The Protective Effect Of FBOX25 On Cardiomyocytes Injury Induced By Hypoxic/Reoxygenation

Background and Objectives:Myocardial ischemia / reperfusion injury(Iischemia/Reperfusion,I/R)refers to the process that the injury of ischemic myocardium should be aggravated with reperfusion when myocardial ischemia occurs.In recent years,with the change of people’s living habits,the motility of coronary heart disease is greatly increased.Myocardial ischemia/reperfusion injury usefully occurs when coronary heart disease is treated,and it has an important influence on treatment effect and prognosis of coronary heart disease.Therefore,how to prevent and treat myocardial I/R injury is one of the hot research fields of cardiovascular disease.It has been reported that the FBXO25(F-box protein-25),a member of F-box protein family,is an E3 ligase and forms a complex with Skp1,Cullin1,Roc1(Skp1-Cullin1-F-box,SCF).The myocardial cells of H9C2 cell line were subjected to hypoxia/reoxygenation(H/R)to mimic the myocardial ischemia/reperfusion.The role of FBXO25 protein in myocardial ischemia/reperfusion injury was studied in order to provide experimental evidence for finding new targets for the treatment of myocardial ischemia/reperfusion injury.Methods:1.The expressions of FBXO25 gene in mice: the expressions of mRNA and protein of FBXO25 were examined in different organs of adult mice using RT-PCR and Western blot analysis.2.Preparations of myocardial hypoxia/reoxygenation injury: The H9c2 cells were exposed to normal or hypoxia culture medium for 2h,4h,6h,8h,10 h and 12 h and the expressions of FBXO25 protein were detected by Western blot for determining the hypoxia time.The myocardial cells were first suffered from 4h hypoxia and then reoxygenation with 0h,3h,6h,9h,12 h and 24 h.The expressions of FBXO25 protein were detected by Western blot.3.The preparations of myocardial cells models with overexpression or knockdown of FBXO25: the plasmids with overexpression of FBXO25 or plasmids with knockdown of FBXO25 were prepared using regular methods and the plasmids were transferred into H9C2 cells for experiments.The experiments were divided into 6 groups: 1)normal control group;2)hypoxia/reoxygenation group;3)hypoxia/ reoxygenation with empty plasmid group;4)hypoxia/reoxygenation with overexpressing FBXO25 group I;5)hypoxia/reoxygenation with overexpressing FBXO25 group II;6)hypoxia/reoxygenation with knockdown of FBXO25 group.The FBXO25 proteins were detected by western blot.Lactate dehydrogenase(LDH)and MTT assay were used to detect cell injury and survival rate,respectively.4.Accesses of the possible mechanisms: the protein of reactive oxygen species(ROS)-associated protein(NOX2、NOX4、SOD2 and CAT)were examined in different group using Western blot analysis.Results1.The mRNA and protein expressions of FBXO25 were detectable in the mouse heart,liver,spleen,lung,kidney,brain and skeletal muscle and other tissues,and the FBXO25 expressions in heart and skeletal muscle were lower than that in the other tissues.2.Hypoxia/reoxygenation significantly increased H9C2 damage and mortality,and markedly reduced the level of FBXO25 expression in cardiomyocytes.3.Overexpression of FBXO25 significantly reduced myocardial cell injury and death induced by hypoxia/reoxygenation in a dose-dependent manner.In contrast,knockdown of this gene significantly increased the hypoxia/reoxygenation-induced myocardial injury and cell death.4.Overexpression of FBXO25 significantly reduced the level of NOX4 expression and markedly increased the level of CAT expression induced by hypoxia/reoxygenation.Conclusion:In the present study,we observed that hypoxia/reoxygenation could induce cell damage and cell death,and reduce FBXO25 protein expression in myocardial cells.We found that overexpression of FBXO25 could significantly reduce myocardial injury induced by hypoxia/reoxygenation in a dose-dependent manner and in contrast,knockdown of FBXO25 gene significantly promoted the myocardial cell injury and cell death induced by hypoxia/reoxygenation.In addition,the overexpression of FBXO25 significantly reduced the level of NOX4 expression and markedly increased the level of CAT expression induced by hypoxia/reoxygenation.Taken together,our results demonstrated that FBXO25 may be an endogenous cellular factor for effectively protecting hypoxia/reoxygenation injury.