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. 2026 Apr 30;34(3):676–688. doi: 10.4062/biomolther.2025.162

Licochalcone E Ameliorates Hepatic Steatosis in Obese Mice by Activating the Sirt1/AMPK Pathway and Reducing Hepatic Lipid Accumulation

Wen-Chung Huang 1,2,3,†, Shu-Ju Wu 4,5,†, Xuan-Min Liu 1, Shu-Chen Cheng 1,2, Po-Ting Lin 6,7, Chun-Ling Kuo 2,6,*, Chian-Jiun Liou 2,8,*
PMCID: PMC13149049  PMID: 42059037

Abstract

Licochalcone E is a chalcone isolated from Glycyrrhiza uralensis and G. inflata Batal. This study explored the effect of licochalcone E on improving hepatic steatosis in obese mice and evaluated the role of licochalcone E in regulating lipid accumulation in hepatocytes. In vitro, oleic acid–induced hepatocytes were treated with licochalcone E to investigate its effect on lipid metabolic pathways. In animal experiments, male C57BL/6 mice were fed with a high-fat diet (HFD) and treated with licochalcone E by intraperitoneal injection for 12 weeks to assess its effects on biochemical indexes and hepatic steatosis. Furthermore, mice were fed a methionine/choline-deficient (MCD) diet and administered licochalcone E, followed by evaluation of liver fibrosis. Licochalcone E effectively reduced body weight, epididymal and inguinal fat weight, and adipocyte size in HFD-induced obese mice. Licochalcone E treatment of obese mice also reduced hepatic lipid accumulation and improved hepatocyte steatosis. Licochalcone E regulated the expression of lipogenesis- and lipolysis-related genes in the livers of obese mice and increased AMPK phosphorylation and Sirt1 expression in the liver. Licochalcone E also attenuated hepatic inflammation and oxidative stress in obese mice. Furthermore, treatment of MCD-induced mice with licochalcone E reduced the number of lipid vacuoles and the extent of fibrosis and inhibited liver inflammation. In FL83B hepatocytes, licochalcone E could regulate lipogenesis and lipolysis, and increase the phosphorylation of AMPK and ACC. These findings provide new insights into the role of licochalcone E in regulating lipid metabolism and preventing hepatic steatosis.

Keywords: AMPK, Hepatic steatosis, Lipogenesis, Lipolysis, Licochalcone E

INTRODUCTION

Metabolic dysfunction–associated fatty liver disease (MASLD), formerly non-alcoholic fatty liver disease (NAFLD), is a common chronic metabolic liver disease that is a global health challenge and whose occurrence and progression are influenced by genetic, epigenetic, and environmental factors (Targher et al., 2024). According to statistics, approximately 25% of the world’s population suffers from MASLD (Huttasch et al., 2024). The progression of MASLD can be divided into two stages: hepatic steatosis and metabolic dysfunction–associated steatohepatitis (MASH). In hepatic steatosis, excessive accumulation of triglycerides within hepatocytes interferes with the regulation of carbohydrate and lipid metabolism (Targher et al., 2024). The MASH stage is characterized by inflammation of the liver lobules and swelling of the hepatocytes. Additionally, severe inflammation and fibrosis may occur in liver cells, leading to liver cell damage. MASH may also worsen and develop into cirrhosis and hepatocellular carcinoma (Li et al., 2024).

The livers of obese people can synthesize fatty acids and triglycerides from carbohydrates through de novo lipogenesis and store excess lipids and lipid derivatives. Excessive lipid accumulation not only interferes with the lipid homeostasis of the liver but is also an important factor in inducing MASLD (Huttasch et al., 2024). The liver is an important energy storage organ that can store excess energy as glycogen and triglycerides and replenish the whole body, depending on the nutritional status of the organism. AMP-activated protein kinase (AMPK) is a metabolic energy–sensing molecule that plays a role in regulating cellular energy homeostasis (Palomer et al., 2025). Activating the Sirt1/AMPK pathway regulates lipid metabolic pathways mainly by inhibiting ATP-consuming synthetic pathways. Therefore, regulating the Sirt1/AMPK pathway is a useful therapeutic strategy for treating metabolic diseases. Activated AMPK can promote the inhibitory phosphorylation of acetyl-CoA carboxylase (ACC), thereby causing the loss of fatty acid synthase (FAS) activity and reducing lipid synthesis (Fang et al., 2022). Furthermore, AMPK can also accelerate the decomposition of triglycerides by activating hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) (Göransson et al., 2023). Therefore, the Sirt1/AMPK pathway plays a critical role in maintaining lipid homeostasis.

Licochalcone E can be isolated from Glycyrrhiza uralensis and G. inflata Batal and has multiple biological activities, including antioxidant and anti-tumor effects (Cao et al., 2021; Deng et al., 2023; Zhang et al., 2022). Licochalcone E could attenuate hepatotoxicity in carbon tetrachloride–induced acute liver injury in mice by activating PPARγ and inhibiting the NF-κB pathways (Han et al., 2017). Licochalcone E can also restore palmitate-induced insulin sensitivity and reduce inflammation in hepatocytes by inhibiting the NLRP3 signaling pathway (Cao et al., 2021). However, it is unclear whether licochalcone E can improve MASLD. Here, we assessed the effects of licochalcone E on liver steatosis, inflammation, and fibrosis in mice induced by high-fat diet (HFD) and methionine/choline-deficient (MCD) diet. We also treated oleic acid–induced hepatocytes with licochalcone E in vitro to evaluate its role in regulating lipid accumulation and the AMPK pathway.

