Fig 1.
Effect of tanshinone IIA on the cell viability in HepG2 cells.
(A) Chemical structure of tanshinone IIA. (B) HepG2 cells were cultured in LPDS medium and treated with the vehicle (0.1% DMSO) or tanshinone IIA (1–20 μM) for 24 h. Cell viability was measured using an MTT assay. The data represent the mean ± SD from three independent experiments.
Fig 2.
Effect of tanshinone IIA on the LDLR mRNA and protein expression.
(A) HepG2 cells were cultured in LPDS medium and treated with the vehicle (0.1% DMSO) or tanshinone IIA (5 and 10 μM) for 24 h. The LDLR mRNA was measured by RT-Q-PCR. (B) The LDLR promoter-luciferase reporter construct and a Renilla luciferase control plasmid were co-transfected into HepG2 cells for 24 h, and the cells were then treated with the vehicle (0.1% DMSO) or tanshinone IIA (5 and 10 μM) for 24 h. The luciferase activities were measured, and the data represent the mean ± SD from three independent experiments. (C) The level of LDLR protein was determined by western blot analysis. A representative blot is shown. (D) The normalized intensity of LDLR versus β-actin is presented as the mean ± SD of three independent experiments. (E) The HMG-CoA reductase mRNA was measured by RT-Q-PCR. *p<0.05 and **p<0.01 represent significant differences compared to the vehicle group.
Fig 3.
Effect of tanshinone IIA on the cell-surface LDLR levels and LDL uptake in HepG2 cells.
HepG2 cells were cultured in LPDS medium and treated with the vehicle (0.1% DMSO) or tanshinone IIA (5 and 10 μM) for 24 h. (A) The level of cell-surface LDLR was determined by flow cytometry analysis. A representative histogram is shown. (B) Summary of cell-surface LDLR levels. (C) BODIPY®-FL-LDL uptake was determined by flow cytometry analysis. A representative histogram is shown. (D) Summary of LDL uptake. The data represent the mean ± SD from three independent experiments. ** p<0.01 represent significant differences compared to the vehicle group.
Fig 4.
Effect of tanshinone IIA on PCSK9 mRNA and protein expression.
(A) HepG2 cells were cultured in LPDS medium and treated with the vehicle (0.1% DMSO) or tanshinone IIA (5 and 10 μM) for 24 h. PCSK9 mRNA expression was measured by RT-Q-PCR. The data represent the mean ± SD from three independent experiments. ** p<0.01 represent significant differences compared to the vehicle-treated cells. (B) The cells were transfected with a pGL3-basic plasmid (as a vector control) or PCSK9 promoter reporter construct (PCSK9-p(-1459/-4)) and a Renilla control plasmid for 24 h. The cells were then treated with the vehicle (0.1% DMSO) or tanshinone IIA (5 and 10 μM) for 24 h. The luciferase activities were measured, and the data represent the mean ± SD from three independent experiments. ** p<0.01 represent significant differences compared to the pGL3-basic vector-transfected cells. ## p<0.01 represent significant differences compared to the vehicle-treated cells. (C) PCSK9 precursor (p) and mature form (m) were measured by western blot analysis. A representative blot is shown. (D) The normalized intensity of mature PCSK9 (m) versus β-actin is presented as the mean ± SD of three independent experiments. ** p<0.01 represent significant differences compared to the vehicle-treated cells.
Fig 5.
Effect of tanshinone IIA on PCSK9 promoter activity.
(A) Serially deleted PCSK9 promoter reporter constructs. (B) HepG2 cells were transfected with the serial deleted PCSK9 promoter reporter constructs for 24 h and then treated with the vehicle (0.1% DMSO) or tanshinone IIA (10 μM) for 24 h. The luciferase activities were determined and normalized to their respective Renilla luciferase control activities. The data represent the mean ± SD from three independent experiments. **p<0.01 represents a significant difference compared to the pGL3-basic vector-transfected cells. ## p<0.01 represents a significant difference compared to the respective vehicle-treated group. (C) The analysis of the tanshinone IIA-response element within the PCSK9 promoter. The previously characterized HNF-1α, SREBP2 and FoxO3a binding sites are indicated as HNF-1α site, SRE and FoxO3a site, respectively.
Fig 6.
Effect of tanshinone IIA on the nuclear accumulation of HNF-1α, SREBP2 and FoxO3a proteins.
(A) Levels of nuclear HNF-1α and SREBP2 proteins measured by Western blot analysis. A representative blot is shown. The normalized intensities of HNF-1α (B) and SREBP2 (C) versus HDAC2 are presented as the mean ± SD of three independent experiments. *p<0.05 represents a significant difference compared to the vehicle-treated group. (D) The nuclear FoxO3a protein was detected by western blot analysis. A representative blot is shown. The normalized intensity of FoxO3a versus HDAC2 is presented as the mean ± SD of three independent experiments. ** p<0.01 represents a significant difference compared to the vehicle-treated group. (E) The ChIP assay was performed as described in the Materials and Methods section. The PCSK9 promoter complex was measured by RT-Q-PCR. The data were expressed as the fold enrichment of the FoxO3a antibody (FoxO3a Ab) or HNF-1α antibody (HNF-1A Ab) over the control IgG. The data represent the mean ± SD from three independent experiments. # p<0.05 and ## p<0.01 represents a significant difference compared to the vehicle-treated control IgG group.
Fig 7.
Effect of the combination of tanshinone IIA and statin treatments on PCSK9 mRNA expression.
The HepG2 cells were cultured in LPDS medium and pretreated with the vehicle or tanshinone IIA (10 μM) for 1 h and then incubated with 1 μM lovastatin or simvastatin for an additional 24 h. (A) Cell viability was measured using an MTT assay. (B) The PCSK9 mRNA was measured by RT-Q-PCR. The data represent the mean ± SD from three independent experiments. **p<0.01 represents a significant difference compared to the vehicle-treated cells. ##p<0.01 represents a significant difference compared to the statin (lovastatin or simvastatin) alone treatment group.
Fig 8.
Effect of the combination of tanshinone IIA and statin treatments on PCSK9 protein expression and LDLR activity.
The HepG2 cells were cultured in LPDS medium and pretreated with the vehicle or tanshinone IIA (10 μM) for 1 h and then incubated with 1 μM lovastatin or simvastatin for an additional 24 h. (A) The PCSK9 protein level was measured by western blot analysis. The immunoblot experiments were repeated three times, and a representative blot is shown. The normalized intensity of mature PCSK9 (m) versus β-actin is presented as the mean ± SD of three independent experiments. **p<0.01 represents a significant difference compared to the vehicle-treated cells. ##p<0.01 represents a significant difference compared to the statin (lovastatin or simvastatin) alone treatment group. (B) The LDL uptake by the cells was measured by flow cytometry analysis, and the data are expressed as the geometric mean fluorescence intensity. The data represent the mean ± SD from three independent experiments. **p<0.01 represents a significant difference compared to the tanshinone IIA-untreated vehicle group. ##p<0.01 represents a significant difference compared to the tanshinone IIA-untreated cells.
Fig 9.
Hypothetical mechanism by which the tanshinone IIA increased LDL uptake via down-regulation of PCSK9 expression in hepatic cells.
Tanshinone IIA increases the nuclear abundance of the FoxO3a and its interaction with PCSK9 promoter, which may result in the inhibition of HNF-1α/PCSK9 promoter complex formation and the decreases of PCSK9 gene expression. Down-regulation of PCSK9 gene expression by tanshinone IIA is associated with increases in the amount of cell-surface LDLR and its LDL-uptake activity in hepatic cells.