Rhein alleviates hepatic steatosis in NAFLD mice by activating the AMPK/ACC/SREBP1 pathway to enhance lipid metabolism

All experimental animals in this study were used exclusively for medical research purposes. All procedures were carried out after discussion and approval by the Animal Committee of The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University. All operations strictly adhered to the regulations for the management of laboratory animals.

A total of 56 six-week-old specific pathogen free male C57BL/6J mice (weighing 18–22 g) were available from Shanghai Model Organisms Center, Inc. The mice were acclimatized for one week under standard laboratory conditions: constant temperature (20–26 °C), relative humidity (40–70%), a natural light/dark cycles, and adequate air circulation before the experimental procedures were initiated.

To establish the NAFLD model, all mice except those in the normal group were fed a high-fat diet (HFD) for 8 consecutive weeks. The HFD (Catalog No.: D12492, Research Diets, Inc., New Jersey, USA) consisted of 40% fat, 20% protein, and 40% carbohydrates by caloric composition. The normal group was maintained on a standard laboratory diet (12% fat, 23% protein, 65% carbohydrates). Successful NAFLD modeling was confirmed through increased body weight, elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and histopathological assessment of liver tissues using Hematoxylin and Eosin (H&E) and Oil Red O staining. Mice were randomly divided into 10 groups (n = 8 per group). Randomization was accomplished using a random number table generated by Excel, and the entire process was carried out by independent researchers who did not participate in subsequent experimental operations to avoid bias due to human intervention. The groups were as follows: Normal group (regular diet); Rhein group (150 mg/kg) (regular diet + high-dose Rhein group (150 mg/kg); NAFLD group; NAFLD + Low Rhein group (25 mg/kg); NAFLD + Mild Rhein group (50 mg/kg); NAFLD + High Rhein group (150 mg/kg); NAFLD + dimethyl sulfoxide (DMSO) group (1%, vehicle control, intraperitoneal injection); NAFLD + AMPK-IN-3 group (10 mg/kg); NAFLD + High-Rhein + DMSO group; and NAFLD + High-Rhein + AMPK-IN-3 group group (10 mg/kg). Rhein (Rhein, R7269, Sigma, purity ≥ 98%, HPLC) was dissolved in phosphate-buffered saline and administered via oral gavage once every 4 days for 8 weeks. The dosage was determined based on previous animal studies and preliminary pilot experiments (Wei et al. 2016; Sheng et al. 2011), designed to cover a pharmacologically relevant dose range. The dosing interval (once every four days) was chosen to maintain pharmacokinetic stability while minimizing animal stress and interference with normal feeding behavior. The AMPK inhibitor AMPK-IN-3 (HY-151361, MCE) was dissolved in 1% DMSO (Sigma, 34869) and administered intraperitoneally at 10 mg/kg, also once every 4 days for 8 weeks. Body weight was recorded before each administration. At the end of the experiment, peripheral blood, liver, kidneys, and retroperitoneal fat pads were collected for subsequent analysis (Wei et al. 2016; Sheng et al. 2011, 2019).

Following HFD feeding and rhein treatment, mice were anesthetized, and 1 mL of blood was gathered from the abdominal aorta into anticoagulant-free tubes. Samples were centrifuged at 3000 rpm for 15 min at 4 °C to acquire the serum. Serum levels of AST, triglycerides (TG), ALT, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were determined usinga Hitachi 7020 Automatic Clinical Analyzer (Tokyo, Japan) (Sheng et al. 2019; Wu et al. 2022).

Following HFD and rhein treatment, serum samples were gathered to assess inflammatory cytokine secretion. Serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were assessed using commercia ELISA kits (TNF-α: BMS607, Thermo; IL-6: 88–7064-88, eBioscience). Absorbance was read at 450 nm and 570 nm using a SpectraMax^®i3 spectrophotometer. To account for background interference, absorbance at 570 nm was subtracted from that at 450 nm. Cytokine concentrations were quantified based on the standard curve generated in each assay (Wu et al. 2022).

After euthanizing the mice, liver tissues were harvested and fixed in 4% paraformaldehyde. Tissues were embedded in paraffin and sectioned at a thickness of 3 μm. Sections were then dewaxed, rehydrated, and stained using an HE staining kit (G1121, Solarbio). After mounting with neutral resin, the stained sections were examined under a Zeiss optical microscope (Zeiss AG, Oberkochen, Germany) at 200× magnification to evaluate liver histopathological changes (Fang et al. 2023).

