Synergistic effect of phytochemicals on cholesterol metabolism and lipid accumulation in HepG2 cells

CGA, GEN and QUE (purity > 98%, as determined by HPLC) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). CRO was extracted and purified as previously reported (purity > 98%, as determined by HPLC) [17]. All drugs were dissolved in 0.1% dimethyl sulfoxide (DMSO). The chemical structures of purified CRO, CGA, GEN, and QUE are illustrated in Fig. 1. All other chemicals used were of molecular biology and analytical grade.

The HepG2 human hepatoma cell line was purchased from the China Center for Type Culture Collection. HepG2 cells were grown and maintained in RPMI-1640 medium (Invitrogen, Waltham, MA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO₂. Cells were subcultured upon reaching approximately 80–90% confluency.

HepG2 cell growth was determined using the MTT assay. Cells (2 × 10⁴) were seeded into 96 well micro-titer plates containing 100 μL complete culture medium per well, and incubated for 24 h at 37 °C. Cultures were transferred into serum-free medium 12 h prior to performing the assay. A range of concentrations of CRO (0.01, 0.1, 1, 10, and 100 μmol/L), CGA (0.3, 3, 30, 300, and 3000 μmol/L), GEN (0.1, 1, 10, 100, and 1000 μmol/L), and QUE (1, 10, 20, 40, 60, 80, and 100 μmol/L) were added. Cells were incubated for a further 24 or 48 h in an incubator (37 °C at 5% CO₂), and then treated with MTT for 4 h. DMSO (150 μL) was added to each well, and plates were then shaken for 10 min. Absorbance was measured at 490 nm using a microplate reader (Bio-Rad). Cell viability in each group was presented as a percentage of the control.

HepG2 cells (2 × 10⁴) were seeded into 6- or 24-well plates and cultured. After incubation in the presence or absence of the different drugs for 48 h, cells were fixed with 4% polyformaldehyde, and then incubated in Oil red O working solution for 30 min. Microscopic images were taken to visualize red oil droplets with an Olympus microscope (Olympus Corporation, Tokyo, Japan). After incubation in the presence or absence of the different drugs for either 24 or 48 h, cell supernatant was used for analysis of total cholesterol (TC) and triacylglycerol (TG) levels. TC and TG kits (Beihuakangtai, Beijing, China) were used according to the manufacturer’s instructions.

Expression of the following genes was studied using semi-quantitative RT-PCR: ATP-binding cassette transporter (ABCA1), sterol regulatory element binding protein 2 (SREBP2), cholesterol 7α-hydroxylase (CYP7A1), liver X receptor alpha (LXRα), AMP-activated protein kinase 2α (AMPKα2) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). Primer pairs (Sangon, Shanghai, China) are listed in Table 1. RNA isolation (Total RNA Extractor, Sangon, Shanghai, China), cDNA synthesis (TaKaRa PrimeScript™ RT reagent Kit, Baosheng, Dalian, China), and amplification (TaKaRa Ex Taq PCR Kit, Baosheng, Dalian, China) were performed as per manufacturer’s instructions. Amplification was performed using a three step temperature cycle as follows: ABCA1, AMPKα2, and HMGCR: 32 cycles at 95 °C for 20 s, 57 °C for 30 s, and 72 °C for 45 s; SREBP2, LXRα, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 32 cycles at 95 °C for 20 s, 56 °C for 30 s, and 72 °C for 45 s; and CYP7A1: 32 cycles at 95 °C for 20 s, 58 °C for 30 s and 72 °C for 30 s. Polymerization was performed at 72 °C for 5 min. Amplified product was analyzed in 1% agarose gel, photographed, and analyzed using Quality One Software (SPSS Inc., Chicago, IL). Each relative densitometric value was calculated after normalization to GAPDH.

HepG2 cells were plated into 6-well chamber slides. After treatment with the different drugs for 48 h, the slides were fixed in 4% neutral buffered paraformaldehyde for 30 min at room temperature, subsequent HMGCR detection. Immunostaining was carried out using a kit (Boaoseng, Beijing, China, SP-0023). The endogenous peroxidase was inactivated by treated the slides with 0.3% hydrogen peroxide for 10 min. After washed with PBS, the slides were added into normal goat serum to incubate for 10 min at 37 °C. Rabbit polyclonal HMGCR antibody (1:50) (Boaoseng, Beijing, China, LOT. YE0905W, bs-5068R) was used as the primary antibody to incubated cells at 4 °C for overnight after washed. The slides were washed and incubated with biotinylated secondary antibody for 30 min. And then slides were added into peroxidase-labeled streptavidin for another 30 min. Slides were stained with 3,3′-diaminobenzidine for 25 min. Cells were incubated with nonimmune serum or PBS to control antibody specificity, and then the nuclei were counterstained with H&E.

