| BackgoundThe incidence of coronary heart disease continues to rise and presents a trend of getting younger over the designated period in recent years in China owing to the increase of the major risk factors of coronary heart disease,such as population aging, hypertension, diabetes, dyslipidemia, obesity, lack of physical activity and unreasonable diet According to "Report on Cardiovascular diseases in China", the mortality of coronary heart disease shows upward trend from 2002 to 2011,which was 95.97/100,000 in urban while 75.72/100,000 in rural in 2011. Interestingly, the mortality of coronary heart disease in urban is higher than in rural,and is higher in men than women. Acute myocardial infarction as the most dangerous type of coronary heart disease also showed the same trend in incidence. Then finding timely and effective treatments of acute myocardial infarction has become the key to reduce the mortality of coronary heart disease and improve prognosis.Generally believe that timely restoration of coronary blood flow is a fundamental measure to save the acute ischemic myocardium. Reperfusion including thrombolysis, percutaneous coronary intervention, coronary artery bypass graft surgery, which can reduce infarct size, improve cardiac function and reduce mortality, has become an important treatment of acute myocardial infarction.However, the rapid restoration of coronary blood supply in the same time can cause severe myocardial injury, ie, myocardial ischemia-reperfusion injury (MIRI), showed severe arrhythmia, endothelial dysfunction, myocardial stunning, cell death, reducing the benefits brought about by reperfusion of blood flow.Therefore, how to effectively mitigate such damage has been the biggest challenge in reperfusion therapy for maximum benefit in acute myocardial infarction treatment.Erythropoietin (EPO) is a glycoprotein hormone major secreted by kidney, which is widely used in clinical practice because of its function of promoting hematopoietic. Over the past decade a large number of experiments confirmed that EPO can also serve as a protective factor, act on different organs and tissues, including the nervous system, heart, kidney, etc. EPO exerts its protective role in organizations by reducing apoptosis, anti-oxidation, inhibiting inflammatory response and promoting angiogenesis.Recently EPO is widespread concerned for its good myocardial protection in myocardial infarction, heart failure and myocardial ischemia-reperfusion injury model. However, EPO will cause excessive stimulation of bone marrow hematopoietic system while it exerts a protective effect on organizations,which resulted in the increase of red blood cells and thrombocytosis,followed with increased blood viscosity and vascular resistance,increasing the risk of hypertension and thrombosis. The clinical practice of EPO is limited due to thrombosis, hypertension and other adverse reactions showed in several large clinical studies in recent years.Fortunately, recent studies have demonstrated that EPO exerted its tissue-protective effects and its erythropoietic functions by different EPO receptors (EPORs). The erythropoietic receptor of EPO is a homodimer of EPOR subunits with a high affinity to EPO, corresponding to low concentmouseions of circulating EPO. In contrast, the tissue-protective receptor of EPO is a heterodimer consisting of one EPOR subunit disulfide-linked to the β common receptor. Research have confirmed that EPO can be completely eliminated its protective effect on nervous system and heart tissue after βcR gene knockout.According to this feature, Brines screened 11 amino acids (sequence:QEQLERALNSS) from helix B segment hydrophilic surface which is not bind to the classic erythropoietin receptor,and synthesized helix B surface peptide (HBSP),followed research confirmed that it has a protective effect on the nerve tissue without the side effects of erythropoietin.HBSP draws extensive attention owing to its good prospects for clinical application, and its role in various pathophysiological conditions has been widely studied. One study have shown that HBSP can significantly reduce the injury and apoptosis of renal tubular epithelium cells of mice subjected to renal ischemia-reperfusion.Ueba H found HBSP can inhibit TNF-a-induced cardiomyocyte apoptosis. Ahmet I builded the rat model of heart failure after myocardial infarction by ligating the left anterior descending artery and found that HBSP can slow the process of ventricular remodeling and heart failure after myocardial infarction,improving heart function and reducing mortality amazingly. Another study showed that HBSP can reduce myocardial apoptosis, inflammation and infarct size in myocardial infarction model,which is related to the activation of ERK1/2 and STAT3 pathway. However,whether HBSP plays protective effect on tissues and cells after myocardial ischemia-reperfusion injury has not been reported,and the mechanism is not clear.Hence, this study was to establish a mouse model of myocardial ischemia-reperfusion injury in order to investigate the role and possible mechanisms of HBSP aftertreatment in MIRI, and these may provide new ideas and theoretical basis for the clinical application of HBSP.Objectives1.To establish the model of myocardial ischemia-reperfusion injury in mice to explore the effects of HBSP aftertreatment on myocardial ischemia-reperfusion injury.2. To explore the possible myocardial protection mechanisms of HBSP aftertreatment on myocardial ischemia-reperfusion injury.Methods1. Animals and groupsIn this experiment, the SPF ICR mice (male, weighing 25-30g) were randomly assigned to 4 group:(1) sham group:mice underwent threading without ligating, saline(10ml/kg) was administered intraperitoneal 40min after threading;(2) ischemia-reperfusion (MIRI) group:mice underwent 45 min of left anterior descending coronary artery occlusion, followed by 2 h of reperfusion,saline (10ml/ kg) was administered intraperitoneal 5min before reperfusion;(3) HBSP group:mice underwent 45 min of left anterior descending