Lunasin Improves the LDL-C Lowering Efficacy of Simvastatin via Inhibiting PCSK9 Expression in Hepatocytes and ApoE-/- Mice

Abstract

Statins are the most popular therapeutic drugs to lower plasma low density lipoprotein cholesterol (LDL-C) synthesis by competitively inhibiting hydroxyl-3-methyl-glutaryl-CoA (HMG-CoA) reductase and up-regulating the hepatic low density lipoprotein receptor (LDLR). However, the concomitant up-regulation of proprotein convertase subtilisin/kexin type 9 (PCSK9) by statin attenuates its cholesterol lowering efficacy. Lunasin, a soybean derived 43-amino acid polypeptide, has been previously shown to functionally enhance LDL uptake via down-regulating PCSK9 and up-regulating LDLR in hepatocytes and mice. Herein, we investigated the LDL-C lowering efficacy of simvastatin combined with lunasin. In HepG2 cells, after co-treatment with 1 μM simvastatin and 5 μM lunasin for 24 h, the up-regulation of PCSK9 by simvastatin was effectively counteracted by lunasin via down-regulating hepatocyte nuclear factor 1α (HNF-1α), and the functional LDL uptake was additively enhanced. Additionally, after combined therapy with simvastatin and lunasin for four weeks, ApoE^-/- mice had significantly lower PCSK9 and higher LDLR levels in hepatic tissues and remarkably reduced plasma concentrations of total cholesterol (TC) and LDL-C, as compared to each monotherapy. Conclusively, lunasin significantly improved the LDL-C lowering efficacy of simvastatin by counteracting simvastatin induced elevation of PCSK9 in hepatocytes and ApoE^-/- mice. Simvastatin combined with lunasin could be a novel regimen for hypercholesterolemia treatment.

Keywords: hepatocyte nuclear factor-1α; low density lipoprotein cholesterol; low density lipoprotein receptor; lunasin; proprotein convertase subtilisin/kexin type 9; simvastatin.

Figure 1

Effects of simvastatin combined with lunasin treatment on PCSK9 and HNF-1α expressions at the mRNA and protein levels in HepG2 cells. HepG2 cells were treated with simvastatin and/or lunasin for 24 h. The mRNA (A) and protein (B) levels of intracellular precursor PCSK9 (PCSK9-P) and mature PCSK9 (PCSK9-M), as well as the mRNA (C) and protein (D) level of HNF-1α were determined by qRT-PCR and Western blot using β-actin as an internal control, respectively. After transient transfection with siRNA for 4 h, EA.hy 926 cells were maintained in fresh medium for 48 h and treated with 1 μM lunasin for an additional 24 h. Then, the levels of HNF-1α (E) and PCSK9 (F) protein expression were analyzed by Western blot analyses, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the simvastatin group (n = 3, means ± SEM).

Figure 2

Effects of simvastatin in combination with lunasin treatment on the LDLR and LDL uptake levels in HepG2 cells. HepG2 cells were treated with simvastatin and/or lunasin for 24 h. The mRNA (A) and protein (B) levels of LDLR were analyzed by qRT-PCR and Western blot using β-actin as an internal control, respectively. * p < 0.05, ** p < 0.01 vs. the control group; ^# p < 0.05, ^### p < 0.001 vs. the simvastatin group. (C) LDL uptake was assessed in HepG2 cells after treatment with simvastatin and/or lunasin for 24 h on a fluorescence plate reader. ^ΔΔΔ p < 0.001 vs. the negativecontrol group; ^# p < 0.05 vs. the simvastatin group; *** p < 0.001 vs. the 20 μg/mL Dil-LDL group (n = 3, means ± SEM). Dil-DLD: LDL labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate.

Figure 3

The combination of simvastatin with lunasin suppresses the up-regulation of PCSK9 induced by simvastatin in ApoE^−/− mice. ApoE^−/− mice were administrated with 10 mg/kg simvastatin and/or 0.5 μmol/kg lunasin on a daily basis for four weeks. The expression levels of PCSK9 andHNF-1α mRNA (A) and protein (B) in ApoE^−/− mice were determined by qRT-PCR and Western blot, respectively. The levels of PCSK9 secreted in hepatic tissues (C,D) were detected by immunohistochemistry staining (a: C57BL/6; b: C57BL/6 + 0.5 μmol/kg lunasin; c: ApoE^−/−; d: ApoE^−/− + 0.5 μmol/kg lunasin; e: ApoE^−/− + 0.5 μmol/kg lunasin + 10 mg/kg simvastatin; f: ApoE^−/− + 10 mg/kg simvastatin). ^### p < 0.001 vs. C57BL/6 mice administrated with normal saline (NS); ** p < 0.01, *** p < 0.001 vs. ApoE^−/− mice administrated with NS; ^ΔΔΔ p < 0.001 vs. ApoE^−/− mice administrated with simvastatin (n = 8, means ± SEM). WT: wild type.

Figure 4

Effects of simvastatin in combination with lunasin on LDLR abundance in ApoE^−/− mice. ApoE^−/− mice were administrated with 10 mg/kg simvastatin and/or 0.5 μmol/kg lunasin on a daily basis for four weeks. The levels of LDLR mRNA (A) and protein (B) in liver tissues of ApoE^−/− mice were determined by qRT-PCR and Western blot, respectively. ^# p < 0.05 vs. C57BL/6 mice treated with normal saline (NS); *** p < 0.001 vs. ApoE^−/− mice treated with NS; ^ΔΔ p < 0.01, ^ΔΔΔ p < 0.001 vs. ApoE^−/− mice treated with simvastatin (n = 8, means ± SEM).

Figure 5

Effects of combined drug on serum cholesterol levels in ApoE^−/− mice fed with HFD. ApoE^−/− mice were i.p. administrated with 10 mg/kg simvastatin and/or 0.5 μmol/kg lunasin on daily basis for four weeks. LDL-C (A) and TC (B) concentrations in serum samples were measured by biochemical kits. ^### p < 0.001 vs. C57BL/6 mice administrated with normal saline (NS); *** p < 0.001 vs. ApoE^−/− mice administrated with NS; ^ΔΔΔ p < 0.001 vs. ApoE^−/− mice administrated with simvastatin (n = 8, means ± SEM).

Figure 6

Schematic diagram of the molecular mechanism by which lunasin enhances the LDL-C lowering efficacy of simvastatin via down-regulating PCSK9 expression.