| Objective To investigate the protective effect of preconditioning with Puerarin on myocardial ischemia reperfusion in rats and the inhibition of apoptotic effect during myocardium ischemia reperfusion as well as the relevance of PI3K/Akt signaling pathway.Methods40 healthy male SD rats were randomly divided into 5 groups (n=8): sham-operated group (SH group); ischemia-reperfusion group (IR group); puerarin preconditioning group (PU group); puerarin preconditioning + LY294002 group (PU+LY group); ischemia-reperfusion+LY294002 group (IR+LY group). The model of acute myocardium ischemia reperfusion injury was performed by 30 min occlusion of left descending coronary artery followed by 120 min reperfusion in open-chest rats.(1) SH Group: a non-ischemic control group of sham-operated rats, normal saline was administered i.v (2.5 ml/kg) 15 min before threading from the left cervical vein, continuous observation of 150 min. (2) IR Groups: normal saline was administered i.v (2.5 ml/kg) 15 min before the ligation, 30 min of myocardial ischaemia followed by 120 min reperfusion. (3) PU Group: puerarin was administered i.v (2.5 ml / kg) 15 min before ischemia through the left cervical vein, the other with the same of IR group. (4) PU+LY group: the selective PI3K inhibitor LY29400 was administered i.v (0.3 mg/kg) 15 min before using puerarin from the left cervical vein, the other with the same of PU group. (5) IR+LY Group: LY29400 was administered i.v (0.3 mg/kg) 15 min before ischemia, the other with the same of IR group. The left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximum rates of left ventricular pressure development and decay (±dp/dtmax) were recorded at before ischemia, 30 min ischemia and reperfusion 60, 90, 120 min. Arterial serum nitric oxide (NO) and nitric oxide synthase (NOS) were determined by chemical colorimetry. The myocardial p-Akt signaling protein expression was determined by Western blot analysis. Myocardial apoptosis was determined by terminal deoxynucleotidyl transfease-mediated dUTP nick end labeling(TUNEL) methods.Results1. Hemodynamic effects of puerarin preconditioningThe hemodynamic was no significant difference before ischemia in each group (P>0.05). Compared with group SH, LVSP and +dp/dtmax were decreased progressively, LVEDP and -dp/dtmax were progressively increased at each time point in other groups (P<0.05). In puerarin preconditioning group, the LVSP and +dp/dtmax were increased, LVEDP and -dp/dtmax were lower (P <0.05), PU+LY group and IR+LY group were no significant difference (P>0.05) as compared with those in group IR. Compared with group PU, the LVSP and +dp/dtmax decreased significantly, LVEDP and -dp/dtmax significantly higher in groups PU+LY and IR+LY (P<0.05). These results suggest that puerarin preconditioning improves myocardial contractility and diastolic function.2. The NO and NOS levels of puerarin preconditioningCompared with IR group, the NO and NOS levels were significantly increased in group PU (P<0.05), PU+LY group and IR+LY group were no significant difference (P>0.05). The NO and NOS levels were significantly lower in groups PU+LY and IR+LY as compared with those in group PU (P<0.05). These results suggest that puerarin pretreatment can increase the expression of NO and NOS, and this effect was abrogated by PI3K/Akt signaling pathway inhibitor LY294002.3. Antiapoptotic effect of puerarin preconditioning in CardiomyocytesCompared with SH group, the other four groups increased cardiomyocytes apoptosis significantly (P<0.05). Compared with group IR, PU group reduced cardiomyocytes apoptosis (P<0.05), PU+LY group and IR+LY group cell apoptosis was no significant difference. In groups PU+LY and IR+LY, the cardiomyocytes apoptosis was increased significantly as compared with group PU (P <0.05).These results suggest that puerarin preconditioning exerted antiapoptotic effects through the PI3K-dependent signal.4. Akt phosphorylation induced by puerarin preconditioning in myocardial tissueCompared with SH group, the expression of myocardial p-Akt protein was increased in groups IR and PU (P<0.05). In PU group, the expression of myocardial p-Akt protein was increased significantly as compared with group IR (P<0.05). Compared with PU group, the expression of myocardial p-Akt protein level was decrease in groups PU+LY and IR+LY group (P<0.05). These results suggest that puerarin preconditioning acts directly on myocardium increased the expression of myocardial p-Akt protein level and induce cardioprotective effects through the activation of PI3K/Akt signaling pathway.ConclusionPuerarin preconditioning significantly attenuated myocardial ischemia/reperfusion injury. These cardioprotective effects are attributed mainly to antiapoptotic effects of Puerarin via a PI3K/Akt signaling pathway. |
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