Between January 2018 and December 2018, 494 patients with or without hypercholesterolemia were enrolled at Anzhen Hospital, Beijing, China. Inclusion criteria were age >18 years, diagnosis of hypercholesterolemia, and use of standard lipid-lowering therapy. Exclusion criteria included any current or prior conditions affecting other bodily systems, such as familial hypercholesterolemia, severe cardiovascular and cerebrovascular diseases (e.g., myocardial infarction, stroke, and heart failure), respiratory diseases, infectious diseases, chronic kidney disease, pregnancy, and malignancies. The diagnostic criteria for hypertension were an average systolic blood pressure (SBP) ≥140 mmHg or diastolic blood pressure (DBP) ≥90 mmHg (measured three times at 5-min intervals), or 24-h SBP ≥130 mmHg or 24-h DBP ≥80 mmHg, or a history of hypertension with treatment using antihypertensive medications (12). As per the American Diabetes Association, the diagnostic criteria for diabetes included symptoms of diabetes (polyuria, polydipsia, and unexplained weight loss) and random blood glucose concentrations ≥200 mg/dL (11.1 mmol/L) from two samples, fasting glucose ≥126 mg/dL (7.0 mmol/L), or glucose concentrations ≥200 mg/dL (11.1 mmol/L) 2 h after a 75-g oral glucose tolerance test (13). According to the Chinese Guideline for Lipid Management (primary care version 2024), the diagnostic criteria for hypercholesterolemia were plasma total cholesterol (TC) ≥5.2 mmol/L or LDL-C ≥3.4 mmol/L. All participants signed written informed consent. This study adhered to the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Anzhen Hospital, Capital Medical University.

Demographic characteristics, including age, sex, body mass index (BMI), and use of lipid-lowering therapy, were recorded for each participant. Fasting blood samples were collected, and plasma TC, triglyceride (TG), LDL-C, and high-density lipoprotein cholesterol (HDL-C) concentrations were measured using an automated biochemical analyzer (AU5400, Beckman Coulter, Brea, CA, United States).

A 100-μL aliquot of plasma was mixed with 400 μL of ice-cold chloroform-methanol (2:1, v/v) containing stable isotope-labeled internal standard L-carnitine-d3. The mixture was vortexed for 5 min at 4°C and centrifuged at 15,000 rpm for 15 min at 4°C. After protein precipitation, the lower organic phase was collected in a clean, dry tube and evaporated to dryness. The dried residue was stored at −80°C until further analysis. Each plasma sample was characterized for lipidomic profiling. Briefly, the residue was reconstituted in 200 μL of chloroform:methanol (1:1, v/v) and diluted threefold with isopropanol:acetonitrile:water (2:1:1, v/v/v). Analysis was performed using a reverse-phase X-select CSH C18 column (2.1 × 100 mm, 2.5 μm; Waters Corp., Milford, MA, United States). The mobile phase consisted of a linear gradient system of phase A (60% acetonitrile/40% water solution containing 10 mM ammonium acetate and 0.1% formic acid) and phase B (isopropanol:acetonitrile, 9:1, v/v): 0 min 60% A, 2 min 57% A, 12 min 40% A, 12.1 min 25% A, 18 min 1% A, 19 min 1% A, and 20 min 40% A. The injection volume was 10 μL. The column temperature was maintained at 40°C at a flow rate of 0.4 mL/min. Mass spectrometry analysis was performed on a Q-Exactive HF MS (Thermo Fisher Scientific, Waltham, MA, United States) using a data-dependent acquisition model. The spray voltage was set to 3.3 kV for the positive ion mode and 2.5 kV for the negative ion mode. Each acquisition cycle included one survey scan from 150 to 1,200 m/z at a resolution of 60,000, followed by 10 MS/MS scans at a resolution of 15,000, using stepped normalized collision energy values of 15, 30, and 45. Dynamic exclusion was set to 10 s, with a sheath gas flow rate of 40 L/h and an auxiliary gas flow rate of 10 L/h. The probe heater temperature and capillary temperature were set at 300°C and 320°C, respectively.

