Cholesterosis is a disease of cholesterol metabolism characterized by the presence of excessive lipid droplets in the cytoplasm. These lipid droplets are mainly composed of cholesterol esters derived from free cholesterol. The removal of excess cholesterol from gallbladder epithelial cells (GBECs) is very important for the maintenance of intracellular cholesterol homeostasis and the preservation of gallbladder function. Several lines of evidence have indicated that the activation of either peroxisome proliferator-activated receptor gamma (PPAR gamma) or liver X receptor a (LXR alpha) relates to cholesterol efflux. While pioglitazone can regulate the activation of PPAR gamma, 22(R)-hydroxycholesterol can activate LXR alpha, and is a metabolic intermediate in the biosynthesis of steroid hormones. However, the effect of 22(R)-hydroxycholesterol in combination with pioglitazone on cholesterosis of the gallbladder is unclear. GBECs were treated with pioglitazone, 22(R)-hydroxycholesterol or PPAR gamma siRNA followed by Western blot analysis for ATP-binding cassette transporter Al (ABCA1), PPAR gamma and LXR alpha. Cholesterol efflux to apoA-I was determined, and Oil Red 0 staining was performed to monitor variations in lipid levels in treated GBECs. Our data showed that 22(R)-hydroxycholesterol can modestly up-regulate LXR alpha while simultaneously increasing ABCA1 by 56%. The combination-of 22(R)-hydroxycholesterol and pioglitazone resulted in a 3.64-fold increase in ABCA1 expression and a high rate of cholesterol efflux. Oil Red 0 staining showed an obvious reduction in the lipid droplets associated with cholesterosis in GBECs. In conclusion, the present findings indicate that the anti-lipid deposition action of 22(R)-hydroxycholesterol combined with pioglitazone involves the activation of the PPAR gamma-LXR alpha-ABCA1 pathway, increased ABCA1 expression and the efflux of cholesterol from GBECs. Thus, 22(R)-hydroxycholesterol synergistically combined with pioglitazone to produce a remarkable effect on lipid deposition in cholesterosis GBECs. (C) 2014 Elsevier Inc. All rights reserved.