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Dexmedetomidine post-treatment reduces hypoxia/reoxygenation injury in primary cultured neonatal rat cardiomyocytesObjective:To observe the effects of dexmedetomidine post-treatment on hypoxia/reoxygenation(H/R)-induced injury in primary cultured neonatal rat cardiomyocytes and investigate the underlying mechanisms.Methods:(1)Extraction and primary culture of neonatal rat cardiomyocytes.Sprague Dawley(SD)rats within 48 h of newborn were used for extraction and primary culture of cardiomyocytes,and the purity of the extracted cardiomyocytes was identified by immunofluorescence staining.(2)Effects of gradient concentration of dexmedetomidine on normal primary cultured cardiomyocytes.Cells were randomly divided into 5 groups(n=6)and treated with 0,0.1,1,10,and 50?M dexmedetomidine for 24 h,respectively.Cell cytotoxicity of different concentrations of dexmedetomidine was tested.(3)Effects of different H/R time on the viability of primary rat cardiomyocytes.Cells were randomly divided into 7 groups(n=6),and an oxygen glucose deprivation/reoxygenation(OGD/R)model was established.To determine the time point of cell hypoxia and reoxygenation,the cell counting kit-8(CCK-8)kit was used to detect the cell viability after hypoxia for 3,6,and 12 h followed by reoxygenation for 3,6,12,and 24 h.(4)Effects of gradient concentration of dexmedetomidine post-treatment on cell viability and cytotoxicity after H/R injury in primary rat cardiomyocytes.Cells were randomly divided into 5 groups(n=6),and dexmedetomidine of 0.1,1,10?M was added to the culture medium at the beginning of reoxygenation,respectively.To determine the optimal concentration of dexmedetomidine post-treatment in this experiment,CCK-8 and lactate dehydrogenase(LDH)kits were used to detect the cell viability and cytotoxicity.(5)Effects of dexmedetomidine post-treatment on apoptosis of primary rat cardiomyocytes during H/R injury.Cells were randomly divided into 3 groups(n=6):control group,H/R group,and H/R+DEX group.At the beginning of reoxygenation,dexmedetomidine 1?M was added to the culture medium.After the end of reoxygenation,the rate of cell apoptosis was measured by using the Annexin V-FITC/PI staining and flow cytometry.(6)Effects of dexmedetomidine post-treatment on the protein expression levels of HIF-la and apoptosis-related genes BCL-2 and BAX in primary rat cardiomyocytes during H/R injury.Cells were randomly divided into 3 groups(n=6):control group,H/R group,and H/R+DEX group.Western Blot was used to detect the protein expression of HIF-1?,BCL-2,and BAX.(7)Effects of dexmedetomidine post-treatment on intracellular localization of HIF-1? and BAX of primary rat cardiomyocytes during H/R injury.Cells were randomly divided into 3 groups(n=6):control group,H/R group,and H/R+DEX group.The immunofluorescence was used to detect the localization and expression levels of HIF-1?and BAX proteins in cardiomyocytes.The above data were expressed as mea±standard error of the mean,and the statistical analyses were performed by using the GraphPad 7 software.Results:(1)Primary rat cardiomyocytes were cultured at normal conditions for 72 h.The purity of cardiomyocytes was 98%by using immunofluorescence staining,which was used in the following cell experiments.(2)Dexmedetomidine 0.1,1,10?M had no significant effect on the viability of normally cultured cells,but high concentration of dexmedetomidine(50?M)reduced cell viability to 82.42±2.39%(P<0.01)compared to the control group,indicating the cytotoxicity of dexmedetomidine with a concentration of higher than 50?M on cardiomyocytes.(3)Cell viability significantly decreased at different time points from 3 to 12 h of hypoxia and 3 to 24 h of reoxygenation compared with the control cells(P<0.01).With 63.21±1.74%cell viability of the control group,6 h of hypoxia and 3 h of reoxygenation was selected for the subsequent cell experiments.