Study On Membrane Injury Of H9c2 Cardiomyocytes Induced By N,N-dimethylformamide

Objective: To explore the effects of membrane injury in H9c2 cardiomyocytes induced by N,N-dimethylformamide(DMF)and its tentative mechanisms.Methods: H9c2 cardiomyocytes were cultured in vitro.Cell Counting Kit-8(CCK-8)method was used to detect the cell viability after the cells were treated with 0,25,50,75,100,125,150,175 and 200 mmol/L DMF for 24 hours respectively.According to the result of CCK-8 experiment,based on the prinple that the cell viability was more than 70%,H9c2 cardiomyocytes were divided into 4 groups: control group,80 mmol/L-group,110 mmol/L-group and 140 mmol/L-group,and these 4 groups of cells were treated with 0,80,110,140 mmol/L DMF for 24 hours respectively to observe the dose-effect relationship.The cells were also divided into 5 groups and treated with 110 mmol/L DMF for 0,2,6,12,24 hours respectively to observe the time-effect relationship: control group,2 h-group,6 h-group,12 h-group and 24 h-group.The LDH activity was detected by chemical colorimetry to reflect the membrane integrity.The fluorescence polarization was detected by fluorescence polarization method to reflect the membrane fluidity.The FDA+/PI+ rate was detected by flow cytometry to reflect the membrane permeability.The cholesterol level was detected by fluorescence method.The Ca2+ level was detected by calcium imaging technology.Results: The cell viability in the 100,125,150,175,200 mmol/L-group were lower than that of the control group(P<0.01)and showed a significant dose-effect(P<0.01).The LDH activity in the 80,110,140 mmol/L-group were higher than that of the control group(P<0.01)and showed a significant dose-effect(P<0.01).The LDH activity in the 2,6,12 h-group were higher than that of the control group(P<0.05)and showed a significant time-effect(P<0.01).The fluorescence polarization in the 80,110,140mmol/L-group were higher than that of the control group(P<0.01)and showed a significant dose-effect(P<0.01).The fluorescence polarization in the 6,12,24 h-goup were higher than that of the control group(P<0.01)and showed a significant time-effect(P<0.01).The FDA+/PI+ rate in the 80,110 mmol/L-group were higher than that of the control group(P<0.01).The FDA+/PI+ rate in the 6,12,24 h-group were higher than that of the control group(P<0.05)and showed a significant time-effect(P<0.01).The PI+ rate in the 110,140 mmol/L-group were higher than that of the control group(P<0.01)and showed a significant dose-effect(P<0.01).The PI+ rate in the 12,24 h-group were higher than that of the control group P<0.05)and showed a significant time-effect(P<0.01).The cholesterol level in the 80,110,140 mmol/L-group were higher than that of the control group(P<0.01)and showed a significant dose-effect(P<0.01).The cholesterol level in the 6,24 h-group were higher than that of the control group(P<0.05)and showed a significant time-effect(P<0.01).The Ca2+ level in the 80,110,140 mmol/L-group were higher than that of the control group(P<0.01)and showed a significant dose-effect(P<0.01).The Ca2+ level in the 2,6,12,24 h-group were higher than that of the control group(P<0.05)and showed a significant time-effect(P<0.01).When the range of the DMF exposure concentration was 0 to 200 mmol/L,the LDH activity was positively correlated with the fluorescence polarization(r=0.97,P<0.01),the cholesterol level(r=0.94,P<0.01)and the Ca2+ level(r= 0.68,P<0.05).When the range of the DMF exposure time was 0 to 24 h,the LDH activity was positively correlated with the fluorescence polarization(r=0.89,P<0.01),the FDA+/PI+ rate(r=0.95,P<0.01),the cholesterol level(r=0.88,P<0.01)and the Ca2+ level(r= 0.66,P<0.05).Conclusion: DMF leads to membrane injury in H9c2 cardiomyocytes,and decreased-membrane fluidity,increased-membrane permeability,disordered-cholesterol homeostasis and calcium overload are involved in the process.