Fucoidan JHCF4s from Hizikia fusiformis against ethanol-induced damage in vitro and in vivo

Highlights

• Hepatoprotective effect of fucoidan

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-025-01865-4.

Abstract

Fucoidan has strong antioxidant and free radical scavenging activities. We successfully isolated the active fucoidan, JHCF4, from edible Hizikia fusiformis. The effects of high-temperature water-extracted (T-JHCF4) and cellulase-assisted extraction (C-JHCF4) of JHCF4 were compared and to demonstrate those potential for treating alcoholic liver disease (ALD). C-JHCF4 contained higher amounts of fucose, arabinose, rhamnose, mannose, and sulfate groups, whereas T-JHCF4 had a higher galactose content. Importantly, C-JHCF4 downregulated the expression of Bax, Caspase 3, and cytochrome C in alcohol-treated LO2 cells and upregulated Bcl-2 expression, indicating its protective effect via an apoptosis-related pathway. In vivo, C-JHCF4 protected alcohol-induced zebrafish from reduced reactive oxygen species production and cell death, further regulating malondialdehyde and glutathione levels. Collectively, T-JHCF4 has advantages in extraction, such as short extraction time and low cost, making it suitable for large-scale industrial production. C-JHCF4 exhibited a stronger hepatoprotective effect than T-JHCF4, indicating its suitability for treating ALD.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-025-01865-4.

Introduction

Hizikia fusiformis, a brown seaweed commonly consumed in East Asian countries, particularly Japan and Korea, is rich in bioactive compounds such as fucoidan, fucoxanthin, alginic acid, and iodine (Salido et al., 2024). Fucoidan is a sulfated polysaccharide that has gained considerable attention owing to its diverse biological activities and potential therapeutic applications in various diseases, including cancer (Dayanidhi et al., 2021), infection, diabetes, cardiovascular disease, and inflammation (Wang et al., 2016). Our recent reports have shown that fucoidan displays antioxidant and anti-apoptotic activities (Dai et al., 2019) and effectively protects the liver via reducing the content of reactive oxygen species (ROS), the activation of the Bax/Bcl-2/Caspase 3 apoptotic signaling pathway is inhibited (Dai et al., 2020a). However, only a few studies have investigated the potential of fucoidan as a hepatoprotective agent against ethanol-induced liver injury.

Alcoholic liver disease (ALD) is a primary global health concern responsible for a large number of liver-related deaths worldwide. This illness encompasses a range of liver conditions, from simple fatty liver to more severe forms such as alcoholic hepatitis, fibrosis, and cirrhosis. The development of ALD is attributed to a complex interplay of factors, including hepatocellular injury, inflammation, oxidative stress, and impaired liver function (Zhu et al., 2022). ROS regulate oxidative stress and react with intracellular molecules such as lipids and DNA, resulting in liver cell apoptosis or injury (Kang et al., 2024). Recent reports have revealed a relationship between the apoptotic signaling pathway and the mechanism of ALD, suggesting a vital role for Bax/Bcl-2 and the Caspase family. Current treatment options for advanced ALD, such as abstinence from ethanol, nutritional support, and liver transplantation, are limited and primarily supportive (Singal and Mathurin, 2021). Thus, future studies should focus on discovering novel therapeutic agents capable of preventing or reversing ethanol-induced liver damage.

Extraction methods such as high-temperature water extraction and ethanol precipitation are the most used protocols for fucoidan extraction. These methods offer advantages such as short processing times and low cost, making them suitable for large-scale industrial production (Men et al., 2024). The extraction cost is an essential factor in industrial extraction. However, toxic compounds are released into the environment during the extraction, which generates wastewater. The environmental impact is worsened when organic solvents are used. Recently, researchers have demonstrated that enzyme-assisted extraction is an environmentally friendly technology with low energy consumption and high yield. Enzyme-assisted methods can extract various materials, including polysaccharides, proteins, and natural pigments (Malvis Romero et al., 2023). Therefore, scientists are eager to seek a natural active fraction with a high yield and strong active properties.

In this study, we used in vitro and in vivo models of ethanol-induced liver damage to assess the hepatoprotective effects of the fucoidans. Understanding the potential liver protective mechanisms of fucoidans can provide valuable insights into developing novel treatment strategies for ALD.

