Wild-type (WT) C57BL/6J mice were acquired from the Animal Institute of Southern Medical University (Guangzhou, China). The mice were housed under specific pathogen-free conditions at a constant temperature (19–23°C) and humidity (55% ± 10%). All animal experiments were approved by the Welfare and Ethical Committee for Experimental Animal Care of Southern Medical University.

Male mice (10–12 weeks old) were chronically fed a high-fat diet and administered binge ethanol to develop a murine model of MetALD. We modified the previous ALD model (Bertola et al., 2013) with a 35% liquid fat diet and alcohol feeding. All mice were fed a liquid control diet (Lieber-DeCarli formulation; Bioserv, Flemington, NJ) for 5 days. The MetALD groups were then fed a liquid diet containing 35% liquid fat diet and 3% v/w ethanol for 10 days, while control mice were pair-fed with the MetALD-fed counterparts for 10 days. On day 11, the MetALD groups received a single oral gavage of ethanol (3 g/kg body weight, 31.25% ethanol), whereas control mice received an isocaloric gavage of dextrin maltose. Fucoidan or phosphate-buffered saline (PBS) was administered via oral gavage. After euthanasia, blood and tissue samples were collected for further analysis.

Antibodies against HIF-1α (20960-1-AP), PHD2 (19886-1-AP), PDK1 (18262-1-AP) and β-actin (20536-1-AP) were obtained from Proteintech (Rosemont, IL, United States). Anti- Ubiquitin antibody (sc-8017) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Anti-Na-K-ATPase (3010S) was purchased from Cell Signaling Technology (Danvers, MA, United States). Percoll (P1644), Tyramine (T90344), Sodium Cyanoborohydride (190020), FITC (1.24546), fucoidan from F. vesiculosus (F5631) and Dexamethasone (D4902) were purchased from Sigma-Aldrich (St. Louis, MO, United States). SiRNA targeting HIF-1α (si-HIF-1α) and siRNA targeting PHD2 (si-PHD2) were obtained from RiboBio (Shanghai, China). The fucoidan (F5631) is derived from F. vesiculosus, a type of brown algae. The fucoidan is in crude powder form, and it is typically extracted from the brown algae using appropriate purification techniques: Extraction involves treatment of the weed with hydrochloric acid at 70° for one hour at pH 2.0–2.5. The crude fucoidan is isolated by fractional precipitation with alcohol, and purified by treatment with formaldehyde (Black et al., 1953). The monosaccharide composition is not explicitly listed in the certificate, but fucoidan from Fucus vesiculosus typically contains fucose, along with small amounts of galactose, glucose, xylose, mannose, and sulfate groups. The molecular weight of this batch of fucoidan is measured by Multi-angle Laser Light Scattering (MALLS), and the range is between 20,000 and 200,000 Da, with a measured value of 39,700 Da for this specific batch.

THP-1 cells (ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% FBS, 100 μg/mL streptomycin, and 100 U/mL penicillin in a humidified 5% CO₂ atmosphere. THP-1 cells were seeded in a 6-well plate and stimulated with 50 nM EtOH and 100 ng/mL LPS for 24 h (Wu et al., 2023). Fucoidan was added to THP-1 cells 6 h before EtOH and LPS treatment. Dexamethasone (5 uM, Sigma) was used for 1 h before stimulation with EtOH and LPS as positive control (Chen et al., 2024).

The effect of fucoidan on cell apoptosis was detected using an Annexin V-FITC/PI apoptosis detection kit (MultiSciences, Hangzhou, China), according to the manufacturer’s instructions. THP-1 cells were seeded into 6-well plates overnight and then cultured in media containing fucoidan. The cells were collected and resuspended in 100 μL of Binding Buffer containing 5 μL Annexin-V and 10 μL PI for 15 min in the dark. Data were acquired on LSRII/Fortessa flow cytometer (BD Biosciences, Heidelberg, Germany) and analyzed with FlowJo software (Tree Star, Ashland, OR, United States).

The effect of EtOH and LPS on cell apoptosis was detected using CCK-8 kit (40203ES60, YEASEN, Shanghai, China), according to the manufacturer’s instructions. THP-1 cells were seeded into 96-well plates overnight and then cultured in media containing different contents of EtOH (0–100 nM) and LPS (0–200 ng/mL). The cells were collected and resuspended in 100 μL of CCK-8 kit in 37°C for 45 min in the dark. Data were acquired on Bio TEK Microplate Reader (Bio Tek Instruments, Inc., United States) and analyzed with FlowJo software (Tree Star, Ashland, OR, United States).