MATERIALS AND METHODS

Cell culture and cell viability

Murine FL83B hepatocytes were purchased from the Bioresource Collection and Research Center (BCRC, Taiwan). Cell viability was analyzed using the MTT assay and cells treated with various concentrations of licochalcone E for 24 h. MTT solution was incubated with FL83B cells for 4 h at 37°C. Next, cells were treated with DMSO to solubilize the formazan crystals. Afterward, cell viability was measured using a microplate reader (Thermo Fisher Scientific, MA, USA) and the optical density (OD) measurements were performed at 550 nm.

Fatty liver cells and licochalcone E treatment

Murine hepatocytes were exposed to 0.5 mM oleic acid for 48 h. Afterward, cells were treated with vehicle (0.1% DMSO) or licochalcone E (1-10 μM) for 24 h to analyze lipid accumulation. In other liver cell experiments, 10 μM licochalcone E–treated FL83B cells were cocultured with AMPK inhibitor compound C (Sigma Aldrich, St. Louis, MO, USA) to evaluate the lipid metabolism pathway.

Oil red O staining analysis

FL83B hepatocytes incubated with oleic acid were treated with (0-10 μM) licochalcone E. The cells were stained with oil red O, and oil droplets were observed using an inverted microscope (Olympus, Tokyo, Japan). Afterwards, oil drops were dissolved and released with isopropanol and experimental values were measured using a microplate reader (Thermo) at OD 490 nm (Liou et al., 2024).

Lipid accumulation and lipoperoxidation in hepatocytes

Fatty liver FL83B cells treated with licochalcone E, and formalin-fixed cells stained with fluorescent probes BODIPY 493/503 and BODIPY 581/591 C11 (Invitrogen, Waltham, MA, USA) were used to detect lipid accumulation and lipoperoxidation, respectively. Nuclei were stained with DAPI, and a fluorescence microscope (Olympus) was used to record cell morphology (Cheng et al., 2024).

Fatty acid uptake

FL83B hepatocytes were incubated with oleic acid for 48 h. Next, cells were treated with licochalcone E for 24 h. The cells were then incubated with BODIPY FL C12 (Invitrogen), and fatty acid uptake was assayed using a fluorescence microscope (Olympus) (Wu et al., 2021).

Experimental animals

This study was approved by the Laboratory Animal Care Committee of Chang Gung University of Science and Technology (IACUC approval number: 2021-004). Male C57BL/6 mice were obtained from the National Laboratory Animal Center in Taiwan. All mice were raised in animal housing rooms with food and water ad libitum and maintained at 22°C and 50% humidity on a 12-h light/dark cycle.

Animal experiment design and licochalcone E treatment

In high-fat diet (HFD)–induced obese mode, male C57BL/6 mice (20-22 g, 5-6 weeks old) were randomly divided into four groups of 12 mice each. The normal group (N) was fed a normal diet (4.5 kcal% fat), the HFD group was fed a high-fat diet (34.9% fat, supplying 60% of energy) and the licochalcone E–treated groups were fed a HFD and treated with 5 mg/kg (the LE5 group) or 10 mg/kg (the LE10 group) licochalcone E. After 4 weeks of HFD feeding, the HFD control group and the LE5 and LE10 groups were maintained on the HFD and treated with DMSO or licochalcone E (dissolved in DMSO) twice a week for 12 weeks by intraperitoneal injection. Furthermore, the normal group also treated with DMSO by intraperitoneal injection.

In MASH animal model, male C57BL/6 mice (8 weeks old) were randomly divided into three groups of 12 mice each. These included the normal group (N) fed a normal diet, a methionine/choline-deficient (MCD) group, and a second MCD group treated with 10 mg/kg licochalcone E (the LE10 group). Mice were administered DMSO or 10 mg/kg licochalcone E by intraperitoneal injection twice a week for 4 weeks. During the animal experiments, the food intake of each mouse was recorded daily, and the body weight of each mouse was recorded twice a week.

Serum and hepatic biochemical assays

Glycogen, insulin, free fatty acids, and LDL-cholesterol (LDL-C) were detected using a colorimetric assay kit (Sigma) according to the manufacturer’s instructions. Total cholesterol (TC), triglyceride (TG), HDL-cholesterol (HDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose were quantified using a biochemical analyzer (DRI-CHEM NX500, Fuji, Tokyo, Japan) (Liou et al., 2020).

Histological analysis

Tissues were fixed in 4% formalin and embedded in paraffin. Tissue sections (5 μm) were made for H&E, Masson’s trichrome, and periodic acid–Schiff (PAS) staining. MASLD pathological scores were assigned on a scale from 0 to 8 (steatosis 0-3, lobular inflammation 0-3, and hepatocyte ballooning 0-2) (Kleiner et al., 2005; Stefan et al., 2025).

Immunofluorescence staining

Paraffin sections were performed with antigen retrieval by baking at 60°C for 1 h. Sections were incubated with anti–IL-1β antibody and anti–HO-1 antibody (1:50) (Cell Signaling Technology, MA, USA) at 4°C overnight. The slides were incubated with secondary antibody and treated with DAB substrate to detect specific protein expression by light microscopy (Cheng et al., 2024).

