ELG and chemicals preparation

Dry G. pentaphyllum (Thunb.) Makino, C. cassia (L.) J. Presl, and A. grossedentata (Hand-Mazz) W.T. Wang were purchased from Anhui Huangshan Greenxtract Co., Ltd. (Huangshan, China). ELG was prepared by grinding 450 g of G. pentaphyllum (Thunb.) Makino, 100 g of C. cassia (L.) J. Presl, and 450 g of A. grossedentata (Hand-Mazz) W.T. Wang. Subsequently, the three herbs were combined and subjected to boiling in a 10X volume of 75% ethanol at 100 °C for 2 h for the initial extraction, followed by 1.5 h for the ubsequent extraction. The combined ethanol extract was filtered and concentrated using a rotary evaporator (RE-2000A, Yarong, Shanghai, China) at 50 °C under reduced pressure. After cooling down, the extract was freeze-dried into 100 g of ELG powder using a freeze dryer (LGJ-10N, Yaxingyiqi, Beijing, China). ELG powder was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mg.mL−1 for subsequent experiments. Lipopolysaccharides (LPS) and compound C (CC) were purchased from Sigma–Aldrich (St. Louis, MO, United States), with a purity of > 98% for both LPS and CC.

Ultra-Performance Liquid Chromatography Quadrupole Time of Flight Mass Spectrometer (UPLC-Q-TOF–MS)

ELG powder (250 mg) was dissolved in 25 mL of 75% ethanol. To facilitate dissolution, this solution underwent sonication twice at 300 W and 40 kHz, followed by centrifugation at a speed of 12, 000 rpm for 5 min. The UPLC was performed using UPLC/Q-TOF–MS (Sciex Triple TOF 4600 high resolution mass spectrum coupled with an Agilent 1290 UPLC system; AB Sciex, Darmstadt, Germany; Agilent, Los Angeles, CA, USA). For separation, the UPLC system was utilized with HSS T3 columns from Acquity (2.1 × 100 mm, 1.8 μm; Waters, Milford, CT, USA), and 2 µL of the supernatant was injected. A solvent system comprising 0.1% formic acid-acetonitrile (phase A) and 0.1% formic acid–water (phase B) was used for gradient elution with a flow rate of 0.3 mL/min. The elution program was as follows: 10% A-20% A 0–5 min, 20% A 5–9 min, 20% A-30% A 9–16 min, 30% A-45% A 16–26 min, 45% A-95% A 26–32 min, 95% A 32–36 min, 95% A-10% A 36–37 min, and 10% A 37–40 min. The specific gradient elution program of the mobile phase was illustrated in Additional file 1: Table S1.

Treatment and culture of cells in vitro

The AML-12 liver cell line derived from mice was obtained from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were grown in DMEM-F12 medium (Biological Industry) containing 10% fetal bovine serum (FBS), penicillin, streptomycin, 0.1 mM dexamethasone, and a combination of insulin-transferrin-selenium (ITS). The cells were incubated in a cell culture incubator (Heracell Series, Thermo Fisher Scientific, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37 °C with 5% CO2.

For in vitro ethanol or inflammation modeling and subsequent cell experiments, the AML-12 cells were cultured in DMEM-F12, and then subjected to ethanol (500 mM) [33] or LPS (100 ng.mL−1) [34] with or without the addition of DMSO (0.1%), CC (10 μM), and the ELG at specified concentrations for 24 h.

