Alantolactone was purchased from Macklin (Shanghai, China) and dissolved in DMSO for experiments in vitro and in 0.5% CMC-Na for studies in vivo. The chemical structure of Alantolactone is shown in Fig. 1A. Antibodies against inhibitor of IκB-α, NF-κB P65 subunit, p-P65 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-TGF-β1 antibody and anti-Lamin B were acquired from Abcam (Cambridge, MA, USA). Antibodies against COL1, α-SMA, Nrf-2, HO-1 and Keap1 were purchased from Proteintech (Wuhan, China). Total cholesterol (TCH), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Total superoxide dismutase assay kit (SOD (superoxide dismutase)) and lipid Peroxidation MDA assay kit (MDA (Malondialdehyde)) were purchased from Shanghai Beyotime Biotechnology (Shanghai, China). Masson’s trichrome staining kit, hematoxylin and eosin (H&E) staining kit, Sirius Red staining kit and Oil red O staining kit were purchased from Solarbio Life Sciences (Beijing, China). Sodium palmitate (PA) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

A Chemical structure of Ala. B Body weight levels in mice. Serum levels of liver function markers AST and ALT (C), and lipids (D–G). H Representative H&E-stained images of liver tissues [scale bar = 100 μm]. I Representative images showing Oil red O staining of liver tissues [scale bar = 100 μm]. Data are expressed as Mean ± SEM, n = 6 per group. ***p < 0.001, ns not significant.

The AML mouse-derived liver cell line was obtained from the Shanghai Institute of Biochemistry and Cell Biology (cat# SCSP-550, Shanghai, China). The cells were cultured in DMEM-F12 medium (Gibco, Eggenstein, Germany) supplemented with 10% FBS (Gibco, Eggenstein, Germany), 100 U/mL penicillin/streptomycin (Gibco, Eggenstein, Germany), 0.1% insulin-transferrin-selenium (ITS, Sigma, Burligton USA), and 40 ng/mL dexamethasone (Sigma, Burligton USA). The culture was maintained in a humidified incubator at 37 °C with 5% CO2.

Male C57BL/6 mice weighing 18–20 g were sourced from the Animal Center of Wenzhou Medical University. All animal care and experimental procedures were conducted in accordance with the guidelines set forth by the Wenzhou Medical University Animal Policy and Welfare Committee (Approval Document No. wydw2019-0145). The mice were housed at a temperature of 22 °C with a relative humidity of 60% and provided with a standard rodent diet and purified water.

Mice were randomly divided into 5 groups: (i) untreated mice receiving 0.5% CMC-Na solution (CON, n = 6) ; (ii) untreated mice receiving 10 mg/kg/2d Ala (Ala 10 mg/kg; n = 6) ; (iii) high-fat diet mice receiving 0.5% CMC-Na solution (HFD; n = 6) ; (iv) high-fat diet mice treated of 5 mg/kg/2d Ala (HFD + Ala 5 mg/kg; n = 6) ; and (v) high-fat diet mice treated of 10 mg/kg/2d Ala (HFD + Ala 10 mg/kg; n = 6).

CON group mice and Ala 10 mg/kg group mice were fed with the low-fat diet. The LFD comprised 10 kcal% fat, 20 kcal% protein, and 70 kcal% carbohydrates (MediScience Yangzhou, China; cat. #MD12031). The HFD consisted of 60 kcal% fat, 20 kcal% protein, and 20 kcal% carbohydrates (cat. #MD12033). The mice were weighed weekly. After 16 weeks of continuous feeding, Ala and 0.5% CMC-Na were administered as oral gavage once every 2 days for another 8 weeks. At the end of treatment, mice were sacrificed under sodium pentobarbital (intraperitoneal [i.p.] injection of 0.2 mL sodium pentobarbital at 100 mg/mL) for anesthesia and pain mitigation. Liver tissues and serum were harvested.