All animal care and use procedures were approved by the Institutional Animal Care and Use Committee of Capital Medical University and were conducted in a specific-pathogen-free facility at Anzhen Hospital, Capital Medical University, Beijing, China. Eight-week-old male C57BL/6J mice and LDLR^−/− global knockout mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). C57BL/6J mice were housed at 27°C in a controlled environment with a 12-h/12-h light/dark cycle, with free access to water and a high-cholesterol diet (HCD; Research Diets, D12108 C, 1.25% cholesterol) or an ALC-supplemented diet (300 mg/kg, Changzhou Shuiyushu’er Company, Changzhou, China) for 4 weeks (n = 6 mice per group). LDLR^−/− global knockout mice were housed in the same environment as C57BL/6J mice, with free access to water and a high-cholesterol diet (HCD; Research Diets, D12108 C, 1.25% cholesterol) or an ALC-supplemented diet (600 mg/kg, Changzhou Shuiyushu’er Company, Changzhou, China) for 8 weeks (Control group, n = 4; Experimental group, n = 10). Food intake was recorded during this period. At the end of this phase, all mice were euthanized, and body and liver weights were recorded. Tissues were dissected, cut into small pieces, and stored at −80°C.

After fasting for 4 h, blood was collected to extract serum, and serum TC and TG concentrations were measured using kits provided by Nanjing Jiancheng Bioengineering Institute, Nanjing, China. Lipids were extracted from 20–30 mg of frozen liver samples, then 0.18–0.27 mL of anhydrous ethanol was added (volume ratio 9:1), and the tissue was homogenized at 4°C, 2,500 rpm for 10 min. The supernatant was collected, and TC and TG contents were determined using enzymatic assays from Nanjing Jiancheng Bioengineering Institute.

Plasma lipoprotein profiles were analyzed using fast protein liquid chromatography (FPLC). Plasma samples were collected from mice after a 12-h fasting period. Equal volumes of plasma from individual mice within each experimental group were pooled to obtain sufficient sample volumes. The pooled plasma (200 μL) was filtered through a 0.22-μm syringe filter to remove particulates. Samples were loaded onto a Superose 6 10/300 GL column (GE HealthCare) pre-equilibrated with phosphate-buffered saline (PBS) containing 0.15 M NaCl and 0.02% sodium azide. The column was connected to an ÄKTA FPLC system (GE HealthCare) and operated at a flow rate of 0.5 mL/min at room temperature. Fractions were collected every minute for 60 min. The cholesterol content in each fraction was determined using an enzymatic cholesterol assay kit (ab65390, Abcam) per the manufacturer’s instructions. The absorbance was measured at 570 nm using a microplate reader (Model 680, Bio-Rad). Lipoprotein fractions were identified based on the elution profile: very-low-density lipoprotein (VLDL) eluted in fractions 10–20, LDL in fractions 21–30, and HDL in fractions 31–50.

For en face analysis of atherosclerotic lesions, the entire aorta was excised and stained with oil red O, and the lesion area was quantified using Fuji ImageJ software. Hearts were fixed in 4% paraformaldehyde and embedded in paraffin or OCT for histological analysis. Five-micron sections of the heart valve region were cut and stained with hematoxylin and eosin (H&E), and the atherosclerotic lesions were analyzed using Fuji ImageJ software across six consecutive sections from four to six littermates per group.

Liver samples were fixed in 4% paraformaldehyde for ≥24 h, rapidly frozen, and embedded in OCT compound. Eight-micron frozen sections were prepared and stained with H&E and oil red O.

Huh-7 cells were obtained from the National Collection of Authenticated Cell Cultures and cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, United States) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 μg/mL penicillin, and 100 μg/mL streptomycin in a 37°C incubator with 5% CO₂.

Protein samples were extracted from mouse liver tissue lysates and Huh-7 cells. Twenty milligrams of liver tissue samples were lysed in RIPA buffer (Solarbio, Beijing, China) using ultrasonic disruption. After centrifugation at 12,000 rpm for 20 min, the supernatant was collected for protein quantification via western blot analysis. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat milk and incubated sequentially with the appropriate primary and secondary antibodies. Supplementary Table S1 lists the antibodies used.

Real-time PCR was performed to quantify specific mRNA. RNA was isolated from liver and cell samples using TRIzol reagent (Life Technologies, Thermo Fisher Scientific, Waltham, MA, United States) and converted to cDNA using a reverse transcription kit from Thermo Fisher Scientific. RT-PCR was conducted using the SYBR Green method on a real-time PCR system. The relative abundance of each mRNA species was evaluated using the 2 − Δ Δ C T method. Supplementary Figure S1 lists the primers used.

Cells were seeded in 6-well chamber slides and incubated in complete DMEM for 24 h, then treated for another 24 h. Cells were fixed with 4% paraformaldehyde for 30 min and washed with non-permeabilizing or permeabilizing PBS containing 0.025% Triton-X. Cells were then blocked with 1% bovine serum albumin for 30 min and stained with anti-GFP antibody in PBS containing 1% bovine serum albumin for 1 h. After washing, cells were incubated with FITC-labeled goat anti-rabbit IgG (H + L) (Beyotime, Shanghai, China) and DAPI for nuclear staining. Cells were subsequently mounted with PermaFluor and visualized using an inverted fluorescence microscope (Nikon Eclipse Ti) at 20× or 40× magnification.

