Platycodin D enhances LDLR expression and LDL uptake via down-regulation of IDOL mRNA in hepatic cells

The root of Platycodon grandiflorum (PG) has long been used as a traditional herbal medicine in Asian country. Platycondin D (PD), triterpenoid saponin that is a main constituent of PG, exhibits various biological activities such as anti-inflammatory, anti-oxidant, anti-diabetic, and anti-cancer effects. A previous study showed that PD had cholesterol-lowering effects in mice that develop hypercholesterolemia, but the underlying molecular mechanisms have not been elucidated during the last decade. Here, we demonstrated that both PG and PD markedly increased levels of cell surface low-density lipoprotein receptor (LDLR) by down-regulation of the E3 ubiquitin ligase named inducible degrader of the LDLR (IDOL) mRNA, leading to the enhanced uptake of LDL-derived cholesterol (LDL-C) in hepatic cells. Furthermore, cycloheximide chase analysis and in vivo ubiquitination assay revealed that PD increased the half-life of LDLR protein by reducing IDOL-mediated LDLR ubiquitination. Finally, we demonstrated that treatment of HepG2 cells with simvastatin in combination with PG and PD had synergistic effects on the improvement of LDLR expression and LDL-C uptake. Together, these results provide the first molecular evidence for anti-hypercholesterolemic activity of PD and suggest that PD alone or together with statin could be a potential therapeutic option in the treatment of atherosclerotic cardiovascular disease.


Luciferase reporter assay. Hepatic cells were co-transfected with pLDLR-Luc plasmid (a gift from Axel
Nohturfft, Addgene #14940) and Renilla luciferase reporter plasmid (pRL-TK) using Lipofectamine 3000 (Thermo Scientific, USA). After 24 h, the transfected cells were treated with 2.5 μM PD or 250 μg/mL PG for additional 24 h. Cell lysates were collected and luciferase activities were measured using Fluoroskan FL Microplate Luminometer (Thermo Scientific, USA) and Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer's instructions. Luciferase activities were normalized to the Renilla luciferase expression.
In vivo ubiquitination assay. HepG2 cells were transfected with HA-ubiquitin using Lipofectamine 3000 reagent (Thermo Scientific, USA). After overnight incubation, the cells were treated or co-treated with 2.5 μM PD and 10 μM T0901317 for 18 h and added with 50 nM Baf A1 for 6 h prior to harvesting the cells. The cell pellets were freeze-thawed twice before resuspension in 100 μl SDS lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1% SDS and protease inhibitor) and boiled for 10 min. The boiled lysates were diluted with 900 μl NP-40 buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40) and rotated for 10 min at cold room. Following centrifugation at 13,000 rpm for 10 min at 4 °C, 25 μl of lysate was used for input sample and the remaining lysate was immunoprecipitated with anti-LDLR overnight at 4 °C, followed by further incubation with protein A/G agarose (Thermo Scientific, USA) for 1 h at 4 ℃. The beads were washed three times with 1 ml of lysis buffer, re-suspended in 2 × SDS sample buffer, boiled at 95℃, and then separated by SDS-PAGE. Scientific Reports | (2020) 10:19834 | https://doi.org/10.1038/s41598-020-76224-w www.nature.com/scientificreports/ Statistical analysis. Data is presented as the mean ± standard deviation (SD). Statistical analysis was performed using student's t-test or one-way ANOVA followed by Tukey's post hoc test. p values < 0.05 were considered to represent the significant difference.

Results
Cytotoxic effects of PG and PD on HepG2 cells. To investigate whether PG and PD display toxic effects on HepG2 cells, cell viability was determined by the WST-8 assay following treatment with different concentrations of PG (10, 50, 100, 250, 500 μg/mL) and PD (1, 2.5, 5 μM) for 24 h. The results showed that 10-250 μg/mL of PG and 1-2.5 μM of PD have no significant cytotoxic effects on HepG2 cells, but 500 μg/mL PG and 5 μM PD reduce cell viability around 15% compared to control (Fig. 1A,B).

