Effect Of DFMG Intervention On TLR4 Signaling Pathway On Endothelial Tubule Formation

Objective:To study the effect of DFMG on endothelial tubule formation of human umbilical vein endothelial cells(HUVE-12)induced by LPC via the regulation of Toll like receptor 4(TLR4)signal pathway.Methods:1.The HUVE-12 endothelial cells were incubated with different concentration(0,10,20,30,40?M)of phosphatidylcholine for oxidation-damaged endothelial cells.Cell proliferation activity was detected by CCK-8 assay.The content of LDH in the supernatant was detected by lactate dehydrogenase(LDH)kit.The effect of LPC on endothelial cell migration was detected by scratch test.Western blotting was used to detect TLR4 and VEGF protein expression,to determine optimum LPC concentration.2.HUVE-12 endothelial cells were builded by the optimum concentration of LPC(30 ?M).HUVE-12 endothelial cells treated with different doses of DFMG(0.3,3 M)were incubated treated with LPC for 30 min.cell proliferation was measured by CCK-8 assay,The content of LDH in the supernatant was measured bylactate dehydrogenase(LDH)kit.The mRNA expression of TLR4,VEGF was used detected by qPCR and the protein expression of TLR4,VEGF was detected by Western blotting.The effects of DFMG on the migration and endothelial tubule formation of HUVE-12 endothelial cells were detected by scratch test and Matrigel matrix gelatin test.3.HUVE-12 stable cell lines with over expression of TLR4 and TLR4 suppression expression were established,detected the mRNA expression of TLR4 by qPCR in stable cell lines.and DFMG and LPC to carry on the processing,Effect of DFMG on the endothelial tubule formation in the TLR4 overexpression lentivirus or in the Si-TLR4-lentivirus HUVE-12 cell lines was detected by Matrigel matrix gelatin test.Results:1?LPC inhibited the proliferation of HUVE-12 cells in a concentration dependent manner,promoted the release of LDH and the migration of endothelial cells,and up-regulated the protein expression of TLR4 and VEGF.30 ?M LPC was selected as the optimal model concentration for inducing HUVE-12 oxidative damage model.2?DFMG antagonized the inhibitory effect of LPC on HUVE-12 proliferation,inhibited of its migration and angiogenesis,down regulation of TLR4 and VEGF mRNA and protein expression3?DFMG inhibits the endothelial tubule formation effect of TLR4 overexpressed cell lines after oxidative damage,and the effect is consistent with the TLR4 inhibiting expression strain,and the difference is not statistically significant.Conclusions:1?DFMG could inhibit endothelial tubule formation via the TLR4 signaling pathway.