Cholesterol-modulating effects of Korean red ginseng (KRG) targeting PCSK9 in hyperlipidemia

Cholesterol-modulating effects of Korean red ginseng (KRG) targeting PCSK9 in hyperlipidemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cholesterol-modulating effects of Korean red ginseng (KRG) targeting PCSK9 in hyperlipidemia Chang Hwan Lee, Yong Yook Lee, Sun Hee Hyun, Jaehoon Lee, Ji-Hye Park, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6525946/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Hyperlipidemia is a major global health concern, closely linked to cardiovascular disease (CVD) and metabolic syndrome. Effective regulation of blood lipid and cholesterol levels is essential for preventing and managing this condition. Korean red ginseng (KRG), a traditional medicinal plant, possesses diverse pharmacological properties, including anti-hyperlipidemic, immune-enhancing, anti-fatigue, and antistress effects. While previous studies suggest that KRG reduces lipid levels and may lower the risk of hyperlipidemia and CVD, its precise molecular mechanisms remain unclear. In this study, proteomic analysis revealed that KRG modulates proprotein convertase subtilisin/kexin type 9 (PCSK9) in the blood of rats administered with KRG. In hyperlipidemic animal models induced by Triton WR-1339 and a high-fat diet (HFD), KRG significantly reduced total cholesterol (TCHO), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) levels. Furthermore, KRG regulated the expression of PCSK9 and low-density lipoprotein receptor (LDLR)—key regulators of LDL metabolism—in liver tissues. These findings indicate that KRG exerts lipid-lowering effects by modulating PCSK9 and LDLR expression, regulating cholesterol metabolism through the SREBP2/PCSK9/LDLR signaling pathway. This study highlights KRG’s potential as a novel therapeutic agent for preventing and managing hyperlipidemia and CVD. Health sciences/Diseases/Cardiovascular diseases/Dyslipidaemias Biological sciences/Biological techniques/Proteomic analysis Health sciences/Medical research/Drug development Health sciences/Medical research/Experimental models of disease Korean red ginseng hyperlipidemia SREBP2 PCSK9 LDLR proteomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Hyperlipidemia is a condition characterized by abnormally elevated levels of lipids or cholesterol in the blood caused by impaired lipid metabolism or function. It can result from dietary imbalances, obesity, genetic disorders, such as familial hypercholesterolemia, or other conditions like diabetes 1 . Hyperlipidemia is marked by high low-density lipoprotein cholesterol (LDL-C), low high-density lipoprotein cholesterol (HDL-C), and elevated triglyceride (TG) levels. It is one of the most common risk factors for cardiovascular diseases (CVD), including atherosclerosis, myocardial infarction, stroke, and coronary artery disease 2 . CVD remains the leading cause of mortality worldwide, with 20.5 million deaths attributed to CVD in 2021, accounting for approximately one-third of global deaths 3 . Given the projected rise in CVD cases, there is growing interest in the early management of hyperlipidemia. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key protein involved in hepatic cholesterol metabolism through its interaction with LDL receptors (LDLR) 4 . LDL-C binds to LDLR and is subsequently transported to the liver, where LDLR is recycled to regulate blood cholesterol levels 5 . However, under hyperlipidemic conditions, excessive PCSK9 binds to LDLR, leading to its degradation in the liver and a consequent increase in circulating cholesterol levels 6 . Thus, PCSK9 serves as a critical target for lipid and cholesterol regulation. Statins, the first-line lipid-lowering agents for hyperlipidemic patients, paradoxically increase serum PCSK9 levels 7 . Despite maximum doses and combination therapy with other lipid-lowering agents, some high-risk patients fail to achieve target cholesterol levels . In addition, statins may be unsuitable for the treatment of severe hypercholesterolemia and can cause various muscle-related side effects 8 . Recently, PCSK9 inhibitors (evolocumab, alirocumab, and bococizumab) have been used for patients unresponsive to statins or experiencing adverse effects. These inhibitors can reduce LDL-C levels by up to 60% without inducing muscle pain or hepatic dysfunction, making them a crucial alternative for patients with statin tolerance 9 . However, PCSK9 inhibitors are associated with high costs (ranging from USD 7,000 to USD 12,000 annually) 10 and require frequent injections, leading to local injection site reactions 11 . As a result, there is increasing interest in natural compounds that can inhibit PCSK9 in a cost-effective, side-effect–free, and orally administrable manner. Panax ginseng Meyer has been used for centuries in traditional medicine 12 for its diverse therapeutic properties, including cardiovascular benefits 13 . Korean red ginseng (KRG), a steamed and dried form of ginseng, undergoes a transformation during processing that enhances its bioactive compounds, such as ginsenosides and polysaccharides 14,15 , and extends its shelf life. Since the Goryeo Dynasty (734 AD), KRG has been widely traded with China and has gained popularity. The Ministry of Food and Drug Safety has recognized six functional benefits of KRG, including cognitive enhancement, blood sugar regulation, improved circulation, and reduced fatigue, with anti-diabetic effects added in 2024. Notably, KRG has been reported to reduce TG levels, enhance HDL-C levels, activate lipoprotein lipase (LPL), and improve fatty liver conditions 16–18 . However, the precise mechanisms underlying KRG’s cholesterol- and TG-lowering effects remain unclear. Investigating the mechanisms of natural compounds such as KRG is challenging because of their complex composition and multiple biological targets. With advancements in proteomics, studies have utilized mass spectrometry–based approaches to analyze multiple targets simultaneously and elucidate the efficacy of KRG. Previous studies have identified biosignatures related to immune function using proteomics-based analysis of KRG 19 . In this study, proteomics analysis was applied to identify biosignatures associated with KRG’s various known therapeutic effects, and it was found that KRG modulates PCSK9, a key biosignature for hyperlipidemia and lipid metabolism. Based on these findings, further animal studies were conducted to explore the role of KRG in lipid regulation and its potential contribution to CVD prevention. Results 2.1. Identification of proteins in rats administered KRG A large-scale quantitative iTRAQ analysis was conducted to identify differentially expressed proteins (DEPs) to elucidate the molecular signatures underlying the effects of KRG. The analysis included control samples (vehicle, 114) and samples from rats administered KRG at doses of 500, 1,000, and 2,000 mg/kg (115, 116, and 117, respectively). A total of 587 unique proteins were identified in rat serum using MS analysis (protein probability: >99.0%; peptide probability: >95%) based on the target-decoy database (UniProt rat database). An ingenuity pathway analysis (IPA)–based serum analysis was performed to determine the cellular distribution and functional classifications of the identified proteins. Most proteins were associated with the extracellular space (36%) and cytoplasm (27%), with a smaller proportion localized to the nucleus (13%) (Fig. 1). Molecular classification revealed that the identified proteins primarily consisted of undefined proteins (44%), enzymes (16%), and peptidases (10%) (Fig. 1). Next, the quantitative values of the iTRAQ analysis were evaluated for DEPs (Supplemental Table X). From the pool of DEPs between normal and KRG-treated samples, proteins exhibiting consistent upregulation or downregulation trends were selected, with log2 fold changes greater than those specified in Tables 1 (a) and (b). Proteins were considered significant if they showed a consistent trend at least at two different KRG concentrations (500, 1,000, or 2,000 mg/kg), with statistical significance determined using a permutation test ( p < 0.01), Mann–Whitney test ( p 0.2. An IPA was performed for biological functions to assess the biological relevance of the selected proteins. The top disease and biofunction analysis indicated that DEPs were primarily involved in CVD and lipid metabolism. Further functional analysis predicted a decrease in hyperlipidemia. Notably, PCSK9, APOB, and PLTP were consistently downregulated, as shown in Table 1 (b). In addition, machine learning–based disease pathway analysis, which integrates known disease-associated proteins and infers novel participants 20 , predicted the downregulation of hypercholesterolemia and hyperlipidemia. Based on these findings, PCSK9 was identified as a potential biosignature molecule mediating the effects of KRG on dyslipidemia. 2.2. Inhibitory effect of KRG on simvastatin-induced PCSK9 expression in HepG2 cells The cytotoxicity of KRG in HepG2 cells was evaluated using the EZ-Cytox assay (Fig. 1 (a)). The data showed that KRG at concentrations of 50, 100, 200, and 400 µg/mL did not exhibit cytotoxic effects, indicating that KRG was not associated with cell death or inhibition of cell growth. Therefore, the highest non-cytotoxic dose was selected for subsequent experiments. To determine whether simvastatin affects PCSK9 expression, HepG2 cells were treated with increasing concentrations of simvastatin, and PCSK9 expression was analyzed by Western blotting (Fig. 1 (b)). The results confirmed that simvastatin increased PCSK9 expression in a dose-dependent manner ( p < 0.05). Furthermore, in the presence of simvastatin-induced PCSK9 overexpression, treatment with KRG significantly suppressed the simvastatin-induced increase in PCSK9 levels, as demonstrated by both confocal microscopy and Western blot analysis. 2.3. Hypolipidemic effect of KRG in a Triton WR-1339–induced acute hyperlipidemia model Based on the in vitro results, the effects of KRG on lipid profiles and PCSK9 secretion in a Triton WR-133–induced acute hyperlipidemia model were further investigated. Compared to the normal control group, mice administered Triton WR-1339 (400 mg/kg) exhibited significant increases in TGs (40-fold), total CHOs (1.5-fold), and LDL-C (2.4-fold), while HDL-C significantly decreased (0.72-fold) ( p < 0.05) (Fig. 3). Administration of KRG at 200, 400, and 800 mg/kg (human equivalent doses: 1 g, 2 g, 4 g) significantly reduced TG, CHO, and LDL-C levels while increasing HDL-C levels ( p < 0.05). In addition, Triton WR-1339 increased PCSK9 secretion in the blood, whereas KRG significantly inhibited PCSK9 secretion ( p < 0.05). The atherogenic index (AI) was also significantly reduced in the KRG-treated groups (200, 400, and 800 mg/kg) compared to the control group. Overall, these results suggest that KRG improves lipid profiles, suppresses PCSK9 secretion, and may prevent CVDs in an acute hyperlipidemia animal model. 