Ischemic Preconditioning Or Zinc Sulfate Pretreatment Protects Heart Against Ischemic Reperfusion Injury Via Nrf2-ARE Pathway

Objective: The purpose of this study is to observe the effects of ischemic preconditioning or zinc sulfate pretreatment on myocardial ischemia reperfusion injury and to explore whether the Nrf2-ARE pathway is involved in the protective effects of these two treatments. Method: Sixty healthy male Sprague-Dawley rats were randomly divided into 6 groups(each group n=10). Twenty-four(24) hours after intraperitoneal injection of zinc sulfate(200μmol/L in Zn and Zn+L groups) or 1.5ml/kg triple-distilled water(the rest 4 groups), hearts were removed and perfused with a Langendorff perfusion system. Hearts in normal group(Nor Group) were subjected to 120 min of continuous Krebs-Henseleit buffer(KHB) perfusion without ischemia. Hearts in ischemia/reperfusion group(I/R Group) were perfused with KHB for a 20 min equilibration period followed by 40 min global ischemia and 60 min KHB reperfusion. Hearts in zinc sulfate + luteolin pretreatment group(Zn+L Group) and zinc sulfate alone pretreatment group(Zn Group) accepted zinc sulfate with or without administration of luteolin(50μmol/L) before ischemia followed by the same procedures of I/R group. Ischemic preconditioning group(IPC Group) was induced by 6 cycles of 10-sec occlusion/10-sec reperfusion before global ischemia. In IPC+L group, luteolin was used before ischemia. Heart rate(HR), left ventricular developed pressure(LVDP), left ventricular end-diastolic pressure(LVDEP), and the maximum rising rate of the left ventricular internal pressure(+ dp/dtmax) were measured at the end of equilibration and reperfusion. Furthermore, at the end of reperfusion, left ventricular myocardial tissue was taken for ultrastructural change analysis by transmission electron microscopy, in which mitochondrial Flameng score was used.. Western blot and Real-Time PCR studies were performed to measure m RNA and protein expression levels of the heme oxygenase 1(HO1), quinone oxidoreductase(NQO1), superoxide dismutase 1(SOD1), and nuclearfactor-related factor-2(Nrf2) of left ventricular myocardial tissue. Coronary effluent was collected to measure the levels of lactate dehydrogenase(LDH) and malondialdehyde(MDA). Results: 1. The cardiac function indexes : At the point of equilibrium, there was no significant difference in cardiac function in all groups(P>0.05). However, at the end of reperfusion the best heart function was found in Nor group(P <0.05); the cardiac function of Zn group and IPC group was greater than that of the I/R group(P <0.05) and the respective luteolin group(P <0.05); there was no difference between Zn group and IPC group(P>0.05). 2. Ultrastructure study of myocardial tissue and the mitochondrial Flameng score. 2.1 The ultrastructure of myocardial tissue: The ultrastructural morphology in Nor group was close to normal. In I/R group, the ultrastructure had the most serious injury. The ultrastructural change in Zn group and IPC group were greater than that of the I/R group and the respective luteolin group. 2.2 The mitochondrial Flameng score: Comparing with Nor group, the scores of I/R, Zn+L, and IPC+L groups increased significantly(P<0.05); the scores of Zn and IPC groups were lower than that of the I/R group and the respective luteolin groups(P<0.05). There was no difference between Zn group and IPC group(P>0.05). 3. Cardiac effluent changes: The concentration of LDH in I/R, Zn+L, and IPC+L groups were higher than that of the Nor group(P<0.05). The LDH concentration in Zn and IPC groups were lower than that of the respective luteolin groups(P<0.05). There was no difference between Zn group and IPC group(P>0.05). The results of MDA showed the same trend as LDH. 4. The m RNA expressions of HO1, NQO1, SOD1 and Nrf2 increased in all other groups comparing with Nor group(P <0.05). When compared with I/R group, all target gene m RNA expressions of Zn group and IPC group increased significantly(P <0.05). When compared with the IPC group, all target gene m RNA expressions in IPC+L group reduced significantly except Nrf2(P <0.05). When compared with Zn group, all targetgene m RNA expressions in Zn+L group decreased significantly except Nrf2(P<0.05). Each target gene m RNA expressions was equal between Zn group and IPC group(P> 0.05). 5. The protein expressions of HO-1、NQO1、SOD1 and Nrf2 showed the same trend as that of m RNA. Conclusion: 1. Both zinc sulfate pretreatment and ischemic preconditioning exert protective effects on langendorff-perfused rat hearts, which suffered from ischemia/reperfusion, by maintaining cardiac function and protecting cardiac ultrastructure. 2. The protective effect of zinc sulfate pretreatment is equal to the effect of ischemic preconditioning. 3. The activation of Nrf2-ARE pathway by zinc sulfate pretreatment and ischemic preconditioning may be involved in the protective mechanisms by alleviating the peroxidation damage induced by IR.