PG and PD induce cell surface LDLR expression and LDL-C uptake in HepG2 cells. LDLR is a cell
surface receptor that mediates the uptake of LDL-C from plasma, lowering blood cholesterol level 23 . A previous study reported that PD has a cholesterol-lowing effect in mouse model which develop hypercholesterolemia 22 . Thus, we first explored whether PG and PD upregulate LDLR expression in HepG2 cells. Western blot analysis showed that the level of LDLR expression was stimulated from 100 μg/ml of PG and 0.5 μM of PD and further increased in a dose-dependent manner, reaching maximum induction of 2.6 and 3 folds relative to controls at 500 μg/ml of PG and 5 μM of PD, respectively ( Fig. 2A,B). For the next experiments, we choose to use the concentration of 250 μg/ml PG and 2.5 μM PD, since cytotoxic effect was not exhibited at this concentration ( Fig. 1). Next, we examined whether PG and PD increase LDLR expression on cell surface. FACS analysis revealed that cell surface level of LDLR was enhanced by 1.2 ± 0.06 and 1.2 ± 0.07 compared to control upon treatment of PG and PD, respectively (Fig. 2C). Finally, to investigate the effect of PG and PD on the uptake of LDL-C, we incubated PG or PD-treated HepG2 cells with BODYPI-labeled LDL particles for 1 h. The uptake of LDL particles was visualized by confocal microscopy and quantified by measuring the fluorescent intensity per cells. As shown in Fig. 2D, treatment of PG and PD caused a similar 1.7-fold increase in the uptake of LDL particles in HepG2 cells. Taken together, these results demonstrate that PG and its derived compound, PD lead to the induction of LDLR cell surface expression, thereby enhancing the uptake of LDL-C in HepG2 cells.
PG and PD reduce IDOL mRNA expression in hepatic cells. LDLR expression is tightly regulated at multiple steps to maintain cholesterol homeostasis in cells. Especially, LDLR gene transcription is controlled by SREBP-2, a transcription factor whose proteolytic activation is dependent on cellular cholesterol levels 24 . LDLR protein is also known to undergo lysosomal degradation by PCSK9 or IDOL 25 . To define the molecular mechanisms underlying the increased LDLR expression by PG and PD, we first determined the effects of PG and PD on LDLR mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) and quantitative realtime PCR. Unexpectedly, LDLR mRNA was not changed by the treatment of PG and PD (Fig. 3A,B). Furthermore, we confirmed that PG and PD did not increase LDLR promoter activity by the luciferase reporter assay (Fig. 3C). Consistent with these results, the amount of mature form of SREBPs were not altered upon treatment of each drug (Fig. 3D). In addition, we could not observe any significant changes in other proteins involved in regulating cholesterol metabolism, such as HMGCR, an enzyme for cholesterol synthesis and ABCA1/ABCG1, membrane transporters mediating cholesterol efflux (Fig. 3D). Therefore, we reasoned that the elevated levels of LDLR by PG and PD might be due to a post-translational mechanism. To test this, we investigated whether PG and PD have an inhibitory effect on PCSK9 that binds to LDLR and direct it to lysosome for degradation and found that both drugs had little effect on precursor and mature form of PCSK9 (Fig. 3D). We then tested their effects on IDOL, an E3 ubiquitin ligase that targets LDLR for its lysosomal degradation. Remarkably, both PG and PD decreased IDOL mRNA levels, as determined by RT-PCR and quantitative real-time PCR (Fig. 3E,F). In addition, we examined the effects of PG and PD on LDLR and IDOL expression in another hepatic cell lines including SNU-387 and Hep3B. Similar to HepG2 cells, upon treatment with PG and PD, LDLR protein was upregulated without a statistically significant increase in its mRNA level and promoter activity in both cells ( Fig. 4A-C). Importantly, real-time PCR revealed that IDOL gene was down-regulated after treatment with both drugs in these hepatic cells (Fig. 4D). Collectively, these results suggest that IDOL, but not other cholesterol regulatory proteins, is involve in the PG and PD-mediated upregulation of LDLR in hepatic cells.