2.4. Hypolipidemic effect of KRG in an HFD-induced chronic hyperlipidemia model Next, the effects of KRG on lipid profiles and PCSK9 expression/secretion in a chronic hyperlipidemia model induced by an eight-week high-fat diet (HFD) were evaluated. Compared to the normal control group, HFD-fed mice exhibited significant increases in TG (1.4-fold), CHO (1.45-fold), and LDL-C (2.08-fold) ( e < 0.05) (Fig. 4). Administration of KRG at 100, 200, and 400 mg/kg significantly reduced TG, CHO, and LDL-C levels ( p < 0.05). Moreover, HFD-fed mice exhibited increased PCSK9 secretion in the blood, which was significantly suppressed by KRG treatment ( p < 0.05). These findings confirm that KRG exerts lipid-lowering effects and inhibits PCSK9 secretion in both acute and chronic hyperlipidemia animal models. 2.5. Involvement of the SREBP2/PCSK9/LDLR signaling pathway in the hypolipidemic effect of KRG Western blot analysis was performed to determine whether the in vitro and in vivo blood analysis results were consistent at the tissue level. In the liver tissues of the chronic hyperlipidemia model induced by eight weeks of HFD, KRG reduced SREBP2 and PCSK9 expression in a dose-dependent manner while increasing LDLR expression compared to the control group ( p < 0.05). These results suggest that the hypolipidemic effect of KRG is associated with the SREBP2/PCSK9/LDLR signaling pathway. Discussion Previously, a proteomic analysis was conducted to evaluate the biological activities of KRG in immune-enhancing responses involving immune organs. Because natural products contain multiple components and target various pathways, identifying the key bioactive signatures of KRG was challenging. Our results revealed DEPs associated with immune response stimulation, demonstrating a molecular functional strategy of KRG 19 . In this study, proteomics was utilized to assess the biological effects of KRG in rat sera administered with the same concentration of KRG. As a result, a total of 587 unique proteins were identified. Among them, 21 proteins exhibited consistent expression trends based on the selection criteria. IPA revealed that the DEPs were primarily associated with CVD and lipid metabolism in the top-ranked diseases and biofunction analysis. Among these findings, the main focus is on hyperlipidemia, which was commonly observed across the analyses and predicted to be downregulated by KRG. Notably, PCSK9 and apolipoprotein B (APOB), both associated with hyperlipidemia, were downregulated (Fig. 1c). In particular, to the best of our knowledge, PCSK9 is the first identified biosignature candidate linked to the lipid-lowering effects of KRG in dyslipidemia. Although several studies have explored the efficacy of KRG 14–18 , the pathway associated with PCSK9 has not been thoroughly investigated. Based on the proteomic results, further investigations were conducted to confirm the type of dyslipidemia affected by KRG and the role of PCSK9. Hyperlipidemia is one of the most common disorders characterized by abnormal blood lipid levels and is a major risk factor for CVD 22 . Previous studies have shown that KRG reduces blood TG and CHO levels by inhibiting pancreatic lipase 23 and HMG-CoA reductase activity in HFD-fed animals 16 . In addition, KRG has been found to prevent lipid accumulation in the liver by improving lipid metabolism 16 . In patients with myocardial infarction, KRG was shown to enhance coronary blood flow 24 , alleviate increased platelet aggregation in hypercholesterolemic conditions, and reduce the incidence of atherosclerosis 25 . These findings suggest that KRG may contribute to reducing CVD risk by improving lipid metabolism; however, no studies have provided a clear mechanistic explanation for this effect. To investigate the PCSK9-inhibitory effect of KRG, immortalized hepatocytes (HepG2), which are known to express and secrete PCSK9, were utilized 26 . In hyperlipidemic animal models, oral administration of KRG at doses of 200, 400, and 800 mg/kg/day (HED = 0.9, 1.8, and 3.6 g/day 27 , Fig. 2) significantly reduced TG, total cholesterol (TC), and LDL-C levels in a dose-dependent manner while increasing high-density lipoprotein cholesterol (HDL-C) levels ( p < 0.05). Furthermore, liver tissue and blood analysis demonstrated that KRG inhibited PCSK9 expression while upregulating LDLR, which regulates cholesterol metabolism and transport. This effect of KRG is associated with the inhibition of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase 28 . In addition, the major ginsenosides in KRG, including Rb1, Rg1, Rh2, Rb2, and CK, have been reported to suppress TG and CHO synthesis, thereby alleviating hyperlipidemia and exerting beneficial effects against CVD 29 , consistent with our findings. Statins, the primary treatment for hyperlipidemia, target the mevalonate (MVA) pathway by inhibiting HMG-CoA reductase (HMGCR) 30 . However, recent studies indicate that statins induce SREBP2 activation, which enhances the transcription of PCSK9 by binding to its proximal promoter containing the SRE motif, leading to an increase in PCSK9 levels 31 . Hyperlipidemia is also accompanied by elevated PCSK9 expression 32 , which may explain why some patients fail to achieve adequate LDL-C reduction with statin therapy. Consequently, PCSK9 inhibitors have emerged as a key therapeutic target for hyperlipidemia and CVD treatment 33 . PCSK9 promotes the degradation of LDLR in hepatocytes, leading to increased circulating LDL-C levels and the maintenance of high LDL-C concentrations 6 . Unlike statins, our findings demonstrate that KRG suppresses the SREBP2/PCSK9 signaling pathway. Elevated TG and CHO levels in hyperlipidemia increase the risk of atherosclerosis, myocardial infarction, and stroke by promoting lipid deposition in blood vessel walls, making them critical markers for clinical diagnosis. However, long-term statin use can cause various adverse effects, and elevated PCSK9 levels accelerate LDLR degradation, thereby contributing to the progression of CVD. This presents a major challenge in current therapeutic strategies 34 . The development of safe and effective agents that suppress PCSK9 expression in hepatocytes could help prevent secondary complications associated with hyperlipidemia. Our study provides evidence that KRG may serve as a promising candidate for hyperlipidemia treatment, ultimately reducing the risk of CVD. This study demonstrates that KRG exerts lipid-lowering effects in hepatocytes and hyperlipidemic animal models. This effect is associated with the protection of LDLR and enhanced LDL uptake activity via the SREBP2/PCSK9 signaling pathway in liver tissue. Given its long-term safety profile, KRG may serve as a functional ingredient for hyperlipidemia prevention and could be used either alone or in combination with statins for therapeutic purposes. However, further studies are required to validate the clinical effects of KRG’s PCSK9-inhibitory properties. Materials and methods 4.1. Global proteomic profiling analysis 4.1.1. Preparation of the KRG water extract and general chemicals The extraction procedure for KRG followed the international standard production process (ISO 19610). The six-year-old P. ginseng root extract (body 75% and root 25%) was prepared through a repeated steaming and drying process by the Korea Ginseng Corporation (Daejeon, Republic of Korea). The extract was freeze-dried, yielding a dark-brown powder (KRG). Ammonium bicarbonate, dithiothreitol (DTT), formic acid (FA), trifluoroacetic acid, ammonium formate, and urea were purchased from Sigma-Aldrich (St Louis, MO, USA). The HPLC-grade acetonitrile (ACN) and water were purchased from JT Baker (Phillipsburg, NJ, USA). Lyophilized trypsin/lys-C were obtained from Promega (Madison, WI, USA). 4.1.2. Animal model Experiments were performed on adult male Sprague Dawley (SD) rats (180–200 g) obtained from Daehan Biolink (DBL, Seoul, Korea) and housed under controlled environmental conditions (22°C–24°C, 12-hour light/dark cycles) for one week prior to the start of the study. The procedures were approved by the Institutional Animal Care and Use Committee of the Korean Ginseng Research Institute (Daejeon, Republic of Korea), in accordance with the Guide for the Care and Use of Laboratory Animals (Approval No. KGC-2015-008). All experiments were carried out using the same method as Lee et al. 19 Briefly, eight-week-old male rats were randomly divided into groups of six rats each. The rats were administered KRG at doses of 0, 500, 1,000, and 2,000 mg/kg for six weeks. Only a vehicle was used for the negative control group (0 mg/kg). Before necropsy, all rats were fasted overnight and euthanized by exsanguination under isoflurane. Blood samples were drawn from the inferior vena cava. After blood collection, serum was obtained in ethylenediaminetetraacetic acid (EDTA)–free vacutainer by centrifugation at 3,000 rpm for 10 min. The remaining serum was stored at −70°C until the global proteomic profiling analysis was performed. 4.1.3. Serum depletion Serum samples were thawed on ice and centrifuged at 3,000× g for 5 min at 4°C. The centrifuged serum samples were pooled in equal volumes. After mixing with buffer A (Agilent, Cat# 5185–5987), the mixed samples were filtered through 0.22-mm Spin-X filters. The mixture was loaded onto the MARS-MS3 column (Agilent Technologies, Wilmington, DE, USA) on a Dionex UltiMate 3000 UHPLC+ system (Thermo Scientific, Germering, Germany). The depleted serum was buffer-exchanged into 50 mM Tris-HCl (pH 8.0) and concentrated through ultrafiltration using the Amicon Ultra-0.5 mL 3 kDa cutoff filter (Millipore, Darmstadt, Germany). The concentration of the collected flow-through sample was determined using a micro bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, IL, USA). 