For lipid deposition assessment, fresh liver tissues were embedded in optimal cutting temperature (OCT) compound and frozen. Frozen liver sections (5 μm thick) were prepared and stained with Oil Red O (O8010, Solarbio). Prior to staining, sections were immersed in anhydrous propylene glycol for 5 min to prevent water contamination. A 0.5% w/v Oil Red O solution, pre-dissolved in isopropanol and cooled, was used to stain the sections for 10 min. Sections were then differentiated in 85% propylene glycol for 5 min, rinsed, and mounted with glycerol gelatin. Lipid droplets were observed under a Zeiss optical microscope at 200× magnification (Fang et al. 2023; Lee et al. 2023).

Protein were extracted from liver tissues using RIPA lysis buffer supplemented with PMSF (Catalog No.: P0013C, Beyotime, Shanghai, China). After incubation, the lysates were centrifuged to collect the supernatant. Total protein concentration was quantified using a BCA assay kit (Catalog No.: 23227, ThermoFisher, USA). Equal amounts of protein (50 µg) were mixed with 2× SDS loading buffer, boiled, and then separated by electrophoresis on a 10% SDS-PAGE gel (Catalog No.: G2177-50T, Servicebio, Wuhan, China). Proteins were transferred onto PVDF membranes (Catalog No.: ISEQ00010, Millipore). Membranes were blocked with 5% skimmed milk for 1 h at room temperature to prevent non-specific binding and incubated overnight at 4 °C with primary antibodies: AMPKα1 (1:2000, ab32047, Abcam, Cambridge, UK), p-AMPKα1 (1:2000, ab131357, Abcam), p-ACC (1:1000, #11818, CST, USA), ACC (1:1000, #3676, CST, USA), SREBP1 (1:2000, ab313881, Abcam), and GAPDH (ab9485, 1:2500, Abcam) as an internal reference. After being washed with TBST, membranes were incubated with HRP-conjugated secondary antibody, specifically a (goat anti-rabbit IgG H&L, ab97051, 1:2000, Abcam) for 1 h. After that, the membranes were washed and developed using ECL reagent (Catalog No.: abs920, Abxin Biotechnology Co., Ltd., Shanghai, China), prepared by mixing equal volumes of solutions A and B in the dark. Images were captured with a Bio-Rad imaging system (Bio-RAD, USA), and band intensities were quantified using Quantity One software version 4.6.2. Protein expression levels were normalized to GAPDH and expressed as the ratio of gray values. Each experiment was repeated three times, and average values were reported (Lee et al. 2023; Ma et al. 2021; Ahuja et al. 2022; Lin et al. 2022; Zeng et al. 2022).

Active compounds of rhein were retrieved from the TCMSP database (https://old.tcmsp-e.com/tcmsp.php↗), applying screening thresholds of oral bioavailability > 30% and drug-likeness > 0.18. Target genes corresponding to these compounds were annotated using the UniProt database (https://www.uniprot.org/↗). NAFLD-related genes were identified from the GeneCards database using “Non-alcoholic fatty liver disease” as the search term. The intersection between drug targets and disease-associated genes was used to build the network. This information was imported into Cytoscape software (version 3.8.0) to construct a drug-ingredient-target-disease network (Liu et al. 2020).

Candidate target genes were subjected to KEGG pathway enrichment using the “ClusterProfiler” package (http://www.bioconductor.org/packages/ClusterProfiler/↗) in R language, with P-value < 0.05 setting as the threshold for significant enrichment. The top enriched signaling pathways were visualized using the “ggplot2” package (http://www.bioconductor.org/packages/ggplot2/↗) (Zhong et al. 2023).

All data were analyzed using SPSS statistical software version 24.0 (SPSS, Inc., USA). Normality and homogeneity of variances were assessed. Normally distributed measurement data were presented as mean ± standard deviation. For comparisons between two groups, independent-sample t-tests were utilized, while for multiple-group comparisons, one-way analysis of variance with Tukey’s post hoc test was employed. Statistical significance was considered at a P-value < 0.05 (De et al. 2024).

These results indicate that rhein effectively alleviates HFD-induced lipid accumulation and improves fat metabolism.

In brief, rhein alleviates liver injury in NAFLD mice by improving liver function and inhibiting inflammation.

To determine whether these regulatory effects are AMPK-dependent, an AMPK-IN-3 intervention group was established. Comparison between the NAFLD + DMSO group and the AMPK-IN-3 group revealed that AMPK-IN-3 further decreased p-AMPKα1 and p-ACC levels and upregulated SREBP1 expression, indicating suppression of AMPK activity under NAFLD conditions (Fig. 5B).

