Effect Of Dexmedetomidine On Autophagy Of Cardiomyocytes Following High Glucose Hypoxia/Reoxygenation Injury

Part I Effects of dexmedetomidine pretreatment on normal H9c2 cardiomyocytesObjective:To investigate the effects of different concentrations of dexmedetomidine on rat H9c2 cardiomyocytes and find the appropriate intervention concentration.Methods:The H9c2 embryonal rat heart-derived cells were cultured in a humidified incubator with 90%O₂and 10%CO₂ at 37?.Cells were seeded on 6-well plates at a density of 1×10⁶ cells/ml,randomly divided into 5 groups(n=9):low glucose normoxia group(LG group,D-glucose concentration of 5.5 mmol/L),dexmedetomidine pretreatment group 1(DEX-1 group),dexmedetomidine pretreatment group 2(DEX-2 group),dexmedetomidine pretreatment group 3(DEX-3group)and dexmedetomidine pretreatment group 4(DEX-4 group).When the cell density reached about 70%,dexmedetomidine pretreatment groups were treated with DEX at different concentrations of 0.10?mol/L,1.00?mol/L,5.00?mol/L,and 10.00?mol/L for 7 h.After the treatment,an inverted microscope was used to observe the overall condition of H9c2 cells,cell viability and lactate dehydrogenase(LDH)activity in culture medium were assessed by CCK8 assay and LDH kit individually.Result:1.Compared with the LG group,the cell viability of the other groups decreased significantly(P<0.05),and the release of LDH in the culture medium of the DEX-1,DEX-2,and DEX-4 groups increased significantly(P<0.05);2.Compared with the DEX-1 group,the cell survival rate of the DEX-3 group increased(P<0.05);3.Compared with the DEX-3 group,the release of LDH in the culture medium of the DEX-2 and DEX-4 groups increased significantly(P<0.05).Conclusion:5.00?mol/L is the appropriate pretreatment concentration of DEX in all groups.Part II Effect of dexmedetomidine on macroautophagy of H9c2 cells following high glucose hypoxia/reoxygenation injuryObjective: To evaluate the protective role of dexmedetomidine(DEX)in H9c2 cells following high glucose hypoxia/reoxygenation(H/R)injury by effecting macroautophagy.Methods: The H9c2 embryonal rat heart-derived cells were cultured in a humidified incubator with 90% O2 and 10% CO2 at 37 ?.Cells were seeded on 6-well plates at a density of 1×106 cells/ml,and divided into 5 groups(n = 15)randomly: low glucose normoxia group(LG group),high glucose hypoxia/reoxygenation group(HG+H/R group),high glucose hypoxia/reoxygenation dexmedetomidine pretreatment group(DEX group),high glucose hypoxia/reoxygenation dexmedetomidine atipamezole treatment group(ATI group)and high glucose hypoxia/reoxygenation dexmedetomidine 3-methyladenine treatment group(3-MA group).All cells were cultured in normal glucose with normoxia condition.When the cell density reached 50%,all groups were given starvation treatment with 1% FBS culture medium for 24 h.Then LG group continued culturing normally,the cells of HG+H/R,ATI and 3-MA groups were cultured in a high-glucose medium(D-glucose concentration of 33.0 mmol/L)for 24 h,and then placed in a 37°C three-gas incubator(95% N2 and 5% CO2)for 4 h.Then placed the cells in a normoxia chamber(90% O2 and 10% CO2)at 37 ? for 2 h to establish a high glucose H/R injury model of cardiomyocytes.DEX group,ATI group and 3-MA group were given DEX(5.00 ?mol/L)for 1 h before H/R,while the ATI group and 3-MA group were given ATI(25 ?mol/L)and 3-MA(5 ?mol/L)at the beginning of H/R treatment separately.Autophagosomes,lysosome and mitochondria were observed by projection electron microscope,cell viability and lactate dehydrogenase(LDH)activity in culture medium were assessed by CCK8 assay and LDH kit individually.Autophagy-related protein Beclin1,LC3 II,LC3 I and P62 expression were detected by Western Blot.Gene expression of P62 and Beclin1 were detected by rt-PCR,the expression of Beclin1 in cells was observed via immunofluorescence.Results: 