Effect Of BFGF On The Expression Of HIF-1αmRNA And VEGFmRNA In Ischemic And Ischemic Reperfusion Myocardium In Rats

Coronary heart disease(CHD) is the major cause of high morbidity and mortality that severely threats to human health. It is reported that the annual increasing rate of the disease in the United States alone has exceeded 100,000 per year. Traditional therapies such as medication, percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) are effective means for the treatment of CHD. However, when it comes to some patients with diffuse coronary artery stenosis or microvascular lesion, these methods tend to inefficiency . For the past few years, considerable amounts of research have been devoted to the myocardial ischemia-induced cell protections which supply some new ideas for the treatment of myocardial ischemia. Therefore, study on how to strengthen the tolerance ability of myocardium and how to improve the myocardium self-regulatory ability to ischemia and hypoxia are very important for the treatment of CHD.ObjectiveTo explore the significance of the changes of HIF-1αand VEGF in ischemic and reperfusion myocardium, and to investigate the effect of bFGF on ischemic and ischemic/reperfusion myocardium in rat.Methods100 healthy male SD rats, weighing 250 ~ 300g,were chosen, after an adaptive feeding week, they were divided into four groups randomly:(1) sham-operated group (sham,n=10), the left anterior descending (LAD) coronary artery was only exposed without ligation and randomly allocated to groups control, bFGF50μg/kg, and bFGF200μg/kg.(2) Myocardial infarction group (AMI,n=30), the LAD was ligated near its origin by a 7.0 prolene suture ,and randomly allocated to groups control, bFGF50μg/kg, and bFGF200μg/kg.(3) ischemia/reperfusion group (I/R,n=30) : ischemia for 30 min and followed by reperfusion ,and randomly allocated to groups control, bFGF50μg/kg, and bFGF200μg/kg.(4) ischemic preconditioning group (IP,n=30): three cycles of 5min occlusion-5min reperfusion was performed prior to ischemia for 30 min and followed by reperfusion, and randomly allocated to groups control, bFGF50μg/kg, and bFGF200μg/kg.Rats were treated immediately after ligation with bFGF to bFGF50μg/kg group(50μg/kg diluted to 0.5 ml with normal saline), bFGF200μg/kg group (200μg/kg diluted to 0.5 ml with normal saline) or saline to sham group (0.5ml) and control group(0.5ml), administered by tail vein injection, every day for 7d or 14d.Lead II was selected to record ECG , paper speed was 50mm/s.The expression of myocardial HIF-1αand VEGF were detected by immunohistochemistry (IHC); and the HIF-1αmRNA and VEGFmRNA expression was determined by reverse transcription polymerase chain reaction (RT-PCR).Results1 General condition of the heartThe ventricular anterior wall myocardial of rat, with LAD ligation 30 minutes, become green purple. After relaxation of coronary artery ligation line, myocardial color returned to normal; The LAD was ligated for 50 minutes, then the regional myocardial dominated by LAD darken, showing infarction changes.2 ECG observationAfter ligated the LAD, the R wave immediately increased, broadening, followed by ST segment elevation gradually. ST segment gradually declined after reperfusion and reperfusion arrhythmias occurred, ventricular premature beat(VP),ventricular tachycardia(VT), ventricular fibrillation(VF) were mostly observed. 25 rats in 30 cases of I/R group occurred in reperfusion arrhythmias, the incidence was 83.3%; 18 in 30 cases of IP group of occurred cardiac reperfusion disorders, the incidence of 60%.3 Expression of HIF-lαmRNA and VEGFmRNA3.1 Expression of HIF-lαmRNA3.1.1 Sham group, the relative expression level of HIF-lαmRNA on 7d were less than that of 14d (0.3978±0.0328/0.4118±0.0326, P> 0.05). 3.1.2 Control group, the relative expression level of HIF-lαmRNA of AMI, I/R, IP on 7d were less than that of 14d(0.9065±0.0395/0.9306±0.0328, P>0.05; 0.9633±0.0380/1.0225±0.0332,P<0.05;1.0131±0.0273/1.0779±0.0405,P<0.05). The relative expression level of HIF-lαmRNA on 7d and 14d of AMI group were higher than that of sham group, respectively (P <0.01), group I/R was higher than group AMI (P<0.05, P<0.01), group IP was higher than group I/R (P <0.05).3.1.3 bFGF50μg/kg group, the relative expression level of HIF-lαmRNA of AMI, I/R, IP on 7d were less than that of 14d (0.8960±0.0735/0.9235±0.0317, P>0.05; 0.9891±0.0456/1.0681±0.0305,P<0.05; 1.0471±0.0271/1.1161±0.0310, P<0.01).The relative expression level of HIF-lαmRNA of I/R group were higher than AMI group on 7d and 14d, respectively (P<0.05, P<0.01) , group IP was higher than group I/R (P<0.05, P<0.05). No significant difference was observed in the myocardial tissue between bFGF50μg/kg group and control group.3.1.4 bFGF200μg/kg group, the relative expression level of HIF-lαmRNA of AMI, I/R, IP on 7d are less than that of 14d (1.1433±0.0426/1.2049±0.0300, P<0.05; 1.2170±0.0309/1.2658±0.0304,P<0.05; 1.2726±0.0257/1.315±0.0273, P<0.05). The relative expression level of HIF-lαmRNA on 7d and 14d of I/R group were higher than AMI group, respectively(P<0.05, P<0.05), group IP was higher than group I/R (P<0.05, P<0.05). The relative expression of HIF-lαmRNA increased significantly in bFGF200μg/kg group as compared to bFGF50μg/kg