Dexmedetomidine Protects Against H₂O₂-induced Oxidative Stress And Apoptosis Via Mitochondria-and ER-medicated Singnal Pathway In Neonatal Rat Cardiomyocytes

In recent years,dexmedetomidine?DEX?,a new highly selective?₂adrenoceptor agonist,was widely used in clinical anesthesia and the intensive care unit?ICU?because of its special advantages such as sedative,anxiolytic,sympatholytic,and analgesic-sparing effects and minimal depression of respiratory function.With the deepening of the research,many Studies have confirmed that DEX provided protective effect to many vital organs such as the myocardium,brain,liver,kidney,lung and so on through inhibiting the inflammatory reaction or regulating the immune system.While exploring for the mechanisms,most people agreed that DEX played a protective role through activating the central or peripheral?₂ adrenoceptors.There were many studies suggested that DEX can provide protection to ischemic myocardium,but a few other studies Showed that DEX may deteriorate the injury of the myocardium instead of providing protection.So,it is still a controversial issue whether DEX can attenuate myocardial ischemia/reperfusion?I/R?injury or not and how does it work.It was well known that apoptosis was a very important mechanism in the early pathophysiologic process of myocardial I/R injury.The mechanisms of apoptosis which were well known in the past included death receptor signal pathway and mitochondrial signal pathway and both of them led to apoptosis through activating cascade reaction.But in recent years,researches have shown that endoplasmic reticulum stress?ERS?was also closely related to cell apoptosis.As two crucial structures to maintain life activities in cells,mitochondria and endoplasmic reticulum would produce stress reaction when the myocardial cells encountered with oxidative damage.Researches suggested that mitochondria oxidative stress played a very important role in myocardial protection because it was the end effector of many drugs which can reduce myocardial I/R injury.A few studies have suggested that the protective effects of DEX may be related to the regulation of opening the mitochondrial permeability transition pore?m-PTP?and upstream mitochondrial ATP sensitive potassium channel(mito-K_ATP)activity in the mitochondrial membrane,but there was no systematic study reported about the roles that mitochondrial-and ER-mediated oxidative stress and apoptosis signal pathways played in the protection of DEX on cardiomyocytes.This is what we want to explore in the study.Objective:Our aim of this study was to investigate the effect of DEX on mitochondrial-and ER-mediated oxidation stress and apoptosis signal pathway in neonatal rat cardiomyocytes?NRCMs?.Methods:The original generation of cultured neonatal rats?1-3 days?cardiomyocytes were used as subjects in the study.The oxidative stress injury model was built by H₂O₂.After a normal culture of 48 hours,different drugs were added to the NRCMs and the cells were divided into the following four groups:control group?group C?:the cells were cultured with normal medium for 6 hours;H₂O₂ group?group H?:H₂O₂was added to the medium for 6 hours at a final concentration of500?mol/L;DEX group?group D?:DEX was added to the medium for 6hours at a final concentration of 5?mol/L;DEX and H₂O₂ group?group DH?:cells pretreated with DEX?5?mol/L?for 2 hours and then exposed to H₂O₂?500?mol/L?for 6 hours.In the study,we first used a fluore-scence microscopy to observe the changes of the microstructures in NRCMs.The important indexes which can reflect the lever of cellular oxidative stress such as LDH,GSH and Caspase-3,8,9 and Caspase-12were detected with ELISA.Flow cytometry?FCM?was used to measure the effect of DEX on the ROS level,apoptosis and mitochondrial membrane potential???m?of NRCMs and the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 and ERS-related proteins GRP78 and IRE1?were detected with Western blotting.Results:1.The changes of the microstructures in the NRCMs:after the treatment of drugs,compared with group C,the changes of the cellular structure was not obvious in group D;but the changes in group H and group DH were apparent because of their disordered arrangement of muscle cells horizontal stripes??-actin?and cytoskeleton?F-actin?;and compared with group H,the broken of the cellular structure was lighter in group DH.2.The activity of LDH,ROS and GSH?n=6?:Compared with group C,the levels of LDH and ROS in group H were significantly increased?P<0.05?,and the activity of GSH decreased at the same time?P<0.05?;In group D,the LDH and ROS levels decreased and the GSH activity elevated,but there was no statistical significance;and compared with group H,the levels of LDH and ROS decreased and GSH activity increased in group DH?P<0.05?.3.The activity of Caspase-3,8,9 and Caspase-12?n=6?:Compared with group C,the activity of Caspase-3,8,9 and Caspase-12 decreased but with no statistical significance in group D;and the levels of these four indicators in group H increased significantly?P<0.01?;and compared with group H,the activity of Caspase-3,8,9 and Caspase-12 decreased in group DH?P<0.05?.4.The cell apoptosis rate?n=5?:Compared with group C,the apoptosis rate change was not obvious in group D but elevated from19.36?6.38%to 57.49?10.52%?P<0.05?in group H;And compared with group H,the apoptosis rate dropped to39.86±9.35%in group DH?P<0.05?.5.The mitochondrial membrane potential???m??n=5?:Compared with group C,the change of the mitochondrial membrane potential was not obvious in group D but there was a depolarization in group H?P<0.01?;And compared with group H,the depolarization of mitochondrial membrane potential in group DH was significantly inhibited?P<0.05?.6.The expression of Bax and Bcl-2?n=5?:Compared with group C,the ratio of pro-apoptotic molecule Bax and the anti-apoptotic protein Bcl-2?Bax/Bcl-2?elevated in group H?P<0.05?;Compared with the group H,the ratio of Bax/Bcl-2 decreased in group DH?P<0.05?.7.The expression of GRP78 and IRE1??n=5?:Compared with group C,the ERS-related proteins GRP78 and IRE1?expression increased signify-cantly in group H?P<0.05?;And compared with group H,the expression of GRP78 and IRE1?decreased significantly in group DH?P<0.05?.Conclusion:1.DEX can inhibite partially the depolarization of mitochondrial membrane potential and the reduced activity of GSH and the increased activity of LDH,ROS,Caspase-3,8,9 and the rising ratio of Bax/Bcl-2and cell apoptosis rate induced by H₂O₂ in NRCMs.2.DEX can recede the increased activity of Caspase-12 and the increased expression of GRP78 and IRE1?caused by H₂O₂ in NRCMs.3.DEX protects NRCMs from H₂O₂-induced oxidative stress and apoptosis via inhibiting the mitochondria-and ER-mediated oxidative stress and apoptosis signal pathways.