scFOS including 1-Kestose, Nystose and 1^F-Fructofuranosylnystose were obtained from FUJIFLM Wako Pure Chemical Corporation (Osaka, Japan). The purity of scFOS measured by high performance liquid chromatography (Wakoosil 5NH₂, Shimadzu LC-20, Japan) was ≥99%.

GES-1 cell was obtained from the American Type Culture Collection (ATCC). Cells were cultured in HyClone RPMI medium with 10% fetal bovine serum (FBS, PAN-Seratech, Heilbronn, Germany) and 0.5% penicillin/streptomycin solution (Gibco, NY, United States) in a humidified incubator (Thermo Fisher Scientific, Langenselbold, Germany) at 37°C, 5% CO₂. GES-1 cells were seeded in 6-well plates, 96-well plates or confocal microplates at a density of 1.5 × 10⁵ cells/mL for the subsequent treatment. After being treated, GES-1 cells were lysed with RIPA Lysis Buffer (Beyotime, Beijing, China) to collect cell lysates for subsequent experimental assays.

To explore the method of establishing ethanol-induced GES-1 cell damage model, cells were exposed to ethanol at various concentrations: 5, 6, 7, 8, and 9% for 2 h to determine cell viability. The ethanol concentration corresponding to a cell viability of approximately 60% was selected as the optimal modeling concentration. To determine the administration concentration of scFOS on GES-1 cells, cells were exposed to 1-Kestose, Nystose and 1^F-Fructofuranosylnystose (dissolved in PBS) at various concentrations: 25, 50, 100 and 200 μg/mL for 24 h, following cell viability measurement. GES-1 cells were pretreated with scFOS at the selected concertation for 24 h, following ethanol exposure at the selected concertation for 2 h. The ameliorative effects of scFOS on cell viability, oxidative stress and inflammation were subsequently evaluated.

Cell counting kit-8 assay kit (CCK-8, Beyotime, Beijing, China) was used to determine cell viability. GES-1 cells after treatment were incubated with100 μL of CCK-8 working solution (fresh medium and CCK-8 reagent were mixed in a 10:1 ratio) at 37°C and 5% CO₂ for 60 min in the dark, the absorbance of each well was determined at 450 nm by a microplate reader (Spectra Max plus³⁸⁴; Molecular Devices, United States).

The MDA content and SOD activity were determined separately using commercial kits according to the instructions (Beyotime, Beijing, China). The protein level of GES-1 cells was determined using KeyBio BCA Protein Assay Kit (KeyBionet, Nanjing, China) to normalize the MDA and SOD activity levels.

The intracellular ROS levels and superoxide anion (O²⁻) production were detected by using dichloro-fluorescein (DCFH-DA) and dihydroethidium (DHE) probes, respectively, (Beyotime, Beijing, China).

After being treated with scFOS and ethanol, GES-1 cells were exposed to 10 μM DCFH-DA or 5 μM DHE, and then incubated at 37°C under light protection for 30 min. The ROS levels were measured using a fluorescent enzyme labeling instrument and DHE fluorescent images were taken under a confocal microscope (Zeiss LSM 700, Carl Zeiss AG, Germany).

ELISA kits (Ruixin Biotechnology, Shanghai, China) were utilized to quantify the quantities of TNF-α, IL-1β and IL-6 in the cell lysates following the manufacturer’s instructions.

Using a NO assay kit (Jiancheng Bioengineering, Nanjing, Jiangsu, China), NO concentration in cells is gauged by the transformation of NO in cell lysates into nitrate and nitrite with a NO assay kit. The absorbance of each well was measured at 550 nm using a microplate reader.

Mitochondrial Membrane Potential Assay Kit with JC-1 (Solarbio, Beijing, China) was used to measure changes in mitochondrial membrane potential caused by ethanol in GES-1 cells. JC-1 monomer fluoresces (green) were observed at excitation/emission (Ex/Em) 515 nm/529 nm, and JC-1 polymer fluoresces (red) were detected at excitation/emission (Ex/Em) 585/590. Changes in the potential of the mitochondrial membrane were detected by the shift in color of the fluorescence. Treated GES-1 cells in confocal dishes were incubated with JC-1 reagent (10 μM, 1 mL/well) for 20 min at 37°C protected from light and then imaged with confocal microscopy. The ratio of red to green fluorescence was utilized for determining changes in the mitochondrial membrane potential.

