Tripterygium regelii decreases the biosynthesis of triacylglycerol and cholesterol in HepG2 cells

Abstract

In the course of screening to find a plant material decreasing the activity of triacylglycerol and cholesterol, we identified Tripterygium regelii (TR). The methanol extract of TR leaves (TR-LM) was shown to reduce the intracellular lipid contents consisting of triacylglycerol (TG) and cholesterol in HepG2 cells. TR-LM also downregulated the mRNA and protein expression of the lipogenic genes such as SREBP-1 and its target enzymes. Consequently, TR-LM reduced the TG biosynthesis in HepG2 cells. In addition, TR-LM decreased SREBP2 and its target enzyme HMG-CoA reductase, which is involved in cholesterol synthesis. In this study, we evaluated that TR-LM attenuated cellular lipid contents through the suppression of de novo TG and cholesterol biosynthesis in HepG2 cells. All these taken together, TR-LM could be beneficial in regulating lipid metabolism and useful preventing the hyperlipidemia and its complications, in that liver is a crucial tissue for the secretion of serum lipids.

The methanol extract of Tripterygium regelii leaves reduced intracellular contents of TG and cholesterol in HepG2 cells.

Key words:

• Tripterygium regelii
• triacylglycerol
• cholesterol
• SREBP1c
• SREBP2

Cardiovascular disease (CVD) is the major cause of death worldwide. According to World Health Organization, 30 percent of all global deaths are due to CVD in 2008, and the number of deaths from CVD will steady increase.¹⁾ Excessive levels of plasma low-density lipoprotein (LDL) and TG but diminished levels of high-density lipoprotein (HDL) are risk factors for atherosclerotic CVD, particularly in patients with metabolic diseases and fatty liver disease.²⁾ Of note, hyperlipidemia, which is the elevation of hepatic lipid secretion such as cholesterol and TG, is a significant risk factor for the development of atherosclerotic CVD.

Statins, one of the most widely prescribed medications for hyperlipidemia, effectively reduce serum cholesterol levels not only by inhibiting endogenous cholesterol synthesis via inhibition of HMGCR,^3,4) but by decreasing intravascular cholesterol through increase of LDL receptors in the peripheral cells.⁵⁾ Fibrates have also been used for the treatment of hypertriglyceridemia or mixed hyperlipidemia. The decrease in plasma TG induced by fibrates is caused by an inhibition of the synthesis and secretion of TGs in the liver.⁶⁾ These pharmaceutical lipid-lowering drugs have been associated with adverse effects including rhabdomyolysis, cramps, and myalgia.⁷⁾ Therefore, dietary and herbal supplements may be an attractive alternative to prescription pharmaceuticals in that traditional herbal medicines are regarded as acceptably safe.

TR has been cultivated extensively in China, Japan, Korea, and Taiwan, and used in traditional medicine as a remedy for diverse range of diseases from sores to fever and inflammation.⁸⁾ TR has been validated to have various pharmacological properties including anti-cancer,⁹⁾ anti-inflammatory effects,¹⁰⁾ and inhibition of polycystic kidney disease.¹¹⁾ The plants in genus Tripterygium of family Celastraceae are well reported to be rich sources of sesquiterpenoids, diterpenoids, triterpenoids, and sesquiterpene pyridine alkaloids.⁸⁾ Representatively, celastrol, one of the bioactive terpenoids isolated from TR, exhibited potent anti-cancer activity,¹²⁾ anti-inflammatory,¹³⁾ and neuroprotective effects.¹⁴⁾ Recently, celastrol has been reported to be a powerful anti-obesity agent as a leptin sensitizer.¹⁵⁾

The aim of this study was to evaluate the effect of TR focused on cholesterol and TG biosynthesis in HepG2 cells because the effect of total methanol extract of TR on lipid synthesis in hepatocyte has not yet been elucidated. Moreover, we proposed that the molecular mechanism of TR on anti-lipogenic activity is associated with sterol response element binding proteins (SREBPs) and their downstream gene expression in HepG2 cells.

