Rosa roxburghii tratt juice (freshly squeezed, 100% purity) was obtained from East China Institute of Medicinal Plants (Lishui, China), and stored in −4°C refrigerator for use. Lactobacillus plantarum HH-LP56 was purchased from Xian Miser Biotechnology Co., Ltd. Proanthocyanidins from grape seeds were purchased from Shandong Saint Jia De Biotechnology Co., Ltd. Dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) and 95% ethanol were purchased from MilliporeSigma. RIPA Lysis Buffer (cat. no. P0013C), Increased Bicinchoninic Acid (BCA) Protein Concentration Kit (cat. no. P0009), Bodipy 500/510 C1, C12 (Fatty Acid Green Fluorescence Probe) (cat. no. C2055), Hoechst 33342 (cat. no. C1022), SOD (cat. no. S0101S), malondialdehyde (MDA) (cat. no. S0131S) and reactive oxygen species (ROS) assay kit (cat. no. S0033S) were obtained from Beyotime Institute of Biotechnology. 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity assay kit (cat. no. BL897A) and Hydroxyl free radical (·OH) scavenging capacity assay kit (cat. no. BL1065A) were purchased from Biosharp, sourced from Langjiako Technology Co., Ltd. Vitamin C (VC; cat. no. A009-1-1), TG (cat. no. A110-2-1) and CAT assay kit (cat. no. A007-1-1) were purchased from Nanjing Jiancheng Bioengineering Institute. Horseradish peroxidase (HRP)-conjugated goat anti-mouse (cat. no. sc-2354) and goat anti-rabbit (cat. no. sc-2357) antibodies were purchased from Santa Cruz Biotechnology, Inc. Primary antibodies against NAD(P)H: quinone oxidoreductase 1 (NQO1) (cat. no. 11451-1-AP), kelch like ECH associated protein 1 (KEAP1) (cat. no. 80744-1-RR) and GAPDH (cat. no. 60004-1-lg) (all 1:1,000 ratio, respectively) were purchased from Proteintech Group, Inc. Primary antibodies against AMPK (cat. no. PAB44300), NRF2 (cat. no. PAB37815), SCD1 (cat. no. RMAB49932), and SREBP-1c (cat. no. PAB39550) (1:1,000, respectively) were purchased from Bioswamp; Wuhan Bienle Biotechnology Co., Ltd.

A total of 50 ml of Rosa roxburghii Tratt juice were enzymatically treated with tannase and pectinase at 45°C for 100 min, while stirring and filtering to remove precipitates. Prior to preparing the additives, 0.012% (v/v) trichloro-sucrose was dissolved in water at 70°C. A total of 1 g each of lotus leaf extract and grape seed proanthocyanidin powder were separately dissolved in water at ~45°C to form colloidal solutions. When mixing, the procedure started by adding the dissolved trichloro-sucrose, followed by the lotus leaf extract and grape seed proanthocyanidin colloids, followed by stirring for 20 min to ensure uniformity. For particle microencapsulation, the mixture was heated to 65°C, homogenized under a pressure of 200 MPa, then cooled to ~37°C before inoculating with the plant lactobacillus HH-LP56 for fermentation. After 24 h, the fermentation juice production process was completed with ultrasonic treatment (20–22 kHz).

The fermented product was chemically characterized by determining the active substance concentration and antioxidant activity in FRRT. The biological activity of the fermented product was assessed by measuring the VC and SOD content. The SOD content in the fruit juice samples was determined using the WST-1 method in an enzyme-linked immunosorbent assay reader, while the VC content was assessed using UV–Visible spectrophotometry (UV–Vis) at a wavelength of 536 nm. The process involved extracting the VC content, leveraging its specific absorption characteristics at a defined wavelength, and measuring the absorbance intensity to quantify its concentration in the fruit juice. While the antioxidant properties were determined by evaluating OH and DPPH radical scavenging ability in FRRT and RRT juice using specific assay kits according to the manufacturer's protocol.

AML-12 cells were obtained from the Cell Bank of the Chinese Academy of Sciences in Shanghai, China. The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; HyClone; Cytiva) supplemented with 10% fetal bovine serum (FBS; Hangzhou Dacheng Biotechnology Co., Ltd.), penicillin (100 U/ml), streptomycin (100 µg/ml), dexamethasone (40 ng/ml), insulin (5 mg/ml), transferrin (5 µg/ml) and sodium selenite (5 ng/ml) at 37°C under a 95% air and 5% CO₂ atmosphere. Passaging was performed when the cells covered approximately the entire surface of the culture flask, at a ratio of 1:3 to 1:5. Cells from the 3rd to 8th passages in the logarithmic growth phase were selected for subsequent experiments. Before cell treatment, both the FRRT and RRT were filtered using a 0.22-µm membrane.

