趋化因子受体5在大鼠心肌缺血再灌注损伤中的作用及机制研究
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摘要
冠心病(coronary heart disease)是一种常见,多发的内科疾病,其导致的急性心肌梗塞(acute myocardial infarction AMI)是在急诊抢救中非常常见的危急重症疾病,是人类常见的死亡原因之一。具不完全统计我国目前有近4000万的冠心病患者,而其中每年有近100余万人死于急性心肌梗死等冠心病的相关并发症。冠心病发病的主要机制就是心肌缺血,当心肌对氧的需求失去平衡时,心肌的结构和功能就会发生不同程度的损伤,因此目前对其治疗抢救成功有效的方法主要是迅速改善冠脉供血,恢复心肌的血液灌注,主要措施有严格的内科药物溶栓治疗,外科搭桥手术治疗,心导管介入手术治疗等,以此挽救“濒死”心肌,缩小心肌梗死面积,提高心脏功能,改善患者的预后。但有研究发现在心肌缺血复通实现再灌注后的一部分患者,却出现了缺血所导致的心肌代谢,心脏功能,心肌结构以及电生理等方面损伤加重的表现,人们将其称之为心肌缺血再灌注损伤(MIRI)。其临床症状主要为在缺血再灌注后的一段时间内,一部分病人出现恶性心律失常,休克,心衰甚至猝死等病情恶化的表现。因此,目前对冠心病及急性心肌梗塞防治的研究重点是在尽快恢复缺血心肌组织血流并改善其供血的情况下,尽量减轻对心肌的缺血再灌注损伤。
引起心肌缺血再灌注损伤的因素很多,目前公认度最高的是氧自由基损伤学说、钙超载学说、细胞凋亡等学说,但近年来研究显示,炎症在冠心病的发生与发展中的作用越来越明显。有研究表明缺血性心脏病人心肌细胞坏死情况和心脏功能损伤程度的大小及炎症因子水平的多少密切相关,可见各种炎症调节因子以及炎症反应在心肌缺血再灌注损伤中起着越来越重要的作用。
趋化因子(chemokines)是一类对白细胞有趋化作用的小分子分泌蛋白,其具有多种生物学功能,最主要的是趋化白细胞的定向移动,以参与炎症及免疫调节作用,而其该项功能的行使主要是通过与细胞表面特异性的受体相结合而实现的。缺血再灌注损伤是机体的一种应激反应,其病理生理过程就是一种炎症反应。而炎症过程就是趋化因子与其受体结合而募集各种炎症细胞通过黏附、穿越血管内皮细胞向炎症部位聚集而发挥生物学作用的。因此,通过干预趋化因子与其受体的结合,以减轻其对心肌缺血再灌注中炎症的趋化作用,减少缺血再灌注对心肌的损伤,可以起到保护心肌的作用。
研究目的：
本课题我们通过给大鼠建立在体的心肌缺血再灌注模型,用来观察大鼠急性心肌梗死实行再灌注后而引起的心肌缺血再灌注损伤的各种病理生理变化情况,然后使用特异性趋化因子受体5(CCR5)抗体和CCR5的配体RANTES作为激动剂在造模前予以干预,观察其对心肌细胞的结构,功能和心脏血流动力学的影响,及其通过抑制炎症反应的作用机制的研究,为有效防治心肌缺血再灌注损伤提供新的思路和方法。本研究分为二个部分进行：第一部分制作大鼠心肌缺血再灌注模型和缺血预处理模型,观察大鼠心脏血流动力学,心肌组织结构,心肌损伤标记物以及CCR5的变化情况,说明CCR5在心肌缺血再灌注中有着明显变化。第二部分通过使用CCR5抗体和激动剂干预后,观察缺血再灌注后大鼠心脏血流动力学,心肌组织结构,心肌损伤标记物,炎症相关因子TNF-α, NF-κB,ICAM-1的变化情况,以及CCR5的变化,说明CCR5抗体能够通过抑制各种炎症因子和趋化因子的表达,进而抑制心脏组织中的炎症反应,使得心肌缺血再灌注损伤的程度减轻,保护和改善心脏功能。
研究方法：
第一部分：
采用结扎(30min)和松开(2h)冠状动脉左前降支的方法制作大鼠心肌缺血再灌注模型,通过先行结扎左前降支5分钟后,松开5分钟共2个循环,然后再结扎10分钟松开10分钟共一个循环,最后结扎30分钟后实行再灌注2h的方法制作心肌缺血预处理模型。随机将36只健康雄性Wistar大鼠分成3组,分别为假手术组(12只),缺血再灌注组(12只),预缺血组(12只)。使用多道生理记录系统观察大鼠心脏的各项血流动力学指标：心率(heart rate HR)、左心室收缩压(left ventricular systolic pressure LVSP)、左心室舒张末压(left ventricular end-diastolic pressure LVEDP)、舒张期左心室内压下降的最大变化速率(-dp/dtmax)和收缩期左心室内压上升的最大变化速率(+dp/dtmax)在再灌注2h时的变化。血样本的采集：于再灌注末从颈总动脉处取血4毫升,静置30分钟,离心10分钟,取出上层血清在冰箱中保存(-70℃),用于LDH,AST,CK的检测。心肌标本的采集：在心底部离断心脏后,取出心脏,剪去左心房和右心房,并马上用冰生理盐水冲洗干净心脏上的血液后,用拭纸拭干后,用4%多聚甲醛将一部分心肌固定,并用乙醇梯度脱水,然后经透明处理后进行石蜡包埋,使用苏木素伊红(HE)染色法观察心肌组织病理结构。另一部分用冰0.9%氯化钠溶液冲洗干净后深低温保存后用于丙二醛(MDA),超氧化物歧化酶(SOD)的测定,以及使用WesternBlot测定趋化因子受体5(CCR5)蛋白的表达。
第二部分：
60只雄性Wistar大鼠随机分成5组。假手术组12只(SHAM),缺血再灌注组12只(I/R),预缺血组12只(I/R+Pre),CCR5抗体组12只(I/R+CCR5Ab), CCR5激动剂组12只(I/R+CCR5Ago)。在再灌注之前CCR5抗体组从颈外静脉注射CCR5抗体,激动剂组则注射RANTES进行处理。再灌注结束后通过多道生理记录系统,监测大鼠的心率、LVSP、LVEDP、±dp/dtmax等血流动力学指标。再灌注末再次结扎冠状动脉左前降支,用1%伊文蓝染色正常心肌组织,用1%TTC染色缺血心肌组织分别称重心肌缺血区重量(AR),左室壁总重量(LV)和梗死区心肌(IS)进行心肌梗死范围测定和MPO活力的测定。血样标本的采集：于再灌注末从颈总动脉处取血4毫升,静置30分钟后,离心10分钟,取出上层血清在冰箱中保存(-70℃),用于TNF-α和CK的检测,心肌标本的采集：在心底部离断心脏后,取出心脏,剪去左心房和右心房,并马上用冰生理盐水冲洗干净心脏上的血液后,用拭纸拭干后,用4%多聚甲醛将一部分心肌固定,并用乙醇梯度脱水,然后经透明处理后进行石蜡包埋,使用苏木素伊红(HE)染色法观察心肌组织病理结构及使用免疫组化方法检测黏附分子-1(ICAM-1)的表达。另一部分心肌用冰生理盐水冲洗干净,进行深低温保存后进行NF-κB的活性的测定以及CCR5蛋白的表达检测。各项指标的检测方法为：酶联免疫吸附测定法(ELISA)法测定血清TNF-α水平,使用免疫组织化学法测定心肌组织中ICAM-1的表达,电泳迁移率变动分析法(EMSA)法测定NF-kB的活性以及使用WesternBlot检测心肌细胞内CCR5表达。