Notably, while high-dose rhein alone increased p-AMPKα1 and p-ACC and reduced SREBP1 levels, co-administration with AMPK-IN-3 markedly weakened or reversed these effects. This suggests that rhein’s modulation of ACC and SREBP1 is dependent on AMPK pathway activation (Fig. 5B).

These results imply that rhein alleviates hepatic lipid accumulation in NAFLD mice by activating the AMPK signaling pathway, thereby enhancing ACC phosphorylation and downregulating SREBP1 expression.

To determine whether the improvement of lipid accumulation in NAFLD by Rhein is mediated through the AMPK signaling pathway, four experimental groups were established: NAFLD + DMSO, NAFLD + AMPK-IN-3, NAFLD + High-Rhein + DMSO, and NAFLD + High-Rhein + AMPK-IN-3, to evaluate the impact of AMPK inhibition on the metabolic benefits of Rhein.

Organ mass analysis (Fig. 6 C) revealed that compared with the NAFLD + DMSO group, AMPK-IN-3 treatment further increased the weights of retroperitoneal fat, liver, and kidneys. Rhein treatment effectively reduced these organ weights, but its protective effects were partially reversed when co-administered with AMPK-IN-3.

Oil Red O staining (Fig. 6D) showed extensive lipid droplet accumulation and pronounced vacuolar degeneration in the AMPK-IN-3 group. In contrast, rhein treatment reduced lipid droplet accumulation and decreased the area of adipocytes, while the co-treatment with AMPK-IN-3 significantly attenuated these improvements (P < 0.001).

In terms of blood lipid levels (Fig. 6E), compared with the NAFLD + DMSO group, AMPK-IN-3 treatment increased the levels of TG and LDL-C, while decreasing HDL-C (P < 0.001). In contrast, rhein intervention showed a trend of decreasing TG and LDL-C levels and increasing HDL-C levels. However, in the NAFLD + High-Rhein + AMPK-IN-3 group, the regulatory effect of rhein on blood lipids was attenuated.

Taken together, these findings confirm that the AMPK signaling pathway plays a key role in mediating the beneficial metabolic effects of rhein. The reversal of these effects by AMPK-IN-3 suggests that rhein’s action in improving lipid metabolism in NAFLD is AMPK-dependent.

The results of inflammatory cytokine analysis (Fig. 7B) further supported this conclusion: the levels of TNF-α and IL-6 in the NAFLD + AMPK-IN-3 group were higher than those in the NAFLD + DMSO group (P < 0.05), while rhein could effectively reduce these inflammatory cytokines. However, co-treatment with AMPK-IN-3 significantly weakened this anti-inflammatory effect (P < 0.001).

HE staining (Fig. 7 C) revealed that liver tissues in the NAFLD + DMSO and AMPK-IN-3 groups exhibited disorganized cellular structure and extensive lipid vacuolization. In contrast, the high-dose rhein group displayed more organized hepatocyte architecture and reduced inflammatory infiltration. However, this improvement was reversed in the group treated with both rhein and AMPK-IN-3, indicating that AMPK inhibition counteracts rhein’s beneficial histological effects.

To sum up, these results provide further evidence that rhein alleviates hepatic injury and inflammation in NAFLD mice primarily through activation of the AMPK signaling pathway.

Although this study provides compelling evidence that rhein exerts hepatoprotective effects in a HFD-induced NAFLD mouse model by activating the AMPK signaling pathway—promoting ACC phosphorylation and inhibiting SREBP1 expression—several limitations should be noted. First, the study utilized only male mice, without assessing the influence of sex-based differences on NAFLD progression or the therapeutic efficacy of rhein. This is particularly relevant given the well-established gender dimorphism in NAFLD incidence and pathology observed in clinical settings. Second, while the HFD-induced mouse model is widely employed for NAFLD research, it does not fully replicate the pathological complexity of human NAFLD, particularly in patients with coexisting metabolic syndrome. This may limit the translational applicability of the findings. Third, the lack of comparison with classical AMPK agonists such as AICAR or Metformin precludes a direct evaluation of rhein’s relative efficacy and pharmacological advantages. Finally, the absence of validation using human clinical samples—such as liver tissue or serum from NAFLD patients—prevents confirmation of whether rhein modulates AMPK signaling and its downstream targets in a clinically relevant context. Future studies should incorporate both male and female animal models, develop composite diet-induced models that better resemble human NAFLD pathology, and integrate multi-omics analyses alongside human samples to enhance the clinical relevance of the findings. Additionally, the potential synergistic effects of rhein in combination with established AMPK activators warrant further investigation.