In recent years, increasing attention has been paid to diseases caused by excessive accumulation of lipids in the liver with therapeutic agents derived from natural products offering an alternative treatment to conventional therapies. Among these therapeutic agents, apigenin, a natural flavonoid, has been proven to exert various beneficial biological effects. In the present study, the antiadipogenic effects of apigenin in HepG2 cells was investigated. It was demonstrated that the treatment of cells with different concentrations of apigenin for 24 h significantly decreased the palmitic acid-induced increases in total cholesterol (TC) and triglyceride (TG) levels as well as intracellular lipid accumulation. In addition, apigenin increased the phosphorylated-AMP-activated protein kinase (AMPK) levels but decreased the expression levels of 3-hydroxy-3-methylglutaryl CoA reductase, sterol regulatory element-binding protein (SREBP)-1, fatty acid synthase, and SREBP-2 in a concentration-dependent manner. The present findings suggested that apigenin might improve lipid metabolism by activating the AMPK/SREBP pathway to reduce lipid accumulation in the liver.
In recent years, obesity has received increasing attention because it leads to various metabolic diseases, such as hyperlipidemia, hypertension, atherosclerosis and type II diabetes mellitus (
For the development of novel antilipidemic drugs with high efficacy and few adverse effects, attention has gradually shifted to the natural plant flavonoids in recent years. Apigenin, a type of plant flavonoid with the chemical name 4′,5,7-dihydroxyflavone, is recognized as a bioactive flavonoid that has antioxidant, anticancer and anti-inflammatory properties that can also lower blood pressure (
The present study established a common hyperlipidemia model by using appropriate doses of palmitic acid to determine whether apigenin lowered lipid levels and, to explore the underlying mechanism in depth.
Palmitic acid and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; Merck KGaA. The apigenin standard was purchased from Beijing Solarbio Technology Co. The AMPK inhibitor compound C was purchased from Selleck Chemicals LLC. Anti-phospho-AMPK (P-AMPK; Thr172; cat. no. 2535) was purchased from Cell Signaling Technology, Inc., anti-AMPKα1 (cat. no. 10929-2-AP), SREBP-2 (cat. no. 14508-1-AP), FAS (cat. no. 13098-1-AP), HMGCR (cat. no. 13533-1-AP) and β-actin (cat. no. 66009-1-lg) were purchased from Proteintech Group Inc., SREBP-1 (cat. no. ab3259) was purchased from Abcam. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L; cat. no. SA00001-1) and HRP-conjugated goat anti-rabbit IgG (H + L; cat. no. SA00001-2) secondary antibodies were obtained from Proteintech Group Inc.
Liver cancer HepG2 cells, purchased from the American Type Culture Collection, were cultured in high-glucose DMEM (GE Healthcare Life Sciences) with 10% fetal bovine serum (Clark Bioscience) and 1% penicillin/streptomycin at 37°C in an atmosphere containing 5% CO2. The cells were dissociated with 0.25% trypsin (w/v) and 0.52 mM EDTA [M&C Gene Technology (Bejing) Ltd.] and routinely sub-cultured at 80% confluency.
Cell cultures were treated with various concentrations of apigenin in DMSO as the carrier solvent. Palmitic acid binds to fatty-acid-free BSA (Beijing Solarbio Science & Technology Co., Ltd.). In brief, palmitic acid was dissolved in 1X PBS and a 250 mM stock solution was obtained following various cycles of incubation in a water bath at 70°C and vortexing. The stock solution was then added to serum-free DMEM containing 5% fatty-acid-free BSA to obtain a 250 µM palmitic acid solution, and the resulting diluted solution was used for the cell treatments (
Cells were seeded in 96-well plates at a concentration of 1×104 cells/well (Corning Inc.) and allowed to adhere overnight at 37°C. Cells were then treated with different concentrations of apigenin (0, 20, 40, 80, 160, 320, 640 and 1,280 µM) or palmitic acid (0, 62.5, 125, 250, 500 and 1,000 µM) for 24 h, and 20 µl of MTT (Sigma-Aldrich; Merck KGaA) solution (5 mg/ml in 1% PBS) was added to each well and incubated for 4 h at 37°C prior to the end of the culture. The culture medium was subsequently removed and the formazan crystals were dissolved using 150 µl DMSO/well. Absorbance values at 570 nm were determined using a microplate reader (BioTek Instruments, Inc.).