Animals treatment

The animal experiment protocols were authorized by the Shanghai University of TCM (approval number: PZSHUTCM211115002). All animal experiments were based on the ARRIVE animal experimentation guidelines [35]. C57BL/6 wild-type (WT) male mice, aged 10 weeks and weighing 20–22 g, were obtained from the SLAC Laboratory in Shanghai, China. The mice were kept in a controlled environment with a temperature of 22–23 °C and a 12-h light/dark cycle, ensuring specific pathogen-free conditions. To generate an acute ethanol-binge model, the mice were randomly assigned to the normal WT control (control, n = 7), AALI model (AALI, n = 7), low-dose ELG (50 mg.kg−1 i.g.) treatment (AALI + ELGL, n = 7), and ELG (100 mg.kg−1 i.g.) treatment (AALI + ELG, n = 7) groups according to their body weights. ELG was administered to the experimental mice once a day for 2 weeks before ethanol administration. ELG dosage was determined by preliminary studies [28, 36, 37] and the results of our pilot studies. Following the conclusion of the previous ELG administration, the mice were subjected to an overnight fasting period and subsequently gavaged with 56% (v/v) ethanol at a dose of 5 g.kg−1 body weight. The normal control mice received an equivalent volume of double-distilled water. After a 16-h interval, the mice were euthanized under the influence of 20% urethane (Sigma, St. Louis, MO) anesthesia, and samples of their cardiac blood and liver tissue were collected. The mouse body weight and liver weight were measured to calculate relevant ratios.

Serum lipids analysis

To separate the serum from the plasma, the blood samples from mice were centrifuged at 3000 rpm using a freezing centrifuge (5810R, Eppendorf, Eppendorf AG., Hamburg, Germany) for 15 min. The levels of serum triglyceride (TG) and total cholesterol (TC) levels were measured utilizing an automatic biochemical analyzer (Hitachi 7020, Tokyo, Japan).

Detection of serum liver enzyme levels

The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in both mouse serum and cell culture supernatant were measured utilizing commercial kits (Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions. After the specified treatment for 24 h, the AML-12 cell culture supernatants were collected from different groups.

Liver histopathology

The paraffin-embedded liver tissues were sliced into sections measuring 5 μm in thickness. These sections were then subjected to staining with hematoxylin and eosin (H&E), following established protocols. To perform the Oil Red O staining, the tissue sections were first prepared with the OCT and subsequently stained with Oil Red O. Finally, all sections were examined under a light microscope (CX23, Olympus, Olympus Corporation., Tokyo, Japan).

Determination of liver TG and TC content

The hepatic lipid content was measured in accordance with previous descriptions [38]. Briefly, the liver tissues were weighed and homogenized, followed by lipid extraction with addition of a combination of chloroform and methanol (2:1, v/v) in equal proportions. The resulting lipids-containing layer was collected and dissolved with isopropanol. The hepatic levels of TC and TG were then measured using commercial kits (Jiancheng Bioengineering Institute, Nanjing, China). The weight of liver tissues served as the standard.

Analysis of relative mRNA expression

The total RNA from mouse tissues and cells was extracted using TRIzol reagent (Invitrogen Life Technologies, MA, USA). The first-strand cDNA was synthesized and subjected to quantitative real-time PCR (qRT-PCR) analysis using SYBR qPCR Master Mix (ABclonal Technology Co., Ltd., Wuhan, China). The ABI StepOnePlus real-time PCR system (Applied Biosystems, USA) was used to analyze the gene expression levels based on the 2-ΔΔCt method. β-Actin was used as the internal reference for normalizing all gene expression data. All primer sequences are provided in Additional file 1: Table S3.

Western blotting

10% SDS-PAGE was used to separate the cells and mouse liver whole protein, and the isolated protein was subsequently transferred onto nitrocellulose membranes. These membranes were blocked with 5% skim milk powder and then incubated with primary antibodies against β-Actin (#3700), phosphorylation of JNK (p-JNK, #4668) (Cell Signaling Technology), AMPK (#AF6423), cleaved Caspase-1 (cle-CASP1, #AF4022), phosphorylation of AMPK (p-AMPK, #AF3423), cleaved Caspase-3 (cle-CASP3, #AF7022) (Affinity Biosciences), C/EBP homologous protein (CHOP, #15204-1-AP), JNK (#15204-1-AP) (Proteintech Group), and NLRP3 (ABclonal Technology, Wuhan, China, #A12694) at a dilution of 1:1000, and incubated overnight at 4 °C. Following this, the membranes were exposed to secondary antibodies labeled with horseradish peroxidase, diluted at a ratio of 1:5000, and incubated for 2 h at room temperature. The protein bands were visualized through chemiluminescence.

