LPS Effect RL95-2Cells Make β-1,4-GalT-I Expression Changes And Correlation Of MAPK Signal Pathway

Objective: β-1,4-galactose transferase–I (β-1,4-GalT-I) can play aglycosyltransferase and cell recognition and adhesion, migration and metastasis intumor cells, synaptic growth, sperm egg recognition and binding process.LPS (Lipopolysaccharides, lipopolysaccharide) with the activation of macrophages andneutrophils, T lymphocytes, reacted and produced inflammatory. Research shows thatLPS could up regulate endothelial cell adhesion molecules such asICAM-1(intercellular adhesion molecule-1), PECAM-1(platelet endothelial celladhesion molecule-1), and adhesion molecules CD11b. MAPKs have four subfamiliesERK1/2, p38-MAPKMAPK, JNK, ERK5, ERK1/2and ERK5signal transductionpathway regulating cell growth and differentiation, JNK and p38-MAPK in MAPKsignal transduction pathway may play an important role in inflammation and apoptosisin response to stress. Increased transcription factor CREB as target moleculedownstream of p38-MAPK and ERK signal pathway, its phosphorylation can regulatetarget gene. In this study, throughing the detection of β-1,4-GalT-I`s expression inRL95-2cells after stimulated by LPS, and the relationship between CREB and β-1,4-GalT-I, find out the correlation between MAPK signaling pathway and themechanism of LPS effect β-1,4-GalT-I expression change.Methods: MTT assay the influence of different concentrations of LPS on RL95-2cell viability; RL95-2cells were divided into different group, LPS effect different time(0,2,4,6,8h) and different concentration (0μg/ml,10μg/ml,100μg/ml,1000μg/ml)gradient group culture, RT-PCR, Western Blot detect the expression of β-1,4-GalT-I after LPS stimulated, find out the optimum conditions of LPS in RL95-2cells; WesternBlot detect MAKPs pathway ERK1/2, p38-MAPK molecular phosphorylation changesand phosphorylation of CREB which is one in the downstream of ERK1/2andp-38-MAPK; transfected the interference sequence of CREB in RL95-2cells, WesternBlot detect the differential expression of β-1,4-GalT-I; add p38-MAPK inhibitorSB203580, ERK1/2inhibitor U0126role in RL95-2cells analyzed β-1,4-GalT-I`sexpression and phosphorylation of CREB, find out the correlation between them.Results: MTT results showed that LPS inhibited RL95-2cell viabilitysignificantly, compared with the control group, the concentration1000μg/mlLPScultured24h, cell viability was significantly inhibited (p<0.05), except0μg/ml and10μg/ml for48h, other concentrations showed significant inhibition of cell viability(p<0.01); the expression of β-1,4-GalT-I was increased after LPS stimulated, and thedose and time-dependence effects. When LPS concentration100μg/ml effect4hβ-1,4-GalT-I gene expression reaches its maximum level (p<0.05), LPS concentration100μg/ml6h β-1,4-GalT-I reaches its maximum expression at the protein level (p<0.01); LPS can upregulate phosphorylation of CREB(p<0.05), p38(p<0.05) andERK1/2(p<0.01) in RL95-2cells; Western Blot result shows CREB sequence effective,transfected the sequence in RL95-2cells CREB expression decreased obviously(p<0.01); Western Blot detect after interferenced CREB expression of β-1,4-GalT-I wasdecreased (p<0.01); in p38-MAPK inhibitor SB203580group and ERK1/2inhibitorU0126group, CREB phosphorylation and β-1,4-GalT-I no significant increase inexpression (p <0.05) compare with the LPS group.Conclusion: LPS effect RL95-2cells make β1,4-GalT-I expression increased,and CREB, p38-MAPK, ERK1/2phosphorylation levels increased; p38-MAPK andERK1/2take a part of the process of LPS make β1,4-GalT-I expression changesthrough adjust the phosphorylation of CREB, and LPS on the activity of the RL95-2cells was inhibited.