Mechanisms Of The Protective Effect Of Dexmedetomidine Preconditioning Against H₂O₂-induced Cardiomyocyte Injury

[Background]Cardiomyocyte injury mainly result from the process of ischemia/reperfusion,severe cardiomyocyte injury can lead to cardiomyocyte death.Cardiomyocyte injury and death seeondary to ischemia/reperfusion is a primary cause of heart remodeling and dysfunction,known as ischemia/reperfusion?I/R?injury.Many factors?such as oxidative stress,Intracellular Ca2+ overload,and Inflammation etc.?have been validated to contribute to myocardial I/R injury.Among these factors,an increasing number of studies have underlined a fundamental role of oxidative stress in I/R injury.Oxidative stress triggers numerous negative effects on eells,causing cell necrosis and apoptosis.Apoptosis is considered to be a form of programmed cell death which is tightly controlled,therefore it has obtained much attention over the past 30 years.Necrosis has always been considered to be an accidental or passive form of cell death.However,growing evidence uncovered the existence of a form of necrosis termed necroptosis,which is precisely programmed and regulated.Oxidative stress has been validated to induce apoptosis and necroptosis.The prevention or reduction of apoptosis and necroptosis has a signifieant protective effect against I/R injury.The previous studies have indicated dexmedetomidine?Dex?could protect rat hearts against I/R injury.However,it is still unclear what the effect of Dex on oxidative stress in cardiomyocytes is.[objective]Exposing H9C2 cells to H₂O₂ to imitate oxidative stress followed by cardiac I/R injury and to identify mechanisms of the protective effect of dexmedetomidine preconditioning against H₂O₂-induced cardiomyocyte injury[Methods]1)H9C2 cardiomyocytes were exposed to different concentration of H₂O₂?100 ?M,500 ?M and 1000 ?M?for 12 hours,identified the extent of cell injury by CCK8 and LDH release assay,and chose an appropriate concentration of H₂O₂ exposure for the following experiments.H9C2 cells were pretreated with various concentrations of Dex?0.001 ?M,0.01 ?M,0.1 ?M,1 ?M and 10 ?M?for 2 hours,and then exposed to H₂O₂?500 ?M?for 12 hours,identified the effect of each concentration of Dex on cell injury by cell viability and LDH release assay at last.After the completion of the above study,employed the most effectively concentration?10 ?M?and the most extensively used concentration of Dex?0.01 ?M?to explore the underlying mechanism of the effect of which Dex ameliorated H₂O₂-induced cardiomyocyte injury.Determination of the intracellular ROS content by DCFH-DA;Cell necrosis and apoptosis were examined by PI-DAPI staining and TUNEL-DAPI assay respectively;The intracellular content of SOD was detected by ELISA and intracellular expression of GAPDH,HO-1,cleaved caspase3,RIPK1 and RIPK3 were detected by WB.2)In order to investigate whether the protective effects of Dex are related to the activation of ?2-adrenoceptor??2-AR?.YOH?1?M?was added into the cell culture medium to inhibit the activation of ?2-AR before Dex?10 ?M?preconditioning.After H₂O₂ exposure,detecting the change of CCK8,LDH release,cell necrosis and apoptosis,intracellular content of SOD and intracellular expression of GAPDH,HO-1,cleaved caspase3,RIPK1 and RIPK3.[Results]1)Accompanied with the elevation of H₂O₂ concentration,cell viability reduced and LDH release increased,Dex dose-dependently elevated cell viability and decreased LDH release.Compared with the pretreatment of Dex?0.01 ?M?,pretreated with Dex?10 uM?before H₂O₂ exposure significantly reduced the level of intracellular ROS?increased the content of SOD and HO-1,and prevented cell apoptosis?TUNEL-positive cells and expression of cleaved caspase3?and necrosis?PI-positive cells and expression of RIPK1 and RIPK3?.2)Compared to Dex preconditioning,administation of YOH prior to Dex preconditioning significantly decreased cell viability,increased LDH release and the level of intracellular ROS,reduced the content of SOD and HO-1,and increased necroptosis?PI-positive cells and expression of RIPK1 and RIPK3?,while there is no alteration in cell apoptosis?TUNEL-positive cells and expression of cleaved caspasei?[Conclusion]1.Dex dose-dependently attenuates H₂O₂-induced cardiomyocytes injury.2.Dex ameliorates H₂O₂-induced oxidative stress in cardiomyocytes by activating ?2-AR.3.Protective effect of Dex on H₂O₂-induced cardiomyocytes apoptosis is independent of the activation of ?2-AR.4.Dex prevents H₂O₂-induced cardiomyocytes necroptosis via the activation of ?2-AR.