Western blot analysis

Proteins were separated using SDS acrylamide gels and transferred to a PVDF membrane. Next, the membrane was incubated with primary antibodies overnight at 4°C. The membrane was washed and incubated with secondary antibodies, followed by exposure to Luminol/enhancer solution to detect and quantify protein expression with a BioSpectrum 600 (UVP, Upland, CA, USA).

Real-time PCR

RNA from liver tissue was isolated using TRI reagent (Sigma). RNA was quantified and treated with cDNA synthesis reagents for cDNA synthesis. The QuantStudio 3 real-time PCR system (Thermo) was used to amplify specific RNA sequences to measure gene expression in mouse liver. Gene expression was quantified after normalization to β-actin.

Proinflammatory cytokine assay

An ELISA assay kit (R&D Systems, Minneapolis, MN, USA) was used to detect pro-inflammatory cytokines in serum according to the instructions. Cytokine values were measured using a microplate reader (Thermo) at OD 450 nm.

Determination of enzymes in liver tissues

Liver tissues were homogenized and the minced tissue slurry was centrifuged at 12000 rpm for 10 min. The supernatant was collected, and malondialdehyde (MDA) and superoxide dismutase (SOD) were assayed using a specific assay kit (Sigma) according to the instruction manual.

Statistical analysis

All data are expressed as mean ± SEM. GraphPad Prism 9.0 software was used to make statistical charts. Ordinary one-way ANOVA followed by Tukey’s post hoc test was used to evaluate significant differences between groups. P-values <0.05 were defined as statistically significant. Cell and animal experiments were repeated at least three independent times.

RESULTS

FL83B cell viability

The cytotoxicity of licochalcone E in FL83B cells was assessed using the MTT assay. Licochalcone E concentrations ≤15 μM had no toxic effect on FL83B cells (Fig. 1A). Therefore, licochalcone E was used at a concentration of 0-10 μM for cell experiments.

Fig. 1.

Fig. 1

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Licochalcone E (LE) treatment reduced lipid accumulation in FL83B cells. (A) Effect of LE treatment on FL83B cell viability. Data presented as mean ± SEM; **p<0.01 compared with FL83B cells treated without LE. (B) Oleic acid (OA)–induced FL83B cells treated with LE for 24 h before staining with BODIPY FL C12 (green) to detect fatty acid uptake. (C) Oil red O staining showing lipid accumulation. (D) FL83B cells were treated with isopropanol and lipid accumulation was measured using the absorbance at OD 490 nm. Data are presented as mean ± SEM; *p<0.05, **p<0.01 compared with mice with oleic acid (OA)–induced FL83B cells. (E) BODIPY 493/503 (green) staining to detect hepatic lipid droplets. Staining with BODIPY 581/591C11 (red) revealed hepatic lipoperoxidation. Nuclei were stained with DAPI (blue) (200× magnification). Scale bar, 100 μm. (F) Oleic acid (OA)–induced FL83B cells were treated with LE and incubated with DCFH-DA to evaluate ROS production by flow cytometry and percentage of ROS. All experiments were repeated at least three times as independent replicates.

Licochalcone E attenuated lipid accumulation and lipoperoxidation in hepatocytes

The cells incubated with BODIPY FL C12 fluorescent dye solution to detect fatty acid uptake. Licochalcone E treatment could mitigate fatty acid uptake compared with that in control oleic acid–induced FL83B cells (the OA group) (Fig. 1B). Oil red O staining revealed that licochalcone E treatment also reduced the accumulation of lipid droplets in fatty liver cells relative to that in untreated control cells (Fig. 1C). An isopropanol solution was used to treat oil red O–stained hepatocytes to further confirm that licochalcone E can significantly reduce lipid accumulation in fatty liver cells (Fig. 1D). In addition, staining with BODIPY 493/503 dye also demonstrated that licochalcone E alleviated lipid droplet accumulation compared with that in the OA group (Fig. 1E). Next, lipoperoxidation was detected using BODIPY 581/591 C11 dye, and the result demonstrated that licochalcone E reduced lipoperoxidation relative to that in the OA group (Fig.1E). Licochalcone E was also used to treat fatty liver cells incubated with DCFH-DA to analyze ROS levels by flow cytometry. Licochalcone E treatment reduced ROS levels relative to those in the control OA group (Fig. 1F).

Licochalcone E modulated lipid accumulation in hepatocytes

Licochalcone E effectively lowered Srebp-1c and FAS expression relative to their expression in the OA group (Fig. 2A, 2B). Licochalcone E increased ATGL and CPT2 (but not CPT1) production (Fig. 2A, 2B). Treatment with licochalcone E also enhanced Sirt1 expression and increased the phosphorylation of ACC and AMPK relative to their levels in the OA group (Fig. 2C, 2D). Oleic acid–induced FL83B cells co-treated with 5 μM licochalcone E and 10 μM compound C were stained with oil red O and BODIPY 493/503 fluorescent dye. The results demonstrated that the licochalcone E–treated cells contained fewer and smaller lipid droplets than did the compound C/OA cells (Fig. 3A-3C). However, quantitative analysis of Oil Red O staining showed that, compared with the 5 μM LE/OA group, co-treatment with 5 μM LE plus 10 μM Compound C tended to increase lipid accumulation, although this difference did not reach statistical significance (p=0.062) (Fig. 3B). Co-treatment of cells from the OA group with 5 μM licochalcone E and 10 μM compound C restored ATGL and CPT2 gene expression and more effectively suppressed FAS and CD36 gene expression compared with levels in OA cells treated with 10 μM compound C alone (Fig. 3D). We also found that there were no significant differences in the mRNA expression levels of ATGL, CPT-2, and CD36 between the 5 μM LE/OA group and the group co-treated with 5 μM LE plus 10 μM Compound C (Fig. 3D).

