Effect of methylene blue on hydrogen peroxide-induced apoptosis in macrophages through mitochondria-dependent pathway in mice / 中华麻醉学杂志

ABSTRACT

Objective To evaluate the effect of methylene blue (MB) on hydrogen peroxide (H2O2)-induced apoptosis in macrophages through mitochondria-dependent pathway in mice.Methods Mouse peritoneal macrophage line RAW264.7 cells were cultured in DMEM culture medium containing 10％ fetal bovine serum.Cells were divided into 6 groups (n =24 each) using a random number table method:control group (group C),H2O2 group,prophylactic different concentrations of MB groups (MB1,2 groups) and therapeutic different concentrations of MB groups (MB3.4 groups).H2O2 50 μmol/L was added to the culture medium in group H2O2.MB was added to the culture medium with the final concentrations of 0.1 μmol/L (in MB1 and MB3 groups) and 1.0 μmol/L (in MB2 and MB4 groups) at 30 min before adding H2O2 in MB1.2 groups and 30 min after adding H2O2 in MB3.4 groups.At 24 h of culture or incubation in each group,the cell survival rate was measured by methyl thiazolyl tetrazolium assay,the activity of reactive oxygen species (ROS) in cells was determined with the fluorescent probe,the lactate dehydrogenase (LDH) activity in supernatant was detected by spectrophotometry,the activity of superoxide dismutase (SOD) in cells was detected by colorimetric method,mitochondrial membrane potential (MMP) was measured using rhodamine 123 staining,the content of ATP was determined by an ATP bioluminescent method,the expression of pro-caspase-3 and spliceosomes P20 protein and P 18 protein was detected by Western blot,and cell apoptosis was detected using flow cytometry.Results Compared with group C,the cell survival rate,SOD activity and contents of MMP and ATP were significantly decreased,the ROS activity and activity of LDH in supernatant were increased,the expression of pro-caspase-3 and spliceosomes P20 protein and P18 protein was up-regulated,and early and late apoptosis rates were increased in the other five groups (P＜0.05).Compared with group H2O2,the cell survival rate,SOD activity and contents of MMP and ATP were significantly increased,the ROS activity and activity of LDH in supernatant were decreased,the expression of pro-caspase-3 and spliceosomes P20 protein and P18 protein was down-regulated,and early and late apoptosis rates were decreased in MB1-4 groups (P＜0.05).Compared with group MB1,the cell survival rate was significantly decreased,and the expression of caspase-3 spliceosome P 18 was down-regulated in group MB2,and the cell survival rate and SOD activity were significantly decreased,and the activity of ROS was increased in group MB3 (P＜0.05).Compared with group MB4,the expression of caspase-3 spliceosome P 18 was significantly down-regulated,early and late apoptosis rates were decreased,and the activity of ROS was increased in group MB2,and the activity of ROS was significantly increased in group MB3 (P＜0.05).Conclusion The mechanism by which MB attenuates H2O2-induced oxidative damage to macrophages is related to inhibiting cell apoptosis in macrophages through mitochondria-dependent pathway in mice.