摘要
研究背景：
心力衰竭(心衰)是缺血性心脏病、高血压病、瓣膜性心脏病等多种心血管疾病的共同终末阶段。在中国,成年人心衰的患病率约为0.9%,随着人口老龄化社会的发展,人群中心衰患者人数将不断上升。目前针对心衰发病机制的多种治疗手段虽能改善其症状,但心衰患者的5年生存率仍然低于50%,提示在心衰的发生发展过程中可能还有其他未知因素的参与。
心衰的病理生理机制极其复杂,多种因素参与其发生发展。近年来发现,在部分心衰患者血清中存在有抗G-蛋白偶联受体家族中几种主要受体(如肾上腺素β1受体、α1受体、胆碱能M2受体、血管紧张素Ⅱ1型受体)的自身抗体。进一步的研究提示,这些自身抗体在心衰的发生发展中具有一定的病理生理意义。
β3肾上腺素受体属于G蛋白偶联受体家族,是继β1、β2肾上腺素受体后发现的又一β肾上腺素受体。2005年本研究室发现,心衰患者血清中存在有抗β3肾上腺素受体的自身抗体(Autoantibodies against theβ3-adrenoceptor,β3AA),其阳性率明显高于正常人；对心衰患者的临床资料进行分析发现,β3AA阳性的心衰患者其心脏左室射血分数(Ejection Fraction, EF)明显高于β3AA阴性者,且β3AA阳性的心衰患者其纽约心脏病协会心功能分级(NYHA)相对较低,即β3AA阳性的心衰患者心功能较阴性者好。这些分析结果提示：β3AA可能具有一定的心脏保护作用,但尚需实验研究进一步证实。
此外,慢性心衰急性失代偿发作是心衰患者住院及死亡的主要原因,而心肌缺血是慢性心衰失代偿和急性发作的主要诱发因素之一。那么β3AA长期作用于慢性心衰大鼠,-是否能够降低心衰大鼠对心肌缺血／再灌注损伤的敏感性,从而降低心衰导致的死亡率,尚待进一步研究。
为此,本研究以慢性心衰大鼠为切入点,通过静脉长期给予β3AA,以大鼠心功能、心重／体重比、血清中脑钠肽水平及心肌细胞凋亡情况为指标,从β3AA是否能够减轻大鼠心衰程度以及是否能降低心衰大鼠对心肌缺血／再灌注损伤的敏感性两方面,观察β3AA对心衰大鼠是否具有心脏保护作用。
目的：
1.观察β3AA能否减轻大鼠心衰的程度；
2.观察β3AA是否能降低心衰大鼠对心肌缺血／再灌注损伤的敏感性。
方法：
1.β3AA的制备
选用8周龄、重量为140-160g、健康雄性Wistar大鼠20只,随机分为对照组与免疫组。以合成的β3AR细胞外第二环氨基酸肽段为抗原(176aa-200aa,R V G A D A E A Q ECHSNPRCCSFASNMP,中国医学科学院上海生物化学院)进行免疫,每隔两周免疫一次,共免疫8周。对照组只注射相应佐剂,方法同免疫组。采用本实验室改良的ELISA方法检测免疫鼠血清中β3AA的水平。利用IgG亲和纯化试剂盒(Mab.Trap Kit)纯化β3AA阳性的免疫大鼠血清中的IgG。将纯化后的IgG进行定量、定性,并常规SDS-PAGE凝胶电泳法检测其纯度。
2.腹主动脉缩窄手术：
选用10周龄、重量为200-220g、健康雄性Wistar大鼠。用10%水合氯醛(0.3mL／100g)腹腔注射麻醉后,在剑突下1 cm沿正中线作一切口,分离腹主动脉,将7号针头与腹主动脉一并结扎,然后抽出探针形成腹主动脉狭窄(约50%),同时腹腔一次性注射庆大霉素抗感染,分层缝合关腹。
3.β3AA输入心衰大鼠模型的制备
将提纯的β3AA阳性的IgG,以2μg/g的剂量,通过尾静脉给入腹主动脉缩窄手术后3天的大鼠体内,每10天加强免疫一次,共免疫50天；免疫前1天,经鼠尾取血样,分离血清,用SA-ELISA法检测其中β3AA的水平。对照组以2μg／g的剂量通过尾静脉给入β3AA阴性的IgG,免疫程序和检测方法同上。
4.在体心功能的检测
应用生物信号记录分析系统MS2000记录各心功能参数,包括左心室收缩压(LVSP),左心室舒张末压(LVEDP)以及左心室内压上升和下降的最大速率(±dp／dtmax)。