Fig. 2.

Fig. 2

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Effects of licochalcone E (LE) on lipid metabolism in FL83B cells. (A) Licochalcone E regulates the expression of proteins in lipid metabolism pathways. (B) Changes in expression levels were measured relative to β-actin expression. (C) Licochalcone E regulates protein expression in the AMPK pathway. (D) Changes in expression levels were measured relative to β-actin expression. Three independent experiments were analyzed using β-actin as an internal control. Data are presented as mean ± SEM; *p<0.05, **p<0.01 in comparison with oleic acid (OA)–induced FL83B cells.

Fig. 3.

Fig. 3

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Effects of licochalcone E (LE) on lipid metabolism in oleic acid (OA)/AMPK inhibitor-induced FL83B cells. LE-treated FL83B cells were cocultured with an AMPK inhibitor (compound C) to evaluate the lipid metabolism pathway. (A) Oil red O staining showing lipid accumulation. (B) FL83B cells were treated with isopropanol, and lipid accumulation was measured using the absorbance at OD 490 nm. (C) BODIPY 493/503 (green) staining to detect hepatic lipid droplets. Nuclei were stained with DAPI (blue) (200× magnification). Scale bar, 100 μm. (D) Effects of LE on the AMPK pathway in FL83B cells. Expression of FAS, ATGL, CPT - 2 and CD36 was assayed by real-time PCR, and the fold-change values were calculated relative to β-actin expression. The data are presented as the mean ± SEM. *p<0.05, **p<0.01 in comparison with the oleic acid (OA) group. #p<0.05 in comparison with the compound C/OA group. All experiments were repeated at least three times as independent replicates.

Licochalcone E inhibits body weight gain and adipose tissue weight gain in obese mice

At the end of the HFD-induced obese mouse experiment, the body weight of the HFD group was statistically significantly higher than that of the Normal group. Obese mice treated with licochalcone E showed a significant reduction in body weight compared with the HFD group (LE5: 42.68 ± 0.86 g, p<0.05; LE10: 40.30 ± 0.98 g, p<0.01 vs. HFD: 47.66 ± 1.66 g; Fig. 4A). Obese mice given licochalcone E for 12 weeks showed significantly less weight gain than those in the HFD group (Fig. 4B), but their food intake did not differ (Fig. 4C). Interestingly, adipocyte size and weight in epididymal and inguinal adipose tissue were significantly lower in obese mice administered licochalcone E than in the HFD group (Fig. 4D-4I).

Fig. 4.

Fig. 4

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Licochalcone E (LE) suppressed body weight gain in obese mice. (A) Body weight recorded over 16 weeks. (B) Weight gain calculated in the last week. (C) Daily food intake. (D) H&E staining of epididymal adipose tissue (200× magnification). (E) Adipocyte size in epididymal adipose tissue. (F) Weight of the epididymal adipose tissue. (G) H&E staining of inguinal adipose tissue (200× magnification). Scale bar, 100 μm. (H) Adipocyte size in inguinal adipose tissue. (I) Weight of the inguinal adipose tissue. The data are presented as mean ± SEM; n=12. *p<0.05, **p<0.01 in comparison with HFD-induced obese mice. All experiments were repeated at least three times as independent replicates.

Licochalcone E ameliorated liver steatosis in obese mice

10 mg/kg licochalcone E significantly mitigated liver weight gain in obese mice (Fig. 5A). 10 mg/kg licochalcone E treatment also resulted in lower TG and TC levels and greater glycogen accumulation in the livers of obese mice (Fig. 5B-5D). H&E and Oil Red O staining of liver tissues revealed that licochalcone E treatment reduced hepatic lipid droplet accumulation in obese mice compared with untreated HFD-fed controls (Fig. 5E, 5F). This histological improvement was consistent with the reduced lipid vacuole area (%) in the licochalcone E–treated group (Fig. 5G). Obese mice treated with licochalcone E treatment also had lower MASLD scores than mice in the HFD group (Fig. 5H).

Fig. 5.

Fig. 5

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Biometrics of MASLD in mice treated with licochalcone E (LE). (A) Liver weight, (B) triglyceride (TG), (C) total cholesterol (TC), and (D) glycogen levels in liver tissue. (E) H&E staining of liver tissues (200× magnification). (F) Oil red O staining of liver tissues (200× magnification). Scale bar, 100 μm. (G) Percentage of lipid vacuole area in liver tissues. (H) MASLD scores in liver tissues. The data are presented as mean ± SEM of three independent experiments; n=12. *p<0.05, **p<0.01 in comparison with HFD-induced obese mice.

