To evaluate the effect of methylene blue (MB) on hydrogen peroxide (H₂O₂)-induced apoptosis in macrophages through mitochondria-dependent pathway in mice.

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 (MB_{1, 2} groups) and therapeutic different concentrations of MB groups (MB_{3, 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 MB_{1, 2} groups and 30 min after adding H₂O₂ in MB_{3, 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 P18 protein was detected by Western blot, and cell apoptosis was detected using flow cytometry.

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 MB_1-4 groups (P<0.05). Compared with group MB₁, the cell survival rate was significantly decreased, and the expression of caspase-3 spliceosome P18 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 P18 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).

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.

Methylene blue; Hydrogen peroxide; Mitochondria; Apoptosis; Macrophages