4.1.4. Protein digestion Each depleted serum sample was modified as previously described 19 . Briefly, the proteins in the depleted serum were denaturized with 6 M urea in 10 mM ammonium bicarbonate (ABC) for 2 hr at 37°C. The sample was then treated with 10 mM dithiothreitol (DTT) for 1 hr at 70°C and alkylated with iodoacetamide (IAA) for 30 min at room temperature in the dark. The sample was diluted with 50 mM ABC to achieve a final concentration of 1 M urea. After the alkylation step, Lys-C/Trypsin solution was added at 37°C for overnight incubation (1:30; Promega). Trypsin digestion was performed at 37°C for an additional overnight incubation (1:100). The peptide sample was desalted using a Macro SpinColumn (C-18; Harvard Apparatus, Holliston, MA, USA) according to the manufacturer’s instructions. After sample drying, peptide quantification was performed using BCA before the iTRAQ labeling. iTRAQ labeling and high pH reversed-phase fractionation steps were performed according to the previously described method 19 . 4.1.5. LC-MS 2 experiment and bioinformatic analysis The peptide samples were analyzed using Q-Exactive mass spectrometry (Thermo Fisher Scientific, Bremen, Germany) equipped with a nano-UHPLC Dionex system (Thermo Scientific) using an Easy nanospray source. For the quantitative analysis of iTRAQ-labeled datasets, the SEQUEST algorithm (Thermo Fisher Scientific) was used against the decoy UniProt database. Scaffold Q+ was also used to calculate the quantification values for each protein. Based on the data, the biological functions were evaluated using IPA (IPA; Ingenuity Systems Analysis; Redwood City, CA, USA) and DAVID (https://david-d.ncifcrf.gov/). All experimental conditions were the same as previously described 19 . 4.2. Pharmacology study 4.2.1. Preparation of KRG (G1899) KRG was manufactured from the roots of six-year-old fresh ginseng, P. ginseng C. A. Meyer, harvested in the Republic of Korea by the Korea Ginseng Corporation (Daejeon, Republic of Korea). The preparation process involved steaming the fresh ginseng roots at 90°C–100°C for 3 hr and then drying at 50°C–80°C. KRG was derived from a KRG water extract, which was produced through three eight-hour cycles of circulating hot water (85°C–90°C). The KRG extract underwent a series of processes, including water extraction, concentration, filtration, solution preparation, spray drying, sieving, and other techniques, resulting in a yield of 50%. The standardized, productized form of this extract is known as G1899. 4.2.2. Cell culture Human hepatoblastoma HepG2 cells were obtained from the American Type Culture Collection and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin solution. All cells were incubated in a cell culture chamber at 37°C under a humidified atmosphere with 5% CO 2 . 4.2.3. Cytotoxicity assay Cell viability was examined using the EZ-Cytox assay kit (Dogen, Suwon, Korea). To determine the nontoxic concentration of KRG, the following concentrations were added to each well (2×10 4 cells/well): 50, 100, 200, and 400 µg/mL. The plates were incubated for 24 hr. After incubation, EZ-Cytox (10 µl/well) was added to each well, and the cells were cultured for an additional 2 hr. Finally, a microplate reader was used to measure the absorbance at 450 nm. 4.2.4. Simvastatin-mediated PCSK9 expression HepG2 cells were seeded in collagen-coated six-well plates (2×10 5 cells/well). After a 24-hr incubation, the cells were pretreated with various concentrations of KRG (100, 200, 400 μg/mL) for 1 hr and stimulated with simvastatin (3 nM/mL) for 24 hr. Following treatment, Western blot analysis was performed to quantify the PCSK9 protein levels in each cell lysate. 4.2.5. Western blotting analysis The total protein from cells and liver tissue was harvested and lysed using 1X RIPA buffer containing a mixture of protease and phosphatase inhibitors (MedChemExpress, Monmouth Junction, NJ, USA). The insoluble debris was removed via centrifugation at 12,000× g and 4°C for 30 min, and the total protein concentration of the isolated supernatants was quantified using the Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific). A total of 10 µg of each protein was resolved by SDS–PAGE (8%–12% acrylamide) and then transferred onto PVDF membranes. The membranes were blocked for 2 hr at room temperature with a 5% skim milk solution. Then, the membranes were incubated overnight at 4℃ with primary antibodies specific to PCSK9, SREBP2, LDLR, or actin. The blots were washed three times for 10 min each with Tris-buffered saline with Tween-20 (TBST). The membranes were then incubated for 2 hr with a secondary antibody. ECL Western blotting detection reagents were used to detect the proteins using the Fusion FX6.0 system (Vilber, Collégien, France). The membranes were stripped and re-probed with an anti-β-actin monoclonal antibody to ensure equal protein loading. Protein bands were quantified using ImageJ 1.52a software (NIH, Bethesda, MD, USA) and normalized to β-actin. 4.2.6. Immunocytochemistry For immunocytochemistry, HepG2 cells were cultured with drugs at a concentration of 1 × 10 5 cells/mL in 48-well tissue culture plates. Cells were also fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 10 min. After blocking with 10% BSA for 1 hr, the plate was incubated with primary antibodies diluted in 10% BSA for 4 hr. Subsequently, the plate was washed three times with TBST and incubated with Alexa Fluor 488–conjugated secondary antibodies for 1 hr. After washing with TBST, the plate was stained with DAPI for 10 min, followed by thorough washing. Finally, the plate was analyzed using LSM 900 with Airyscan 2 confocal microscopes (Carl Zeiss, Oberkochen, Germany). 4.2.7. Acute hyperlipidemia model All experiments were performed on adult male ICR mice (22 ± 2 g) obtained from Daehan Biolink (DBL, Seoul, Korea) and kept under controlled environmental conditions (22°C–24°C and 12 hr light/dark cycles) for one week before the start of the experiments. All the mice were randomly divided into six groups when experiments began: normal control group, model group, and KRG treatment groups (100, 200, 400, and 800 mg/kg). After administering the mice for seven days and fasting them overnight, Triton WR-1339 (400 mg/kg B.W) was injected intraperitoneally. At 24 hr after the Triton WR-1339 injection, blood and liver were collected directly from the anesthetized animals. All experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the Korean Ginseng Research Institute (Daejeon, Republic of Korea) in accordance with the Guide for the Care and Use of Laboratory Animals (Approval No. KGC-2023-014). 4.2.8. Chronic hyperlipidemia model All experiments were performed on adult male Sprague Dawley (SD) rats (180–200 g) obtained from Daehan Biolink (DBL, Seoul, Korea) and kept under controlled environmental conditions (22°C–24°C and 12 hr light/dark cycles) for one week before the start of the experiments. After acclimatization for one week, the rats were randomly assigned to five groups (n = 10 per group) as follows: (i) normal diet, (ii) HFD, (iii) HFD + KRG 50 mg/kg, (iv) HFD + KRG 100 mg/kg, and (v) HFD + KRG 200 mg/kg. The rats in the normal diet group were fed a normal diet (No. Teklad Global Diets 2918, Envigo), while the rats in the other four groups were fed an HFD (No. D12452, Research Diets, Inc.). The rats were fed for eight weeks and were allowed free access to food and water throughout the entire study period. KRG, dissolved in 0.9% saline, was administered daily by oral gavage for eight weeks. The rats in the Nor and HFD groups received an equal volume of saline by oral gavage. At eight weeks, blood and liver were collected directly from the anesthetized animals. All experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the Korean Ginseng Research Institute (Daejeon, Republic of Korea) in accordance with the Guide for the Care and Use of Laboratory Animals (Approval No. KGC-2023-020). 4.2.9. Biochemical analysis Blood samples were separated by centrifugation at 3,000× rpm for 15 min at 4°C. The blood tests for T-CHO, TG, HDL-C, and LDL-C were measured using a Hitachi 7100 Automatic Analyzer (Hitachi, Tokyo, Japan), while serum PCSK9 levels were measured using ELISA kits (Abcam, USA). 4.2.10. Statistics Statistical comparisons between multiple groups were compared using one-way ANOVA with the Tukey HSD post-hoc test. Differences between groups were considered significant at p < 0.05, and all values are expressed as mean ± standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA). Declarations Data availability The authors confirm that the data supporting the findings of this study are available within the article Author contributions The authors confirm their contributions to this paper as follows: study conception and design: C.H.L, Y.Y.L, S.H.H, S.H.L. experimental execution, data collection, interpretation, and analysis: C.H.L, Y.Y.L, S.H.H, J.H.L, J.H.P, S.K.P and S.H.L. draft manuscript preparation, data analysis, and resources: C.H.L. and Y.Y.L. All authors discussed the results and commented on the manuscript. Funding This study was funded by the Korea Ginseng Corporation. Competing interests The authors declare the following competing interests: C.H.L., Y.Y.L., S.H.H., and S.H.L. have filed an institution-owned patent entitled “A Composition for Preventing or Treating Dyslipidemia with the Effect of Inhibiting PCSK9 Expression” (Patent No. 10-2023-0194713), related to the development of KRG for hyperlipidemia treatment. No other authors declare any competing interests. Ethics approval The procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Ginseng Corporation (Approval Nos. KGC-2015-008, KGC-2023-014, and KGC-2023-020), and the experiments were conducted in accordance with institutional guidelines and the principles outlined in the ARRIVE 2.0 guidelines to ensure transparency, reproducibility, and adherence to ethical standards in animal research. References Ezeh, K. J. & Ezeudemba, O. Hyperlipidemia: A review of the novel methods for the management of lipids. 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Ther. 27 , 188–199, https://doi.org/10.1016/j.ymthe.2018.10.016 (2019). Chen C. F., Chiou W. F. & Zhang J. T. Comparison of the pharmacological effects of Panax ginseng and Panax quinquefolium. Acta Pharmacol Sin . 29, 1103–1108, https://doi.org/10.1111/j.1745-7254.2008.00868.x (2008). Radad, K., Gille, G., Liu, L. & Rausch, W.-D. Use of ginseng in medicine with emphasis on neurodegenerative disorders. J. Pharmacol. Sci. 100 , 175–186, https://doi.org/10.1254/jphs.crj05010x (2006). Bae, J.-S., Park, H.-S., Park, J.-W., Li, S.-H. & Chun, Y.-S. Red ginseng and 20(S)-Rg3 control testosterone-induced prostate hyperplasia by deregulating androgen receptor signaling. J. Nat. Med. 66 , 476–485, https://doi.org/10.1007/s11418-011-0609-8 (2012). Choi, Y.