Galectin-3对急性心梗后心衰发生的预测价值及阿托伐他汀对其干预作用

发布时间：2018-05-23 22:19

本文选题：Galectin-3 + 急性心肌梗死　；参考：《吉林大学》2013年博士论文

【摘要】：心衰是各种心血管疾病的终末阶段,具有较高的死亡率,如何减慢或延缓心室重塑的进展是心衰治疗领域中的研究热点。同时,人们一直在寻找新的生物学标志物,该标志物不仅要能够反应心衰本身的发生、进展,还要能更早的识别和诊断心衰患者,具有疾病监测及指导治疗的价值,这将有利于改善患者预后,降低死亡率,从而使患者更多获益。心肌重塑是心衰发生与发展重要的病理基础,是心功能由代偿向失代偿演变的关键步骤,BNP、NT-proBNP[2]能很好的反映心室重构的情况,因而被应用于诊断心衰、评估心衰治疗效果及判断心衰预后的指标。当BNP或NT-proBNP不高时，有助于排除左心收缩功能不全的诊断。但由于BNP和NT-proBNP水平受多种因素影响,如心动过速,右心负荷过重,心肌缺血,低氧血症,’肾功能不全,高龄,肝硬化,感染等,使其诊断心衰的价值有所下降。此外,BNP和NT-proBNP与心力衰竭疾病本身的进展无关,监测NT-proBNP并不能改善患者的预后[2]。因此,亟待我们寻找特异性的生化指标或与其配合使用来指导患者的治疗。目前,半乳凝素-3(Galectin-3)被认为是心肌细胞纤维化的新型生物标志物[3]。Galectin-3是由巨噬细胞激活分泌的,可以反映炎症和纤维化激活,具有诱导成纤维细胞增殖、Ⅰ型胶原沉积的作用。有研究表明[4],给予健康SD大鼠连续低剂量心包注射Galectin-3,4周后大鼠出现左心功能不全,进一步研究发现Galectin-3影响胶原蛋白的增生,表现为Ⅰ型胶原、Ⅲ型胶原增加的比例不协调,Ⅰ型胶原的增多率是Ⅲ型胶原增多率的3倍,而Ⅰ型胶原、Ⅲ型胶原的表达增加且增加比率不协调是导致左心功能不全发生的原因之一。另外,Galectin-3可通过促进心脏巨噬细胞浸润、刺激纤维母细胞活化进而参与心肌纤维化的形成,而巨噬细胞和纤维母细胞在心肌纤维化过程中具有重要作用。同样,急性心肌梗死(acute myocardial infarction, AMI)发生后,大量心肌细胞坏死,炎症因子大量释放,诱导巨噬细胞迁移、纤维母细胞活化、成纤维细胞增生,由此我们推断,急性心肌梗死发生后,可能存在Galectin-3的过度表达,导致不适当的心肌纤维化,进而引发心功能不全的出现。但目前对于Galectin-3在急性心梗发病过程中的作用研究较少,且受那些因素影响也尚不清楚。此外,Galectin-3还可以通过基质金属蛋白酶(matrix metalloproteinases, MMPs)和金属蛋白酶组织抑制剂(tissue inhibitors of metalloproteinases,TIMPs)抑制细胞外基质的降解,从而导致心肌成纤维细胞增生,胶原蛋白合成增加。有研究表明[5]，MMPs/TIMPs平衡是维持心肌成纤维细胞胶原合成与降解代谢平衡的关键。如果MMPs的活性过度增加或者MMPs/TIMPs(比例)严重失调,将导致心室重构、心肌纤维化的发生[6]。心力衰竭时,心肌MMPs/TIMPs的平衡状态被破坏,不仅使MMPs持续激活,还可引起细胞外基质(Extracellular matrixc, ECM)的蛋白水解和相应的心肌重构。有实验证明,除MMPs/TIMPs比例失调会导致心肌重构外,MMPs还可通过其他途径导致心肌重构的发生。如心肌细胞受损后,ECM退化,导致心肌排列紊乱,收缩功能异常,为进一步的细胞外基质重构奠定了基础。而且在ECM退化的刺激下,机体通过增加ECM的合成以期维持平衡,但这种增生往往是过度的,组成成分也是不恰当的。胶原和纤连蛋白等ECM组分在数量上的增长使心肌僵硬,收缩无力,舒张障碍。此外,MMPs通过趋化作用使纤维细胞进入其作用后的区域,介导TNF-α、TGF-β等细胞因子活化,刺激心肌纤维细胞大量增生,启动胶原及纤黏连蛋白基因的转录。上述细胞因子可促进胶原合成、破坏心肌细胞,这将导致MMPs表达增加,由此形成恶性循环,加速心室重构。因此,MMPs及MMPs/TIMPs平衡对心室重塑的发展有着重要的影响。Galectin-3既能影响MMPs的活性,也能引起TIMPs功能改变,那么Galectin-3对MMPs/TIMPs是如何影响的,这也是本研究关注的另一焦点。