ABSTRACT
OBJECTIVE: To explore the effect of N,N-dimethylformamide( DMF)-induced inflammatory injury in H9c2 cardiomyocytes and its mechanism. METHODS: H9c2 cardiomyocytes were cultured in vitro and randomly divided into 4different groups: control group,50 mmol / L-group,100 mmol / L-group,200 mmol / L-group. These 4 groups of cells were treated with different DMF concentrations( 0,50,100,200 mmol / L) for 12 hours. The cells were also divided into 6groups and treated with 200 mmol / L DMF at different time points( 0,2,4,6,8,12 h) : control group,2 h-group,4 hgroup,6 h-group,8 h-group and 12 h-group. The level of lactate dehydrogenase( LDH) was detected by colorimetry. The levels of creatine kinase( CK) and isoenzyme of creatine kinase( CK-MB) were detected by ultraviolet spectrometry. The levels of tumor necrosis factor-α( TNF-α),interleukin( IL)-1β,IL-6,and IL-8 were detected by enzyme linked immunosorbent assay. The level of reactive oxygen species( ROS) was detected by fluorescence probe. The location of nuclear factor-kappa B( NF-κB) p65 protein was detected by immunofluorescence cytochemistry( IFC) staining. RESULTS: The levels of LDH,CK and CK-MB in the 50 mmol / L-group,100 mmol / L-group and 200 mmol / L-group were higher than that of the control group( P < 0. 05) and showed a significant dose-effect( P < 0. 05). The levels of LDH,CK and CK-MB in the 6 h-group,8 h-group and 12 h-group were higher than that of the control group( P < 0. 01) and showed a significant time-effect( P < 0. 01). The levels of TNF-α,IL-1β,IL-6 and IL-8 of the 200 mmol / L-group were higher than the control group( P < 0. 05). Compared with the control group,the levels of TNF-α of the 4 h-group,12 h-group were higher( P < 0. 05),the levels of IL-1β of the 2 h-group,4 h-group,6 h-group,8 h-group and 12 h-group were higher( P < 0. 05),the levels of IL-6 of the 2 h-group and 4 h-group were higher( P < 0. 05),the level of IL-8 of the 2 h-group was higher( P < 0. 05). In addition,the levels of TNF-α,IL-1β and IL-6 reached a peak at 4 h-group and the level of IL-8 reached a peak at 2 h-group. The ROS levels of the 2 h-group,4 h-group and 6 h-group were higher than the control group( P < 0. 01),and the level of ROS reached a peak at 2 h-group. Furthermore,IFC staining showed that the fluorescence intensity of NF-κB p65 protein in nucleus of the 2h-group and 4 h-group increased after treatment with DMF,comparing with the control group. CONCLUSION: DMF leads to inflammatory injury in H9c2 cardiomyocytes. ROS and NF-κB might be involved in the process.
ABSTRACT
Objective@#To observe the change levels of nuclear factor-kappa B (NF-κB) p65 protein in cytoplasm and nuclear, phosphorylation of inhibitor of kappa B (p-IκB) protein and cytochrome C (Cyt-c) , cleaved cysteinyl aspartate specific proteinase-3 (Cleaved caspase-3) , B-cell lymphoma/leukemia-2 (Bcl-2) in cytoplasm in the process of N, N-dimethylformamide (DMF) -induced apoptosis in H9c2 cardiomyocytes, and explore the tentative mechanism of apoptosis.@*Methods@#H9c2 cardiomyocytes were exposed to 200 mmol/L DMF. Western blotting was used to detect the protein expression levels of p65 in cytoplasm and nuclear, p-IκB after exposure for 0, 2, 4, 6, 8, 12 h, and the protein expression levels of Cyt-c, Cleaved caspase-3, Bcl-2 in cytoplasm after exposure for 0, 2, 4, 8, 12, 24 h. Immunofluorescencecytochemistry (IFC) was used to observe the location of Cyt-c after 200 mmol/L DMF exposure for different times.@*Results@#The levels of p65 in cytoplasm and nuclear and p-IκB among groups were statistically significant (F were 7.79, 33.11, 90.25, respectively, all P<0.01) . Compared with the control group, the levels of p65 in cytoplasm of 2, 4, 6 h group were significantly decreased (all P<0.01) ; the levels of p65 in nuclear of 2, 4, 6, 8 h were significantly increased (all P<0.01) ; the levels of p-IκB of 2, 4, 6 h group were significantly increased (all P<0.01) . The levels of Cyt-c, Cleaved caspase-3 and Bcl-2 among groups were statistically significant (F were 51.42, 503.68, 73.37, respectively, all P<0.01) . Compared with the control group, the levels of Cyt-c of 8, 12 h group were significantly increased (both P<0.01) ; the levels of Cleaved caspase-3 of 2, 4, 8, 12, 24 h were significantly increased (all P<0.01) ; the levels of Bcl-2 of 2, 4, 8, 12, 24 h group were significantly decreased (all P<0.01) . IFC showed that Cyt-c was released from the mitochondria to the cytoplasm gradually as the extension of the exposure time.@*Conclusion@#NF-κB signaling pathway and mitochondrial pathway are involved in the mechanism of DMF-induced apoptosis in H9c2 cardiomyocytes.