Effect Of Preconditioning On Myocardial Ischemia Reperfusion Injury In Diabetic Rats

ObjectiveThe purpose of this study was to explore the pathophysiological mechanisms underlying ischemia reperfusion injury in diabetic and non diabetic rat heart.To investigate the protective effects of pharmacological preconditioning (PPC) and ischemia preconditioning(IPC) on myocardial ischemia reperfusion injury in diabetic and non diabetic rats and to search the clinical value.Methods(1)The diabetic rat model was established by streptozotocin injection to the abdominal cavities of Spraque-Dawlay rats.Four weeks later, the myocardial ischemia reperfused models were established in anesthetic SD rats. The rats were randomly divided into two groups(diabetic group and non diabetic group),and each group divided into two subgroups including sham group and ischemia reperfusion(IR) group. In sham group,there were made no procedures after surgery. In IR group, the injury was induced by occlusion of the left anterior descending coronary after for 30 min followed by reperfusion for 120 min. A micro-catheter was inserted into left ventricular through right carotid artery to measure the left ventricular pressure.Hemodynamic data were continuously monitored and simultaneously digitized.Blood samples were drawn from caudal vein to measure blood glucose. At the end of the experiment, ventricular blood was sampled to measure triglyceride and cholesterol.The myocardial infarct size and ventricle weight index were determined,After embedded in paraffin, heart was formed into sections, then which were stained with hematoxylin and eosin.(2)The model and protocol were performed as previously described.The changes of plasma angiotensinâ…¡(Angâ…¡) and aldosterone(ALD) and serum insulin-like growth factor-1 (IGF-1)were examined with RIA.The contents of malonaldehyde(MDA) and nitric oxide(NO) in serum and myocardial tissues were detected. The activities of nitric oxide synthase(NOS) and superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) in serum and myocardial tissues were measured.The activities of Na⁺,K⁺-ATPase,Mg²⁺-ATPase, Ca²⁺-ATPase in myocardial mitochondria were measured.The intercellular adhesion molecule-1(ICAM-1),matrix metalloproteinase-2(MMP-2) protein expressions in myocardium were evaluated by immunohistochemical method. The RhoA and Rho correlation kinase(ROCK) protein expressions were assessed by western blot assay. (3)The effect of Vitamin E administrating orally for 4 weeks against myocardial ischemia reperfusion injury in diabetic and non diabetic rats was investigated.The contents of MDA in serum and myocardial tissues were detected. The activities of SOD and GSH-Px in serum and myocardial tissues were measured. The activities of Na⁺,K⁺-ATPase,Mg²⁺-ATPase, Ca²⁺-ATPase in myocardial mitochondria were measured.The ICAM-1 protein expressions were evaluated. The myocardial infarct size was determined.(4)The diabetic and non diabetic rats were given Breviscapine by stomach perfusion to investigate the protective effect of in vivo pharmacological preconditioning of Breviscapine.The changes of plasma Angâ…¡,ADD and serum IGF-1 were examined by RIA. The contents of MDA in serum and myocardium were detected. The activities of SOD and GSH-P_X in serum and myocardium were measured. The activities of Na⁺,K⁺-ATPase, Mg²⁺-ATPase, Ca²⁺-ATPase in myocardial mitochondria were measured.The ICAM-1 protein expressions were evaluated. The myocardial infarct size was determined.(5)Following 4 weeks of streptozotocin-induced diabetic rats,all hearts were divided randomly into four groups:non-diabetic ischemia reperfusion group(C_IR group),non-diabetic ischemia preconditioning group(C_IP group),diabetic IR group(D_IR group),diabetic ischemia preconditioning group(D_IP group).In IR group ,the injury was induced by occlusion of the left anterior descending coronary after for 30 min followed by reperfusion for 120 min.In IP group,preconditioning consisted of three cycles of 5 min ischemia and 5 min reperfusion,prior to ischemia.At the end of the experiment,checked infarct size.The protein expressions of MMP-2 were evaluated by immunohistochemical method. The RhoA and ROCK protein expressions were assessed by western blot assay.The contents of MDA and NO in serum and myocardium were detected. The activities of SOD, GSH-Px and NOS in serum and myocardium were measured. The activities of Na⁺,K⁺-ATPase,Mg²⁺-ATPase, Ca²⁺-ATPase in myocardial mitochondria were measured.Results(1) The effects of diabetes on myocardial ischemia reperfusionAfter four weeks of diabetic duration, blood glucose levels significantly increased (P<0.001).while body weight significantly decreased in diabetic group compared with non diabetic group (P<0.01). There were no significant differences in Hemodynamic data(such as HR,LVSP,LVEDP)among all groups.Compared with that of C_IR group, the myocardial infarct size was increased significantly(P<0.001),ventricular weight index was increased at 4 weeks of D_IR group as compared to C_IR group (P<0.001).By HE staining:in the myocardial ischemia reperfusion of diabetic rats we observed disorganized cardiac muscle.