Materials and methods

Plant material

The fresh leaves of TR were collected from Baekun-Myeon, Jecheon-Si, and Chungbuk (Korea) in July, and identified by a botanist, Prof. K. H. Bae (College of Pharmacy, Chungnam National University, Daejeon, Korea). Its voucher specimen (Herbarium No. 027-048) has been preserved at the Korea Plant Extract Bank (Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea). The plant material was dried naturally in open air, and the powdered leaves of TR (3 kg) were extracted with MeOH (72 L) by maceration for 2 weeks at room temperature. TR extract was filtered through Whatman filter paper, and concentrated at 40 °C under reduced pressure to obtain the crude methanol extract of TR (500 g, 16.7%). The extract was sealed and stored in dark at –20 °C.

Cells culture and sample preparation

Human HepG2 hepatocyte cells were obtained from ATCC. Cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium) high glucose medium (Welgene, Korea), supplemented with 10% fetal bovine serum (Gibco, USA), 1% antibiotic-antimicrobial (Gibco, USA) at 37 °C, 100% humidity, and 5% CO₂. TR-LM was dissolved in DMSO. The final DMSO concentration in culture medium was 0.05%. Control cells were treated 0.05% DMSO without TR-LM. All measurements were performed in duplicate for each treatment.

RNA isolation and quantitative RT-PCR

Total RNA was extracted from HepG2 cells using TRIzol reagent (Invitrogen). Complementary DNAs were synthesized from 5 μg of RNA using ImProm-ll Reverse Transcriptase (Promega). After cDNA synthesis, RT-PCR was performed in 20 μL of PCR master mix (Promega) using a surecycler 8800 (Agilent Technologies).

Western blot analysis

HepG2 cells were treated with TR-LM 12.5, 25, 50, and 100 μg/mL for 2 h. After incubation, cells were lysed with lysis buffer (pro-prep, iNtRON) on ice for 30 min. After centrifugation (13,200 g for 25 min at 4 °C), collected proteins were quantified using Bredford reagent. Equal amounts of protein were resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked with Chemi block (ATTO) for 1 h at room temperature. After that, the membranes were incubated overnight at 4 °C with specific primary antibodies of interest in chemi block. Membranes were washed several times with TBST before the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies were treated. Specific immune complexes were detected by chemiluminescence regent (Thermo).

Measurement of cell viability

Cell viability was assessed using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay. Cells (5 × 10⁵ cells/ml) were incubated at various concentrations of TR-LM (12.5, 25, 50, and 100 μg/ml) for 24 h. After incubation, MTT solution was treated and it dissolved the insoluble purple formazan product in dimethyl sulfoxide (DMSO). The absorbance of the colored solution was measured at 570 nm using the ELISA plate reader.

Intracellular TG and cholesterol staining

HepG2 cells were seeded at 5 × 10⁵ cell/mL in μ-slide plates. After sample treatment, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min. After rinsing, the cells were stained with Filipin lll (50 μg/mL) for 2 h and Bodipy 493/503 (10 μg/mL) with Hoechst33342 (10 μg/mL) for 15 min at room temperature. Fluorescence images were obtained using a fluorescent microscope (ZEISS).

Measurement of newly synthesized TG

HepG2 cells were incubated with various concentrations of TR-LM in the presence of [¹⁴C] acetate (46 kBq) in serum-free medium for 2 h. Cell were washed twice with PBS, and total lipids were extracted with hexane: 2-propanol (3:2, v/v). Lipids were separated by thin-layer chromatography by using hexane: diethyl ether: acetic acid (80: 20: 1, v/v/v) and visualized by exposure to a bio-imaging analyzer (FLA-7000, FUJIFILM).

In vitro cell-free HMG-CoA reductase activity assay

The HMG-CoA reductase assay kit from Sigma-Aldrich based on the catalytic domain of the human enzyme was used, following the manufacture protocol.

