BACKGROUND: In the early stage of cerebral ischemia-reperfusion, there is a tremendous amount of inflammatory factor expression in the spleen. These inflammatory factor cause oxidative stress damage which leads to cell apoptosis in the spleen after cerebral ischemia-reperfusion.
OBJECTIVE: To investigate the effect of exogenous hydrogen peroxide on spleen cell viability and the effect of N-acetyl-L-cytokine on protection of induced oxidative stress in the spleen of metallothionein-Ⅰ/Ⅱ knockout mice.
METHODS: Metallothionein-Ⅰ/Ⅱ knockout mice spleen cell suspension was prepared and treated with various concentrations of hydrogen peroxide (0.1, 0.2, 0.5, 1, 2 mmol/L) for 2 hours, and the cell viability was detected by MTT colorimetric method. Based on the cells and mitochondria level, hydrogen peroxide induced spleen cells were divided into six group: control group, N-acetyl-L-cytokine group, 0.5 mmol/L hydrogen peroxide group, 1 mmol/L hydrogen peroxide group, N-acetyl-L-cytokine+0.5 mmol/L hydrogen peroxide group, N-acetyl-L-cytokine+1 mmol/L hydrogen peroxide group. Then, the cell viability was detected by MTT colorimetric method, lactate dehydrogenate activity were assayed by microplate reader; mitochondrial permeability transition pore was evaluated by ultraviolet spectrophotometer after 2 hours.
RESULTS AND CONCLUSION: With the increase of the concentration of hydrogen peroxide, the spleen cell viability was significantly decreased (P < 0.01), and 0.2, 0.5, 1, 2 mmol/L hydrogen peroxide groups had the greatest reduction. Compared with the control group, N-acetyl-L-cytokine could significantly increase spleen cell viability (P < 0.01), decrease lactate dehydrogenate activity (P < 0.01) and decrease the opening state of mitochondrial permeability transition pore (P < 0.01). Compared with 0.5 mmol/L hydrogen peroxide group and 1 mmol/L hydrogen peroxide group, N-acetyl-L-cytokine+0.5 mmol/L hydrogen peroxide group and N-acetyl-L-cytokine+1 mmol/L hydrogen peroxide group could also increase spleen cell viability (P < 0.01), decrease lactate dehydrogenate activity (P < 0.01) and decrease the opening state of mitochondrial permeability transition pore (P < 0.01), respectively. These findings suggest that with the increase of the concentration of hydrogen peroxide, the spleen cell viability is significantly decreased in a dose-dependent manner, especially for 0.2, 0.5, 1, 2 mmol/L hydrogen peroxide; N-acetyl-L-cysteine can relieve the oxidative stress damage induced by hydrogen peroxide in metallothionein-Ⅰ/Ⅱ knockout mice spleen cells, through reducing the lactate dehydrogenase release and the opening state of mitochondrial permeability transition pore, and increasing spleen cell viability.