To further verify the effect of BBR on ACAT protein
To further verify the effect of BBR on ACAT2 protein expression, we have conducted an in vitro permeability assay using Caco-2 monolayer. This assay has long been used to predict the in vivo Caspase-5, human recombinant protein and bioavailability of a compound. It was observed that the amount of cholesterol esters in the medium of basolateral chamber was significantly decreased in the presence of BBR and the effect was dose-dependent. A similar effect was seen on the permeability of total cholesterol while no effect was found on free cholesterol. It is indicated by the results that cholesterol esterification in Caco-2 cells was inhibited by BBR, in agreement with the reduced gene and protein expressions of ACAT2 in both the Caco-2 cells and small intestine. As there were no significant differences of cholesterol-lowering efficacy among the three BBR doses, a lower dose than 50mg/kg·d might be equally effective. Thus, a dose optimization study should be conducted prior to clinical application. According to the dose translation from animals to humans , the dosage of 50mg/kg·d in rats is equivalent to 567mg/d for a 70kg adult, which is highly achievable. Various dosages were used in different in vitro assays. For the micellarization assay, a series of dosage was applied. The results were presented only for those that showed significant effects and a few higher concentrations that showed no further improvement. For cell-based assays, cell toxicity was performed first and then treatment concentrations were chosen within the non-toxic range. These concentrations are physiologically relevant in rats as estimated on the dosages of BBR and water consumption , the primary dilutor in the gastro-intestinal lumen, and the bioavailability of BBR . However, a pharmacokinetic study is required to determine whether the concentrations used in the present studies are physiologically relevant. In human subjects, cholesterol absorption is partially determined by genetic traits and increased body weights were also reported to lower cholesterol absorption efficiency due to increased biosynthesis . In the current study, there was not a huge difference in the body weights of rats fed the AC and NC diets and no significant correlation was found between the body weights and cholesterol absorption rates in rats of these two diet groups. It was suggested that if there was any impact of body weight on cholesterol absorption, the effect might be minimal and confound with the effects of dietary cholesterol and bile acid supplementation. Moreover, the NC was used to determine whether rats fed the AC diet significantly increased blood cholesterol levels and thus validated the experimental model. The primary focus was to determine the efficacy and mechanisms of action of BBR on cholesterol metabolism by comparing among the AC and three treatment groups. The significant difference in blood lipid levels but no significant difference in body weights occurred among the AC and three treatment groups provided meaningful determinations on the mechanism of action of BBR in cholesterol absorption and homeostasis. The present study investigated the intestinal cholesterol absorption rate, which was measured in a biology system using the plasma dual isotope ratio method. This method was well-established and cross-validated against several other methods that have long been used to quantitatively measure cholesterol absorption . The rate of cholesterol absorption in a biology system is a result of comprehensive effects of multiple steps involved in the absorption process and a reliable parameter for the effect of a given compound on cholesterol absorption in vivo. Further in vitro and in vivo experiments on these steps using biochemical and molecular approaches consistently supported the inhibitory effect of BBR on cholesterol absorption. The effect of BBR on cholesterol micellarization and absorption could be further evaluated in vivo by measuring cholesterol content in the intestinal lumen micelles and lymphatic chylomicrons, respectively, which become the limitations of the present study.