Statistical analysis

The data are presented as the mean ± standard deviation. Statistical analysis was performed using Student’s t-test. Differences were considered significant when p < 0.05.

Results

Tentative identification of phytochemicals in TR by UPLC–QTOF–MS

The methanol extract of TR leaves is characterized using analytical reverse-phase UPLC–QTOF–MS analysis. As presented in Fig. 1, a complete chromatographic separation of phytochemicals was reached within 14 min under base peak ion (BPI). The five phytochemicals were identified by MS analysis as the following known phytochemicals: 3-O-[α-D-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]quercetin (1), 3-O-β-rutinosylquercetin (2), 3-O-β-D-galactopyranosylquercetin (3), 3-O-β-D-glucopyranosylquercetin (4), and celastrol (5). (Supplementary 1). The tentative identification of compounds were characterized using spectroscopic data, including retention times, mass spectral data of molecular ions, and UV–Vis absorption maxima, in comparison with previously published data.^16–18)

Fig. 1. UPLC-PDA-QTOF-MS chromatograms of TR: 3-O-[α-D-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]quercetin (1), 3-O-β-rutinosylquercetin (2), 3-O-β-D-galactopyranosylquercetin (3), 3-O-β-D-glucopyranosylquercetin (4) and celastrol (5).

Effect of TR-LM on intracellular lipid contents in HepG2 cells

In order to verify the inhibitory effect of TR-LM on intracellular neutral lipids and cholesterols, we carried out the staining with BODIPY 493/503 and Filipin after treatment of the indicated concentrations of TR-LM for 24 h in HepG2 cells. BODIPY 493/503 are lysochromes, so they bind directly into hydrophobic neutral lipids such as TG and cholesterol ester. In contrast, Filipin binds specifically to sterols, sequestering with the 3β-hydroxy group of cholesterol. As shown in Fig. 2(A), fluorescence has weakened in TR-LM treated cells in a dose-dependent manner. The cells, when counter-stained with Hoechst 33342, showed that TR-LM did not have a cytotoxic effect. The quantitative data of BODIPY staining present that the exposure of 50 and 100 μg/mL TR-LM could reduce intracellular neutral lipid contents approximately by 25% and 50% compared to control cells, respectively (Fig. 2(B)). Further analysis of the effect of TR-LM on total cholesterol quantitative levels showed that TR-LM gradually decreased cholesterol level up to 50% at 100 μg/mL vs. control cells (Fig. 2(C)). Collectively, the results demonstrate that TR-LM inhibits intracellular content of neutral lipids and cholesterols in HepG2 cells.

Fig. 2. Effect of TR-LM on intracellular neutral lipid and cholesterol contents in HepG2 cells.

Notes: (A) Cells were treated with various concentrations (12.5, 25, 50 and 100 μg/mL) for 24 h. Then, neutral lipids detected by using Bodipy 493/503, and cholesterols stained by cholesterol-sequestering agent Filipin lll. The counter staining of nucleus was performed with Hoechst 33342. (B) Fluorescence of Bodipy 493/503 was quantified by flow cytometry at FITC channel. (C) Total cholesterol quantification assay was carried out by manufacturer’s instructions. The bar graphs are presented as mean ± S.D. (n = 2). Significance: **p < 0.01 vs. control.