HepG2 cells were inoculated at a density of 4×105 cells per well in 6-well plates and cultured with different concentrations of apigenin (10, 20 and 40 µM) and 250 µM palmitic acid for 24 h. Cells were then washed twice with cold 1X PBS and fixed with 4% paraformaldehyde for 40 min at 37°C. Subsequently, the residual paraformaldehyde was washed with double-distilled water, and cells were incubated with 60% isopropanol for 3–5 sec. Cells were then stained with freshly prepared 0.5% Oil Red O solution in 60% isopropanol for 1 h at 37°C in the dark. Lipid droplets in the cells were observed and imaged using a bright-field microscope (Olympus Corporation) at a magnification of ×400. In addition, the accumulated lipids were extracted with 100% isopropanol for 3–5 min at room temperature, and the absorbance at 570 nm was determined using a microplate reader.
HepG2 cells were inoculated at a density of 4×105 cells/well in 6-well plates and pretreated with 10 µM AMPK inhibitor compound C for 1 h at 37°C then cultured with different concentrations of apigenin (10, 20 and 40 µM) and 250 µM palmitic acid for 24 h at 37°C in a culture chamber with 5% CO2. The intracellular TC and TG contents were measured using a TC assay kit (cat. no. E1015; Applygen Technologies Inc.) and a TG assay kit (cat. no. E1013; Applygen Technologies Inc.) according to the manufacturer's instructions. The absorbance values were measured at 570 nm. Bicinchoninic acid (BCA) protein quantitative kit (Beyotime Institute of Biotechnology) was used to determine the protein concentration of the samples, and the absorbance value of the proteins was measured at 562 nm. The intracellular TC and TG contents are presented as µM/mg cellular protein.
HepG2 cells were seeded in 6-well plates at a concentration of 6×105 cells per well and pretreated with 10 µM AMPK inhibitor compound C for 1 h and then cultured with different concentrations of apigenin (10, 20 and 40 µM) and 250 µM palmitic acid (
Data were processed using SPSS v18.0 statistical software (SPSS Inc.), and the measurement data were expressed as the mean ± standard deviation. Statistical analysis was performed by one-way analysis of variance with Tukey's test and Dunnett's test used for post hoc analysis. Histograms were plotted by Prism software v5.0 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
To analyze the effect of apigenin and palmitic acid on the viability of HepG2 cells, the cells were exposed to different concentrations of apigenin or palmitic acid for 24 h. Results demonstrated that apigenin significantly decreased the viability of HepG2 cells at concentrations from 320 to 1,280 µM (P<0.01;
The cells were exposed to different concentrations of palmitic acid (150, 250 and 350 µM) for 24 h and cellular TC and TG contents were subsequently measured. The results demonstrated that the intracellular TC and TG levels were significantly elevated when cells were treated with 250 and 350 µM palmitic acid compared with control cells (P<0.05;
To verify the Oil red O staining results, the cellular TC and TG contents were measured. Results demonstrated that the intracellular TC and TG levels were significantly elevated when cells were treated with 250 µM palmitic acid compared with the control cells (P<0.01;
The activity of AMPK is closely associated with phosphorylation of the Thr172 site in the α subunit (P-AMPKα1). Therefore, the activity of AMPK can be evaluated by measuring the levels of P-AMPKα protein. Results demonstrated that the P-AMPK/AMPK ratio in the cells treated with 250 µM palmitic acid was significantly lower than in the normal group (P<0.01;
To investigate the mechanism underlying the apigenin-mediated inhibition of lipid production and regulation of the AMPK signaling pathway in HepG2 cells, the expression levels of proteins associated with fatty acid synthesis and cholesterol synthesis were determined.