THP-1 cells (2 × 10⁵ cells per well) in 100 μL of serum-free medium containing ethanol (EtOH), lipopolysaccharide (LPS), or fucoidan were seeded in the upper chamber. The lower chamber contained the same medium with 15% FBS. After 24 h of incubation, the migrated cells in the lower chamber were collected, counted, and photographed.

Paraffin-embedded liver tissue blocks were cut into 5-μm-thick slices and mounted onto poly-L-lysine-coated glass slides. Mouse liver sections were processed for hematoxylin and eosin (H&E) staining and Oil Red O staining according to the standard protocols.

The mononuclear cells (MNCs) in the liver were assayed using an LSRII/Fortessa flow cytometer (BD Biosciences, Heidelberg, Germany). Briefly, hepatic MNCs were prepared as previously described (Tang et al., 2018). Liver MNCs or peripheral blood cells were blocked by CD16/32 (14–0161–82, Invitrogen, Waltham, MA, United States) and then stained with Pacific Blue- or FITC-conjugated anti-mouse CD11b antibody (eBioscience, San Diego, CA, United States), PE-conjugated anti-F4/80 antibody (eBioscience) or FITC-conjugated anti-Ly6G antibody (eBioscience). Finally, 7-AAD viability staining solution (eBioscience) was used. FlowJo software (Tree Star, Ashland, OR, United States) was used to analyze the flow cytometric data.

RIPA lysis buffer containing protease inhibitors (Beyotime) was used to extract total protein. Protein samples were separated on polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, United States). Nonspecific binding sites on the membranes were blocked with 5% bovine serum albumin (BSA) for at least 1 h at room temperature. Subsequently, the membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Lastly, the membranes were washed three times, and the target proteins were detected using enhanced chemiluminescence (Thermo Fisher, Carlsbad, CA, United States). ImageJ software (NIH, Bethesda, MD) was used to quantify the densities of protein blots.

The cell lysates were incubated for immunoprecipitation with the indicated antibody and protein A/G PLUS-agarose (sc-2003, Santa Cruz Biotechnology) at 4°C overnight. The beads were washed three times with PBS, resuspended in loading buffer, and boiled for 5 min. Eluted immunoprecipitants were resolved by SDS-PAGE and analyzed for the associated proteins using specific antibodies.

Trizol (ET101-01, Transgen, Beijing, China) was used to extract total RNA from liver tissues or cultured cells. Subsequently, RNA was transcribed into cDNA using the reverse transcription kit (TaKaRa, Dalian, China), as instructed by the manufacturer. SYBR Green quantitative RT-PCR was performed to determine the gene expression levels using a 7900HT fast real-time PCR system (Applied Biosystems, San Francisco, CA, United States), according to the protocol provided with SYBR Premix EX Taq (TaKaRa, Dalian, China). The levels of the target gene were normalized to β-actin gene expression. The primer sequences for the genes are presented in Table 1.

Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TCH), and hepatic TG levels were measured according to the instructions of commercial assay kits from the manufacturer (Jiancheng Biotech, Nanjing, China).

The commercial ELISA kits for TNF-α (88–7324–22), MCP-1 (88–7503–22), and IL-1β (88–7013–22) were purchased from eBioscience. The levels of cytokines in mouse sera and culture supernatants were detected using the ELISA kits, following the manufacturer’s protocols.

FITC labeling of fucoidan was performed as previously described (Luo et al., 2022). In brief, 100 mg of fucoidan (F5631, Sigma Aldrich, United States) was dissolved in 3.75 mL of 0.2 M PBS (pH = 8), followed by the addition of tyramine (100 mg) and sodium cyanoborohydride (37.5 mg). After a 96-h incubation at 37°C with intermittent shaking, ethanol (80% v/v) was added to the supernatant, and the precipitate was collected by centrifugation. The precipitate (100 mg) was dissolved in 5 mL of 0.5 M NaHCO₃, followed by the addition of FITC (12.5 mg). After overnight incubation at room temperature in the dark, ethanol (80% v/v) was added, and the precipitation was collected by centrifugation to obtain the FITC-labeled fucoidan.