统计方法：所有定量检测实验结果以均数±标准差(SD)表示。非正态分布的变量采用非参数"Mann-Whitney U"检验。各组间均数比较采用单因素方差分析方法。以P<0.05为差异有统计学意义。所有实验数据均采用SPSS13.0版软件包进行统计学处理。
研究结果
第一部分：
血流动力学的结果显示缺血再灌注组的心率、LVSP、±dp/dtmax等血流动力学指标,均较假手术组和缺血预处理组明显降低,同时左心室舒张期末压明显升高,而假手术组和缺血预处理组之间的差距不明显,说明大鼠心脏在缺血再灌注后血流动力学水平明显下降,而经过缺血预处理后能改善大鼠心脏的血流动力学水平。心肌损伤标记物CK, LDH,AST的活性以缺血再灌注组最高,其他2组的差别不明显,同时与假手术组相比,缺血再灌注组的SOD明显下降,MDA明显增高,说明缺血再灌注后会引起心肌的损伤。心肌组织的病理学检查显示假手术组心肌组织未见明显病理变化,预缺血组部分心肌细胞有轻度肿胀,心肌纤维变性和间质水肿轻微,而缺血再灌注组可见心肌细胞排列紊乱,肿胀明显,细胞核浓缩且形态不规则,心肌纤维肿胀,间质水肿,并有炎性细胞(以中性粒细胞为主)的浸润。说明缺血再灌注损伤会导致心肌组织结构的变化。WestemBlot测定的CCR5蛋白水平以缺血再灌注组的最高。说明经缺血再灌注处理后会导致心肌组织中CCR5表达水平的增高。
第二部分：
血流动力学指标中LVSP,±dp/dtmax由高到低依次是假手术组,CCR5抗体组,预缺血组,缺血再灌注组和CCR5激动剂组,说明CCR5抗体可以改善大鼠心脏的血流动力学水平。心梗范围测定结果显示经过缺血预处理和CCR5抗体处理后能够明显减少心肌梗死范围,而起到保护心肌的作用。大鼠心肌组织中MPO活性由高到低的循序是CCR5激动机组,缺血再灌注组,缺血预处理组,CCR5抗体组。说明CCR5抗体能够明显降低MPO的活性,保护心肌功能。心肌组织病理变化程度由重到轻的循序是缺血再灌注组,CCR5激动机组,缺血预处理组,CCR5抗体组和假手术组。说明CCR5抗体可以减轻心肌细胞的肿胀变性及炎性反应。免疫组化检测ICAM-1的表达由重到轻的循序是CCR5激动机组,缺血再灌注组,缺血预处理组,CCR5抗体组和假手术组。说明CCR5抗体能够减轻心肌细胞中ICAM-1的表达。用EMSA检测的NF-kB的活性由高到低的循序为,CCR5激动剂组,缺血再灌注组,缺血预处理组,CCR5抗体组和假手术组。说明缺血再灌注组和CCR5激动机组的NF-kB活性明显高于CCR5抗体组和预缺血组。’WesternBlot测定的CCR5蛋白水平显示CCR5抗体组的CCR5蛋白表达明显低于其他各组,说明CCR5抗体能够明显减少心肌组织中CCR5的表达,起到保护心肌的作用。
结论
1.本研究成功地建立了在体大鼠的心肌缺血再灌注模型,并通过该模型我
们证实了缺血再灌注可以导致心肌结构的破坏,血流动力学的变化,心肌损伤标记物的变化以及心脏功能损伤。
2.通过缺血预处理后可以一定程度的改善缺血再灌注对大鼠心肌的损伤。
3.CCR5抗体能够减轻缺血再灌注对大鼠心肌组织结构,心脏功能以及血流动力学的的损伤。
4.CCR5抗体保护心脏作用的机理是：通过抑制细胞因子’TNF-a的表达,从而使心肌细胞内NF-κB的活化减轻,进而抑制了粘附因子ICAM-1的表达,使心肌组织中炎症细胞(主要为中性粒细胞)的聚集减少,减轻了心肌的损伤。
Coronary heart disease (CHD) is a common and high prevalent medical disorder. Acute myocardial infarction (AMI), as the most severe complication of CHD, is not only a common emergency in intensive care unit (ICS), but also has a high mortality rate. According to a recent survey, China currently has nearly40million patients with coronary heart disease, of which there are nearly100million people a year died from AMI or CHD-related complications. The main mechanism for the pathogenesis of CHD is myocardial ischemia. Different degrees of damage would occur on myocardial structure and function when myocardial oxygen provision is in deficient. Therefore, the most effective treatment is to improve coronary blood supply and restore myocardial blood reperfusion. Treatments used include thrombolytic therapy, surgical bypass surgery, and cardiac catheterization interventional procedures, in order to save the "dying" myocardium, reduce myocardial infarct size, improve patient prognosis. However, some studies found that the ischemia induced cardiac injury represented by myocardial metabolism, cardiac function, myocardial structure and electrophysiological injury, becomes aggravated after recanalization. This is called myocardial ischemia-reperfusion injury. The clinical manifestations include malignant arrhythmia, shock, heart failure, and even sudden death etc during a period time after ischemia-reperfusion. Therefore, the main focus on CHD and AMI treatment is to restore and improve the myocardial blood supply to the ischemia area but minimize myocardial ischemia-reperfusion injury as much as possible at the same time.
