Extra Virgin Olive Oil Phenol Extracts Exert Hypocholesterolemic Effects through the Modulation of the LDLR Pathway: In Vitro and Cellular Mechanism of Action Elucidation

This study was aimed at investigating the hypocholesterolemic effects of extra virgin olive oil (EVOO) phenols and the mechanisms behind the effect. Two phenolic extracts were prepared from EVOO of different cultivars and analyzed using the International Olive Council (IOC) official method for total phenols, a recently validated hydrolytic procedure for total hydroxytyrosol and tyrosol, and 1H-NMR analysis in order to assess their secoiridoid profiles. Both of the extracts inhibited in vitro the 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGCoAR) activity in a dose-dependent manner. After the treatment of human hepatic HepG2 cells (25 µg/mL), they increased the low-density lipoprotein (LDL) receptor protein levels through the activation of the sterol regulatory element binding proteins (SREBP)-2 transcription factor, leading to a better ability of HepG2 cells to uptake extracellular LDL molecules with a final hypocholesterolemic effect. Moreover, both of the extracts regulated the intracellular HMGCoAR activity through the increase of its phosphorylation by the activation of AMP-activated protein kinase (AMPK)-pathways. Unlike pravastatin, they did not produce any unfavorable effect on proprotein convertase subtilisin/kexin 9 (PCSK9) protein level. Finally, the fact that extracts with different secoiridoid profiles induce practically the same biological effects suggests that the hydroxytyrosol and tyrosol derivatives may have similar roles in hypocholesterolemic activity.


HPLC-DAD analysis of phenols from EVOO
Phenolic compounds were analyzed both before and after acid hydrolysis [16]. The phenols extraction was carried out according to the IOC method [17] in the presence of syringic acid as internal standard. The chromatographic analyses were carried out with a HP 1100 system provided with a quaternary pump and a DAD detector (Agilent Technologies, Santa Clara, CA, USA). Phenols were separated using a SphereClone ODS (2), 5 μm, 250 x 4.6 mm id column; the elution was obtained by H2O (at pH 2.0 by phosphoric acid), acetonitrile and methanol as eluents, applying the gradient reported in the IOC method (IOC/T.20/Doc No. 29); flow rate, 1 mL/min, injection volume 20 μL. Chromatograms were registered at 280 nm and syringic acid was used as an internal standard for the quantitative analysis, thus expressing the results as mg tyrosol/kg oil.
The hydroalcoholic extracts obtained as described above were treated by the acid hydrolysis method previously proposed in order to evaluate the total contents of free and bound tyrosol and hydroxytyrosol [16]. Briefly, 300 μL of the extract were heated at 80 °C for 2 h in the presence of 300 μL of H2SO4 1.0 M, then the solution was diluted with 400 μL of water. The following chromatographic analysis was carried out in a HP1200 liquid chromatograph equipped with a DAD detector (Agilent Technologies, Santa Clara, CA, United States) and a reverse phase (RP) C18 column, 150×3 mm (5 μm) Gemini (Phenomenex, Torrance, CA, USA); flow rate, 0.4 mL/min. Eluents: H2O acidified to pH 3.2 with formic acid (A) and acetonitrile (B). The linear solvent gradient was applied as follow: solvent A varied 95% to 70% in 5 min, then to 50% in 5 min, then varied to 2% in 5 min and stayed in this condition for 5 more min; finally, solvent A came back to 95% in 2 min. The total time of analysis was 22 min, equilibration time, 10 min. The total content of tyrosol was evaluated using a calibration line built using an authentic standard (purity grade 98%), and considering the chromatographic areas at 280 nm. Regarding hydroxytyrosol, its amount was evaluated again using the calibration line of tyrosol at 280 nm, but applying the following formula for keeping into account that it is overestimated by about 35%: mg OH-tyrosol = mg tyrosol × 0.65 [18]. All data were expressed as mg/kg oil.

Preparation of the phenolic extracts for the biological testing
Phenolic compounds were extracted from the two EVOO samples according to the following procedure: 50 g of each EVOO sample were exactly weighted and put in a 500 mL flask together with 150 mL of MeOH:H2O 80:20 solution. The mixture was vigorously hand-shaken for 1 min. The extraction was then performed with the aid of an ultrasound bath for 15 min. The obtained mixture was centrifuged at 5,000 rpm for 25 min; the supernatant was recovered and filtered using PVDF type 0.45 µm 13 mm diameter filters with a 60 mL syringe. The obtained solution was defatted twice with 75 mL of hexane and evaporated under vacuum at room temperature. The dried extract was then dissolved in ethanol up to a total volume of 10 mL. The obtained solution was then split in 10 vials, the solvent evaporated, thus obtaining 10 aliquots of dried extract (each corresponding to 5 mL of EVOO), to be used for the chemical and biological analysis. By this way, each BUO vial contained about 10.1 ± 0.16 mg dry weight, whereas each OMN vial 5.6 ± 0.12 mg of dry weight.

