X染色体骨髓酪氨酸激酶在内毒素／脂多糖诱导RAW264．7小鼠单核巨噬细胞促炎性细胞因子产生中的作用及机制

Role of bone marrow tyrosine kinase on chromosome X in the production of pro-inflammatory cyto- kines from mouse mononuclear-macrophages RAW264.7 induced by endotoxin/lipopolysaccharide and its mechanism

目的 探讨X染色体骨髓酪氨酸激酶（BMX）在LPS诱导小鼠单核巨噬细胞产生TNF－α和IL－1β中的作用及相关机制。 方法 将RAW264.7小鼠单核巨噬细胞接种于6孔板中，常规培养，用于以下实验。（1）取细胞，按随机数字表法分为空白对照组、LPS对照组及75、750、7 500、75 000 nmol/L BMX－IN－1预处理组，每组8孔。空白对照组细胞常规培养25 h；LPS对照组细胞常规培养24 h后，用终质量浓度（下同）为0.1 μg/mL LPS刺激1 h；后4组细胞分别用终物质的量浓度（下同）为75、750、7 500、75 000 nmol/L的BMX－IN－1处理24 h后，用0.1 μg/mL LPS刺激1 h。实时荧光定量RT－PCR法检测各组细胞中TNF－α mRNA表达量，筛选BMX－IN－1最佳作用浓度。（2）取细胞，按随机数字表法分为LPS对照组及BMX－IN－1预处理2、4、8、12、18 h组，每组8孔。LPS对照组细胞用0.1 μg/mL LPS刺激1 h；后5组细胞用最佳作用浓度的BMX－IN－1分别处理2、4、8、12、18 h后，用0.1 μg/mL LPS刺激1 h。实时荧光定量RT－PCR法检测各组细胞中TNF－α mRNA表达量，筛选BMX－IN－1最佳作用时间。（3）取细胞，按随机数字表法分为空白对照组、BMX－IN－1对照组、LPS对照组和BMX－IN－1＋LPS组，每组16孔。空白对照组细胞常规培养最佳作用时间加1 h；BMX－IN－1对照组细胞用最佳作用浓度BMX－IN－1处理最佳作用时间后，常规培养1 h；LPS对照组细胞常规培养最佳作用时间后，用0.1 μg/mL LPS刺激1 h；BMX－IN－1＋LPS组细胞用最佳作用浓度BMX－IN－1处理最佳作用时间后，用0.1 μg/mL LPS刺激1 h。采用实时荧光定量RT－PCR法检测各组细胞中TNF－α和IL－1β mRNA表达量，蛋白质印迹法检测各组细胞中BMX和p38MAPK活性，样本数均为8。对数据行单因素方差分析和LSD检验。结果 （1）与空白对照组比较，其余5组细胞中TNF－α mRNA表达量显著增高（P值均小于0.01）。与LPS对照组比较，各BMX－IN－1预处理组细胞中TNF－α mRNA表达量均下降，但仅75 000 nmol/L BMX－IN－1预处理组细胞中TNF－α mRNA表达量显著降低（P〈0.05）。BMX－IN－1最佳作用浓度为75 000 nmol/L。（2）与LPS对照组比较，BMX－IN－1预处理2、4 h组细胞中TNF－α mRNA表达量无明显变化（P值均大于0.05），BMX－IN－1预处理8、12、18 h组细胞中TNF－α mRNA表达量显著降低（P〈0.05或P〈0.01）。其中，BMX－IN－1预处理12 h组细胞中TNF－α mRNA表达量最低。BMX－IN－1最佳作用时间为12 h。（3）BMX－IN－1对照组细胞中TNF－α和IL－1β mRNA表达量分别为0.97±0.13、0.98±0.06，与空白对照组的1.00相近（P值均大于0.05）。LPS对照组细胞中TNF－α和IL－1β mRNA表达量分别为2.97±0.17和3.07±0.60，BMX－IN－1＋LPS组细胞中TNF－α和IL－1β mRNA表达量分别为2.31±0.94和2.55±0.73，均显著高于空白对照组（P值均小于0.01）。BMX－IN－1＋LPS组细胞中TNF－α和IL－1β mRNA表达量明显低于LPS对照组（P值均小于0.05）。BMX－IN－1对照组细胞中BMX和p38MAPK活性分别为0.95±0.19和0.98±0.18，均与空白对照组的1.00±0.14和1.00±0.22相近（P值均大于0.05）；LPS对照组细胞中BMX和p38MAPK活性分别为1.98±0.33和2.05±0.34，均显著高于空白对照组（P值均小于0.01）。BMX－IN－1＋LPS组细胞中BMX和p38MAPK活性分别为1.00±0.17和1.67±0.27，明显低于LPS对照组（P〈0.05或P〈0.01）。结论 BMX可促进LPS诱导小鼠单核巨噬细胞促炎性细胞因子TNF－α和IL－1β的产生，该作用可能与BMX活化p38MAPK途径有关。

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.