Many theories have been proposed to be responsible for myocardial ischemia-reperfusion injury:oxygen free radical, calcium overload, apoptosis etc. Recent studies have shown that inflammation has played a more and more important role in the occurrence and development of CHD. It has been reported that the size of myocardial necrosis, the damage extent of myocardial function are high associated with the level of various inflammatory factors. The critical role of inflammation regulation factors and inflammatory responses in ischemia-reperfusion injury becomes more and more being recognized. Chemotactic factors (chemokines) is a group of small molecules which have various biological functions other than chemotactic effect on leukocytes. It mainly achieves its functions by binding to its receptors on specific cell surface. Ischemia-reperfusion injury is believed to be a stress reaction caused by inflammation. The whole process is characterized by accumulation of inflammatory cells in ischemic area and adhesion and migration of inflammatory cells through vascular walls via the binding of chemokines and their receptors. Therefore, the myocardial protection and prevention of ischemia-reperfusion injury can be achieved through the intervention on the binding of chemotactic factors and their receptors.
Objective:
In this study, we set up an in vivo rat model of myocardial ischemia-reperfusion. With this model, we have observed pathophysiological changes associated with ischemia reperfusion injury after AMI. Chemokine receptor5(CCR5) antibody and CCR5ligand RANTES were used to pre-treat the rat model to study their effects on myocardial structure, function and cardiac hemodynamics. This study would provide a new strategy and ideology to prevent myocardial ischemia-reperfusion injury via inhibition of inflammatory reaction. The study is divided into two parts:1) Set up ischemia preconditioning rat model and myocardial ischemia-reperfusion rat model, examine cardiac hemodynamics, myocardial tissue structure, markers of myocardial injury as well as CCR5changes in those models, and determine whether there was an increase of CCR5in ischemia-reperfusion rat.2) Examine the cardiac hemodynamics, myocardial tissue structure, markers of myocardial injury, inflammatory factors (TNF-a, NF-κB, ICAM-1) as well as CCR5changes after the interventions with CCR5antibody or CCR5receptor antagonist and determine whether CCR5antibody can protect heart function and prevent ischemia-reperfusion injury via suppression of chemokines expression and inflammatory cytokines.
