| Background:World Health Organization (WHO)"2012World Health Statistics Report" showed that ischemic heart disease has become a hazard to human health,"the number one killer". Coronary heart disease (CHD) was one of the major diseases. Early recovery after coronary artery occlusion of ischemic area blood reperfusion is the most effective mean to treat the myocardial ischemia injury nowadays, However, evidences suggested that reperfusion can cause additional cell death, which partially offset the favorable effects of coronary artery to restore the blood supply to the heart, so the myocardial tissue injury caused by ischemia and reperfusion was defined as myocardial ischemia/reperfusion injury (MIRI). How to effectively reduce MIRI to minimize the loss of myocardial cells has become an important research topic for the treatment of ischemic heart disease.In1986Murry et al. first proposed the ischemic preconditioning (IPC), a mechanical myocardial protective mechanism, which was to conduct several briefly interrupted ischemia/reperfusion cycles in advance, can significantly reduce the infarct size and ischemia/reperfusion injury. Then Zhao et al. found another way of protecting the ischemia myocardium:ischemic postconditioning. But due to the lack of the established standards in pretreatment and posttreatment concerning implementation number, frequency and so on, the clinical application of these measures has certain limitation. Therefore, to clarify the MIRI protection mechanism of these mechanical regulations and to explore specific molecular mechanism as well as functional target spot appears very important. Our and other research teams found that ischemic postconditioning myocardial protection may be associated with promoting the overexpression of HIF-la. Thus we speculated that if to give HIF-la or increase its expression directly may also has the myocardial protective effect. But there have no related reports recently.Tissue ischemia or hypoxia can launch a series of response, while Hypoxia-inducible factor-1(HIF-1) was involved in ischemic responses as a critical transcription factor that mediated adaptation to hypoxia by regulating nearly200genes transcriptional activation. HIF-1contained the oxygen-dependent a-subunit (120kDa) as well as a constitutively expressed β subunit (91-94kDa). Under normoxic conditions, HIF-1was subsequently degradated by VHL (von Hippel-Lindau) for the ubiquitin and proteasome to inactive followed by prolyl hydroxylase (PHD) hydroxylation; In hypoxia, PHD activity was inhibited, HIF-1dimerization and translocation into the nucleus, binding to target genes by hypoxia response element promoter sequences to active a series of hypoxic stress protein expression, thereby participating metabolic processes of the body, such as glucose metabolism, cell proliferation, angiogenesis, etc.. However, whether the HIF-1α had myocardial protection in the process of MIRI and how to act remains unclear.Because of there is no commercial HIF-1α currently can be bought, meanwhile endogenous HIF-1α was easy to degradate and inactivate extremely under normoxic conditions, HIF-1α degradation inhibition may be the primary path to study it. The2-methyl-ethyl-2-acyl glycine (Dimethyloxalyglycine, DMOG), as an inhibitor of proline hydroxylase, can stabilize HIF-1α expression and inhibit its degradation under normoxic conditions, which has become the ideal HIF-1α activator until now. Therefore in this study, in rat ischemia/reperfusion model, we administered HIF-1α activator DMOG and inhibitor YC-1to observe whether the regulation of HIF-1α expression affected myocardial injury degree after ischemia/reperfusion, and to discuss the underlying pathway, in order to provide experimental consideration and theoretical foundation for HIF-α as a new target for preventing the MIRI.Objective:1. To identify whether the HIF-1α expression upregulation had protective effect on myocardial MI/RI.2. To investigate probable pathway how HIF-1α to protect myocardium.Methods:1. The animal model and experiment guoup:Healthy male Wistar rats were randomly divided into five groups:(1) Ordinary pseudo-operation group (Sham, n=10):threading under the left anterior descending coronary artery (LAD), without ligation;(2) HIF-1α activator+sham group (DMOG+Sham, n=10):intraperitoneal injection of HIF-la activator DMOG (40mg/kg)24h before ischemia, LAD just to thread but not ligate;(3) Myocardial ischemia/reperfusion group (MI/R, n=40):LAD ligation caused myocardial