-J., Choi, H., Cho, C.-H. & Park, J.-W. Red ginseng deregulates hypoxia-induced genes by dissociating the HIF-1 dimer. J. Nat. Med. 65 , 344–352, https://doi.org/10.1007/s11418-010-0504-8 (2011). Kwak, Y.-S., Kyung, J.-S., Kim, J. S., Cho, J. Y. & Rhee, M.-H. Anti-hyperlipidemic effects of red ginseng acidic polysaccharide from Korean red ginseng. Biol. Pharm. Bull. 33 , 468–472, https://doi.org/10.1248/bpb.33.468 (2010). Inoue, M., Wu, C. Z., Dou, D. Q., Chen, Y. J. & Ogihara, Y. Lipoprotein lipase activation by red ginseng saponins in hyperlipidemia model animals. Phytomedicine 6 , 257–265, https://doi.org/10.1016/S0944-7113(99)80018-X (1999). Yamamoto, M., Uemura, T., Nakama, S., Uemiya, M. & Kumagai, A. Serum HDL-cholesterol-increasing and fatty liver-improving actions of Panax ginseng in high cholesterol diet-fed rats with clinical effect on hyperlipidemia in man. Am. J. Chin. Med. 11 , 96–101, https://doi.org/10.1142/S0192415X83000161 (1983). Lee, Y. Y. et al. Proteomic studies of putative molecular signatures for biological effects by Korean Red Ginseng. J. Ginseng Res. 43 , 666–675, https://doi.org/10.1016/j.jgr.2019.05.001 (2019). Krämer, A. et al. Mining hidden knowledge: embedding models of cause-effect relationships curated from the biomedical literature. Bioinform. Adv. 2 , vbac022, https://doi.org/10.1093/bioadv/vbac022 (2022). Haglund, O., Luostarinen, R., Wallin, R., Wibell, L. & Saldeen, T. The effects of fish oil orides, cholesterol, fibrinogen and malondialdehyde in humans supplemented with vitamin E. J. Nutr. 121 , 165–169, https://doi.org/10.1093/jn/121.2.165 (1991). Garcia, C. & Blesso, C. N. Antioxidant properties of anthocyanins and their mechanism of action in atherosclerosis. Free Radic. Biol. Med. 172 , 152–166, https://doi.org/10.1016/j.freeradbiomed.2021.05.040 (2021). Min, S.-W., Jung, S.-H., Cho, K.H. & Kim D.-H. Antihyperlipidemic effects of red ginseng, crataegii fructus and their main constituents ginsenoside Rg3 and ursolic acid in mice. Biomolecules & Therapeutics 16(4) , 364–369 (2008). Ahn, C. M. et al. Red ginseng extract improves coronary flow reserve and increases absolute numbers of various circulating angiogenic cells in patients with first ST-segment elevation acute myocardial infarction. Phytother. Res. 25 , 239–249, https://doi.org/10.1002/ptr.3250 (2011). Hwang, S.-Y. et al. Korean red ginseng attenuates hypercholesterolemia-enhanced platelet aggregation through suppression of diacylglycerol liberation in high-cholesterol-diet-fed rabbits. Phytother. Res. 22 , 778–783, https://doi.org/10.1002/ptr.2363 (2008). Lebeau, P. et al. Endoplasmic reticulum stress and Ca2+ depletion differentially modulate the sterol regulatory protein PCSK9 to control lipid metabolism. J. Biol. Chem. 292 , 1510–1523, https://doi.org/10.1074/jbc.M116.744235 (2017). Nair, A. B. & Jacob, S. A simple practice guide for dose conversion between animals and human. J. Basic Clin. Pharm. 7 , 27–31, https://doi.org/10.4103/0976-0105.177703 (2016). Lee, M.-S., Kim, C.-T., Kim, I.-H. & Kim, Y. Effects of Korean red ginseng extract on hepatic lipid accumulation in HepG2 cells. Biosci. Biotechnol. Biochem. 79 , 816–819, https://doi.org/10.1080/09168451.2014.997186 (2015). Jin, W. et al. Hypolipidemic effect and molecular mechanism of ginsenosides: a review based on oxidative stress. Front Pharmacol. 14 , 1166898, https://doi.org/10.3389/fphar.2023.1166898 (2023). Endo, A. A historical perspective on the discovery of statins. Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci 86 , 484–493, https://doi.org/10.2183/pjab.86.484 (2010). Lammi, C. et al. Virgin olive oil extracts reduce oxidative stress and modulate cholesterol metabolism: comparison between oils obtained with traditional and innovative processes. Antioxidants (Basel) 9 , https://doi.org/10.3390/antiox9090798 (2020). Di Donna, L. et al. Statin-like principles of bergamot fruit (Citrus bergamia): isolation of 3-hydroxymethylglutaryl flavonoid glycosides. J. Nat. Prod. 72 , 1352–1354, https://doi.org/10.1021/np900096w (2009). Sobati, S. et al. PCSK9: A key target for the treatment of cardiovascular disease (CVD). Adv Pharm. Bull. 10 , 502–511, https://doi.org/10.34172/apb.2020.062 (2020). Seidah, N. G. The PCSK9 revolution and the potential of PCSK9-based therapies to reduce LDL-cholesterol. Glob. Cardiol. Sci. Pract. 2017 , e201702, https://doi.org/10.21542/gcsp.2017.2 (2017). Table 1 Table 1 is available in the Supplementary Files section. Supplemental Table X Supplemental Table X is not available with this version Additional Declarations Competing interest reported. The authors declare the following competing interests: C.H.L., Y.Y.L., S.H.H., and S.H.L. have filed an institution-owned patent entitled “A Composition for Preventing or Treating Dyslipidemia with the Effect of Inhibiting PCSK9 Expression” (Patent No. 10-2023-0194713), related to the development of KRG for hyperlipidemia treatment. No other authors declare any competing interests. Supplementary Files SupplementFigure.pdf Table1.docx Cite Share Download PDF Status: Published Journal Publication published 20 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 01 Jul, 2025 Reviews received at journal 24 Jun, 2025 Reviewers agreed at journal 18 Jun, 2025 Reviewers agreed at journal 17 Jun, 2025 Reviewers invited by journal 16 Jun, 2025 Editor assigned by journal 16 Jun, 2025 Editor invited by journal 09 Jun, 2025 Submission checks completed at journal 25 May, 2025 First submitted to journal 25 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6525946","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":472801524,"identity":"3211cf10-23c8-49b1-a8ba-60ede8e0a1de","order_by":0,"name":"Chang Hwan Lee","email":"","orcid":"","institution":"Korea Ginseng Corporation","correspondingAuthor":false,"prefix":"","firstName":"Chang","middleName":"Hwan","lastName":"Lee","suffix":""},{"id":472801525,"identity":"c4a28bba-9bc1-46ed-a3ed-6db9ea741162","order_by":1,"name":"Yong Yook Lee","email":"","orcid":"","institution":"Korea Ginseng Corporation","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"Yook","lastName":"Lee","suffix":""},{"id":472801526,"identity":"60891386-727a-43d0-94dc-7c3b87e99262","order_by":2,"name":"Sun Hee Hyun","email":"","orcid":"","institution":"Korea Ginseng Corporation","correspondingAuthor":false,"prefix":"","firstName":"Sun","middleName":"Hee","lastName":"Hyun","suffix":""},{"id":472801527,"identity":"77974e94-cff4-437b-b2ae-228a2af622ca","order_by":3,"name":"Jaehoon Lee","email":"","orcid":"","institution":"Korea Ginseng Corporation","correspondingAuthor":false,"prefix":"","firstName":"Jaehoon","middleName":"","lastName":"Lee","suffix":""},{"id":472801528,"identity":"b8a5e1e9-2a9e-4b3a-8dcb-b94c7b713f66","order_by":4,"name":"Ji-Hye Park","email":"","orcid":"","institution":"Korea Ginseng Corporation","correspondingAuthor":false,"prefix":"","firstName":"Ji-Hye","middleName":"","lastName":"Park","suffix":""},{"id":472801529,"identity":"6d50d86c-537a-458d-8543-3064e59c34c0","order_by":5,"name":"Soo Kyung Park","email":"","orcid":"","institution":"Korea Ginseng Corporation","correspondingAuthor":false,"prefix":"","firstName":"Soo","middleName":"Kyung","lastName":"Park","suffix":""},{"id":472801530,"identity":"61485b30-a9f3-49b5-9b57-b4acf669a4ee","order_by":6,"name":"Seung Ho Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYBAC9gYwZWMA5ScQ1sJzgBlEpZGu5TApWqT7D378UXHeWN79AOOHDwxp+YS1yBxmluY5c9vM8EwCs+QMhhzLBkJa7CWSGaQZ227bGM5gYGPmYagwIKSDgUcimfnnz3/nSNPCJsHbcMBMXgKsJYcILTKHzax5jiUbG/AkNkvOMEgjQot04+ObP2rsDOe3Hz744UNFMmEtDBJQ2uAAYwOQJKwBoUW+gRjVo2AUjIJRMCIBALAwMtO3LnGLAAAAAElFTkSuQmCC","orcid":"","institution":"Korea Ginseng Corporation","correspondingAuthor":true,"prefix":"","firstName":"Seung","middleName":"Ho","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2025-04-25 06:38:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6525946/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6525946/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-15863-3","type":"published","date":"2025-08-20T16:29:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85098486,"identity":"7f404607-451e-4719-8d90-3aa1d2830dc3","added_by":"auto","created_at":"2025-06-21 05:09:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":581929,"visible":true,"origin":"","legend":"\u003cp\u003eIdentified proteins and bioinformatic analysis in rat sera after Korean red ginseng (KRG) administration.\u003c/p\u003e\n\u003cp\u003eIdentified proteins and bioinformatic analysis in rat sera after Korean red ginseng (KRG) administration. (a) Subcellular localization; (b) molecular type of the identified proteins; and (c) selected downregulated cholesterol- and hyperlipidemia-related proteins by IPA (green and blue in (c) indicate a decreasing trend).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6525946/v1/7f72d38d485c055f0d0639ef.png"},{"id":85099110,"identity":"f61b4b98-4947-44f7-983b-c636d1c4f40d","added_by":"auto","created_at":"2025-06-21 05:33:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":932945,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Korean red ginseng (KRG) on the inhibition of PCSK9.\u003c/p\u003e\n\u003cp\u003eThe effect of Korean red ginseng (KRG) on the inhibition of PCSK9. Cell viability following KRG treatment was assessed using the EZ-Cytox assay (Fig. 1 (a)). HepG2 cells were treated with different concentrations of simvastatin, and PCSK9 protein expression was detected by Western blot (Fig. 1 (b)). PCSK9 expression inhibition by KRG was assessed via (c) immunohistochemistry (DAPI: blue; PCSK9: green) and (d) Western blot analysis. Scale bars; 25 µm. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e vs. Nor group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e vs. Con group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6525946/v1/22381ebcfcde2d82fcf55cb3.png"},{"id":85098491,"identity":"ab9022b5-b8cc-4f41-9b08-6409d1e1f016","added_by":"auto","created_at":"2025-06-21 05:09:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":310931,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of Korean red ginseng (KRG) on serum components in Triton WR-1339–induced hyperlipidemic model mice.\u003c/p\u003e\n\u003cp\u003eThe effects of Korean red ginseng (KRG) on serum components in Triton WR-1339–induced hyperlipidemic model mice. Serum component levels were evaluated by measuring (a) TG, (b) CHO, (c) HDL-C, (d) LDL-C, (e) PCSK9, and (f) the atherogenic index. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e vs. Nor group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e vs. Con group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6525946/v1/82aa31d7613c0eba4c47a894.png"},{"id":85098489,"identity":"f136eea0-ad8d-4183-9618-12290f56b014","added_by":"auto","created_at":"2025-06-21 05:09:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":209120,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of Korean red ginseng (KRG) on serum components in hyperlipidemic model rats fed a high-fat diet (HFD).\u003c/p\u003e\n\u003cp\u003eThe effects of Korean red ginseng (KRG) on serum components in hyperlipidemic model rats fed a high-fat diet (HFD). Serum components were evaluated by measuring (a) TG, (b) CHO, (c) HDL-C, (d) LDL-C, and (e) PCSK9. The atherosclerotic index (AI)(f) was calculated as AI = (TC –HDL-C) / HDL-C\u003csup\u003e21\u003c/sup\u003e. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. Nor group. #\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs. Con group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6525946/v1/bdc47b9bb8d0538b30e212d7.png"},{"id":85098488,"identity":"1ba2536e-6a15-42bd-b3d3-970464e03e63","added_by":"auto","created_at":"2025-06-21 05:09:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":296898,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of SREBP2, PCSK9, and LDLR proteins in the liver after Korean red ginseng (KRG) treatment in a high-fat diet (HFD) model.\u003c/p\u003e\n\u003cp\u003eThe expression of SREBP2, PCSK9, and LDLR proteins in the liver after Korean red ginseng (KRG) treatment in a high-fat diet (HFD) model. Protein expression levels were analyzed by quantification analysis, showing the expression levels of (a) SREBP2, (b) PCSK9, and (c) LDLR after KRG treatment in an HFD model. Western blot analysis (d) demonstrated that in the KRG group, PCSK9 and SREBP2 expression decreased, while LDLR expression significantly increased in the liver. Nor: normal; CON: control; KRG: Korean red ginseng. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e vs. Nor group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e vs. Con group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6525946/v1/f5ca59c50e14ab00f8ee6bda.png"},{"id":89847267,"identity":"9359060a-3e27-445d-8517-046235d710e6","added_by":"auto","created_at":"2025-08-25 16:42:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3611441,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6525946/v1/ae06ed0d-36ff-40ce-aae7-a8e6593a560a.pdf"},{"id":85099109,"identity":"06e9d8c0-0eac-44ab-9286-7ac82ad79de9","added_by":"auto","created_at":"2025-06-21 05:33:31","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":221964,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementFigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6525946/v1/079f4f925ec686e653dbd127.pdf"},{"id":85098485,"identity":"f5ef0a1b-e24f-49dc-ab26-80f635a442d7","added_by":"auto","created_at":"2025-06-21 05:09:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22977,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6525946/v1/ac03eb4f4de35ec2ec3c8dc3.docx"}],"financialInterests":"Competing interest reported. The authors declare the following competing interests: C.H.L., Y.Y.L., S.H.H., and S.H.L. have filed an institution-owned patent entitled “A Composition for Preventing or Treating Dyslipidemia with the Effect of Inhibiting PCSK9 Expression” (Patent No. 10-2023-0194713), related to the development of KRG for hyperlipidemia treatment. No other authors declare any competing interests.","formattedTitle":"Cholesterol-modulating effects of Korean red ginseng (KRG) targeting PCSK9 in hyperlipidemia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHyperlipidemia is a condition characterized by abnormally elevated levels of lipids or cholesterol in the blood caused by impaired lipid metabolism or function. It can result from dietary imbalances, obesity, genetic disorders, such as familial hypercholesterolemia, or other conditions like diabetes\u003csup\u003e1\u003c/sup\u003e. Hyperlipidemia is marked by high low-density lipoprotein cholesterol (LDL-C), low high-density lipoprotein cholesterol (HDL-C), and elevated triglyceride (TG) levels. It is one of the most common risk factors for cardiovascular diseases (CVD), including atherosclerosis, myocardial infarction, stroke, and coronary artery disease\u003csup\u003e2\u003c/sup\u003e. CVD remains the leading cause of mortality worldwide, with 20.5 million deaths attributed to CVD in 2021, accounting for approximately one-third of global deaths\u003csup\u003e3\u003c/sup\u003e.\u0026nbsp;Given the projected rise in CVD cases, there is growing interest in the early management of hyperlipidemia.\u003c/p\u003e\n\u003cp\u003eProprotein convertase subtilisin/kexin type 9 (PCSK9) is a key protein involved in hepatic cholesterol metabolism through its interaction with LDL receptors (LDLR)\u003csup\u003e4\u003c/sup\u003e.\u0026nbsp;LDL-C binds to LDLR and is subsequently transported to the liver, where LDLR is recycled to regulate blood cholesterol levels\u003csup\u003e5\u003c/sup\u003e.\u0026nbsp;However, under hyperlipidemic conditions, excessive PCSK9 binds to LDLR, leading to its degradation in the liver and a consequent increase in circulating cholesterol levels\u003csup\u003e6\u003c/sup\u003e.\u0026nbsp;Thus, PCSK9 serves as a critical target for lipid and cholesterol regulation. Statins, the first-line lipid-lowering agents for hyperlipidemic patients, paradoxically increase serum PCSK9 levels\u003csup\u003e7\u003c/sup\u003e.\u0026nbsp;Despite maximum doses and combination therapy with other lipid-lowering agents, some high-risk patients fail to achieve target cholesterol levels\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eIn addition, statins may be unsuitable for the treatment of severe hypercholesterolemia and can cause various muscle-related side effects\u003csup\u003e8\u003c/sup\u003e. Recently, PCSK9 inhibitors (evolocumab, alirocumab, and bococizumab) have been used for patients unresponsive to statins or experiencing adverse effects. These inhibitors can reduce LDL-C levels by up to 60% without inducing muscle pain or hepatic dysfunction, making them a crucial alternative for patients with statin tolerance\u003csup\u003e9\u003c/sup\u003e.\u0026nbsp;However, PCSK9 inhibitors are associated with high costs (ranging from USD 7,000 to USD 12,000 annually)\u003csup\u003e10\u003c/sup\u003e and require frequent injections, leading to local injection site reactions\u003csup\u003e11\u003c/sup\u003e. As a result, there is increasing interest in natural compounds that can inhibit PCSK9 in a cost-effective, side-effect\u0026ndash;free, and orally administrable manner.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePanax ginseng\u003c/em\u003e Meyer has been used for centuries in traditional medicine\u003csup\u003e12\u003c/sup\u003e for its diverse therapeutic properties, including cardiovascular benefits\u003ccite\u003e\u003csup\u003e13\u003c/sup\u003e\u003c/cite\u003e\u003ccite\u003e.\u0026nbsp;\u003c/cite\u003eKorean red ginseng (KRG), a steamed and dried form of ginseng, undergoes a transformation during processing that enhances its bioactive compounds, such as ginsenosides and polysaccharides\u003csup\u003e14,15\u003c/sup\u003e,\u0026nbsp;and extends its shelf life. Since the Goryeo Dynasty (734 AD), KRG has been widely traded with China and has gained popularity.\u003c/p\u003e\n\u003cp\u003eThe Ministry of Food and Drug Safety has recognized six functional benefits of KRG, including cognitive enhancement, blood sugar regulation, improved circulation, and reduced fatigue, with anti-diabetic effects added in 2024. Notably, KRG has been reported to reduce TG levels, enhance HDL-C levels, activate lipoprotein lipase (LPL), and improve fatty liver conditions\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e.\u0026nbsp;However, the precise mechanisms underlying KRG\u0026rsquo;s cholesterol- and TG-lowering effects remain unclear. Investigating the mechanisms of natural compounds such as KRG is challenging because of their complex composition and multiple biological targets.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWith advancements in proteomics, studies have utilized mass spectrometry\u0026ndash;based approaches to analyze multiple targets simultaneously and elucidate the efficacy of KRG. Previous studies have identified biosignatures related to immune function using proteomics-based analysis of KRG\u003csup\u003e19\u003c/sup\u003e. In this study, proteomics analysis was applied to identify biosignatures associated with KRG\u0026rsquo;s various known therapeutic effects, and it was found that KRG modulates PCSK9, a key biosignature for hyperlipidemia and lipid metabolism. Based on these findings, further animal studies were conducted to explore the role of KRG in lipid regulation and its potential contribution to CVD prevention.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e2.1. Identification of proteins in rats administered KRG\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA large-scale quantitative iTRAQ analysis was conducted to identify differentially expressed proteins (DEPs) to elucidate the molecular signatures underlying the effects of KRG. The analysis included control samples (vehicle, 114) and samples from rats administered KRG at doses of 500, 1,000, and 2,000 mg/kg (115, 116, and 117, respectively). A total of 587 unique proteins were identified in rat serum using MS analysis (protein probability: \u0026gt;99.0%; peptide probability: \u0026gt;95%) based on the target-decoy database (UniProt rat database).\u003c/p\u003e\n\u003cp\u003eAn ingenuity pathway analysis (IPA)\u0026ndash;based serum analysis was performed to determine the cellular distribution and functional classifications of the identified proteins. Most proteins were associated with the extracellular space (36%) and cytoplasm (27%), with a smaller proportion localized to the nucleus (13%) (Fig. 1). Molecular classification revealed that the identified proteins primarily consisted of undefined proteins (44%), enzymes (16%), and peptidases (10%) (Fig. 1).