Apoptosis of GES-1 cells was assessed using the One Step TUNEL Apoptosis Assay Kit (Beyotime, Beijing, China). Briefly, cells were fixed with 4% paraformaldehyde (Beyotime, Beijing, China) for 30 min and then incubated with Terminal Deoxynucleotidyl Transferase containing fluorescein-dUTP for 60 min at 37°C. Finally, an Antifade Mounting Medium (Beyotime, Beijing, China) was added and then visualized by confocal microscopy.

GES-1 cells were treated and immobilized with 4% paraformaldehyde for 30 min, then blocked with 5% skim milk powder (Wako Pure Chemical, Osaka, Japan) for 2 h. Cells were incubated with anti-Nrf2 antibody (16396-1-AP, 1:1000; Proteintech, Chicago, IL, United States) at 4°C overnight. The next day, they were incubated with a fluorescent secondary antibody for 1 h. Antifade Mounting Medium with DAPI (Beyotime, Beijing, China) was added and images were taken with confocal microscopy.

The protein concentration in the cell lysate was quantified using KeyBio BCA Protein Assay Kit (Beyotime, Beijing, China). Following thorough mixing with loading buffer, lysates were boiled for 5 min. Following a 10% SDS-PAGE separation, the proteins were electrophoresed at 300 mA for 1.5 h, and subsequently transferred to a PVDF membrane (Millipore, MA, United States). The PVDF membranes were blocked with 5% skim milk powder for 2 h at room temperature and then co-cultured with the primary antibody overnight at 4°C. The next day, the PVDF membranes were co-incubated with secondary antibody for 1.5 h at room temperature. Finally, the membranes were immersed in ECL chemiluminescent solution, detected by Tanon Imager 4,600 system (Tanon, Shanghai, China) and analyzed by Image J software (NIH, Bethesda, MD, United States).

The experimental data were analyzed by Graph Pad Prism version 8.0 (Graph Pad Software, San Diego, CA, United States). Results were expressed as means ± SEM, and the differences were analyzed by One-way ANOVA with Dunnett’s multiple comparison test, where p < 0.05 was considered statistically significant.

The chemical structures of the three scFOS were shown in Figure 1A. Figure 1B showed that the viability of GES-1 cells decreased with the increase of ethanol concentration from 5 to 9% with incubation for 2 h. According to our results and literature search, we selected 7% ethanol with an inhibition rate of about 40% as (Model, M) concentration for our further study (28).

The cell viability of GES-1 cells treated with different concentrations of scFOS for 24 h was shown in Figure 1C. scFOS at the concentration from 0 mg/mL (Control, C) to 200 mg/mL for 24 h did not affect GES-1 cells viability. scFOS at concentrations of 50 and 100 μg/mL were selected for subsequent experiments.

As shown in Figure 1D, GES-1 cells treated with the three scFOS at 50 μg/mL and 100 μg/mL for 24 h markedly improved cell viability compared to that in the M group. After the aforesaid ethanol treatment, the cells were found to be shrunken and rounded. However, the morphology of cells pretreated with scFOS was significantly improved (Figure 1E). These results indicated that scFOS improved cell viability and morphology in ethanol-exposed GES-1 cells.

The O²⁻production in GES-1 cells detected using DHE staining was shown in Figure 2A. O²⁻ over-production was observed in GES-1 cells under 7% ethanol, and significantly reversed under scFOS administration. The DCFH-DA staining showed that ethanol exposure promoted intracellular ROS production, which was attenuated after scFOS treatment (Figure 2B). As shown in Figure 2C, the SOD activity of the M group was significantly reduced, while scFOS treatment significantly elevated SOD activity. Meanwhile, Nystose (N1, N2) and 1^F-Fructofuranosylnystose (F1, F2) pretreatment exhibited better enhancement of SOD activity in ethanol-damaged GES-1 cells. As shown in Figure 2D, scFOS pretreatment significantly decreased the MDA content in comparison to that in M group. Among them, Nystose (N1, N2) and 1^F-Fructofuranosylnystose (F1, F2) pretreatment could better reduce the MDA content in ethanol-exposed GES-1 cells. These findings suggested that pretreated with scFOS could attenuate ethanol-induced oxidative stress by suppressing ROS production and lipid peroxidation, as well as increasing intracellular SOD activity, of which Nystose and 1^F-Fructofuranosylnystose showed a better improvement effect.