ischemia30min, reperfusion by Oh,3h,6h,9h respectively;(4) HIF-1α activator+myocardial ischemia/reperfusion group (DMOG+MI/R, n=40):injection intraperitoneally of HIF-la activator DMOG24h before ischemia (40mg/kg), following same to (3); (5) HIF-1α inhibitor+myocardial ischemia/reperfusion group (YC-1+MI/R, n=40):femoral vein injection of HIF-1α inhibitor YC-1(2mg/kg)30min before ischemia, following same to (3).2. Myocardial HIF-1α expression level test:to detect the HIF-1α mRNA expression level of myocardial tissue at different time point in rats using real-time PCR.3. Myocardial histopathology test:HE staining.4. Myocardial infarction area measurement:Evan’s blue and TTC double staining.5. Serum myocardial enzyme marker levels detection:Fully automatic biochemical analyzer rate method.6. Myocardial cell apoptosis detection:Using TUNEL staining and Caspase-3activity assay.Results:1. The establishment of myocardial ischemia/reperfusion model was successful:ECGⅡ showed ST-segment arched elevation (≥0.15mV) after the left anterior descending coronary artery ligation, and when reperfusion ST segment gradually returned to baseline level (Fig.1), which showed myocardial I/R model was established successfully.2. HIF-1α activator DMOG and inhibitor YC-1effect on the HIF-1α expression of the myocardial tissue:Real-time PCR detection results showed, HIF-1α mRNA expression in simple MI/R group presented a trend of gradually reducing in the0-9h reperfusion, at Oh reperfusion, MI/R group was1.3times than the Sham group (P<0.05); HIF-1α activator DMOG upregulated the HIF-1α expression significantly, and up to peak at3h reperfusion, which was2.1times than the MI/R group(P<0.05); However, HIF-1α inhibitors YC-1reduced HIF-a mRNA expression compared with the MI/R group at Oh,3h,6h,9h (P<0.05, Fig.2). So in our study we choosed the3h point reperfusion to detect related indicators.3. The determination results of myocardial tissue morphology:HE dyeing showed, compared with the MI/R group, in DMOG+MI/R group the myocardial fibers were complete, arrange relative orderliness, coloring wasrelatively uniform, interstitial mild edema, inflammatory cells infiltration; but in YC-1+MI/R group, myocardial fiber fracture, disordered arrangement, myocardial cell necrosis, boundary was not clear, the nucleus concentration, fracture, severe inflammatory cells infiltration(Fig.3).4. The determination results of myocardial infarction area:There were no significant differences of the percentage of at risk area accounts for left ventricular area (AAR/LV ratio) between all groups (P>0.05). This showed equal roughly ischemia degree when the ischemia/reperfusion model was established, which excluded the error caused by operation. Compared with the Sham group, the myocardial infarction area significantly increased in MI/R group [(48.76±2.50)%vs.(4.23±1.84)%, P<0.01]; While HIF-la activator DMOG reduced significantly the myocardial infarction area compared with the MI/R group [(28.32±2.25)%vs.(4.23±1.84)%, P<0.05](Fig.4-1,2).5. The determination results of Serum myocardial enzyme marker:The simple MI/R group CK activity was up to0.85±0.15U/ml, which significantly higher than0.12±0.08U/ml of Sham group (P<0.01); however HIF-1α activator DMOG partially reversed the raised LDH level caused by simple MI/R, LDH reduced to0.46±0.03U/ml, compared with the MI/R group, P<0.05; In addition, HIF-1α activator also partially reversed the raised LDH level caused by simple MI/R (0.98±0.11U/ml vs.2.17±0.16U/ml, P<0.05)(Fig.5).6. The determination results of myocardial cell apoptosis:TUNEL staining showed, in simple Ml/R group, myocardial apoptosis index was (8.23±2.45)%, HIF-1α activator DMOG made this index down to (2.68±0.84)%(P<0.01); whereas HIF-1α inhibitor YC-1made myocardial apoptosis index rised to(27.2±2.22)%, compared with the MI/R group, P<0.01)(Fig.6). In simple MI/R group, OD value of the myocardial tissue Caspase-3activity is0.19±0.03, which was higher significantly than0.09±0.02in Sham group (P<0.05), while HIF-1α activator DMOG reverse obviously increased activity of Caspase-3due to MI/R (OD value was0.11±0.01), compared with the simple MI/R group, P<0.05(Fig.7).Conclusion:The upregulation of HIF-1α using DMOG significantly alleviated rat ischemia/reperfusion injury and reduced the number of cardiac myocyte apoptosis, suggesting that the cardioprotective role of HIF-1α may be related to inhibiting the myocardial cell apoptosis. |
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