Licochalcone E regulates lipogenesis and lipolysis in liver tissue

Compared with the HFD group, obese mice treated with licochalcone E had reduced expression of the lipogenesis-related genes SREBP-1c and FAS in liver (Fig. 6A, 6B). Licochalcone E treatment also promoted the expression of lipolysis-related genes, including ATGL and HSL, in liver tissue (Fig. 6C, 6D). Furthermore, obese mice treated with licochalcone E also increased the expression of CPT2 and Sirt1, genes involved in fatty acid β-oxidation and the AMPK pathway, respectively (Fig. 6E, 6F). Therefore, we examined protein expression in the AMPK pathway and found that, compared with HFD group mice, HFD mice treated with licochalcone E had significantly higher Sirt1 expression and AMPK phosphorylation (Fig. 6G, 6H).

Fig. 6.

Fig. 6

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Licochalcone E (LE) modulated lipogenesis and lipolysis gene expression in liver tissue. Real-time RT-PCR to measure the expression of the genes encoding SREBP-1C (A), FAS (B), ATGL (C), HSL (D), CPT-2 (E), and Sirt1 (F). The fold changes in expression were calculated relative to the level of β-actin (internal control). (G) LE treatment group had enhanced phosphorylated AMPK and Sirt1 protein expression in the liver. (H) Changes in expression levels were measured relative to β-actin expression. The data are presented as mean ± SEM from three independent experiments; n=12. *p<0.05, **p<0.01 in comparison with HFD-induced obese mice.

Licochalcone E regulates lipid homeostasis in serum

HFD mice treated with licochalcone E had lower serum TG, TC, LDL, and free fatty acid levels than the untreated controls (Fig. 7A-7D). However, licochalcone E treatment did not increase the serum HDL level in obese mice (Fig. 7E). Therefore, we analyzed the LDL/HDL ratio, and the results showed that it was lower in the LE10 group than in the HFD group (Fig. 7F). Furthermore, compared with the HFD group, obese mice given licochalcone E had markedly lower serum ALT, AST, insulin, glucose, and leptin levels and increased adiponectin expression (Fig. 7G-7L).

Fig. 7.

Fig. 7

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Serum biochemical analysis in mice. Levels of free fatty acid (A), TG (B), TC (C), LDL (D), HDL (E), LDL/HDL ratio (F), AST (G), ALT (H), glucose (I), insulin (J), adiponectin (K), and leptin (L). The data are presented as mean ± SEM from three independent experiments; n=12. *p<0.05, **p<0.01 in comparison with HFD-induced obese mice.

Licochalcone E reduced liver inflammation and oxidation in obese mice

Compared with HFD group mice, 10 mg/kg licochalcone E–treated obese mice had markedly higher SOD and lower liver MDA activity and levels (Fig. 8A, 8B). IHC staining revealed that licochalcone E treatment led to higher HO-1 expression than that seen in the HFD group (Fig. 8C). Moreover, obese mice treated with 10 mg/kg licochalcone E had lower serum TNF-α and IL-6 levels than did the untreated obese mice (Fig. 8D, 8E). In liver tissue, 10 mg/kg licochalcone E–treated mice had lower TNF-α and IL-6 gene expression than did untreated obese mice (Fig. 8F, 8G).

Fig. 8.

Fig. 8

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Effects of licochalcone E (LE) on oxidation and the inflammatory response in mice. (A) SOD and (B) MDA levels in liver tissue. (C) Immunohistochemical staining reveals HO-1 expression (brown color) in liver tissue. Scale bar, 100 μm. (D) LE suppressed TNF-α and (E) IL-6 levels in serum. (F) TNF-α and (G) IL-6 gene expression in liver tissue. The fold-change values were measured relative to β-actin expression. The data are presented as mean ± SEM of three independent experiments; n=12. *p<0.05, **p<0.01 in comparison with HFD-induced obese mice.

Licochalcone E improved liver fibrosis and inflammation in MCD diet–fed mice

HFD-induced obese mice are less likely to develop macrophage accumulation and fibrosis in the liver. Therefore, mice were fed an MCD diet to induce the MASH model, with or without concomitant licochalcone treatment. At the end of the MASH model experiment, in comparison with the untreated MCD mice, MCD mice treated with 10 mg/kg licochalcone E did not gain significant weight (Fig. 9A-9C) and did not have altered food intake (Fig. 9D). Grossly, the livers of normal mice appeared dark brown-red, whereas the livers of MCD-induced mice appeared light red. Those of 10 mg/kg licochalcone E–treated MCD-induced mice appeared dark brown (Fig. 10A) and did not differ in weight from the livers from untreated MCD mice (Fig. 10B). Furthermore, licochalcone E treatment reduced ALT and AST levels to below the levels in the untreated MCD mice (Fig. 10C, 10D). Interestingly, H&E staining of liver sections demonstrated that licochalcone E treatment reduced the numbers of fat vacuoles and macrophage aggregates compared with those in the MCD group (Fig. 10E). Compared with the untreated MCD-induced mice, those treated with licochalcone E also had significantly lower scores for lobular inflammation, ballooning, and MASH (Fig. 10F), and less liver fibrosis as detected by Masson’s trichrome staining (Fig. 10E, 10F). Additionally, MCD-induced mice treated with licochalcone E accumulated glycogen to higher levels than did the untreated MCD group (Fig. 10E). IHC demonstrated that licochalcone E–treated mice had lower levels of inflammatory cytokine IL-1β in the liver than did the untreated MCD mice (Fig. 10G). Compared with the MCD group, licochalcone E–treated mice showed decreased hepatic expression of the M1 macrophage marker CD80, accompanied by increased expression of the M2 macrophage marker CD163. In addition, licochalcone E reduced the hepatic expression of the pro-inflammatory cytokines IL-1β and TNF-α, as well as the fibrotic marker α-SMA (Fig. 10H). Protein analysis further showed that licochalcone E increased hepatic SIRT1 and phosphorylated AMPK levels (Fig. 10I).

