| Part I Expression of IncRNA H19 on myocardial ischemia-reperfusion injury and sevoflurane postconditioningObjective:To investigate the effect of sevoflurane postconditioning on myocardial I/R injury and to observe the expression of IncRNA H19 on myocardial I/R injury and sevoflurane postconditioningMethods:1.Rat isolated heart experiments:Forty-eight male SD rats weighing 200-250g were randomly divided into 4 groups(n=12):control group(group C),ischemia-reperfusion group(I/R group),ischemic postconditioning group(IPC group)and sevoflurane postconditioning group(SPC group).Langendorff isolated cardiac I/R model was prepared by ischemia for 30 min and reperfusion for 120min.the K-H liquid was continuously perfused for 150 min in group C.After 30 min of ischemia,I/R group reperfusion with K-H liquid for 120min,IPC group was given 4 cycles of 20s reperfusion/20s ischemia before K-H liquid reperfusion,SPC group was perfused with K-H liquid saturated with 2.5%sevoflurane for 15min,and then reperfusion with K-H liquid.The total reperfusion time of IPC group and SPC group was 120min.At the end of equilibrium perfusion(basic state)(TO)and at 30,60,90 and 120min of reperfusion(T1～4).,HR,LVSP,LVEDP,±dp/dtmax were measured,and LDH and CPK activity in myocardial perfusion fluid were measured by ELISA.At the end of reperfusion,myocardial infarct size was measured by TTC,apoptotic index(AI)was measured by TUNEL,and the expression of H19 on myocardial tissue was measured by real-time quantitative PCR.2.Cell experiments:H9c2 cardiomyocytes were randomly divided into 4 groups:control group(group C),anoxia/reoxygenation group(A/R group),anoxia postconditioning group(APC group)and sevoflurane postconditioning group(SPC group).The A/R injury model of H9c2 cardiomyocytes was established by anoxia and A/R device,anoxia for 3h and reoxygenation for 3h.After 3 hours of anoxia,APC group was given 3 cycles of 5min reoxygenation/5min anoxia,and then reoxygenated;SPC group was treated with 2.5%sevoflurane prefilled DMEM serum-free medium for 30min,and then reoxygenated.The total reoxygenation time of APC group and SPC group was 3h.At the end of the experiment,LDH and CPK activity in cardiomyocyte culture medium were measured by ELISA.Cell viability was detected by MTT assay,apoptosis rate was detected by flow cytometry,and the expression of H19 on cardiomyocytes was measured by real-time quantitative PCR.Results:1.Results of rat isolated heart experiment:Compared with T0,HR,LVSP and±dp/dtmax were significantly decreased while LVEDP was increased at T1～4 in all groups except group C(P<0.05);Compared with group C,HR,LVSP and ±dp/dtmax in I/R group were significantly decreased while LVEDP increased.At each time point,LDH and CPK activity in myocardial perfusion fluid were significantly increased,myocardial infarct size and apoptotic index were increased(P<0.05);the relative expression of H19 on myocardial tissue showed an upward trend,but no statistical significance(P>0.05);Compared with the I/R group,LVSP and ±dp/dtma,in IPC group and SPC group increased while LVEDP decreased at each time point,LDH and CPK activity in myocardial perfusion fluid were decreased,myocardial infarct size and apoptotic index decreased,the relative expression of H19 up-regulated(P<0.05).2.Results of cell experiment:compared with group C,LDH and CPK activity in cardiomyocyte culture medium and apoptosis rate were significantly increased,cell viability was significantly decreased in group A/R(P<0.05);the relative expression of H19 on cardiomyocytes showed an upward trend,but no statistical significance(P>0.05);Compared with A/R group,cell viability in APC group and SPC group was significantly increased,LDH and CPK activity in cardiomyocyte culture medium and apoptosis rate were significantly decreased,the relative expression of H19 up-regulated(P<0.05).Conclusion:1.The results of rat isolated heart experiment and H9c2 cardiomyocytes experiment showed that sevoflurane postconditioning can play a role in alleviating myocardial I/R injury by improving cardiac function indexes,reducing myocardial infarct size,decreasing LDH and CPK activity,decreasing apoptotic index,apoptosis rate,and improving cell viability.2.Sevoflurane postconditioning can significantly up-regulate the expression of H19,suggesting that H19 may participate in the myocardial protection of sevoflurane