MTT powder (cat. no. M8180, Solarbio; Beijing, China) was dissolved in phosphate-buffered saline. AML-12 cells (4000 per well) were seeded in 96-well plates. After cell adherence, Ala was added to the wells at various doses (0.2–50 μM), followed by incubation for 48 h. Next, MTT was added to each well (1 mg/mL) and incubated for 4 h. The formazan crystals were then dissolved in dimethyl sulfoxide (DMSO; 150 μL/well), and absorbance was examined at 490 nm using SpectraMax M5 microplate reader (Molecular Devices, CA, USA).

Liver tissues of each group were fixed with 4% paraformaldehyde, embedded in paraffin wax and sliced into 5 μm sections. H&E, Sirius red, Masson’s trichrome staining and Oil red O staining were performed using specific commercial kits (Solarbio, Beijing, China).

To assess the ROS level in liver tissues, we conducted DHE staining. DHE powder was dissolved in PBS (20 mM) (Vigorous, Beijing, China). Following PBS washing of frozen liver tissue sections, they were incubated with a DHE dilution (2 μM). Subsequently, the sections were counterstained with DAPI. Images were captured, and analysis was performed using Image J (NIH, Bethesda, MD, USA). The resulting data was presented as relative fluorescence intensity.

Purified total RNA of mouse liver tissues was extracted using the TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s guidelines. Using NanoDrop ND-1000, the amount and purity of RNA in each cardiac sample were measured (NanoDrop, DE). The integrity of the RNA was examined by a Bioanalyzer 2100 (Agilent, CA) and then validated by electrophoresis assay on a denaturated agarose gel. Utilizing Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA), poly (A) RNA is isolated from 1 μg total RNA. The magnesium RNA Fragmentation Module (NEB, cat. e6150, MA) was then utilized to fragment poly(A) RNA into small fragments. SuperScriptTM II reverse transcriptase was then used to convert the cleaved RNA fragments into cDNA (Invitrogen, cat. 1896649, CA, USA). As per the manufacturer guidelines, the sequencing analysis was completed using an Illumina NovaseqTM 6000. After the whole transcriptome generation, all transcripts’ expression levels were quantified. mRNAs with differential expression were identified utilizing the following criteria: fold change < 0.5 or fold change > 2, with p-value < 0.05. Pathway enriched was analyzed using modEnrichr (https://maayanlab.cloud/modEnrichr/).

Cultured cells or liver tissue samples were lysed with Trizol (cat. no. 15596026, Thermo Fisher; CA, USA). Total RNA was extracted and separated using chloroform and isopropanol and purified with ethanol. Reverse transcription was performed using the PrimeScript™ RT reagent Kit (cat. no. DRR037A, Takara Bio Inc., Kusatsu, Japan). Quantitative PCR was performed using TB Green® Premix Ex Taq™ II (cat. no. RR820B, Takara Bio Inc.). Primers (Supplementary Table S1) were obtained from Sangon Biotech (Shanghai, China). mRNA levels were detected and normalized using Actb as the loading control.

Cells or liver tissue samples were lysed using RIPA buffer (cat# P0013B; Beyotime Biological Technology, Shanghai, China). Total proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electro-transferred to polyvinylidene fluoride (PVDF) membranes (cat. no. 1620177, Bio-Rad Laboratory; Hercules, CA). Membranes were blocked in 5% non-fat milk for 2 h at room temperature and cut into bands, followed by incubation with respective primary antibodies. After overnight incubation, protein bands were incubated with HRP-conjugated secondary antibodies and scanned using an image analyzer (Quantity One System; Bio-Rad, Richmond, CA, USA).

All data were presented as mean ± SEM. Prism 8.0 software (GraphPad, San Diego, CA, USA) was used for the statistical analysis. Shapiro–Wilk test was used to assess the normality of our data distribution. Student’s t test was used to compare two groups of data. One-way ANOVA followed by Dunnett’s post-hoc test was used to compare more than two groups of data. A p value < 0.05 was considered as significant.