coronary artery occlusion, followed by 2 h of reperfusion,HBSP (90μg/kg, 10ml/kg) was administered intraperitoneal 5min before reperfusion;(4) HBSP+PD98059 group:mice underwent 45 min of left anterior descending coronary artery occlusion, followed by 2 h of reperfusion, PD98059 (lmg/kg) was administered intraperitoneal 20min before reperfusion while HBSP (90μg/kg, 10ml/kg) was administered intraperitoneal 5min before reperfusion.2. Establishment of MIRI model in miceThe mice were anesthetized with xylazine (5mg/kg) and ketamine (100mg/kg) by intraperitoneal injection before endotracheal intubation. Under aseptic conditions, the mice were placed in a supine position, and surface leads were placed subcutaneously. The electrocardiogram (ECG) monitoring was synchronously continued until the end of this experiment. After a left thoracotomy incision, a 8-0 nylon suture was placed around the left anterior descending coronary artery (LAD) from the tip of the left auricle and tied for 45 min to perform myocardial ischemia, which was then removed to initiate reperfusion for 120 min. Ischemia was confirmed by the appearance of blanching of the myocardium and continuous elevation of ST segment. Successful reperfusion was indicated by descending ST segment and a restoration of normal rubor. Sham operation mice underwent identical surgical procedures, but without ligation of the LAD.3. Assessment of infarct sizeAfter 2 h of reperfusion, the chest was reopened and the coronary artery was retightened in situ, Evans blue and 2,3,5-triphenyl tetrazolium chloride (TTC) staining were used to detect myocardial infarct size in mice. Myocardium outside the occluded vessel stained blue, whereas the area at risk(AAR) remained unstained. After stained with TTC,the infarcted area was white while the viable myocardium was stained brick red.Infarct size (IS) and AAR were measured manually with Image J,and the ratio of AAR/LV (left ventricle) and IS/AAR were calculated.4. Detection of serum LDH and CKAfter 2 h of reperfusion,0.5-1.0ml blood was collected transapical before the heart was removed from the chestBlood was centrifuged by 3000r/min for 10min,and the serum was stored at -20℃. Lactate dehydrogenase (LDH) and creatine kinase (CK) were detected by ELISA method.5. Determination of myocardial apoptosisAfter 2 h of reperfusion, the heart was removed from the chest,followed by fixing, embedding and slicing. Myocardial apoptosis was detected by TUNEL assay kit,then recorded the results with LSM710 laser scanning confocal microscope. The number of TUNEL-positive nuclei and the total number of nuclei was counted in 10 random high-power fields each mouse. The apoptotic rate was calculated as the percentage of TUNEL-positive cells out of the number of total cell nuclei.6. The detection of ERK1/2, phospho-ERK1/2 and cleaved caspase3After 2 h of reperfusion, the heart was removed from the chest,take the left ventricular myocardium following ligature.Heart tissue was ground into a fine powder using a mortar and pestle and then lysed with RIPA Lysis buffer.Then cell lysates were subjected to SDS-PAGE, and transferred to PVDF membranes.The blots were incubated overnight at 4℃ with a primary antibody against ERK1/2, phospho-ERKl/2 and cleaved caspase3 after blocking with 5% skim milk, followed by incubation for 1 h at 37℃ with a secondary antibody. Immunoreactive bands were visualized using ECL,and densitometric analysis was performed using Image pro plus 6.0 to assess the levels of protein expression.Results1. HBSP aftertreatment decreases infarct size of the heart of mouse subjected to MIRIAfter 2 h of reperfusion, area of myocardial infarction in HBSP group significantly smaller compared to MIRI group, which increased after administration of PD98059 [(47.1 ± 7.4)% in MIRI group vs. (24.2±3.8)% in HBSP group vs. (38.4 ± 2.2)% in HBSP+PD98059 group, P<0.05], explaining that HBSP aftertreatment can reduce infarct size in mice after MIRI and this protective effect can be partially inhibited by ERK1/2 specific inhibitor PD98059. No significant difference of AAR/LV was found among each groups (P> 0.05).2. HBSP aftertreatment reduces the release of LDH and CK after mouse subjected to MIRIAfter 2 h of reperfusion, LDH and CK in MIRI group significantly increased compared to sham group with P<0.05, and HBSP significantly decreased the serum levels of LDH and CK compared with MIRI group. However, this reduction of cardiac enzyme release by HBSP was largely alleviated by PD98059 with P<0.05.3. HBSP aftertreatment decreases cardiomyocyte apoptosis in mouse subjected to MIRIAfter 2 h of reperfusion, compared to sham group, myocardial apoptosis in MIRI group was significantly increased. Apoptosis of cardiocyte was significantly lower in HBSP group than in MIRI group, which increased after administration of PD98059[(5.5±1.1)% in sham group vs. (29.4±5.1)% in MIRI group vs. (15.0± 2.3)% in HBSP group vs. (21.8±2.5)% in HBSP+PD98059 group, P<0.05].4. HBSP aftertreatment decreases the expression of cleaved caspase3 in mouse subjected to MIRIAfter 2 h of reperfusion,the expression of cleaved caspase3 in MIRI group was significantly higher compared to sham group (1.75±0.21 vs.0.29±0.06, P<0.05), which decreased after administration of HBSP(0.60±0.12 vs.1.75±0.21, P<0.05). However, this effect was largely alleviated by PD98059(1.04±0.12 vs.0.60±0.12, P <0.05).5. HBSP aftertreatment increases the phosphorylation of ERK1/2 in mouse subjected to MIRIAfter 2 h of reperfusion, the phosphorylation of ERK1/2 in MIRI group increased compared to sham group with P<0.05. Compared to MIRI group, HBSP can upregulate the expression of phospho-ERKl/2 (PO.05). This increase phosphorylation of ERK1/2 was abolished by administration of PD98059 with P< 0.05.ConclusionsHBSP aftertreatment exerts a protective effect against MIRI in a mouse model by reducing the damage and apoptosis of cardiocyte hence decreasing area of myocardial infarction, which is related to the activation of ERKl/2 pathway. |
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