Mechanism Of Dexmedetomidine Protection Against Hypoxia/Reoxygenation Injury Of Cardiomyocytes Based On Stress Regulation Of Endoplasmic Reticulum

Objective: To investigate whether the protective effect of Dexmedetomidine(DEX)on Hypoxia/Reoxgenation(H/R)cells is linked to Endoplasmic Reticulum Stress(ERS)and whether dexmedetomidine may regulate ERS through the P38-MAPK signaling pathway by constructing an in vitro hypoxia/reoxygenation(H/R)model of H9C2 cardiomyocytes.Methods: 1.The H9C2 cells were cultured in vitro,and the H/R model was established.The H9C2 cells were randomly divided into control group(the cells were placed in an incubator containing 95% air and 5% CO2)and Hypoxia/Reoxygenation group(the cells were rapidly placed in a closed hypoxia tank containing 1% O2,5% CO2 and94% N2 for 3 h and then replaced with DMEM containing 10% fetal bovine serum)and cultured under normoxic conditions for 3,6,12,and 24 h,respectively.The survival rate of each group of cells was detected by CCK-8 method to determine the time of hypoxia/reoxygenation of H9C2 cells,and the H/R model was prepared.2.The relationship between the protective effect of dexmedetomidine on H/R myocardial cells and ERS H9C2 cells were randomly divided into control group;DEX group;H/R group;DEX + H/R group;4-PBA group(4-phenylbutyrate group);4-PBA+ H/R group;DEX+ 4-PBA group + H/R group.The cell supernatant was used to detect the concentration of LDH;CCK-8method was used to observe the survival rate of cardiomyocytes;flow cytometry was used to detect the apoptosis,and RT-PCR and Western blot analysis were used to detect the m RNA and protein concentrations of Grp78,CHOP,and Caspase-12 in each experimental group.3.The relationship between dexmedetomidine and P38-MAPK signaling pathway The H9C2 cells were randomly divided into: control group;TG group (thapsigargin group);DEX + TG group;DEX + TG + 4-PBA group;SB202190(P38-MAPK phosphorylation inhibitor)+ TG group;DEX + TG + SB202190 group;and DEX + TG + 4-PBA + SB202190 group.The concentration of LDH was detected by cell supernatant;the survival rate of cardiomyocytes was observed by CCK-8 method;the apoptosis was detected by flow cytometry,and the m RNA and protein concentrations of Grp78,CHOP,and Caspase-12 were detected by RT-PCR and Western blot analysis.Result 1.H9C2 cells were cultured in vitro to construct the H/R model.In addition to the control group,cardiomyocytes had the lowest survival rate(H3/R3)at 3 h after hypoxia and 3 h after reoxygenation(P < 0.01),and the survival rate of cardiomyocytes at 3 h after hypoxia was the highest at 12 h after reoxygenation(P < 0.05),while the cell survival rate was decreased after 24 h of reoxygenation(compared with 12 h of reoxygenation andcompared with 6 h of reoxygenation,P < 0.05).Thus,the H/R model of H9C2 cells was constructed by reoxygenation for 3 h under hypoxia.2.The relationship between the protective effect of dexmedetomidine on H/R cardiomyocytes and ERS The results of CCK-8,LDH and flow cytometry showed that the number of apoptotic cells in H/R group was the highest(P < 0.01).Compared with H/R group,the proportion of apoptotic cells in DEX + H/R group was significantly lower than that in 4-PBA + H/R group and DEX + 4-PBA + H/R group(P < 0.05);while the number of apoptotic cells in DEX + 4-PBA + H/R group was lower than that in DEX+ H/R group and 4-PBA + H/R group(P < 0.05).The RT-PCR and Western blot analysis of Grp78,CHOP,Caspase-12 m RNA and protein concentration expression in each experimental group showed that: H/R group significantly up-regulated gene and protein expression(P < 0.01).Compared with H/R group,DEX + H/R group and 4-PBA + H/R group and DEX + 4-PBA + H/R group were significantly down-regulated(P < 0.05).,while DEX + 4-PBA + H/R group was down-regulated compared with DEX + H/R group and 4-PBA + H/R group(P < 0.05).3.The relationship between dexmedetomidine and P38-MAPK signaling pathway The results of CCK-8,LDH and flow cytometry showed that the number of apoptotic cells in TG group was the highest(P < 0.01).The number of apoptotic cells in DEX + TG + 4-PBA group was lower than that in DEX +TG group(P < 0.05).The number of apoptotic cells in DEX + TG + SB202190 group was lower than that in DEX + TG group and SB202190 + TG group(P < 0.05).The number of apoptotic cells in DEX + TG + SB202190 + 4-PBA group was significantly lower than that in DEX + TG + 4-PBA group and DEX + TG +SB202190 group(P < 0.05).The RT-PCR and Western blot analysis of Grp78,CHOP,Caspase-12 protein concentration and m RNA expression in each experimental group showed that: TG group gene and protein expression was up-regulated(P < 0.01),DEX + TG + 4-PBA group was down-regulated compared with DEX + TG group(P < 0.05),DEX + TG +SB202190 group was down-regulated compared with DEX + TG group and SB202190 + TG group(P < 0.05),while DEX + TG + SB202190 + 4-PBA was significantly down-regulated compared with DEX + TG + 4-PBA group,DEX + TG +SB202190 group(P < 0.05).Conclusion: 1,The H/R model of H9C2 cells was used to verify that the cardiomyocytes treated with H/R could induce ER stress and thus reduce cell viability.2,After dexmedetomidine pretreatment,it inhibited the ER stress effect and thus exerted the effect of reducing cell damage.3,The inhibition of ER stress-induced cell injury by dexmedetomidine pretreatment was exerted through the P38-MAPK signaling pathway.