| Objective Myocardial ischemia-reperfusion(I/R)injury is the further injury of the ultrastructure,function,metabolism and electrophysiology of the myocardium after the recovery of the blood supply,which usually occurs in acute myocardial infarction and other ischemic heart diseases.I/R injury will lead to the expansion of the original infarct area,severe arrhythmia and inhibited ventricular function.Therefore,prevention and treatment of I/R have always been a problem that needs to be solved in cardiovascular research.7,8-dihydroxyflavone(7,8-DHF)is a flavonoid compound that plays a potent neuroprotective and nutritional role as a tyrosine kinase B(Trk B)receptor agonist.In cardiovascular studies,it has been found that 7,8-DHF can relax the thoracic aorta of rats,an effect which is related to the activation of NO/c GMP signaling pathway and the blocking of intracellular calcium release and extracellular calcium influx.7,8-DHF can also inhibit H2O2-induced endothelial cell injury,but the protective effect of 7,8-DHF on ischemic myocardium has not been reported.In this study,a rat model of myocardial I/R injury was used to observe the protective effect of 7,8-DHF and its underlying mechanism.Methods and Contents Wistar rats were randomly divided among three groups: sham group,I/R group and I/R+7,8-DHF group with 5-6 rats in each group.In the sham group,thoracotomy was performed only by puncture without ligation,and rats in I/R group were treated with ischemia for 30 min then the blood supply was restored for 3 h.7,8-DHF(10 mg/kg,i.p)was given 10 min before reperfusion.After the reperfusion of blood supply for 3 h,left anterior descending coronary(LAD)arteries of two rats from I/R group were ligated again,and Evans blue was injected into the left ventricle of the heart.Sections from the apex to the bottom of the heart were cut for TTC staining to determine whether the ischemia-reperfusion model of the rats was successful.After the animals were killed,blood was taken from the heart and myocardial tissue below the ligation line was taken for the following observations: 1.Serum myocardial enzyme assays: the serum lactate dehydrogenase(LDH)was determined by the rate method,and the serum creatine kinase isoenzyme(CK-MB)was determined by immunosuppression method.2.Estimation of oxidative stress injury: wst-8 method was used to detect the activity of superoxide dismutase(SOD)in myocardial tissue,TBA method was used to detect the MDA content,and Western blot method was used to detect p-H2 A.X protein level.3.Estimation of cell apoptosis: the protein levels of Bcl-2,Bax,and cleaved caspase-3 were measured by Western blot method.4.Estimation of signaling proteins: the changes of Akt and Erk proteins were determined by Western blot method.5.Morphological examination: the frozen sections of heart tissues in each group were observed,and the histological changes of myocardium were observed by HE staining.Results 1.The results of Evans blue-TTC staining showed that the heart was stained into three parts: the blue part was normal myocardium,the red part was the ischemic risk area,and the white part was the myocardial infarction area,indicating the successful establishment of myocardial ischemia-reperfusion injury model.2.The serum levels of both LDH and CK-MB in I/R group were significantly increased compared with that of the sham group(LDH: 2415.17±80.96 U/L v.s 1596.00±145.46 U/L,P<0.01;CK-MB: 1019.40±63.48 U/L v.s 557.50±111.40 U/L,P<0.01),while 7,8-DHF pretreatment inhibited these changes(LDH: 1868.50±90.15 U/L v.s 2415.17±80.96 U/L,P<0.01;CK-MB: 647.50±84.59 U/L v.s 1019.40±63.48 U/L,P<0.05),suggesting that 7,8-DHF has a protective effect on ischemia-reperfusion injury in rats.3.The results of oxidative stress study showed that the content of MDA in myocardial tissue of I/R group was 1.78 fold compared with the sham group(1.21±0.22 ?mo L/mg v.s 0.68±0.20 ?mo L/mg,P<0.01),while the activity of SOD was significantly decreased(13.07±2.40 U/mg v.s 22.22±0.57 U/mg,P<0.01).In addition,the level of p-H2 A.X in myocardial tissue of the I/R group was 2.64 fold compared to that of the control group(1.11±0.13 v.s 0.42±0.20,P< 0.01),suggesting severe oxidative stress in the reperfused myocardium.7,8-DHF significantly decreased the MDA content(0.88±0.15 ?mo L/mg v.s 1.21±0.22 ?mo L/mg,P<0.05),increased SOD activity(18.51±0.46 U/mg v.s 13.07±2.40 U/mg,P<0.05),and inhibited protein expression of p-H2 A.X(0.71±0.12 v.s 1.11±0.13,P<0.05).These results indicate that the protective effect of 7,8-DHF is related to its antioxidant effect.4.The results of apoptosis were as follows: compared with their respective controls,the level of Bcl-2 in I/R group was significantly decreased(0.65±0.12 v.s 1.40±0.13,P<0.01),the expression of Bax was increased(1.85±0.52 v.s 0.59±0.35,P< 0.01),and the level of cleaved caspase-3 was increased(1.14± 0.19 v.s 0.58±0.19,P<0.01),indicating that ischemia-reperfusion induced significant myocardial cell apoptosis.However,after 7,8-DHF pretreatment,Bcl-2 expression level was significantly increased(1.17±0.03 v.s 0.65±0.12,P<0.05),Bax expression was decreased(1.00±0.18 v.s 1.85±0.52,P< 0.01),and cleaved caspase-3 expression was decreased(0.76±0.18 v.s 1.14±0.19,P<0.05).Therefore,the reduced apoptosis by 7,8-DHF might be contributed to its protective effect.5.The Erk protein expression in I/R group was significantly reduced(0.49±0.22 v.s 1.12±0.22,P<0.05),while 7,8-DHF treatment significantly enhanced the Erk protein expression(0.97±0.18 v.s 0.49±0.22,P<0.05).6.Compared with the sham group,the Akt level in I/R group was significantly enhanced(1.90±0.46 v.s 0.89±0.28,P<0.01),while 7,8-DHF reduced the Akt expression(1.20±0.21 v.s 1.90±0.46,P<0.05).7.HE staining showed that myocardial injury was severe in the I/R group,as evidenced by loss of myocardial nuclei and disordered arrangement of myocardial fibers.These changes were partially attenuated after 7,8-DHF treatment.Conclusions In summary,7,8-DHF treatment is effectve against ischemia-reperfusion injury in rats.This might be related to its anti-oxidative and anti-apoptosis effects of 7,8-DHF. |
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