RCT pathway plays a critical role
RCT pathway plays a critical role in the maintenance of cholesterol homeostasis by facilitating the cholesterol clearance and cholesterol efflux (Levinson & Wagner, 2015). In the process of RCT, ABCA1 and ABCG1 played as crucial regulators in cholesterol efflux (Yasuda et al., 2011). Compared with high cholesterol-treated cells, treatment with persimmon tannin significantly up-regulated the expressions of ABCA1 and ABCG1 in both HepG2 and Caco-2 cells. Similar results were also reported in cacao polyphenols (Yasuda et al., 2011) in HepG2 and Caco-2 cells and resveratrol in human macrophages (Sevov, Elfineh, & Cavelier, 2006). A randomised controlled trail in humans found that olive oil polyphenols enhanced ABCA1 gene expression in white blood cells at postprandial (Farras et al., 2013). Besides, ABCG5 and ABCG8, which work as sterol efflux pumps in the small intestine and liver, can promote efflux of cholesterol and plant sterols from the enterocyte into the intestinal lumen for excretion. Treatment with persimmon tannin up-regulated the gene expression of ABCG5/G8 in both cell lines significantly, illustrating that persimmon tannin could promote the efflux of cholesterol. SR-BI is a cell surface receptor that mediates selective HDL uptake and also facilitates the efflux of free cholesterol toward HDL (van der Wulp et al, 2013, Valacchi et al, 2011). Our data indicated that persimmon tannin up-regulated SR-BI gene expression in both cell lines, suggesting that cholesterol ester can be transported to the liver for the further excretion. It was reported that cacao liquor procyanidins significantly increased SR-BI mRNA expression in HepG2 cells (Yasuda et al., 2011), which was in line with our results well. In addition, conversion of cholesterol to bile acids is also an important regulatory point for elimination of cholesterol from the body. CYP7A1 plays a critical role in bile ONO-8711 biosynthesis. The activation of CYP7A1 may reduce the cholesterol accumulation in hepatocytes. Our previous study indicated that up-regulation of CYP7A1 gene of persimmon tannin in high fat diet rats partially accounted for its cholesterol lowering effect (Zou et al., 2014). Our present data demonstrated that persimmon tannin up-regulated the expression of CYP7A1 in both cells in both protein and mRNA levels, which was in agreement with previous studies well (Jiao et al, 2010, Zou et al, 2014). Taken together, our results suggested that the cholesterol-lowering effect of persimmon tannin was a synthesised result of its combined regulation on cholesterol homeostasis related genes, especially cholesterol efflux related genes, such as SR-BI, ABCA1, ABCG1, ABCG5/G8 and CYP7A1. It was reported that LXRα may be essential for the up-regulation of the cholesterol efflux and conversion related genes such as ABCA1, ABCG1, ABCG5/G8, and CYP7A1 genes in response to high dietary cholesterol (Chen et al, 2012, Rossmeisl et al, 2004). However, in the present study, we observed that persimmon tannin significantly enhanced the expression of ABCA1 and CYP7A1 both on the protein and mRNA levels, but decreased the expression of LXRα (Fig. 3 and Fig. 4). To further confirm whether the molecular mechanism of the inhibitory effect of persimmon tannin on cholesterol homeostasis is LXRα independent, we employed siRNA to block the expression of LXRα in both cells, and the results indicated that siRNA did not abrogate the up-regulation of persimmon tannin on the expression of CYP7A1 and ABCA1 in both transcriptional and translational levels (Fig. 5 and Fig. 6), confirming that the hypocholesterolaemic effect of persimmon tannin was LXRα independent. The regulation of the cholesterol metabolism-related genes by LXRα is complex and might differ for different compounds or in different systems. It has been found that LXRα stimulates the expression of CYP7A1 in mice but not in primary human hepatocytes (Goodwin et al, 2003, Peet et al, 1998). Grape-seed proanthocyanidin has also been found to enhance the excretion of bile acid by up-regulating CYP7A1 but lowering the expression of LXRα in hamsters (Jiao et al., 2010). Some previous studies demonstrated that other receptors, such as farnesoid X receptor (FXR) and PPARγ, or microRNAs may also regulate CYP7A1 and ABCA1 transcription and be responsible for the cholesterol homeostasis (Davalos, Fernandez-Hernando, 2013, Fu et al, 2014, Tu et al, 2013).