Inflammatory Injury Of The Myocardial Cells(H9c2) Induced By N,N-dimethylformamide

Objective To investigate Dimethylformamide(DMF) is caused by the inflammatory injury of the H9c2 myocardial cells,and to explore whether the inflammatory response due to activation of NF-κB signaling pathway.Method The LDH、CK、CK-MB kit was used to detect the injury of myocardial cells after different doses of DMF(0 m M, 50 m M, 100 m M, 200 m M) exposure for 12 h, the injury of myocardial cells after 200 m M DMF exposure for different time intervals(0h, 2h, 4h, 6h, 8h, 12h).The Elisa kit was used to detect the level of TNF-α、IL-1β、IL-6、IL-8 from myocardial cells after different doses of DMF(0 m M, 50 m M, 100 m M, 200 m M) exposure for 12 h, the level of TNF-α、IL-1β、IL-6、IL-8 from myocardial cells after 200 m M DMF exposure for different time intervals(0h, 2h, 4h, 6h, 8h, 12h).The ROS kit was used to detect the level of ROS after 200 m M DMF exposure for different time intervals(0h, 2h, 4h, 6h, 8h, 12h).Western Blotting was used to detect the levels of p65 in nucleus and cytoplasm and the levels of p-IκB in total cell protein after 200 m M DMF exposure for different time intervals(0h, 2h, 4h, 6h, 8h, 12h).Immunofluorescence(IFC) was used to observe the location of p65 after 200 m M DMF exposure for different time intervals.Results After different doses of DMF(0 m M, 50 m M, 100 m M, 200 m M) exposure for 12 h,the levels of LDH、CK、CK-MB increased.The differences between groups were statistically different(LDH:F=564.426,P<0.0001;CK:F=938.695,P<0.0001;CK-MB:F=15.636,P=0.001)。The levels of LDH 、CK and CK-MB increased after 50 m M、100 m M and 200 m M DMF exposure compared with control group(P<0.01).After different doses of DMF(0 m M, 50 m M, 100 m M, 200 m M) exposure for 12 h,the levels of TNF-α、IL-1β、IL-6、IL-8 increased.The differences between groups were statistically different(TNF-α:F=19.810,P<0.001; IL-1β: F=50.638, P<0.001; IL-6:F=13.265, P<0.01; IL-8:F=11.521,P<0.001).The levels of TNF-α increased after 200 m M DMF exposure compared with control group(P<0.001)), while the difference between 50 m M and 100 m M DMF exposure and control group were not statistically significant. The levels of IL-1β increased after 50 m M、100 m M and 200 m M DMF exposure compared with control group(P<0.01).The levels of IL-6 increased after 50 m M exposure compared with control group were not statistically significant,while the difference between 100 m M and 200 m M DMF exposure were statistically significant(P<0.05).IL-8 increased after 50 m M、100 m M and 200 m M DMF exposure compared with control group(P<0.001).The levels of LDH、CK、CK-MB in cell culture supernatant increased after 200 m M DMF exposure for different time intervals(0h, 2h, 4h, 6h, 8h, 12h).The differences between groups were statistically different(LDH:F=1618.106,P<0.0001;CK:F=609.899,P<0.001;CK-MB:F=119.176,P<0.001)。The levels of TNF-α、IL-1β、IL-6,4h higher than other groups,the differences were statistically significant(P<0.05).The levels of IL-8,2h higher than other groups,the differences were statistically significant(P<0.05).The levels of ROS different after 200 m M DMF exposure for different time intervals(0h, 2h, 4h, 6h, 8h, 12h).The differences between groups were statistically different(F=54.722,P<0.001).The 2h group of active oxygen level is the highest, compared with the control group, the differences were statistically significant(P<0.05).The levels of p65 in nucleus increased after2 h,4h,6h exposure compared with control group and 8h,12 h exposure.The differences were statistically different(P<0.01).The levels of p65 in cytoplasm,2h exposure group decreased compared with the control group.The difference is statistical significance(P< 0.05). 2h exposure group decreased compared with 12 h exposure group.The differences were statistically different(P < 0.01).2h exposure group decreased compared with 4h, 6h, 8h exposure group showed no statistical significance.The levels of p-IκB increased after2h,4h,6h exposure compared with control group and 8h,12 h exposure.The difference is statistical significance(P < 0.05).Immunofluorescence(IFC) showed that the fluorescence of in nucleus obvious of DMF 2h exposure group compared with other group.P65 enter the nucleus.Conclusion DMF exposure up-regulated the levels of LDH、CK、CK-MB; TNF alpha, IL-1, IL-6, IL-8 increased in short time after DMF exposure; P65 enter the nucleus in the 2h after DMF. Dimethylformamide in H9c2 cells caused by inflammatory injury, NF-κB signaling pathway involved in inflammatory reaction of H9c2 myocardial cells.