Methods:
Part1:
Ischemia-reperfusion model was achieved via three cycles of occlussion and reperfusion. In the first cycle, left anterior descending artery was ligated for5min and then released for5min and repeat; the second cycle, the ligation lasted10min and release for10min; the third cycle, ligation lasted30min followed by2hours reperfusion.36male Wistar rats were randomly divided into three groups respectively: sham group (12), ischemia-reperfusion group (12), ischemic preconditioning group (12). Multi-channel physiological recording system was used to observe rat heart hemodynamic parameters:left ventricular systolic pressure (LVSP); left ventricular end-diastolic pressure (LVEDP); heart rate (HR),+dP/dtmax and-dP/dtmax during the2h of reperfusion. Blood samples collection:In the end of reperfusion,4ml of blood was drawn from the carotid artery. After standing for30minutes and centrifugation for10min, the upper layer of serum was collected and stored in the-70℃refrigerator for the detection of CK, AST, LDH. Myocardial specimen collection:In the end of the experiment the heart was isolated. The left atrium and right atrium as removed and the blood was washed away with ice saline rinse. It was then dried with wipes and fixed with4%paraformaldehyde, dehydrated with ethanol gradient dehydration, embedded with paraffin, use hematoxylin-eosin (HE) staining myocardial tissue pathological structures. Another part is rinsed with0.9%ice cold sodium chloride solution and cryopreserved for malondialdehyde (MDA), superoxide dismutase (SOD) determination, as well as to use WesternBlot Determination of chemokine receptor5(CCR5) protein expression
Part2:
60male Wistar rats were randomly divided into five groups, sham-operated group (SHAM,12), ischemia-reperfusion group (I/R,12), ischemic preconditioning group (I/R+Pre,12), the CCR5antibody group (I/R+CCR5Ab,12), of CCR5agonist group (I/R+CCR5Ago,12). CCR5antibody group before reperfusion from the external jugular vein injection CCR5antibody and agonist group were injected with RANTES for processing.Left ventricular systolic pressure (LVSP); left ventricular end-diastolic pressure (LVEDP); heart rate (HR),+dP/dtmax and-dP/dtmax) were measured through a short segment of saline-filled PE50tubing which was advanced to left ventricle through right carotid artery and connected to a multi-channel physiological monitoring system (LEAD2000, Sichuan, China) before LAD ligation or sham operation. Hemodynamic parameters were obtained immediately after2hours reperfusion. At the end of reperfusion, the LAD was ligated and normal myocardial tissue was marked with1%Evans blue but ischemic myocardial tissue was marked with1%TTC. Myocardial ischemic area (AR), left ventricular wall (LV) and infarcted myocardium (IS) were weighed with the help of staining and MPO activities in different areas were also measured. The collection of blood samples and myocardial samples were the same as part1. Detection method of the indicators are as follows:enzyme-linked immunosorbent assay (ELISA) method for measuring serum TNF-alpha levels, using immunehistochemical method determination of ICAM1expression in myocardial tissue, the electrophoretic mobility of variation analysis (EMSA)method for determining the activity of NF-kB and use the WesternBlot detection CCR5expression in the cells
Statistical methods:
Quantitative data were expressed as mean±standard deviation (SD). One-way ANOVA was used for the analysisof normal distribution. Non-normal distribution of variables was analyzed with non-parametric "Mann-Whitney U" test. P<0.05was considered statistically significant. SPSS (version13.0) was used for all statistical analysis.
Results
Part Ⅰ:
The hemodynamic results showed that LVSP,+dP/dtmax and-dP/dtmax were significantly decreased in I/R group than SHAM and I/R-Pre groups. Meanwhile, LVEDP was significantly higher in I/R group than SHAM and I/R-pre groups but no difference was observed between SHAM and I/R-Pre group. It suggested that cardiac hemodynamics were significantly damaged after ischemia-reperfusion, but were improved with pre-conditioning. Myocardial injury markers CK, LDH, AST activity were highest in ischemia-reperfusion group and not significant difference between other two groups suggesting that I/R would lead to myocardial injury. However, Myocardial tissue pathology examination showed no significant pathological changes in the myocardial tissue of the SHAM group. In ischemic preconditioning group, there were only mild cardiomyocytes swelling, mild myocardial fibrosis and interstitial edema in comparison, visible myocardial cell derangement, obvious swelling, nuclear condensation and irregular swelling of the myocardial fibers, interstitial edema, and inflammatory cells (mainly neutrophils) infiltration were observed in I/R group. It is therefore concluded that Ischemia-reperfusion injury will lead to changes in the structure of the myocardial tissue. Again, CCR5protein levels were highest in I/R group measured by western blot suggesting that CCR5expression was upregulated in the myocardial tissue after I/R injury.
Part2:
The level of hemodynamic parameters, LVSP and±dP/dtmax descended from group SHAM, I/R+CCR5Ab, I/R-Pre, I/R and I/R+CCR5Ago. It suggested that CCR5antibody can improve rat heart hemodynamic level. AR measurements showed ischemic preconditioning and CCR5antibody treatment significantly reduced myocardial infarct size so as to confer myocardial protection. Rat myocardial tissue MPO activity descended from I/R+CCR5Ago, I/R, I/R+Pre, I/R+CCR5Ab, SHAM suggesting that CCR5antibody can protect myocardial via suppression of MPO activity. The pathology assay showed that the severity of myocardial injury descended from I/R, I/R+CCR5Ago, I/R+Pre, I/R+CCR5Ab, SHAM showing that CCR5antibody can reduce the swelling of myocardial cells and degeneration and inflammatory response. The expression level of ICAM-1detected by immunohistochemical descended from I/R+CCR5Ago, I/R, I/R+Pre, I/R+CCR5Ab, SHAM suggesting CCR5antibody can reduce ICAM-1expression in the myocardial cells during I/R. The NF-kB activity detected by EMSA descended from descending I/R+CCR5Ago, I/R, I/R+Pre, I/R+CCR5Ab, SHAM demonstrating that NF-kB activity is much higher in I/R+CCR5Ago, I/R than other groups. The CCR5protein levels measured by Western blot was significantly reduced in I/R+CCR5Ab groop suggesting that CCR5antibody can inhibit CCR5protein expression to protect myocardial cells.
