[Soluble egg antigen of induces macrophage apoptosis in mice].

OBJECTIVE

To investigate the dynamic changes of macrophage numbers and apoptosis during infection, and to investigate the possible mechanisms of macrophage apoptosis induced by soluble egg antigen (SEA).

METHODS

C57BL/6 mice at ages of 6~8 weeks were randomly divided into 4 groups, including three experimental groups and a normal control group. Each mouse in the experimental groups was infected with (12 ± 1) cercariae of via the abdominal skin, and all mice in an experimental group were sacrificed 3, 5, 8 weeks post-infection, respectively, while mice in the control group were not infected with cercariae and sacrificed on the day of infection in the experimental group. Mouse liver specimens and peritoneal exudation cells were sampled in each group, and the dynamic changes of macrophage numbers and apoptosis were detected. Mouse peritoneal macrophages were isolated, purified and treated with SEA, PBS and ovalbumin (OVA) , and the macrophage apoptosis was detected using flow cytometry. The mRNA and protein expression of BCL-2 protein family members were determined in macrophages using real-time quantitative PCR (qP-CR) and Western blotting assays, and the activation of caspase 3 was determined using flow cytometry and Western blotting. In addition, macrophages were treated with SEA in presence of a caspase inhibitor, HO or N-acetyl-L-cysteine, and the apoptosis of macrophages was detected using flow cytometry.

RESULTS

The total macrophage numbers continued to increase in mouse liver [(0.873 ± 0.106) × 106, (2.737 ± 0.460) × 10 and (3.107 ± 0.367) × 10 cells, respectively; = 81.900, < 0.01] and peritoneal specimens [(5.282 ± 1.136) × 105, (7.500 ± 1.200) × 10 and (12.800 ± 0.800) × 10 cells, respectively; = 55.720, < 0.01] 3, 5 and 8 weeks post-infection with , and the numbers of apoptotic macrophages also continued to increase in mouse liver [(0.092 ± 0.018) × 10, (0.186 ± 0.025) × 10 and (0.173 ± 0.0270) × 10 cells; = 57.780, < 0.01] and peritoneal specimens [(0.335 ± 0.022) × 10, (0.771 ± 0.099) × 10 and (1.094 ± 0.051) × 10 cells; = 49.460, < 0.01] 3, 5 and 8 weeks post-infection with . The apoptotic rate of SEA-treated macrophages [(24.330 ± 0.784)%] was significantly higher than that of PBS-[(18.500 ± 1.077)%] and OVA-treated macrophages [(18.900 ± 1.350)%] (both values < 0.01). There were no significant differences in the mRNA or protein expression of Bcl-2 [ mRNA expression: (1.662 ± 0.943) vs. (1.000 ± 0.000), = 1.215, > 0.05; BCL protein expression: (0.068 ± 0.004) vs. (0.070 ± 0.005), = 0.699, > 0.05], [ mRNA expression: (0.711 ± 0.200) vs. (1.000 ± 0.000), = 2.507, > 0.05; BAX protein expression: (0.089 ± 0.005) vs. (0.097 ± 0.003), = 2.232, > 0.05] and Bak [ mRNA expression: (1.255 ± 0.049) vs. (1.00 ± 0.00), = 0.897, > 0.05; BAK protein expression: (0.439 ± 0.048) vs. (0.571 ± 0.091), = 2.231, > 0.05] between in SEA- and PBS-treated macrophages. SEA induced macrophage apoptosis in the presence of a caspase inhibitor ( = 0.411, > 0.05); however, SEA failed to induce macrophage apoptosis in the presence of HO or NAC ( = 11.880 and 9.897, both values < 0.05).

CONCLUSIONS

SEA may induce macrophage apoptosis through promoting reactive oxygen species expression during infections in mice.