Role of bone marrow tyrosine kinase on chromosome X in the production of pro-inflammatory cytokines from mouse mononuclear-macrophages RAW264.7 induced by endotoxin/lipopolysaccharide and its mechanism / 中华烧伤杂志

ABSTRACT

Objective@#To investigate the role of bone marrow tyrosine kinase on chromosome X (BMX) in the production of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) from mouse mononuclear-macrophages induced by endotoxin/lipopolysaccharide (LPS) and its related mechanism.@*Methods@#Mouse mononuclear-macrophages RAW264.7 were inoculated in 6-well plates and routinely cultured for the following experiments. (1) Cells were collected and divided into blank control group, LPS control group, and 75, 750, 7 500, 75 000 nmol/L BMX-IN-1 pretreatment groups according to the random number table, with 8 wells in each group. Cells in blank control group were routinely cultured for 25 h. Cells in LPS control group were routinely cultured for 24 h and stimulated by LPS in the final mass concentration (the same below) of 0.1 μg/mL for 1 h. Cells in the latter 4 groups were pretreated with BMX-IN-1 in the final molarity (the same below) of 75, 750, 7 500, 75 000 nmol/L for 24 h and stimulated by 0.1 μg/mL LPS for 1 h. The mRNA expression of TNF-α of cells in each group was determined by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) to screen the optimum concentration of BMX-IN-1. (2) Cells were collected and divided into LPS control group and 2, 4, 8, 12, 18 h BMX-IN-1 pretreatment groups according to the random number table, with 8 wells in each group. Cells in LPS control group were stimulated by 0.1 μg/mL LPS for 1 h. Cells in the latter 5 groups were pretreated with optimum concentration of BMX-IN-1 for 2, 4, 8, 12, 18 h respectively and stimulated by 0.1 μg/mL LPS for 1 h. The mRNA expression of TNF-α of cells in each group was determined by real-time fluorescent quantitative RT-PCR to screen the optimum time for BMX-IN-1 pre-treatment. (3) Cells were collected and divided into blank control group, BMX-IN-1 control group, LPS control group, and BMX-IN-1+ LPS group according to the random number table, with 16 wells in each group. Cells in blank control group were routinely cultured for the optimum time plus 1 h. Cells in BMX-IN-1 control group were pretreated with BMX-IN-1 in the optimum concentration for the optimum time and then routinely cultured for 1 h. Cells in LPS control group were routinely cultured for the optimum time and then stimulated by 0.1 μg/mL LPS for 1 h. Cells in BMX-IN-1+ LPS group were pretreated with BMX-IN-1 in the optimum concentration for the optimum time and then stimulated by 0.1 μg/mL LPS for 1 h. The mRNA expressions of TNF-α and IL-1β were determined by real-time fluorescent quantitative RT-PCR, and the activity of BMX and p38 mitogen-activated protein kinase (MAPK) were determined by Western blotting, with 8 samples in each determination. Data were processed with one-way analysis of variance and LSD test.@*Results@#(1) Compared with that in blank control group, the mRNA expression of TNF-α of cells was significantly increased in the other 5 groups (with P values below 0.01). Compared with that in LPS control group, the mRNA expression of TNF-α of cells was decreased in each BMX-IN-1 pretreatment group, but only the mRNA expression of TNF-α of cells in 75 000 nmol/L BMX-IN-1 pretreatment group was significantly decreased (P<0.05). The optimum concentration of BMX-IN-1 was 75 000 nmol/L. (2) Compared with that in LPS control group, the mRNA expression of TNF-α of cells was not significantly changed in 2 and 4 h BMX-IN-1 pretreatment groups (with P values above 0.05) but significantly decreased in 8, 12, and 18 h BMX-IN-1 pretreatment groups (P<0.05 or P<0.01). The mRNA expression of TNF-α of cells in 12 h BMX-IN-1 pretreatment group was the lowest. The optimum time for BMX-IN-1 pre-treatment was 12 h. (3) The mRNA expressions of TNF-α and IL-1β of cells in BMX-IN-1 control group were 0.97±0.13 and 0.98±0.06, respectively, which were similar to 1.00 of blank control group (with P values above 0.05). The mRNA expressions of TNF-α and IL-1β of cells in LPS control group were 2.97±0.17 and 3.07±0.60, respectively, while those in BMX-IN-1+ LPS group were 2.31±0.94 and 2.55±0.73, respectively, with the 4 values significantly higher than those in blank control group (with P values below 0.01). The mRNA expressions of TNF-α and IL-1β of cells in BMX-IN-1+ LPS group were significantly lower than those in LPS control group (with P values below 0.05). The activity values of BMX and p38MAPK of cells in BMX-IN-1 control group were 0.95±0.19 and 0.98±0.18, respectively, which were close to 1.00±0.14 and 1.00±0.22 of blank control group (with P values above 0.05). The activity values of BMX and p38MAPK of cells in LPS control group were 1.98±0.33 and 2.05±0.34, respectively, which were significantly higher than those of blank control group (with P values below 0.01). The activity values of BMX and p38MAPK of cells in BMX-IN-1+ LPS group were 1.00±0.17 and 1.67±0.27, respectively, which were obviously lower than those of LPS control group (P<0.05 or P<0.01).@*Conclusions@#BMX can increase the production of pro-inflammatory cytokines TNF-α and IL-1β from mouse mononuclear-macrophages induced by LPS, which may be associated with the activation of the p38MAPK pathway by BMX.