Materials and methods

Materials

Glucans (160, 80, 50, 30, 10, and 5 kDa), potassium bromide, 1-phenyl-3-methyl-5-pyrazolone (PMP), cell counting Kit-8 (CCK-8), malondialdehyde (MDA), glutathione (GSH) assay kits, monosaccharide standards (mannose, galactose, xylose, fucose, and glucuronic acid), and commercial fucoidans were purchased from Yuanye Bio-Technology Co. Ltd. (Shanghai, China). Propidium Iodide (PI) dual fluorescence staining kit, Hoechst 33342 fluorescence staining kit, oil Red O reagent, 2′,7′-dichlorofluorescein-diacetate (DCFH-DA), diphenyl-1-pyre-nylphosphine (DPPP), and Acridine Orange (AO) reagents were obtained from Solarbio (Beijing, China). Roswell Park Memorial Institute medium (RPMI) medium, penicillin–streptomycin (P/S), and fetal bovine serum (FBS) were obtained from Gibco Inc. (Grand Island, NY, USA). All chemical reagents were acquired through commercial channels and used in scientific research.

Extraction of fucoidan

Hizikia fusiformis was harvested from Jeju Island, Korea, in September 2024. In accordance with our previous protocol (Dai et al., 2019), high-temperature water and cellulase-assisted extractions were performed with slight improvements. Briefly, Hizikia fusiformis was soaked in a 1% citric acid solution for 30 min, washed with distilled water until the pH reached 7.0, and dried. Heavy metals, including as were extracted from Hizikia fusiformis by heating it in water at 90 °C for 10 min. The solid matter was collected, dried using a freeze dryer, and powdered. Distilled water was added to the powder at a 1:10 (w/v) ratio.

High-temperature water extraction involved heating the mixture to above 95 °C and refluxing for 3 h. For cellulase-assisted extraction, the pH of the mixture was adjusted to 4.5, and the mixture was digested with 0.5% cellulase at 50 °C for 24 h. To inactivate the cellulase, the mixture was boiled for 10 min.

Both methods require centrifugation of the mixture and discarding of the precipitation. Proteins were removed from the supernatant using sevage reagent, then acidified with HCl to pH 5.0. Calcium chloride solution (4 M) was added dropwise until no more precipitation were formed. After a second centrifugation, the pH of the supernatant increased to 7.0. Three volumes of 95% ethanol were used to precipitation the supernatant at 4 °C for 8 h after it had been concentrated to one-third of its original volume. Crude polysaccharides (CPs) were obtained by centrifuging the precipitation, washing with ethanol, and drying. The CPs were dissolved in distilled water, dialyzed against distilled water using a dialysis membrane (molecular weight cutoff, 8–10 kDa) for 72 h at 4 °C, with the replacement of fresh distilled water every 12 h, and then lyophilized. The crude fucoidan (CF) was obtained. JHCF4 was isolated and purified from CFs. The products obtained by high-temperature water and cellulase-assisted extractions were named T-JHCF4 and C-JHCF4, respectively.

Chemical analysis

The samples' sulfate, polyphenol and uronic acid contents were determined using barium sulfate turbidimetry, Folin-Ciocalteu test and the m-hydroxybiphenyl method, respectively.

Composition and content of monosaccharides

High-performance liquid chromatography (HPLC) system (Agilent 1260, Agilent Technologies, Santa Clara, CA, USA) was used to detect the types and contents of monosaccharides in the samples after PMP derivatization. The processing methods followed the published literature (Tao et al., 2023) and were used for all monosaccharide standards and samples.

Molecular weight analysis

The molecular weight of fucoidan was analyzed using agarose gel electrophoresis, as previously described (Wang et al., 2019). Fucoidans were electrophoresed on 1% agarose gel at 100 V for 20 min. Dextran sulfate with molecular weights of 160, 80, 50, 30, 10, and 5 kDa was used as a molecular weight marker (D8906, Sigma). The gel was fixed in 0.1% cetrimonium bromide for 4 h, stained with a 0.1% toluidine blue solution for 30 min, and decolorized with acetic acid: water: ethanol (1:49:50).

Flourier-transform infrared spectroscopy (FT-IR)

Thermo Scientific Nicolet 6700 FT-IR spectrometer (MA, USA) was used to perform IR spectroscopy analysis on fucoidans in the range of 500–4000 cm⁻¹.