group (P<0.01) .3.2 Expression of VEGFmRNA3.2.1 Sham group, the relative expression level of VEGFmRNA on 7d were less than that of 14d(0.2487±0.0345/0.2610±0.0310,P>0.05).3.2.2 Control group, the relative expression level of VEGFmRNA of AMI, I/R, IP on 7d were less than that of 14d(0.8507±0.0305/0.8885±0.0326,P>0.05;0.9115±0.0345/0.9671±0.0293,P<0.05;0.9744±0.0314/1.0343±0.0334,P<0.05). The relative expression level of VEGFmRNA of AMI group were higher than that of sham group on 7d and 14d, respectively (P<0.01), group I/R was higher than group AMI (P<0.05, P<0.01), group IP was higher than group I/R (P<0.05,P<0.01).3.2.3 bFGF50μg/kg group, the relative expression level of VEGFmRNA of AMI, I/R, IP on 7d were less than that of 14d( 0.8391±0.0278/0.8741 ±0.0301,P>0.05;0.9045±0.0299/0.9853±0.0309,P<0.01;0.9680±0.0351/1. 0457±0.0307,P<0.01).The relative expression level of VEGFmRNA on 7d and 14d of I/R group were higher than that of AMI group, respectively (P<0.01) , group IP was higher than group I/R (P<0.05). No significant difference was observed in the myocardial tissue between bFGF50μg/kg group and control group.3.2.4 bFGF200μg/kg group, the relative expression level of VEGFmRNA of AMI, I/R, IP on 7d are less than that of 14d(1.1040±0.0314/1.1590±0.0320,P<0.05;1.1566±0.0314/1.2115±0.0344,P<0.05;1.2098±0.0302/1.2616±0.0298,P<0.05).The relative expression level of VEGFmRNA on 7d and 14d of I/R group were higher than that of AMI group, respectively (P <0.05), group IP was higher than group I/R (P<0.05). The relative expression of HIF-lαmRNA increased significantly in bFGF200μg/kg group as compared to bFGF50μg/kg group (P<0.01).3.3 Correlative analysisThere was a significant positive correlation between the relative expression level of VEGFmRNA and HIF-lαmRNA (r=0.98541, P<0.0001, n=100).4 Immunohistochemical expression of HIF-lαand VEGF protein4.1 Expression of HIF-lαprotein4.1.1 Sham group, a slight increase of the protein expression of HIF-lαwas observed.4.1.2 Control group, the protein expression of HIF-lαof AMI ,I/R , IP on 7d were lower than that of 14d (P>0.05, P<0.05, P<0.05). The protein expression of HIF-lαof AMI group were significantly higher than sham group on 7d and 14d, respectively (P<0.01), group I/R was higher than group AMI (P<0.05, P<0.01), group IP was higher than group I/R (P<0.05 ).4.1.3 bFGF50μg/kg group, the protein expression of HIF-lαof AMI , I / R and IP on 7d were lower than that of 14d (P>0.05, P<0.05, P<0.05). The positive expression of HIF-lαprotein on 7d and 14d of I/R group were significantly higher than AMI group, respectively (P<0.01), group IP was higher than group I/R (P<0.05). The positive expression of HIF-lαprotein increased but no significantly difference in bFGF50μg/kg group as compared to control group (P>0.05)4.1.4 bFGF200μg/kg group, the protein expression of HIF-lαof AMI and I/R and IP on 7d were lower than that of 14d(P<0.05). The positive expression of HIF-lαprotein of I/R group were higher than that of AMI group on 7d and 14d, respectively (P<0.05), group IP was higher than I/R group (P<0.05). HIF-lαprotein expression of AMI, I/R, IP, were higher than that of bFGF50μg/kg group (P<0.01, P<0.05, P<0.05).4.2 Expression of VEGF protein4.2.1 Sham group, a slight increase of the protein expression of VEGF was observed.4.2.2 Control group, the protein expression of VEGF of AMI and I/R and IP on 7d were lower than that of 14d (P>0.05, P<0.05, P<0.05). The protein expression of VEGF on 7d and 14d of AMI group were significantly higher than sham group, respectively (P<0.01), group I/R was higher than group AMI (P<0.05, P<0.01), group IP was higher than group I/R (P<0.05 ).4.2.3 bFGF50μg/kg group, the protein expression of VEGF of AMI and I/R and IP on 7d were lower than that of 14d (P>0.05, P<0.05, P<0.05). The positive expression of VEGF protein on 7d and 14d of I/R group were higher than AMI group, respectively(P<0.05,P<0.01), group IP was higher than I/R (P<0.05). The protein expression of VEGF increased but no significantly difference of bFGF50μg/kg group as compared to control group (P>0.05).4.2.4 bFGF200μg/kg group, the protein expression of VEGF of AMI and I/R and IP on 7d were lower than that of 14d (P<0.05). The positive expression of VEGF protein on 7d and 14d of I/R group were higher than AMI group, respectively (P<0.05), group IP was higher than group I/R (P<0.05). HIF-lαprotein expression of AMI and I/R and IP, were higher than that of bFGF50μg/kg group (P<0.01, P<0.05, P<0.05).Conclusions1. The expression of HIF-1αmRNA, VEGFmRNA and their proteins increased both in ischemic and I / R myocardium, which maybe one of the protective mechanism of myocardial cell to hypoxia and ischemia.2. Ischemic preconditioning could significantly increase the expression of HIF-1αmRNA, VEGFmRNA and their proteins in ischemic reperfusion myocardium. It suggested that increases of HIF-1αand VEGF expression may be one mechanism of myocardial protection induced by ischemic preconditioning.3. The expression of HIF-1αmRNA, VEGFmRNA and related protein could be significantly up regulated by bFGF in rats ischemic/reperfusion myocardium .4. The expression of HIF-1αand VEGF increased and this may be the protective mechanism of bFGF on rats ischemic and I / R myocardium.