The impact of BMX on tumor necrosis factor-α secretion and phagocytic function in lipopolysaccharide-stimulated macrophages

Hu Ying; Han Linian; Qiu Le; Wang Fei; Chen Xulin

doi:10.3969/j.issn.1672-8521.2025.03.003

Abstract：Objective:To investigate the effects of non-receptor tyrosine kinase,bone marrow tyrosine kinase on chromosome X(BMX),on lipopolysaccharide(LPS)stimulated macrophage secretion of the pro-inflammatory cytokine tumor necrosis factor-α(TNF-α)and phagocytic function against Escherichia coli.Methods:The macrophage cell line RAW264.7 cells were used for the following experiments.(1)The effect of BMX on macrophage secretion of pro-inflammatory cytokine TNF-α:experimental group included blank control group,LPS group,25 μmol/L BMX-IN-1+LPS group,and 75 μmol/L BMX-IN-1+LPS group.The LPS group was stimulated with 10 ng/mL LPS for 1 h.The 25 μmol/L BMX-IN-1+LPS group and the 75 μmol/L BMX-IN-1+LPS groups were pre-treated with the corresponding concentration of the BMX inhibitor BMX-IN-1 for 12 h,and then stimulated with 10 ng/mL LPS for 1 h.The secretion of TNF-α in macrophages was detected by flow cytometry,and the expression of BMX and phosphorylated c-Jun amino-terminal kinase(p-JNK)proteins were detected by Western blotting.(2)The impact of BMX on macrophage phagocytic function under inflammatory conditions:green fluorescent Escherichia coli(E.coli)was shaken overnight and the absorbance value was measured spectrophotometrically.The experiment was divided into blank control group,LPS group,and BMX-IN-1+LPS group.The BMX-IN-1+LPS group was pre-treated with 75 μmol/L BMX-IN-1 for 12 h,while the LPS group and the BMX-IN-1+LPS group were stimulated with 10 ng/mL LPS.Meanwhile,the three groups were added with an equal amount of E.coli simultaneously,RAW264.7 cells∶E.coli=1∶50.After 2 h of action,macrophage phagocytic rate was detected by flow cytometry.Results:(1)There were statistically significant differences in TNF-α secretion among the blank control group,LPS group,and different concentrations of BMX-IN-1+LPS groups(F=151.1,P＜0.05).The LPS group showed the highest TNF-α secretion level[(78.68±3.67)%].BMX-IN-1 intervention significantly reduced TNF-α secretion,with the most pronounced decrease observed in the 75 μmol/L BMX-IN-1+LPS group(P＜0.05).(2)Western blotting analysis revealed that LPS stimulation significantly upregulated intracellular BMX and p-JNK expression,which could be inhibited by pretreatment with BMX-IN-1.Statistical analysis demonstrated significant intergroup differences in BMX(F=319.2,P＜0.05)and p-JNK(F=1,897.0,P＜0.05)expression levels.(3)Flow cytometry analysis of macrophage phagocytic rate against E.coli showed no statistically significant differences among the PBS control group,LPS group,and BMX-IN-1+LPS group(F=0.46,P＞0.05).Conclusions:The non-receptor tyrosine kinase BMX promotes LPS-stimulated TNF-α secretion in macrophages through the JNK pathway,but does not affect their phagocytic function.