另外,还有实验研究发现,在心力衰竭治疗中联合应用血管紧张素转换酶抑制剂和MMPs抑制剂可减轻左室扩张而不引起心肌硬化,说明有效控制MMPs的功能,是防治心肌重塑的手段之一。Hayashidani等[7]发现氟伐他汀可抑制左室胶原酶、MMP-2、MMP-13的表达,减少心脏胶原容积分数,减轻心梗后大鼠心肌肥厚和间质纤维化,说明他汀可能会引起MMPs/TIMPs比率的变化,进而影响心肌纤维化。这也是本研究要探讨的另外一个重要问题。本研究中,首先选取106例急性心梗患者为研究对象,常规接受PCI治疗。根据患者的症状、体征及NT-proBNP、EF值等结果,诊断其心功能分级,然后分析心功能与Galectin-3的关系。其次,通过结扎雌性大鼠冠状动脉左前降支(LAD)建立心梗后心衰动物模型,4w后灌胃大鼠阿托伐他汀,设立假手术组,模型对照组及药物干预组。再连续给药4w后,进行如下检测：ELISA法检测血清中BNP、Galectin-3水平；HE染色和Masson染色观察心肌病理形态学；Western-blot检测MMP-2和TIMP-2的表达；明胶酶谱法检测MMP-2的活性；实时定量PCR检测胶原蛋白Ⅰ、胶原蛋白Ⅲ、MMP-2和TIMP-2的mRNA表达。最后,体外分离培养大鼠心肌成纤维细胞,以AngⅡ诱导心肌成纤维细胞增殖模型,并给予外源性阿托伐他汀进行干预,进行如下检测：MTT法检测细胞增殖；ELISA法检测血清中羟脯氨酸、PⅠCP和PⅢNP含量；实时定量PCR检测胶原蛋白Ⅰ、胶原蛋白Ⅲ、MMP-2和TIMP-2的mRNA表达。本研究结果显示：①随着患者心功能的不断恶化,Galectin-3呈逐渐升高的趋势,应用阿托伐他汀干预4周后,Galectin-3水平下降。②成功建立心梗后心衰的大鼠模型。与假手术组比较,模型对照组大鼠血请BNP水平增高；心肌细胞排列紊乱,大量纤维组织增生；与模型对照组比较,阿托伐他汀干预组大鼠血清BNP、Galectin-3水平下降；心肌细胞排列较整齐,纤维增生程度减弱；MMP-2活性下降；胶原蛋白Ⅰ、胶原蛋白Ⅲ、MMP-2和TIMP-2的mRNA表达有所下降。进一步研究发现,经阿托伐他汀干预后,MMP-2/TIMP-2比率升高,心肌纤维化程度降低。③体外成功培养大鼠心肌成纤维细胞,并建立AngⅡ诱导的大鼠心肌成纤维细胞增殖模型。与模型组相比,AngⅡ组心肌成纤维细胞分泌脯氨酸、PⅠCP和PⅢNP含量增加,胶原蛋白Ⅰ、胶原蛋白Ⅲ、MMP-2和TIMP-2的mRNA表达增多,且经阿托伐他汀干预后,MMP-2/TIMP-2比率升高。综上,Galectin-3可能预测急性心梗后心衰的发生,应用阿托伐他汀后,Galectin-3水平下降,心肌纤维化程度降低,说明Galectin-3可用于或与其他指标联合用于评价心功能：初步探讨了阿托伐他汀抑制心肌纤维化的机制可能为：①抑制Galectin-3表达,导致MMP-2、TIMP-2的功能及表达量降低,减轻心肌纤维化。②抑制心肌成纤维细胞合成胶原蛋白Ⅰ和胶原蛋白Ⅲ的合成,减轻心肌纤维化。③应用阿托伐他汀后，，MMP-2/TIMP-2的表达比率上调,使胶原的降解大于合成,进而减轻心肌纤维化。
[Abstract]:Heart failure is the final stage of all kinds of cardiovascular diseases, with high mortality. How to slow down or delay the progress of ventricular remodeling is a hot spot in the field of heart failure. At the same time, people have been looking for new biological markers, which not only reflect the occurrence and progress of heart failure itself, but also be able to identify and diagnose earlier. Patients with heart failure have the value of monitoring and guiding treatment. This will help improve the prognosis and reduce the mortality rate, so that patients can benefit more.
