Roles Of Aquaporin-4(AQP-4) And Adenosine Receptor(ADR) In The Protection Of Remifentanil Post-conditioning On Human Cardiac Myocytes After Hypoxia/Reoxygenation Injury

Objective To investigate the protective effect of remifentanil post-conditioning on human cardiac myocytes（HCM）hypoxia/reoxygenation（H/R）injury by establishing an in vitro model of H/R injury in HCM,and further to explore the role of aquaporin-4（AQP-4）in this process.To observe the effect of adenosine receptor（ADR）in the protective mechanism of HCM after remifentanil post-conditioning.Methods HCM from ventricle of adult heart were purchased from Promo Cell Gmb H,which were cultured to 4～10 generations for subsequent experiments.According to the research designs and requirements,the specific groups were these:（1）Control group（CON group）:HCM were cultured in incubator with 5%CO₂ at 37℃for 8 h+2 h.（2）Hypoxia/Reoxygenation group（H/R group）:HCM were subjected to 8 h of hypoxia in hypoxystation and 2 h of reoxygenation in incubator with 5%CO₂.（3）Hypoxia/Reoxygenation+0.1,1.0 or 10.0μM Remifentanil post-conditioning group（H/R+0.1,1.0 or 10.0μM Rem group）:HCM were subjected to 8 h of hypoxia and 2 h of reoxygenation,and then remifentanil（0.1,1.0 or 10.0μM）was given 10 min before reoxygenation until the end,respectively.（4）AQP-4 inhibitor group（H/R+TGN group）:HCM were subjected to 8 h of hypoxia and 2 h of reoxygenation,furthermore,an AQP-4 specific inhibitor,TGN-020（10μM）was given 15 min before reoxygenation until the end.（5）AQP-4 inhibitor+Remifentanil post-conditioning group（H/R+Rem+TGN group）:HCM were subjected to 8 h of hypoxia and 2 h of reoxygenation,furthermore,TGN-020（10μM）and remifentanil（10μM）were given 15 min and 10min before reoxygenation until the end,respectively.（6）Adenosine receptor（ADR）antagonist group（H/R+Theo group）:HCM were subjected to 8 h of hypoxia and 2 h of reoxygenation,furthermore,an ADR non-selective antagonist,theophylline（1 m M）was given 15 min before reoxygenation until the end.（7）ADR antagonist+Remifentanil post-conditioning group（H/R+Rem+Theo group）:HCM were subjected to 8 h of hypoxia and 2 h of reoxygenation,furthermore,theophylline（1 m M）and remifentanil（10μM）were given 15 min and 10 min before reoxygenation until the end,respectively.HCM viability was measured by cell counting kit-8（CCK-8）assay.Annexin V-FITC/PI apoptosis detection kit and flow cytometry were used to detect HCM apoptosis rate.Intracellular Ca²⁺level was assessed by using Fluo-3 AM fluorescence probe.Western blot was used to analyze the expression level of AQP-4,phosphorylated and total extracellular signal-regulated kinase（p-,t-ERK1/2）.Results（1）The comparison of HCM viability:Compared with CON group,HCM viability was decreased in H/R group（P<0.01）.Compared with H/R group,HCM viability was improved in H/R+1.0μM Rem group,H/R+10.0μM Rem group and H/R+TGN group（all P<0.01）,but there were no significant differences in H/R+0.1μM Rem group and H/R+Theo group（all P>0.05）.HCM viability in H/R+10.0μM Rem group was higher than that in H/R+1.0μM Rem group（P<0.05）.（2）The comparison of HCM apoptosis rate:Compared with CON group,HCM apoptosis rate in H/R group was increased（P<0.01）.Compared with H/R group,HCM apoptosis rate was decreased in H/R+1.0μM Rem group and H/R+10.0μM Rem group（all P<0.01）,but there were no significant differences in H/R+0.1μM Rem group,H/R+TGN group and H/R+Theo group（all P>0.05）.（3）The comparison of intracellular Ca²⁺level:Compared with CON group,the level of intracellular Ca²⁺in H/R group was increased（P<0.01）.Compared with H/R group,the level of intracellular Ca²⁺were both decreased in H/R+1.0μM Rem group and the H/R+10.0μM Rem group（all P<0.05）,while the H/R+0.1μM Rem group was not statistically significant（P>0.05）.（4）The comparison of AQP-4expression level:Compared with CON group,the expression level of AQP-4 in H/R group was increased（P<0.01）.Compared with H/R group,the expression level of AQP-4 was decreased in H/R+Rem group（P<0.01）,but there was no change in H/R+Theo group（P>0.05）.The level of AQP-4 in H/R+Rem+Theo group was higher than that in H/R+Rem group（P<0.05）.（5）The comparison of ERK1/2 expression level:Compared with CON group,the expression level of p-ERK1/2 in H/R group was decreased（P<0.01）.Remifentanil post-conditioning could increase the expression level of p-ERK1/2（P<0.05）.However,the application of Theophylline could not change the level of p-ERK1/2（P>0.05）.There were no significant differences in the expression level of t-ERK1/2 among different groups（P>0.05）.Conclusions（1）Remifentanil post-conditioning could alleviate HCM H/R injury.（2）AQP-4 might be involved in this effect which might be mediated by ERK1/2 signaling pathway.（3）There might be cross-linking effect between ADR and opioid receptors,which together regulated the expression level of AQP-4,thus achieving anti-hypoxia/reoxygenation injury effect.