INTRODUCTION
Non-alcoholic fatty liver disease (NAFLD) is a disease characterized by the presence of different degrees of fatty liver in individuals who do not have a habit of use of alcohol. NAFLD is one of the important health problems with its prevalence estimated to be around 25% worldwide. Triglyceride accumulation in hepatocytes is increased in NAFLD patients1,2. Palmitate, a 16-carbon saturated fatty acid, is found in the structure of triglycerides in the liver and is one of the most abundant fatty acids in the livers of both healthy individuals and NAFLD patients3.
NAFLD is not just a liver disease, but it is a multisystem disease. NAFLD has also been shown to be associated with metabolic syndrome and atherosclerosis4,5. Paraoxonase (PON) 1 and PON3 enzymes are anti-atherogenic enzymes mainly synthesized in the liver. PON1 and PON3 are secreted from the liver into the circulation and are transported in the circulation bound to high-density lipoprotein (HDL)6.
HepG2 cells are commercially available human-derived hepatoma cells and are frequently used to induce an experimental NAFLD model with free fatty acids because of their similarity to hepatocytes7-10. It has been reported that the steatosis induced by 1 mM palmitate in HepG2 cells is a cellular model of NAFLD that can be used to investigate acute and toxic effects in liver cells due to fat accumulation7.
Rosmarinic acid (RA) is phenolic acid, the ester of caffeic acid and 3,4 dihydroxyphenyllactic acid synthesized from L-phenylalanine and L-tyrosine amino acids11. RA is a polyphenol found naturally in plants such as rosemary, sage, and perilla steak grass, with shown antioxidant and antiinflammatory effects12. In the literature, we did not come across any study investigating the effect of RA on PON1 and PON3 enzymes.
In this study, it was aimed to investigate the effect of RA on cell viability, steatosis, PON1 and PON3 protein levels in palmitate-induced NAFLD model in human-derived hepatoma cells. With this study, the effect of RA on PON1 and PON3 levels both when administered directly and in NAFLD was shown for the first time.
MATERIALS AND METHODS
Chemical and Consumables
RA, sodium palmitate, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and Oil Red O were purchased from Sigma-Aldrich (St. Louis, MO, USA). Eagle’s minimal essential mediums were purchased from Wisent (St-Bruno, QC, Canada). Fetal bovine serum (FBS), antibiotic-antimycotic and chemiluminescent substrate were purchased from Thermo Fisher (Waltham, MA, USA). Primary antibodies to PON1, PON3, and alpha tubulin and secondary antibodies were purchased from Abcam (Cambridge, England). Fatty acid-free bovine serum albumin was purchased from Gold Biotechnology (MO, USA). Radioimmunoprecipitation lysis buffer (RIPA) was purchased from Santa Cruz (Heidelberg, Germany). Polyvinylidene difluoride (PVDF) membrane was purchased from Bio-Rad (Hercules, CA, USA). All chemicals were of analytical purity.
Cell Culture Applications
This study was approved by Trakya University Deanship of Faculty of Medicine Scientific Research Ethics Committee on 29.03.2017 with the protocol code of TUTF-BAEK 2017/95 and decision number 06/05. Cells were kept in an incubator at 37 °C in 5% CO2 with Eagle’s minimum essential medium containing 10% FBS and 1% antibiotic-antimycotic. For the experiments, cells between passages of 10 and 20 were used.
HepG2 cells were incubated with 1 mM palmitate for 24 hours to induce steatosis7-9. Sodium palmitate was dissolved in sterile water at 70 °C13,14 and conjugated for at least 3 hours 15 with 0.7 mM fatty acid-free bovine serum albumin, which was dissolved in medium and its concentration was similar to human serum albumin concentrations and cells were incubated with mixture for 24 hours. In the control group, only medium containing 0.7 mM albumin was applied.
MTT Test
MTT test was performed to assess cell viability16. 104 cells were seeded in 96-well plates. RA alone and together with 1 mM palmitate was applied to the cells at different concentrations for 24 hours. After 24 hours, the medium was removed and replaced with MTT (5 mg/mL) dissolved in 10 µL of PBS and 100 µL of Eagle’s minimum essential medium without phenol red, and incubated for 4 hours. The medium containing MTT was taken and formazan, was dissolved with 200 µL dimethyl sulfoxide and 25 µL Sorenson buffer (0.1 M glycine and 0.1 M sodium chloride; adjusted to pH: 10.5 with 0.1 M sodium hydroxide) and the resulting color was measured spectrophotometrically at 570/630 nm in a microplate reader17. The measured absorbances were divided by the mean of the control group on the same plate, and the results were given as a percentage of the control group.