Fig. 9.

Fig. 9

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Effects of licochalcone E (LE) on body weight in MCD-induced MASH mice. (A) Weight gain was measured for 4 weeks. (B) At the end of MASH model experiment, mice treated with LE had not gained significant weight compared with that in the MCD group. (C) Gross appearance of the animals. (D) Caloric intake monitored each day for 4 weeks. The data are presented as mean ± SEM of three independent experiments; n=12. **p<0.01 in comparison with the MCD group.

Fig. 10.

Fig. 10

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Licochalcone E (LE) modulated inflammation and fibrosis in the liver. (A) Gross appearance of the livers. (B) Liver weight. (C) Serum ALT and (D) AST levels. (E) H&E staining, PAS staining, and Masson’s trichrome stain detected pathology in the liver (200× magnification). Scale bar, 100 μm. (F) The scores for hepatic steatosis, lobular inflammation, ballooning, MASH, and fibrosis score were evaluated from H&E staining and Masson’s trichrome staining of liver tissues. (G) IL-1β expression was analyzed by immunohistochemistry (brown). 200× magnification. Scale bar, 200 μm. (H). Relative mRNA expression of macrophage markers, inflammatory, and fibrotic genes in the liver. (I) Effects of licochalcone E on protein expression in the hepatic AMPK signaling pathway. The data are presented as mean ± SEM of three independent experiments; n=12. *p<0.05, **p<0.01 in comparison with the MCD group.

DISCUSSION

High-calorie diet and alcoholism are important factors that lead to the continued increase in the prevalence of MASLD (Chan et al., 2023). Although early hepatic steatosis does not require special clinical treatment, busy city people who do not have proper diet management and moderate exercise may aggravate the deterioration of hepatic steatosis and progress toward the MASH pathway (Targher et al., 2024). Some MASH patients may even develop more dangerous liver diseases, including cirrhosis and hepatocellular carcinoma. (Schwärzler et al., 2024). Therefore, it is crucial to develop specific therapeutic drugs to slow the progression of MASLD. In in vitro and in vivo experiments, purified compounds of medicinal plants are reported to prevent or alleviate MASLD. For example, maslinic acid and phloretin improve hepatic steatosis in obese mice, mainly through regulating lipogenesis and the AMPK pathway (Liou et al., 2019, 2020). Here, in our in vitro experiments, licochalcone E regulated lipogenesis and lipolysis and increased the phosphorylation of AMPK and ACC. In addition, in obese mice, licochalcone E inhibited gains in body weight and epididymal and inguinal fat weight and adipocyte size. Licochalcone E also suppressed liver lipid accumulation and hepatocyte steatosis and promoted Sirt1 expression and the phosphorylation of AMPK in the liver. Additionally, licochalcone E inhibited liver fibrosis and inflammation in MCD diet-induced mice.

Since MASLD is closely related to various metabolic abnormalities of metabolic syndrome, including obesity, hypertension, and insulin resistance, the development of MASLD is often considered to be a manifestation of excessive lipid accumulation in the liver caused by obesity (Syed-Abdul, 2023). Our experiments showed that mice fed a HFD not only gained weight, but also accumulated large amounts of lipid in their epididymal and inguinal adipose tissue. We hypothesized that licochalcone E treatment in obese mice would reduce hepatic lipid accumulation and thereby ameliorate the progression of MASLD. Our results show that licochalcone E attenuated liver weight gain, reduced hepatic lipid accumulation, and decreased hepatocellular vacuolation in obese mice. However, macrophage aggregation in the livers of HFD-induced obese mice was not obvious by H&E staining. Macrophage accumulation in the liver is one of the items in the MASLD score, and activated macrophages are important immune cells that cause liver cell inflammation. Therefore, we also used the MCD diet to induce hepatic macrophage accumulation and fibrosis in mice to cause more severe MASH. Since the MCD diet lacks methionine and bile acid, mice are unable to transport triglycerides out of the liver and thus develop severe fatty liver (Alshawsh et al., 2022). More activated macrophages can be observed in the livers of MCD-induced mice. Administration of licochalcone E to MCD-induced mice reduced macrophage accumulation in the liver. Licochalcone E promoted M2 macrophage polarization in the livers of MCD-induced mice and mitigated hepatic inflammation, as indicated by decreased hepatic levels and distribution of the pro-inflammatory cytokines IL-1β and TNF-α. In addition, licochalcone E reduced α-SMA expression, supporting its anti-fibrotic potential. Taken together, these results suggest that licochalcone E may concurrently alleviate liver inflammation and fibrosis, thereby helping to slow the progression of MASH. In addition, in HFD model mice and MCD-induced mice, serum ALT and AST values were significantly lower with licochalcone E treatment. We believe that licochalcone E can restore liver function in MASLD mice.

