Effects Of MiR-146a-3p Targeting IRAK1/TRAF6 Axis On Myocardioprotection Induced By Dexmedetomidine

Objectives:Reperfusion can improve the clinical outcome of patients,while it can cause myocardial ischemia-reperfusion injury(MIRI).Although ischemic preconditioning can alleviate MIRI injury,its extensive clinical application is in limitations.Pharmacological preconditioning,in terms of mimicing roles of ischemic preconditioning in attenation of myocardial injury,is prone to performance in the clinical scenario in patients.Dexmedetomidine(DEX)provides myocardioprotection through lowering the incidence of perioperative cardiovascular events,but the mechanism is not well known.MicroRNAs(miRNAs)play important roles in the pathogenesis of myocardial injury,and miR-146a is a potent regulator of myocardial function.While mechanisms of miR-146a-3p in DEX preconditioning induced-cardioprotection are not well documented.Therefore,it is of great importance to elucidate the potential roles and mechanisms of miR-146a-3p in myocardioprotection induced by DEX preconditioning.Methods:In vivo,a myocardial ischemia-reperfusion injury(MIRI)model was established in the Sprague-Dawley rat by transient occlusion of the left anterior descending coronary artery(LAD)for 30 minutes and subsequent opening for 120 minutes.Some of rats were preconditioned with DEX at a dosage of 20 ?g/kg before onset of ischemia.Myocardial infarct sizes were evaluated by 2,3,5-triphenyltetrazolium chloride(TTC)staining.Effects of DEX preconditioning on changes of differentialy-expressed miRNAs were analyzed by miRNA microarray analysis.Key miRNAs were checked by bioinformatic analysis.Target gene(s)of miR-146a-3p were analyzed by dual-luciferase reporter gene assay.In vitro,in rder to mimic the MIRI in vivo,a cellular anoxia/reoxygenation(A/R)model was established in H9C2 cells incubated with anoxia for 3 hours and reoxygenation for 2 hours.H9C2 cells in several groups were incubated with 10 nM of DEX for 1 hour before initiation of anoxia.Apoptosis rates and reactive oxygen species(ROS)emission of H9C2 cells were determined by flow cytometry.Cell viabilities of H9C2 cells were checked by CCK-8 assay,and relative expression of miR-146a-3p in H9C2 cells were analyzed by quantitative real-time polymerase chain reaction(qRT-PCR).Expressions of target proteins(interleukin-1 receptor-associated kinase 1,IRAK1;tumor necrosis factor receptor-associated factor 6,TRAF6),apoptosis related proteins(Caspase 3,cleaved Caspase 3,and BAX),and anti-apoptotic protein(BCL-2)were determined after overexpression(with mimics)or inhibition(with inhibitor)of miR-146a-3p.Furthermore,following overexpression of miR-146a-3p and knockdown of IRAK1 or TRAF6 with siRNAs,expressions of NF-?B p65,I?B?,and their phosphorylation(p-NF-?B p65,p-I?B?),and ROS emission were determined to elucidate roles and mechanisms of miR-146a-3p in DEX preconditioning-induced myocardioprotection.Results:1.DEX preconditioning can significantly reduce myocardial infarction sizes in rats with MIRI(P<0.05).Compared with those in IR group,29 miRNAs are up-regulated and 40 miRNAs down-regulated in DEX preconditioning group.DEX preconditioning upregulates relative expression of miR-146a-3p in rat myocardia from MIRI group(P<0.05).2.Bioinformatics analysis shows that 3'-UTR of IRAK and TRAF6 have binding sites of miR-146a-3p.The dual-luciferase reporter assay results denote that IRAKI and TRAF6 are target genes of miR-146a-3p.3.DEX preconditioning significantly reduces the apoptosis rates and restores cardiomyocyte vability of H9C2 cells undergoing A/R injury(P<0.05).DEX preconditioning could dramatically decreases protein expressions of IRAK1,TRAF6,and apoptosis related proteins(BAX,cleaved Caspase 3),while increases protein expression of BCL-2(P<0.05).But the expression of Caspase 3 was not in difference(P>0.05).4.DEX preconditioning can significantly upregulate relative expression of miR-146a-3p,while reduce protein expression of NF-?B p65,p-NF-?B p65,and emission of ROS(P<0.05).Furthermore,following combination of transfection with miR-146a-3p mimics and siIRAK1 or siTRAF6,DEX preconditioning lowers protein expressions of NF-?B p65,p-NF-?B p65,and p-I?B?,and emission of ROS(P<0.05).Conclusions:1.DEX preconditioning plays important roles in reducing myocardial infarction sizes derived from MIRI and lowering H9C2 cell injury due to A/R.2.Mechanisms of myocardial protective effects of DEX preconditioning may be associated with upregulating the expression of miR-146a-3p,inhibiting the expression of target proteins IRAK1 and TRAF6,and then inhibiting the activation of NF-?B signaling pathway.3.DEX preconditioning can also reduce the emission of ROS,lower levels of which reduces the activation of NF-?B,preventing myocardia from injuries.4.miR-146a-3p is a novel target leading to drug discovery in cardiovascular diseases(particlularly in conditions of ischemia or hypoxia injury).