H₂O₂ was purchased from Sigma-Aldrich (St. Louis, MO, USA). The insulin content assay kit was purchased from EZAssay (Shenzhen, China). The β-galactosidase (β-gal) activity kit was purchased from GenMed Scientific (Shanghai, China). The ATP assay kit was purchased from Beyotime (Shanghai, China). The reactive oxygen species (ROS) and superoxide dismutase (SOD) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

The antibodies against Ki-67, γH2A.X, Kif5b, Snap25, SIRT1, PGC1-α, Tfam, and Nrf2 were purchased from Abcam (Cambridge, UK). Stx1, Aqp2, Atp5α, Uqcrc2, Mtco1, Sdhb and Ndufb8 were purchased from ABclonal (Wuhan, China). Kir6.2 was purchased from origene (Maryland, USA).

Fucoidan extracted from L. japonica was prepared according to a previous study (20). Then, 1 g fucoidan was degraded in 0.1 M HCl for 2 h at 80°C. The degradation solution was neutralized, concentrated, and precipitated by ethanol. Then, the precipitation was re-dissolved and degraded in 0.5 M HCl. The degradation solution was again neutralized, concentrated and precipitated by ethanol. The precipitation was re-dissolved, purified on a Bio Gel P10 column (2.6 × 100 cm) (Bio-Rad, CA, USA), and eluted with 0.2 M NH₄HCO₃ to obtain SFG. SFG powder was dissolved in phosphate-buffered saline (PBS) as stock solution with a concentration of 20 mg/ml. Polysaccharide composition and molecular weight (MW) were determined according to previous studies (21–23). In brief, 1-Phenyl-3-methyl-5-pyrazolone (PMP) is a common sugar derivatization agent used in high-performance liquid chromatography (HPLC). Polysaccharide composition was determined by PMP derivatization HPLC, including the total sugar content, fucose (Fuc) content, uronic acid (UA) content, sulfate content. The gel permeation chromatography–high-performance liquid chromatography (GPC-HPLC) analysis are used to estimate the MW.

The MIN6 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium containing 0.05% 2-mercaptoethanol, 15% embryonic stem-cell screened fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere under 5% CO₂. Cells were exposed to 125 μM H₂O₂ for 2 h. Then, the H₂O₂-containing medium was replaced with fresh medium containing 0, 25, 50, 100, or 200 μg/ml SFG for an additional 24 h before harvesting.

The MIN6 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well. Cells were treated with 125 μM H₂O₂ for 2 h, then replaced with fresh medium containing various concentrations of SFG and incubated for 24 h. After treatment, the culture supernatant was removed, and the cells were incubated in culture medium containing 10 μl Cell Counting Kit-8 (CCK-8, Yeasen Biotech, Shanghai, China) solution for 1.5 h at 37°C in the dark. The absorbance in each well was measured at 450 nm using a Multiskan GO microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

The MIN6 cells were plated in 12-well plates at a density of 2 × 10⁵ cells per well. Cells were treated with 125 μM H₂O₂ for 2 h, then replaced with fresh medium containing various concentrations of SFG and incubated for 24 h. After treatment, the cells were incubated with glucose-free Krebs-Ringer bicarbonate buffer (KRBH, containing 0.1% BSA) for 30 min before 1 h incubation in KRBH buffer containing 2 mM or 25 mM glucose. The supernatant was collected, and the insulin content was measured by enzyme-linked immunosorbent assay. Results of insulin secretion were normalized by protein concentration and expressed as fold change related to control.

The method of isolating islets was referred to the previous study (O'Dowd et al., 2020). Islets were exposed to 125 μM H₂O₂ for 6 h and picked up to the fresh medium containing 50 and 100μg/ml SFG for an additional 24 h. Subsequently, islets were incubated with KRBH containing 0.1% BSA for 30 min and stimulated at 2.8 mM and 16.7 mM glucose KRBH buffer for 1 h separately. The supernatant was collected, for detecting insulin content of islets, the steps were the same as for the detection of MIN6 cells.