Conclusions
1. We have successfully established an I/R rat model through which we confirmed previous reports that I/R would induce myocardial structural damage, hemodynamic deterioration, myocardial injury and heart function impairment.2. I/R preconditioning or pretreatment can improve the I/R damage to myocardial to a certain degree.
3. CCR5antibody can reduce I/R induced cardiac tissue structure damage, heart function impairment and blood flow dynamics deterioration.
4. CCR5antibody protect heart and attenuate myocardial injury via the following mechanism/route:Inhibit the expression of cytokines to reduce the activation of NF-κB in myocardial cells; consequently the adhesion molecule ICAM-1expression was inhibited in the myocardial tissue and finally inflammatory cells (mainly neutrophils) aggregation was reduced.
引起心肌缺血再灌注损伤的因素很多,目前公认度最高的是氧自由基损伤学说、钙超载学说、细胞凋亡等学说,但近年来研究显示,炎症在冠心病的发生与发展中的作用越来越明显。有研究表明缺血性心脏病人心肌细胞坏死情况和心脏功能损伤程度的大小及炎症因子水平的多少密切相关,可见各种炎症调节因子以及炎症反应在心肌缺血再灌注损伤中起着越来越重要的作用。
趋化因子(chemokines)是一类对白细胞有趋化作用的小分子分泌蛋白,其具有多种生物学功能,最主要的是趋化白细胞的定向移动,以参与炎症及免疫调节作用,而其该项功能的行使主要是通过与细胞表面特异性的受体相结合而实现的。缺血再灌注损伤是机体的一种应激反应,其病理生理过程就是一种炎症反应。而炎症过程就是趋化因子与其受体结合而募集各种炎症细胞通过黏附、穿越血管内皮细胞向炎症部位聚集而发挥生物学作用的。因此,通过干预趋化因子与其受体的结合,以减轻其对心肌缺血再灌注中炎症的趋化作用,减少缺血再灌注对心肌的损伤,可以起到保护心肌的作用。
研究目的：
本课题我们通过给大鼠建立在体的心肌缺血再灌注模型,用来观察大鼠急性心肌梗死实行再灌注后而引起的心肌缺血再灌注损伤的各种病理生理变化情况,然后使用特异性趋化因子受体5(CCR5)抗体和CCR5的配体RANTES作为激动剂在造模前予以干预,观察其对心肌细胞的结构,功能和心脏血流动力学的影响,及其通过抑制炎症反应的作用机制的研究,为有效防治心肌缺血再灌注损伤提供新的思路和方法。本研究分为二个部分进行：第一部分制作大鼠心肌缺血再灌注模型和缺血预处理模型,观察大鼠心脏血流动力学,心肌组织结构,心肌损伤标记物以及CCR5的变化情况,说明CCR5在心肌缺血再灌注中有着明显变化。第二部分通过使用CCR5抗体和激动剂干预后,观察缺血再灌注后大鼠心脏血流动力学,心肌组织结构,心肌损伤标记物,炎症相关因子TNF-α, NF-κB,ICAM-1的变化情况,以及CCR5的变化,说明CCR5抗体能够通过抑制各种炎症因子和趋化因子的表达,进而抑制心脏组织中的炎症反应,使得心肌缺血再灌注损伤的程度减轻,保护和改善心脏功能。
研究方法：
第一部分：
采用结扎(30min)和松开(2h)冠状动脉左前降支的方法制作大鼠心肌缺血再灌注模型,通过先行结扎左前降支5分钟后,松开5分钟共2个循环,然后再结扎10分钟松开10分钟共一个循环,最后结扎30分钟后实行再灌注2h的方法制作心肌缺血预处理模型。随机将36只健康雄性Wistar大鼠分成3组,分别为假手术组(12只),缺血再灌注组(12只),预缺血组(12只)。使用多道生理记录系统观察大鼠心脏的各项血流动力学指标：心率(heart rate HR)、左心室收缩压(left ventricular systolic pressure LVSP)、左心室舒张末压(left ventricular end-diastolic pressure LVEDP)、舒张期左心室内压下降的最大变化速率(-dp/dtmax)和收缩期左心室内压上升的最大变化速率(+dp/dtmax)在再灌注2h时的变化。血样本的采集：于再灌注末从颈总动脉处取血4毫升,静置30分钟,离心10分钟,取出上层血清在冰箱中保存(-70℃),用于LDH,AST,CK的检测。心肌标本的采集：在心底部离断心脏后,取出心脏,剪去左心房和右心房,并马上用冰生理盐水冲洗干净心脏上的血液后,用拭纸拭干后,用4%多聚甲醛将一部分心肌固定,并用乙醇梯度脱水,然后经透明处理后进行石蜡包埋,使用苏木素伊红(HE)染色法观察心肌组织病理结构。另一部分用冰0.9%氯化钠溶液冲洗干净后深低温保存后用于丙二醛(MDA),超氧化物歧化酶(SOD)的测定,以及使用WesternBlot测定趋化因子受体5(CCR5)蛋白的表达。
第二部分：
60只雄性Wistar大鼠随机分成5组。假手术组12只(SHAM),缺血再灌注组12只(I/R),预缺血组12只(I/R+Pre),CCR5抗体组12只(I/R+CCR5Ab), CCR5激动剂组12只(I/R+CCR5Ago)。在再灌注之前CCR5抗体组从颈外静脉注射CCR5抗体,激动剂组则注射RANTES进行处理。再灌注结束后通过多道生理记录系统,监测大鼠的心率、LVSP、LVEDP、±dp/dtmax等血流动力学指标。再灌注末再次结扎冠状动脉左前降支,用1%伊文蓝染色正常心肌组织,用1%TTC染色缺血心肌组织分别称重心肌缺血区重量(AR),左室壁总重量(LV)和梗死区心肌(IS)进行心肌梗死范围测定和MPO活力的测定。血样标本的采集：于再灌注末从颈总动脉处取血4毫升,静置30分钟后,离心10分钟,取出上层血清在冰箱中保存(-70℃),用于TNF-α和CK的检测,心肌标本的采集：在心底部离断心脏后,取出心脏,剪去左心房和右心房,并马上用冰生理盐水冲洗干净心脏上的血液后,用拭纸拭干后,用4%多聚甲醛将一部分心肌固定,并用乙醇梯度脱水,然后经透明处理后进行石蜡包埋,使用苏木素伊红(HE)染色法观察心肌组织病理结构及使用免疫组化方法检测黏附分子-1(ICAM-1)的表达。另一部分心肌用冰生理盐水冲洗干净,进行深低温保存后进行NF-κB的活性的测定以及CCR5蛋白的表达检测。各项指标的检测方法为：酶联免疫吸附测定法(ELISA)法测定血清TNF-α水平,使用免疫组织化学法测定心肌组织中ICAM-1的表达,电泳迁移率变动分析法(EMSA)法测定NF-kB的活性以及使用WesternBlot检测心肌细胞内CCR5表达。