TR-LM regulates the lipid metabolism related gene expression

To reveal further mode of action on reduction of hepatic lipid content, the expression levels of genes related to lipid metabolism were determined by RT-PCR and immunoblot analysis. SREBP-1c is known to regulate genes required for de novo lipogenesis including fatty acid, TG, and phospholipid biosynthesis. Exposure of TR-LM for 2 h on HepG2 cells strongly decreased SREBP-1c expression at transcriptional and translational levels (Fig. 3(A) and (B), top panel). Consistently, TR-LM suppressed the expression of several target genes of SREBP-1c including fatty acid synthase (FAS), glycerol-3-phosphate acyltransferase 1 (GPAT1), diacylglycerol acyltransferase 1 (DGAT1), DGAT2, and stearoyl-CoA desaturase 1 (SCD1) (Fig. 3(A) and (B)). Next, the effect of TR-LM on gene expression of SREBP-2 was examined. SREBP-2 has been closely associated with cholesterol synthesis and accumulation. TR-LM downregulated gene expression of SREBP-2 and its target HMGCR at both transcriptional and translational levels in a concentration-dependent manner (Fig. 3(C) and (D)). Additionally, we observed the changes of PPAR-α which is one of the representative genes involved in fatty acid oxidation in hepatocytes. TR-LM significantly enhanced the expression of PPAR-α at both mRNA and protein levels (Fig. 3(E) and (F)). Our results that SREBP-1c and SREBP-2 gene expression were both suppressed by TR-LM suggests an anti-lipogenic effect and the increased PPAR-α expression reflects the enhancement of fatty acid oxidation. Consequently, the inhibited TG and cholesterol biosynthesis and increased fatty acid oxidation may be the cause for the decrease in intracellular lipid contents in TR-LM-treated HepG2 cells.

Fig. 3. Effect of TR-LM on transcriptional and translational expression of lipid metabolism-associated genes in HepG2 cells.

Notes: Cells were incubated with indicated concentrations of TR-LM and incubated for 2 h. RT-PCR and western blot were carried out to analysis of the genes expression related to lipogenesis (A and B), cholesterogenesis (C and D), and lipolysis (E and F).

TR-LM blocks de novo TG and cholesterol synthesis in HepG2 cells

Since we assumed that reduction of lipogenic gene expression in TR-LM-treated HepG2 cells contribute to the inhibition of intracellular lipid content, further examination of TR-LM on virtual activity-associated TG and cholesterol biosynthesis was performed. To examine whether TR-LM can inhibit newly synthesized TG, we analyzed isotope-labeled TG contents after treatment of [¹⁴C] acetate as a substrate in the presence or absence of TR-LM in HepG2 cells. Treatment of TR-LM gradually reduced newly synthesized TG contents compared to the control (inhibition of 31.3% at 50 μg/mL, and 59.3% at 100 μg/mL) (Fig. 4(A)). Next, we carried out the in vitro cell-free enzymatic activity assay of HMG-CoA reductase, which is the key enzyme of the mevalonate pathway that produces cholesterol. As shown in Fig. 4(B), TR-LM attenuated HMG-CoA reductase activity in a concentration-dependent manner (inhibition about 50% at 100 μg/mL). TR-LM not only decreased HMG-CoA gene expression in HepG2 cells, but also inhibited HMG-CoA reductase enzymatic activity in a cell-free system. These results present that TR-LM inhibits virtual activity-associated TG and cholesterol biosynthesis.

Fig. 4. TR-LM blocks de novo TG and cholesterol synthesis.

Notes: (A) Lipid profile analyzed by using TLC after treatment of [¹⁴C] acetate as radiolabeled substrate. Relative percentage of newly synthesized [¹⁴C] TG was shown as a graph. C75 (15 μg/mL), a FAS inhibitor, was used as a positive control. (B) In vitro HMG-CoA reductase activity. Pravastatin, a specific HMG-CoA reductase inhibitor (500 nM), was used as apositive control. Data are presented as mean ± S.D. (n = 3). Significance: *p < 0.05; **p < 0.01 vs. control.

