| Objective: To evaluated the effects of alprostadil injection on cardiac protection in a rat model of coronary microembolization(CME)and explored the underlying mechanisms.Methods: SD male rats were selected and randomly divided into four groups:sham operation group,coronary artery microembolization group,alprostadil2μg/kg treatment group and alprostadil 4μg/kg treatment group,with 10 rats in each group,40 in total.A rat model of CME was established by injecting polyethylene microspheres into the left ventricle.After injection of microspheres,rats in the alprostadil group received alprostadil via tail vein within 2 minutes.The low-dose treatment group was given 2μg/kg,the high-dose group was given4μg/kg,the sham operation group and the model group were given the same dose of saline.After 12 hours of modeling,echocardiography was performed to detect the cardiac function of the rats,and then the rats were anesthetized.The serum troponin I(c Tn I)level of rats was detected by ELISA method with blood from the abdominal aorta.Cut off the heart and wash the remaining blood with ice normal saline and then the heart were divided into three part at the level parallel to the atrioventricular sulcus.The bottom part of the heart was immersed and fixed in formaldehyde solution for 72 hours and then dehydrated,embedded in paraffin and sectioned,which used for HE,HBFP and TUNEL staining to observe the degree of myocardial tissue damage,myocardial microinfarction area and myocardial apoptosis in rats myocardium,respectively.The immunofluorescence double-labeled staining method was used to detect the co-expression of Nrf2 and HO-1 in myocardium.In the middle part of the heart,left ventricular anterior wall was cut and fixed in pre-cooled glutaraldehyde solution at 4°C and protected from light,for electron microscopy examine the ultrastructural changes of myocardium and myocardial autophagosomes obesevation used.The rest of heart were stored in a refrigerator at-80℃,which used to detect myocardial adenosine triphosphate(ATP),malondialdehyde(MDA)and superoxide dismutase(SOD)content,and Western blot method was apply to detect myocardial Bax,Bcl-2,Cyt-c,Cleaved-caspase-3,Cleaved-caspase-9,Nrf2,HO-1,p-GSK-3β(Ser9),GSK-3β,LC3b/LC3 a and p62 protein expression levels.The apex of the heart was put into the RNA preservation solution for extraction of RNA from the myocardium,and the expression levels of Nrf2 and HO-1 m RNA are detected by fluorescence quantitative PCR.Results: 1.Compared with the sham group,ATP concentrations,SOD activity in the myocardium,and cardiac function were significantly decreased in the CME group.In addition,serum c Tn I levels,MDA content,pro-apoptotic proteins expression level,and the number of TUNEL-positive nuclei were remarkably higher in CME group than those in the Sham group.The activity of GSK-3βincreased,and the protein and m RNA expression levels of Nrf2 and HO-1 were decreased significantly(P<0.05).2.Compared with the CME group,the heart function of rats in the alprostadil 2,4μg/kg treatment group was significantly improved,the myocardium ATP content increased,the pro-apoptotic protein level decreased,and myocardial autophagy was activated.GSK-3β activity was inhibited,p-GSK-3β(Ser9)/GSK-3β protein expression ratio increased,Nrf2 and HO-1 protein and m RNA expression levels significantly increased(P<0.05).3.Compared with the sham group,myocardial microinfarction area and histological abnormalities in the CME group were significantly increased.While microinfarction size and histological abnormalities was significantly alleviated in the alprostadil treatment groups.4.Compared with the CME group,alprostadil treatment group could significant reduced myocardium MDA content and improved SOD activity.Conclusion: Alprostadil injection seems to significantly suppress oxidative stress,alleviate myocardial apoptosis and activated myocardial autophagy in the myocardium following CME by regulating the GSK-3β/Nrf2/HO-1 signaling pathway. |
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