Inhibitory Effect of Isoliquiritigenin in Niemann-Pick C1-Like 1-Mediated Cholesterol Uptake

Figures

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  Figure 1. (A) Schematic diagram of the mechanism of NPC1L1 in small intestine and liver cholesterol transport. (B) Schematic diagram of NPC1L1-mediated cholesterol uptake process. (PL) phospholipids, (BA) bile acids, (FC) free cholesterol, and (PM) plasma membrane.

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  Figure 2. SPR assay of the interaction of ISL with NPC1L1. (A) Binding sensing map showing the interaction of EZ with immobilized NPC1L1. (B) Binding sensing map showing the interaction of ISL with immobilized NPC1L1. (Inhibitor concentrations were 0, 12.5, 25, 50, 75 and 100 μM).

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  Figure 3. (A) NPC1L1 protein levels in HepG2, Caco-2, U-2OS, SW1990, MCF-7, and Hela with β-Actin as the internal reference protein. (B) Analysis of the optical densitometry of the protein blots.

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  Figure 4. Inhibitory effect of ISL on cholesterol uptake in HepG2/Caco-2 cells. Cells were pretreated with different concentrations of ISL for 4 h and then incubated with radioactive micellar cholesterol for 1 h. In the absence of ISL, cholesterol uptake was normalized to 100%. Results are the mean ± SEM of three determinations in three independent experiments. (A) Inhibitory effect of ISL on cholesterol uptake in HepG2 cells. (B) Inhibitory effect of ISL on cholesterol uptake in Caco-2 cells. * p < 0.05, ** p < 0.001, *** p < 0.0001.

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  Figure 5. ISL inhibited cellular absorption of cholesterol by filipin bacteriocin staining. (A) The procedure used to treat the cells. (B,C) Quantification of total cholesterol of cells in (D). Error bars represent standard deviations. * p < 0.05, ** p < 0.001, *** p < 0.0001. (D) Cells were treated as shown in (A). Images were captured with Nikon confocal microscope after fixing cells and staining with filipin at different time points. Scale bar = 20 μm.

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  Figure 6. ISL is a noncompetitive inhibitor of NPC1L1. (A) Schematic diagram of the mode of action of competitive inhibitors. (B) Lineweaver-Burk plots in the presence of different concentrations of ISL.

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  Figure 7. The result of Molecular Docking. (A) The cholesterol binding cavity (red) in the NTD. (B) Binding modes of ISL to NPC1L1 (PDB: 6V3F). The inhibitor ISL was shown color by element (carbon in cyan). The critical amino acid residues were shown color by element (carbon in yellow).

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  Figure 8. Effect of ISL on NPC1L1 protein expression in HepG2 cells. (A) Cells were treated with different concentrations of ISL for 24 h, and whole cell lysates were analyzed by protein blotting. (B) Analysis of the optical densitometry of the protein blots. ** p < 0.001, *** p < 0.0001.

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  Figure 9. (A) Diagram of ISL molecular structure. (B) Effect of 100 μM ISL on cell viability. 100 μM ISL had a minimal effect on cell viability. The viability of HepG2 cells was approximately 87%, and that of Caco-2 cells was approximately 93%. Experiments were performed in triplicate and expressed as mean ± standard deviation.