5.心重／体重比的测定
开胸,迅速取出心脏,洗净心腔内积血,去除心脏周围组织及血管,用滤纸吸干水分后,称量全心重,计算心脏重量与体重的比值。
6.血清中脑钠肽(BNP)的测定
血清中BNP的测定采用ELISA试剂盒(ADL,美国)进行测定,具体操作按照说明书进行。
7.大鼠心肌缺血／再灌注手术
选取进行腹主动脉缩窄术制备的心衰大鼠,用氨基甲酸乙酯(20%,1g／kg,i.p.)腹腔注射麻醉后,左冠脉前降支结扎30 min后,松开结扎线再灌注3h；假手术组大鼠手术操作相同,将缝合针穿过左冠脉前降支下方,但不结扎。
8.心梗面积测定
应用TTC染色法,测定β3AA输入的心衰大鼠经历心肌缺血／再灌注手术后心肌梗死面积。
9.心肌组织Caspase 3活性测定
应用Caspase 3测定试剂盒,检测大鼠心肌组织中caspase 3的活性。
10.原位末端标记法(TUNEL)测定心肌细胞凋亡
利用原位细胞死亡检测试剂盒(In Situ Cell Death Detection Kit,POD),检测大鼠心肌组织中细胞凋亡数量,并计算心肌细胞凋亡指数。
结果：
1.β3AA向腹主动脉缩窄术后大鼠转移的时间点选择
1.1腹主动脉缩窄术后大鼠心功能随时间变化情况
腹主动脉缩窄术后8周,大鼠LVSP、+dp／dtmax和-dp／dtmax明显下降,LVEDP明显升高,心功能开始下降；腹主动脉缩窄术后12周、16周,大鼠心功能进行性恶化(表1)。
1.2腹主动脉缩窄术后大鼠血清中β3AA随时间变化情况
腹主动脉缩窄术后8周,大鼠血清中β3AA明显升高(0.314±0.02vs.0.22±0.05,P<0.05),术后12周达到高峰,之后开始下降(图1)。
以上结果提示,腹主动脉缩窄术后0-8周是大鼠心功能从正常到明显下降的过程,是大鼠心衰发生发展非常重要的时期；而且此期间,是β3AA产生的过程,体外给予β3AA可以单纯研究其对大鼠心脏的保护作用。因此本研究选择在腹主动脉缩窄术后0-8周,研究在大鼠心衰发生发展过程中,p3AA对心脏功能的影响。
2.大鼠心衰模型制备成功
2.1腹主动脉缩窄术后8周大鼠在体心功能明显下降
腹主动脉缩窄术后8周,大鼠LVSP和+dp／dtmax明显下降,LVEDP和-dp／dtmax显著升高。说明,腹主动脉缩窄术后8周,大鼠心衰模型制备成功(图3-6)。2.2腹主动脉缩窄术后8周大鼠心重／体重比增大
与伪手术组相比,腹主动脉缩窄术后8周大鼠心重／体重比明显增大。提示,心衰大鼠心脏发生了心肌重构(图7)。
以上结果提示,腹主动脉缩窄术后8周,大鼠心脏已经发生明显的心肌重构,而且心功能已经表现出失代偿现象。
3.β3AA输入心衰大鼠模型制备成功
3.1β3AA输入心衰大鼠的剂量选择
分别以0.5μg/g、1μg/g、2μg／g的剂量,将p3AA输入腹主动脉缩窄术后的大鼠,免疫后第1天,采血检测大鼠血清中β3AA的水平。以2μg/g输入后,大鼠血清中β3AA的平均OD值为0.67,与临床相匹配,故作为最终免疫剂量(图8)。3.2β3AA的IgG亚类测定
应用ELISA法对β3AA的IgG亚类鉴定结果表明,免疫鼠中的β3AA主要属于IgG2b,还有极少部分属于IgG2a。有研究表明不同的IgG亚类具有不同的代谢半衰期,IgG2b亚类的代谢半衰期为6天左右,IgG2a亚类的代谢半衰期为8天左右。明确β3AA的主要IgG亚类则可以为输入周期的选择提供一定的理论依据(图9)。3.3β3AA输入腹主动脉缩窄术后大鼠,体内β3AA的消除时间测定
β3AA输入腹主动脉缩窄术后大鼠第1天,大鼠血清中抗体水平增高,平均OD值为0.67,与对照组相比增高了0.35。抗体水平下降其增高值的一半所需的时间大约为10天。因此本研究将免疫周期确定为10天(图10)。3.4β3AA输入腹主动脉缩窄术大鼠后,血清中β3AA水平维持稳定
