The human hepatoma (HepG2) cell line was grown in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose, supplemented with 10% fetal bovine serum (FBS), in a humidified atmosphere at 37°C with 5% CO₂. The cells were allowed to grow until they reached approximately 80% confluence before the start of the experiments.

The preparation of ML was reported previously (Wang et al., 2022). Briefly, the dried herb of K. indica (2.5 kg) was soaked in water for 60 min and refluxed with water (10 L) for 3 times (1.5 h each). After combining the ML extraction, 0.43 kg of the extract was obtained under vacuum.

Dried K. indica (10.0 g) was powdered and soaked in 100 mL 70% methanol solution (v/v) for 0.5 h and extracted by reflux condensation for 1 h. The mixture was then filtered, and the filter residue was treated with 50 mL of a 70% ethanol aqueous solution and refluxed for an additional 0.5 h. The two filtrates were combined and concentrated to a final volume of 15 mL. The extract of K. indica was subsequently placed in a 4°C environment for 24 h, after which the supernatant was obtained through filtration. The extraction was centrifuged at 3,000 rpm for 15 min and the supernatant was collected after filtering through 0.22 μm membrane for HPLC-QTOF-MS analysis.

Sample separation was carried on an Agilent 1260 HPLC system coupled with XBridge BEH C18 Column (5 μm, 4.6 mm × 250 mm) with a mobile phase consisting of water 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The gradient elution program was set as follows: 0–5 min 3% (B), 5–35 min 3%–99% (B), 35–40 min 99%–10% (B). Agilent 6530 QTOF MS was used to achieve the identification, positive and negative mode was used in the analysis and the injection volume was 5 μL at the flow rate of 0.6 mL/min.

Reactive oxygen species (ROS) levels were measured in HepG2 cells using a commercial kit protocol. Cells were pre-incubated with different concentrations of ML or without ML, and then exposed to 5% alcohol. After treatment, cells were washed and incubated with a ROS probe. Fluorescence intensity was measured using a microplate reader. Results were presented as fluorescent intensity, indicating ROS levels under different treatment conditions.

The study protocol received approval from the Ethics Committee for Animal Experiments of Liaoning University. All mice were housed in a controlled environment with regulated temperature and lighting. Following a one-week acclimation period, pathogen-free (SPF) grade male C57BL/6 mice were randomly divided into five groups (n = 7 for each group): Control (CON), Model (MOD), Low dose group (ML-L), High dose group (ML-H), and Silymarin (Sil). All mice were fasted for 2 h before daily dosing. Mice in the LD/HD/SD groups received ML or silymarin via gavage for 15 days. From the 10th days, 56% ethanol (5 g/kg bw) was administered to the mice, except for those in the control group, 2 h after ML/silymarin/saline administration. ML and silymarin were prepared using saline.

The liver tissue samples, each weighing 60 mg, were mixed with 540 μL of saline in a cold environment to create a homogenate. The mixture was then centrifuged at 3,500 rpm and 4°C for 15 min to separate the components. The resulting supernatant was carefully collected for further analysis. Levels of reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) were measured using commercially available kits (Shanghai Enzymelinked Biotechnology Co., Ltd., Shanghai, China). Additionally, the protein concentration was determined using a BCA reagent kit.

The levels of serum ALT and AST, which are indicators of liver function, were measured using commercially available kits (Biosino Bio-Technology and Science Inc. Beijing, China). Fresh livers were carefully collected, and a small portion of each mouse liver was preserved in 4% phosphate-buffered paraformaldehyde for subsequent staining with a combination of hematoxylin and eosin (H&E). Following a 24-h fixation period, the livers were embedded in paraffin blocks and cut into thin 9-μm slices. These slices were then stained with H&E to visualize the cellular structure and were observed under a bright-field light microscope for further analysis.

To assess the expression of Nrf2 and HO-1 proteins, paraffin-embedded liver tissue samples were sectioned into 4 μm slices and then dewaxed and rehydrated. The sections were treated with a 10 μmol/L citrate buffer, microwaved for 15 min, cooled, and rinsed with PBS thrice. A peroxidase blocking solution (50 μL) was applied, followed by a 20-min incubation at room temperature, and another three rinses with PBS. The sections were then serum-blocked for 30 min. After removing the serum, diluted antibodies against HO-1 and Nrf2 (servicebio, China) were added and incubated overnight at 4°C. After three additional PBS rinses, the sections were incubated with horseradish peroxidase-labeled secondary antibodies for 50 min. DAB chromogenic solution was applied for staining, followed by reverse hematoxylin staining. The sections were dehydrated and sealed.