(4)Compared with the H/R group,0.1,1,10?M dexmedetomidine post-treatment significantly improved cell viability and reduced LDH release after H/R injury(P<0.01).Among these,1?M dexmedetomidine had the best effects.With cell viability of 75.21±2.23%vs.60.00±1.11%(P<0.01)and LDH of 200.8±7.37%vs.267.5±5.57%(P<0.01)compared to the H/R group,1?M dexmedetomidine was used in the subsequent cell experiments.(5)Flow cytometry showed that compared with the control group,the process of H/R significantly increased the cell apoptosis rate to 38.60±0.90%(P<0.01).Dexmedetomidine post-treatment reduced H/R-induced cell apoptosis to 20.94±0.96%(P<0.01).(6)Western Blot results showed significantly increased HIF-1? and BAX protein expression(P<0.01)and decreased BCL-2 protein level and BCL-2/BAX ratio in the H/R group(P<0.01).Dexmedetomidine post-treatment significantly reduced HIF-la and BAX protein levels(P<0.01)and restored BCL-2 protein expression(P<0.01)and the BCL-2/BAXratio(P<0.01).(7)The results of immunofluorescence experiments showed the colocalization of HIF-1?and BAX proteins in primary cardiomyocytes.In the H/R group,the levels of HIF-1? and BAX fluorescence intensity were significantly higher than that of the control group(P<0.01).Dexmedetomidine post-treatment reduced the fluorescence of HIF-la and BAX during H/R injury(P<0.01).Conclusion:Post-treatment with 1?M dexmedetomidine significantly reduced H/R injury in primary cultured rat cardiomyocytes,evidenced by reduced cell viability and cytotoxicity and inhibition of cell apoptosis.Regarding the mechanism,the protective effects of dexmedetomidine post-treatment against H/R-induced injury at the cellular level was likely to be related to inhibition of HIF-la protein expression,regulation of the expression of apoptosis-related proteins BCL-2 and BAX and the ratio of BCL-2/BAX,and inhibition of cell apoptosis.Part ? Molecular mechanism of dexmedetomidine post-treatment to reduce hypoxia/reoxygenation-induced apoptosis in cardiomyocytes by inhibiting HIF-la at the post transcriptional levelObjective:To further explore the underlying mechanism of dexmedetomidine post-treatment to reduce hypoxia/reoxygenation injury in primary cultured neonatal rat cardiomyocytes by regulating transcriptional activity of HIF-1? and inhibiting apoptosis.Methods:(1)Effects of HIF-1? prolyl hydroxylase 2(PHD2)inhibitor IOX2 on the expression of HIF-la and apoptosis-related proteins in primary rat cardiomyocytes under normoxic and H/R conditions.Cells were randomly divided into 4 groups(n=6):control group,control+IOX2 group,H/R group,and H/R+IOX2 group.IOX2,a specific inhibitor of HIF-1? PHD2,can inhibit the degradation of HIF-1? and enhance its transcriptional activity.IOX2 50?M was added to the culture medium before the use of dexmedetomidine.The protein expression of HIF-1?,Bcl-2/adenovirus E1B 19kDa interacting protein 3(BNIP3),and cleaved caspase-3 were tested by using Western Blot.(2)Effects of IOX2 on cell viability of dexmedetomidine post-treated primary cardiomyocytes during H/R injury.Cells were randomly divided into 6 groups(n=6):control group,control+IOX2 group,H/R group,H/R+DEX group,H/R+DEX+IOX2 group,and H/R+DEX+vehicle group.The vehicle was 0.1%dimethyl sulfoxide(DMSO),which was used to dissolve IOX2.CCK-8 kit was used to detect the viability of cardiomyocytes in each group.(3)Effects of IOX2 on mitochondrial membrane potential in dexmedetomidine post-treated primary cardiomyocytes during H/R injury.Cells were randomly divided into 5 groups(n=6):control group,H/R group,H/R+DEX group,H/R+DEX+IOX2 group,H/R+DEX+vehicle group.JC-1 fluorescence staining was used to detect the mitochondrial membrane potential(??m)of cardiomyocytes in each group.In the healthy mitochondrial membrane,JC-1 aggregates show red fluorescence under a fluorescence microscope;while in the apoptotic cells with decreased ??m,JC-1 monomers show green fluorescence.The relative values of ??m were obtained by analyzing the ratio of red/green fluorescence of JC-1.