以2μg/g为免疫剂量,10天为免疫周期,β3AA输入腹主动脉缩窄术后大鼠,定期监测大鼠血清中β3AA水平,发现该抗体水平能够稳定维持(图11)。提示β3AIA输入心衰大鼠模型制备成功。
4.p3AA对心衰大鼠具有心肌保护作用
4.1β3AA降低心衰大鼠血清中脑钠肽(BNP)水平
血清BNP水平与心力衰竭的临床严重程度相平行。实时监测心衰大鼠血清中BNP的水平,腹主动脉缩窄术后2周的心衰大鼠,血清中BNP开始有轻度升高；术后6周,β3AA输入心衰大鼠组血清中BNP水平升高程度低于未进行βsAA输入的心衰大鼠组；术后8周,β3AA输入心衰大鼠组血清中BNP的水平较β3AA未免疫心衰大鼠组明显降低。提示,β3AA输入组大鼠心衰程度相对较轻(图12)。4.2β3AA改善心衰大鼠的心功能
腹主动脉缩窄术后8周,β3AA输入组的+dp／dtmax及-dp／dtmax降低幅度均低于未免疫组(图13-16)。
4.3β3AA对心衰大鼠的心重／体重比没有明显影响
腹主动脉缩窄术后8周,β3AA输入组的心重／体重比较未免疫组没有明显改变(图17)。
5.1β3AA降低心衰大鼠对心肌缺血／再灌注损伤的敏感性
5.1β3AA改善心衰大鼠经历心肌缺血／再灌注损伤后的生存情况
β3AA输入心衰大鼠组经历心肌缺血／再灌注手术后,发现p3AA的输入可以明显延长大鼠心肌缺血／再灌注手术后的生存时间(图19)。
5.2β3AA使心衰大鼠经历心肌缺血／再灌注损伤后心梗面积减小
β3AA输入心衰大鼠组,经历心肌缺血／再灌注手术后,心梗面积较对照组明显减小(图20)。
5.3β3AA使心衰大鼠经历心肌缺血／再灌注损伤后心肌细胞凋亡减少
β3AA输入心衰大鼠组,经历心肌缺血／再灌注手术后,TUNEL染色阳性细胞明显减少,心肌组织caspase-3活性明显降低(图21、22)。
结论：
1.β3AA输入压力后负荷性心衰大鼠后,可以使其心功能恶化程度明显减轻、血清中BNP释放减少；
2.当心衰大鼠经历心肌缺血／再灌注损伤时,β3AA可以改善其生存情况,并可以减少心肌缺血／再灌注引起的心肌梗死面积和心肌细胞凋亡。
Background:
Chronic heart failure (CHF) is the final clinical manifestation of a variety of human heart diseases, including dilated cardiomyopathy (DCM), coronary artery disease (CAD), hypertensive heart disease (HHD) and rheumatic heart disease (RHD).In China, the prevalence of the heart failure is about 0.9% in adult population and with the aging of population the prevalence of CHF will increase substantially. Although current treatment against some pathogeniesis of heart failure can improve some symptoms, the 5-year survival rate of patients with heart failure remained less than 50%. It indicates that, there may be some other unknown factors involved in the occurrence and development process of heart failure.