Triglyceride Measurement
Cells were seeded into 25 cm2 flasks for triglyceride measurement. Different concentrations of RA were applied to the cells simultaneously with 1 mM palmitate for 24 hours. After 24 hours, cells were washed with phosphate buffered saline (PBS) and then scraped with PBS buffer containing 1% protease inhibitor and 0.1% Triton X-100. After homogenization using glass beads, the homogenate was centrifuged at 10,000 g for 10 minutes. The supernatant in the upper part was used in the experiments. Intracellular triglyceride levels were measured using the autoanalyzer’s original kits. Protein levels were measured according to the method of Lowry et al.18, and triglyceride levels were divided to protein results and results of groups were given as fold of control by dividing the results to the mean value of control group.
Oil Red O Staining
For staining with Oil Red O, cells were seeded in 6-well plates. Different concentrations of RA were applied to the cells simultaneously with 1 mM palmitate for 24 hours. After 24 hours, cells were washed with PBS and fixed with 10% paraformaldehyde. After fixation, it was washed with 60% isopropanol and incubated with 60% isopropanol containing Oil Red O [stock 0.35% (w/v) Oil Red O dissolved in 100% isopropanol] and washed with distilled water and photographed under a phase contrast invert microscope using a microscope camera9.
Western Blot Method
Cells were seeded in 75 cm2 flasks. Different concentrations of RA were applied to the cells simultaneously with 1 mM palmitate for 24 hours. After the experimental procedure, cells washed with PBS were scraped with RIPA containing 1% of protease inhibitor cocktail, phenyl methyl sulfonyl fluoride and sodium orthovanadate solutions. After homogenization using glass beads, the homogenate was centrifuged at 10,000 g for 10 minutes. The supernatant in the upper part was used in the experiments. Protein measurement was performed according to the method of Lowry et al.18. A 4-12% polyacrylamide gel was prepared according to the Laemmli19 method, and 20 µg of protein was separated by vertical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were transferred to a polyvinylidene difluoride membrane using a semi-wet transfer system. Membranes were blocked with 5% milk powder for 1 hour and incubated with primary antibodies (1:1,000 dilution for PON1 and PON3, 1:10,000 dilution of tubulin) overnight at 4 °C. Then, membranes incubated for 1 hour with secondary antibody (1:10,000 dilution) were visualized using a chemiluminescent substrate. Band intensity for each protein was calculated using the Image J program20. PON1 and PON3 protein levels were divided to the level of alpha tubulin protein used as the loading control of the same sample, and the results were given as a fold of control by dividing value to the value of control group in the same membrane.
Statistical Analysis
Statistical analyzes were performed using the Statistical Package for the Social Sciences 20 program. One-way ANOVA was used to compare experimental parameters, and Tukey and Tamhane tests were used for comparison between groups. Results were expressed as mean±standard deviation, and p<0.05 was considered statistically significant.
RESULTS
Cell viabilities after RA treatment to HepG2 cells for 24 hours were found as: 100±3 in the control group, 96±6% in cells treated with 5 μM RA, 106±8% in cells treated with 10 µM RA, 107±6% in cells treated with 25 µM RA, 122±9% in cells treated with 50 µM RA, 131±8% in cells treated with 100 µM RA, 119±8% in cells treated with 200 µM RA, 77±7% in cells treated with 300 µM RA, 73±5% in cells treated with 400 µM RA, and 68±6% in cells treated with 500 µM RA. 5, 10 and 25 µM RA did not significantly change cell viability compared to the control group (p>0.05 for all). 50, 100 and 200 µM RA significantly increased cell viability compared to the control group, 5, 10 and 25 µM RA (p<0.05 for all). 200 µM RA significantly decreased cell viability compared to 100 µM RA (p<0.05). 300, 400 and 500 µM RA significantly decreased cell viability compared to control and 5, 10, 25, 50, 100 and 200 µM RA (p<0.05 for all) (Figure 1).
Cell viabilities after 24 hours treatment to HepG2 cells were found as: 100±6% in the control group, 54±4% in cells treated with only 1 mM palmitate; 54±6% in cells treated with 1 mM palmitate and 5 µM RA, 55±6% in cells treated with 10 µM RA, 59±6% in cells treated with 25 µM RA, 66±6% in cells treated with 50 µM RA, 75±7% in cells treated with 100 µM RA and 83±5% in cells treated with 200 µM RA. Cell viability was significantly reduced in all cells treated with 1 mM palmitate compared to the control group (p<0.05 for all). Treatment of 50 µM RA with 1 mM palmitate significantly increased cell viability compared to treatment of only 1 mM palmitate and 5 and 10 µM RA with 1 mM palmitate (p<0.05 for all). Treatment of 100 µM RA with 1 mM palmitate significantly increased cell viability compared to treatment of only 1 mM palmitate and 5, 10, and 25 µM RA with 1 mM palmitate (p<0.05 for all). Treatment of 200 µM RA with 1 mM palmitate significantly increased cell viability compared to treatment of only 1 mM palmitate and 5, 10, 25 and 50 µM RA with 1 mM palmitate (p<0.05 for all) (Figure 2).