Results demonstrated that compared with the control cells, the levels of SREBP-1 (P<0.01;
To further validate the previous findings, the intracellular TC and TG contents were measured following pretreatment with 10 µM compound C for 1 h. The compound C treatment significantly reversed the apigenin-induced reductions in lipid accumulation and led to increases in TC and TG contents (P<0.01;
In addition, the protein expression of AMPK and P-AMPK were examined. The increase in P-AMPK/AMPK ratio induced by apigenin was reversed by pretreatment with compound C in HepG2 cells (P<0.01;
The present findings indicated that apigenin effectively inhibited the effects of palmitic acid-induced lipid accumulation via the AMPK/SREBP signaling pathway in HepG2 cells (
Numerous flavonoids display anti-obesity and hypolipidemic effects in animals and humans. It has been demonstrated that apigenin exerts anticancer, anti-inflammatory and hypotensive effects (
The present study identified that apigenin served an essential role in the reduction of excessive lipid accumulation induced by palmitic acid in HepG2 cells. Results demonstrated that apigenin was not toxic to HepG2 cells at concentrations of <320 µM, whilst palmitic acid exhibited no apparent toxicity to HepG2 cells at concentrations <250 µM. Of note, apigenin has been determined to be cytotoxic to 3T3-L1 cells at concentrations as low as 10 µM (
To further elucidate the molecular mechanism underlying the apigenin-mediated suppression of adipogenesis, the present study investigated the activation of AMPK, which is an important kinase that regulates energy homeostasis. This key protein is involved in various cell signal transduction pathways, senses the intracellular energy state and has an important role in the regulation of glucose and lipid metabolism (
Changes in proteins associated with the AMPK/SREBP pathway were investigated to fully elucidate the hypolipidemic mechanism of apigenin. Apigenin treatment activates AMPK thereby further inhibiting the synthesis of fatty acids and cholesterol (
In summary, apigenin alleviated the palmitic acid-induced increases in lipid accumulation in HepG2 cells thereby reducing the lipid content. The underlying mechanism might be associated with the activation of the intracellular AMPK/SREBP signaling pathway. These findings provide a novel approach for the development of natural hypolipidemic drugs to prevent lipid accumulation.
Not applicable.
This study was funded by the Graduate Innovation Fund of Jilin University, China (grant no. 419020201348).
All data generated or analyzed during this study are included in this published article.
SG, JL, ZM and BC conceived the study and participated in its design. ZM and BC performed the experiments. ZM, BC, ML and ST participated in the acquisition and interpretation of the data. SG, JL, ZM and BC participated in manuscript drafting. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Effects of apigenin and palmitic acid on the viability of HepG2 cells, and analysis of apigenin-mediated inhibition of lipid accumulation in HepG2 cells. (A) Cell viability following treatment with various concentrations of apigenin (0, 20, 40, 80, 160, 320, 640 and 1,280 µM) and (B) palmitic acid (0, 62.5, 125, 250, 500 and 1,000 µM) (n=6). (C) TG and (D) TC contents following treatment with various concentrations of palmitic acid (150, 250 and 350 µm). (E) Photomicrographs of Oil Red O-stained, intracellular lipids (red staining) were obtained by light microscopy (magnification, ×400) and (F) quantification of Oil Red O absorbance. (G) TG and (H) TC contents following treatment with palmitic acid (0 or 250 mm) various concentrations of apigenin (0, 10, 20 and 40 mm; n=3). *P<0.05 and **P<0.01 vs. control; #P<0.05 and ##P<0.01 vs. treatment with palmitic acid alone. TG, triglyceride; TC, total cholesterol.
Effect of apigenin on the expression of AMPK, P-AMPK, SREBP-1, FAS, SREBP-2 and HMGCR proteins in HepG2 cells. (A) Representative western blots and (B) quantification of P-AMPK/AMPK, (C) P-AMPK/β-actin, (D) SREBP-1, (E) FAS, (F) SREBP-2 and (G) HMGCR protein expression measured by western blot analysis (n=3). *P<0.05 and **P<0.01 vs. control; ##P<0.01 vs. treatment with palmitic acid alone. AMPK, AMP-activated protein kinase; P-AMPK, phosphorylated AMPK; SREBP, sterol regulatory element-binding protein; FAS, fatty acid synthase; HMGCR, 3-hydroxy-3-methylglutaryl CoA reductase.
Effect of an AMPK inhibitor compound C on AMPK and P-AMPK protein levels, and the apigenin-mediated inhibition of lipid accumulation in HepG2 cells. (A) TC and (B) TG contents following treatment with palmitic acid (0 or 250 mm), apigenin (0 or 40 mm) and compound C (0 or 10 mm). (C) Representative western blots and (D) quantification of P-AMPK protein levels determined by western blot analysis (n=3). **P<0.01 vs. control; ##P<0.01 vs. treatment with palmitic acid alone; ^P<0.05 and ^^P<0.01 vs. treatment with apigenin and palmitic acid. AMPK, AMP-activated protein kinase; p, phosphorylated; TG, triglyceride; TC, total cholesterol.
Hypothetical mechanism underlying the inhibition of palmitic acid-induced lipid accumulation by apigenin in HepG2 cells. AMPK, AMP-activated protein kinase; SREBP, sterol regulatory element-binding protein; FAS, fatty acid synthase; HMGCR, 3-hydroxy-3-methylglutaryl CoA reductase.