To analyze the components of EB and the target gene network, we used the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Cytoscape. EB characteristics analyze of EB was analyzed as previously described [15]. The TCMSP (http://ibts.hkbu.edu.hk/LSP/tcmsp.php) was used to identify the active components of EB. As recommended by TCMSP, bioactive substances were analyzed based on their pharmacokinetic properties (absorption, distribution, metabolism, and excretion (ADME)), including oral bioavailability (OB), Caco-2 permeability (Caco-2) for intestinal epithelial permeability, and drug-likeness (DL). For further analysis, the active compounds with OB ≥ 30%, Caco-2 ≥ -0.4, and DL ≥ 0.18, were selected as candidate compounds. The active components of EB exert their biological functions through specific targets. After directly obtaining the potential targets of the core active ingredients in EB from the TCMSP database, the protein names of all target genes were converted into corresponding gene symbols using the UniProt database. Gene ontology (GO) analysis was performed to identify the biological processes related to the collected genes using the DAVID 6.8 Gene Functional Classification Tool (https://david.ncifcrf.gov/). The p-values from the enrichment results underwent correction through the Benjamini-Hochberg method, with significance set at p < 0.01. To decipher the mechanisms of Ellagic Acid (EB) based on active ingredients and target networks, we utilized Cystoscope visualization software version 3.7.2 to construct compound-target networks (https://cytoscape.org/). The selected candidate ingredients and targets were input into the software and the network was analyzed. The relationships between various active EB ingredients and target genes, as well as the biological metabolic processes related to exercise metabolism, were selected, and a process-target network (PT network) was subsequently constructed.

Cells were cultured as described previously [16]. Briefly, HepG2 cells ( human hepatoma cell line) were obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were maintained at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium containing 200 mg/L of l-glutamine (Hyclone, Logan, UT, USA), 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin (Invitrogen), and 100 μg/mL of streptomycin (Invitrogen).

HepG2 cells (1×10⁴ cells/well) were seeded in 96-well plates and incubated for 24 h. The cells were treated with diverse concentration of EB (10, 100, 1000 and 10000 µg/mL) for 24 h. HepG2 cells were treated with WST-1 reagent for 2 h, and the cells were measured using a microplate reader at 450 nm.

Cells were cultured as described previously [16]. Brief, HepG2 cells (1×10⁵ cells/well) were seeded in 60-mm Tomodishes and treated with 500 µg/mL EB at 36°C for 24 h. 3D holotomography images of live cells were obtained using an HT-1 microscope (magnification, ×600; Tomocube, Inc.). Cell structures were scanned using a digital micromirror and reconstructed using a 3D refractive-index tomogram. The masses of the cytoplasm and lipid droplets were analyzed based on densities of 0.135 and 0.178 g/ml, respectively.

Six-week-old C57BL/6 mice (6 weeks) were purchased from Orient Bio (Gyeonggi-do, Korea). Animal care was performed as previously described [17]. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Semyung University (approval number 20-10-02), and the experimental protocols complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All the experiments were conducted over 32 weeks. A them high-fat diet was provided for 24 weeks to create animal models, and the untreated group was fed a standard diet. The mice were divided into three groups as follows: HF group, high-fat diet for 8 weeks; ME group, high-fat diet with the positive control (orally administered 17 mg of metformin/kg/day) for 8week, EB group, high-fat diet with natural products (orally administered 26 mg of Erigeron breviscapus /kg/day) for 8 weeks. The untreated (UN) group received the AIN 93G stock diet for 32 weeks, while the HF, ME, and EB groups were provided with a high-fat diet comprising 60% fat, 20% carbohydrates, and 20% protein (5.21 Kcal) based on lard. Subsequently, all mice underwent anesthesia via an intraperitoneal injection of sodium pentobarbital (25 μg/10 g body weight).

Immunohistochemistry was performed as previously described [18]. Briefly, the mice were perfused and fixed in 10% formalin, and then the liver specimens were prepared according to the routine procedures for cryosection. The sections were stained with hematoxylin and eosin. One set of specimens underwent a 3-minute treatment with 0.3% hydrogen peroxide to inhibit endogenous peroxidase activity, followed by a 1 h blocking step using 5% goat serum. The remaining specimens were then incubated with different primary antibodies in 3% goat serum at 4 °C for 72 h. Antibodies to SREBP-1 and PPAR-γ were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The tissues were subsequently washed three times using phosphate-buffered saline, and the Vectastain ABC Kit was used to induce avidin-biotin complex interactions, according to the manufacturer’s instructions. Immunostaining analysis was performed using a substrate solution of 0.05% DAB, and the slides were counterstained with hematoxylin. All the sections were evaluated under a light microscope (BX50; Olympus Co., Ltd., Hachioji-shi, Tokyo, Japan) at × 200 magnification.

Statistical data are expressed as mean ± standard error, based on at least three independent experiments. All data were analyzed using GraphPad Prism software (Prism 4.00 Windows, GraphPad, San Diego, CA, USA), based on Student’s t-test for intergroup differences and one-way analysis of variance with Tukey’s test for multiple comparisons. Statistical significance was set at P < 0.05. significant.

To identify the active compounds in EB, we used the TCMSP database. Table 1 lists 17 bioactive EB compounds. Next, we identified the active molecule-related genes and analyzed the gene network. A total of 111 genes were associated with 17 active compounds. As shown in Figure 1, the gene networks were analyzed using Cytoscape. A total of 111 genes were associated with the top 31 gene ontologies.

To test whether EB regulates lipogenesis, we assessed the cell viability and 3D holograms. As shown in Figure 2A, HepG2 cells were treated with diverse concentrations of EB (1, 10, 100, 1,000 and 10,000 μg/mL) for 24 h. Cell viability was not toxicity up to 1,000 μg/mL. Next, we confirmed that EB downregulates PA-induced lipid accumulation in HepG2 cells. As shown in the Figure 2B, palmitic acid (PA) increased the lipid accumulation to 0.16 μm³ ± 0.02 μm³, as compared to the untreated group. Whereas the EB significantly reduced the lipid accumulation to 0.12 μm³ ± 0.01 μm³ compared to the group treated with only PA.

To investigate whether EB can control lipid accumulation in mice, mice were fed a high-fat diet for 24 weeks and were administered a combination of a high-fat diet and EB for an additional 8 weeks. The liver tissues were then stained with hematoxylin and eosin. As shown in Figure 3, the HF group had more mesh-like cells than the UN group. Furthermore, the EB and ME groups had fewer mesh-like cells than the HF group.

To explore EB’s potential in regulating lipid accumulation in mice, we assessed the expression levels of SREBP-1 and PPAR-γ using immunohistochemistry and ImageJ. As shown in Figure 4, HF diet increased the expression of PPAR-γ up to 520 % ± 43.59 % compared with UN group. However, the ME and EB group indicated the expression of PPAR-γ to 370.0 % ± 36.06 % and 256.6 % ± 30.5 %, respectively. Moreover, the ME and EB group showed the expression of SREBP-1 to 316.67 % ± 30.55 % and 266.6 % ± 47.2 %, respectively (Figure 5).