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Both aging and diabetes can weaken the ability of ischemic postconditioning to protect myocardium. Adropin can potentially activate reperfusion injury salvage kinase (RISK) signaling pathway in according with the protection mechanism of ischemic postconditioning to myocardium. This study aims to investigate whether Adropin combined with ischemic postconditioning contribute to alleviation of ischemia-reperfusion injury of aged diabetic myocardium and whether RISK signaling pathway play a role.
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: 5 groups receiving 45-minute ischemia and then 7-minute or 24-hour reperfusion except the sham group: 1)Sham: threading without occluding, persisting perfusion; 2)Ischemia/Reperfusion group (I/R Group): 45-minute ischemia then 24-hour reperfusion; 3) Ischemic/postconditioning group (Ipost Group)：three-cycle of seperated 10s' I and R before persisting reperfusion; 4) Adropin group: additional injection of Adropin 0.2mg/kg in external jugular vein slowly in 5 mins before reperfusion; 5) Adropin+Ischemic/postconditioning group (A+Ipost Group):adropin and postconditioning come befoer reperfusion; 6) Adropin+ Ischemic/postconditioning +LY294002(A+IPost+LY group): injected of PI3K specific inhibitor LY294002(40mg/kg) i.p. 15mins before reperfusion. After 24-hour reperfusion, several mice (n=3-6) were sacrificed to contribute hearts. The infarct size was evaluated by triphenyltetrazolium chloride (TTC), caspase-3 by imumunohistochemistry; the rest (n=3-6) (after 45-min I/7-min R) were used to detected the level of Akt and endothelial nitric oxide synthase (eNOS) by Western blot.
Compared with I/R group, neither Adropin group nor Ipost group succeeds in alleviating IS/AAR in aging and diabetes mellitus mice (P>0.05). But A+Ipost group can downsize IS/AAR of 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 myocardium apoptosis caused by MIRI (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-eNOS/eNOS (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). LY294002 can weaken the ability of Adropin+Ipost to downsize the infarct size and minus myocardium apoptosis (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) by inhibiting the phosphorylation of Akt and eNOS signaling proteins.
Aging diabetes aggravated mouse MIRI, and Adropin or Ipost failed to alleviate aged/diabetic mice myocardial ischemic/reperfusion injury (MIRI). Adropin combined with Ipost contributed to relieve MIRI by reducing myocardium apoptosis, in which PI3K/Akt/eNOS signaling pathway may be involved.