统计方法：所有定量检测实验结果以均数±标准差(SD)表示。非正态分布的变量采用非参数"Mann-Whitney U"检验。各组间均数比较采用单因素方差分析方法。以P<0.05为差异有统计学意义。所有实验数据均采用SPSS13.0版软件包进行统计学处理。
研究结果
第一部分：
血流动力学的结果显示缺血再灌注组的心率、LVSP、±dp/dtmax等血流动力学指标,均较假手术组和缺血预处理组明显降低,同时左心室舒张期末压明显升高,而假手术组和缺血预处理组之间的差距不明显,说明大鼠心脏在缺血再灌注后血流动力学水平明显下降,而经过缺血预处理后能改善大鼠心脏的血流动力学水平。心肌损伤标记物CK, LDH,AST的活性以缺血再灌注组最高,其他2组的差别不明显,同时与假手术组相比,缺血再灌注组的SOD明显下降,MDA明显增高,说明缺血再灌注后会引起心肌的损伤。心肌组织的病理学检查显示假手术组心肌组织未见明显病理变化,预缺血组部分心肌细胞有轻度肿胀,心肌纤维变性和间质水肿轻微,而缺血再灌注组可见心肌细胞排列紊乱,肿胀明显,细胞核浓缩且形态不规则,心肌纤维肿胀,间质水肿,并有炎性细胞(以中性粒细胞为主)的浸润。说明缺血再灌注损伤会导致心肌组织结构的变化。WestemBlot测定的CCR5蛋白水平以缺血再灌注组的最高。说明经缺血再灌注处理后会导致心肌组织中CCR5表达水平的增高。
第二部分：
血流动力学指标中LVSP,±dp/dtmax由高到低依次是假手术组,CCR5抗体组,预缺血组,缺血再灌注组和CCR5激动剂组,说明CCR5抗体可以改善大鼠心脏的血流动力学水平。心梗范围测定结果显示经过缺血预处理和CCR5抗体处理后能够明显减少心肌梗死范围,而起到保护心肌的作用。大鼠心肌组织中MPO活性由高到低的循序是CCR5激动机组,缺血再灌注组,缺血预处理组,CCR5抗体组。说明CCR5抗体能够明显降低MPO的活性,保护心肌功能。心肌组织病理变化程度由重到轻的循序是缺血再灌注组,CCR5激动机组,缺血预处理组,CCR5抗体组和假手术组。说明CCR5抗体可以减轻心肌细胞的肿胀变性及炎性反应。免疫组化检测ICAM-1的表达由重到轻的循序是CCR5激动机组,缺血再灌注组,缺血预处理组,CCR5抗体组和假手术组。说明CCR5抗体能够减轻心肌细胞中ICAM-1的表达。用EMSA检测的NF-kB的活性由高到低的循序为,CCR5激动剂组,缺血再灌注组,缺血预处理组,CCR5抗体组和假手术组。说明缺血再灌注组和CCR5激动机组的NF-kB活性明显高于CCR5抗体组和预缺血组。’WesternBlot测定的CCR5蛋白水平显示CCR5抗体组的CCR5蛋白表达明显低于其他各组,说明CCR5抗体能够明显减少心肌组织中CCR5的表达,起到保护心肌的作用。
结论
1.本研究成功地建立了在体大鼠的心肌缺血再灌注模型,并通过该模型我
们证实了缺血再灌注可以导致心肌结构的破坏,血流动力学的变化,心肌损伤标记物的变化以及心脏功能损伤。
2.通过缺血预处理后可以一定程度的改善缺血再灌注对大鼠心肌的损伤。
3.CCR5抗体能够减轻缺血再灌注对大鼠心肌组织结构,心脏功能以及血流动力学的的损伤。
4.CCR5抗体保护心脏作用的机理是：通过抑制细胞因子’TNF-a的表达,从而使心肌细胞内NF-κB的活化减轻,进而抑制了粘附因子ICAM-1的表达,使心肌组织中炎症细胞(主要为中性粒细胞)的聚集减少,减轻了心肌的损伤。
Coronary heart disease (CHD) is a common and high prevalent medical disorder. Acute myocardial infarction (AMI), as the most severe complication of CHD, is not only a common emergency in intensive care unit (ICS), but also has a high mortality rate. According to a recent survey, China currently has nearly40million patients with coronary heart disease, of which there are nearly100million people a year died from AMI or CHD-related complications. The main mechanism for the pathogenesis of CHD is myocardial ischemia. Different degrees of damage would occur on myocardial structure and function when myocardial oxygen provision is in deficient. Therefore, the most effective treatment is to improve coronary blood supply and restore myocardial blood reperfusion. Treatments used include thrombolytic therapy, surgical bypass surgery, and cardiac catheterization interventional procedures, in order to save the "dying" myocardium, reduce myocardial infarct size, improve patient prognosis. However, some studies found that the ischemia induced cardiac injury represented by myocardial metabolism, cardiac function, myocardial structure and electrophysiological injury, becomes aggravated after recanalization. This is called myocardial ischemia-reperfusion injury. The clinical manifestations include malignant arrhythmia, shock, heart failure, and even sudden death etc during a period time after ischemia-reperfusion. Therefore, the main focus on CHD and AMI treatment is to restore and improve the myocardial blood supply to the ischemia area but minimize myocardial ischemia-reperfusion injury as much as possible at the same time.
