The Effect Of FXR On ApoM Expression And The Initial Research Of Its Mechanism

Background:Atherosclerosis (AS) is a cardiovascular disease, it's a serious threat to the human health. Plasma cholesterol level is closely related to the formation of atherosclerotic plaque. Farnesoid X Receptor (FXR) was found as an orphan nuclear receptor, as the sensor of the endogenous bile acids, which plays an important role to maintain the metabolic balance of cholesterol. And the chenodexycholic acid (CDCA) is the optimal natural ligands for FXR, which plays an extremely important role in regulating the metabolism of cholesterol, lipids and glucose, by regulating many target genes. FXR is closely related with AS. However, so far, it is difficult to determine whether FXR promotes the development of AS.Apolipoprotein M (apoM) is a newly discovered apolipoprotein which belongs to the Lipocalin superfamily members. ApoM contains a specific hydrophobic ligand-binding box, and mature apoM reserves the signal peptide having "the hydrophobic anchor" effect. Studies have been found that it is quite possible that apoM anchor around the phosphatide single layer of high-density lipoprotein (HDL) by this signal peptide, and apoM is extremely rich in content in HDL. The research results show that apoM may be involved in the process of HDL metabolism by influencing the reverse transport process of cholesterol, by which apoM plays important role in regulatory function.FXR and apoM both have a high expression in the liver, and which are closely related with the AS. Now, we have a question whether apoM is a new target gene of FXR? In order to Solve the problem, we will further clarify the role of FXR and apoM in the occurrence and development of AS, then finally provide a new scientific evidence for finding a new target to prevent and cure the AS. Objective:To investigate FXR effects to apoM expression, and the possible mechanism of FXR.Methodology:(1) Select hepatoma cell HepG2 and fetal liver cell L02 as the cell model. Test 1 treated the cell model by CDCA for 24h, then detect the changes of apoM expression by using RT-PCR; test 2, treated by CDCA for 12h, then treated by inhibitor Guggulsterones for 24h, detect the changes of apoM expression by using RT-PCR.(2) Select hepatoma cell HepG2 as cell model, detect the differences of apoM expression by using Western Blot.(3) Select C57BL/6 mice as animal model, feed them with CDCA, and detect the changes of apoM expression by RT-PCR, and then detect the differences of protein by western blot method.(4) Through bioinformatics analysis, online Projected apoM gene 5'flanking promoter region-1863bp Department may exist FXR binding sites (DR2); HepG2 cells extracted genomic DNA, using PCR amplification of recombinant plasmids were apoM report PGL3-basic/apoM (-1900～+165) with DR2 sequence PGL3-basic/apoM (-1800～+165) non-DR2 sequence. Use of liposomes, with the FXR and RXR plasmid were transfected into HepG2 cells in transient, by luciferase reporter gene assay to observe the FXR gene expression on the activity of apoM.Conclusion.Results:(1) FXR agonist (CDCA) treatment of human HepG2 hepatoma cells and fetal liver cells L02, we found that FXR ligands was dose-dependent reduction in apoM expression;(2) In FXR inhibitor (Guggulsterones) treated HepG2 human hepatoma cells and fetal liver cells L02, we found that FXR ligands was dose-dependent increases apoM expression(3) C57BL/6 mice were fed food containing CDCA, FXR activation in vivo reduced the expression of mouse apoM.(4) Immunohistochemistry showed that mice:With the increasing concentration of CDCA, mouse liver and kidney apoM expression in a dose-dependent decrease.(5) Determination of lipids in mice results:HDL and TC concentrations decreased with CDCA, LDL concentration and increased with the CDCA. This is consistent with the experimental results. TG concentrations with CDCA first increasing and then decreasing. (6) According to bioinformatics analysis of human apoM gene transcription start site upstream 3000bp region, there is obtained in-1863bp Department may FXR binding sites (DR2). By cotransfection results showed that:FXR can reduce the human apoM (-1900～+165) promoter fragment containing the DR2 sequence of transcriptional activity, and the reduction from 40% of the control. And FXR sequences do not contain DR2 people apoM (-1800～+165) promoter fragment had no effect on activity.Conclusion:(1)With the FXR agonist (CDCA) treatment cells, discover FXR ligands was dose-dependent reduction in the expression of apoM;(2) With FXR inhibitor (Guggulsterones) treated cells and found that FXR ligands was dose dependent increases the expression of apoM(3) FXR activation in vivo reduced the expression of mouse apoM.(4) results were transfected by draw:FXR may be human apoM gene promoter through specific binding sequence (DR2) down apoM gene expressionStudy discussed above, and apoM FXR in lipid metabolism and related diseases, AS provides an important role in the experimental evidence...