The Role Of LXRs Agonists In The Regulation Of Cholesterol Homeostasis And Release Of Cytokines In RAW 264.7 Cell

Objective: Liver X receptors (LXRαand LXRβ) are ligand-dependent transcriptional regulators, belong to nuclear receptors superfamily. LXRs are'cholesterol sensors'that regulate the expression of many proteins involved in lipid (especially cholesterol) metabolism and innate immune response. They play an important role in lipid homeostasis and inflammation. LXRs constitute with DNA-binding and ligand-binding domain. LXRs binding with retinoid X receptors (RXRs) to form LXR-RXR heterodimer, which can be activated by ligands of LXRs and/or RXRs. LXR-RXR heterodimer regulate the expression of target genes by interacting with LXR response elements (LXRE). Physiologic activators for the LXRs include oxysterols and intermediates in the cholesterol biosynthetic pathway such as 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27-hydroxycholesterol and 24(S),25-epoxycholesterol. The synthetized LXR agonists include T-1317 and GW3965. Methyl 3β-hydroxy-5α,6α-epoxycholanate (MHEC) was synthetized by our university. Recent studies have demonstrated that LXR agonists have an important atheroprotective effect in macrophages. The transcriptional activation of LXRs induces the expression of genes, such as ABCA1 and ApoE, which facilitate the efflux of cholesterol and reverse cholesterol transport (RCT). Recent studies have also revealed the existence of crosstalk between macrophage inflammatory pathways and nuclear receptor signaling. Synthetic ligands for LXR have been reported to inhibit inflammatory gene expression,such as iNOS, COX-2, IL-6 and MMP-9. In this study, the effects of T-1317 and MHEC on RAW 264.7 cells were observed in vitro.Methods: RAW264.7 murine macrophage-like cells were cultured at 37°C in a humidified incubator (95% air with 5% CO2) in DMEM medium with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin. Adherent cells were subcultured over night in 6-well or 24-well Costar tissue culture plates at a concentration of 500,000 cells per well in serum-free DMEM. The cells were treated with T-1317 and MHEC with or without 9-cRA. Immunohistochemical Streptavidin/Peroxidase method was used to assess the expression of ABCA1 protein and fluorometric assay was used for the quantitative determination of the...