| OBJECTIVE: To simulate the endotoxin tolerance model and to investigate the expression of TREM-1 in endotoxin tolerance monocytes by stimulating THP-1 cells with LPS.METHODS: THP-1 cells were cultured in serum-free medium. They were randomly divided into groups A(negative control group), B, C and D(experiment groups), divided groups B, C and D into B1, B2, B3, C1, C2, C3, D1, D2, D3 sub-groups. Cultured groups A, B, C and D in serum-free medium with 0, 10,100,500 ng/mL LPS for 24 h, Cultured group A in serum-free medium with 0 ng/mL LPS, sub-groups B1, C1, D1 in serum-free medium with 10 ng/mL LPS, sub-groups B2, C2, D2 in serum-free medium with 100 ng/mL LPS, sub-groups B3, C3, D3 in serum-free medium with 500 ng/mL LPS respectively for 6 h. The expression of TREM-1 mRNA was detected by RT-PCR. The expression of TREM-1 and NF-κB p65 protein was detected by Western Blot. TNF-αand IL-10 was determined by ELISA.RESULTS: The results showed that the A values of TREM-1/β-actin in sub-groups A, B1, B2, B3, C1, C2, C3, D1, D2 and D3 were 0.5641±0.0031, 0.6112±0.0012, 0.6860±0.0041, 0.8360±0.0034, 0.4687±0.0031, 0.5862±0.0038, 0.5958±0.0032, 0.4387±0.0038, 0.5762±0.0044, 0.5894±0.0041 the total gray values of TREM-1 protein/GAPDH in sub-groups A, B1, B2, B3, C1, C2, C3, D1, D2 and D3 were 0.4698±0.0096, 0.5962±0.0044, 0.6541±0.0043, 0.7617±0.0050, 0.4700±0.0042, 0.4878±0.0021, 0.5204±0.0010, 0.3985±0.0036, 0.4393±0.0015, 0.4953±0.0051, the total gray values of NF-κB p65/GAPDH in sub-groups A, B1, B2, B3, C1, C2, C3, D1, D2 and D3 were 0.7627±0.0009, 0.8146±0.0047, 0.8661±0.0034, 0.9720±0.0065, 0.5548±0.0047, 0.7852±0.0061, 0.8037±0.0052, 0.5214±0.0026, 0.7742±0.0039, 0.7855±0.0041, the O.D. values of TNF-αin sub-groups A, B1, B2, B3, C1, C2, C3, D1, D2 and D3 were 0.0746±0.0032, 0.0826±0.0053, 0.1201±0.0135, 0.1904±0.0135, 0.0722±0.0037, 0.0821±0.0033, 0.0859±0.0031, 0.0654±0.0047, 0.0748±0.0050, 0.0812±0.0042, the total gray values of il-10 in sub-groups A, B1, B2, B3, C1, C2, C3, D1, D2 and D3 were 0.0929±0.0052, 0.1290±0.0053, 0.0966±0.0037, 0.0802±0.0044, 0.1940±0.0091, 0.1124±0.0078, 0.0849±0.0047, 0.2573±0.0055, 0.2553±0.0087, 0.2259±0.0074. The differences were significant among groups A,B,C,D, the differences among subgroups B,C and D were sigmnificant, the differences among subgroups B1, C1 and D1, B2, C2 and D2, B3, C3 and D3 were sigmnificant. The TREM-1 mRNA expression was positively correlated with the TREM-1 protein expression(r=0.922, P=0.000), the TREM-1 mRNA expression was positively correlated with the NF-κB p65 protein expression(r=0.936, P=0.000), the TREM-1 protein expression was positively correlated with the NF-κB p65 protein expression(r=0.795, P=0.000), the TREM-1 mRNA expression was positively correlated with the TNF-αexpression(r=0.901, P=0.000), the TREM-1 protein expression was positively correlated with the TNF-αexpression(r=0.897, P=0.000), the TREM-1 mRNA expression was negatively correlated with the IL-10 expression(r=-0.627, P=0.000), the TREM-1 protein expression negatively correlated with the IL-10 expression(r=-0.661, P=0.000). In na?ve THP-1 cell, high LPS concentration could result in the higherexprssion of TREM-1. In endotoxin tolerance THP-1 cells, when the LPS concentrations for the first stimulation were the same, high LPS concentration for the second stimulation could result in the higher expression of TREM-1, when the LPS concentrations for the second stimulation were the same, high LPS concentration for the first stimulation could result in the higher expression of TREM-1. In THP-1 cells, expreesion of TREM-1 was correlated with NF-κB p65 and TNF-αpositively, with IL-1 negatively.CONCLUSIONS: This suggested that TREM-1 could amplify the signal in the singal transduction when monocytes and macrophages were stimulated by LPS. It was concluded that the down-regulation of the expression of TREM-1 mRNA and TREM-1 protein might play an important role in the development of endotoxin tolerance cells and sepsis.
|
PDF Full Text Request