Cholesterol-Lowering Activity of Vitisin A Is Mediated by Inhibiting Cholesterol Biosynthesis and Enhancing LDL Uptake in HepG2 Cells

Abstract

Pyranoanthocyanins have been reported to possess better chemical stability and bioactivities than monomeric anthocyanins in some aspects. The hypocholesterolemic activity of pyranoanthocyanins is unclear. In view of this, this study was conducted to compare the cholesterol-lowering activities of Vitisin A with the anthocyanin counterpart Cyanidin-3-O-glucoside(C3G) in HepG2 cells and to investigate the interaction of Vitisin A with the expression of genes and proteins associated with cholesterol metabolism. HepG2 cells were incubated with 40 μM cholesterol and 4 μM 25-hydroxycholeterol with various concentrations of Vitisin A or C3G for 24 h. It was found that Vitisin A decreased the cholesterol levels at the concentrations of 100 μM and 200 μM with a dose-response relationship, while C3G exhibited no significant effect on cellular cholesterol. Furthermore, Vitisin A could down-regulate 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) to inhibit cholesterol biosynthesis through a sterol regulatory element-binding protein 2 (SREBP2)-dependent mechanism, and up-regulate low-density lipoprotein receptor (LDLR) and blunt the secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein to promote intracellular LDL uptake without LDLR degradation. In conclusion, Vitisin A demonstrated hypocholesterolemic activity, by inhibiting cholesterol biosynthesis and enhancing LDL uptake in HepG2 cells.

Keywords: HMGCR; LDL uptake; Vitisin A; cholesterol biosynthesis; pyranoanthocyanins.

Figure 1

Several common pyranoanthocyanins’ structures. (A) Basic structure of anthocyanins. Structures of Vitisin A- or Vitisin B- (B), Phenolic- (C), Flavanol- (D), Methyl- (E), Portisin A- or Portisins B- (F), Oxovitisins- (G), and Dipolymer- (H) pyrananthocyanins. The structure of Cyanidin-3-O-glucoside (I) and Vitisin A (J) used in the current research.

Figure 2

Identification and quantification of Vitisin A. HPLC-DAD profiles of Vitisin A at 280 nm (A) and 520 nm (B). (C) Vitisin A characteristic absorption curve. (D) The LC-MS spectrogram of Vitisin A.

Figure 3

Effects of C3G, Vitisin A and Ator on the viability of HepG2 cells. (A,B) Cells were cultured with 50, 100, and 200 μM of C3G and Vitisin A for 24 h. (C) Cells were cultured with 10, 20, and 40 μM of Ator for 24 h. The cell viability of control was set at 100%. (One-way ANOVA in comparison between all groups, mean ± SD. ns, no significance) (D) Cells’ morphological changes with anthocyanins and Ator treatment (100×).

Figure 4

Effect of anthocyanins on the total cholesterol level in HepG2 cells. (A) Effects of 200 μM C3G and Vitisin A on TC in HepG2 cells. Cells were incubated with 40 μM cholesterol and 4 μM 25-HC, and 200 μM C3G, Vitisin A or 10 μM Ator for 24 h. (B) Effect of 0, 50, 100, and 200 μM Vitisin A on TC in HepG2 Cells. Cells were incubated with 40 μM cholesterol + 4 μM 25-HC and multiple concentrations of Vitisin A (50, 100 and 200 μM) and 10 μM Ator for 24 h. (One-way ANOVA in comparison between all groups. The results are expressed as mean ± SD, ^# p < 0.05, and ^### p < 0.001, compared with the blank group. * p < 0.05 and ** p < 0.01, *** p < 0.001, compared with the model group.).

Figure 5

Effects of Vitisin A on mRNA expression of cholesterol metabolism in HepG2 cells. Cells were incubated with 40 μM cholesterol, 4 μM 25-HC, and different concentrations of Vitisin A (50, 100, and 200 μM) or 10 μM Ator for 24 h. The mRNA expression of SREBP2 (A), HMGCR (B), LDLR (C), PCSK9 (D), LXRα (E), PPARγ (F), ABCA1 (G), ABCG5 (H), and ABCG8 (I). (One-way ANOVA in comparison between all groups. The results are expressed as mean ± SD, ^### p < 0.001, compared with the blank group. * p < 0.05 and ** p < 0.01, compared with the model group. ns, no significance).

Figure 6

Effects of Vitisin A on protein expressions of cholesterol metabolism in HepG2 cells. Cells were incubated with 40 μM cholesterol, 4 μM 25-HC, and different concentrations of Vitisin A (50, 100, and 200 μM) and 10 μM Ator for 24 h. Representative photographs and grayscale analysis of proteins involved in SREBP2 (A), HMGCR (B), LDLR (C), PCSK9 (D), LXRα (E), PPARγ (F), ABCA1 (G), ABCG5 (H), and CYP7A1 (I). (One-way ANOVA in comparison between all groups. The results are expressed as mean ± SD, ^# p < 0.05, compared with the blank group. * p < 0.05 and ** p < 0.01, *** p < 0.001, compared with the model group. ns, no significance).

Figure 7

A hypothetical mechanism of Vitisin A in cholesterol metabolism.