Lipopolysaccharide induced synthesis of CD14 proteins and its gene expression in hepatocytes during endotoxemia.

aimTo observe synthesis of CD14 protein and expression of CD14 mRNA in hepatic tissue and hepatocytes of rats during endotoxemia.

methodsThe endotoxemia model of Wistar rat was established by injection of a dose of lipopolysaccharide (LPS) (5mg x kg(-1), Escherichia coli O111:B4) via the tail vein, then the rats were sacrificed after 3, 6, 12 and 24 h in batches respectively. Hepatocytes were isolated from normal and LPS-injected rats by in situ collagenase perfusion technique and were collected to measure the expression of CD14 mRNA and synthesis of CD14 protein by reverse transcript-polymerase chain reaction (RT-PCR) or Western blot analysis. The binding of fluorescein isothiocyanate (FITC)-CD14 polyclonal antibody to isolated hepatocytes was also assessed by flow cytometric analysis (FCM).

resultsIn the rats with endotoxemia, the expressions of CD14 mRNA in hepatic tissue and isolated hepatocytes were stronger at 3, 6, and 12 h than that in control rats (3.48+/-0.15, 5.89+/-0.62, 4.33+/-0.18, vs 1.35+/-0.14 in hepatic tissue, P<0.01; 4.12+/-0.17, 6.24+/-0.64, 4.35+/-0.18, vs 1.87+/-0.15 in hepatocytoes, P<0.01). The synthesis of CD14 protein in hepatic tissue and isolated hepatocytes increases also obviously in 6 and 12 h when compared to that in control rats (13.27+/-1.27, 17.32+/-1.35, 11.42+/-1.20, vs 7.34+/-0.72 in hepatic tissue, P<0.01; 14.68+/-1.30, 17.95+/-1.34,11.65+/-1.19, vs 7.91+/-0.70 in hepatocytes, P<0.01). FCM showed that mean fluorescence intensity (MFI) and numbers of FITC-CD14 positive cells in the rats with endotoxemia increased obviously at 3,6,12 and 24h when compared with normal control group (43.4%, 70.2%, 91.4%, 32.6% vs 4.5%, P<0.01).

conclusionLPS can markedly promote the synthesis of CD14 protein and up-regulate the expression of CD14 mRNA in isolated hepatocytes and hepatic tissue. Liver might be a main source for soluble CD14 production during endotoxemia.