Myocardial remodeling is an important pathological basis for the occurrence and development of heart failure. It is the key step of cardiac function from compensatory to decompensation. BNP, NT-proBNP[2] can reflect the condition of ventricular remodeling, so it is used to diagnose heart failure, evaluate the effect of heart failure and determine the prognosis of heart failure. When BNP or NT-proBNP is not high, it is helpful to exclude BNP and NT-proBNP levels are affected by many factors, such as tachycardia, excessive right heart load, myocardial ischemia, hypoxemia, kidney dysfunction, age, cirrhosis, infection and so on, and the value of the diagnosis of heart failure has been reduced. In addition, BNP and NT-proBNP are not related to the progress of heart failure itself, Monitoring NT-proBNP can not improve the prognosis of [2].. Therefore, we need to find specific biochemical indicators or cooperate with them to guide the treatment of patients.
At present, galactoin -3 (Galectin-3) is considered to be a new biomarker of myocardial fibrosis, [3].Galectin-3 is activated by macrophages, which can reflect the activation of inflammation and fibrosis, which can induce fibroblast proliferation and type I collagen deposition. A study showed that [4] was given to healthy SD rats for a continuous low dose of pericardium. After Galectin-3,4 weeks, left ventricular dysfunction was found in rats. Further study found that Galectin-3 affects collagen proliferation, which is type I collagen, the proportion of type III collagen is not coordinated, and the increase rate of type I collagen is 3 times that of type III collagen increase, while type I collagen and type III collagen increase and increase the ratio is incongruous One of the causes of left ventricular dysfunction is that Galectin-3 can promote the formation of myocardial fibrosis by stimulating macrophage infiltration and stimulating fibroblast activation, and macrophages and fibroblasts play an important role in the process of myocardial fibrosis. The same, acute myocardial infarction (acute myocardial infarction). After the occurrence of AMI) a large number of myocardial cells were necrotic and inflammatory factors were released, inducing macrophage migration, fibroblast activation, fibroblast proliferation. Therefore, we infer that after acute myocardial infarction, there may be overexpression of Galectin-3, causing discomfort of myocardial fibrosis and leading to the appearance of cardiac dysfunction. There is less study on the role of Galectin-3 in the pathogenesis of acute myocardial infarction, and the influence of those factors is not yet clear.
In addition, Galectin-3 can also inhibit the degradation of extracellular matrix through the matrix metalloproteinases (matrix metalloproteinases, MMPs) and metalloproteinase tissue inhibitors (tissue inhibitors of metalloproteinases, TIMPs), resulting in the proliferation of myocardial fibroblasts and the increase of collagen synthesis. There is a study showing [5], MMPs/TIMPs equilibrium. It is the key to maintain the metabolic balance of collagen synthesis and degradation in cardiac fibroblasts. If excessive increase in MMPs activity or severe imbalance of MMPs/TIMPs (proportion), it will lead to ventricular remodeling, and when [6]. heart failure occurs in myocardial fibrosis, the balance of MMPs/TIMPs in the myocardium is broken, which not only causes the MMPs to continue to activate, but also causes the extracellular matrix. The protein hydrolysis of Extracellular matrixc (ECM) and the corresponding myocardial remodeling. It has been proved that MMPs can also lead to myocardial remodeling in other ways except for the imbalance of MMPs/TIMPs, such as the degeneration of the myocardium, the degeneration of ECM, the disorder of the myocardium, the abnormal contractile function, and the further extracellular matrix. Mass remodeling lays the foundation. And under the stimulation of ECM degradation, the body maintains a balance by increasing the synthesis of ECM, but the proliferation is often excessive and the components are not appropriate. The increase in ECM components, such as collagen and fibronectin, makes myocardial stiffness, contraction weakness, and diastolic dysfunction. In addition, MMPs is made by chemotaxis. Fibrous cells enter the area after its action, mediate the activation of TNF- a, TGF- beta and other cytokines, stimulate the proliferation of myocardial fibroblasts and activate the transcription of collagen and fibronectin gene. These cytokines can promote collagen synthesis and destroy cardiac myocytes, which will lead to an increase in the MMPs table, thus forming a vicious cycle and accelerating ventricular remodeling. Therefore, MMPs and MMPs/TIMPs balance have an important influence on the development of ventricular remodeling..Galectin-3 can affect both the activity of MMPs and the change of TIMPs function, then how Galectin-3 affects MMPs/TIMPs, which is another focus of this study.