The pathophysiologic mechanism of heart failure is extremely complex, there are several mechanisms involved in this syndrome. Resent yeas, some autoanbibodies against the G-protein-coupled receptors were detected in patients of heart failure, such as autoantibodies againstβ1 adrenoceptors/α1 adrenoceptors/M2-muscarinic receptors/angiotensinⅡ-1 receptors. Some studies showed that, autoanbibodies against the G-protein-coupled receptors have important pathophysiologic role in the occurrence and development process of heart failure.
P3-adrenoceptor is a member of G-protein-coupled receptors. In 2005, our research group found that there is a import autoantibody named autoantibodies against the second extracellular loop ofβ3-adrenoceptor (β3AA) existing in patients of heart failure, and the positive rate ofβ3AA in patients of heart failure is higher than that in normal people. Resent study from our research group showed, there is positive correlation between the titer ofβ3AA and cardiac left ventricular ejection fraction (EF) and negative correlation between the titer ofβ3AA and NYHA (New York Heart Association) function class in patients of heart failure, that is the cardiac function of patients withβ3AA positive is better than patient withβ3AA negative. This result indicates that theβ3AA may have some protect effect on heart and the function is different from the injury effect induced by autoantibodies againstβ1 adrenocepters andα1 adrenoceptors. However, because of the limitation of clinical research, the exact role ofβ3AA in the process of occurrence and development of heart failure is still unknown, so some animal experiments are needed to further confirm the real function ofβ3AA.
In addition, Transition from Chronic Compensated to Acute Decompensated Heart Failure is the main reason for hospitalization and death, and myocardial ischemia is one of the main triggers. Then whether the susceptibility to ischemic/reperfusion injury of heart failure rats can be decreased ifβ3AA long term exist in chronic heart failure rats.
Therefore, in this study we chosed chronic heart failure rats as starting point, givenβ3AA through intravenous, with cardiac function, heart weight/body weight ratio, serum brain natriuretic peptide and apoptosis of myocardial cells as indicators, from two aspects whetherβ3AA can reduce the degree of heart failure and whetherβ3AA can reduce the susceptibility to ischemic/reperfusion injury of heart failure rats to abserve whetherβ3AA have cardio protective effect to the heart failure rats.
Objectives:
1.To observe whetherβ3AA can reduce the occurrence of heart failure;
2. To observe whetherβ3AA have protect effect on the heart of rats with heart failure undergoing ischemia/reperfusion injury.
Methods:
1. The preparation ofβ3AA
20 healthy Wistar rats (male, initially weighing 140-160g,8 weeks old) were randomly divided into immunize group and control group. Rats were immunized with the second extracellular loops of theβ3AR (176aa～200aa,RVGADAEAQECHSNPRCCSFA S N M P) for 8 weeks. The level ofβ3AA in the sera was detected by ELISA which was improved by our research group.β3AA positive IgG and IgG withoutβ3AA were purified, quantified and qualified. SDS-PAGE was used to detect the purity.
2. Aortic banding surgery
Wistar rats(10 weeks old, weighing 200-220g) were chose and anaesthetized with 10% chloral hydrate solution (30 mg/kg) by peritoneal injection. Below the xiphoid lcm, a small horizon incision was made. Separate the abdomen aortic artery, band the artery with a needle (size7), then draw out the needle and form strictness (approximate 50%).At the same time inject Gentamicin and close the abdomen.