Cells were plated on poly-D-lysine-coated glass coverslips and exposed to LPS (100 ng/mL) and alcohol (50 nM) for 12 h, with pretreatment of FITC-FUC for 3 h. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 (Solabio, Beijing, China) in PBS for 8 min, and stained with primary antibodies. The primary antibodies were detected using Alexa Fluor 647-conjugated donkey anti-rabbit IgG, Alexa Fluor 568-conjugated donkey anti-rabbit IgG, or Alexa Fluor 488-conjugated donkey anti-goat IgG. All the images were acquired with a ×20 objective on an Olympus IX81 FV1000 Laser Scanning confocal microscope (Shinjuku, Tokyo, Japan).

All results are expressed as mean ± SD. Statistical significance between two groups was evaluated using the Student’s t-test, while comparisons between multiple groups were assessed by two-way analysis of variance (ANOVA), followed by the Student-Newman-Keul’s test. P < 0.05 was considered significant.

To explore the potential role of fucoidan from F. vesiculosus in MetALD, mice were chronically fed a high-fat chow diet plus binge ethanol feeding to establish a MetALD murine model, along with fucoidan administration via intragastric gavage (20 mg/kg, 100 mg/kg) (Lim et al., 2015; Zhao et al., 2021). Our previous experiments have also demonstrated that these concentrations do not impair liver function in mice (Luo et al., 2022). Chronic binge ethanol feeding (Figure 1A) induced hepatocyte damage and hepatic steatosis, along with an increased liver-to-body weight ratio. Notably, fucoidan significantly attenuated these effects in a dose-dependent manner (Figures 1B–D). Moreover, fucoidan supplementation led to a marked reduction in serum alanine aminotransferase (ALT) and aspartate transaminase (AST) levels (Figures 1D,E). Hepatic triglycerides (TG), serum total cholesterol (TCH), and triglycerides (TG) were also significantly decreased in fucoidan-treated mice, with the 100 mg/kg fucoidan showing superior efficacy (Figures 1F–H). The serum levels of IL-1β, TNF-α and MCP-1 were elevated in the MetALD group mice, while they decreased with fucoidan supplementation (Figures 1I–K). Together, these findings suggest that fucoidan has the potential to protect against MetALD injury in mice.

In alcoholic liver disease, the infiltration of macrophages into the liver during chronic injury is closely associated with the progression of liver inflammation in both mice and humans (Duffield et al., 2005; Karlmark et al., 2009; Karlmark et al., 2010; Zimmermann et al., 2010). We investigated the effects of fucoidan on macrophage infiltration in MetALD mice models. Flow cytometry analysis (Figures 2A,B) identified infiltrating macrophages (CD11b⁺F4/80^int). Chronic binge ethanol feeding increased infiltrating macrophages, while fucoidan remarkably reduced the frequency of infiltrating macrophages in the livers of mice. Consistently, the frequency of monocytes (CD11b⁺Ly6G⁻) was lower in fucoidan-treated mice compared to the MetALD group in blood (Figures 2C,D), indicating that fucoidan decreased the infiltration of monocytes into the liver during MetALD progression.

Inflammatory monocytes play a key role in promoting inflammation during alcohol-related liver disease (Tacke, 2012). Inhibition of hepatic monocyte infiltration is a promising therapeutic approach for MetALD. To explore the pharmacological effects of fucoidan on monocyte migration, we conducted in vitro experiments using THP-1 cells. First, we tested the effect of fucoidan on cell apoptosis by flow cytometry. Fucoidan treatment did not increase the apoptosis rate in THP-1 cells at doses of 20, 100 or 200 μg/mL (Figure 3A). Then, the effect of EtOH and LPS on cell apoptosis by CCK-8 was tested. We selected 50 nM of EtOH and 100 ng/mL of LPS as safe concentrations to stimulate THP-1 cells. (Figure 3B). Given the disrupted intestinal barrier and increased LPS levels in the liver microenvironment during MetALD (Wu et al., 2023), we hypothesized that EtOH may promote cell migration via activation of chloride channels (Wei et al., 2015), and that lipopolysaccharide (LPS)-induces migration in RAW264.7 cells and bone marrow-derived macrophages (BMDMs) in vitro (Gao et al., 2021). We stimulated THP-1 cells with EtOH and LPS, dexamethasone (4 uM, Sigma) was used for 1 h before stimulation with EtOH and LPS as positive control. Transwell assays revealed that fucoidan dose-dependently inhibited the migration of THP-1 cells under EtOH and LPS treatment (Figure 3C). Images and cell counts both confirmed fewer migrated THP-1 cells in the presence of fucoidan compared to the EtOH and LPS group (Figures 3D,E). Overall, these results suggest that fucoidan inhibits monocyte migration induced by EtOH and LPS.