In addition, experimental studies have found that combined use of angiotensin converting enzyme inhibitors and MMPs inhibitors in the treatment of heart failure can reduce left ventricular dilatation without causing myocardial sclerosis, indicating that effective control of MMPs function is one of the means to prevent myocardial remodeling, such as.Hayashidani, [7] found that fluvastatin can inhibit left ventricular collagenase, MMP-2, M. The expression of MP-13 reduces cardiac collagen volume fraction, reduces myocardial hypertrophy and interstitial fibrosis in rats after myocardial infarction, indicating that statins may cause changes in the ratio of MMPs/TIMPs to myocardial fibrosis, which is also an important issue to be discussed in this study.
In this study, first of all, 106 patients with acute myocardial infarction were selected as the subjects and received PCI treatment routinely. According to the symptoms, signs and NT-proBNP, EF values, the classification of cardiac function was diagnosed and the relationship between cardiac function and Galectin-3 was analyzed. Secondly, by ligating the left anterior descending branch of the coronary artery (LAD) of the female rats, the heart failure animals were established. Model, 4W after gastric perfusion of atorvastatin, set up a sham operation group, model control group and drug intervention group. After continuous administration of 4W, the following tests were carried out: ELISA method was used to detect the serum BNP, Galectin-3 level, HE staining and Masson staining to observe the myocardial pathomorphology; Western-blot detected the expression of MMP-2 and TIMP-2; gelatin zymogram detection MMP-2 activity; real-time quantitative PCR was used to detect the mRNA expression of collagen I, collagen III, MMP-2 and TIMP-2. Finally, the rat myocardial fibroblasts were isolated and cultured in vitro, and the proliferation model of myocardial fibroblasts was induced by Ang II, and the exogenous atorvastatin was given the following detection: MTT method was used to detect cell proliferation; ELISA Methods the contents of hydroxyproline, P I CP and P III NP in serum were detected. The mRNA expression of collagen I, collagen III, MMP-2 and TIMP-2 was detected by real-time quantitative PCR.
The results of this study showed that: (1) with the continuous deterioration of cardiac function, Galectin-3 showed a tendency to increase gradually. After 4 weeks of atorvastatin intervention, the level of Galectin-3 decreased. (2) the rat model of heart failure after myocardial infarction was successfully established. Compared with the sham operation group, the level of BNP in the model control group was higher; the myocardial cells were arranged in disorder, Compared with the model control group, the serum BNP, Galectin-3 level of the rats in the Atorvastatin intervention group decreased, the myocardial cells were arranged neatly, the degree of fibrous proliferation was weakened, the activity of MMP-2 decreased, and collagen I, collagen III, MMP-2 and TIMP-2 decreased. Further studies found atropine. The MMP-2/TIMP-2 ratio increased and the degree of myocardial fibrosis decreased. (3) the rat myocardial fibroblasts were successfully cultured in vitro, and the rat fibroblast proliferation model induced by Ang II was established. Compared with the model group, the content of proline, P I CP and P III NP in Ang II group was increased, collagen I, collagen eggs The expression of mRNA in white III, MMP-2 and TIMP-2 increased, and the ratio of MMP-2/TIMP-2 increased after atorvastatin intervention.
To sum up, Galectin-3 may predict the occurrence of heart failure after acute myocardial infarction. After the use of atorvastatin, the level of Galectin-3 decreased and the degree of myocardial fibrosis decreased, indicating that Galectin-3 can be used to evaluate cardiac function combined with other indicators: the mechanism of atorvastatin to inhibit myocardial fibrosis may be: (1) inhibition of Galectin- The expression of 3 led to the decrease of function and expression of MMP-2, TIMP-2 and the reduction of myocardial fibrosis. 2. Inhibit the synthesis of collagen I and collagen III of myocardial fibroblasts and reduce myocardial fibrosis. (3) after atorvastatin, the expression ratio of MMP-2/TIMP-2 was up-regulated, the degradation of collagen was greater than that of synthesis, and then myocardial fibrosis was alleviated.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R541.61

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