients achieved weight loss and only 1 patient stayed unchanged. Overall, the weight loss was 3.17±2.10 kg on average(P<0.01). With respect to safety profile, administration of prednisone in relatively short period was safe and there were no severe side effects (exacerbation of angina, infection and poor controlled hypertension etc.) were observed in the study period.Conclusions: 1 Establishing animal models successfully is an important premise of experiment. We can raise animal model survival rate only by training frequently to accumulate experience, so that experiment can be proceed smoothly. 2 Hemodynamics is one of means to appraise animal model heart function. Amelioration of hemodynamics is one of markers appraising heart function improvement. Compared with rats treated with saline,symptom of CHF and hemodynamics improved in rats treated with dexamethasone. Dexamethasone is effective to therapy of CHF. 3 Myocardialβ1-AR density down-regulation andα1、β2、β3-AR up-regulation obviously in rats with CHF compared with normal rats. 4 Compared with rats treated with saline, myocardialβ1、β3-AR density up-regulation obviously,α1-AR down-regulation,β2-AR no change in rats treated with dexamethasone .Seen from clinical effect, haemodynamics and general condition both better in rats treated with dexamethasone than rats treated with saline. We had a presumption on these grounds that the sum of negativity myodynamia generated byβ3-AR up-regulation andα1-AR down-regulation possibly less than positive myodynamia generated byβ1-AR up-regulation. 5 For patients with DCHF, adding prednisone to the usual care resulted in significant relief of the congestive symptoms, which was accompanied with a potent diuresis with time and an improvement in renal function. There were no severe side effects were observed in the study period and administration of prednisone in relatively short period was safe.更多还原