| Background The reactive oxygen species(ROS)burst followed myocardial ischemia reperfusion was a contributing factor to myocardial ischemia/reperfusion injury(MI/RI).Although mi R-327 put protective effect on cardiomyocytes as described previously,the potential mechanism still needs further exploration.Objective The aim of this study is to investigate the role and mechanism of mi R-327 on oxidative stress in MI/RI process.Methods In vitro: In the hypoxia/reoxygenation(H/R)injury model,H9C2 rat cardiomyocytes were randomly divided into five groups: Control group,H/R group,Ad-EGFP-NC+H/R(Ad-NC)group,Ad-mi R-327-RNAi+H/R(Ad-mi R-327i)group and Ad-mi R-327+H/R(Ad-mi R-327)group.In the oxidative damage model induced by tert-butyl hydrogen peroxide(TBHP),H9C2 cardiomyocytes were randomly divided into four groups:Control group,TBHP group,Ad-EGFP-NC+TBHP(Ad-NC)group and Ad-mi R-327-RNAi+TBHP(Ad-mi R-327i)group.After H9C2 cells were successfully transfected with recombinant adenovirus,the H/R model(hypoxia 4 h,reoxygenation 2 h)and/or TBHP model(TBHP stimulated for 4 h)were established.The cell viability was measured by CCK-8 assay.The superoxide dismutase(SOD)activity,malondialdehyde(MDA)concentration,glutathione peroxidase(GSH-Px)activity and lactate dehydrogenase(LDH)level were measured using thermo scientific microplate reader.Intracellular ROS generation was detected by fluorescence confocal microscope.The m RNA levels of mi R-327 and FGF10 were determined by quantitative real-time polymerase chain reaction(q PCR).The protein expression levels of FGF10,p-PI3 K,t-PI3 K,p-Akt,t-Akt,nuclear Nrf2,HO-1,Bax,Bcl-2and cleaved Caspase-3 were detected by Western blot.In vivo: Seventy healthy male SD rats were randomly divided into five groups:(1)Sham group: normal nonischemic;(2)I/R group: myocardial I/R;(3)Ad-NC group: myocardial I/R with Ad-EGFP-NC;(4)Ad-mi R-327 i group: myocardial I/R with Ad-mi R-327-inhibition;(5)Ad-mi R-327 group: myocardial I/R with Ad-mi R-327.After successfully transfected with adenovirus through myocardial multi-point injection,the MI/RI model was established by ligation of left anterior descending coronary artery for 30 min,and then reperfused for 2 hours.The lactate dehydrogenase(LDH)level was detected by automatic biochemical analyzer.Fluorescent probe dihydroethidium(DHE)was used to evaluate superoxide production in the reperfusion area of left ventricle myocardium.Oxidative stress and cardiomyocytes injury were detected in rats model of MI/RI.The m RNA levels of mi R-327 and FGF10 were detected by q PCR,and the expression levels of signaling pathway and apoptosis related proteins in myocardial tissues were determined by Western blot.Results 1.In vitro:(1)H/R induced oxidative stress and mi R-327 expression was up-regulated: compared with Control group,the H/R group showed the lower levels of the antioxidant enzymes SOD and GSH-Px,higher MDA concentrations,along with a significant increase in mi R-327 expression(P<0.05).(2)Down-regulation of mi R-327 repressed H/R-induced oxidative stress injury in H9C2 cells: in Ad-mi R-327 i group,the mi R-327 expression was obviously decreased,while the cell viability was restored,the level of LDH activity decreased,the levels of SOD and GSH-Px increased,the MDA concentration decreased,and the intracellular ROS level significant decreased(P < 0.05).(3)Down-regulation of mi R-327 promoted activation of FGF10/Akt/Nrf2 signaling pathway under H/R treatment: in the Ad-mi R-327 i group,the FGF10 m RNA and protein levels were significantly increased,the phosphorylation levels of PI3 K,Akt and the protein expression levels of nucleus Nrf-2,HO-1 were remarkably increased.Meanwhile,the expression levels of pro-apoptotic related protein cleaved Caspase-3 were decreased(P<0.05).(4)TBHP induced mi R-327 up-regulation and oxidative stress in H9C2 cells: The cell activity decreased to about 58.84±4.10%,intracellular ROS level was significantly increased,and mi R-327 expression level was increased after THP stimulation(P<0.05).(5)The oxidative stress injury in H9C2 cells induced by TBHP was inhibited by mi R-327 down-regulation: compared to Control group,the cell viability of the Ad-mi R-327 i group elevated,while the level of LDH activity decreased,the SOD and GSH-Px activity increased,the MDA concentration and the intracellular ROS level were obviously decreased(P<0.05).(6)FGF10/Akt/Nrf2 signaling pathway was activated by mi R-327 down-regulation under TBHP stimulation: The protein expression levels of FGF10,p-PI3 K,p-Akt,nuclear Nrf-2 and HO-1 were significantly increased in the Ad-mi R-327 i group,whereas cleaved caspase-3 were decreased.2.In vivo:(1)Down-regulation of mi R-327 suppressed myocardial I/R-induced oxidative stress: The mi R-327 expression was obviously increased in the I/R group compared to Sham group,and decreased in the Ad-mi R-327 i group compared to Ad-NC group(P<0.05).In the Ad-mi R-327 i group,the activities of SOD and GSH-Px in myocardial increased,the MDA concentration decreased,and the proportion of ROS positive cells stained by DHE decreased(P<0.05).(2)Down-regulation of mi R-327 alleviated MI/RI in rats: The serum LDH level was decreased and the myocardial infarction size was reduced in the Ad-mi R-327 i group when compared to Ad-NC group(P<0.05).(3)Down-regulation of mi R-327 promoted the activation of FGF10/Akt/Nrf2 signaling pathway under I/R induction: In line with the trend of in vitro results,the protein expression levels of FGF10,p-PI3 K,p-Akt,nucleus Nrf-2 and HO-1 in myocardial tissue were significantly increased,while the expression levels of cleaved Caspase-3 was reduced in Ad-mi R-327 i group.Conclusion Inhibition of mi R-327 could alleviate oxidative stress induced by MI/RI.And the mechanism mediates this effect was possibly by regulating FGF10/Akt/Nrf2 pathway,which could be a promising therapeutic agent for MIRI. |
PDF Full Text Request