Many scholars have confirmed that the AMPK signaling pathway can promote lipolysis and suppress lipogenesis, thereby limiting lipid accumulation in hepatocytes (Day et al., 2017; Fang et al., 2022). The AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) can inhibit hepatosteatosis and liver tumorigenesis in HFD-induced obese mice (Börgeson et al., 2017). AICAR also enhances Sirt1 expression to reduce obesity-induced inflammation and insulin resistance (Fang et al., 2022). In addition, some researchers have found that compound C (an AMPK inhibitor) can exacerbate obesity and fatty liver disease in mice induced by an HFD (Wang et al., 2019). Therefore, we used compound C and licochalcone E co-treatment on oleic acid-induced hepatocytes. Compared with the compound C–treated oleic acid hepatocytes, licochalcone E/compound C–treated oleic acid hepatocytes had not only lower lipid droplet accumulation, but also restored ATGL and CPT2 gene expression and inhibited FAS and CD36 gene expression. Most cellular fatty acid uptake is coordinated by multiple transport-related proteins, including CD36, fatty acid transport proteins, and caveolin-1 (Samovski et al., 2023). Among these, CD36 is a key mediator of long-chain fatty acid uptake in adipocytes, hepatocytes, and myocytes, thereby playing a central role in lipid homeostasis (Garcia et al., 2025). Notably, previous studies have shown that AMPK activation can suppress hepatic CD36 expression by promoting FOXO1 nuclear signaling, which in turn reduces fatty acid uptake and alleviates lipid accumulation (Zhang et al., 2024). Recent studies have suggested that the Sirt1/AMPK pathway plays a central role in the regulation of intracellular energy homeostasis (Göransson et al., 2023). Activation of AMPK has been shown to inhibit lipid synthesis in hepatocytes and adipocytes, particularly under conditions of energy excess. Furthermore, AMPK phosphorylation promotes ACC phosphorylation, thereby suppressing fatty acid synthesis (Smith et al., 2016). Resveratrol, a well-known Sirt1 activator, has been reported to attenuate lipid accumulation and improve MASLD through activation of the Sirt1/AMPK pathway in HFD-induced obese mice (Tejada et al., 2021). Consistent with these observations, licochalcone E treatment significantly increased hepatic Sirt1 expression and AMPK phosphorylation in obese mice. Collectively, these findings suggest that the inhibitory effect of licochalcone E on hepatic lipid accumulation may be mediated, at least in part, through activation of the Sirt1/AMPK pathway.

The liver can synthesize excess free fatty acids and triglycerides from excess carbohydrates via de novo lipogenesis. Excessive accumulation of triglycerides can cause steatosis and interfere with liver function (Alves-Bezerra and Cohen, 2017). Srebp-1c is a transcription factor required for the lipogenesis process. When Srebp-1c is activated, it can enter the nucleus from the cytoplasm and initiate FAS gene and protein expression to synthesize long-chain fatty acid chains (Wang et al., 2015). ACC is the rate-limiting enzyme for fatty acid chain synthesis. ACC can catalyze the carboxylation of acetyl-CoA to produce malonyl-CoA to initiate the synthesis of fatty acid chains. Furthermore, ACC activation will also promote FAS activation (Mateo-Marín and Alves-Bezerra, 2024). AMPK phosphorylation can also induce ACC phosphorylation (ACC will lose its activity) to inhibit FAS activity and reduce fatty acid chain synthesis (Jeon et al., 2023). Cell experiments and animal experiments have shown that licochalcone E can suppress the activation of Srebp-1c and FAS by activating Sirt1/AMPK and ACC, thereby reducing hepatocyte lipogenesis and alleviating hepatic steatosis. AMPK signaling pathway can also activate ATGL and HSL phosphorylation to improve lipolysis (Smith et al., 2016). ATGL and phosphorylated HSL can decompose TG to produce free fatty acids and glycerol, reducing TG levels in liver (Grabner et al., 2021). Licochalcone E can also activate ATGL and HSL phosphorylation in hepatocytes. In addition, we also found that licochalcone E has the ability to reduce the TC and TG concentrations in the livers of obese mice. However, free fatty acids released by the decomposition of TC in liver will cause inflammation of liver cells. Therefore, the excess free fatty acids in the liver must be decomposed to produce energy through fatty acid β-oxidation and reduce the inflammatory response caused by fatty acids (Ding et al., 2021). The AMPK pathway can also activate the fatty acid β-oxidation-related proteins CPT1 and CPT2 in hepatocytes (Fang et al., 2022). Our experiments revealed that licochalcone E could increase CPT2 (but not CPT1) production in oleic acid-induced hepatocytes. CPT2 is crucial for long-chain fatty acid oxidation in mitochondria. CPT2 can catalyze acyl-carnitine through the mitochondrial inner membrane and the release of fatty acid chains for fatty acid β-oxidation (Fritzen et al., 2020). These results support a model in which licochalcone E activates AMPK signaling, thereby reducing hepatic lipid accumulation through coordinated changes in lipid synthesis and utilization.

