The olive constituent oleuropein triggers a postconditioning-like effect in anesthetized rabbits through GSK3beta inhibition and JAK/STAT activation

Abstract

Background: Nutritional dose of oleuropein (oleu), which is the main polyphenolic constituent of olive, attenuates ischemia (isc) - reperfusion (rep) injury. Aim: We investigated the effect of pharmacological dose of oleu on infarct size, the intracellular signaling pathway changes and its potential role as postconditioning (PostC) analogue in vivo. Methods: Anesthetized male rabbits were subjected to 30-min regional myocardial isc followed by 3-hour rep. The animals were divided into 6 groups with the following additional interventions: (i) Control group: no further intervention; (ii) PostC group: 8 cycles of 30-sec isc/rep at the onset of rep; (iii) Oleu-50 group and (iv) Oleu-100 group: intravenous bolus administration of 50 and 100 mg.kg-1 oleu at the 20th min of isc, respectively; (v) Oleu-100 +L-NAME group and (vi) Oleu-100+Wortmannin group: administration of the NOS inhibitor L-NAME at a dose of 10 mg.kg-1 and of the PI3K/Akt inhibitor wortmannin at a dose of 60 μg.kg-1, respectively, one min before oleu administration. Circulating oleu plasma levels were assessed at the 1st and 20th min of rep by UHPLC-ESI-HRMS/MS. The percent infarct to area at risk ratio (%I/R) was estimated. In a second series of experiments, rabbits were subjected to the same initial interventions and then tissue samples were quickly excised from the ischemic regions of the heart at the 10th min of rep for eNOS, Akt, GSK3β and STAT3 assessment. Results: A significant %I/R reduction was observed in the Oleu-100 and PostC groups compared to the Control and Oleu-50 groups (20.0±2.5%, 26.4±0.3% vs 49.2±2.0% and 44.2±2.0 respectively, p<0.05). L-NAME and wortmannin did not abolish the cardioprotection afforded by Oleu-100 (23.8±4.42% and 24.5±2.4% respectively, p=NS vs Oleu-100 group).The phosphorylation of e-NOS, Akt, GSK3β and STAT3 was also significantly higher in the Oleu-100 and PostC groups (p<0.05 vs Control). Circulating oleu levels have been found to exhibit a 20-fold increase in the Oleu-100 group compared to the Oleu-50 group at both time points. Conclusion: Higher dose of oleu, given acutely at the index isc and prior to the onset of rep, induced a postconditioning-like effect in anesthetized rabbits, reducing the final infarct size through GSK3β inhibition and STAT3 activation, independently of eNOS and Akt. Cardioprotection from oleu is a dose-dependent effect that is possibly correlated with the increased circulating plasma levels.