The MIN6 cells were plated in 35-mm MatTek imaging dishes(Cellvis, CA, USA) at a density of 4 × 10⁵ cells per well. After 18 h culture for adhesion, the cells were transiently transfected with VAMP2-pHluorin using Lipofectamine 3000 according to the manufacturer’s protocol. At 24 h after transfection, the cells were treated with 125 μM H₂O₂ for 2 h and the medium was replaced with fresh medium containing various concentrations of SFG for 24 h. The cells transfected with VAMP2-pHluorin were serum-starved in KRBH buffer for 1 h prior to microscopy. Throughout the imaging experiment, the cells were kept in an Air-Therm (WPI) temperature-regulated environmental chamber (Shanghai JingHong Laboratory Equipment Co., Shanghai, China) at 37°C. Basal insulin secretion state of cells were imaged for 2 min in KRBH containing 2 mM glucose. Then glucose stock (50 mM) was added to the edge of the MatTek dish on the microscope stage to reach a final concentration of 25mM glucose. The cells were imaged for 6 min in the glucose-stimulated state. TIRFM was performed using an Olympus objective-type IX-70 inverted microscope fitted with a 60×/1.45 NA TIRFM lens (Olympus, Center Valley, PA, USA) controlled by Andor iQ software (Andor Technologies, South Windsor, CT, USA) and detected with a back-illuminated Andor iXon 897 EMCCD (electron multiplying charge-coupled device) camera (512 × 512, 14 bit; Andor Technologies) (24).

The MIN6 cells were plated in 12-well plates at a density of 2 × 10⁵ cells per well. Cells were treated with 125 μM H₂O₂ for 2 h, then replaced with fresh medium containing various concentrations of SFG and incubated for 24 h. Senescence was assessed by X-Gal staining for detecting β-gal activity with a commercially available kit after 24 h incubation in the fixative solution provided in the kit.

The MIN6 cells were plated in 12-well plates at a density of 2 × 10⁵ cells per well. Cells were treated with 125 μM H₂O₂ for 2 h, then replaced with fresh medium containing various concentrations of SFG and incubated for 24 h. The MIN6 cells were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100/PBS for 15 min, and blocked with 5% BSA for 1 h. Cells on coverslips were incubated overnight at 4°C with the detection antibodies. After several washes in PBS, the fixed cells were incubated for 1 h with a goat anti-rabbit secondary antibody (Invitrogen, CA, USA; 1: 200) and counterstained for 5 min with DAPI. After mounting with fluorescence decay-resistant medium, the cells were observed and photographed under a confocal microscope (IX83-FV3000, Olympus, Tokyo, Japan).

The MIN6 cells were plated in 6-well plates at a density of 4 × 10⁵ cells per well. Cells were treated with 125 μM H₂O₂ for 2 h, then replaced with fresh medium containing various concentrations of SFG and incubated for 24 h. RNA was isolated using AG RNAex Pro reagent and reversed-transcribed using an Evo M-MLV RT Premix kit (Accurate Biotechnology, Hunan, China) according to the manufacturers’ protocols. Real-time PCR was performed using the LightCycler 480 II system (Roche, Basel, Switzerland) and a SYBR Green Premix Pro Taq HS qPCR kit (Accurate Biotechnology, Hunan, China). After normalization to actin, the relative gene expression was determined using the comparative threshold cycle (2^−ΔΔCT) method. The primer sequences are shown in the Supplementary Data .

The MIN6 cells were plated in 6-well plates at a density of 4 × 10⁵ cells per well. Cells were treated with 125 μM H₂O₂ for 2 h, then replaced with fresh medium containing various concentrations of SFG and incubated for 24 h. The MIN6 cells were lysed with cell lysis buffer containing a protease inhibitor cocktail. Protein concentrations were determined by a bicinchoninic acid protein assay kit (FdBio Science, Hangzhou, China). Total protein extracts were denatured and separated by 10% or 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The membranes were incubated with primary antibodies for detection at 4°C overnight. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody. The proteins were visualized using an enhanced chemiluminescence kit (FdBio Science, Hangzhou, China).

The MIN6 cells were plated in 6-well plates at a density of 4 × 10⁵ cells per well. Cells were treated with 125 μM H₂O₂ for 2 h, then replaced with fresh medium containing various concentrations of SFG and incubated for 24 h. The ATP levels were measured using an ATP assay kit. The method is based on a luciferase-luciferin reaction assay. The results were normalized by protein concentration.

