br Materials and methods br Results br Discussion Persimmon

Materials and methods
Results
Discussion Persimmon tannin has been reported to have the bile acid binding ability in vitro (Matsumoto et al., 2011) and hypolipidaemic effects in animals (Matsumoto et al, 2008, Zou et al, 2012). Our previous study also proved that persimmon tannin promoted the elimination of cholesterol from the body (Zou et al, 2012, Zou et al, 2014). However, the underlying molecular mechanisms of the cholesterol-lowering effect of persimmon tannin have not been fully elucidated. In the present study, we investigated whether persimmon tannin could affect cholesterol metabolism and modulate the expression of genes involved in cholesterol biosynthesis, cholesterol GSK591 receptor and cellular cholesterol efflux in HepG2 and Caco-2 cell lines. We found that persimmon tannin significantly (P < 0.05) reduced the cholesterol and 25-hydroxycholesterol induced cellular cholesterol accumulation in both HepG2 and Caco-2 cells. Moreover, persimmon tannin notably increased HDL-C levels, and decreased LDL-C and ApoB levels in HepG2 and Caco-2 cells, providing further support for hypocholesterolaemic effect of persimmon tannin. In addition, we found that persimmon tannin extensively modulates the expression of genes involved in cholesterol absorption and efflux in both HepG2 and Caco-2 cells. The intestine acts as a gate keeper for cholesterol absorption. Inhibition of cholesterol absorption has been demonstrated to contribute to the reduction of plasma cholesterol levels. The inhibition effects of persimmon tannin on the uptake of cholesterol in both cell lines were directly proved by the 22-NBD-cholesterol uptake experiment (Fig. 2), this novel fluorescent substrate has replaced radioactive substances labelled cholesterol for tracing cholesterol absorption (Sengupta et al., 2013). NPC1L1 transporter, located at the brush border membrane of enterocytes in the proximal ileum and jejunum, is identified as the key transporter for uptake of cholesterol and stanols (Lee-Rueckert et al, 2013, van der Wulp et al, 2013). Inhibitors of NPC1L1 could provide protection against diet-induced hypercholesterolaemia and hepatic steatosis. In the present study, we observed that persimmon tannin significantly decreased mRNA level by about 13% in Caco-2 cells, implying that the inhibitory effects of persimmon tannin on cholesterol uptake is mediated by down-regulation of NPC1L1 expression. Curcumin (Feng, Ohlsson, & Duan, 2010) and polyphenol-rich black chokeberry extract (Kim, Park, Wegner, Bolling, & Lee, 2013) have been reported to lower cholesterol levels by inhibiting NPC1L1 expression. SREBP-2 was reported to be tightly related with cholesterol biosynthesis. It regulates the expression of cholesterol synthesis-related genes, such as HMGR and LDLR, thus increasing the cellular cholesterol levels. Inhibitor of SREBP-2 was regarded to be a therapy to treat metabolism diseases such as atherosclerosis and type II diabetes (Xiao & Song, 2013). In the present study, persimmon tannin intervention inhibited the expression of SREBP-2 by about 36% and 57% in HepG2 and Caco-2 cells, respectively. Our results indicated that the inhibitory effect of persimmon tannin on SREBP-2 expression was close to that of polyphenol-rich black chokeberry extract (CBE), but stronger than that of grape seed procythocyanidin (Jiao et al., 2010). Additionally, it was reported that overexpression of SREBP-2 results in an enhanced transcription of NPC1L1 (Kumar et al., 2011), As seen in Fig. 3, compared to normal cells, the gene expression of SREBP-2 in high-cholesterol induced Caco-2 cells increased by about 7.8-fold, accompanying a notable up-regulation on the expression of NPC1L1 gene. However, after treatment with persimmon tannin, the increased expression of SREBP-2 was inhibited by about 61.57% in and Caco-2 cells, respectively. Simultaneously, the raised expression of NPC1L1 was also significantly down-regulated. These results demonstrated that the regulation effect of persimmon tannin on NPC1L1 gene might be mediated by SREBP-2.