Analysis of the phenolic extract
The samples prepared for the biological testing were submitted to the HPLC-DAD analysis as indicated above as well as to 1H-NMR analysis using a literature method [19], by using a 400 MHz instrument Advance 400 (Bruker, Bremen, Germany). For the NMR analysis, each defatted phenolic extract, obtained from 5 mL of EVOO, was dissolved in 1 mL of CDCl3.

Cell culture conditions and treatment
HepG2 cell line was bought from ATCC (HB-8065, ATCC from LGC Standards, Milan, Italy) and was cultured in DMEM high glucose with stable L-glutamine, supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin (complete growth medium) with incubation at 37 °C under 5% CO2 atmosphere. HepG2 cells were used for no more than 20 passages after thawing, because a higher number of passages may change the cell characteristics and impair assay results.
BUO and OMN extracts were tested separately. Briefly, each EVOO extract was diluted using DMSO in order to prepare a stock solution (50 mg/mL), which was diluted in order to reach the final concentration of 25.0 ug/mL in complete growth DMEM. The growth medium of adherent HepG2 cells was discarded and each diluted EVOO extract in complete DMEM was replaced and incubated for the desirable incubation time based on the experiments.

HMGCoAR activity assay
The assay buffer, NADPH, substrate solution, and HMGCoAR were provided in the HMGCoAR Assay Kit (Sigma). The experiments were carried out following the manufacturer's instructions and conditions previously optimized at 37 °C [20]. In particular, each reaction (200 µL) was prepared adding the reagents in the following order: 1 X assay buffer, pravastatin 1.0 µM or BUO and OMN EVOO extracts (10.0 -250.0 μg/mL or vehicle (C), the NADPH (4 µL), the substrate solution (12 µL), and finally the HMGCoAR (catalytic domain) (2 µL). Subsequently, the samples were mixed and the absorbance at 340 nm read by the microplate reader Synergy H1 at time 0 and 10 min. The HMGCoAR-dependent oxidation of NADPH and the inhibition properties of samples were measured by absorbance reduction, which is directly proportional to enzyme activity.

Western blot analysis
Experiments were performed following conditions previously described [21]. In particular, a total of 1.5 × 10 5 HepG2 cells/well (24-well plate) were treated with 25.0 μg/mL of BUO or OMN EVOO extracts or pravastatin 1.0 µM for 24 h. After each treatment, cells were scraped in 30 µL ice-cold lysis buffer [RIPA buffer + inhibitor cocktail + 1:100 PMSF + 1:100 Na-orthovanadate] and transferred in an ice-cold microcentrifuge tube. After centrifugation at 13,300g for 15 min at 4 °C, the supernatant was recovered and transferred into a new ice-cold tube. Total proteins were quantified by Bradford method and 50 μg of total proteins loaded on a pre-cast 7.5% Sodium Dodecyl Sulfate -Polyacrylamide (SDS-PAGE) gel at 130 V for 45 min. Subsequently, the gel was pre-equilibrated with 0.04% SDS in H2O for 15 min at room temperature (RT) and transferred to a nitrocellulose membrane (Mini nitrocellulose Transfer Packs,) using a trans-Blot Turbo at 1.3 A, 25 V for 7 min. Target proteins, on milk or BSA blocked membrane, were detected by primary antibodies as follows: anti-SREBP2, anti-LDLR, anti-HMGCoAR, anti-phospho AMPK (Thr172), anti-phospho HMGCoAR (Ser872), anti-PCSK9, anti-HNF1-α and anti-β-actin. Secondary antibodies conjugated with HRP and a chemiluminescent reagent were used to visualize target proteins and their signal was quantified using the Image Lab Software (BioRad). The internal control β-actin was used to normalize loading variations.

In-Cell Western (ICW) assay
Experiments were performed following the conditions previously described by us elsewhere [22]. Briefly, a total of 3 x 10 4 HepG2 cells/well were seeded in 96-well plate and, the following day, they were treated with 25 μg/mL of BUO and OMN extracts or Pravastatin 1.0 μM in complete growth medium for 24 h. Subsequently, they were fixed in 4% paraformaldehyde for 20 min at room temperature (RT). Cells were washed 5 times with 100 µL of PBS/well (each wash was for 5 min at RT) and the endogenous peroxides activity quenched adding 3% H2O2 for 20 min at RT. Non-specific sites were blocked with 100 µL/well of 5% Bovine Serum Albumin (BSA, Sigma) in PBS for 1.5 h at RT. LDLR primary antibody solution (1:3000 in 5% BSA in PBS, 25 µL/well) was incubated O/N at +4 °C. Subsequently, the primary antibody solution was discarded and each sample was washed 5 times with 100 µL/well of PBS (each wash was for 5 min at RT). Goat anti-rabbit Ig-HRP secondary antibody solution (Santa Cruz) (1:6000 in 5% BSA in PBS, 50 µL/well), was added and incubated 1