# Polyphenol-Rich Black Elderberry Extract Stimulates Transintestinal Cholesterol Excretion
## 4 Discussion
ACAT2 esterifies cholesterol for absorption [57], and MTTP assembles and secretes chloromicon from enterocytes to the basolateral lumen, facilitating intestinal cholesterol absorption [10]. The expression of ACAT2 and MTTP was significantly decreased in BEE-treated Caco-2 cells. Consistent with our results, apple polyphenols decreased lipoprotein secretion, contributing to its hypolipidemic effects in Caco-2/TC7 enterocytes [58]. All of the genes involved in the flux of cholesterol from the apical to basolateral side of enterocytes were decreased by BEE treatment. Intestine-specific stimulation of TICE can be an alternative pathway for cholesterol excretion. The underlying mechanisms of TICE are still unknown, but intestinal SR-B1 and HDL are not involved in the process of TICE [41]. In contrast, LDL-derived cholesterol and LDLR have been reported to be involved in the alteration of TICE. Notably, we observed significant increases in both mRNA and protein levels of LDLR in BEE-treated Caco-2 cells. To investigate the underlying mechanisms of LDLR alteration by BEE, we measured whether BEE could induce PCSK9. PCSK9, responsible for LDLR protein degradation, was not altered by BEE. The heterodimer ABCG5/G8 is a well-known transporter for cholesterol flux to the intestinal lumen. Furthermore, LDL-derived cholesterol taken up by LDLR can be effluxed to the intestinal lumen via ABCG5/G8, stimulating TICE. In the process of TICE, the cholesterols can be transported from the basolateral to the apical side of the enterocyte for subsequent excretion from the body. BEE increased both mRNA and protein levels of ABCG5, which may decrease intestinal cholesterol absorption. ABCG5/G8 transporters mediate the flux of free cholesterol to the intestinal lumen [59]. ABCB1, the apical transporter, acts as a cholesterol floppage and plays an essential role in TICE [60]. BEE markedly increased the mRNA and protein levels of ABCB1. These results indicate that BEE may stimulate the transporters involved in cholesterol flux from the basolateral to apical side of enterocytes. The availability of triglycerides regulates the assembly of lipoproteins [9; 10]. Therefore, we measured the genes involved in fatty-acid metabolism in BEE-treated Caco-2 cells. BEE decreased the expression of lipogenic transcription factor SREBP1c and its downstream genes such as _FAS_ and _SCD-1_. According to our results, there was a marked alteration of genes involved in intestinal cholesterol and lipid metabolism. Mammalian sirtuins (SIRTs), nicotinamide adenine dinucleotide-dependent deacetylases, are known as epigenetic and physiological regulators of biological metabolism [61; 62]. Mounting evidence supports that SIRTs can regulate cholesterol and lipid metabolism [63; 64]. Resveratrol, a well-known polyphenol present in wine, is known to modulate SIRT [65; 66]. Therefore, we investigated whether BEE could alter SIRT expression. We observed an induction of SIRT1, SIRT3, and SIRT6 and a reduction in SIRT2 expression by BEE in Caco-2 cells. Further study is warranted to understand the mechanisms of SIRT alteration by BEE. All of the gene changes, which occurred in a direction to flux cholesterol from the basolateral to the apical side of the enterocyte, by BEE indicate that black elderberry may lower plasma cholesterol by increasing the uptake of LDLR-mediated cholesterol circulation in the enterocyte. Furthermore, BEE altered the expression of SIRTs. These results indicate that black elderberry might have hypocholesterolem effects via alteration of the TICE pathway.