Cell viability was assessed using the MTT assay. Hepatocytes were seeded in a 96-well culture plate at a density of 2×10⁴ cells per well and cultured until reaching 80% confluency. Subsequently, the cells were divided into different groups. The negative control (NC) group received culture medium without FRRT or ethanol, while the ethanol (EtOH) group was exposed to various concentrations of ethanol (25, 50, 100, 200, 400 and 800 mM) for 24, 36 and 48 h. The experimental group was treated with different concentrations of FRRT (2.5, 5, 10, 20, 40, 80 and 160 mg/ml) for 24 h in a 37°C incubator. The positive control group was treated with different concentrations of RRT (2.5, 5, 10, 20, 40 mg/ml) for 24 h. After the respective incubation periods, MTT solution was added to each well, and the cells were further incubated in darkness at 37°C for 4 h. Following the dissolution of formazan crystals with DMSO (150 µl/well), the absorbance was measured at 490 nm using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = AsampleAcontrol× 100% (1)

AML-12 cells were inoculated on 24-well plates at a density of 5×10⁵ cells per well. After incubation for 24 h in a 37°C incubator, the cells were divided into different groups: NC group, EtOH group (200 mM ethanol), lower dose FRRT group (2.5 mg/ml), higher-dose FRRT group (5 mg/ml) and RRT positive control group (10 mg/ml). AML-12 cells were pretreated with FRRT (2.5, 5 mg/ml) and RRT (10 mg/ml) for 2 h, respectively, and then stimulated with or without EtOH (200 mM) for 24 h. After discarding the supernatant of the prepared cells, they were washed with PBS and the cell pellet were retained. A total of 0.2 to 0.3 ml of homogenization medium was added for homogenization. Following the manufacturer's protocol, TG assay kit was used to measure the TG content in the homogenate. A BCA protein assay kit was used to determine the protein concentration in the homogenate.

After inoculating AML-12 cells on 6-well plates at a density of 2×10⁵ cells per well, they were grouped as per section according to the aforementioned method. Following the cultivation period, the supernatant was discarded and the activity levels of MDA, CAT, GSH and SOD were assessed in the liver cell homogenate using specific assay kits. Additionally, a BCA protein assay kit was used to determine the protein concentration in the liver cell homogenate.

The following culture methods were performed as aforementioned. After the intervention, cells were stained with Hoechst 33342 staining at 37°C for 10 min. Following staining, the cells were washed twice with PBS, ensuring that enough PBS was added to evenly cover them. Finally, the results were examined under an inverted fluorescence microscope.

Following the intervention, cells were stained with Bodipy dye at 37°C for 20 min and then examined under an inverted fluorescence microscope.

After being cultured, the cell culture medium was treated with the fluorescent dye DCFH-DA at a final concentration of 10 µmol/l. The cells were then cultured in a cell incubator at 37°C for 20 min. Subsequently, the cells were washed three times with a serum-free cell medium. Finally, images of three different areas of each well were captured using the Olympus IX84 inverted fluorescence microscope.

After isolating total RNA from AML-12 cells using the TRIzol^® method (Invitrogen; Thermo Fisher Scientific, Inc.), a reverse transcription reaction [Accurate Biotechnology (Hunan) Co., Ltd.] was performed following the cDNA synthesis kit protocol. qPCR was performed using the SYBR Green premix Pro Taq HS qPCR Kit (Accurate Biotechnology (Hunan) Co., Ltd.) under the following conditions: Initial denaturation at 95°C for 30 min, denaturation at 95°C for 5 sec, annealing/extension at 60°C for 30 sec and 40 cycles. A periodic threshold (Cq) was determined and the relative expression of the target mRNA was quantified by the 2^−ΔΔCq method (17) with GAPDH as a normalized reference gene. The qPCR primers used in the present study were synthesized by Hunan Accurate Biotechnology Co., Ltd. and their sequences are listed in Table I.