PD enhances LDLR stability by inhibiting LXR-IDOL pathway in HepG2 cells. Liver X receptors
(LXRs) are cholesterol-sensing transcription factors that are activated in response to excessive intracellular cholesterol, inducing key genes involved in regulating cholesterol homeostasis including IDOL. Thus, LXR-IDOL Figure. 2. PG and PD induce LDLR expression and LDL-C uptake in HepG2 cells. (A, B) HepG2 cells were treated with indicated concentration of PG or PD for 24 h. Cell lysates were subjected to western blotting with anti-LDLR and anti-GAPDH antibodies. (C) HepG2 cells were treated with 250 μg/ml PG and 2.5 μM PD for 24 h, followed flow cytometry to determine the amount of cell surface LDLR expression. Data were analyzed using CellQuest Pro software version 5.2 and the average fluorescence intensity of LDLR was shown as fold change. Error bar represented the mean ± SD. *P < 0.05 by Student's t tests. (D) HepG2 cells were treated with 250 μg/ml PG and 2.5 μM PD for 24 h, followed by incubation with 5 μg/mL Bodipy FL dye-labeled LDL for 1 h. The internalization of the fluorescence labeled LDL (green) was imaged using confocal microscopy. DAPI (blue) was used for nuclear DNA staining. Quantification of LDL fluorescence intensity per cells was analyzed using Image J. Bar graph represents the mean ± SD. *P < 0.05 by one-way ANOVA with Tukey's post hoc test. www.nature.com/scientificreports/ pathway represents a mechanism for feedback inhibition of LDLR expression and cholesterol uptake. To investigate the effect of PD on the LXR-IDOL pathway, HepG2 cells were treated with T0901317, a synthetic LXR agonist in the presence or absence of PD and IDOL mRNA expression was examined. As shown in Fig. 5A,B, IDOL mRNA expression was increased by T0901317, but which was attenuated by the addition of PD. Meanwhile, there was no significant change in the protein levels of LXR α after PD treatment (Fig. 5C). These results indicate that PD inhibits the LXR-dependent IDOL expression, but not changed in levels of LXR protein.

Scientific Reports
Since IDOL promotes LDLR degradation through ubiquitination, the reduced expression of IDOL mRNA by PG and PD may contribute to enhance LDLR protein stability. To test this possibility, we chased LDLR protein levels after treatment of cycloheximide (CHX) which blocks new protein synthesis and found that the half-life of LDLR protein was increased in PD-treated cells compared to untreated cells (Fig. 5D). We next analyzed whether PD leads to changes in LDLR ubiquitination level by in vivo ubiquitination assay and found that, indeed, PD reduced the ubiquitination level of LDLR compared to untreated control. We further observed that PD can suppress the LDLR ubiquitination, which was enhanced by the activation of LXR-IDOL pathway upon T0901317 treatment (Fig. 5E). Taken together, these results indicate that PD enhances LDLR stability by inhibiting LXR-IDOL-mediated ubiquitination and degradation of LDLR.

PG and PD exhibit a synergistic effect with simvastatin on hepatic LDLR expression and LDL-C uptake in HepG2 cells.
Statins are widely used for lowering blood levels of LDL-C, because it upregulates hepatic LDLR expression and enhances the subsequent uptake of LDL-C in the blood. To investigate whether PG and PD have a synergistic effect with statins on LDLR expression, we treated HepG2 cells with PG or PD together with simvastatin for 24 h and examined the expression levels of LDLR by western blot. Our results showed that simvastatin alone exhibits approximately 2.2-fold increase in hepatic LDLR level compared to untreated control cells. Importantly, the combined treatment with simvastatin and PG or PD synergistically increased LDLR levels to an average of 5.5 and 4.19-fold compared to control cells (Fig. 6A). In addition, a synergistic increase in LDL-C uptake was also examined upon co-treatment of PG or PD with simvastatin. As shown in Fig. 6B, LDL uptake assay showed that simvastatin alone promoted approximately 1.6-fold increase in the uptake of LDL particles compared to untreated cells. Meanwhile, PG or PD with simvastatin markedly increased the uptake of LDL particles around 3 and 3.5-fold, respectively, which were in accordance with the increased LDLR expression in Fig. 6A. These results suggest that PG and PD can be used in combination with statins for cholesterol-lowering therapy.

