Protective Effect Of CORM-2 On Oxygen-glucose Deprived Injury In CHO Cells And The Related Mechanism

To investigate the protective effect of carbon monoxide-releasing molecule-2（CORM-2）on injury of CHO cells induced by oxygen-glucose deprived,and to explore the underlying mechanism.Na₂S₂O₄with no suger Earle,s balanced salt solution were used to induce oxygen-glucose deprived damage of CHO cells.Various concentrations of CORM-2(5,20,40,60,80,100μmol?L^-1)were added into the medium for pretreatment of CHO cells for 2 hours,and then Na₂S₂O₄with no suger Earle,s balanced salt solution were used in place of the original medium for co-culturing for another 3 hours.Cell viability was measured by MTT assay.Intracellular reactive oxygen species（ROS）level was measured using 2,7-dichlorfluorescein diacetate probe under florescence microplate reader.Contents of Cyt C,AIF,Endo G and protein expression level of Cleaved Caspase-3 were detected by Western Blot.AIF and Endo G translocation from the mitochondria were examined by immunofluorescence staining.The number of mitochondrial-derived vesicle（MDV）were examined by immunofluorescence staining.Oxygen-glucose deprived had a significant inhibitory effect on the viability of CHO cells（P<0.01）.Oxygen-glucose deprived induced CHO cells significant increased intracellular ROS levels（P<0.01）.Oxygen glucose deprived increased the content of Cyt C in the cytoplasm,decreased the content of Cyt C in the mitochondria and up regulated protein expression of Cleaved Caspase-3（P<0.05）.Oxygen-glucose deprived decreased the content of AIF and Endo G in the cytoplasm,increased the content of AIF and Endo G in the nuclei（P<0.05）.Oxygen-glucose deprived induced the translocation of AIF and Endo G into the nuclei.Oxygen-glucose deprived decreased the number of MDV in the CHO cells（P<0.05）.Compared with the oxygen-glucose deprived group,CORM-2 at 5,20,40,60,80,100μmol?L^-1markedly improved the survival rate of CHO cell（P<0.05）;CORM-2 at 60 and 100μmol?L^-1cleared cellular ROS（P<0.05）;CORM-2 at 5,60,100μmol?L^-1restrained the release of Cyt C and activation of Caspase-3（P<0.05）;CORM-2 at60μmol?L^-1restrained the translocation of AIF and Endo G into the nuclei（P<0.05）.Compared with the control group,pretreatment with CORM-2(60μmol?L^-1)increased the number of MDV in the CHO cells（P<0.05）.In conclusion,CORM-2 could alleviate injury of CHO cells induced by oxygen-glucose deprived.The mechanism of the protective effect could be through increasing the number of MDV,decreasing the level of cellular oxidative stress and inhibiting apoptosis via the endogenous mitochondrial pathway.