The Protective Effect Of Oleuropein On Myocardial Ischemia Reperfusion Injury And Its Related Mechanism

Background: Coronary heart disease is one of the leading causes of death and disability worldwide.The effects of coronary heart disease are usually attributable to the detrimental effects of acute myocardial ischemia-reperfusion injury(MIRI).So far,however,the exact pathogenesis of the disease remains unclear.The present study focuses on oxygen free radical formation,intracellular calcium overload and cell apoptosis,oxygen and energy utilization obstacle,etc.,especially that oxidative stress injury and apoptosis have been identified to play important roles in the process of myocardial reperfusion injury.Oleuropein,one of the representative predominant phenolic oleosides in Olea europaea,has been shown to display several pharmaceutical properties,including antioxidant,anti-inflammatory,and anticancer effects.In 2004,Manna reported that Oleuropein showed certain protective effect on cardiac oxidative myocardial injury caused by ischemia reperfusion in vitro,but few experimental study in vivo were conducted,and the mechanism need to further study.Objectives: To evaluate the protective effect of oleuropein on MIRI and to investigate its related mechanisms.Methods: A rat model of MIRI and a primary cultured neonatal rat MIRI model were established,the level of CK,LDH,MDA,SOD,CAT and GSH-PX were tested using ELISA assay.The infarct area was observed by TTC staining.The apoptosis was detected utilizing flow cytometry assessment or TUNEL.Detection of intracellular ROS level was carried out by fluorescent probe DCFH-DA.Mitochondrial membrane potential(MMP)was determined by cationic dye JC-1.In addition,expression of cytochrome c(Cyt-c),Caspase-3,Caspase-9,Bcl-2 family proteins,Akt,and ERK1/2 were measured by western blot.Results: 1)Compared with the model group,Oleuropein(100,200,400 mg/kg)significantly inhibited the increase of CK,LDH and MDA(P < 0.01;P < 0.05;P < 0.05)and decrease of SOD,CAT and GSH-PX(P < 0.05;P < 0.05;P < 0.05),induced by ischemia reperfusion injury in rats.Myocardial infarction ratio of model group is 18.9%,and the difference was statistically significant(P < 0.001)compared with the control group.Oleuropein significantly decreased the rates of infarction(P < 0.05)and myocardial apoptosis index(P < 0.05).Compared with model group,Oleuropein(400 mg/kg)significantly increased Bcl-2 / Bax ratio(P < 0.01),also increased the expression of p-ERK1/2 and p-Akt(p < 0.05,p < 0.05).2)SI/R injury model was established in neonatal rat cardiomyocytes.Compared with the model group,Oleuropein(100,200,400?g/mL)reduced cell injury(P<0.001),and decreased cellular apoptosis with a dose-dependent manner(P<0.001).Oleuropein reduced CK,LDH and ROS levels(P < 0.01,P < 0.01,P < 0.001),inhibited the excessive release of MDA(P < 0.001),increased SOD,CAT and gsh-px activity(P < 0.05,P < 0.05,P < 0.05).Oleuropein reduced the green/red fluorescent ratio(P < 0.001).Also Oleuropein inhibited the expression of Bax,cyt-c,Caspase-3 and Caspase-9(P < 0.05,P < 0.05,P < 0.05,P < 0.01),increased bcl-2 expression(P < 0.05)and Bcl-2 / Bax ratio(P < 0.05).The high expression of Akt and ERK phosphorylation induced by Oleuropein(400 g/mL)and elevated p-Akt/t-Akt ratio(P < 0.01)and p-ERK1/2 /t-ERK1/2 ratio(P < 0.05),which were reversed by LY294002(Specific inhibitor of PI3K/Akt)and U0126(Specific inhibitor of ERK),respectively(P < 0.05,P < 0.05).Conclusions: Oleuropein has significant protective effects on MIRI,and its mechanism may be related to the inhibition of mitochondrial apoptosis pathway induced by oxidative stress and activation of RISK pathway.