postconditioning.Part II Effects of down-regulation expression of lncRNA H19 on myocardial protection of sevoflurane postconditioningObjective:To investigate the effect of down-regulation of IncRNA H19 expression on myocardial protection of sevoflurane postconditioning.Methods1.Specific silencing of H19 with RNA interference technology was used to construct pGreenpuro-shlncRNA H19 recombinant plasmid,then transfected into H9c2 cardiomyocytes for interference efficiency detection.2.H9c2 cardiomyocytes were randomly divided into 5 groups:group C,A/R group,sevoflurane postconditioning group(Sevo group),sevoflurane postconditioning+ short-hairpin H19 group(Sevo+shH19 group)and sevoflurane postconditioning+short-hairpin H19 negative control group(Sevo+H19-shNC group).The A/R model of H9c2 cardiomyocytes was constructed by anoxia for 3h and reoxygenation for 3h.Sevo group was treated with 2.5%sevoflurane prefilled DMEM serum-free medium for 30min after 3h of anoxia,and then reoxygenation for 3h.In Sevo+shH19 group and Sevo+ h19-shNC group,H9c2 cardiomyocytes were transfected with interference vector shH19 or interference empty pGreenpuro plasmid 48h before A/R,the remaining steps were the same as those in Sevo group.At the end of the experiment,the activity of LDH and CPK in the cardiomyocyte culture medium was measured by ELISA,the levels of MDA,SOD and caspase-3 in cardiomyocytes were detected,and the cell viability was detected by MTT assay.The mitochondrial membrane potential and apoptosis rate were detected by flow cytometry.Mitochondria and cytoplasm of cardiomyocytes were isolated and the expressions of Cyt c in cytoplasm and mitochondria were measured by western blot.Results:1.The results showed that the effect of shH19-3 interference H19 expression was the most significant,so it was selected as the interference vector;pGreenpuro itself as the interference empty carrier.2.Compared with group C,LDH and CPK activity in cardiomyocyte culture medium were increased in A/R group,MDA content in cardiomyocytes was increased,SOD activity was decreased,caspase-3 activity and apoptosis rate were increased,the cell viability and mitochondrial membrane potential was decreased,the expression of Cyt c in mitochondrial was down-regulated while Cyt c in cytoplasm up-regulated(P<0.05);Compared with A/R group,LDH and CPK activity in cardiomyocyte culture medium were decreased in Sevo group,MDA content in cardiomyocytes was decreased,SOD activity was increased,caspase-3 activity and apoptosis rate were decreased,the cell viability and mitochondrial membrane potential was increased,the expression of Cyt c in mitochondrial was up-regulated while Cyt c in cytoplasm down-regulated(P<0.05);Compared with the Sevo group,after silencing the expression of H19,LDH and CPK activity in cardiomyocyte culture medium were increased,MDA content in cardiomyocytes was increased,SOD activity was decreased,caspase-3 activity and apoptosis rate were increased,the cell viability and mitochondrial membrane potential was decreased,the expression of Cyt c in mitochondrial was down-regulated while Cyt c in cytoplasm up-regulated(P<0.05)Conclusion:1.pGreenpuro-shlncRNAH19-3 can effectively down-regulate the expression of H19 on H9c2 cardiomyocytes.2.Sevoflurane postconditioning can play a role in protecting myocardial by reducing the oxidative stress level of cardiomyocytes,stabilizing mitochondrial membrane potential,inhibiting the mitochondrial apoptosis pathway.But the specific silencing of H19 expression eliminated the myocardial protection of sevoflurane postconditioning,indicating that the myocardial protection of sevoflurane postconditioning is mediated by H19.PartⅢ Significance of PI3K/AKT signaling pathway in H19-mediated sevoflurane postconditioning for myocardial protectionObjective:To explore the relationship between H19 and PI3K/AKT signaling pathway to elucidate the mechanism of myocardial protection of sevoflurane postconditioning.Methods1.Construction of H19 expression vector:NCBI searched for H19 gene sequence,synthesize gene fragment with enzyme cleavage site of BmHI/XbaI;then the target gene fragment was recombined with plvxpuro plasmid and verified by double enzyme digestion to obtain H19 expression vector,then transfected into H9c2 cardiomyocytes for verification,and the expression of H19 was measured by RT-PCR.2.H9c2 cardiomyocytes were randomly