(2)The effects of diabetes on myocardial ischemia reperfusionCompared with that of C_IR group, the contents of MDA and NO in myocardium were increased significantly in D_IR group(P<0.05),the activities of NOS in myocardium were increased significantly in D_IR group(P<0.01).The levels of IGF-1 were decreased significantly in D_IR group(P<0.05). And the activities of Na⁺-K⁺-ATPase, Mg²⁺-ATPase, Ca²⁺-ATPase were decreased significantly in D_IR group (P<0.05). Compared with C_IR group, the ICAM-1 and MMP-2 and RhoA and ROCK protein expressions were increased significantly in D_IR group.(3) The effects of Vitamin E on myocardial ischemia reperfusion in diabetic ratsCompared with that of D_IR group, the activities of Na⁺-K⁺-ATPase, Mg²⁺-ATPase in myocardial mitochondria were increased significantly in D_VE group(P<0.05).The levels of MDA in serum and myocardium were decreased significantly (P<0.05).whereas the activities of SOD and GSH-P_X increased. Compared with that of C_IR group, the levels of ICAM-1 in myocardium were decreased significantly in Dve group (P<0.05). The myocardial infarct size were not reduced significantly(P>0.05).(4)The effects of Breviscapine on myocardial ischemia reperfusion in diabetic ratsCompared with that of D_IR group, the levels of Angâ…¡and ALD in plasma were decreased significantly and the levels of IGF-1 were increased significantly in D_Bre group.The activities of Na⁺,K⁺-ATPase,Mg²⁺-ATPase,Ca²⁺-ATPase in myocardial mitochondria were increased significantly in D_Bre group compared with D_IR group.The levels of MDA in serum and myocardium were decreased significantly in D_Bre group, whereas activities of SOD and GSH-Px in serum and myocardium increased significantly.The expression levels of ICAM-1 protein were decreased significantly in D_Bre group compared with D_IR group.And the myocardial infarct size were not reduced significantly(P>0.05).(5)The effects of ischemia preconditioning on myocardial ischemia reperfusion in diabetic ratsCompared with that of D_IR group, the levels of MDA in serum and myocardium were decreased significantly, the activities of SOD and GSH-Px in serum and myocardium were increased significantly in D_IP group. Compared with that of Dm groups.the expression of MMP-2 and RhoA and ROCK in D_IP group were decreased.Compared with that of D_IR group, the contents of NO in serum and myocardium were decreased significantly,the activities of NOS in serum and myocardium were decreased significantly in D_IP group. The activities of Na⁺,K⁺-ATPase,Mg²⁺-ATPase in myocardial mitochondria were increased significantly in D_IP group compared with D_IR group.And the myocardial infarct size were reduced significantly (P<0.05). Conclusion(1) Early diabetic during ischemia significantly exacerbates myocardial ischemia reperfusion injury, as evidenced by significantly enlarged infarct size, and worse myocardial structure following myocardial IR.(2) Early diabetic during ischemia significantly exacerbates myocardial ischemia reperfusion injury by increasing MDA,Angâ…¡,ALD,NO,NOS,MMP-2,ICAM-1,RhoA and ROCK,decreasing Na⁺,K⁺-ATPase,Mg²⁺-ATPase, Ca²⁺-ATPase,SOD,GSH-P_X and IGF-l.(3) Vitamin E could relieve myocardial ischemia reperfusion injury and the damage of lipid peroxidation and free radical induced by MIR in diabetic rats, and this effect was mediated by reduction of the expression of ICAM-1 protein.(4) Breviscapine had obvious protective effects on myocardial ischemic and reperfusion injury in diabetic rats probably by scavenging hydroxyl free radicals, decreasing the level of plasma Angâ…¡and ALD, the level of MDA in serum and myocardium, the ICAM-1protein expression in myocardium and increasing the level of serum IGF-1,the level of SOD and GSH-Px in myocardium, the level of Na⁺,K⁺-ATPase,Mg²⁺-ATPase,Ca²⁺-ATPase inmyocardial mitochondria.(5) IPC could reduce the severity of reperfusion induced myocardial infarct size,Protects myocardial structure in non-diabetic rats.In 4 weeks diabetes afford the same myocardium protection as in non-diabetic rats. IPC had obvious cardioprotective effects on myocardial ischemic and reperfusion injury in diabetic rats probably by scavenging hydroxyl free radicals, increasing the level of Na⁺,K⁺-ATPase,Mg²⁺-ATPase,Ca²⁺-ATPase in myocardial mitochondria and decreasing the protein expression of MMP-2 and RhoA and ROCK in myocardium.