Total cell protein from each sample was extracted using a kit (Protease inhibitor cocktail set I, Calbiochem, USA) and quantified using a Bio-Rad protein kit (Pierce, Rockford, IL). Equal quantities of protein were separated using 10% SDS-PAGE and transferred to PVDF membrane (Millipore Corporation, USA) overnight at 4 °C: the membrane was then blocked in 5% non-fat milk. The membrane was probed separately with the primary antibodies for GAPDH-HRP (1:1000, Kangcheng, Shanghai, China) and rabbit antihuman HMGCR (1:1000, Cell Signaling), and secondary antibodies (1:10000, Santa Cruz). Signal was detected using an ECL Kit (Millipore, USA) and developed on X-ray film. Protein bands were detected by ChemiDoc XRS systems and Quality One 4.6.1 (Bio-Rad, Hercules, CA) software, and GAPDH antibody was used as the internal control.

We examined the cytotoxicity of CRO, CGA, GEN, and QUE to determine suitable concentrations for the treatment of HepG2 cells. Results showed that 100 μM CRO, CGA, and GEN, and ≤ 10 μM QUE had no inhibitory effect on HepG2 cell viability after 24 or 48 h of treatment (Fig. 2). Therefore, subsequent experiments used the following concentrations: 10 μmol/L SIM, 1 μmol/L CRO, 30 μmol/L CGA, 10 μmol/L GEN, 10 μmol/L QUE, and a combination of 1 μmol/L CRO, 30 μmol/L CGA, 10 μmol/L GEN, and 10 μmol/L QUE (CCGQ), consistent with concentrations used in previous studies [18‐21].

To test whether drugs affected lipid deposition, we examined lipid droplets in HepG2 cells using Oil red O staining. Compared with the control group, we found prominent lipid accumulation in the cytoplasm of HepG2 cells, using 0.1 mmol/L of oleate. Compared with the oleate group, treatment with SIM, CRO, CGA, GEN, and CCGQ significantly attenuated the accumulation of lipid droplets, however, QUE alone did not induce notable inhibition (Fig. 3a, b). To further assess lipid deposition, we determined TC and TG content in the culture medium of HepG2 cells (Fig. 3c, d). Consistent with Oil red O findings, all drugs elevated extracellular TC and TG levels after treatment for 24 or 48 h. CCGQ and CRO induced significant increases in concentrations of secreted TC in cells treated for 48 h, which were greater than effects produced by any other single drug. Compared with the SIM group, treatment with CCGQ for 24 or 48 h significantly increased extracellular TC and TG content (p < 0.05). These results indicate that combined treatment with CCGQ results in a much greater decrease in lipid accumulation in HepG2 cells than treatment with the drugs individually.

In order to further clarify the mechanism responsible for the changes in cholesterol levels of HepG2 cells, we next examined the mRNA expression levels of cholesterol metabolism-related enzymes (results shown in Fig. 4). All drugs increased ABCA1 mRNA expression in comparison with the control group, and had significantly greater effects than SIM treatment. CYP7A1 mRNA expression was significantly enhanced by treatment with SIM, CCGQ, CGA, and GEN, but was comparable to the control group in CRO-treated cells, and was lower than levels in all other groups in QUE-treated cells. All individual drugs except GEN significantly decreased HMGCR mRNA expression.

Numerous studies indicate that AMPK, LXRα and SREBP2 play key roles in regulating cholesterol homeostasis. As shown in Fig. 4, treatment with SIM, CCGQ, or CRO significantly inhibited SREBP2 expression compared to the control group, while the other drugs had no significant effect on SREBP2 expression. Treatment with SIM significantly up-regulated LXRα mRNA expression, while the opposite effect was observed in cells treated with all other drugs. All drugs except SIM enhanced AMPK2α expression compared with the control group.

We also examined intracellular localization and expression of HMGCR protein by immunostaining and western blotting, after treatment for 48 h with the different drugs. Figure 5 demonstrates prominent accumulation of brown HMGCR granules localized in the cytoplasm of control, CRO, and QUE cells. Treatment with SIM, CGA, GEN, or CCGQ markedly reduced HMGCR expression compared with untreated cells. Consistent with these results, as shown in Fig. 6, HMGCR protein expression decreased in all treatment groups compared to the control group, but not as much as the SIM group. In particular, CCGQ combined treatment resulted in a much greater decrease in HMGCR expression than individual treatments.