Cell culture

The human hepatic cell line, LO2, was used to evaluate the protective effects of fucoidan against ethanol-induced cytotoxicity, oxidative stress, and inflammation. LO2 cells were purchased from Procell Life Science & Technology Co., Ltd. LO2 cells were cultured in a medium containing 89% RPMI, 10% FBS, and 1% P/S. The cell samples were cultured at 37 °C in a 5% CO₂ environment.

Screening of ethanol and JHCFs

The cells were cultured in a 96-well plate at a density of 1 × 10⁴ cells/well. Different concentrations of ethanol were added when the cell density reached 80%, and CCK-8 reagent was added after 24 h.

Comparison of antioxidant activities of JHCFs

In a 96-well plate, 1 × 10⁴ cells/well were added to each well. When cell density reached 80%, different concentrations of JHCFs were added to the plate. After 1 h of cultivation, ethanol was added to reach a concentration of 200 mM in the culture medium. After 24 h, CCK-8 and DCFH-DA were added for detection.

Hoechst 33342 staining

The cells were seeded in a 6-well plate. When the cell density reached 80%, different sample concentrations (0, 6.25, 12.5, and 25 µg/mL) were added to the plate. After 1 h of incubation, 200 mM ethanol was added. After 24 h, the cells were stained with Hoechst 33342 staining reagent, and images were captured under a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera (Meyer Instruments, Inc., Houston, TX, USA) at 37 °C in the dark.

PI staining

Based on a previous study with minor modifications (Li et al., 2025), LO2 cells were incubated in a 6-well plate. When the cells reached 80% confluence, different sample concentrations were added to the plates. After 1 h of cultivation, 200 mM ethanol was added, and the cells were incubated for 24 h. PI reagent (10 µL) was added to each well, and photos were captured using a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera (Meyer Instruments, Inc., Houston, TX, USA) in the dark after 10 min.

Transmission electron microscope (TEM)

TEM was used to examine mitochondrial morphological changes in LO2 cells after drug treatment at the indicated concentrations. LO2 cells were trypsinized, harvested, and fixed in 2.5% glutaraldehyde (EM Grade) at 4 °C overnight. Appropriate areas were selected, and ultrathin sections of 0.08 µm were stained with lead citrate and uranyl acetate. Sections were examined under a TEM (JEM-1400Plus, JEOL, Ltd., Tokyo, Japan).

Flow cytometry analysis

LO2 cells were cultured in 6-well plates and treated with different concentrations of the sample when the cell density reached 80%. After incubation for 1 h, 200 mM ethanol was added to the plates. The cells were collected after 24 h and washed twice with PBS. Each cell precipitation was suspended in 200 µL of buffer solution and 5 µL fluorescein isothiocyanate and 5 µL of propidium iodide were added to it. The mixture was incubated in the dark for 1 h, and apoptotic cells were quantified using flow cytometry (BD Biosciences, San Jose, CA, USA).

Western blotting

LO2 cells were seeded in a cell culture dish (60 mm × 15 mm) and treated with different sample concentrations until the cell density reached 80% confluence. After 1 h of incubation, ethanol was added, and cells were harvested for 24 h after incubating. Cell lysates were used to lyse cells, extract proteins, and detect the protein concentrations in each sample. A total of 30 µg of each protein was electrophorized in a running buffer. The protein imprint was transferred to a polyvinylidene difluoride membrane, blocked with skim milk for 3 h, and incubated with the primary antibody for 8 h and the secondary antibody for 3 h. A chemical substrate luminescent reagent was dripped onto the protein imprint and fluorescence imaging was performed using the FUSION SOLO Vilber Lourmat system (FUSION, Paris, France).Image J software was used to calculate the strength of the blots.

Immunofluorescence staining

To determine the expression levels of cytochrome C and Bcl-2, immunofluorescence staining was performed in each group of LO2 cells. Each group of LO2 cells was fixed with 4% paraformaldehyde for 20 min, incubated with cytochrome C and Bcl-2 primary antibodies for 24 h, incubated with a secondary antibody for 3 h, stained with 4',6-diamidino-2-phenylindole (DAPI), and imaged under a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera (Meyer Instruments, Inc., Houston, TX, USA).