(4)Effects of IOX2 on the expression of HIF-1? and apoptosis-related proteins in dexmedetomidine post-treated primary cardiomyocytes during H/R injury.Cells were randomly divided into 4 groups(n=6):H/R group,H/R+DEX group,H/R+DEX+IOX2 group,and H/R+DEX+vehicle group.Western Blot was used to detect the protein expression of HIF-1?,BCL-2,BAX,BNIP3,cleaved caspase-3,and cleaved poly ADP-ribose polymerase 1(PARP-1).(5)Effects of dexmedetomidine post-treatment on the mRNA expression levels of HIF-1?and its target gene BNIP3 during H/R injury in primary cardiomyocytes.Cells were randomly divided into 3 groups(n=6):control group,H/R group,and H/R+DEX group.Quantitative polymerase chain reaction(qPCR)was used to detect the mRNA expression levels of HIF-1? and its target gene BNIP3 in each group.(6)Normally primary cultured cardiomyocytes were transfected with plasmid to confirm the activation of BNIP3 promoter by HIF-1?.The HIF-1? DNA was synthesized and constructed into the plasmid vector pLenti-GIII-CMV-GFP-2A-Puro;and the BNIP3 promoter and BNIP3 promoter mutants were synthesized and constructed into a dual luciferin reporter plasmid vector pLenti-miniCMV-RenLuc-PGK-Luc-SV40-GFP-2A-Puro.Cells were randomly divided into 4 groups(n=6):BNIP3 promoter group,BNIP3 promoter+empty plasmid group,BNIP3 promoter+HIF-1? plasmid group,and BNIP3 promoter mutation+HIF-1? plasmid group.After cells were transfected with plasmids for 24 h,the expression of green fluorescent protein(GFP)was observed under a fluorescent microscope,and the activation of BNIP3 promotor by HIF-1? was detect by using a dual luciferase reporter gene kit.(7)Effects of IOX2 on the activity of BNIP3 promoter in dexmedetomidine post-treated primary cardiomyocytes during H/R injury.Cells were randomly divided into 6 groups(n=6):control group,control+DEX group,H/R group,H/R+DEX group,H/R+DEX+IOX2 group,and H/R+DEX+vehicle group.After cells were transfected with the reporter gene BNIP3 promoter for 24 h,cells were treated with H/R,DEX,and IOX2.The dual luciferase reporter gene kit was used to detect the activity of the BNIP3 promoter.The above data were expressed as mean±standard error of the mean,and the statistical analyses were performed by using the GraphPad 7 software.Results:(1)Western Blot results showed that IOX2 significantly increased the protein expression level of HIF-1? in normally cultured primary cardiomyocytes to 254.4±22.37%compared to the control group(P<0.01).During H/R,IOX2 still tended to increase HIF-1?,but the difference was not statistically significant(431.1±37.86%vs.353.2±35.61%,P>0.05).In addition,IOX2 did not affect the protein expression of BNIP3 or cleaved caspase-3 in normally cultured cells or during H/R.These results indicated that IOX2 could effectively stabilize the expression level of HIF-1? without increasing apoptosis.(2)IOX2 had no significant effect on the viability of normally cultured cardiomyocytes.H/R caused a significant decrease in cell viability to 60.26±2.17%compared to the control group(P<0.01).Post-treatment with 1?M dexmedetomidine increased the cell viability to 77.72±1.63%(P<0.01),while IOX2 decreased the cell viability to 63.10±2.36%(P<0.01).Therefore,IOX2 reversed the effects of dexmedetomidine post-treatment,indicating that dexmedetomidine post-treatment reduced H/R-induced decrease in cell viability by regulating HIF-1?.(3)JC-1 staining results showed that H/R significantly reduced the mitochondrial membrane potential(??m)in primary cardiomyocytes,with a higher ratio of green JC-1 monomer/red JC-1 aggregates.Post-treatment with 1?M dexmedetomidine attenuated the decrease in ??m,resulting in an increase in red JC-1 aggregates and a decrease in green JC-1 monomers.However,IOX2 reversed the effects of dexmedetomidine on ??m(P<0.01).These results suggested that dexmedetomidine post-treatment could alleviate mitochondrial structure damage during H/R-induced cell apoptosis by regulating HIF-1?.