\u003c/p\u003e\n\u003cp\u003eNext, the quantitative values of the iTRAQ analysis were evaluated for DEPs (Supplemental Table X). From the pool of DEPs between normal and KRG-treated samples, proteins exhibiting consistent upregulation or downregulation trends were selected, with log2 fold changes greater than those specified in Tables 1 (a) and (b). Proteins were considered significant if they showed a consistent trend at least at two different KRG concentrations (500, 1,000, or 2,000 mg/kg), with statistical significance determined using a permutation test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), Mann\u0026ndash;Whitney test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), and |log\u003csub\u003e2\u003c/sub\u003e(\u003cem\u003ex\u003c/em\u003e)| \u0026gt; 0.2.\u003c/p\u003e\n\u003cp\u003eAn IPA was performed for biological functions to assess the biological relevance of the selected proteins. The top disease and biofunction analysis indicated that DEPs were primarily involved in CVD and lipid metabolism. Further functional analysis predicted a decrease in hyperlipidemia. Notably, PCSK9, APOB, and PLTP were consistently downregulated, as shown in Table 1 (b). In addition, machine learning\u0026ndash;based disease pathway analysis, which integrates known disease-associated proteins and infers novel participants\u003csup\u003e20\u003c/sup\u003e, predicted the downregulation of hypercholesterolemia and hyperlipidemia. Based on these findings, PCSK9 was identified as a potential biosignature molecule mediating the effects of KRG on dyslipidemia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Inhibitory effect of KRG on simvastatin-induced PCSK9 expression in HepG2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cytotoxicity of KRG in HepG2 cells was evaluated using the EZ-Cytox assay (Fig. 1 (a)). The data showed that KRG at concentrations of 50, 100, 200, and 400 \u0026micro;g/mL did not exhibit cytotoxic effects, indicating that KRG was not associated with cell death or inhibition of cell growth. Therefore, the highest non-cytotoxic dose was selected for subsequent experiments.\u003c/p\u003e\n\u003cp\u003eTo determine whether simvastatin affects PCSK9 expression, HepG2 cells were treated with increasing concentrations of simvastatin, and PCSK9 expression was analyzed by Western blotting (Fig. 1 (b)). The results confirmed that simvastatin increased PCSK9 expression in a dose-dependent manner (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Furthermore, in the presence of simvastatin-induced PCSK9 overexpression, treatment with KRG significantly suppressed the simvastatin-induced increase in PCSK9 levels, as demonstrated by both confocal microscopy and Western blot analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Hypolipidemic effect of KRG in a Triton WR-1339\u0026ndash;induced acute hyperlipidemia model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the in vitro results, the effects of KRG on lipid profiles and PCSK9 secretion in a Triton WR-133\u0026ndash;induced acute hyperlipidemia model were further investigated. Compared to the normal control group, mice administered Triton WR-1339 (400 mg/kg) exhibited significant increases in TGs (40-fold), total CHOs (1.5-fold), and LDL-C (2.4-fold), while HDL-C significantly decreased (0.72-fold) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) (Fig. 3).\u003c/p\u003e\n\u003cp\u003eAdministration of KRG at 200, 400, and 800 mg/kg (human equivalent doses: 1 g, 2 g, 4 g) significantly reduced TG, CHO, and LDL-C levels while increasing HDL-C levels (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). In addition, Triton WR-1339 increased PCSK9 secretion in the blood, whereas KRG significantly inhibited PCSK9 secretion (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The atherogenic index (AI) was also significantly reduced in the KRG-treated groups (200, 400, and 800 mg/kg) compared to the control group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall, these results suggest that KRG improves lipid profiles, suppresses PCSK9 secretion, and may prevent CVDs in an acute hyperlipidemia animal model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Hypolipidemic effect of KRG in an HFD-induced chronic hyperlipidemia model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, the effects of KRG on lipid profiles and PCSK9 expression/secretion in a chronic hyperlipidemia model induced by an eight-week high-fat diet (HFD) were evaluated. Compared to the normal control group, HFD-fed mice exhibited significant increases in TG (1.4-fold), CHO (1.45-fold), and LDL-C (2.08-fold) (\u003cem\u003ee\u003c/em\u003e \u0026lt; 0.05) (Fig. 4).\u003c/p\u003e\n\u003cp\u003eAdministration of KRG at 100, 200, and 400 mg/kg significantly reduced TG, CHO, and LDL-C levels (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Moreover, HFD-fed mice exhibited increased PCSK9 secretion in the blood, which was significantly suppressed by KRG treatment (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eThese findings confirm that KRG exerts lipid-lowering effects and inhibits PCSK9 secretion in both acute and chronic hyperlipidemia animal models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Involvement of the SREBP2/PCSK9/LDLR signaling pathway in the hypolipidemic effect of KRG\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot analysis was performed to determine whether the in vitro and in vivo blood analysis results were consistent at the tissue level. In the liver tissues of the chronic hyperlipidemia model induced by eight weeks of HFD, KRG reduced SREBP2 and PCSK9 expression in a dose-dependent manner while increasing LDLR expression compared to the control group (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eThese results suggest that the hypolipidemic effect of KRG is associated with the SREBP2/PCSK9/LDLR signaling pathway.\u003c/p\u003e\n"},{"header":"Discussion","content":"\u003cp\u003ePreviously, a proteomic analysis was conducted to evaluate the biological activities of KRG in immune-enhancing responses involving immune organs. Because natural products contain multiple components and target various pathways, identifying the key bioactive signatures of KRG was challenging. Our results revealed DEPs associated with immune response stimulation, demonstrating a molecular functional strategy of KRG\u003csup\u003e19\u003c/sup\u003e. In this study, proteomics was utilized to assess the biological effects of KRG in rat sera administered with the same concentration of KRG. As a result, a total of 587 unique proteins were identified. Among them, 21 proteins exhibited consistent expression trends based on the selection criteria. IPA revealed that the DEPs were primarily associated with CVD and lipid metabolism in the top-ranked diseases and biofunction analysis.\u003c/p\u003e\n\u003cp\u003eAmong these findings, the main focus is on hyperlipidemia, which was commonly observed across the analyses and predicted to be downregulated by KRG. Notably, PCSK9 and apolipoprotein B (APOB), both associated with hyperlipidemia, were downregulated (Fig. 1c). In particular, to the best of our knowledge, PCSK9 is the first identified biosignature candidate linked to the lipid-lowering effects of KRG in dyslipidemia. Although several studies have explored the efficacy of KRG\u003csup\u003e14\u0026ndash;18\u003c/sup\u003e, the pathway associated with PCSK9 has not been thoroughly investigated. Based on the proteomic results, further investigations were conducted to confirm the type of dyslipidemia affected by KRG and the role of PCSK9.\u003c/p\u003e\n\u003cp\u003eHyperlipidemia is one of the most common disorders characterized by abnormal blood lipid levels and is a major risk factor for CVD\u003csup\u003e22\u003c/sup\u003e. Previous studies have shown that KRG reduces blood TG and CHO levels by inhibiting pancreatic lipase\u003csup\u003e23\u003c/sup\u003e and HMG-CoA reductase activity in HFD-fed animals\u003csup\u003e16\u003c/sup\u003e. In addition, KRG has been found to prevent lipid accumulation in the liver by improving lipid metabolism\u003csup\u003e16\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn patients with myocardial infarction, KRG was shown to enhance coronary blood flow\u003csup\u003e24\u003c/sup\u003e,\u0026nbsp;alleviate increased platelet aggregation in hypercholesterolemic conditions, and reduce the incidence of atherosclerosis\u003csup\u003e25\u003c/sup\u003e.\u0026nbsp;These findings suggest that KRG may contribute to reducing CVD risk by improving lipid metabolism; however, no studies have provided a clear mechanistic explanation for this effect.\u003c/p\u003e\n\u003cp\u003eTo investigate the PCSK9-inhibitory effect of KRG, immortalized hepatocytes (HepG2), which are known to express and secrete PCSK9, were utilized\u003csup\u003e26\u003c/sup\u003e.\u0026nbsp;In hyperlipidemic animal models, oral administration of KRG at doses of 200, 400, and 800 mg/kg/day (HED = 0.9, 1.8, and 3.6 g/day\u003csup\u003e27\u003c/sup\u003e, Fig. 2) significantly reduced TG, total cholesterol (TC), and LDL-C levels in a dose-dependent manner while increasing high-density lipoprotein cholesterol (HDL-C) levels (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Furthermore, liver tissue and blood analysis demonstrated that KRG inhibited PCSK9 expression while upregulating LDLR, which regulates cholesterol metabolism and transport. This effect of KRG is associated with the inhibition of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase\u003csup\u003e28\u003c/sup\u003e. In addition, the major ginsenosides in KRG, including Rb1, Rg1, Rh2, Rb2, and CK, have been reported to suppress TG and CHO synthesis, thereby alleviating hyperlipidemia and exerting beneficial effects against CVD\u003csup\u003e29\u003c/sup\u003e, consistent with our findings.\u003c/p\u003e\n\u003cp\u003eStatins, the primary treatment for hyperlipidemia, target the mevalonate (MVA) pathway by inhibiting HMG-CoA reductase (HMGCR)\u003csup\u003e30\u003c/sup\u003e. However, recent studies indicate that statins induce SREBP2 activation, which enhances the transcription of PCSK9 by binding to its proximal promoter containing the SRE motif, leading to an increase in PCSK9 levels\u003csup\u003e31\u003c/sup\u003e. Hyperlipidemia is also accompanied by elevated PCSK9 expression\u003csup\u003e32\u003c/sup\u003e, which may explain why some patients fail to achieve adequate LDL-C reduction with statin therapy. Consequently, PCSK9 inhibitors have emerged as a key therapeutic target for hyperlipidemia and CVD treatment\u003csup\u003e33\u003c/sup\u003e. PCSK9 promotes the degradation of LDLR in hepatocytes, leading to increased circulating LDL-C levels and the maintenance of high LDL-C concentrations\u003csup\u003e6\u003c/sup\u003e. Unlike statins, our findings demonstrate that KRG suppresses the SREBP2/PCSK9 signaling pathway.