3. Preparation ofβ3AA passive immunized model
Administrate theβ3AA (2μg/g) which was purified to the rats through the tail vein 3 days after the aortic banding surgery. Every 10 days to repeat the immunity and the total period of immunization is 50 days.1 day before the immunization get the blood sample from the rat tail and separate the serum. SA-ELISA is used to detect the level ofβ3AA.In the control group, IgG withβ3AA negative (2μg/g) is administrated to the rats through the tail vein, the immunization process and detect methods is as same as the procedure described above.
4. Measurement of the in vivo cardiac function
MS2000, a computerized biological signaling system is used to monitor changes of the cardiac function parameters, such as left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and maximal rate of rise and decline of ventricular pressure (±dp/dtmax).
5.Measurement of the Heart weight to body weight ratio
Thoracotomy, quickly remove the heart and wash the remained blood in the cardiac cavities.Wash intracardiac hematoma s excised with all the cavities being washed clean with normal saline immediately. Remove the blood vessels and tissues around the heart; absorb the water around the heart by filter paper. Weigh the whole heart and calculate the ratio of heart weight to body weight.
6. Detect the level of Brain natriuretic peptide (BNP) in the sera
ELISA kit is used to detect the level of BNP in the sera; the specific operations are carried out in accordance with the instructions.
7. The ischemia/reperfusion surgery
Ligating the left anterior descending coronary artery for 30 minutes, then loose the ligature for reperfusion 3 hours;in the sham group, suture needle go through the bottom of the left anterior descending coronary artery, but not ligation, other operation is the same as described above.
8. Measurement of the myocardial infarct size
TTC coloration is used to detect the myocardial infarct size ofβ3AA passive immunized heart failure rats after ischemia/reperfusion surgery.
9. Caspase 3 ability assay
Caspase 3 ability assay kit is used to detect the ability of caspase 3 in rat cardiac tissues.
10. Detected Myocardial apoptosis by TdT-mediated dUTP nick end labeling (TUNEL)
In Situ Cell Death Detection Kit is used to detect the apoptosis of cardiomyocytes and calculate the ratio.
Results:
1. Choose the time of transferringβ3 AA to the heart failure rats
1.1 The cardiac function changed over time after the aortic banding surgery
As shown in Table 1,8 weeks after the aortic banding surgery, LVSP and±dp/dtmax were remarkably decreased, LVEDP was significantly increased, the cardiac function begins to decline; 12 weeks and 16 weeks after the aortic banding surgery, the cardiac function progressively deteriorate.
1.2β3AA in the sera of the rat changes over time after the aortic banding surgery
8 weeks after the aortic banding surgery,β3AA in the sera of rats was remarkably increased (0.31±0.02 vs.0.22±0.05,P<0.05); 12 weeks after the aortic banding surgery, the level ofβ3AA reached the peak; after the 12th week, the level ofβ3AA began to decline (Figure 1).
All the data above indicate that, the phrase of 0-8 weeks after the aortic banding surgery is a process of changes of cardiac function from normal to significantly decrease, that means this phrase is very important to the occurrence and development of heart failure; more important, during this phrase theβ3AA was produced and the protective effect ofβ3AA on the rat heart could be singly studied. So in this study, we chose the phrase of 0-8 weeks after the aortic banding surgery to study the protective effect ofβ3AA on the rat heart in the process of occurrence and development of heart failure.
2. Successfully prepared heart failure model
2.1 cardiac functions decreased in 8 weeks after the aortic banding surgery
8 weeks after the aortic banding surgery, LVSP and±dp/dtmax were remarkably decreased, LVEDP was significantly increased. The result showed that the heart failure model was successfully prepared (Figure 3-6).
2.2 The ratio of in 8 weeks after the aortic banding surgery
8 weeks after the aortic banding surgery, the ratio of heart weight to body weight significant increased compared with the sham group. Suggest that there is reconstruction of heart in heart failure rats (Figure 7).
All the data of result 2 showed that there was notably reconstruction of heart and decompensation of cardiac function in 8 weeks after the aortic banding surgery. So 0-8 weeks after the aortic banding surgery is a important phrase in the process of occurrence and development of heart failure.