Triglyceride levels after 24 hours treatment to HepG2 cells were found as 1.00±0.07 in the control group, 2.54±0.06 in cells treated with only 1 mM palmitate; 2.55±0.04 in cells treated with 1 mM palmitate and 5 µM RA, 2.47±0.12 in cells treated with 1 mM palmitate and 10 μM RA 2.44±0.15 in cells treated with 1 mM palmitate and 25 μM RA 2.27±0.05 in cells treated with 1 mM palmitate and 50 μM RA 1.94±0.17 in cells treated with 1 mM palmitate and 100 μM RA and 1.77±0.27 in cells treated with 1 mM palmitate and 200 μM RA. Triglyceride levels increased significantly in all cells treated with 1 mM palmitate compared to the control group (p<0.05 for all). Treatment of 50 µM RA with 1 mM palmitate significantly reduced triglyceride levels compared to treatment of only 1 mM palmitate and treatment of 5 µM RA with 1 mM palmitate (p<0.05 for both). Treatment of 100 µM RA with 1 mM palmitate significantly reduced triglyceride levels compared to administration of only 1 mM palmitate and administration of 5, 10, and 25 µM RA with 1 mM palmitate (p<0.05 for all). Treatment of 200 µM RA with 1 mM palmitate significantly reduced triglyceride levels compared to treatment of only 1 mM palmitate and treatment of 5, 10, and 25 µM RA with 1 mM palmitate (p<0.05 for all) (Figure 3A).
The effect of RA on steatosis was examined microscopically by Oil Red O staining in HepG2 cells, in which palmitate steatosis was created. It was observed that the staining with oil red O was higher in the cells treated with 1 mM palmitate compared to the control group. It was observed that RA concentrations applied together with 1 mM palmitate reduced staining with Oil Red O, and this decrease was more pronounced especially at higher concentrations (Figure 3B).
PON1 protein levels after RA treatment to HepG2 cells for 24 hours were: 1.11±0.13 fold of control in cells treated with 100 µM RA, and 1.07±0.07 fold of control in cells treated with 200 µM RA. PON3 protein levels after 24 hours treatment to HepG2 cells were: 1.12±0.16 fold of control in cells treated with 100 µM RA and 1.25±0.28 fold of control in cells treated with 200 µM RA. There was no significant difference in PON1 and PON3 levels between 100 µM and 200 µM RA treated and control cells (p>0.05 for all) (Figure 4A).
After RA treatment to HepG2 cells with 1 mM palmitate for 24 hours, PON1 levels were 0.94±0.05 fold of control group in cells treated with only 1 mM palmitate; it was found to be 1.01±0.10 fold of control group in cells treated with 1 mM palmitate and 100 µM RA, and 0.92±0.05 fold of control group in cells treated with 200 µM RA. PON3 levels were 1.03±0.11 fold of control group in cells treated with only 1 mM palmitate; it was found to be 0.95±0.08 fold of control group in cells treated with 1 mM palmitate and 100 µM RA, and 1.04±0.17 fold of control group in cells treated with 200 µM RA. There was no significant difference in PON1 and PON3 protein levels between treatment of 1 mM palmitate, 100 µM and 200 µM RA with 1 mM palmitate and control cells (p>0.05 for all) (Figure 4B).
DISCUSSION
NAFLD is a multisystemic disease whose effects are not limited to the liver and is an important health problem with an increasing incidence all over the world4,21. NAFLD is an independent risk factor for atherogenic dyslipidemia and has also been shown to be associated with atherosclerosis2,5. Although diet and exercise are recommended primarily in the treatment of NAFLD, it has been reported that antioxidant therapy, especially vitamin E, can be added to the treatment in these patients1,22. RA is a plant-derived polyphenol that protects against oxidative damage and scavenges free radicals12,23. The most common cause of death in NAFLD patients is cardiovascular diseases24. PON1 and PON3 enzymes are antioxidant enzymes mainly synthesized in the liver. These enzymes take part in the structure of HDL in the circulation and play a role in preventing the development of atherosclerosis6.
The aim of our study was to investigate the effect of RA on cell viability, steatosis, PON1 and PON3 protein levels in palmitate-induced experimental NAFLD model in HepG2 cells.
