| BackgroundWhen acute myocardial infarction happened,the blood vessels are urgently opened to restore blood perfusion,and the ischemia-reperfusion injury caused can lead to malignant arrhythmia,heart failure,etc.It increases the risk of death,which greatly reduces the benefit.Therefore,acute myocardial infarction should not be only restored perfusion as soon as possible,but also reduced the risk of myocardial injury caused by ischemia-reperfusion.PI3K/Akt is an important intracellular signaling pathway,and activation of the PI3K/Akt signaling pathway can prevent apoptosis and necrosis of cells and reduce ischemia-reperfusion injury.Studies have found that microRNA is closely related to the occurrence and development of cardiovascular diseases,and microRNA-499 may play a role in myocardial protection through PI3K/Akt.Objectives1.To study the effect of miRNA-499 gene knockdown on myocardial ischemia-reperfusion injury2.To study regulating effect of miR-499 and PI3K/Akt signaling pathway on myocardial ischemia/reperfusion injury MethodsC57BL/6 mice were randomly divided into sham operation group(Sham+AAV9-miR-499),myocardial ischemia-reperfusion group(MIRI+AAV9-miR-499),PI3 K inhibitor(LY294002)pretreatment ischemia-reperfusion group(LY294002+MIRI+AAV9-miR-499)and negative control group(MIRI+AAV9).Firstly,adenovirus9(AAV9)carrying miR-499 gene knockdown sequence was injected into mice by tail vein for constructing the model of miR-499 knockdown gene.Secondly,with myocardial ischemia-reperfusion model established,the coronary artery was ligated for 30 min and then the blood flow was restored after loosening for 2h,and not ligatured in the sham-operation group.The PI3 K inhibitor intervention group was injected with PI3 K inhibitor 1 hour before the establishment of ischemia-reperfusion model.Finally,the hearts were harvested at the end of myocardial ischemia-reperfusion of mice,and miRNA-499 expression was detected by Real Time PCR,Akt and phosphorylated Akt(P-Akt)expression was detected by Western Blot,and myocardial infarction area was measured by TTC staining,so as to study the correlation between myocardial infarction area,miR-499 and Akt expression.Results1.The expression of miR-499 of each group Compared with the control group(1.50±048),the expression of miR-499 in the sham group(0.61±0.26)decreased(P<0.05).Compared with the control group,the expression in miR-499 gene knockdown surgery group decreased(0.84±0.39)(P<0.05).The expression in LY294002(0.69±0.19)intervention group was also significantly lower than that in the control group(P<0.05).Meanwhile,there was no statistical difference in the expression of miR-499 in the sham group,the miR-499 gene knockdown surgery group and the LY294002 intervention group(P>0.05)2.The phosphorylation activity of Akt of each group The phosphorylation activity of Akt deponds on the ratio of P-Akt and Akt.Compared with the sham group(0.36±0.35),the phosphorylation activity of Akt significantly reduced in the miR-499 knockdown surgery group(0.17±0.29)(P<0.05).Compared with the sham group,the phosphorylation activity of Akt in the LY294002 intervention group(0.16±0.87)significantly decreased(P<0.05),while there was no statistical difference compared withthe phosphorylation activity of Akt in the miR-499 knockdown surgery group(P>0.05)3.The infarction size of each group Compared with the miR-499 gene knockdown surgery group(12.43±1.77)%,the myocardial infarction area in the control group(8.47±1.67)%reduced(P<0.05).Compared with the sham operation group(0±0.00)%,the myocardial infarction area of the miR-499 gene knockout surgery group significantly increased(P<0.05);however,the myocardial infarction area decreased compared with the LY294002 intervention group(18.67±5.09)%(P<0.05)ConclusionMiRNA-499 knockdown and the down-regulation of PI3K/Akt signaling pathway activity aggravated myocardial ischemia-reperfusion injury. |
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