3. Successfully establishedβ3AA passive immunized heart failure rats
3.1 Dose selection of P3AA passive immunization
0.5μg、1μg/g、2μg/gβ3AA was respectively used to passive immunize rats after the aortic banding surgery. First day after the immunization, collect the sera and detect the level ofβAA. After 2μg/gβ3AA passive immunized, the average OD value ofβ3AA is 0.67, matched to the clinical level. So we used 2μg/g as the final immunize doze (Figure 8).
3.2 Detection the IgG subclass ofβ3AA
Figure 9 showed that the main IgG subclass ofβ3AA in the immunized rats is IgG2b subclass, and little belongs to IgG2a subclass. Some studies showed that different IgG subclass have different metabolic half-life. IgG2b has a half-life of 6 days; the average half-life of serum IgG2a is about 8 days. So to determine the main IgG subclass of P3AA can provide a theoretical basis for the cycle of passive immunization (Figure 9).
3.3 Determine the elimination time ofβ3AA in the rats after the aortic banding surgery
First day after theβ3AA passive immunization, the level ofβ3AA in the sera significantly increased, the average OD value was 0.67, it increased by 0.35 compared with the control group. Approximately 10 days were needed to make sure the OD value ofβ3AA decline by half of the increasing value. So in this study 10 days were chose as the immunizing cycle (Figure 10).
3.4 The level of P3AA kept stable in the sera after passive immunized the aortic banding rats
2μg/g as the immunizing doze,10 days as the immunizing cycle,β3AA was used to immunize the aortic banding rats. Regular monitoring the level ofβ3AA in the sera showed that the level ofβ3AA could keep stable (Figure 11).
4. Cardiac protect effect of P3AA on the heart failure rats
4.1β3AA decrease the level of Brain natriuretic peptide (BNP) in the sera
Serum BNP levels and clinical severity of heart failure are parallel. Real-time monitoring the level of BNP showed that there was light increase in 2 weeks after theβ3AA was passive immunized to the post-aortic banding rats; 6 weeks later, the level of BNP in theβ3AA passive immunized post-aortic banding rats was lower than the control group in the same time;8 weeks later, the level of BNP was significantly lower. These results indicate that the degree of heart failure inβ3AA passive immunized group is more slight (Figure 12).
4.2β3AA improve the cardiac function of heart failure rats
8 weeks after the the aortic banding surgery, inβ3AA passive immunized group, reduction ranges of+dp/dtmax and-dp/dtmax were remarkably lower than that in control group which is P3AA negative (Figure 13～16).
4.3 P3AA have no effect on the ratio of heart weight to body weight
8 weeks after the the aortic banding surgery, in P3AA passive immunized group, heart weight to body weight had no significant changes to the control group (Figure 17).
5. Cardiac protect effect of P3AA to the rats with heart failure undergoing ischemia/reperfusion injury
5.1β3AA improve the survival situation of the rats with heart failure undergoing ischemia /reperfusion injury
After the rats passive immunized byβ3AA underwent the ischemia/reperfusion injury, the survival time could be obviously extended compared with the control group whichβ3AA not passive immunized (Figure 19).
5.2β3AA reduce the myocardial infarct size of the rats with heart failure undergoing ischemia/reperfusion injury
After the rats passive immunized by P3AA, the myocardial infarct size induced by ischemia/ reperfusion injury was smaller than that in the control group (Figure 20).
5.3 P3AA decrease the cardiomyocyte apoptosis of the rats with heart failure undergoing ischemia/reperfusion injury
After the rats passive immunized byβ3AA, TUNEL-positive cells and caspase-3 activity in myocardiocytes decreased significantly which were induced by ischemia/reperfusion injury (Figure 21,22).
Conclusions:
1.P3AA passive immnized the heart failure rats induced by pressrue overload can significantly reduce the degree of deterioration of cardiac function and reduce the level of BNP in the sera;
2.β3AA can improve the survival situation of the rats with heart failure undergoing ischemia/reperfusion injury and can also decrease the myocardial infarct size and cardiomyocyte apoptosis induced by ischemia/reperfusion injury.
引文
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