(4)Western Blot showed that dexmedetomidine post-treatment significantly reduced HIF-1?,BNIP3,cleaved caspase-3,and cleaved PARP-1 protein expression in primary cardiomyocytes during H/R(P<0.01),and increased the ratio of BCL-2/BAX protein(P<0.01).IOX2 reversed the effects of dexmedetomidine on these proteins(P<0.05 or P<0.01).These results confirmed that dexmedetomidine post-treatment reduced H/R-induced changes in apoptosis-related proteins by regulating HIF-1?.(5)The qPCR results showed that HIF-la and BNIP3 mRNA expression levels were significantly increased during H/R(P<0.01).Post-treatment with 1?M dexmedetomidine significantly reduced H/R-induced BNIP3 activation(P<0.01),but did not significantly change the mRNA level of HIF-1?.These results suggested that dexmedetomidine post-treatment inhibited the activation of HIF-1? target gene BNIP3 at the post-transcriptional level.(6)After normally cultured cardiomyocytes were transfected with the BNIP3 promoter/promoter mutant and HIF-la plasmids for 24 h,the dual luciferase reporter gene results showed that the luciferase activity of the BNIP3 promoter+HIF-1? plasmid group was significantly increased(P<0.01),BNIP3 promoter mutation significantly reduced the luciferase activity(P<0.01).These results indicated that the synthetic BNIP3 promoter could be successfully activated by HIF-1? DNA.(7)The results of dual luciferase reporter gene showed significantly increased luciferase activity and activated BNIP3 promoter during H/R(P<0.01).Post-treatment with 1?M dexmedetomidine reduced theH/R-induced high luciferase activity(P<0.01).However,IOX2 blocked the inhibitory effect of dexmedetomidine on BNIP3 promoter activity(P<0.01).These results confirmed that dexmedetomidine post-treatment inhibited H/R-induced BNIP3 promoter activation by regulating the transcriptional activity of HIF-1?.Conclusion:The specific HIF-1? prolyl hydroxylase-2 inhibitor IOX2 stabilized the protein expression level of HIF-1?,without adverse effects on cell viability and apoptosis of primary cultured neonatal rat cardiomyocytes.Dexmedetomidine post-treatment improved cell viability,restored mitochondrial membrane potential and mitochondrial structural damage,regulated apoptosis-related proteins BCL-2,BAX,BNIP3,cleaved caspase-3,and cleaved PARP-1,and inhibited the activation of HIF-1? target gene BNIP3 at the post-transcriptional level.However,the use of IOX2 reversed the above-mentioned effects and blocked the protective effects of dexmedetomidine post-treatment on H/R-induced injury in primary cardiomyocytes.Based on these mechanism studies,dexmedetomidine post-treatment could alleviate H/R-induced injury at the cellular level by inhibiting the activation of downstream target genes and suppressing apoptosis through acting on the key target of HIF-1?.Part ? Dexmedetomidine post-treatment reduces myocardial ischemia/reperfusion injury in rats by inhibiting apoptosis through regulating HIF-la signalingObjective:To determine whether dexmedetomidine post-treatment exerts protective effects against ischemia/reperfusion(I/R)-induced myocardial injury through regulating HIF-1? signaling in a rat myocardial I/R injury model.Methods:(1)Changes of HIF-1? protein expression over reperfusion time during myocardial I/R injury in rats.Twenty-four male SD rats were randomly divided into 4 groups(n=6):sham group,and reperfusion 2,6,and 24 h groups.For the myocardial I/R injury model,rats underwent left anterior descending artery(LAD)ligation for 30 min,after which the ligature was released for myocardial reperfusion.Sham rats underwent only thoracotomy but not LAD ligation.Western Blot was used to detect the expression level of HIF-1? protein in left ventricular tissues at 2,6,and 24 h after reperfusion,and the time at which the expression changed most significantly was selected as the optimal time point for the subsequent rat experiments.