To quantify protein expression in cells, RIPA lysis buffer was used to lyse cells (contains 1% phosphatase inhibitor and 1% protease inhibitor), and a BCA assay kit was used to determine protein concentration. Each experimental group was adjusted for protein concentration. A total of 20 µg/lane cell lysates were subjected to electrophoresis on a 10% SDS-polyacrylamide gel. Subsequently, 10% SDS-PAGE gels were electrophoretically separated: 80 V for 30 min and 120 V for 60–90 min. Isolated proteins were transferred to a PVDF membrane at low temperature using a transfer device (200 mA, 120 min). After blocking with 5% skim milk at room temperature for 1 h, the membrane was incubated overnight at 4°C with the primary antibody against the target protein. TBST (0.1% Tween-20 solution was used to prepare) was used for washing membranes to remove unbound antibodies, and secondary antibodies (goat anti-rabbit or anti-mouse IgG-horseradish peroxidase; cat. nos. sc-2357 and sc-2354 respectively; Santa Cruz Biotechnology, Inc.) were applied (1:50,000 dilution;.) at room temperature for 1 h. TBST was used to remove unbound antibodies after incubation with the secondary antibody. SuperPico ECL Chemiluminescence Kit (Vazyme Biotech Co., Ltd.) was used to visualize protein bands, and digital gel imaging systems to capture images. Gray-level analysis of the images was performed using ImageJ software (version 1.53e; National Institutes of Health), and the relative expression of the target protein was normalized to the expression of the valet protein GAPDH.

All experiments were performed in triplicate. The experimental data were analyzed using GraphPad Prism 9.5.1 software (Dotmatics), and the results are presented as the mean ± standard deviation (SD) of ≥3 independent experiments performed under identical conditions. For comparisons involving three or more groups, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to determine differences. In cases where there were two groups being compared, unpaired Student's t-test was conducted. Furthermore, ImageJ software was utilized to quantify the grayscale values of protein bands. P<0.05 was considered to indicate a statistically significant difference.

Changes in pH, VC content, SOD activity, ·OH and DPPH radical scavenging rate during the fermentation process of FRRT are demonstrated in Fig. 1. The results demonstrated that the color of the fermented juice significantly lightens (Fig. 1A), and the production of LAB during fermentation led to a decrease in pH of the FRRT compared with RRT (Fig. 1B). Furthermore, there was a significant increase in VC content and SOD activity (Fig. 1C and D). The ·OH and DPPH free radical scavenging abilities of FRRT were enhanced compared with RRT (Fig. 1E and F), which indicated an improved antioxidant capacity post-fermentation (P<0.05).

The results revealed that, as compared with the control group (cells not exposed to ethanol), cell viability decreased with the increase of ethanol concentration. This trend was consistent across all three exposure periods (24, 36 and 48 h; Fig. 2A-C). Specifically, after a 24 h exposure period, cell viability significantly decreased from 100±1.5% in the control group to 86.4±2.9% in cells exposed to 200 mM ethanol (P<0.001). These findings suggested that ethanol hampers cell viability and inhibits the proliferation of AML-12 cells in a concentration-dependent manner. No significant changes were observed in the data at 36 and 48 h when compared with the 24 h data. This could potentially be attributed to the volatility of ethanol. Therefore, the 24 h exposure period and a concentration of 200 mM ethanol were selected to establish an ethanol-induced hepatocyte injury model. Treatment with lower concentrations of FRRT (FRRT-L) (2.5–40 mg/ml) significantly increased cell viability, while higher concentrations (80–160 mg/ml) led to a decrease in cell viability (P<0.001; Fig. 2D). The results of the preliminary research revealed that the overall antioxidant effects of 2.5 and 5 mg/ml were greater than those of ≥10 mg/ml (Fig. S1). Therefore, in subsequent studies, a total of 2.5 and 5 mg/ml as the intervention concentrations were chosen. Additionally, the results demonstrated that treatment with low concentrations of RRT (~2.5–20 mg/ml) significantly increased cell viability, while high concentration of RRT treatment (40 mg/ml) resulted in a decrease in cell viability (P<0.001; Fig. 2E). Therefore, the concentration of 10 mg/ml was selected, which exhibited the most significant enhancement in cell viability, as the positive control group.

