In vitro assessment of nutraceutical compounds and novel nutraceutical formulations in a liver-steatosis-based model

Abstract

Background: Steatosis is a chronic liver disease that depends on the accumulation of intracellular fatty acids. Currently, no drug treatment has been licensed for steatosis; thus, only nutritional guidelines are indicated to reduce its progression. The aim of this study is to combine different nutraceutical compounds in order to evaluate their synergistic effects on a steatosis in vitro model compared to their separate use. In particular, three different formulations based on silymarin, curcumin, vitamin E, docosahexaenoic acid (DHA), choline, and phosphatidylcholine were assayed.

Methods: Human hepatocellular carcinoma cells (HepG2 cell line) were treated with a mixture of fatty acids in order to induce an in vitro model of steatosic cells, and then the amount of intracellular fat was evaluated by Oil Red O staining. The peroxisome proliferator-activated receptors α and γ (PPARα and γ) expression, closely correlated to lipid metabolism, was evaluated. The efficiency of these receptors was evaluated through the study of LPL mRNA expression, a marker involved in the lipolysis mechanism. Superoxide dismutase (SOD-2) and malondialdehydes (MDA) in lipid peroxidation were assayed as specific biomarkers of oxidative stress. In addition, experiments were performed using human liver cells stressed to obtain a steatosis model. In particular, the content of the intracellular fat was assayed using Oil Red O staining, the activation of PPARα and γ was evaluated through western blotting analyses, and the LPL mRNA expression level was analyzed through qRT-PCR.

Results: All formulations proved effective on lipid content reduction of about 35%. The oxidative stress damage was reduced by all the substances separately and even more efficiently by the same in formulation (i.e. Formulation 1 and Formulation 3, which reduced the SOD-2 expression and induced the PPARs activation). Lipid peroxidation, was reduced about 2 fold by foormulation2 and up to 5 fold by the others compared to the cells pretreated with H₂O₂.Formulation 1, was more effective on PPARγ expression (2.5 fold increase) respect to the other compounds on FA treated hepathocytes. Beside, LPL was activated also by Formulation 3 and resulted in a 5 to 9 fold-increase respect to FA treated control.

Conclusions: Our results proved that the formulations tested could be considered suitable support to face steatosis disease beside the mandatory dietetic regimen.

Keywords: HepG2; Normal liver cells; Nutraceutical compounds; Oxidative stress; PPARs expression; Steatosis in vitro model.

Fig. 1

In vitro steatosis, Oil Red O staining colorimetric assay. a Oil Red O staining pictures for HepG2 cells in presence of fatty acids (FA) 6 mM, different nutraceutical compounds (0.001 mg/mL) and their formulations (0.005 mg/mL). b Spectophotometric quantification and evaluation of lipid amount percentage with the different nutraceutical compounds (concentration range 0.001–0.05 mg/mL) respect to the control of steatotic cells (FA 6 mM). Curcumin, sylimarin vs CTR * that means p < 0.05 at 0.001 mg/ml. Choline vs CTR ** that means p < 0.01 at 0.001 mg/ml, and vs vitamin E ^#, DHA ^# and phosphatidylcholine ^# that means p < 0.05 at 0.001 mg/ml. Curcumin, sylimarin, choline and phosphatidylcholine vs CTR * that means p < 0.05 at 0.005 mg/ml. DHA vs CTR ** that means p < 0.01 at 0.005 mg/ml. DHA vs sylimarin ^# that means p < 0.05 at 0.005 mg/ml. Sylimarin, choline, vitamin E, DHA and phosphatidylcholine vs CTR * that means p < 0.05 at 0.05 mg/ml. Curcumin vs CTR ** that means p < 0.01 at 0.05 mg/ml. Curcumin vs DHA ^# and phosphatidylcholine ^# that means p < 0.05 at 0.05 mg/ml. c The graph reported Oil Red O staining colorimetric assay relative to lipid droplets accumulation in HepG2 cells treated with different nutraceutical formulations (0.005 mg/mL). All values were expressed in the form of mean ± SD (n = 3). b and c are relative to two groups of different experiments in which intracellular fat difference in the averaged data proved not to be significant using t-student test p > 0.05

Fig. 2

In vitro steatosis model, Colorimetric assay (Spectophotometric quantification) of Oil Red O staining for human normal hepatocytes in presence of fatty acids (FA) 6 mM and nutraceutical formulations (0.005 mg/mL) normalized on control

Fig. 3

MTT assay for single nutraceutical compounds (a) and formulations (b). The figure shows the cell viability percentage of HepG2 cells after 30 min of H₂O₂ (50 μM) treatment and after nutraceutical compounds incubation. b and c are relative to two groups of different experiments in wich intracellular fat difference in the averaged data proved not to be significant using t-student test p > 0.05

Fig. 4

a, b Oxidative stress. The figure shows the anti-oxidant effect of nutraceutical compounds in HepG2 cells, after 30 min of H₂O₂ (50 μM) treatment through the  gene expression analyses of SOD-2

Fig. 5

a, b Western blot relative to SOD-2 as marker of oxidative stress. Actin is used to normalize the results. All values were expressed in the form of mean ± SD (n = 3)

Fig. 6

PPARα and PPARγ protein expression level on HepG2 cells determined by Western blotting. Actin is used to normalize the results

Fig. 7

a, b PPARα and γ protein expression level determined by western blotting in human normal liver cells, exposed to FA to induce steatosis and eventually treated with nutraceuticals. Actin is used to normalize the results

Fig. 8

LPL gene expression on human normal liver cells in presence of nutraceutical formulations

Fig. 9

a Effect of single nutraceutical compounds (0.001 mg/mL) on lipid peroxidation after exposure to oxidative stress induced by 50 μM H₂O₂ in vitro on HepG2 cells. b Lipid peroxidation in presence of nutraceutical formulations. Data are the means ± SD (n = 3) and were significantly different: * p < 0.05 and ** p < 0.01 respect to H₂O₂ pre-treatment