(2)Effects of dexmedetomidine post-treatment and IOX2 on heart rate in rats during myocardial I/R injury.Electrocardiogram changes in rats were monitored by using the MedLab biomedical signal processing system.Fourty-two male SD rats were randomly divided into 7 groups(n=6):sham group,sham+DEX group,I/R group,I/R+IOX2 group,I/R+DEX group,I/R+DEX+IOX2 group,and I/R+DEX+vehicle group.The vehicle is 5%dimethyl sulfoxide(DMSO)+30%polyethylene glycol 300(PEG300)for dissolving IOX2.At the beginning of reperfusion,a loading dose of dexmedetomidine 6?g/kg/h was administered via the right internal jugular vein for 10 min,followed by a maintenance infusion of 0.7?g/kg/h for 15 min.In the IOX2 group,25?g/mg IOX2 was given intraperitoneally before dexmedetomidine use.Heart rates were recorded at baseline,15 and 30 min of ischemia,and 15,30,and 60 minutes of reperfusion.In addition,the area under the curve of heart rate was analyzed to observe the overall changes during this period.(3)Effects of dexmedetomidine post-treatment and IOX2 on arterial blood gas,electrolytes,and acid-base balance during myocardial I/R injury in rats.In the above-mentioned 7 groups of rats,blood samples were taken from the abdominal aorta 6 h after reperfusion,and the arterial blood pH,partial pressure of oxygen(PaO2),partial pressure of carbon dioxide(PaCO2),arterial oxygen saturation(SaO2),hemoglobin(Hb),hematocrit(Hct),Na+,K+,Ca2+,Cl-,HCO3-,base excess(BE)were analyzed.(4)Effects of IOX2 on serum cardiac troponin I(cTnI)during myocardial I/R injury in rats treated with dexmedetomidine.Thirty-six male SD rats were randomly divided into 6 groups(n=6):sham group,sham+DEX group,I/R group,I/R+DEX group,I/R+DEX+IOX2 group,and I/R+DEX+vehicle group.To determine the degree of myocardial damage,blood samples were taken from the inferior vena cava at 6 h after reperfusion,and the cTnI levels were tested by using an enzyme linked immunosorbent assay(ELISA).(5)Effects of IOX2 on myocardial tissue apoptosis after myocardial I/R injury in dexmedetomidine-treated rats.The left ventricle tissues were taken from the above 6 groups of rats at 6 h after reperfusion.After the frozen sections were performed,TdT-mediated dUTP Nick-End Labeling(TUNEL)was used to label the 3'-OH end of damaged DNA in apoptotic cells,and the myocardial tissue apoptosis was observed under a fluorescence microscope.(6)Effects of IOX2 on the area of myocardial infarction during myocardial I/R injury in dexmedetomidine-treated rats.Thirty-six male SD rats were randomly divided into 6 groups(n=6):sham group,I/R group,I/R+IOX2 group,I/R+DEX group,I/R+DEX+IOX2 group,and I/R+DEX+vehicle group.Evans blue/2,3,5-triphenyltetrazolium chloride(TTC)staining was performed at 6 h of reperfusion to analyze the area at risk(AAR)and the infarct area(IA).The weight of each myocardial section was measured.The area of myocardial infarction was calculated as the weight percentage of IA area to AAR area(IA weight/AAR weight×100%).(7)Effects of IOX2 on the expression of HIF-1? and apoptosis-related proteins during myocardial I/R injury in dexmedetomidine-treated rats.Thirty-six male SD rats were randomly divided into 6 groups(n=6):sham group,I/R group,I/R+IOX2 group,I/R+DEX group,I/R+DEX+IOX2 group,and I/R+DEX + vehicle group.Western Blot was used to detect HIF-la and apoptosis-related proteins including BCL-2,BAX,BNIP3,cleaved caspase-3,and cleaved PARP-1 in rat myocardial tissues.(8)Comparison of mortality during myocardial I/R injury in rats.Record and compare the death of rats caused by any reason prior to the end of the experiment throughout the study.The above data were expressed as mean±standard error of the mean,and the statistical analyses were performed by using the GraphPad 7 software.Results:(1)Western Blot results showed that the myocardial expression of HIF-1? protein in rats of reperfusion for 30 min and reperfusion for 2,6,and 24 h were significantly higher than that of the sham group(P<0.01),with a significantly higher expression at 6 h than that at 2 h(P<0.01)and 24 h(P<0.05).Therefore,30 min of ischemia and 6 h of perfusion were selected as the optimal time points for the subsequent rat experiments.