Figure 3A showed that the Nrf2 expression level was upregulated and Keap1 expression was downregulated in the scFOS group in comparison with the M group. As shown in Figure 3B, compared with the C group, the expression of HO-1, SOD1 and SOD2 proteins in the M group was significantly decreased. Compared with the M group, HO-1, SOD1 and SOD2 protein expression of the scFOS administration group was significantly reduced to a level close to the normal group. Indeed, Nrf2 immunofluorescence also revealed an accumulation of nuclear Nrf2 in the scFOS treatment group, while Nrf2 expression in the cytoplasm was significantly reduced in comparison with those in the M group (Figure 3C). In addition, nuclear morphology was observed using DAPI staining, while cells with nuclear fragmentation were considered to be a marker of apoptosis (29). Compared to the C group, the nucleus of the ethanol-treated group was fragmented and the morphology was abnormal, which was improved after scFOS pretreatment. Therefore, scFOS could trigger the degradation of Keap1 and promote the entry of Nrf2 into the nucleus, thereby increasing the expression of antioxidants such as HO-1 and SOD to reduce the level of oxidative stress in cells. These results demonstrated that scFOS could improve ethanol-induced oxidative stress in GES-1 cells by regulating the Nrf2/Keap1 signaling pathway.

The production of NO can be used as a predictor of the inflammatory effects in the disease process. As shown in Figures 4A,B, ethanol significantly increased NO and iNOS production in GES-1 cells in comparison to the C group. In the scFOS treatment group, all three scFOS could correct the significant decrease in iNOS content induced by ethanol. However, 1-Kestose and Nystose administration significantly reduced NO production in comparison with the ethanol-treated group, while there was no significant change in the 1^F-Fructofuranosylnystose-treated group, indicating the potential of scFOS to suppress the inflammatory response.

Proinflammatory cytokines, including TNF-α, IL-1β and IL-6, are crucial mediators in the majority of inflammatory pathologies. Therefore, we further determined the effect of scFOS on the release of TNF-α, IL-1β and IL-6 in ethanol-exposed GES-1 cells. As demonstrated in Figure 4C, ethanol treatment considerably increased TNF-α, IL-1β and IL-6 levels in comparison to the C group, while scFOS administration dramatically decreased these inflammatory factor levels. The above results suggested that scFOS may exert excellent anti-inflammatory effects in ethanol-treated GES-1 cells.

Apoptosis can be detected early by observing the change in JC-1’s fluorescence from red to green. The red/green fluorescence ratio of the M group was considerably reduced following ethanol treatment for 2 hours, as seen in Figure 4D. All three scFOS significantly increased the red/green fluorescence ratio in comparison with that in the ethanol-treated GES-1 cells, with 1-Kestose and Nystose exhibiting a better improvement effect than 1^F-Fructofuranosylnystose. JC-1 staining results showed that all scFOS treatment groups could improve ethanol-induced apoptosis in a dose-dependent manner, which was further confirmed by TUNEL analysis. As shown in Figure 4E, the positive staining cells in the M group were significantly elevated than those in the C group and scFOS treatment group.

The NLRP3 signaling pathway is crucial for both apoptosis and the inflammatory response. Western blot analysis showed that ethanol induction increased the expression of NLRP3, apoptosis-associated speck-like adaptor protein (ASC) and cysteinyl aspartate specific proteinase 1 (Caspase-1) (Figure 4F). However, pretreatment with three scFOS significantly inhibited ethanol-induced activation of these proteins, suggesting that scFOS can also prevent ethanol-induced inflammatory response and apoptosis by inhibiting NLRP3 signaling.