| 1 BackgroundAlthough patients with myocardial infarction obtain benefits from intervention therapies,sudden blood flow in the tissue after a quite long period of ischemia leads to irreversible injury on cardiomyocytes,which impairs the recovery of myocardium function.Mesenchymal stem cells(MSCs)is a promising candidate of cell-transplant therapy,and paracrine factors secreted from MSCs play the most important role for the protective effects.Application of secretions of MSCs rather than MSCs themselves could avoid some of the limitation of cell therapy,and reduce side effects and costs.PKC epsilon plays an important role in protective effects of cardiac ischemia preconditioning,and PKC epsilon becomes an essential target of preventing isehemia/reperfusion injury.2 ObjectiveIn this study,we tried to demonstrate protective effects of MPF(MSC paracrine factors)in ischemia/reperfusion-injured myocardium.And we tried to reveal the role of PKCε in MPF-induced protection.3 Methods3.1 We compared protective effects of injection of MPF 2 mg/kg,5 mg/kg and 10 mg/kg with 10 mg/kg FibroPF(fibroblast paracrine factors)via tail vein.We applied Evan,s blue/TTC staining at 24 hours after reperfusion,TUNEL staining at 2 day and echocardiography assessment at 7 day after reperfusion so that we demonstrate the effective dosage and cardio-protection of MPF.We also used western blot to investigate the phosphorylation and translocation of PKCe after MPF treatment.3.2 We investigate apoptosis and ROS level in hypoxia/reoxygenation-injured cardiomyocytes with or without MPF treatment.Meanwhile,we applied εV1-2,a PKCεtranslocation inhibitor,to investigate the role of PKCε in MPF-induced protection in vitro.We also tested the activity of ALDH2 and 4HNE level in mitochondria after reoxygenation injury.3.3 We treated MPF on PKCe dominant negative(PKCε-DN)mice to investigate the importance of PKCe in MPF-induced protection in vivo.We also investigated 4HNE level in mitochondria isolated from myocardium to demonstrate the anti-oxidative stress effects of MPF in PKCε-DN mice.3.4 We tested NO level and phosphorylation of eNOS and expression of NOS in reoxygenation-injured cardiomyocytes with or without MPF treatment.Meanwhile,we applied L-NAME,a NOS inhibitor,and ebselen,a peroxynitrite scavenger,to investigate the mechanism of MPF-induced PKCε translocation.4 Results4.1 Injection of MPF 5 mg/kg via tail vein reduced infarct size,ameliorated cardiac function and reduced apoptosis of cardiomyocytes after reperfusion.MPF activated PKCe phosphorylation and induced PKCε translocation from cytoplasm to mitochondria.4.2 MPF reduced apoptosis and oxidative stress in reoxygenation-injured cardiomyocytes and increase ALDH2 activity and 4HNE level in mitochondria.εV1-2 attenuated MPF-induced protective effects.4.3 Increased infarct size and apoptosis,and reduction of cardiac function was observed in PKCε dominant negative mice treated with MPF.Compared with white type mice,4HNE in mitochondria isolated from myocardium was increased in PKCε-DN mice after MPF treatment.4.4 Phosphorylation of eNOS and production of NO was increased in reoxynation-injured myocytes after MPF treatment,whereas little difference was observed in expression of nNOS and iNOS.Both L-NAME and ebselen attenuated MPF-induced PKCs translocation into mitochondria.§ ConclusionOur study indicated that MPF protect myocardium from ischemia/reperfusion injury and PKCs translocation plays an essential role in MPF-induced protective effects.Our results also suggested that eNOS/NO pathway possibly contributes to MPF-induced PKCε translocation. |
