Study On The Mechanism Of Endoplasmic Reticulum Stress Induced By Dimethylformamide In H9c2 Cardiomyocytes

Objective To investigate whether endoplasmic reticulum stress was involved in H9c2 cardiomyocytes apoptosis induced by dimethylformamide(DMF),and whether oxidative stress mediated the activation of the pathway.Method Cells were subcultured or carried out to exposure experiment when they grew to about 80%,then CCK-8 method was used to detect the cell viability at 24 hours under different concentration gradients.Calculatingthe 24 h IC50 of DMF,the maximum concentration exposure dose of DMF was designedthat based on the IC50 to ensure cell viability upon70%.Protein immunoblotting method(western blotting,WB)was used to detect the level of the target protein(GRP78,P-IRE1?,Caspase-12,Peif2?,CHOP)at different concentrations(0mmol/L,50 mmol/L,100 mmol/L,150 mmol/L)after 24 h,or(0h,4h,8h,24)at 150 mmol/L,or NAC intervention group(Control ? 4mmol/LNAC 1h ? 150 mmol/LDMF ? 4mmol/LNAC 1h +150 mmol/LDMF)after 24 h,the maximum dose was designed according to the 24 h IC50of DMF,the time and the 4mmol / L NAC pre-intervention for1 h were designed according to previous experiments.TUNEL methods was used to detect the apoptosis of NAC cells,and immunofluorescence cytochemistry(IFC)was used to locate intracellular GRP78.Results CCK-8 test illustrated that the IC50 of H9c2 cardiomyocytes induced by DMF at 24 h was 176.378mmol/L,according to the DMF at different doses of exposure 24h(0,50,75,100,125,150,175,200,225 and 250 mmol / L).The results of the protein immunoblotting method showed that,as for different concentration groups(0mmol/L,50 mmol/L,100 mmol/L and 150 mmol/L),the differences of total cellular protein(GRP78,P-eif2?,P-IRE1?,Caspase-12 and CHOP)between groups were statistically significant(GRP78,F=68.517,P<0.001;P-eif2?,F=38.932,P<0.001;P-IRE1?,F=204.660,P<0.001;Caspase-12,F=67.445,P<0.001;CHOP,F=130.587,P<0.001).With the increase of exposure dose,the levels of five stress proteins increasedin different degrees,with the P-eif2? and CHOP protein at the highest level of 150 mmol / L,the other three protein levels were the highest at 100 mmol / L,and the difference was statistically significant(P <0.05),indicating the presence of endoplasmic reticulum stress.As for the levels of total protein(GRP78,Peif2?,P-IRE1?,Caspase-12 and CHOP)in H9c2 cardiomyocytes in different time groups(0h,4h,8h,24h)under the same DMF concentration of 150mmol/L compared with the control group,the levels were ascending and had statistically significant(GRP78,F=92.902,P<0.001;P-eif2?,F=3316.899,P<0.001;P-IRE1?,F=161.780,P<0.001;Caspase-12,F=57.485,P<0.001;CHOP,F=97.513,P<0.001).With the extension of exposure time,five groups of protein levels had increased to varying degrees,and four groups of protein levels were the highest in 8h except Caspase-12 protein which had been increased,and it's still had statistically significant compared with the control group(P<0.05),indicating the presence of endoplasmic reticulum stress,and compared with different dose groups,they were accompanied by a comprehensive dose of toxic effects and time points.After NAC intervention,the cells were retreated,translucent and partially floating in the culture medium after exposure to 150 mmol / L for 24 h,and the cells in the group that NAC pre-intervention 1h then exposuring to 150 mmol / L for 24 h were more improved than the direct exposure group of DMF.TUNEL method was used to detect the apoptosis of NAC intervention(control,NAC pre-intervention for 1 h,DMF,NAC pre-intervention 1 h + DMF)for 24 h,there were statistically significant between four groups(F=65.653,P<0.001).There was statistically significant between the DMFgroup and the control group(P<0.05).Also,there was statistically significant between the NAC+DMF-group and the DMF-group for the cellular apoptosis.This result showed that the cellular apoptosis have been alleviated after the impact of the NAC.The results of western blotting showed that the difference of total protein GRP78,P-IRE1?,Caspase-12,P-eif2? and CHOP in NAC group was statistically significant(GRP78,F=2688.457,P<0.001;P-eif2?,F=847.743,P<0.001;PIRE1?,F=178.206,P<0.001;Caspase-12,F=1931.161,P<0.001;CHOP,F=42.001,P<0.001).There was no statistically significant difference between NAC group and control group,itindicated that NAC itself had no toxic effect on H9c2 cardiomyocytes.Compared with DMF group,the levels of GRP78,P-eif2?,PIRE1?,Caspase-12 and CHOP were decreased in the NAC + DMF group,the difference was statistically significant(P<0.05).It indicated that endoplasmic reticulum stress was associated with changes in oxidative stress.Immunofluorescence cytochemistry method(IFC)showed that,under the microscope observation,the nuclei of the control group and the NAC group were blue,a few cells' GRP78 were red fluorescent markers,which were distributed at both ends of the nucleus.However,some of the nuclei appeared to be broken in the DMF-group,most of the intracellular GRP78 was spotted or cloudy dispersed near the nucleus.The cells in the NAC + DMF-group were more complete than those in the DMF group,and the GRP78 dispersed near the nucleus partially.It had a better performance than the DMF-group.Conclusion Endoplasmic reticulum stress and N,N-dimethylformamide(DMF)could be involved in the apoptosis of H9c2 cardiomyocyte.Oxidative stress may mediate the activation of stress signaling pathways in endoplasmic reticulum.