divided into 7 groups:Group C,A/R group,Sevo group,H19 expression vector group(H19 group),H19 expression negative control group(H19-NC group),sevoflurane postconditioning + LY294002 group(Sevo+LY294002 group)and H19 expression vector +LY294002 group(H19+LY294002 group).H9c2 cardiomyocytes were transfected with H19 expression vector or expression empty plvxpuro plasmid 48h before A/R in H19 group,H19+LY294002 group and H19-NC group.The A/R model of H9c2 cardiomyocytes was constructed by anoxia for 3h and reoxygenation for 3h.After 3h of anoxia,Sevo group was treated with 2.5%sevoflurane prefilled DMEM serum-free medium for 30min,Sevo+LY294002 group was treated with 2.5%sevoflurane prefilled and DMEM serum-free medium containing LY294002(20μmol/L)for 30min,and H19+LY294002 group was treated with DMEM serum-free medium containing LY294002(20 μmol/L)for 30min,and then reoxygenation.The total reoxygenation time was 3h.At the end of the experiment,the activity of LDH and CPK in the cardiomyocyte culture medium was measured by ELISA,the levels of MDA,SOD and caspase-3 in cardiomyocytes were detected,and the cell viability was detected by MTT assay.The mitochondrial membrane potential and apoptosis rate were detected by flow cytometry.Mitochondria and cytoplasm of cardiomyocytes were isolated,the expressions of PI3K,p-PI3K,AKT and p-AKT,Cyt c in mitochondria and cytoplasm were measured by Western blot.Results:1.H9c2 cardiomyocytes were transfected with plvxpuro-lncRNA H19 recombinant plasmid for 48h,the expression of H19 was significantly up-regulated(P<0.05,vs Control);Plvxpuro as expression empty had no effect on the expression of H19.It is suggested that the H19 expression vector was successfully constructed.2.Compared with group C,LDH and CPK activity in cardiomyocyte culture medium were increased in A/R group,MDA content in cardiomyocytes was increased,SOD activity was decreased,caspase-3 activity and apoptosis rate were increased,the cell viability and mitochondrial membrane potential was decreased,the expression of Cyt c in mitochondrial was down-regulated while Cyt c in cytoplasm up-regulated(P<0.05);The expressions of p-PI3K and p-AKT showed an upward trend,but no statistical significance(p>0.05);Compared with A/R group,LDH and CPK activity in cardiomyocyte culture medium were decreased in the Sevo group and H19 group,MDA content in cardiomyocytes was decreased,SOD activity was increased,caspase-3 activity and apoptosis rate were decreased,the cell viability and mitochondrial membrane potential was increased,the expression of Cyt c in mitochondrial was up-regulated while Cyt c in cytoplasm down-regulated,the expressions of p-PI3K and p-AKT were up-regulated(P<0.05);After administration of the PI3K specific inhibitor LY294002,LDH and CPK activity in cardiomyocyte culture medium were increased,MDA content in cardiomyocytes was increased,SOD activity was decreased,caspase-3 activity and apoptosis rate were increased,the cell viability and mitochondrial membrane potential was decreased,the expression of Cyt c in mitochondrial was down-regulated while Cyt c in cytoplasm up-regulated,the expressions of p-PI3K and p-AKT were down-regulated(P<0.05).Conclusion:1.The expression vector plvxpuro-lncRNA H19 can significantly up-regulate the expression of H19 on H9c2 cardiomyocytes,indicating that the H19 expression vector was successfully constructed.2.High expression of H19 can reduce the content of MDA,increase the activity of SOD,stabilize the mitochondrial membrane potential and inhibit the apoptosis of mitochondrial cells,indicating that high expression of H19 has a protective effect on myocardium.3.High expression of H19 can up-regulate the expression levels of p-PI3K and p-AKT proteins in cardiomyocytes,while the expression levels of p-PI3K and p-AKT proteins in cardiomyocytes are significantly down-regulated by the addition of PI3K inhibitor LY294002,suggesting that H19 may play a protective role in myocardium through the PI3K/AKT signaling pathway.In summary,sevoflurane postconditioning can up-regulate the expression of lncRNA H19,activate the PI3K/AKT signaling pathway,reduce oxidative stress,stabilize the mitochondrial membrane potential,inhibit the mitochondrial apoptosis pathway,and ultimately reduce the cell apoptosis and play a protective role in myocardium. |
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