Many theories have been proposed to be responsible for myocardial ischemia-reperfusion injury:oxygen free radical, calcium overload, apoptosis etc. Recent studies have shown that inflammation has played a more and more important role in the occurrence and development of CHD. It has been reported that the size of myocardial necrosis, the damage extent of myocardial function are high associated with the level of various inflammatory factors. The critical role of inflammation regulation factors and inflammatory responses in ischemia-reperfusion injury becomes more and more being recognized. Chemotactic factors (chemokines) is a group of small molecules which have various biological functions other than chemotactic effect on leukocytes. It mainly achieves its functions by binding to its receptors on specific cell surface. Ischemia-reperfusion injury is believed to be a stress reaction caused by inflammation. The whole process is characterized by accumulation of inflammatory cells in ischemic area and adhesion and migration of inflammatory cells through vascular walls via the binding of chemokines and their receptors. Therefore, the myocardial protection and prevention of ischemia-reperfusion injury can be achieved through the intervention on the binding of chemotactic factors and their receptors.
Objective:
In this study, we set up an in vivo rat model of myocardial ischemia-reperfusion. With this model, we have observed pathophysiological changes associated with ischemia reperfusion injury after AMI. Chemokine receptor5(CCR5) antibody and CCR5ligand RANTES were used to pre-treat the rat model to study their effects on myocardial structure, function and cardiac hemodynamics. This study would provide a new strategy and ideology to prevent myocardial ischemia-reperfusion injury via inhibition of inflammatory reaction. The study is divided into two parts:1) Set up ischemia preconditioning rat model and myocardial ischemia-reperfusion rat model, examine cardiac hemodynamics, myocardial tissue structure, markers of myocardial injury as well as CCR5changes in those models, and determine whether there was an increase of CCR5in ischemia-reperfusion rat.2) Examine the cardiac hemodynamics, myocardial tissue structure, markers of myocardial injury, inflammatory factors (TNF-a, NF-κB, ICAM-1) as well as CCR5changes after the interventions with CCR5antibody or CCR5receptor antagonist and determine whether CCR5antibody can protect heart function and prevent ischemia-reperfusion injury via suppression of chemokines expression and inflammatory cytokines.
Methods:
Part1:
Ischemia-reperfusion model was achieved via three cycles of occlussion and reperfusion. In the first cycle, left anterior descending artery was ligated for5min and then released for5min and repeat; the second cycle, the ligation lasted10min and release for10min; the third cycle, ligation lasted30min followed by2hours reperfusion.36male Wistar rats were randomly divided into three groups respectively: sham group (12), ischemia-reperfusion group (12), ischemic preconditioning group (12). Multi-channel physiological recording system was used to observe rat heart hemodynamic parameters:left ventricular systolic pressure (LVSP); left ventricular end-diastolic pressure (LVEDP); heart rate (HR),+dP/dtmax and-dP/dtmax during the2h of reperfusion. Blood samples collection:In the end of reperfusion,4ml of blood was drawn from the carotid artery. After standing for30minutes and centrifugation for10min, the upper layer of serum was collected and stored in the-70℃refrigerator for the detection of CK, AST, LDH. Myocardial specimen collection:In the end of the experiment the heart was isolated. The left atrium and right atrium as removed and the blood was washed away with ice saline rinse. It was then dried with wipes and fixed with4%paraformaldehyde, dehydrated with ethanol gradient dehydration, embedded with paraffin, use hematoxylin-eosin (HE) staining myocardial tissue pathological structures. Another part is rinsed with0.9%ice cold sodium chloride solution and cryopreserved for malondialdehyde (MDA), superoxide dismutase (SOD) determination, as well as to use WesternBlot Determination of chemokine receptor5(CCR5) protein expression
Part2:
