Palmitate Up-regulates The Expression Of Toll-like Receptor4in Vascular Endothelial Cells

Objective:To study the effect of the saturated FFA, palmitate, on toll-like receptor4(TLR4) expression in vascular endothelial cells, explore the effect of TLR4on secretion of interleukin-6and tumor necrosis factor alpha in endothelial cell under palmitic acid stimulation, to explore the molecular mechanism underlying endothelial dysfunction induced by FFAs in metabolic syndrome.Methods:Pig Iliac Endothelial cells were cultured in RPMI1640. the cells were divided intoexperimental groups and control groups. Experimental groups were treated with different concentration palmitate acid(PA100μmol/l、200μmol/l), the control group were treated without palmitate acid. After6hours,12hours and24hours, TLR4gene expression levels were measured in PIECs by quantitative real-time PCR.. After12hours, TLR4protein contents were measured in PIECs by western blotting. Then surface levels of TLR4protein in PIECs were quantified using flow cytometry. The TNF-a and IL-6concentrations in cells supernatants were detected by sandwich ELISA at the6h,12h and24h after200μmol/l PA stimulating, respectivelyResults:1. quantitative real-time PCR:after being stimulated by effective concentration of PA with100μM, the expression of TLR4gene were increased for6h, but there was no significant difference compared with control group (P>0.05); after12h, the expression of TLR4gene compared with6h and the control group had significant difference (P<0.05); after24h, the expression of TLR4mRNA than12h was lower (P<0.05), but there was significant difference compared with the6h and the control group (P<0.05). PA with200μM and PA with100μM groups are basically the same, but the expression of TLR4gene began to increase in PA with200μM group after6h, compared with the control group with significant difference (P<0.05). the expression of TLR4gene was increase in PA with200μM group compared with PA with100μM group at the same time point, with significant difference (P<0.05). The expression of TLR4gene had not significant difference in Each control group.2. western blot:after being stimulated by effective concentration of PA with200μM and100μM, increased TLR4protein content were observed in PIECs of palmitate group compared with control group for12h (P<0.05); each palmitic acid group of binary comparison between had significant difference(P<0.05).3. flow cytometry:after being stimulated by effective concentration of PA with200μM and100μM, caused a significant increase in surface levels of TLR4compared with control group for12h (P<0.05); each palmitic acid group of binary comparison between had significant difference(P<0.05).4. enzyme linked immunosorbent assay:after being stimulated by effective concentration of PA with200μM, the secretion of IL-6and TNF-α were increased significantly for only6h (P<0.05), moreover the effects were increased gradually with the time, the peaks were at24h (P<0.05).Conclusion:1.Palmitate up-regulated the expression of TLR4gene and protein content in pig iliac endothelial cells. Under this experimental condition, the expression level of TLR4gene was in a downward trend in the endothelial cells after12h, this may be related to boosting the cytotoxic effect of palmitic acid, caused by disorders of DNA and protein synthesis in cells.2. Palmitate increases TLR4gene expression and protein content in vascular endothelial cells, leading to a net increase in the number of endothelial cell surface TLR4receptors. In a coordinated fashion, increased TLR4expression and activity could result in the development of insulin resistance in vascular endothelial cells. 3. Palmitate up-regulated the expression of TLR4in pig iliac endothelial cells,further increasing the production of IL-6and TNF-α. The time peak IL-6and TNF-α generated time peak lags behind the peak time of TLR4, show that the enhanced expression of TLR4is related with the production of IL-6and TNF-α.