Discussion

To this day, studies about medicinal values of TR focused on their roots. Because its leaves are high-valued botanical materials due to their sustainable productivity as a perennial plant, we performed the assay to evaluate the possibility of the leaves of TR as alternative lipid-lowering agent. Few reports are available on the lipid-lowering effect of plant extract; however, we focused on TR-LM’s effects on intracellular TG and cholesterol contents at cellular level by the regulation of the expression of hepatic genes involved in lipid metabolism rather than in vitro enzymatic activity such as HMG-CoA.¹⁹⁾ Unfortunately, reports indicate that plant methanol extracts having an inhibitory effect on HMG-CoA reductase showed relatively low efficacy. It is worth noting that the exposure of TR-LM induced the alteration of this protein expression and reflected in the decrease of de novo TG biosynthesis in HepG2 cells. However, further studies are needed to prove its efficacy on cholesterol biosynthesis because TR-LM showed moderate efficacy on HMG-CoA reductase (Fig. 4(B)). In addition, further investigation of in vivo models should be performed in order to confirm its potential as an alternative treatment for hypercholesterolemia and related cardiovascular diseases.

The molecular mechanism decreasing intracellular lipid contents might be related inhibition of hepatic expression of lipogenic enzymes (FAS, SCD1, GPAT1, DGAT1 and 2, and HMGCR) by regulating SREBP-1c and SREBP-2, and assumed increase of fatty acid oxidation by PPAR-α. SREBPs are important transcription factors that regulate the biosynthesis of TG and cholesterol²⁰⁾; SREBP-1c enhances transcription of genes involved in TG biosynthesis, whereas SREBP-2 stimulates expression of genes involved in cholesterol biosynthesis. The post-translational mature form of both SREBPs is closely associated with the changes in mRNA levels of the target genes. In our data, TR-LM reduced the mRNA expression of both SREBP-1c and SREBP2; however, TR-LM only decreased the level of the mature SREBP-1c protein located in the nucleus but not SREBP-2. Accordingly, it is necessary to further determine the protein level of the mature form of SREBP-2, in order to verify the role of this transcription factor in the inhibitory effect of TR-LM on expression of hepatic cholesterogenic genes.

Unfortunately, we could not reveal upstream regulator of both SREBPs. Many studies addressing the transcriptional activity of nuclear SREBP-1c have shown that it is controlled by multiple posttranslational modifications, including phosphorylation, ubiquitination, and acetylation.^21–24) Since AMPK is known to phosphorylate and inhibit SREBP activity,²⁵⁾ we confirmed whether TR-LM could activate AMPK. However, we did not observe TR-LM induced phosphorylation and activation of AMPK (data not shown). Knebel et al. have shown that p38 and JNK phosphorylate SREBP-1a and -c in HepG2 cells.²⁶⁾ Further study should be carried out to reveal the post-translational modifications of SREBPs by treatment of TR-LM.

According to the tentative identification of compounds, 3-O-[α-D-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]quercetin, 3-O-β-rutinosylquercetin, 3-O-β-D-galactopyranosylquercetin, 3-O-β-D-glucopyranosylquercetin, and celastrol are the main compounds in TR-LM. 3-O-[α-D-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]quercetin is reported to ameliorate the hyperlipidemia in high cholesterol-fed rabbit.²⁷⁾ Also, the available literature demonstrated that celastrol improved lipid metabolism in high fat diet-induced obese rats.¹³⁾ Therefore, we suggest some possibilities that 3-O-[α-D-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]quercetin and celastrol contribute to the decreasing activity of TG and cholesterol in HepG2 cells.

In summary, our observations suggest that TR-LM has a specific effect on TG and cholesterol metabolism in human HepG2 cells. Therefore, TR-LM supplementation may offer therapeutic possibility to ameliorate dyslipidemia, and its clinical complications such as atherosclerotic CVD.

Author contributions

Conceived and designed the experiments: MK HL SO SUL DM ML. Performed the experiments and analyzed the data: MK EK HJY HWR SK. Wrote the paper: MK MK.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by grants from the KRIBB Research Initiative Program [KGM1221713], Republic of Korea. This study was also supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government [NRF-2013R1A1A2063964].

Supplemental materials

The supplemental material for this paper is available at https://doi.org/10.1080/09168451.2017.1390392.