The MIN6 cells were plated in 6-well plates at a density of 4 × 10⁵ cells per well. Cells were treated with 125 μM H₂O₂ for 2 h, then replaced with fresh medium containing various concentrations of SFG and incubated for 24 h. ROS production was identified using an ROS assay kit. The treated MIN6 cells were washed with PBS and incubated with DCFH-DA without light for 1 h at 37°C. The cells were washed in PBS three times and the fluorescence intensity was detected using a fluorescence microscope (Nikon Corporation, Tokyo, Japan).

The SOD activity in the cell medium was analyzed with SOD assay kit according to the manufacturer’s protocol. The absorbance at 450 nm was recorded using a microplate reader (Synergy H4, BioTek Instruments, Winooski, VT, USA). The results were normalized by protein concentration.

All experiments were repeated in triplicate. The results are expressed as the mean ± SEM. One-way analysis of variance (GraphPad Prism 7) was used for data comparisons within multiple groups. P < 0.05 was set as the threshold for statistical significance.

The chemical composition analysis indicated that SFG contained 59.9% total sugar, 25.8% Fuc, 10.4% UA, and 13.1% sulfate. The molar ratio of monosaccharides of SFG was 1.25: 1 (galactose: Fuc), demonstrating that SFG it is mainly a sulfated fucogalactan ( Figure 1 ). The GPC-HPLC analysis indicated that the average MW of SFG was approximately 3.9 kDa ( Figure 1 ).

The cytotoxicity of SFG was evaluated in vitro. After SFG (25–200 μg/ml) treatment, MIN6 cell viability was measured using the CCK-8 assay. There were no significant changes in cell viability after SFG treatment at any of the concentrations tested, showing that SFG had no obvious cytotoxic effects on the cells ( Figure 2A ). Compared with the untreated control cells, exposure to H₂O₂ inhibited MIN6 cell viability while treatment with 50–200 μg/ml SFG significantly alleviated this effect (P < 0.05) ( Figure 2B ). The effect of 50 and 100 μg/ml SFG was studied in the follow-up experiments.

The effect of SFG on MIN6 cell insulin secretion was investigated using the GSIS test. Cells that had been exposed to H₂O₂ had decreased insulin secretion in the presence of high glucose levels (25 mM) compared with that of the untreated control cells and SFG treatment enhanced insulin secretion (P < 0.05). Nevertheless, there was no difference under low-glucose stimulation ( Figure 2C ). Meanwhile, consisting with the results in MIN6 cells, GSIS test on islets isolated from C57BL/6 mice have also demonstrated that SFG treatment enhanced insulin secretion in the presence of high glucose levels (16.7 mM) (P < 0.05) ( Figure 2D ).The regulation of fusion pore dynamics is important for insulin secretion, as premature fusion pore closure is a likely mechanism by which insulin release is prevented. A recent study revealed two exocytosis modes: (1) full fusion, in which the exocytic vesicle and the plasma membrane fusion completely; and (2) kiss‐and‐run events, in which insulin granules release their contents through a transiently opened fusion pore (25). Here, H₂O₂-induced MIN6 cells had fewer glucose-stimulated full fusion events compared with the control cells, and SFG treatment significantly increased full fusion events (P < 0.05) ( Figure 2E ). There was no difference in the proportion of kiss-and-run fusion events between the treatment and control groups ( Figure 2F ). Representative MIN6 cells under glucose-stimulated state were observed under TIRFM ( Figure 2G ). Our findings demonstrate that H₂O₂ exposure results in significantly reduced insulin secretion in MIN6 cells, which is alleviated by SFG treatment.

We studied the effect of SFG on MIN6 cell proliferation via immunofluorescence staining of Ki-67, a key cell proliferation marker (26). H₂O₂ exposure significantly inhibited Ki-67 expression in the cells and SFG eliminated this effect (P < 0.05) ( Figure 3A ). We examined the protein expression of the cell cycle regulators CDK4, RB, pRB, E2F1, and EZH2. Compared with the untreated control cells, H₂O₂-treated MIN6 cells had decreased CDK4, pRB, E2F1, and EZH2 protein expression, which was increased with the addition of SFG (P < 0.05) ( Figure 3B ). This indicates that SFG facilitated cell proliferation through cell cycle upregulation in H₂O₂-treated MIN6 cells.

