[Establishment of a toxic injury model induced by carbon monoxide in H9c2 cells].
Source: PubMed, NCBI / U.S. National Library of Medicine
To observe the effect of carbon monoxide on cardiac H9c2 cells and determine the appropriate concentration and exposure time for carbon monoxide-induced injury in H9c2 cells. H9c2 cells were cultured in vitro, passaged upon reaching the logarithmic growth phase, and subjected to different culture conditions according to the experimental requirements. 1) Carbon monoxide fluorescence probe was used for qualitative detection of carbon monoxide in H9c2 cells. H9c2 cells were exposed to different concentrations (1×10, 2×10, 3×10, 4×10) of carbon monoxide for 3 hours, and cell viability was detected by cell counting kit-8 (CCK-8). H9c2 cells were exposed to carbon monoxide at a concentration of 3×10for 1, 2, 3, 4, 6 hours, and cell viability was detected by CCK-8. Subsequent experiments were performed based on the screening results. 2) H9c2 cells were divided into a blank control group and a carbon monoxide poisoning group. The blank control group was cultured under conventional conditions, while the carbon monoxide poisoning group was cultured in a portable cell culture device with a carbon monoxide concentration of 3×10for 3 hours. After 21 hours of reoxygenation, morphological changes of H9c2 cells were observed under inverted phase contrast microscope. The occurrence of reactive oxygen species (ROS) was observed under inverted fluorescence microscope. Mitochondrial damage was detected by JC-1 fluorescence staining method. The levels of MB isoenzyme
Abstract
To observe the effect of carbon monoxide on cardiac H9c2 cells and determine the appropriate concentration and exposure time for carbon monoxide-induced injury in H9c2 cells. H9c2 cells were cultured in vitro, passaged upon reaching the logarithmic growth phase, and subjected to different culture conditions according to the experimental requirements. 1) Carbon monoxide fluorescence probe was used for qualitative detection of carbon monoxide in H9c2 cells. H9c2 cells were exposed to different concentrations (1×10, 2×10, 3×10, 4×10) of carbon monoxide for 3 hours, and cell viability was detected by cell counting kit-8 (CCK-8). H9c2 cells were exposed to carbon monoxide at a concentration of 3×10for 1, 2, 3, 4, 6 hours, and cell viability was detected by CCK-8. Subsequent experiments were performed based on the screening results. 2) H9c2 cells were divided into a blank control group and a carbon monoxide poisoning group. The blank control group was cultured under conventional conditions, while the carbon monoxide poisoning group was cultured in a portable cell culture device with a carbon monoxide concentration of 3×10for 3 hours. After 21 hours of reoxygenation, morphological changes of H9c2 cells were observed under inverted phase contrast microscope. The occurrence of reactive oxygen species (ROS) was observed under inverted fluorescence microscope. Mitochondrial damage was detected by JC-1 fluorescence staining method. The levels of MB isoenzyme of creatine kinase (CK-MB) and cardiac troponin T (cTnT) in H9c2 cells supernatant were detected by enzyme-linked immunosorbent assay (ELISA). 1) Qualitative detection results showed that carbon monoxide could enter H9c2 cells. Cell viability in the 3×10carbon monoxide treatment group was reduced to approximately 50%, and lower than that in the blank control group [(52.27±4.29)% vs. (99.42±0.49)%, P<0.05). Cell viability was slightly decreased at a carbon monoxide concentration of 1×10, while it was excessively inhibited at a carbon monoxide concentration of 4×10. Compared with the blank control group, cell viability was decreased after 3 hours of 3×10carbon monoxide exposure [(52.81±5.28)% vs. 100%, P<0.05], slightly increased before 2 hours, and excessively inhibited after 4 hours. Therefore, exposure of H9c2 cells to carbon monoxide at a concentration of 3×10for 3 hours was selected as the condition for establishing carbon monoxide-induced myocardial cell injury in subsequent experiments. 2) Compared with the blank control group, cells in the carbon monoxide poisoning group showed morphological changes (pyknosis, rough cell membrane, poor adherence, and increased dead cells), and intracellular ROS levels were increased (fluorescence intensity: 195.97±5.16 vs. 98.53±4.08, P<0.05), indicating that carbon monoxide could enhance oxidative stress and increase cell death. MMP in the carbon monoxide poisoning group was decreased as compared with the blank control group (ratio of red/green fluorescence intensity: 0.92±0.02 vs. 1.13±0.08, P<0.05), indicating that carbon monoxide damaged mitochondria in H9c2 cells. Compared with the blank control group, the levels of myocardial injury markers CK-MB and cTnT in H9c2 cells supernatant were increased after carbon monoxide stimulation [CK-MB (μg/L): 33.26±1.93 vs. 20.05±1.43, cTnT (ng/L): 131.18±4.87 vs. 66.40±3.57, both P<0.05], indicating that carbon monoxide induced H9c2 cells injury. A model of carbon monoxide-induced toxic myocardial injury can be established by stimulating H9c2 cells with a carbon monoxide concentration of 3×10for 3 hours.