The Protective Effects And Its Mechanisms Of Chemical Gene Silencer Targeting LOX-1 On Myocardial Ischemia-reperfusion Injury In Rats

Objective: Myocardial ischemia-reperfusion injury(MIRI)reduces the beneficial of reperfusion,and the expression of lectin-like oxidized low-density lipoprotein receptor-1(LOX-1)was significantly increased after MIRI.The pyrrole-imidazole polyamide(PIP)compound is a novel gene silencer that can be readily designed and synthesized to target against any gene.We found that the PIP targeting against LOX-1can inhibit the expression of LOX-1.In this study,we will use the PIP targeting LOX-1in our experiment in which the model of the rat MIRI is used,to investigate the effects of the PIP targeting LOX-1 on attenuating myocardial ischemia-reperfusion injury and the potential mechanisms involved in it.All of these will spark a new idea and method of preventing and treating the MIRI.Methods: 6-week-old male Sprague-Dawley rats,were randomly divided into the following groups(n=16),all groups except the Sham group underwent 45 min ischemia(I)and 3 h or 24 h reperfusion(R).The MIRI animal model was constructed by ligating and releasing left main coronary artery.(1)sham operation group(sham);(2)ischemia-reperfusion group(I/R);(3)ischemia-reperfusion+PIP targeting LOX-1 group(I/R+PIP);(4)ischemia-reperfusion+Mismatch PIP(I/R+ Mismatch PIP);and(5)ischemia-reperfusion+PIP targeting LOX-1+LY294002 group(I/R+PIP+ LY).PIP targeting LOX-1 and Mismatch PIP(5mg/kg)was given by tail vein injection 15 min before reperfusion.PI3 K inhibitor LY294002(0.3mg/kg)was given by tail vein injection 10 min before injection PIP targeting LOX-1.After 3 h of reperfusion,myocardial cellular oxidative stress was evaluated by measuring malondialdehyde(MDA)in plasma,LOX-1 expression was detected by real-time PCR and Western blot,and the activation of phospho-Akt was detected by Western blot.After 24 h reperfusion,cardiac function was determined by echocardiography,myocardial infarct size was evaluated by the Evans Blue and TTC double staining,myocardial apoptosis was determined by immunohistochemistry to detect the expression of cleaved caspase-3.Results:(1)The expression of LOX-1 m RNA and protein was significantly increased in the I/R group,compared with that in the sham group(P<0.05).PIP targeting LOX-1significantly decreased the amount of LOX-1 m RNA and protein(P<0.05,vs.I/R group).There was no difference between I/R+PIP+LY and I/R+PIP group.However,the mismatch polyamide did not affect on LOX-1 expression.(2)The MDA levels were markedly increased in the I/R group,compared with that in the sham group(P<0.05).PIP targeting LOX-1 reduced plasma MDA levels(P<0.05,vs.I/R group).Whereas the mismatch polyamide had no effect.Moreover,PI3 K inhibition abolished the beneficial effects of PIP targeting LOX-1,the plasma MDA levels were significantly increased in the I/R+PIP+LY group,compared with that in the I/R+PIP group(P<0.05).(3)The infarct size and the expression of cleaved caspase-3 were significantly reduced in the I/R+PIP group,compared with that in the I/R group(P<0.05).Whereas the mismatch polyamide have no effect.Moreover,PI3 K inhibition abolished the beneficial effects of PIP targeting LOX-1,the infarct size and the expression of cleaved caspase-3 were significantly increased in the I/R+PIP+LY group,compared with that in the I/R+PIP group(P<0.05).(4)PIP targeting LOX-1 partially preserved the heart function(P<0.05,vs.I/R group).Whereas the mismatch polyamide have no effect.Moreover,PI3 K inhibition abolished the beneficial effects of PIP targeting LOX-1,the heart function was significantly reduced in the I/R+PIP+LY group,compared with that in the I/R+PIP group(P<0.05).(5)The activation of phospho-Akt protein was significantly increased in the I/R+PIP group,compared with that in the I/R group(P<0.05).Moreover,LY significantly blockade the Akt activity(P<0.05,vs.I/R+PIP group).Conclusions:(1)This study shows that myocardial ischemia-reperfusion injury increases LOX-1 expression in rats.(2)PIP targeting LOX-1 significantly decreased the LOX-1 expression.(3)PIP targeting LOX-1 reduce reperfusion injury through reducing lipid peroxidation,apoptosis,infarct size,and cardiac dysfunction.(4)PIP targeting LOX-1 may protect myocardium from ischemia-reperfusion injury through PI3K-Akt signaling pathway.