Huh-7 cells were seeded in black transparent-bottom 96-well plates for 24 h and treated with experimental reagents for an additional 24 h. During the last 5 h of treatment (19–24 h), cells were exposed to 3,3’-dioctadecylindocarbocyanine-labeled low-density lipoprotein (Dil-LDL, 10 μg/mL), then washed twice with pre-warmed (37°C) HBSS containing 20 mM HEPES before analysis. The intracellular fluorescence intensity of the Dil was quantified using the PerkinElmer Operetta CLS imaging system.

Statistical analyses were performed using SPSS (version 26; IBM Corp., Armonk, NY, USA). Measurement data conforming to a normal distribution were expressed as mean ± standard deviation, while data not conforming to a normal distribution were expressed as median (interquartile range). For data conforming to a normal distribution, comparisons between two groups were conducted using the independent samples t-test, and comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). For data not conforming to a normal distribution, comparisons between two groups were conducted using the Mann–Whitney U test, and comparisons among multiple groups were performed using the Kruskal–Wallis (H) test. Categorical data were described using frequencies and percentages, and differences between groups were analyzed using the chi-square test or Fisher’s exact test. A two-sided p-value <0.05 was considered statistically significant.

In total, 494 participants (mean age: 43 years, 71.05% male) were enrolled in this study (Table 1). The mean BMI of the participants was 28.0 (25.6, 31.0) kg/m². The prevalence of hypertension was low (16.80%), as were the historical incidences of cardiovascular disease (9.51%) and prior lipid-lowering therapy (14.37%). The lipid profiles were favorable, with levels of LDL-C at 3.03 mmol/L, HDL-C at 1.15 mmol/L, TC at 4.94 mmol/L, and TGs at 1.67 mmol/L. However, the prevalence of diabetes mellitus (fasting blood glucose concentration of ≥126 mg/dL or 7.0 mmol/L) was relatively high (27.53%). Given these baseline characteristics, we investigated potential metabolic factors influencing lipid levels in this population.

Metabolomic qualitative and quantitative assessments of the patients’ plasma and clinical characteristics revealed that plasma ALC was significantly downregulated. Individuals with hypercholesterolemia exhibited approximately 42% lower plasma ALC levels than those of the general population (Figure 1A). ALC was significantly negatively correlated with TC (r = −0.43, p < 0.0001; Figure 1B) and LDL-C (r = −0.53, p < 0.0001; Figure 1C) but not significantly correlated with TGs (r = −0.0088, p < 0.0001; Figure 1D) or HDL-C (r = −0.0081, p < 0.0001; Figure 1E). These findings suggest a potential role of ALC in modulating cholesterol levels. To further explore this relationship, we conducted animal studies.

Building upon the clinical observations, among the differential plasma metabolites of patients with hyperlipidemia, ALC was significantly downregulated and negatively correlated with plasma LDL-C. To investigate whether ALC could reduce plasma lipid levels in hyperlipidemic mice, we administered ALC to C57BL/6J mice to observe its effect on plasma lipids. ALC was incorporated into D12108 feed containing 1.25% cholesterol to produce an HCD with an ALC concentration of 300 mg/kg. This diet was provided to 8-week-old male C57BL/6J mice (the HCD + ALC group) for 4 weeks (Figure 2A). A control group received the HCD without ALC supplementation. Compared with the HCD group, the HCD + ALC group showed no significant changes in body weight or food intake (Figures 2B,C), indicating that ALC did not affect overall health status. FPLC analysis revealed that plasma LDL-C in the HCD + ALC group exhibited a decreasing trend, whereas HDL-C showed an increasing trend (Figure 2D). TC and TG levels in the mouse plasma and livers were measured using enzymatic assays. Compared with the HCD group, the HCD + ALC group had significantly reduced plasma TC and TG levels (Figures 2E,F). However, hepatic TC and TG levels did not significantly differ (Figures 2G,H). These results indicate that ALC effectively reduced plasma lipid levels without altering hepatic lipid storage.

To clarify the mechanism by which ALC affects plasma lipid levels in mice, we analyzed hepatic gene and protein expression related to cholesterol metabolism. Hepatic protein and mRNA levels were assessed using western blot analysis (Figure 3A) and qRT-PCR (Figure 3H), respectively. Compared with the HCD group, the HCD + ALC group exhibited significantly decreased hepatic protein levels of SREBP2 (Figure 3B), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR; Figure 3C), and APOB (Figure 3E) and significantly increased levels of ABCG5 (Figure 3F), ABCG8 (Figure 3G), and low-density lipoprotein receptor (LDLR; Figure 3D). Furthermore, compared with the HCD group, the HCD + ALC group showed significantly decreased Srebp2, Hmgcr, Ldlr, Apob, and Abcg8 mRNA levels and a significantly increased Abcg5 mRNA level (Figure 3H). These alterations suggest that ALC modulates key regulators of cholesterol synthesis and transport at the transcriptional and translational levels.