Discussion
The link between high blood cholesterol and CVD have been well established, with the clearing of serum LDL-C by upregulation of hepatic LDLR being the therapeutic strategy. Previously, Zhao et al. have demonstrated that platycodin saponins from PG possess the anti-hypercholesterolemia activity on mice fed with high-fat diet 26,27 . In addition to these findings, the authors also revealed that PD is an active component in PG responsible for the cholesterol-lowering effect 22 . However, the molecular mechanisms underlying hypocholesterolemic action of PD have not been explored in the last decades. Here, we provide insight into the mechanism by which PD enhance LDL-C uptake in hepatic cells.
The changes of hepatic LDLR expression are driven by both transcriptional and post-translational regulation. Statins promote the transcriptional activation of hepatic LDLR gene by inhibiting the activity of HMG-CoA reductase, a rate-limiting enzyme in the pathway of cholesterol biosynthesis called mevalonate pathway, which leads to a decrease in intracellular cholesterol levels and subsequent activation of the SREBP-mediated gene expression. But, since the mevalonate pathway is not only essential for synthesis of cholesterol but also isoprenoid lipids including farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), used for protein prenylation process 28 , strategies aimed at blocking PCSK9-or IDOL-mediated post-translational modifications of LDLR have been considered as better approaches for LDLR upregulation 29 . While two anti-PCSK9 monoclonal antibodies, namely alirocumab and evolocumab, were approved for use to treat patients with familial hypercholesterolemia and CVD and ongoing clinical trial of the siRNA targeting PCSK9 named inclisiran yielded favorable outcomes 30 , drug development for anti-IDOL therapies is still at the preclinical stage.
In this study, we identified that PD contributed to upregulation of cell surface LDLR expression in HepG2 cells, importantly which was correlated with increased LDL-C uptake. In our attempt to search for the mechanism underlying the up-regulation of LDLR by PD, we found that PD prolonged the half-life of LDLR protein by down-regulating IDOL mRNA expression rather than promoting SREBP2-dependent induction of LDLR Figure 3. PG and PD inhibit IDOL transcription but not LDLR transcription and promoter activity in HepG2 cells. (A, B, E, F) HepG2 cells were treated with 250 μg/mL PG and 1, 2.5 μM PD for 24 h. RT-PCR and Realtime PCR assay were performed to measure the expression of LDLR mRNA (A, B) or IDOL mRNA (E, F). GAPDH was used as a reference gene for quantification analysis. Quantitative real-time PCR represent the mean ± SD from three independent experiments. *P < 0.05 by one-way ANOVA with Tukey's post hoc test. NS not significant. (C) HepG2 cells were cotransfected with the pRL-TK vector and pLDLR-Luc plasmid. The cells were re-seeded in 12-well plate and treated with PG (250 μg/mL) or PD (1, 2.5 μM) for 24 h. The luciferase activities were measured and normalized with the respective Renilla activity. The data represent the mean ± SD of independent experiments. NS not significant. (D) After treatment of HepG2 cells with 250 μg/mL PG and 1, 2.5 μM PD for 24 h, cell lysates were subjected to western blotting with the indicated antibodies. The figure shows a representative western blot. The intensity of each protein bands from western blotting were determined by Image J software and normalized to that of GAPDH control. Bar graph shows the mean ± SD from three independent experiments.   13,36 . IDOL-null mouse embryonic stem cells displayed a marked increase in LDLR expression and LDL-C uptake independent of SREBP and PCSK9 pathway 37 . Thus, inhibiting the IDOL-mediated LDLR degradation pathway may offer a therapeutic benefit to enhance hepatic LDL-C clearance. www.nature.com/scientificreports/ IDOL belongs to an E3 ubiquitin ligase to trigger specific degradation of LDLR 10 and induced by LXRs that can be activated with LXR ligands such as oxysterols and synthetic agonists 37 . We also found that PD reduced the IDOL mRNA expression induced by synthetic LXR agonist T0901317, demonstrating that PD inhibits the LXR-dependent IDOL gene expression. The LXR-IDOL pathway can be inhibited by down-regulation of LXR expression 38,39 or competitively inhibiting LXR agonist binding into the ligand-binding pocket of LXR protein 16 . Our data showed that LXRα expression was not changed by PD, suggesting that PD may antagonize LXR transcriptional activity without affecting LXR expression. The inhibitory effect of PD on LXR activity can be caused by blocking oxysterol binding to LXR ligand-binding pocket, reducing intracellular oxysterol concentration, or other mechanisms. Further studies are needed to uncover the details of molecular mechanism by which PD inhibits the LXR-IDOL pathway in HepG2 cells.  Figure 6. The synergistic effect of PG or PD with simvastatin to enhance LDLR expression and LDL-C uptake.