60male Wistar rats were randomly divided into five groups, sham-operated group (SHAM,12), ischemia-reperfusion group (I/R,12), ischemic preconditioning group (I/R+Pre,12), the CCR5antibody group (I/R+CCR5Ab,12), of CCR5agonist group (I/R+CCR5Ago,12). CCR5antibody group before reperfusion from the external jugular vein injection CCR5antibody and agonist group were injected with RANTES for processing.Left ventricular systolic pressure (LVSP); left ventricular end-diastolic pressure (LVEDP); heart rate (HR),+dP/dtmax and-dP/dtmax) were measured through a short segment of saline-filled PE50tubing which was advanced to left ventricle through right carotid artery and connected to a multi-channel physiological monitoring system (LEAD2000, Sichuan, China) before LAD ligation or sham operation. Hemodynamic parameters were obtained immediately after2hours reperfusion. At the end of reperfusion, the LAD was ligated and normal myocardial tissue was marked with1%Evans blue but ischemic myocardial tissue was marked with1%TTC. Myocardial ischemic area (AR), left ventricular wall (LV) and infarcted myocardium (IS) were weighed with the help of staining and MPO activities in different areas were also measured. The collection of blood samples and myocardial samples were the same as part1. Detection method of the indicators are as follows:enzyme-linked immunosorbent assay (ELISA) method for measuring serum TNF-alpha levels, using immunehistochemical method determination of ICAM1expression in myocardial tissue, the electrophoretic mobility of variation analysis (EMSA)method for determining the activity of NF-kB and use the WesternBlot detection CCR5expression in the cells
Statistical methods:
Quantitative data were expressed as mean±standard deviation (SD). One-way ANOVA was used for the analysisof normal distribution. Non-normal distribution of variables was analyzed with non-parametric "Mann-Whitney U" test. P<0.05was considered statistically significant. SPSS (version13.0) was used for all statistical analysis.
Results
Part Ⅰ:
The hemodynamic results showed that LVSP,+dP/dtmax and-dP/dtmax were significantly decreased in I/R group than SHAM and I/R-Pre groups. Meanwhile, LVEDP was significantly higher in I/R group than SHAM and I/R-pre groups but no difference was observed between SHAM and I/R-Pre group. It suggested that cardiac hemodynamics were significantly damaged after ischemia-reperfusion, but were improved with pre-conditioning. Myocardial injury markers CK, LDH, AST activity were highest in ischemia-reperfusion group and not significant difference between other two groups suggesting that I/R would lead to myocardial injury. However, Myocardial tissue pathology examination showed no significant pathological changes in the myocardial tissue of the SHAM group. In ischemic preconditioning group, there were only mild cardiomyocytes swelling, mild myocardial fibrosis and interstitial edema in comparison, visible myocardial cell derangement, obvious swelling, nuclear condensation and irregular swelling of the myocardial fibers, interstitial edema, and inflammatory cells (mainly neutrophils) infiltration were observed in I/R group. It is therefore concluded that Ischemia-reperfusion injury will lead to changes in the structure of the myocardial tissue. Again, CCR5protein levels were highest in I/R group measured by western blot suggesting that CCR5expression was upregulated in the myocardial tissue after I/R injury.
Part2:
The level of hemodynamic parameters, LVSP and±dP/dtmax descended from group SHAM, I/R+CCR5Ab, I/R-Pre, I/R and I/R+CCR5Ago. It suggested that CCR5antibody can improve rat heart hemodynamic level. AR measurements showed ischemic preconditioning and CCR5antibody treatment significantly reduced myocardial infarct size so as to confer myocardial protection. Rat myocardial tissue MPO activity descended from I/R+CCR5Ago, I/R, I/R+Pre, I/R+CCR5Ab, SHAM suggesting that CCR5antibody can protect myocardial via suppression of MPO activity. The pathology assay showed that the severity of myocardial injury descended from I/R, I/R+CCR5Ago, I/R+Pre, I/R+CCR5Ab, SHAM showing that CCR5antibody can reduce the swelling of myocardial cells and degeneration and inflammatory response. The expression level of ICAM-1detected by immunohistochemical descended from I/R+CCR5Ago, I/R, I/R+Pre, I/R+CCR5Ab, SHAM suggesting CCR5antibody can reduce ICAM-1expression in the myocardial cells during I/R. The NF-kB activity detected by EMSA descended from descending I/R+CCR5Ago, I/R, I/R+Pre, I/R+CCR5Ab, SHAM demonstrating that NF-kB activity is much higher in I/R+CCR5Ago, I/R than other groups. The CCR5protein levels measured by Western blot was significantly reduced in I/R+CCR5Ab groop suggesting that CCR5antibody can inhibit CCR5protein expression to protect myocardial cells.
Conclusions
1. We have successfully established an I/R rat model through which we confirmed previous reports that I/R would induce myocardial structural damage, hemodynamic deterioration, myocardial injury and heart function impairment.2. I/R preconditioning or pretreatment can improve the I/R damage to myocardial to a certain degree.
3. CCR5antibody can reduce I/R induced cardiac tissue structure damage, heart function impairment and blood flow dynamics deterioration.
4. CCR5antibody protect heart and attenuate myocardial injury via the following mechanism/route:Inhibit the expression of cytokines to reduce the activation of NF-κB in myocardial cells; consequently the adhesion molecule ICAM-1expression was inhibited in the myocardial tissue and finally inflammatory cells (mainly neutrophils) aggregation was reduced.
引文
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