To evaluate ALC’s efficacy in a hypercholesterolemic model prone to atherosclerosis, HCD-fed LDLR^−/− mice provide a well-established animal model of hypercholesterolemia, which is an independent risk factor for atherosclerosis. To establish a mouse model of hypercholesterolemia-induced atherosclerosis, we fed 8-week-old male LDLR^−/− mice D12108 feed containing 1.25% cholesterol for 8 weeks. From week 8 onward, the mice were divided into two groups: one group continued on the established HCD; the other received the same diet supplemented with 300 mg/kg ALC for an additional 8 weeks (HCD + ALC group; Figure 4A). This allowed us to assess the impact of ALC on established hyperlipidemia. Western blot analysis revealed the hepatic LDLR expression levels. Expectedly, LDLR^−/− mice exhibited significantly reduced hepatic LDLR levels compared with those of the wild-type mice (Figures 4B,C). Body weight and food intake did not significantly differ between the HCD and HCD + ALC groups (Figures 4D,E). Enzymatic assays revealed the plasma and hepatic TC and TG levels. The HCD + ALC group showed significantly decreased plasma TC levels compared with those of the HCD group, whereas plasma TG levels remained unchanged (Figures 4F,G). Hepatic TC levels did not significantly differ between the two groups (Figure 4H); however, hepatic TG levels were significantly reduced in the HCD + ALC group (Figure 4I). Liver sections were stained with H&E and oil red O (Figure 4K). Compared with the HCD group, the HCD + ALC group exhibited reduced hepatocyte vacuolation and significantly decreased oil red O-positive staining in the liver sections (Figure 4J). These findings demonstrate that ALC effectively lowers plasma cholesterol and reduces hepatic lipid accumulation even in the absence of LDL receptors.

To investigate whether ALC influences atherosclerotic progression, we analyzed aortic root sections from mice using H&E and oil red O staining. Oil red O en face staining of the entire aorta demonstrated a reduced atherosclerotic plaque area in the HCD + ALC group compared with that of the HCD group (Figure 5A). Similarly, oil red O staining of aortic root cross-sections showed a significantly decreased oil red O-positive staining area in the HCD + ALC group compared with that of the HCD group (Figures 5B,C). H&E staining revealed that the atherosclerotic lesion areas in the aortic root cross-sections were significantly reduced in the HCD + ALC group compared with the HCD group (Figures 5D,E). Additionally, the total necrotic area was significantly decreased in the HCD + ALC group (Figures 5F,G). These results suggest that ALC supplementation mitigates atherosclerosis development in hypercholesterolemic conditions.

To elucidate the cellular mechanisms underlying ALC’s cholesterol-lowering effects, ALC reduced the plasma TC in mice and decreased the hepatic SREBP2 and HMGCR levels. Therefore, we hypothesized that ALC may lower plasma TC levels by reducing cholesterol synthesis in hepatocytes. Thapsigargin is a common inducer of endoplasmic reticulum (ER) stress. Previous studies have shown that thapsigargin-induced ER stress increases SREBP2 levels in Huh-7 hepatocytes (14). We treated Huh-7 cells for 24 h with medium containing 100 nM TG, with or without addition of 100 mM ALC. Cells were divided into the control (CON), ALC, TG, and ALC + TG groups. Western blotting and qRT-PCR were performed to assess the protein and mRNA SREBP2 and HMGCR levels. Compared with the TG group, the ALC + TG group exhibited significantly decreased protein and mRNA SREBP2 and HMGCR levels (Figures 6A–C). Cholesterol uptake was assessed using Dil-LDL; the Dil-LDL fluorescence intensity was significantly increased in the ALC group compared with the CON group and significantly decreased in the ALC + TG group compared with the TG group (Figures 6D,E). Immunofluorescence analysis of SREBP2 expression in Huh-7 cells showed that compared with the CON group, the ALC group had significantly reduced SREBP2 levels, and similarly, compared with the TG group, the ALC + TG group had significantly reduced SREBP2 levels (Figures 6F,G). These in vitro findings support the notion that ALC decreases cholesterol synthesis by downregulating SREBP2, thereby contributing to its lipid-lowering effects observed in vivo.