Previous studies have shown that HIF-1α is associated with cell migration and invasion (Gao et al., 2020). Glycolytic reprogramming promotes macrophage migration via the HIF-1α-PDK1 axis (Semba et al., 2016). Therefore, we investigated whether fucoidan regulates HIF-1α to inhibit monocyte migration. The results showed that EtOH and LPS stimulation significantly increased HIF-1α expression, while fucoidan decreased HIF-1α expression in THP-1 cells in a dose-dependent manner (Figure 4A). Fucoidan also inhibited HIF-1α expression in mononuclear cells (MNCs) isolated from the livers of MetALD mice (Figure 4B). To further confirm that fucoidan regulates HIF-1α, we applied HIF-1α siRNA to interfere with HIF-1α expression. The results of transwell assays (Figure 4C), images of migrated cells (Figure 4D), and the number of cells that migrated to the lower chamber (Figure 4E) confirmed that knockdown of HIF-1α significantly decreased the migration of THP-1 cells and abolished the differences between the groups with or without fucoidan. These findings suggest that the HIF-1α-PDK1 signaling pathway could be a potential target of fucoidan in regulating cell migration. Taken together, these results suggest that fucoidan reduces monocyte migration via the HIF-1α signaling pathway.

An increasing number of proteins and small molecules have been identified to regulate HIF-1α ubiquitination activity by modulating the physical or functional interaction of PHD2, VHL, or HSP90 with HIF-1α (Semenza, 2007). In EtOH and LPS stimulated THP-1 cells treated with or without fucoidan, the mRNA level of HIF-1α showed no significant differences (Figure 5A). Then we focused on the post-translational modifications of HIF-1α. We observed that fucoidan decreased the protein expression of HIF-1α, accompanied by an upregulation of HIF-1α ubiquitination under EtOH and LPS stimulation (Figure 5B). Furthermore, the mRNA level (Figure 5C) and ubiquitination of HIF-1α (Figure 5D) in MetALD liver MNCs treated with fucoidan were confirmed. These results indicate that fucoidan downregulates HIF-1α expression via the ubiquitination pathway, suggesting that fucoidan promotes the ubiquitination of HIF-1α to decrease the activity of the HIF-1α pathway.

Next, we sought to investigate how fucoidan affects the ubiquitination level of HIF-1α. Firstly, we needed to determine whether fucoidan could traverse the cell membrane. In this study, fucoidan was labeled with fluorescein isothiocyanate (FITC). Immunofluorescence results showed that FITC-labeled fucoidan (FITC-FUC) was partly transported into THP-1 cells (Figure 6A), which was supported by previous reports indicating that fucoidan immunostaining in liver tissue appeared in an elliptical or club-shaped pattern in non-parenchymal cells in sinusoids but not in parenchymal cells (Nagamine et al., 2014). Then we investigated potential protein interactions with fucoidan. Initially, we tested whether fucoidan could directly bind to HIF-1α. The results (Figures 6B,C) suggested that fucoidan did not bind directly to HIF-1α. HIF-1α ubiquitination is mediated by several proteins, including the von Hippel-Lindau tumor suppressor gene product (pVHL), prolyl hydroxylase (PHD2), and heat shock protein-90 (HSP90). As reported, fucoidan is negatively charged, which enables it to bind positively charged proteins (Figure 6B). Subsequently, we analyzed the isoelectric points of HIF-1α, pVHL, PHD2, and HSP90. As shown in Figure 6B, only PHD2 was positively charged. Consistently, co-localization of fucoidan with PHD2 was observed in THP-1 cells (Figure 6D). Furthermore, co-immunoprecipitation results showed that FITC-fucoidan could physically interact with PHD2 (Figures 6E,F). To further confirm the role of PHD2 in fucoidan’s action, we examined the expression and ubiquitination levels of HIF-1α after treatment with siPHD2. The results showed no significant differences in HIF-1α expression or ubiquitination between the presence and absence of fucoidan following PHD2 knockdown (Figure 6G). These results indicated that fucoidan may traverse the membrane and interact with PHD2, leading to an increase in HIF-1α ubiquitination and a decrease in HIF-1α protein levels. In summary, these findings suggest that fucoidan reduces macrophage infiltration by interacting with PHD2, thereby enhancing the ubiquitination of HIF-1α and inhibiting the HIF-1α pathway.