The Effect Of Non-receptor Tyrosine Kinase BMX On LPS-stimulated Macrophage In Secretion Of Pro-inflammatory Cytokine TNF-? And Phagocytosis Of E.coli

Background Burn wounds due to the large number of necrotic and degenerative tissues,bacterial colonization is inevitable.Gram-negative bacteria(Pseudomonas aeruginosa,Escherichia coli,Klebsiella,etc.)caused a gradual increase in the trend of infection.The non-receptor tyrosine kinase BMX(bone marrow tyrosine kinase on chromosome X)is referred to as bone marrow kinase X and is one of the Tec family members.The Tec family kinase is a "star protein" researched at home and abroad in recent years and is the second largest family of non-receptor tyrosine kinases.BMX kinase was initially found to be closely related to the development of tumors.It is widely expressed in bone marrow hematopoietic cells,lymphocytes,epithelial cells and endothelial vascular cells and certain malignant tumor cells(prostate cancer,breast cancer,renal cell carcinoma,etc.),and participates in inflammatory reactions and a variety of intracellular signal transduction.It plays an important role in cellular immunity,development,proliferation,and apoptosis.Recent studies have shown that BMX kinase is associated with the occurrence of some chronic inflammatory diseases,immune system diseases(rheumatoid arthritis)and trauma(brain reperfusion injury).Macrophages are present in all tissues and play an important role in development,homeostasis,tissue repair and immunity.As an important part of innate immunity,macrophages play an important role in many tissues.Macrophages are named in different tissues,including microglia,tissue cells,osteoclasts,alveolar macrophages,No cells and other mononuclear-macrophage system is an important antigen presenting system,both(monocytes for macrophage precursors)have different antigen recognition and presentation ability,macrophages through the microfilament involved in the cell Phagocytosis in phagocytosis activates specific immune systems.Studies have shown that the endotoxin of Gram-negative bacteria in the wound infection and the occurrence and development of sepsis has an important role in this experiment macrophage cell line RAW264.7 cells as experimental materials,E.coli LPS-induced cellular inflammatory response The effect of non-receptor tyrosine kinase BMX on the secretion of proinflammatory cytokine TNF-? and phagocytosis by LPS-stimulated macrophages and its possible molecular mechanism were observed by using specific inhibitor BMX-IN-1 Further clarify the role of innate immune macrophages in the inflammatory response after burn,and provide advice on the prevention and treatment of post-burn infection.Purpose To investigate the effect of non-receptor tyrosine kinase BMX on LPS-stimulated macrophage secretion of proinflammatory cytokine TNF-? and phagocytosis of E.coli.Contents PART I Time Effect of Expression of the First Nonreceptor tyrosine Kinase BMX in Inflammatory Circumstances Mouse RAW264.7 macrophages following conventional culture is divided into two dose effect and time effect was studied.the time effect study: Accession to the0.1ug/ml and LPS were stimulated by 0,15 min,30min,60 min and 120min;the expression of P-BMX kinase in cells was detected using Western blot method.The secretion of TNF-? was measured by flow cytometry.PART II The role of non-receptor tyrosine kinase BMX in LPS-induced secretion of proinflammatory cytokine TNF-? by macrophages and its signaling pathway Macrophage cell line RAW264.7 was made into a single cell suspension and cultured in a 48-well plate,1 * 106/ well.Experimental group: blank control group,LPS group,25 ummol / l + LPS group,75 ummol / l + LPS group.LPS group stimulated with10 ng / ml LPS for 1 h.The latter two groups were pretreated with appropriate dose of BMX-IN-1 for 12 h and then stimulated with 10 ng / ml LPS for 1 h.The secretion of TNF-? was measured by flow cytometry.The same experimental method and Western blot were used to detect the expression of BMX,P-JNK protein expression.PART ? The effect of nonreceptor tyrosine kinase BMX on macrophage phagocytosis in an inflammatory environment.Macrophage cell line RAW264.7 was made into a single cell suspension and cultured in a 48-well plate,1 * 106 / well.Shake the bacteria overnight,E.coli with green fluorescence,spectrophotometer OD value.RAW264.7 cells: E.coli = 1:50.Experimental group: blank control group,LPS group,75?mol / L BMX-IN-1+ LPS group.LPS group and 75?mol / LBMX-IN-1 + LPS group were stimulated with LPS for 2 hours in 75?mol / L BMX-IN-1+ LPS group with 75?mol / LBMX-IN-1 for 12 hours.Flow cytometry was used to detect macrophage phagocytosis.Results PART I Time Effect of Expression of the First Nonreceptor tyrosine Kinase BMX in Inflammatory Circumstances TNF-?(7.96%?25.93%?42.61%?53.63%?70.71%)was detected by flow cytometry with 10 ng / ml LPS stimulation(0,15,30,60,120 min).After stimulated with10 ng / ml LPS for 15 min,the P-BMX band was the brightest by Western blot.PART II The role of non-receptor tyrosine kinase BMX in LPS-induced secretion of proinflammatory cytokine TNF-? by macrophages and its signaling pathway(1)Graph Pad Prism software analysis of one-way ANOVA,75?mol / L BMX-IN-1 +LPS group and LPS group,q = 5.337,P <0.01;75?mol / L BMX-IN-1 + LPS group and 25?mol / L BMX-IN-1 + LPS group,q = 3.960,P <0.05;75?mol / L BMX-IN-1+ LPS group and the blank control group,q = 21.07,P <0.001,all with statistical significance.(2)Western blot showed that the brightness of BMX and P-JNK bands were weakened,and the univariate analysis of variance was performed with Image J and Graph Pad Prism software.The results of BMX / ?-Actin and P-JNK / ?-Actin were consistent.PART ? The effect of nonreceptor tyrosine kinase BMX on macrophage phagocytosis in an inflammatory environment.In the inflammatory environment,macrophage phagocytosis percentage of Escherichia coli was detected by flow cytometry.One-way analysis of variance was performed by Graph Pad Prism software.LPS group and 75?mol / L + LPS group,q =0.2535,P> 0.05.There was no statistical significance.Conclusions(1)The non-receptor tyrosine kinase BMX promotes LPS-stimulated macrophages to secrete pro-inflammatory cytokine TNF-? through the JNK pathway.(2)The non-receptor tyrosine kinase BMX had no statistical significance for the phagocytosis of LPS-stimulated macrophages.