Protection of C-JHCF4 in ethanol-induced zebrafish larvae at 72 hours post-fertilization (hpf)

Zebrafish are well-established model vertebrate organisms known for their numerous advantages in biomedical research, including high genetic similarity to human, transparent embryos and larvae, easy reproduction, and short growth cycles. Zebrafish models have been used to screen for potential hepatoprotective agents such as fucoidan, which can attenuate ethanol-induced liver injury in zebrafish by modulating various signaling pathways and molecular mechanisms (Tran et al., 2016). Adult zebrafish were provided by the China Zebrafish Resource Center (Wuhan, China). The zebrafish were cultured in water at 28.5 °C with a light/dark cycle of 14/10 h, and embryos produced through natural fertilization were collected. In this study, zebrafish larvae were treated with different ethanol concentrations for 72 hpf, and the survival rate of the zebrafish was tested to select the most suitable ethanol concentration. At 8 hpf, the embryos were treated or untreated to varying sample concentrations of C-JHCF4 (0, 6.25, 12.5, and 25 µg/mL), and all were incubated for 1 h. Following this, ethanol of the most suitable concentration was added to each group, except control group and the above operation was repeated every 24 h. Zebrafish embryos from each group were collected after 72 h. Varying concentrations of DCFH-DA, DPPP, and AO reagents were added to the embryos (Dai et al., 2020a), which were then incubated in the dark for 1 h at 28.5 °C. Images were obtained using a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera (Meyer Instruments, Inc., Houston, TX, USA), and the fluorescence intensity of each group was calculated using Image J software.

Protective effect of fucoidan in ethanol-induced liver degeneration in 128 hpf zebrafish larvae

Liver damage can be induced by exposing zebrafish larvae to ethanol, allowing for the observation of the harmful effects of ethanol on liver morphology, function, and gene expression (Shimizu et al., 2023). According to previous studies, the zebrafish liver is formed at 120 hpf (Chu and Sadler, 2009), therefore, the 128 hpf zebrafish larvae was selected for this study. According to our previous study (Dai et al., 2020b), 96 hpf zebrafish were treated with different concentrations of C-JHCF4 (0, 6.25, 12.5, and 25 µg/mL) and 200 mM ethanol, and all were incubated for 32 h. The larvae were fixed with 4% paraformaldehyde. The fixed larvae were washed with PBS, added oil Red O reagent for staining, and incubated in the dark for 2 h before imaging. The 128 hpf zebrafish livers were fixed in a 10% neutral phosphate-buffered formalin solution and embedded in paraffin to prepare conventional paraffin sections. Hematoxylin and eosin (H&E) staining was performed to observe pathological changes in liver tissue. Using MDA and GSH assay kits, the levels of MDA and GSH were measured to determine the degree of lipid peroxidation in zebrafish (Gosslau, 2016). Briefly, 10 mg of dried whole zebrafish larvae was homogenized and centrifuged at 15,000 revolutions per minute (rpm), and the supernatant was collected. The reaction reagents, MDA and GSH, were added. The absorbance was measured at 532 and 412 nm, and the corresponding standard curves were plotted. C-JHCF4 pretreated ethanol-induced larvae were subjected to Western blotting. Thirty larvae were collected from each group, and signaling proteins were evaluated by western blotting assay.

Statistical analysis

All experiments were repeated three times, with values expressed as mean ± standard error (SE), and the mean was analyzed using a one-way analysis of variance (using SPSS 11.5 statistical software). The mean values of the significantly different parameters were analyzed using Student's t-test (*P <0.05,**P <0.01, ***P <0.001, ****P <0.0001, #P <0.05, ##P <0.01 and ###P <0.001).

Results and discussions

Chemical composition and structural analysis

According to the molecular weight assay, the molecular weight of C-JHCF4 and T-JHCF4 are close (10–50 kDa). However, the molecular weight of C-JHCF4 was slightly lower than that of T-JHCF4 (Fig. 1A). The large molecular weight range indicates that relatively complex polysaccharides are present in fucoidan, implying its relatively broad development prospects.

Fig. 1.

