| AIM:To investigate neutrophils infiltration and the expression of growth differentiation factor-15(GDF-15) in rat myocardial anatomic no-reflow phenomenon.METHODS:144male Wistar rats were randomly divided into operation group (I/R group) and sham operation group(sham group). Myocardial ischemia was induced by the ligation of left anterior descending coronary artery with the Y-shaped sutures, and the rats were subjucted to ischemia for60min followed by reperfusion for2h,4h,6h,12h,24h or7d, respectively. Sham group wasn’t underwent occlusion of coronary artery. The expression of GDF-15mRNA was measured by Realtime-PCR. The expression of GDF-15protein and ICAM-1were determined by immunohistochemical analysis. The activity of myeloperoxidase (MPO) in myocardial tissue was determined by ELISA enzyme-linked immunosorbent assay. The areas of myocardial anatomy no-reflow and infarction were assessed by Thioflavin S, Phthalocyanine blue, Triphenyltetrazolium chloride (TTC) staining. The cardiac histological infiltration of neutrophils were assessed by hematoxylin-eosin(HE) staining.RESULTS:1、There were apparent no-reflow phenomenon in rats with acute myocardial ischemia/reperfusion. At each time point, ischemic range, myocardial no-reflow range, infarct size are0in sham operation group. I/R group:there were no significant difference in ischemic range of each time point. At R=2h time point, infarct area were obvious, involving the left ventricular and left ventricular anterior and apical areas.The extension of infarct size were gradually increasing with reperfusion time, and the overall progress was moderate(p<0.05). The area of no-reflow had obvious extension trend(p<0.05) in the first three time points. They were significantly different at each time point. But,in the trend of progress, there was a plateau after reperfusion for6hours. Suggesting that the early6hours after reperfusion is important for reducing no-reflow and infarction.2、The expression of GDF-15:The sham group R=2h of GDF-15expression level as a reference, was the unit1. GDF-15mRNA of myocardial infarction and infarct border zone in I/R group were significantly higher compared with the reference group. From R=2h to R=24h, GDF-15mRNA levels maintained almostly an linear growth trend. The peak was at R=24h. There were statistically significant difference (*p <0.05,**p<0.01) in each time point of I/R geoup compared to the results of sham group R=2h. At each time point of sham group, the GDF-15mRNA was slightly increased compared to the reference, but only at R=7d, there was significant difference (p<0.01). In the tissue level, GDF-15protein expression were significantly higher than the sham group at each time point. There were significant difference compared with sham group R=2H (#p<0.001), In the I/R group, GDF-15protein showing a rapid upregulation after reperfusion2h～6h, then the trend slowed down. When R=24h, it reached the peak. It still maintain a high expression Until R=7days. However, GDF-15protein expression was slightly increased in sham group. The peak was R=12h. And there no significant difference (p>0.05) among each time point.3、MPO activity of I/R group showed a gradual decline trend and the peak appeared at R=2h. In the sham group, MPO activity increased slightly, but there were no significant changes in the law. At each time point of I/R group, there were significant difference (p<0.05), and at each time point MPO activity were significantly increased compared with the sham group.4、ICAM-1expression at each time point in I/R group was significantly higher than the sham group, there were significant difference (p<0.001). ICAM-1expressed highly at R=2h and reached to the peak at R=24h.Compared with sham group, there were significant difference (p<0.001) in ICAM-1expression at each time point of I/R group.5、HE staining showed diffuse distribution of neutrophils in myocardial microvascular of I/R group. There were large areas of myocardial necrosis and apoptosis around that. It was more obvious at R=2h、4h、6h. After reperfusion for24hours, large tissue space formed with the death of cardiomyocytes. However, there were only minor and visible injuries at the sites of threading in sham group.6、In the development of no-reflow, there was a correlation between neutrophil infiltration (MPO activity) and no-reflow area (r=-0.46, p<0.01), as well as between ICAM-1level and no-reflow area (r=0.39, p<0.05). From reperfusion for2hours to7days, the MPO activity lowed with GDF-15expression increasing. There was a negative correlation (r=-0.55, p<0.001). There was significant correlation between GDF-15and ICAM-1(r=0.52, p<0.01).CONCLUSION:GDF-15expressed highly with stress-inducing in the occurrence and development no-reflow, and had a certain expression pattern. Both neutrophil infiltration and ICAM-1expression existed in no-reflow phenomenon. And both of them and no-reflow area were correlated. GDF-15expression and neutrophil infiltration (MPO activity) was negatively correlated. Suggesting that GDF-15may be play inhibition of no-reflow effect by inhibition of neutrophil infiltration. |
PDF Full Text Request