1.Compared with the LG group,Cell viability of HG+H/R group,DEX group,ATI group and 3-MA group were decreased,and LDH activity of the HG+H/R group,ATI group and 3-MA group were increased.The cells in the HG+H/R group were severely damaged and there were more autophagosomes in the cells.The autophagosomes in the cells in the DEX group increased significantly and swollen lysosomes were observed.The radio of LC3 II / LC3 I was down-regulated in ATI group and 3-MA group,P62 expression was down-regulated in HG+H/R group and 3-MA group(P<0.05).2.Compared with HG+H/R group,LDH activity of DEX group was decreased,of 3-MA group was increased,radio of LC3 II / LC3 I of DEX group was increased,the expressions of P62 and Beclin1 were increased.The m RNA expressions of Beclin1 and P62 were up-regulated in the DEX group(P<0.05).3.Compared with the DEX group,the cell viabilities of the ATI group and the 3-MA group were decreased,while the LDH activities were increased.There were less autophagosomes observed in cells in ATI group and 3-MA group.In ATI group and 3-MA group,LC3 II / LC3 I ratio decreased,P62 and Beclin1 expressions were decreased,the m RNA expressions of Beclin1 and P62 were down-regulated(P<0.05).Conclusion: The protective effect of dexmedetomidine on cardiomyocytes following high glucose hypoxia/reoxygenation injury is related with macroautophagy regulated by dexmedetomidine via ?2-AR.Part III Dexmedetomidine protects H9c2 cells following high glucose hypoxia/reoxygenation injury via ?2-AR/ AMPK/mTOR pathwayObjective: To investigate the protective effect of dexmedetomidine in high glucose hypoxia/reoxygenation injured H9c2 cardiomyocytes by regulating ?2-AR/AMPK/mTOR pathway.Methods: The H9c2 embryonal rat heart-derived cells were cultured in a humidified incubator with 90% O2 and 10% CO2 at 37 ?.Cells were seeded on 6-well plates at a density of 1×106 cells/ml,and divided into 5 groups(n = 15)randomly: low glucose normoxia group(LG group),high glucose hypoxia/reoxygenation group(HG+H/R group),high glucose hypoxia/reoxygenation dexmedetomidine pretreatment group(DEX group),high glucose hypoxia/reoxygenation dexmedetomidine atipamezole treatment group(ATI group) and high glucose hypoxia/reoxygenation dexmedetomidine 3-methyladenine treatment group(3-MA group).All cells were cultured in normal glucose with normoxia condition.When the cell density reached 50%,all groups were given starvation treatment with 1% FBS culture medium for 24 h.Then LG group continued culturing normally,the cells of HG+H/R,ATI and 3-MA groups were cultured in highglucose medium(D-glucose concentration of 33.0 mmol/L)for 24 h,and placed in a 37 °C three-gas incubator(95% N2 and 5% CO2)for 4 h.Then placed the cells in a normoxia chamber(90% O2 and 10% CO2)at 37 ? for 2 h to establish a high glucose H/R injury model of cardiomyocytes.DEX group,ATI group and 3-MA group were given DEX(5.00 ?mol/L)for 1h before H/R,and the ATI group and 3-MA group were given ATI(25 ?mol/L)and 3-MA(5 ?mol/L)at the beginning of H/R treatment separately.After hypoxia and reoxygenation treatment,signaling pathway related proteins AMPK,p-AMPK,mTOR,p –mTOR were detected by Western Blot.Results: 1.Compared with the LG group,the radio of p-AMPK/AMPK in HG+H/R group,ATI group and 3-MA group were decreased,p-mTOR/mTOR ratio were increased in ATI group and 3-MA group(P<0.05).2.Compared with HG+H/R group,the ratio of p-AMPK/AMPK in DEX group was increased,the ratio of p-mTOR/mTOR of DEX group was decreased.The ratio of p-mTOR/mTOR was increased in ATI group(P<0.05).3.Compared with the DEX group,the p-AMPK/AMPK ratio in ATI group and 3-MA group decreased,p-mTOR/mTOR ratio in ATI group and 3-MA group increased(P<0.05).Conclusion: Dexmedetomidine protects H9c2 cells from high glucose hypoxia/ reoxygenation injury by enhancing autophagy via ?2-AR/AMPK/mTOR signaling pathway.