Comparison of structural characterization between C-JHCF4 and T-JHCF4. A Molecular weight range of C-JHCF4 and T-JHCF4. B Infrared spectra of C-JHCF4 and T-JHCF4. C HPLC chromatogram of standard monosaccharides, C-JHCF4 and T-JHCF4. The first unlabeled peak was PMP

Figure 1B shows the FT-IR spectra of C-JHCF4 and T-JHCF4. A wide hydroxyl stretching vibration absorption peak was observed at 3600–3200 cm⁻¹, commonly observed for polysaccharides with previous study (Huang and Huang, 2021). The C–H bending stretching vibration peak appeared at 2915 cm⁻¹ and that of esterified carboxyl groups at 1730 cm⁻¹. The peaks observed at 1635 and 845 cm⁻¹ indicate stretching vibrations of S=O bonds and bending vibrations of C–O–S in the sulfate group, respectively (Li et al., 2021). In FT-IR scanning, the peaks between 1650 cm⁻¹ in C-JHCF4 showed higher abundance than those in T-JHCF4, indicating the higher sulfate content of C-JHCF4 relative to T-JHCF4. The peak at 1400 cm⁻¹ indicates the presence of C–H bending vibrations, but the absorption peak intensity of C-JHCF4 was stronger than that of T-JHCF4. This was possibly due to the greater number of C–H bonds in C-JHCF4 than in T-JHCF4. The absorption peak of 1040 cm⁻¹ indicates stretching vibrations of the C–O bond in the C–O–C structure (Ikemoto et al., 2022). Overall, both extracts exhibited strong absorption.

HPLC results revealed the presence of various standard monosaccharides in C-JHCF4 and T-JHCF4 (Fig. 1C). Table 1 presents the results of this analysis. C-JHCF4 and T-JHCF4 contain almost the same types of monosaccharides. The most abundant monosaccharides were fucose, galactose, and mannose, with small amounts of rhamnose, arabinose, and fucose accounting for most of the total sugar content. However, C-JHCF4 had higher fucose (63.86%), sulfate group (17.27% ± 0.09%), and uronic acid (7.20%) contents than T-JHCF4 (54.58%, 15.42% ± 0.06%, and 6.50%, respectively). Due to the antioxidant activity of fucoidan is positively correlated with fucose and sulfate groups (Husni et al., 2022), C-JHCF4 may has stronger potential in the therapy of ALD than T-JHCF4.

Table 1.

Chemical compositions of C-JHCF4 and T-JHCF4

Sample	Uronic acid	Polyphenol	Protein	Sugar(%)					Sulfate (%)a
				Mannose	Rhamnose	Galanose	Arabinose	Fucose
C-JHCF4	7.20	1.68	2.86	5.57	1.05	24.25	5.25	63.86	17.27 ± 0.09
T-JHCF4	6.50	1.14	2.20	5.31	1.01	34.02	5.06	54.58	15.42 ± 0.06

^aEach value represents mean ± SD of three determinants of each fraction

Comparison of antioxidant activities of C-JHCF4 and T-JHCF4 in ethanol-treated LO2 cells

First, the appropriate ethanol concentration was selected (Fig. 2A). Compared with the control group, ethanol caused damage to LO2 cells and decreased their survival rates. As the ethanol concentration increased, the cell survival rae gradually decreased. The cell survival rate was 60% in 200 mM ethanol. The concentrations of C-JHCF4 and T-JHCF4 were determined (Fig. 2B and C). When the concentration was higher than 25 µg/mL, the cell survival rate decreased significantly. Thus, C-JHCF4 and T-JHCF4 were significantly toxic to LO2 cells at concentrations greater than 25 µg/mL. Therefore, 6.25, 12.5, and 25 µg/mL were selected as the treatment concentrations of C-JHCF4 and T-JHCF4 for this experiment. As shown in (Fig. 2D and E), ethanol caused notable damage to LO2 cells compared to the control group. After the administration of C-JHCF4 or T-JHCF4, the survival rate of the cells increased considerably and showed a dose-dependent relationship with the concentration of C-JHCF4 or T-JHCF4. Therefore, C-JHCF4 was observed to have a stronger protective effect than T-JHCF4 against ethanol-induced cell injury.

Fig. 2.