The results revealed that the EtOH group exhibited a significant increase in TG content compared with the NC group (P<0.001; Fig. 3A). However, pretreatment with FRRT at concentrations of 2.5 and 5 mg/ml significantly reduced the ethanol-induced increase in TG content in the AML-12 cells when compared with the EtOH group. This reduction occurred in a dose-dependent manner (P<0.001). To further investigate the impact of FRRT on lipid accumulation, BODIPY™ staining was performed. The images obtained from BODIPY staining revealed a substantial accumulation of lipid droplets in the EtOH group (Fig. 3B). However, in the FRRT pretreatment group, both the density and staining intensity of lipid droplets in the cells were significantly reduced compared with the ethanol-treated group. These reductions also exhibited a dose-dependent relationship. These findings suggested that pretreatment with FRRT at concentrations of 2.5 and 5 mg/ml can effectively attenuate ethanol-induced TG accumulation and lipid droplet formation in AML-12 cells. The dose-dependent response indicated that a higher concentration of FRRT (FRRT-H) may provide even stronger protection against lipid accumulation.

To elucidate the molecular mechanism underlying the regulatory effects of FRRT on intracellular lipid metabolism, the expression levels of pivotal lipogenic genes, namely AMPK, SREBP-1c and SCD1 were quantified using qPCR. It was observed that, compared with the NC group, the gene expression of the SREBP-1c and SCD1 gene was significantly increased in the EtOH group, while that of the AMPK gene was decreased (P<0.001). Of note, these alterations in gene expression were effectively reversed by treatment with FRRT, as depicted in Fig. 4B. Furthermore, the protein levels of AMPK, SREBP-1c and SCD1 were examined using western blotting. Compared with the NC group, the group exposed to ethanol exhibited a statistically significant decrease in liver AMPK protein levels (P<0.05). In addition, there was a significant trend towards increased SREBP-1c and SCD1 protein levels in the liver; however, this increase did not reach statistical significance. Markedly, pretreatment with FRRT resulted in a significant reversal of these alterations, particularly with FRRT-H exerting pronounced effects on ethanol-induced changes in AMPK, SREBP-1c and SCD1 protein levels (Fig. 4A).

Superoxide anion is recognized as one of the primary forms of ROS within mitochondria (18). Increased superoxide anion release leads to oxidative stress and apoptosis (19). To visualize hepatocyte damage, DCFH-DA staining was used to detect the distribution and quantity of superoxide free radicals. Antioxidant levels in the cells were assessed by measuring MDA, SOD, CAT and GSH. The results demonstrated that ROS levels in AML-12 cells were significantly increased following ethanol exposure but were reversed following treatment with FRRT, as indicated by fluorescence intensity results (Fig. 5A). Compared with the NC group, the EtOH group exhibited a significant reduction in the intracellular activities of SOD, CAT and GSH, along with a significant increase in MDA levels (Fig. 5B-E; P<0.01). These findings suggested an imbalance in cellular oxidative status, indicating the presence of oxidative injury. However, the pretreatment of cells with FRRT at concentrations of 2.5 and 5 mg/ml resulted in significantly lower MDA levels compared with the EtOH group (Fig. 5B; P<0.001), suggesting that FRRT can suppress the ethanol-induced elevation of MDA in a dose-dependent manner within this concentration range. Furthermore, the addition of different concentrations of FRRT led to increased SOD, GSH and CAT levels compared with the EtOH group (Fig. 5C-E; P<0.01). Of note, the lower dose of FRRT intervention demonstrated a more pronounced reduction in MDA levels and greater enhancement of GSH, CAT and SOD activities compared with the RRT group (Fig. 5B-E). Collectively, these experimental findings confirmed the protective effect of FRRT against ethanol-induced liver cell damage through the modulation of intracellular oxidative stress response within a specific concentration range.

To study the molecular mechanism of the FRRT-induced inhibition of oxidative stress, the total protein levels of NRF2, NQO1, HO-1 and KEAP1 were detected by western blotting. As demonstrated in Fig. 6A, compared with the NC group, the hepatic protein levels of HO-1, NRF2 and NQO1 were decreased, while the protein level of KEAP1 was increased in the EtOH group (P<0.05). However, FRRT pretreatment effectively reversed these changes, leading to the restoration of NRF2, NQO1 and KEAP1 protein levels. In addition, the gene expression levels of NRF2, KEAP1, HO-1 and NQO1 were assessed in AML-12 cells using qPCR. The results included in Fig. 6B revealed that the FRRT-treated groups exhibited a significantly increased mRNA expression of NRF2, HO-1 and NQO1, accompanied by a decreased expression of KEAP1 compared with that in the EtOH group (P<0.05). The dose-dependent response suggested that FRRT-L may potentially exhibit a stronger protective effect against oxidative stress.