Enzyme-linked immunosorbent assay (ELISA)

A volume of 100 μL of mouse serum or AML cell supernatant was taken for IL-1β measurement by using ELISA Assay Kits (ABclonal Technology), following the instructions provided by the manufacturer.

Cell counting kit-8 (CCK8) assessment

Cell viability was assessed using the CCK-8 assay following the guidelines provided by the manufacturer (Yeasen, Shanghai, China). In summary, AML-12 cells were plated in 96-well culture plates with a concentration 1 × 105 cells/well in 100 μL of the medium. The plates were then placed in a CO2 incubator at 37 °C. Following indicated treatments, 10 μL CCK-8 solution was diluted into each well and incubated for another 1 h at 37 °C. The microplate reader (Synergy™4, BioTek, Vermont, USA) was utilized to measure the absorbance values at 450 nm.

Intracellular oxidative stress assay

The total intracellular levels of ROS were measured using dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime, China) according to the manufacturer’s protocol. Briefly, AML-12 cells were rinsed using PBS and subsequently treated with 10 μM DCFH-DA for 30 min at room temperature, while ensuring protection from light. The nuclei were re-stained with 0.5 μg/mL DAPI (Yeasen, Shanghai, China). Following another round of PBS washing, both images and fluorescence intensities were acquired at a wavelength of 488 nm with a Wide Field High Content Screening System (IXM-4, Molecular Devices, USA).

Immunohistochemistry

For immunohistochemistry analysis, the liver Sections (5 μm) were subjected to immunostaining using a primary antibody against NLRP3 (1:50, ABclonal Technology, #A12694) at 4 °C overnight. Following this, secondary antibodies were incubated. The resulting complex was visualized using the DAB reagent for microscopic examination. Hematoxylin was employed to counterstain the nuclei. Finally, the images were photographed with light microscopy (CX23, Olympus, Olympus Corporation., Tokyo, Japan).

Immunofluorescence staining

Immunofluorescence was performed on the 5 μm paraffin-sectioned liver samples fixed in formalin. F4/80 primary antibody (1:100, Affinity Biosciences, #DF2789) was incubated at 4 °C overnight on the sections. Afterward, the sections underwent treatment with secondary antibodies labeled with Alexa Fluor 594 (1:600, Yeasen, Shanghai, China) for 60 min at 25 °C. DAPI (0.5 μg.ml−1, Yeasen, Shanghai, China) was used to stain the nuclei. The images were photographed with an IXM-4 fluorescent microscope (Molecular Devices, USA).

Hepatic malondialdehyde and glutathione determination

The liver tissues (50 mg/mouse) were homogenized with 0.5 mL saline using an ultrasonic method in an ice water bath. Subsequently, the homogenates underwent centrifugation at 12,000 rpm for 10 min at 4 °C. The collected supernatants were used to evaluate hepatic lipid peroxidation by quantifying the production of thiobarbituric acid reactive substances (TBARS) through spectrophotometry, with the outcomes reported as the content of malondialdehyde (MDA) content. The levels of hepatic glutathione (GSH) were determined employing a colorimetric technique. Commercial kits (Jiancheng Bioengineering Institute, Nanjing, China) were employed to measure hepatic MDA and GSH contents following the manufacturer’s guidelines.

Statistical analysis

All data were presented as the means ± SEM. Statistical differences were evaluated using GraphPad 5 (San Diego, CA, US). Significance between two groups with normally distributed data were evaluated by independent two-tailed t-tests. Statistical differences among three or more groups were performed using One-way ANOVA with Bonferroni' s post hoc test. Statistics were considered significant with p < 0.05.