Protective effects of C-JHCF4 and T-JHCF4 on LO2 cells against ethanol damage. A Cytotoxicity of different concentrations of ethanol on LO2 cells. B Cytotoxicity of different concentrations of C-JHCF4 on LO2 cells. C Cytotoxicity of different concentrations of T-JHCF4 on LO2 cells. D Different concentrations of C-JHCF4 inhibit the toxic effect of ethanol on LO2 cells. E Inhibiting the toxic effect of ethanol on LO2 cells at different concentrations of T-JHCF4. F Different concentrations of C-JHCF4 reduced the ability of ethanol induced ROS production in LO2 cells. G The ability of T-JHCF4 at different concentrations to inhibit ethanol induced ROS production in LO2 cells. Data are denoted as the mean ± SE of three independent experiments. #p < 0.05, ##p < 0.01, ###p < 0.001, *p < 0.05, **p < 0.01, ***p < 0.001 (# denotes significance compared with control, while * represents significance compared with ethanol-treated group)

Ethanol caused large amounts of ROS production in LO2 cells compared with the control group (Fig. 2F and G). After treatment with C-JHCF4 or T-JHCF4, ROS production in both groups decreased dose-dependent. Compared to T-JHCF4, C-JHCF4 had a stronger antioxidant capacity at the same concentration. Thus, the bioactivity of C-JHCF4 was selected for further evaluation.

C-JHCF4 inhibits ethanol-induced LO2 cell damage through the regulation of apoptosis-related pathways

As shown in Fig. 3A, compared with the control group, the ethanol-induced group showed a notable increase in blue fluorescence, and C-JHCF4 effectively weakened blue fluorescence intensity. As the concentration of C-JHCF4 increased, the degree of weakening of blue fluorescence intensity increased considerably.

Fig. 3.

Validation of the protective effect of C-JHCF4 on LO2 cells. A Hoechst 33,342 staining, the white arrows point to the nucleus of apoptotic cells. B PI staining, white arrows point to the nucleus of apoptotic cells. C TEM, the red arrows point to the mitochondria. D Immunofluorescence images of cytochrome C (red), Bcl-2 (green) and nuclear staining with DAPI (blue) in LO2 (200×). E Western blotting analyses of LO2 cells for measuring the expression of Caspase 3, Bax and cytochrome C. F Results of cell apoptosis detection by flow cytometry. Data are denoted as the mean ± SE of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (* represents significance compared with ethanol-treated group)

Compared to the control group, the ethanol-induced group showed a notable increase in red fluorescence intensity, and C-JHCF4 effectively weakened red fluorescence intensity (Fig. 3B). As the concentration of C-JHCF4 increased, the degree of weakening of red fluorescence increased.

TEM results revealed that the mitochondria of cells treated with ethanol showed significant contraction and no obvious cristae, a typical feature of apoptosis. Furthermore, C-JHCF4 treatment effectively restored mitochondrial characteristics in cells (Fig. 3C).

As shown in Fig. 3D, C-JHCF4 significantly decreased and increased the immunofluorescence staining intensity of cytochrome C and Bcl-2, respectively. Western blotting results revealed that C-JHCF4 substantially reduced the expression of Bax, Caspase 3, and cytochrome C in ethanol-treated LO2 cells (Fig. 3E). Likewise, similar results in Fig. 3D and E further demonstrated the alleviating effect of C-JHCF4 on ethanol-induced cell damage.

To verify the anti-apoptotic effects of C-JHCF4, flow cytometry was performed to detect the survival of cells in each group. As shown in Fig. 3F, compared with the 0 µg/mL group, after treatment with higher concentrations of C-JHCF4, the number of apoptotic cells was considerably reduced, especially late apoptotic cells. These results indicate that C-JHCF4 effectively inhibited ethanol-induced apoptosis in LO2 cells.

Protective effect of C-JHCF4 against ethanol-treated zebrafish embryos (72 hpf)

As shown in Fig. 4A, the survival rate of the zebrafish decreased when the ethanol concentration reached 200 mM, therefore, 200 mM ethanol was selected for validation in this study. Zebrafish were stimulated with 200 mM ethanol strongly increased cell death (Fig. 4B), ROS production (Fig. 4C), and lipid oxidation levels (Fig. 4D). After treatment with C-JHCF4, the fluorescence intensity of the zebrafish significantly decreased, indicating that treatment with C-JHCF4 substantially reduced the degree of ethanol-induced ROS release, cell death, and lipid peroxidation.