There are conflicting results in the literature regarding the effect of RA on cell viability in HepG2 cells. Adomako-Bonsu et al.25 reported that RA applied to HepG2 cells for 5 hours was not toxic up to 700 µM and caused 25% toxicity at 2800 µM concentration; Wu et al.26 reported that 160 µM RA applied to HepG2 cells for 24 or 48 hours was not toxic. Ozgun and Ozgun27 reported that 100 µM RA applied to HepG2 cells for 24 hours did not cause a decrease in cell viability, while 1000 µM RA reduced cell viability by more than 50%. On the other hand, Ma et al.28 reported that approximately 35 µM of RA applied to HepG2 cells for 24 hours reduced cell viability. For this reason, in our study, the effect of RA on cell viability was investigated by applying RA concentrations up to 500 µM to HepG2 cells for 24 hours. While 50, 100 and 200 µM RA significantly increased cell viability in HepG2 cells, RA concentrations of 300 µM and above significantly decreased cell viability, and therefore RA concentrations up to 200 µM were applied against palmitate-induced steatosis.
It is known that in the palmitate-induced cellular NAFLD model in HepG2 cells, cell viability decreases and intracellular steatosis increases7,8. In our study, palmitate application significantly decreased cell viability and increased intracellular triglyceride level, which was consistent with the literature. It was also seen microscopically that palmitate application increased the intracellular lipid content. These findings prove that palmitate-induced cellular NAFLD model are formed in HepG2 cells in our study.
In our study, 50, 100 and 200 µM RA significantly reduced palmitate-induced cell death. In the literature, we could not find any study investigating the effect of RA on cell viability in the palmitate-induced cellular NAFLD model, but in a different experimental model that supports our findings, it has been reported that RA reduces tert-butyl hydroperoxide-induced cell death in HepG2 cells depending on the dose29. In our study, 50, 100 and 200 µM RA significantly reduced the increase in triglyceride levels caused by palmitate, and it was also seen microscopically with Oil Red O staining that RA reduced steatosis, especially at high doses. We found only one study investigating the effect of RA on palmitate-induced experimental steatosis, and in this study published in 2020, Kim et al.10 reported that RA reduced the steatosis induced by 500 µM palmitate, in line with our study.
In our study, 1 mM palmitate administration in HepG2 cells did not significantly change PON1 and PON3 protein levels. In the literature, Özgün et al.8 supportive of our study, reported that palmitate administration to HepG2 cells, in which an experimental NAFLD model was created with 1 mM palmitate, did not change PON1 and PON3 protein levels. At the same time, Kudchodkar et al.30 reported that dietary tripalmitin did not change PON1 activity in rats, while Boshtam et al.31 reported that there was no difference between palmitic acid content of HDL and PON1 activities.
There is no study in the literature investigating the effect of RA on PON1 or PON3 enzymes. In our study, 100 and 200 µM RA, the most effective doses on cell viability and intracellular steatosis, had no significant effect on PON1 and PON3 protein expressions both directly in HepG2 cells and in the palmitate-induced cellular NAFLD model in HepG2 cells. In the light of these findings, we can say that RA has no significant effect on PON1 and PON3 protein levels in HepG2 cells, neither when administered alone nor in combination with palmitate.
Study Limitations
Examination of the effects of RA only at the cellular level since it is an in vitro experimental study, experimental NAFLD model’s being limited to 24 hours to avoid serum starvation in cells as stated in the literature, and the use of HepG2 cell line instead of primary hepatocytes, although it is frequently used in the literature, are the limitations of this study.
CONCLUSION
Our study showed that RA increased cell viability and decreased steatosis, but did not change PON1 and PON3 protein levels in the palmitate-induced cellular NAFLD model in human hepatoma cells. In the light of our findings, we can say that RA may be effective in the prevention of NAFLD because it reduces steatosis, but it has no effect on the levels of antiatherogenic enzymes PON1 and PON3. However, our study is an in vitro experimental study and our findings should be supported by future animal and human studies.
Ethics
Ethics Committee Approval: This study was approved by Trakya University Deanship of Faculty of Medicine Scientific Research Ethics Committee on 29.03.2017 with the protocol code of TUTF-BAEK 2017/95 and decision number 06/05.
Informed Consent: Commercially available HepG2 cells were used in this study.
Peer-review: Externally and internally peer-reviewed.
Authorship Contributions
Surgical and Medical Practices: E.Y., E.Ö., Concept: E.Y., E.Ö., Design: E.Y., E.Ö., Data Collection or Processing: E.Y., E.Ö., Analysis or Interpretation: E.Y., E.Ö., Literature Search: E.Y., E.Ö., Writing: E.Y., E.Ö.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: This study was supported by Trakya University Scientific Research Projects Unit (TUBAP) with project number TUBAP-2017/134 and was produced from Esra YAKŞİ’s master’s thesis completed at Trakya University Health Sciences Institute. The data of this study were presented as an oral presentation at the International Medical and Health Sciences Research Congress held in Bandırma/Turkey in 2019. We thank TUBAP and Trakya University Technology Research and Development Application and Research Center where chemiluminescence imaging was performed.