Effect Of LPS On TLR4-NF-кB Pathway In Human Intrahepatic Biliary Epithelial Cell

Objective Different time on normal human intrahepatic biliary epithelial cells(Hi BEC) in TLR4-NF-кB pathway TLR4 and NF-кB m RNA expression of inflammatory stimuli to explore different concentrations of lipopolysaccharide(LPS) acts the same time and the same concentration of LPS. In order to understand the possible mechanism of infected cells by changing the direction of the tumor.Methods In vitro culture of human intrahepatic biliary epithelial cells(HIBEC) to 4-6 generations, divided into three experimental groups: A group divided into six groups, one group of normal control group without LPS, the remaining five groups to join the same concentration LPS(4 ug / ml) but different time(respectively 3,6,9,12,24h); group B is divided into six groups, one group of normal control group without LPS, the remaining five groups were added to different concentrations of LPS(followed by 0.1,1,4,8,10 ug / ml) but the same duration of action(6h). Cells in each group were collected using Realtime RT-PCR analysis of the expression levels of two groups of cells within TLR4 m RNA and NF-κB m RNA’s. Group C: divided into two groups, namely the normal control group without LPS, another group using 4ug / ml of LPS, the role of 6h, using Western blot analysis of each team TLR4, NF-κB protein expression levels. The results of each group were using SPSS16.0 statistical software for data analysis, all the experimental data x±s, said many groups were compared using analysis of variance when(ANOVA), pairwise comparisons using the least significant difference method(LSD method) observed a linear relationship between the two variables are closely related to the extent and direction of the use of linear correlation(Pearson product-moment correlation coefficient). All provisions of P <0.05 was considered statistically significant.Results(1) Using LPS(4ug / ml) after stimulation HiBEC TLR4, NF-κB mRNA expression levels were significantly increased(P <0.05), showed that LPS activates Hi BEC in TLR4-NF-κB pathway, which mediates inflammation. Compared with the NC group, TLR4, NF-κB m RNA expression level reached a peak when stimulated with LPS 6h, for the effect of the strongest time of stimulation. TLR4 m RNA statistically significant positive correlation with the expression of NF-κB m RNA between has a significant(r = 0.979, P <0.05).(2) within a certain range(≤4ug / ml), with increasing concentrations of LPS, Hi BEC in TLR4, expression levels of NF-κB m RNA average increased, but the high concentration(≥8ug / ml) LPS stimulation effect gradually reduce, TLR4, NF-κB m RNA expression levels of an average reduction, 4ug / ml concentration for stimulation of the strongest effect. TLR4 m RNA statistically significant positive correlation with the expression of NF-κB m RNA between has a significant(r = 0.975, P <0.05).(3) the use of the most stimulating effect concentration and stimulus duration, namely 4ug / ml LPS stimulation Hi BEC 6h, the protein level detection TLR4, NF-κB protein expression levels, compared with the NC, LPS group TLR4, NF-κB a significant increase in protein expression(P <0.05).Conclusion 1. LPS can activate normal intrahepatic biliary epithelial cells of TLR4-NF-кB pathway, and the effect of the concentration of the strongest stimulus and stimulus duration 4ug / ml LPS stimulation Hi BEC 6h. 2. LPS stimulation at different times with different concentrations of normal human intrahepatic biliary epithelial cells, TLR4 m RNA expression and NF-кB m RNA both time and dose dependent manner. 3.Normal human intrahepatic biliary epithelial cells in 4-6 generations, the cell metabolism, it is appropriate to do experiments. Six generations later, the cells gradually aging apoptosis.