Fig. 4.

Protective effect of C-JHCF4 on zebrafish at 72 hpf. A Screening the optimal ethanol concentration through 72 hpf zebrafish larvae. B AO generation in the ethanol-induced zebrafish treated with C-JHCF4. C ROS production in the ethanol-induced zebrafish under C-JHCF4 treatment. D Degree of lipid peroxidation. Data are denoted as the mean ± SE of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (* represents significance compared with ethanol-treated group)

Protective effect of C-JHCF4 against ethanol-treated zebrafish embryos (128 hpf)

Experimental outline for 128 hpf zebrafish larvae (Fig. 5A). To confirm the effect of C-JHCF4, oil Red O was used to detect the fat degeneration in ethanol-treated 128 hpf larvae. As shown in Fig. 5B, there was no lipid accumulation in the liver tissue of zebrafish in the control group. However, zebrafish treated with ethanol instead of C-JHCF4 showed significant lipid accumulation. As the concentration of C-JHCF4 increased, the degree of lipid accumulation in the zebrafish livers decreased considerably. As shown in Fig. 5C, H&E staining results showed that compared with the control group, the degree of liver tissue lesion in zebrafish was significantly enhanced after ethanol induction, while after treatment with C-JHCF4, the degree of liver tissue lesion in zebrafish was significantly decreased. These findings indicate that C-JHCF4 effectively inhibits ethanol-induced liver damage in zebrafish.

Fig. 5.

Protective effect of C-JHCF4 on 128 hpf zebrafish larvae. A Outline is the ethanol treatment protocol to zebrafish; ethanol exposure began at 96 hpf and continued for 32 h up to 128 hpf. B Degree of degeneration of the liver of zebrafish larvae detected by oil Red O. C Effect of C-JHCF4 on liver histopathology in zebrafish with ethanol-induced liver injury (400×). D MDA content in 128 hpf zebrafish larvae. E GSH content in 128 hpf zebrafish larvae. F Expression of related proteins in zebrafish larvae. Data are denoted as the mean ± SE of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (* represents significance compared with ethanol-treated group)

MDA production in the liver is indirectly associated with lipid peroxidation. In addition, GSH production in the livers treated with C-JHCF4 indicated that antioxidant production in liver tissues is a defense mechanism against oxygen free radicals. As shown in Fig. 5D and E, the MDA level in 128 hpf larvae increased substantially after ethanol treatment, and the GSH level decreased considerably compared to untreated larvae. However, C-JHCF4 reduced MDA production and increased GSH content dose-dependently. Western blotting also showed that C-JHCF4 increased the expression of the anti-apoptotic protein Bcl-2 in 128 hpf larvae and inhibited the expression of the apoptotic proteins Bax, Caspase 3, and cytochrome C (Fig. 5F). These results indicated the protective effects of C-JHCF4 against ethanol-induced damage in 128 hpf larvae.

The prospect of fucoidan as a treatment for ALD

People have long anticipated the use of drugs with low toxicity and abundance for ALD treatment. Recent study have shown that marine algae, especially, brown seaweeds meet these two requirements as they grow in marine environments worldwide and exert anticancer, anti-inflammatory, and other therapeutic effects (Dhahri, 2023). Hizikia fusiformis can effectively inhibit liver tumor growth and metastasis (Kim et al., 2021). Study have shown that padina pavonia, as a member of brown algae, can enhance the antioxidant activity of liver (Abdella et al., 2016). Fucoidan extracted from Costaria costata has antioxidant activity and can be used to treat liver damage (Wang et al., 2014). In our previous study, we extracted fucoidan from Hizikia fusiformis using cellulase-assisted extraction method, and fucoidan has antioxidant activity (Dai et al., 2019). In this study, we extracted fucoidans from Hizikia fusiformis through cellulase-assisted and high-temperature water extraction methods. We purified them to obtain JHCF4s, and we found that C-JHCF4 have strong anti-ethanol damage activity.