Total genomic DNA samples were extracted using the OMEGA Soil DNA Kit (M5635-02) (Omega Bio-Tek, Norcross, GA, United States) following the manufacturer’s instructions, and were stored at −20°C before further analysis. PCR amplification of the bacterial 16S rRNA genes V3–V4 region was performed using the forward primer 338F (5′-ACT​CCT​ACG​GGA​GGC​AGC​A-3′) and the reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′). PCR amplicons were purified with Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, United States). After the individual quantification step, amplicons were pooled in equal amounts, and pair-end 2 × 250 bp sequencing was performed using the Illlumina NovaSeq platform with NovaSeq 6000 SP Reagent Kit (500 cycles) at Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China). The data were analyzed on the online platform of Personalbio Genescloud (https://www.genescloud.cn/).

All data were presented as mean ± SEM. Differences between groups were compared using One-way ANOVA analysis. p value <0.05 was considered statistically significant.

We used HPLC-Q-TOF-MS experiment to reveal the components of ML. Consequently, 16 constituents were identified based on the high-resolution experimental ion peaks, including p-hydroxybenzoic acid, indole-3-carbaldehyde, chlorogenic acid, apigenin-7-O-glucoside, etc (Figure 1; Table 1).

The fluorescence of DCF in each group was measured. As illustrated in Figure 5, a significant increase in absorbance was observed when treating 5% alcohol alone, indicating that alcohol stimulates the production of ROS in HepG2 cells. However, after incubation with moderate and high doses of ML (150 and 300 μg/mL), ROS levels were significantly reduced, as evidenced by the decreased absorbance. Therefore, ML was revealed to be capable of reducing alcohol-induced oxidative stress in HepG2 cells (Figure 2).

To assess the extent of in vivo liver damage caused by excessive alcohol consumption, we analyzed the levels of ALT and AST in the serum with silymarin as the positive drug since it had been reported to show a protective effect against alcohol-induced liver damage. As shown in Figure 3A, alcohol treatment significantly elevated ALT and AST levels in the serum, which were all ameliorated by the ML administration. H&E staining gave a similar result, showing the disordered structure of hepatic lobule, and plenteous large and small lipid vacuoles, which are typical pathological changes of ALD. The protective effect of the high-dose group was evident, showing a relatively normal structure of hepatic lobule and reduced lipid droplets compared with the model group. However, the protective effect of the low-dose group was relatively weak compared with the high-dose group (Figure 3B).

To assess the in vivo effect of ML on liver oxidative stress, the level of GSH, SOD, CAT, and MDA were tested. GSH is a non-enzymatic antioxidant that plays a vital role in eradicating free radicals, which helps to reduce oxidative stress. SOD and CAT are two enzymatic antioxidants. MDA is a byproduct of lipid peroxidation that can be inhibited by GSH, SOD, and CAT. As a result, upregulated GSH, SOD, and CAT levels and downregulated MDA levels were observed after alcohol treatment, while ML administration could partially reverse these changes in GSH, SOD, CAT, and MDA levels (Figure 4).

To further study the mechanism of ML’s protective effect on alcohol-induced liver injury, immunohistochemical experiments were conducted. The results indicated that ML treatment further activated Nrf2 to elicit an anti-oxidative stress effect (Figure 5). Moreover, alcohol treatment suppressed the HO-1 level, while ML administration reversed the HO-1 level (Figure 5).

To assess the effect of ML on microbiota, we conducted 16S rRNA sequencing experiments. The results showed that there is a certain degree of clustering between samples, indicating that the similarity of community composition within groups is relatively close, and there is obvious separation between groups without overlapping parts, suggesting significant differences between populations (Figure 6A). After intervention with ML, there were significant changes in the gut microbiota of mice. Detailed analysis indicated differences in microbial composition and abundance at the phylum, family, genus, and species levels among the blank group, model group, and ML treatment group. In addition to the differences in microbial communities in each group, it was suggested that ML can lead to different secretion of metabolites by microorganisms, thereby indirectly regulating liver metabolism through the “hepatointestinal axis” (Figure 6B). To be specific, the microbiota in the model group, which is significantly different from the blank group and the ML group, is mainly Limosilactobacillus. The ML group has a significantly richer variety of microbial communities compared to the other two groups, with the representative species being the Cryptobacteroides genus, UBA932 family; The genus Helicobacter in the family Helicobacteraceae (Figure 6C).