(A) HepG2 cells were treated with 1 μM simvastatin with or without 250 μg/mL PG and 2.5 μM PD for 24 h. Cell lysates were subjected to western blotting with anti-LDLR and anti-GAPDH antibodies. The intensity of LDLR protein was measured using Image J software and normalized to that of GAPDH control. Bar graph shows the mean ± SD from three independent experiments. *P < 0.05 by one-way ANOVA with Tukey's post hoc test. (B) HepG2 cells were treated with 1 μM simvastatin with or without 250 μg/mL PG and 2.5 μM PD for 24 h, followed by incubation with 5 μg/mL Bodipy FL dye-labeled LDL for 1 h and confocal microscopic imaging. Quantification of LDL fluorescence intensity per cells was analyzed using Image J software. DAPI (blue) was used for nuclear DNA staining. Bar graph represents the mean ± SD. *P < 0.05 by one-way ANOVA with Tukey's post hoc test.
Scientific Reports | (2020) 10:19834 | https://doi.org/10.1038/s41598-020-76224-w www.nature.com/scientificreports/ Is it possible that an effective concentration of PD is reached in the circulation after its dietary intake? A recent study determined that oral administration of PD at 20 mg/kg in rats resulted in the maximum plasma concentration (Cmax) of 44.45 ng/ml 15 . Considering LDLR upregulation occur from 0.5 μM PD ( Fig. 2A) which corresponds to 600 ng/ml, oral dosage of 270 mg/kg of PD is necessary to produce the biological effects. Because PD does not show any signs of toxicity against 14 principle organs upon oral administration of up to 2000 mg/ kg in mice 40 , we can conclude that the serum concentration of PD required for effective hepatic LDLR elevation can be achieved after safe level of intake of PD.
Through more than 25 years of clinical trials, statins were established as the first line treatment for lowering LDL-C levels in CVD patients 41 . When stain monotherapy is not sufficient for achieving treatment goal for LDL-C, the addition of other cholesterol-lowering drugs to statins may be considered. The statin combination therapy also allows for reducing statin doses for patients with statin intolerance or other side effects. Several clinical studies have demonstrated that the combination of statins and ezetimibe, an intestinal cholesterol absorption inhibitor, provided further mean reduction of LDL-C by 15-30% when compared with statin monotherapy [42][43][44][45] . Furthermore, the addition of PCSK9 inhibitors such as alirocumab and evolocumab on a background of statin therapy induced a significant decrease in LDL-C levels and reduced several cardiovascular risk factors 46,47 . In this respect, several natural compounds have been tested for their synergistic effects with statins. The results showed that PCSK9 inhibitors such as curcumin, epigallocatechin gallate, and tanshinone IIA, and IDOL inhibitor such as xanthohumol enhanced the statin-mediated induction of LDLR mRNA and LDL-C uptake 16,[48][49][50] . In the present study, we demonstrated that co-treatment of HepG2 cells with PD and simvastatin resulted in further increase in LDLR expression and LDL-C uptake compared to simvastatin alone. What is the molecular mechanism for the synergistic effect of PD with statins on LDLR upregulation? Statins activate LDLR gene transcription and PD increases LDLR protein stability. Therefore, the combined use of these drugs with different mechanisms of action might show synergistic effects to elevate LDLR levels via prolonging LDLR protein half-life that is transcriptionally induced upon statins. Additionally, because stains were also known to suppress IDOL expression 13 , treatment of both drugs may lead to cumulative effects on IDOL downregulation.
In conclusion, we demonstrated that PD induces LDLR expression and uptake of LDL-C particles by inhibiting the LXR-IDOL pathway. In addition, we showed that PD has synergistic effects with statin for upregulation of LDLR expression and uptake of LDL-C (Fig. 7), suggesting that PD, with its potent inhibitory activity towards IDOL, could be useful as an adjunctive therapy to statins.