A Study On The Protective Effect Of HIF-1/HRE Pathway On Hypoxia-reoxygenation Of Rat Cardiomyocytes After Pinacidil Postconditioning

Objective:1.To observe the protective effect of pinacidil postconditioning on hypoxic-reoxygenated cardiomyocyte injury in rats;2.To investigate the role and activation mechanism of HIF-1/HRE pathway in myocardial protection after pinacidil postconditioning.Methods:Adult rat cardiomyocytes were isolated by an in vitro cardiac perfusion device（MPA）and cultured in a 37℃incubator for 24 h.cardiomyocytes were randomly divided into normal group（group N）,hypoxia-reoxygenation group（group HR）,Pinacidil postconditioning group（group P）,5-hydroxy decanoic acid and Pinacidil postconditioning group（group 5HD+P）,N-2-mercaptopropionyl-glycine and Pinacidil postconditioning group（group MPG+P）,N-2-mercaptopropionyl-glycine and dimethyloxalyl glycine and Pinacidil postconditioning group（group MPG+DMOG+P）.Group N:continuous incubation for 160 min in an incubator;group HR:reoxygenation for 120 min after 40 min hypoxia;group P:Pinacidil treatment for 5 min after 40 min hypoxia,and then in reoxygenation for 115 minutes;group 5HD+P:5HD treatment for 10min then Pinacidil treatment for 5min after 40 min hypoxia,at last in reoxygenation for 105min;group MPG+P:MPG treatment for 10 minutes then Pinacidil treatment for 5 minutes after 40min hypoxia,at last in reoxygenation for 105 minutes;group MPG+DMOG+P:hypoxia for 40 minutes,then MPG treatment for 10 minutes,DMOG treatment for 10 minutes and Pinacidil treatment for 5 minutes,at last in reoxygenation for 95 minutes.Experimental index:（1）Observe the ultrastructure of myocardial cells in each group under transmission electron microscope at the end of reoxygenation,and analyze mitochondrial Flameng score;（2）Detect mitochondrial membrane potential（MMP）in each group at the end of reoxygenation by Laser confocal microscopy;（3）Detect the content of ROS at 25min and the end of reoxygenation by ELISA kit;（4）qRT-PCR and Western-Blot were used to measure the mRNA and protein expression of the target genes:HIF-1α,HO-1,iNOS,and VEGF.Results:1.Ultrastructure under transmission electron microscope and mitochondrial Flameng score in each group:Ultrastructure shows:The ultrastructure of cells in group N was basically normal,the injury in group HR was the most severe;compared with group P,the injury was worse in group 5HD+P and group MPG+P,there was no significant difference in ultrastructure between the two groups,group MPG+DMOG+P were same with group P;compared with group 5HD+P,the injury in group MPG+DMOG+P was significantly reduced,there was no significant difference with group MPG+P.compared with group MPG+P,the injury in group MPG+DMOG+P was significantly reduced.The mitochondrial Flameng score:The score in group N was the lowest,and group HR was the highest,（P<0.05）;compared with group P,the score in group HR,group 5HD+P and group MPG+P was significantly increased（P<0.05）,there was no significant difference among the three groups（P>0.05）,group MPG+DMOG+P increased slightly,and there was no significant difference（P>0.05）;compared with group MPG+P,group MPG+DMOG+P was significantly reduced（P<0.05）.2.Changes of cardiomyocyte MMP（red/green fluorescence ratio）:The red/green fluorescence ratio was the highest in group N and the lowest in group HR（P<0.05）;compared with group P,the ratio in group HR,group 5HD+P and group MPG+P decreased significantly（P<0.05）,there was no significant difference among the three groups（P>0.05）,group MPG+DMOG+P was slightly reduced,there was no significant difference（P>0.05）;Compared with the group 5HD+P,the MPG+P group had no statistically significant decrease（P>0.05）,and the MPG+DMOG+P group increased significantly（P<0.05）;compared with group MPG+P,group MPG+DMOG+P was increased significantly（P<0.05）.3.Changes of ROS content in cardiomyocytes:25 minutes of reoxygenation:The ROS content was the lowest in group N and the highest in group HR and group5HD+P（P<0.05）,there was no significant difference between the two groups（P>0.05）;compared with group P,the content in group MPG+P and group MPG+DMOG+P was reduced（P<0.05）,and there was no significant difference between the two groups（P>0.05）;At the end of reoxygenation:The ROS content was the lowest in group N and the highest in group HR（P<0.05）;Compared with group P,the changes of group MPG+DMOG+P had no statistically significant（P>0.05）,the content in group 5HD+P and group MPG+P was increased（P<0.05）,and there was no significant difference between the two groups（P>0.05）;the content in group 5HD+P and group MPG+P were higher than group MPG+DMOG+P significantly.Compared with the time of reoxygenation at 25minutes,there was no significant difference in ROS content in the group N,group HR,group 5HD+P and group MPG+DMOG+P at the end of reoxygenation（P>0.05）;group P decreased（P<0.05）;group MPG+P increased（P<0.05）.4.The mRNA amount of HIF-1α,HO-1,iNOS and VEGF:Expression of HIF-1αmRNA showed no obvious differences among all the groups（P>0.05）.Compared with the group N,the mRNA expression of HO-1,iNOS and VEGF increased significantly in the other groups（P<0.05）,the expression was highest in group P and MPG+DMOG+P（P<0.05）,there was no significant difference between the two groups（P>0.05）;compared with group P,the mRNA expression of HO-1,iNOS and VEGF was significantly reduced in group HR,group 5HD+P and group MPG+P（P<0.05）,and there was no significant difference among the three groups（P>0.05）;compared with group5HD+P and group MPG+P,the expression in group MPG+DMOG+P was significantly increased（P<0.05）.5.The expression of HIF-1α,VEGF,iNOS and HO-1 protein:Compared with group N,the expression of the target gene protein in the other groups was significantly increased,and the expression levels in group P and group MPG+DMOG+P were the highest（P<0.05）,there was no significant difference between the two groups（P<0.05）;compared with group P,the expression levels in group HR,group 5HD+P and group MPG+P were significantly reduced（P<0.05）;compared with group 5HD+P and group MPG+P,the expression of group MPG+DMOG+P was significantly increased（P<0.05）.Conclusion:1.The HIF-1/HRE pathway participates in the protective effect of pinacidil post-treatment on rat hypoxic-reoxygenated cardiomyocytes.2.Pinacidil postconditioning generates a certain amount of ROS as a signal molecule by opening mitoK_ATP,activates HIF-1/HRE pathway,further regulates downstream HO-1,iNOS,and VEGF genes to play the protective role in hypoxic-reoxygenated cardiomyocytes.