The Protective Effects Of Cygb On Macrophage During Oxidative Damage

[Object]Cytoglobin(CYGB) is a newly fourth oxygen-carrying globulin. The expression of CYGB was significantly induced in hypoxia condition, which protected the nerve cells from oxidative damage. This study is to observe changes of cytoglobin expression and to explore the protective role of CYGB during the process of oxidaive stress via overexpression and downexpression of CYGB in macrophages.[Method]1.Western blot was used to detect CYGB expression in mouse macrophage cell line RAW264.7, human macrophage cell line THP-1, human endothelial cells and human Hep G2 cells under normal physiological conditions. 2.Western blot was used to detect CYGB expression after H2O2 treatment at different time. The RAW264.7 cells were divided into four groups: control group, 0.5mmol/L H2O2 with 6,12, 24 h group. CYGB expression was detected after H2O2 treatment at different concentrations for 12 h. The RAW264.7 cells were divided into four groups: control group, 0.25mmol/L H2O2, 0.5mmol/L H2O2, 1mmol/L H2O2-treated group. 3.The CYGB overexpression and downexpression cell model was constructed, then the role of CYGB during oxidetive stress was observed. The RAW264.7 cells were divided into four groups:control group, 0.25mmol/L H2O2-treated group, 0.5mmol/L H2O2 treatment+CYGB expression group and 0.5mmol/L H2O2 treatment+CYGB interference group. After treated with H2O2 for 12 h, CYGB protein levels, glutathione peroxidase(GSH-PX), lactate dehydrogenase(LDH), malondialdehyde(MDA), reactive oxygen species(ROS), superoxide dismutase(SOD) in each group were detected. Real-time PCR was used to explore CYGB and NF-κB expression.[Result]1.The results of Western blot showed that there was CYGB expression in RAW264.7, THP-1, EC and Hep G2 cells under normal physiological condition. 2.The results of western blot showed that the expression of CYGB of H2O2-treated group was higher than control group. The expression cytoglobin in H2O2 treatment 6,12,24 h group was increased, and the highest group was 12 h group, the difference was statistically significant amount(P<0.05). The expression of cytoglobin in 0.25mmol/L H2O2, 0.5mmol/L H2O2, 1mmol/L H2O2-treated group was increased, the difference was significant(P<0.05). 3. Fluorescence microscopy showed that there was visible green fluorescence in the macrophages transfected with CYGB expression/ interference plasmid. 4.The results of western blot showed that the expression of CYGB in H2O2 treatment group was higher than control group, the difference was significance(P<0.05). The expression of CYGB in 0.5mmol/L H2O2 treatment+CYGB expression group was highest(P<0.05).The expression of CYGB of 0.5mmol/L H2O2 treatment+CYGB interference group was lowest compared to the H2O2-treated group(P<0.05). Enzyme-linked immunosorbent assay(ELISA) and ROS detection showed that GSH-PX, SOD level elevated and LDH, MDA, ROS level decreased in H2O2 treatment+CYGB expression group; GSH-PX, SOD level decreased, LDH, MDA, ROS level increased in H2O2 treatment+CYGB interference group. Compared with control group, CYGB and NF-κB expression was upregulated in H2O2-treated group(P<0.05). Compared with the H2O2-treated group, CYGB expression induced and NF-κB expression decreased in H2O2 treatment+CYGB expression, howerver, CYGB expression dereased and NF-κB induced in H2O2 treatment+CYGB interference group.[Conclusion]1.CYGB was expressed in macrophages, and increased during the process of oxidative stress. 2.The cytoprotecion of CYGB against oxidative injury in macrophage.