| Objective: Ischemic postconditioning is an endogenous protective mechanism to alleviate myocardial ischemia-reperfusion injury(MIRI). Both aging and diabetes can weaken the ability of ischemic postconditioning to protect myocardium, which may result from a decrease in the activation of phosphatidylinositol 3-kinase-Serine/threonine Kinase(PI3K/Akt). Adropin is a novel endogenous biological peptide and can activate PI3K/Akt signaling pathway. This study aims to investigate whether Adropin combined with ischemic postconditioning may alleviat MIRI in the aged diabetic myocardium by activating PI3K/Akt signaling pathway.Methods: Aging mice, both 16-month type 2-diabetes mellitus db/db mice and brood wild C57BL/6J mice, built as myocardial ischemia-reperfusion models, were randomly divided into 6 groups: receiving 45-minute ischemia and then 24-hour or 7-minute reperfusion: 1)Sham: threading without occluding, persisting perfusion; 2)Ischemia/Reperfusion group(I/R group): 45-minute ischemia and then reperfusion for 24 hours; 3) Ischemic postconditioning group(Ipost group):three-cycles of 10 s I and 10 s R before persisting reperfusion; 4) Adropin group: additional injection of Adropin 0.2mg/kg into external jugular vein 5 min before reperfusion; 5) Adropin + Ischemic postconditioning group(A+Ipost group): additional injection of Adropin 0.2mg/kg into external jugular vein 5 min before reperfusion and then three-cycles of 10 s I and 10 s R; 6) Adropin + Ischemic postconditioning +LY294002(A+IPost+LY group): treated with Adropin and I/R and additional injection of PI3 K specific inhibitor LY294002(40mg/kg) i.p. 15 min before reperfusion. After 24-hour reperfusion, some mice(n=3-6) were sacrificed to contribute hearts. The infarct size was evaluated by triphenyltetrazolium chloride(TTC), and caspase-3 by imumunohistochemistry. After 7 min reperfusion, some hearts(n=3-6) were used to detected the level of Akt and endothelial nitric oxide synthase(e NOS) by Western blot. Venous blood was drawn alive for the detection of creatinekinase MB(CKMB).Results: Except for the Sham group, IS/AAR and CKMB of the other groups increased obviously. In the I/R group, IS/AAR of db/db mice significantly increased compared with C57BL/6J mice(50.84±4.16% vs 60.81±4.76%, P<0.05). While compared with I/R group, neither Adropin group nor Ipost group succeeds in alleviating IS/AAR and the level of CKMB in aging and diabetes mellitus mice(C57BL/6J mice: IS/AAR:50.84±4.16% vs 47.43±3.69%, 48.35±3.56%, P>0.05; CKMB:15.64 ±3.46 ng/ml vs 13.11±2.94 ng/ml,14.33±2.10ng/ml, P>0.05; db/db mice: IS/AAR: 60.81 ±4.76% vs 59.66±4.49%, 60.63±4.65%, P>0.05; CKMB:29.14±4.21ng/ml vs 28.03±2.26ng/ml, 27.01±1.96 ng/ml, P>0.05). Compared with the I/R group Adropin combined with Ipost can reduce IS/AAR in aging and diabetes mellitus mice(C57BL/6J mice:50.84±4.16% vs 24.84±3.15%, P<0.05;db/db mice : 60.81±4.76% vs. 28.82±3.46%, P<0.05)and decrease Caspase-3 protein expreesion(C57BL/6J mice: 0.364±0.022 vs 0.178±0.016, P<0.05; db/db mice:0.472±0.033 vs 0.227±0.017, P<0.05), with an increase of p-Akt/Akt and p-e NOS/e NOS(C57BL/6J mice:0.60±0.10 vs 1.29±0.13, P<0.05;0.47±0.14 vs 1.36±0.09, P<0.05; db/db mice:0.57±0.05 vs 1.04±0.20, P<0.05; 0.32±0.08 vs 1.18±0.13, P<0.05, respectively). LY294002 can abolish the cardioprotective effects conferred by the combination of Adropin and Ipost(infarct size,C57BL/6J mice: 46.54±3.67%vs 24.84±3.15%,P<0.05;db/db mice:55.03±5.42% vs 28.82±3.46%, P<0.05) via inhibiting the phosphorylation of Akt and e NOS signaling proteins.Conclusions: Aging diabetes aggravated mouse MIRI, and Adropin or Ipost alone failed to alleviate MIRI in aged diabetic mice. Adropin combined with Ipost alleviate MIRI, in which PI3K/Akt/e NOS signaling pathway may be involved. |
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