Research On The Mechanism Of HIF-1/HRE Pathway Involved In Diazoxide Postconditioning On Hypoxic/reoxygenated Cardiomyocytes

Objective:To observe the protective effect of diazoxide postconditioning in hypoxic/reoxygenation injury on rat cardiomyocytes,and explore the activated mechanism of HIF-1/HRE pathway in diazoxide postconditioning.Methods:1.Experiments were carried out on the SPF adult healthy SD male rats(250-300g).The isolated rat heart were perfused in a MAP model to culture adult rat cardiomyocytes.According to the intervention scheme,the cardiomyocytes were randomly divided into six groups: normal group(N),hypoxia/reoxygenation group(H/R),diazoxide postconditioning group(D),5-HD(mitoKATP blocker)+ diazoxide group(5-HD+D),MPG(active oxygen scavenger)+ diazoxide group(MPG + D)and active oxygen scavenger + HIF1 stabilizer + diazoxide group(MPG + DMOG + D).2.The intervention scheme of each group is as follows: Group N: The cardiomyocytes were cultured in an incubator under normal oxygen condition(95% air + 5% CO2)for 165min;Group H/R: The cardiomyocytes were cultured in hypoxia condition(5% CO2 + 1% O2 + 94% N2)for 45 min,then in reoxygenation condition(95% CO2 + 5% O2)for 120 min;Group D: Based on the Group H/R,the cardiomyocytes were cultured in hypoxia for 45 min,and at the start of reoxygenation,in Diazoxide [50?mol/L] for 3min.Then the cardiomyocytes were cultured in reoxygenation for 117 min;Group 5-HD+D: Based on the Group D,at the start of reoxygenation the cardiomyocytes were cultured in 5-HD [100?mol/L] for 10 min and in Diazoxide [50?mol/L] for 3min,then reoxygenation for 107 min;Group MPG+D: Based on the Group D,at the start of reoxygenation the cardiomyocytes were cultured in MPG [2mmol/L] for 10 min and in Diazoxide [50?mol/L] for 3min,then reoxygenation for 107 min;Group MPG + DMOG + D: Based on the Group D,at the start of reoxygenation the cardiomyocytes were cultured in MPG [2mmol/L] for 10 min,in DMOG[100?mol/L] for 10 min and in Diazoxide [50?mol/L] for 3min,then reoxygenation for 97 min.3.Record and measurement index3.1 Ultrastructural morphology of cardiomyocyte:The ultrastructural changes of cardiomyocyte and mitochondria were observed by transmission electron microscopy at the end of reoxygenation,and the Flamening score of mitochondrial injury was calculated;3.2 Mitochondrial membrane potential: At the end of reoxygenation,JC-1 method is used to observe the mitochondrial membrane potential by confocal microscope;3.3 ROS concentration: The concentration of ROS in cardiomyocytes was measured by ELISA at 25 min and 120 min of reoxygenation;3.4 HIF-1 ? and the downstream gene expression: At the end of reoxygenation,the mRNA and protein expression of hypoxia inducible factor-1 ?(HIF-1 ?),vascular endothelial growth factor(VEGF),apoptosis related genes(Bax and Bcl-2)were measured by qRT-PCR and Western blotting.Results:1.Electron microscope structure of cardiomyocyte1.1 The ultrastructural changes of cardiomyocytes : the ultrastructure of cardiomyocytes in group N were normal,and the ultrastructural damage in group H/R was the most serious;compared with group H/R,group D and group MPG+DMOG+D got improved obviously(P<0.05);compared with group D,group 5-HD+D and MPG+D were more damaged(P<0.05),group MPG+D was superior to group 5-HD+D(P<0.05).1.2 Flameng score of mitochondrial injury: group N got the lowest score,and the group H/R got highest;compared with group H/R,the score of group D was significant lower(P < 0.05);compared with group D,the scores of group 5-HD+D and group MPG+D were higher(P < 0.05);compared with group MPG+D,the scores of group MPG+DMOG+D was lower(P < 0.05)2.ROS concentration of cardiomyocytes measured by ELISA: At 25 min of reoxygenation: the ROS concentration was the lowest in group N.Compared with group N,ROS concentration in group MPG +D and group MPG+DMOG+D were no significantly different(P >0.05);ROS concentration in group D,group 5-HD+D,and group H/R gradually increased(P <0.05);at 120 min of reoxygenation: ROS concentration was the lowest in group N.Compared with group N,ROS concentration in group MPG+DMOG+D,group D,group MPG+D,group 5-HD+D and group H/R gradually increased(P <0.05).3.Measurement of mitochondrial membrane potential by JC-1 method: Potential got highest in group N and lowest in group H/R(P < 0.05).compared with group H/R,group D increased significantly(P < 0.05);group 5-HD+D and MPG+D got lower potential than group D(P < 0.05),and potential of group MPG + D was higher than that of group 5-HD+D(P < 0.05);group MPG+DMOG+D got higher potential than group MPG+D(P < 0.05).4.mRNA and protein expression of HIF-1? and the downstream target gene: The mRNA expression of HIF-1? were no different among all groups(P >0.05);the target gene were the least expressed in group N;Diazoxide postconditioning not only improved VEGF and Bcl-2 mRNA expression and reduced Bax mRNA expression(P<0.05),but also improved HIF-1?,VEGF and Bcl-2 protein expression and reduced Bax protein expression(P<0.05);the protective effect of Diazoxide activating HIF-1 /HRE pathway can be eliminated by mitoKATP channel blocker(5-HD)or ROS scavenger(MPG)(P<0.05);after MPG was added with HIF-1? stabilizer(DMOG),the effect of Diazoxide activating HIF-1/HRE can be restored.Conclusion:1.HIF-1/HRE pathway was involved in myocardial protection of diazoxide postconditioning ameliorating hypoxia-reoxygenation.2.The mitoKATP opened by the diazoxide postconditioning in the early stage of reoxygenation activated the HIF-1/HRE pathway.The downstream HIF-1?,VEGF,Bcl-2 and Bax genes protected cardiomyocytes from hypoxia/reoxygenation injury.3.During Diazoxide postconditioning,the suitable amount of ROS released by opened mitoKATP was the key to activate HIF-1/HRE pathway.The protective effect was depended on the content of ROS.