Methods for extracting fucoidan from Hizikia fusiformis can be divided into cellulase-assisted and high-temperature water extraction methods. The high-temperature extraction of drugs is easy to implement, cost-effective, and widely used in industries worldwide (Hifney et al., 2016). Although cost is an important factor for industrial production, numerous studies have shown that drugs obtained with cellulase-assisted extraction are more active than those obtained with high-temperature water extraction, and the use an efficient extraction method is important for maximizing the activity of fucoidan obtained from Hizikia fusiformis (Husni et al., 2022). Fucoidan extracted from Ecklonia cava with the assistance of enzymes has an excellent anti-inflammatory effect compared to non-enzymes (Lee et al., 2012). Therefore, we analyzed the different compositions and structures of fucoidans extracted using these two methods in this study. In vitro activity comparison experiments were conducted, and the antioxidant and anti-apoptotic effects of fucoidan extracted using the cellulase-assisted extraction method were verified.

Classic apoptotic pathways include the mitochondrial, dead receptor, and endoplasmic reticulum stress pathways. In this study, the Bax/Bcl-2/Caspase 3 pathway was selected as the mitochondrial pathway, which belongs to the classical apoptotic pathway (Liu et al., 2022). The anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax jointly maintain cell stability through mutual regulation (F. S. Al-Zubaidy et al., 2022) and mediation by the Bcl-2/Bax/Caspase 3 apoptotic signaling pathway. In the mitochondria, an increase in Bax expression induces the release of cytochrome C, further stimulating caspase production (Jensen et al., 2019). The downregulation of Bax and upregulation of Bcl-2 resulted in dose-dependent protective effects. Cytochrome C activated the prototype caspase enzyme Caspase 3, which increased its expression and caused cell apoptosis. On treatment with fucoidan, the ethanol-induced expressions of Bax, Caspase 3, and cytochrome C were reduced and those of Bcl-2 increased (Yang et al., 2015). Therefore, C-JHCF4 has the potential to be developed into a drug that confers protection against ethanol-induced liver damage. In addition, C-JHCF4 exhibits significant hepatoprotective effects by exerting antioxidant activity. Therefore, it is speculated that the synergistic use of C-JHCF4 with other substances with antioxidant activity, such as silymarin, can enhance its antioxidant activity and strengthen its hepatoprotective effect (Shaker et al., 2010).

LO2 cells, as a model of normal human liver, have the advantages of rapid growth and low cost, and are often selected as models for studying human liver related diseases. Zebrafish, due to their highly similar genes to humans, rapid reproduction, and transparent embryos, were selected as models for studying ALD in this study (Goessling and Sadler, 2015). However, compared to mammalian models, zebrafish have a different liver anatomy and framework, and their less conservative physiology and morphogenesis are major drawbacks to their use in biomedical research (Zhu et al., 2022). Therefore, it is necessary to use mammalian models to verify the hepatoprotective effect of C-JHCF4 and further clarify its mechanism of action in subsequent studies.

As production cost is a decisive factor for large-scale industrial production, the inexpensive high-temperature water extraction method can be adopted (Singh et al., 2023). Although fucoidan obtained using enzyme-assisted extraction has a stronger therapeutic effect on ALD, its high cost is a major factor limiting large-scale industrial production (Sulyman et al., 2020). The yield of C-JHCF4 can be improved by repeatedly extracting the residue. In addition, cellulase can be produced commercially (Areeshi, 2022), suggesting an essential role for fucoidan extraction using cellulase-assisted extraction in treating ALD.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Tongchuan Wu: Writing-review & editing, Validation, Resources, Methodology. Yichao Liu: Writing-original draft, Investigation, Data curation, Software. Xindan Ai: Writing-original draft, Validation, Investigation, Formal analysis. Sixuan Wang: Writing-original draft, Validation, Investigation, Visualization, Formal analysis. Aixia Hou: Writing-original draft, Formal analysis. Fei Zheng: Writing-review & editing, Data curation. Hao Yue: Writing-review & editing, Supervision, Project administration, Conceptualization. Yulin Dai: Writing-review & editing, Project administration, Funding acquisition, Conceptualization, Supervision and Resources.

Funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This study was supported by the National Natural Science Foundation of China (No. 82204719, China) and the Scientific and Technological Development Program of Jilin Province (No. 20220508063RC, QK2021007, China).

Data availability

The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.

Declarations

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Ethical statement

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. The animal study was approved by the Institutional Animal Care and Use Committee of Changchun University of Chinese Medicine (resolution number: 2023473). The study was conducted in accordance with the local legislation and institutional requirements.