We used an oleic acid–induced hepatocellular steatosis model as an in vitro system. This model mainly reflects intracellular lipid accumulation in hepatocytes as well as changes in the expression of key regulatory proteins involved in lipogenesis and lipolysis. However, we did not employ isotope- or radiotracer-based approaches, such as 13C-acetate or 14C-glucose tracing, to follow the incorporation of labeled substrates into fatty acids for accurate quantification of de novo lipogenesis flux (Carbó et al., 1991; Hellerstein et al., 1991). Therefore, we cannot directly determine the actual fatty acid flux contributing to lipid synthesis in this model. In future studies, we will address this limitation by incorporating tracer-based experiments to more precisely clarify the causal relationship between exogenous fatty acid uptake and de novo lipogenesis in vitro.

Adipocytes secrete a variety of adipokines to maintain the constant function of various organs. Obese people secrete excessive leptin to regulate the hypothalamus to reduce appetite and thus accumulate less excess energy (Zhang and Chua, 2017). In our study, treatment of obese mice with licochalcone E suppressed serum leptin and increased adiponectin levels. Adiponectin improves insulin resistance in obese people (Engin, 2024). We found that fasting blood glucose and insulin levels were lower in obese mice treated with licochalcone E than in untreated obese mice. Therefore, licochalcone E may improve blood sugar and insulin resistance by regulating the constant effects of leptin and adiponectin. Furthermore, licochalcone E can also regulate serum TC and TG and regulate the LDL-to-HDL ratio. The LDL/HDL ratio is a better predictor of coronary atherosclerotic heart disease severity than LDL or HDL (Sun et al., 2022). Therefore, licochalcone E seems to be protective against cardiovascular disease. This is an interesting experimental direction that can be continued in future research.

However, this study did not directly investigate pathways related to cholesterol metabolism. Specifically, we did not assess key regulators of hepatic cholesterol efflux and biliary secretion, such as ABCA1, ABCG5, and ABCG8 (Plummer et al., 2021). Consequently, we cannot determine whether licochalcone E modulates cholesterol transport or clearance in this model. Given the critical role of dysregulated cholesterol homeostasis in MASLD progression, future studies will incorporate a more comprehensive evaluation of cholesterol handling and related pathways to further clarify the mechanisms by which this natural compound ameliorates MASLD.

Obesity is a chronic inflammatory disease. Free fatty acids secreted by adipocytes stimulate macrophages infiltrating adipose tissue to polarize into the inflammatory M1 type (Li et al., 2023). These M1 macrophages continuously secrete low levels of inflammatory cytokines, including TNF-α and IL-6. As a result, the serum of obese mice contains higher levels of TNF-α and IL-6 (Yunna et al., 2020). Licochalcone E not only regulates serum TNF-α and IL-6, but also reduces the gene expression of TNF-α and IL-6 in the livers of obese mice. In MCD-induced mice, licochalcone E treatment was also found to reduce the expression of IL-1β in the liver. Inflammation can also cause oxidative damage and even lipid oxidative toxicity and cell death in liver cells (Clare et al., 2022). In cell experiments, we found that licochalcone E can not only reduce fatty acid uptake but also reduces lipid synthesis and lipid peroxidation in liver cells. MDA is an important marker of lipid peroxidation. It is a peroxidation product produced by cell metabolism of unsaturated fatty acids (Hughey et al., 2022). In the obese mouse experiment, licochalcone E treatment raised the level of liver antioxidant enzyme SOD and reduced MDA levels in obese mice. Liver IHC staining also confirmed that licochalcone E treatment could increase the expression of the antioxidant molecule HO-1. Apparently, licochalcone E can help prevent liver inflammation and oxidation caused by MASLD.

In conclusion, we performed studies using obese mice with MASLD syndrome to explore the therapeutic effects of licochalcone E and elucidate its mechanism of action. Our results suggest that licochalcone E improves metabolism-related fatty liver disease in obese mice by modulating the Sirt1/AMPK pathway and inhibiting lipid accumulation (Fig. 11). In addition, we also found that licochalcone E can reduce liver fibrosis and inflammation in MCD-induced mice. This study provides evidence to support licochalcone E as a potential treatment option for MASLD.

Fig. 11.

Fig. 11

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Licochalcone E ameliorates obesity-induced hepatic steatosis by improving hepatic lipid homeostasis.

ACKNOWLEDGMENTS

This study was supported in part by grants from Chang Gung Memorial Hospital (CMRPG3P0951, CMRP1N0071, CMRPF1N0051, CMRPF1N0081, and CMRPF1L0031), the Chang Gung University of Science and Technology (ZRRPF3N0091, ZRRPF3P0091, and ZRRPF3Q0091), and the Ministry of Science and Technology in Taiwan (112-2320-B-255 -005 -MY3, 112-2320-B-255 -006 -MY3, 112-2320-B-255 -007 -MY3).

Footnotes

CONFLICT OF INTEREST

The authors report no potential conflict of interest.

AUTHOR CONTRIBUTIONS

Wen-Chung Huang: Investigation, validation, formal analysis, methodology. Shu-Ju Wu: Investigation, validation, formal analysis, methodology. Xuan-Min Liu: Investigation, formal analysis. Shu-Chen Cheng: Investigation, formal analysis. Po-Ting Lin: Investigation, formal analysis. Chun-Ling Kuo: Investigation, visualization, writing – original draft. Chian-Jiun Liou: Investigation, visualization, writing – original draft.

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