(2)ECG and heart rate monitoring results showed that myocardial I/R process caused significant ECG changes,including elevated ST segment during the stage of ischemia and rapid arrhythmias such as ventricular tachycardia during the stage of reperfusion.Dexmedetomidine post-treatment had no significant effect on ST segment or arrhythmias.Heart rate of rats in the dexmedetomidine group tended to decrease during the experiment,but the differences between the groups were not statistically significant(P>0.05).The area under the curve(heart rate×time)were similar among groups.(3)The results of blood gas,electrolytes,and hemoglobin showed that the arterial blood pH,PaO2,PaCO2,SaO2,Hb,Hct,Na+,K+,Ca2+,Cl-,HCO3-,and BE values at 6 h of reperfusion of all groups were in the normal range.Compared with the sham group,the values of PaCO2,HCO3-,and BE were lower in rats undergoing myocardial I/R,but the differences between groups were not statistically significant(P>0.05).Therefore,the internal environment of the animals remained stable during the experiment.(4)The serum cTnI test results showed that the serum cTnI concentration in the I/R group significantly increased to 9.78±0.42 ng/ml(P<0.01),and the dexmedetomidine post-treatment reduced the cTnl level to 5.07±0.33 ng/ml(P<0.01),the I/R+DEX+IOX2 group was 8.19±0.43 ng/ml(P<0.01).Dexmedetomidine post-treatment reduced the myocardial I/R-induced cTnI release,and this protective effect was reversed by IOX2,suggesting that dexmedetomidine post-treatment reduced I/R-induced myocardial tissue damage in rats by regulating HIF-1?.(5)TUNEL apoptosis test results showed that the myocardial tissue apoptosis rate in the I/R group was 36.67±1.97%(P<0.01),while dexmedetomidine post-treatment reduced the apoptosis rate to 15.59±0.80%(P<0.01).However,IOX2 increased the apoptosis to 28.74±1.38%in the I/R+DEX+IOX2 group(P<0.01).This result demonstrated that dexmedetomidine post-treatment reduced I/R-induced myocardial tissue apoptosis in rats by regulating HIF-1?.(6)The Evans Blue/TTC double staining test results showed that the area of AAR was comparable among groups(about 45%,except the sham group).The I/R and I/R+IOX2 groups showed significantly increased infarct area of 39.27±0.72%and 41.53±1.50%,respectively(P<0.01).Dexmedetomidine post-treatment significantly reduced the infarct area to 20.20±2.07%(P<0.01),while IOX2 increased the infarct size to 33.90±2.06%(P<0.01).Therefore,dexmedetomidine post-treatment reduced I/R-induced myocardial infarct size in rats by regulating HIF-1?.(7)Western Blot results showed that post-treatment with dexmedetomidine significantly reduced HIF-1?,BNIP3,cleaved caspase-3,and cleaved PARP-1 protein expression in myocardial tissue during I/R(P<0.01),and increased the ratio of BCL-2/BAX(P<0.01),while IOX2 treatment reversed the effects of dexmedetomidine on the above proteins(P<0.05 or P<0.01).These results confirmed that dexmedetomidine post-treatment reduced I/R-induced changes in myocardial apoptosis-related proteins by regulating HIF-1?.(8)A total of 9 rats died prior to the end of the experiment.Causes of death included blood loss,severe arrhythmias(ventricular tachycardia and ventricular fibrillation),and respiratory failure.The mortality rates were:sham group(1/25,4.0%),sham+DEX group(0/6,0%),I/R group(2/38,5.26%),I/R+IOX2 group(2/20,10%),I/R+DEX group(1/19,5.26%),I/R+DEX+IOX2 group(2/20,10%),I/R+DEX+vehicle group(1/19,5.26%).There were no statistically significant differences in mortality among the groups(P>0.05).Conclusion:Dexmedetomidine post-treatment could reduce myocardial I/R injury in rats,as evidenced by reducing serum cTnI levels,inhibiting myocardial tissue apoptosis,decreasing myocardial infarction area,and regulating the expressions of apoptosis-related proteins BCL-2,BAX,BNIP3,cleaved caspase-3,and cleaved PARP-1.The administration of the specific HIF-1? prolyl hydroxylase-2 inhibitor IOX2 before the use of dexmedetomidine reversed the myocardial protection and changes in the above indicators by dexmedetomidine treatment.Based on these results,dexmedetomidine reduced I/R-induced myocardial injury in rats through acting on the key target of HIF-1?. |
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