# Effects of Bilberry Extract on Hepatic Cholesterol Metabolism in HepGG2 Cells
## 4 Discussion  
Hypercholesterolem is a major modifiable risk factor for the prevention of CVD [36]. Plasma cholesterol level is tightly associated with the regulation of cellular cholesterol metabolism [37]. Natural products and their bioactive components have preventative effects on CVD by exerting antioxidant, anti-inflammatory and hypocholesterolemic properties. Among natural products, polyphenol intake improved metabolic syndrome, a cluster of conditions associated with CVD risk factors, in animal and human clinical trials [38; 39; 40; 41]. With high contents of polyphenols and anthocyanins, bilberry is known for its antioxidant and anti-inflammatory effects [42; 43; 44]. Previously, we reported the hypocholesterolemic effects of BE by altering the genes for the stimulation of TICE in Caco-2 cells [34]. The stimulation of TICE is an attractive strategy for the prevention of CVD [8]. However, the primary pathway for cholesterol removal from the body is mediated by hepatoliquality cholesterol driven by RCT [9; 10]. Furthermore, the liver is the principal site of cholesterol metabolism, accounting for 70% of fecal sterol excretion under normal conditions [45]. Therefore, we investigated whether the hypocholesterolemic effects of BE are attributed to the regulation of hepatic genes involved in cholesterol biosynthesis, uptake and efflux in HepG2 cells. The previous study confirmed that BE, the standardized bilberry extract to anthocyanin contents of 25%, contains 237.9 \(\pm\) 17.1 mg cyanidin-3-glucoside equivalents/g of total anthocyanin [34]. Consistent with other studies, BE with high anthocyanin contents exerted antioxidant effects by scavenging DPPH and ABTS radicals and reducing power. The ethanolic extract of bilberry exhibited antioxidant effects by exerting significant DPPH and ABTS radical scavenging activity and suppressing nitric oxide in LPS-induced Raw 264.7 macrophages [46]. These data support that bilberry with high contents of anthocyanin can be used as a natural antioxidant. Regulation of cholesterol biosynthesis is one of the regulatory mechanisms of maintaining cholesterol homeostasis. SREBPs are the transcription factor that regulates lipid metabolism [47]. SREBP2, the master regulator for cholesterol metabolism, is primarily responsible for the transcription of HMGR and LDLR [48]. HMGR, the key rate-limiting enzyme for de novo cholesterol synthesis, converts HMGCoA to mevalonate, the key  
Figure 6: Modulation of SIRTs in BE-treated HepG2 cells. The cells were treated with either 50 or 100 \(\upmu\)g/mL of BE for 24 h. The mRNA expression of SIRT isoforms was measured by qRT-PCR. Bars with different letters are significantly different (\(p\) < 0.05). Values are means \(\pm\) S.E.M. \(n\) = 6.  
intermediate in cholesterol biosynthesis [49]. The most commonly prescribed drug to lower cholesterol is statins, the HMGR inhibitors. Decreasing hepatic cholesterol biosynthesis by statins will lead to the upregulation of LDLR responsible for the clearance of LDL from plasma [11]. In the present study, the maturation of SREBP2 was significantly decreased with concomitant decreases in HMGR mRNA and protein levels by BE treatment. Consistent with these findings, natural products rich in polyphenols, including flavone and anthocyanin, modulated expressions of HMGR and SREBP2. Bergamot rich in flavones decreased HMGR levels and flavone luteolin decreased HMGR transcription by suppressing the nuclear translocation of SREBP2 in HepG2 cells [50; 51]. Mulberry extract rich in anthocyanin ameliorated mRNA and protein abundance of HMGR in HepG2 cells [52]. Anthocyanin-rich red raspberry supplementation lowered HMGR expression, leading to decreased total cholesterol concentrations in high-fat-diet-induced hyperlipidemic mice [53]. Supplementation of Solanum nigrum polyphenols ameliorated the hepatic HMGR and SREBP2 protein levels in high-fat-diet-induced obese mice [54]. These results support that the reduction of HMGR and SREBP2 by BE may be attributed to its high polyphenol contents.