\u003c/p\u003e\n\u003cp\u003eElevated TG and CHO levels in hyperlipidemia increase the risk of atherosclerosis, myocardial infarction, and stroke by promoting lipid deposition in blood vessel walls, making them critical markers for clinical diagnosis. However, long-term statin use can cause various adverse effects, and elevated PCSK9 levels accelerate LDLR degradation, thereby contributing to the progression of CVD. This presents a major challenge in current therapeutic strategies\u003csup\u003e34\u003c/sup\u003e.\u0026nbsp;The development of safe and effective agents that suppress PCSK9 expression in hepatocytes could help prevent secondary complications associated with hyperlipidemia. Our study provides evidence that KRG may serve as a promising candidate for hyperlipidemia treatment, ultimately reducing the risk of CVD.\u003c/p\u003e\n\u003cp\u003eThis study demonstrates that KRG exerts lipid-lowering effects in hepatocytes and hyperlipidemic animal models. This effect is associated with the protection of LDLR and enhanced LDL uptake activity via the SREBP2/PCSK9 signaling pathway in liver tissue. Given its long-term safety profile, KRG may serve as a functional ingredient for hyperlipidemia prevention and could be used either alone or in combination with statins for therapeutic purposes. However, further studies are required to validate the clinical effects of KRG\u0026rsquo;s PCSK9-inhibitory properties.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e4.1. Global proteomic profiling analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.1.1. Preparation of the KRG water extract and general chemicals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe extraction procedure for KRG followed the international standard production process (ISO 19610). The six-year-old \u003cem\u003eP. ginseng\u003c/em\u003e root extract (body 75% and root 25%) was prepared through a repeated steaming and drying process by the Korea Ginseng Corporation (Daejeon, Republic of Korea). The extract was freeze-dried, yielding a dark-brown powder (KRG). Ammonium bicarbonate, dithiothreitol (DTT), formic acid (FA), trifluoroacetic acid, ammonium formate, and urea were purchased from Sigma-Aldrich (St Louis, MO, USA). The HPLC-grade acetonitrile (ACN) and water were purchased from JT Baker (Phillipsburg, NJ, USA). Lyophilized trypsin/lys-C were obtained from Promega (Madison, WI, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.1.2. Animal model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiments were performed on adult male Sprague Dawley (SD) rats (180\u0026ndash;200 g) obtained from Daehan Biolink (DBL, Seoul, Korea) and housed under controlled environmental conditions (22\u0026deg;C\u0026ndash;24\u0026deg;C, 12-hour light/dark cycles) for one week prior to the start of the study. The procedures were approved by the Institutional Animal Care and Use Committee of the Korean Ginseng Research Institute (Daejeon, Republic of Korea), in accordance with the Guide for the Care and Use of Laboratory Animals (Approval No. KGC-2015-008). All experiments were carried out using the same method as Lee et al.\u003csup\u003e19\u003c/sup\u003e Briefly, eight-week-old male rats were randomly divided into groups of six rats each. The rats were administered KRG at doses of 0, 500, 1,000, and 2,000 mg/kg for six weeks. Only a vehicle was used for the negative control group (0 mg/kg). Before necropsy, all rats were fasted overnight and euthanized by exsanguination under isoflurane. Blood samples were drawn from the inferior vena cava. After blood collection, serum was obtained in ethylenediaminetetraacetic acid (EDTA)\u0026ndash;free vacutainer by centrifugation at 3,000 rpm for 10 min. The remaining serum was stored at\u0026nbsp;\u0026minus;70\u0026deg;C until the global proteomic profiling analysis was performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.1.3. Serum depletion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum samples were thawed on ice and centrifuged at 3,000\u0026times; g for 5 min at 4\u0026deg;C. The centrifuged serum samples were pooled in equal volumes. After mixing with buffer A (Agilent, Cat# 5185\u0026ndash;5987), the mixed samples were filtered through 0.22-mm Spin-X filters. The mixture was loaded onto the MARS-MS3 column (Agilent Technologies, Wilmington, DE, USA) on a Dionex UltiMate 3000 UHPLC+ system (Thermo Scientific, Germering, Germany). The depleted serum was buffer-exchanged into 50 mM Tris-HCl (pH 8.0) and concentrated through ultrafiltration using the Amicon Ultra-0.5 mL 3 kDa cutoff filter (Millipore, Darmstadt, Germany). The concentration of the collected flow-through sample was determined using a micro bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, IL, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.1.4. Protein digestion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach depleted serum sample was modified as previously described\u003csup\u003e19\u003c/sup\u003e. Briefly, the proteins in the depleted serum were denaturized with 6 M urea in 10 mM ammonium bicarbonate (ABC) for 2 hr at 37\u0026deg;C. The sample was then treated with 10 mM dithiothreitol (DTT) for 1 hr at 70\u0026deg;C and alkylated with iodoacetamide (IAA) for 30 min at room temperature in the dark. The sample was diluted with 50 mM ABC to achieve a final concentration of 1 M urea. After the alkylation step, Lys-C/Trypsin solution was added at 37\u0026deg;C for overnight incubation (1:30; Promega). Trypsin digestion was performed at 37\u0026deg;C for an additional overnight incubation (1:100). The peptide sample was desalted using a Macro SpinColumn (C-18; Harvard Apparatus, Holliston, MA, USA) according to the manufacturer\u0026rsquo;s instructions. After sample drying, peptide quantification was performed using BCA before the iTRAQ labeling. iTRAQ labeling and high pH reversed-phase fractionation steps were performed according to the previously described method\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.1.5.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eLC-MS\u003csup\u003e2\u003c/sup\u003e experiment and bioinformatic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe peptide samples were analyzed using Q-Exactive mass spectrometry (Thermo Fisher Scientific, Bremen, Germany) equipped with a nano-UHPLC Dionex system (Thermo Scientific) using an Easy nanospray source. For the quantitative analysis of iTRAQ-labeled datasets, the SEQUEST algorithm (Thermo Fisher Scientific) was used against the decoy UniProt database. Scaffold Q+ was also used to calculate the quantification values for each protein. Based on the data, the biological functions were evaluated using IPA (IPA; Ingenuity Systems Analysis; Redwood City, CA, USA) and DAVID (https://david-d.ncifcrf.gov/). All experimental conditions were the same as previously described\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2. Pharmacology study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.1. Preparation of KRG (G1899)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKRG was manufactured from the roots of six-year-old fresh ginseng, \u003cem\u003eP. ginseng\u003c/em\u003e C. A. Meyer, harvested in the Republic of Korea by the Korea Ginseng Corporation (Daejeon, Republic of Korea). The preparation process involved steaming the fresh ginseng roots at 90\u0026deg;C\u0026ndash;100\u0026deg;C for 3 hr and then drying at 50\u0026deg;C\u0026ndash;80\u0026deg;C. KRG was derived from a KRG water extract, which was produced through three eight-hour cycles of circulating hot water (85\u0026deg;C\u0026ndash;90\u0026deg;C). The KRG extract underwent a series of processes, including water extraction, concentration, filtration, solution preparation, spray drying, sieving, and other techniques, resulting in a yield of 50%.\u0026nbsp;The standardized, productized form of this extract is known as G1899.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2. Cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman hepatoblastoma HepG2 cells were obtained from the American Type Culture Collection and cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin solution. All cells were incubated in a cell culture chamber at 37\u0026deg;C under a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3. Cytotoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell viability was examined using the EZ-Cytox assay kit (Dogen, Suwon, Korea). To determine the nontoxic concentration of KRG, the following concentrations were added to each well (2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well): 50, 100, 200, and 400 \u0026micro;g/mL. The plates were incubated for 24 hr. After incubation, EZ-Cytox (10 \u0026micro;l/well) was added to each well, and the cells were cultured for an additional 2 hr. Finally, a microplate reader was used to measure the absorbance at 450 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.4. Simvastatin-mediated PCSK9 expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHepG2 cells were seeded in collagen-coated six-well plates (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well). After a 24-hr incubation, the cells were pretreated with various concentrations of KRG (100, 200, 400 \u0026mu;g/mL) for 1 hr and stimulated with simvastatin (3 nM/mL) for 24 hr. Following treatment, Western blot analysis was performed to quantify the PCSK9 protein levels in each cell lysate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.5. Western blotting analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total protein from cells and liver tissue was harvested and lysed using 1X RIPA buffer containing a mixture of protease and phosphatase inhibitors (MedChemExpress, Monmouth Junction, NJ, USA). The insoluble debris was removed via centrifugation at 12,000\u0026times; g and 4\u0026deg;C for 30 min, and the total protein concentration of the isolated supernatants was quantified using the Pierce\u003csup\u003eTM\u003c/sup\u003e BCA Protein Assay Kit (Thermo Fisher Scientific). A total of 10 \u0026micro;g of each protein was resolved by SDS\u0026ndash;PAGE (8%\u0026ndash;12% acrylamide) and then transferred onto PVDF membranes. The membranes were blocked for 2 hr at room temperature with a 5% skim milk solution. Then, the membranes were incubated overnight at 4℃ with primary antibodies specific to PCSK9, SREBP2, LDLR, or actin. The blots were washed three times for 10 min each with Tris-buffered saline with Tween-20 (TBST). The membranes were then incubated for 2 hr with a secondary antibody. ECL Western blotting detection reagents were used to detect the proteins using the Fusion FX6.0 system (Vilber, Coll\u0026eacute;gien, France). The membranes were stripped and re-probed with an anti-\u0026beta;-actin monoclonal antibody to ensure equal protein loading. Protein bands were quantified using ImageJ 1.52a software (NIH, Bethesda, MD, USA) and normalized to \u0026beta;-actin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.6. Immunocytochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor immunocytochemistry, HepG2 cells were cultured with drugs at a concentration of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL in 48-well tissue culture plates. Cells were also fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 10 min. After blocking with 10% BSA for 1 hr, the plate was incubated with primary antibodies diluted in 10% BSA for 4 hr. Subsequently, the plate was washed three times with TBST and incubated with Alexa Fluor 488\u0026ndash;conjugated secondary antibodies for 1 hr. After washing with TBST, the plate was stained with DAPI for 10 min, followed by thorough washing. Finally, the plate was analyzed using LSM 900 with Airyscan 2 confocal microscopes (Carl Zeiss, Oberkochen, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.7. Acute hyperlipidemia model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed on adult male ICR mice (22 \u0026plusmn; 2 g) obtained from Daehan Biolink (DBL, Seoul, Korea) and kept under controlled environmental conditions (22\u0026deg;C\u0026ndash;24\u0026deg;C and 12 hr light/dark cycles) for one week before the start of the experiments. All the mice were randomly divided into six groups when experiments began: normal control group, model group, and KRG treatment groups (100, 200, 400, and 800 mg/kg). After administering the mice for seven days and fasting them overnight, Triton WR-1339 (400 mg/kg B.W) was injected intraperitoneally. At 24 hr after the Triton WR-1339 injection, blood and liver were collected directly from the anesthetized animals. All experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the Korean Ginseng Research Institute (Daejeon, Republic of Korea) in accordance with the Guide for the Care and Use of Laboratory Animals (Approval No. KGC-2023-014).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.8. Chronic hyperlipidemia model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed on adult male Sprague Dawley (SD) rats (180\u0026ndash;200 g) obtained from Daehan Biolink (DBL, Seoul, Korea) and kept under controlled environmental conditions (22\u0026deg;C\u0026ndash;24\u0026deg;C and 12 hr light/dark cycles) for one week before the start of the experiments. After acclimatization for one week, the rats were randomly assigned to five groups (n = 10 per group) as follows: (i) normal diet, (ii) HFD, (iii) HFD + KRG 50 mg/kg, (iv) HFD + KRG 100 mg/kg, and (v) HFD + KRG 200 mg/kg. The rats in the normal diet group were fed a normal diet (No. Teklad Global Diets 2918, Envigo), while the rats in the other four groups were fed an HFD (No. D12452, Research Diets, Inc.). The rats were fed for eight weeks and were allowed free access to food and water throughout the entire study period. KRG, dissolved in 0.9% saline, was administered daily by oral gavage for eight weeks. The rats in the Nor and HFD groups received an equal volume of saline by oral gavage. At eight weeks, blood and liver were collected directly from the anesthetized animals. All experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the Korean Ginseng Research Institute (Daejeon, Republic of Korea) in accordance with the Guide for the Care and Use of Laboratory Animals (Approval No. KGC-2023-020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.9. Biochemical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples were separated by centrifugation at 3,000\u0026times; rpm for 15\u0026thinsp;min at 4\u0026deg;C. The blood tests for T-CHO, TG, HDL-C, and LDL-C were measured using a Hitachi 7100 Automatic Analyzer (Hitachi, Tokyo, Japan), while serum PCSK9 levels were measured using ELISA kits (Abcam, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.10.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical comparisons between multiple groups were compared using one-way ANOVA with the Tukey HSD post-hoc test. Differences between groups were considered significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and all values are expressed as mean\u0026thinsp;\u0026plusmn; standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA).\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm their contributions to this paper as follows: study conception and design: C.H.L, Y.Y.L, S.H.H, S.H.L. experimental execution, data collection, interpretation, and analysis: C.H.L, Y.Y.L, S.H.H, J.H.L, J.H.P, S.K.P and S.H.L. draft manuscript preparation, data analysis, and resources: C.H.L. and Y.Y.L. All authors discussed the results and commented on the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Korea Ginseng Corporation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare the following competing interests: C.H.L., Y.Y.L., S.H.H., and S.H.L. have filed an institution-owned patent entitled \u0026ldquo;A Composition for Preventing or Treating Dyslipidemia with the Effect of Inhibiting PCSK9 Expression\u0026rdquo; (Patent No. 10-2023-0194713), related to the development of KRG for hyperlipidemia treatment. No other authors declare any competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Ginseng Corporation (Approval Nos. KGC-2015-008, KGC-2023-014, and KGC-2023-020), and the experiments were conducted in accordance with institutional guidelines and the principles outlined in the ARRIVE 2.0 guidelines to ensure transparency, reproducibility, and adherence to ethical standards in animal research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEzeh, K. J. \u0026amp; Ezeudemba, O. Hyperlipidemia: A review of the novel methods for the management of lipids. \u003cem\u003eCureus\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e16412, https://doi.org/10.7759/cureus.16412 (2021).\u003c/li\u003e\n\u003cli\u003eNelson, R. H. Hyperlipidemia as a risk factor for cardiovascular disease. \u003cem\u003ePrim. Care\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 195\u0026ndash;211, https://doi.org/10.1016/j.pop.2012.11.003 (2013).\u003c/li\u003e\n\u003cli\u003eLindstrom, M.\u003cem\u003e \u003c/em\u003eet al. 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Pract.\u003c/em\u003e \u003cstrong\u003e2017\u003c/strong\u003e, e201702, https://doi.org/10.21542/gcsp.2017.2 (2017).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"},{"header":"Supplemental Table X","content":"\u003cp\u003eSupplemental Table X is not available with this version\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Korean red ginseng, hyperlipidemia, SREBP2, PCSK9, LDLR, proteomics","lastPublishedDoi":"10.21203/rs.3.rs-6525946/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6525946/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Hyperlipidemia is a major global health concern, closely linked to cardiovascular disease (CVD) and metabolic syndrome. Effective regulation of blood lipid and cholesterol levels is essential for preventing and managing this condition. Korean red ginseng (KRG), a traditional medicinal plant, possesses diverse pharmacological properties, including anti-hyperlipidemic, immune-enhancing, anti-fatigue, and antistress effects. While previous studies suggest that KRG reduces lipid levels and may lower the risk of hyperlipidemia and CVD, its precise molecular mechanisms remain unclear. In this study, proteomic analysis revealed that KRG modulates proprotein convertase subtilisin/kexin type 9 (PCSK9) in the blood of rats administered with KRG. In hyperlipidemic animal models induced by Triton WR-1339 and a high-fat diet (HFD), KRG significantly reduced total cholesterol (TCHO), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) levels. Furthermore, KRG regulated the expression of PCSK9 and low-density lipoprotein receptor (LDLR)—key regulators of LDL metabolism—in liver tissues. These findings indicate that KRG exerts lipid-lowering effects by modulating PCSK9 and LDLR expression, regulating cholesterol metabolism through the SREBP2/PCSK9/LDLR signaling pathway. This study highlights KRG’s potential as a novel therapeutic agent for preventing and managing hyperlipidemia and CVD.","manuscriptTitle":"Cholesterol-modulating effects of Korean red ginseng (KRG) targeting PCSK9 in hyperlipidemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-21 05:09:27","doi":"10.21203/rs.3.rs-6525946/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-01T08:11:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-24T07:55:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168537962403404036968011171489603587906","date":"2025-06-19T03:40:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285096422430705469849427310761462801936","date":"2025-06-18T01:34:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-17T02:43:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-17T02:42:23+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-10T03:55:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-25T09:14:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-25T09:13:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fc34bdac-9a54-4361-ae31-c3dd4f0747bf","owner":[],"postedDate":"June 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50210492,"name":"Health sciences/Diseases/Cardiovascular diseases/Dyslipidaemias"},{"id":50210493,"name":"Biological sciences/Biological techniques/Proteomic analysis"},{"id":50210494,"name":"Health sciences/Medical research/Drug development"},{"id":50210495,"name":"Health sciences/Medical research/Experimental models of disease"}],"tags":[],"updatedAt":"2025-08-25T16:35:46+00:00","versionOfRecord":{"articleIdentity":"rs-6525946","link":"https://doi.org/10.1038/s41598-025-15863-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-20 16:29:29","publishedOnDateReadable":"August 20th, 2025"},"versionCreatedAt":"2025-06-21 05:09:27","video":"","vorDoi":"10.1038/s41598-025-15863-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-15863-3","workflowStages":[]},"version":"v1","identity":"rs-6525946","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6525946","identity":"rs-6525946","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}