We investigated whether SFG treatment would alleviate MIN6 cell senescence. The expression of senescence-related phenotypes was detected, including the senescence marker γH2A.X; the cellular senescence-associated proteins such as p16, p21, and p53; and SA-β-gal activity. Immunofluorescence staining showed that H₂O₂ increased γH2A.X accumulation in the cells as compared with the untreated control cells while SFG treatment rescued this effect (P < 0.05) ( Figure 3C ). Western blot confirmed that p16, p21, and p53 protein levels were greatly upregulated in the H₂O₂ group and that SFG treatment downregulated all three proteins (P < 0.05) ( Figure 3D ). Compared with the untreated control cells, H₂O₂-treated MIN6 cells had widespread and intense SA-β-gal-positive staining, while only sporadic staining was detected in the cells following SFG treatment (P < 0.05) ( Figure 3E ). Taken together, SFG promotes the inhibited proliferation in H₂O₂-treated MIN6 cells and ameliorates their senescence.

We evaluated several genes and proteins that are essential in β-cells. NeuroD1, Mafa, and Pdx1 are critical for maintaining function in mature β-cells. The decreased expression of these key β-cell markers is associated with cellular dedifferentiation (27). In our study, Neurod1, Mafa, and Pdx1 gene levels were dramatically downregulated in the H₂O₂ group while SFG treatment upregulated all three genes (P < 0.05) ( Figure 4A ). Western blot revealed a similar pattern for the Neurod1, Mafa, Pdx1 mRNA levels (P < 0.05) ( Figure 4B ).

We then examined the genes and proteins crucial to insulin secretion. Glut2 controls glucose entry into cells and is key to maintaining glucose homeostasis (28). Kif5b is responsible for the transport of insulin-containing granules (29). Glut2 mRNA levels was significantly reduced in H₂O₂-treated MIN6 cells and was upregulated by the addition of SFG (P < 0.05) ( Figure 4C ). Glut2 protein expression revealed a similar pattern for the Glut2 mRNA levels (P < 0.05).While there was no difference in Kif5b protein expression ( Figure 4D ). We also evaluated some genes and proteins essential for vesicle fusion with the plasma membrane to release insulin. H₂O₂ resulted in significantly decreased Snap25, Ip3r1, Aqp2, Stx1, and Kir6.2 mRNA levels, while SFG increased these mRNA expression (P < 0.05) ( Figure 4C ). Western blot revealed a similar pattern for the Snap25, Aqp2, Stx1, and Kir6.2 mRNA levels (P < 0.05) ( Figure 4D ). These data indicate that SFG potentiates β-cell function in MIN6 cells by regulating β-cell identity and the insulin secretion-related genes and proteins.

As the powerhouse of the cell, the mitochondria play a central role in maintaining β-cell health and function, which is involved in integrating and generating metabolic signals to control insulin secretion (5). Here, we explored the effect of SFG on mitochondrial function by detecting mitochondrial respiratory chain related proteins and genes and ATP content in MIN6 cells. H₂O₂-treated MIN6 cells had decreased levels of mitochondrial respiratory chain related proteins and mRNA, but SFG supplementation restored the decrease (P < 0.05) ( Figures 5A, B ). Meanwhile, the ATP content is decreased when exposed to H₂O₂, and increased when supplied with SFG (P < 0.05) ( Figure 5C ). The mitochondria are the primary site of ROS production and are susceptible to oxidative stress (30). Here, the H₂O₂-treated MIN6 cells had higher ROS levels and lower SOD levels than the untreated control cells. SFG markedly reduced ROS levels while significantly increasing SOD activity (P < 0.05) ( Figures 5D, E ). Our data indicate that H₂O₂ promotes mitochondrial dysfunction and ROS accumulation in MIN6 cells, leading to β-cell failure, and that SFG treatment alleviates these effects.

PGC1-α is a critical regulator of oxidative metabolism and is involved in maintaining mitochondrial biogenesis and function (31). SIRT1 acts as an upstream kinase and activates PGC1-α expression directly (32). In the present study, H₂O₂-treated MIN6 cells had markedly decreased Sirt1 and Pgc1-α mRNA expression compared with the untreated control group, and SFG increased Sirt1 and Pgc1-α mRNA expression (P < 0.05) ( Figure 6A ). PGC1-α exerts effects through the coactivation of many nuclear receptors and factors outside the nuclear receptor family. Nrf2 and Tfam are key targets of PGC1-α in mitochondrial biogenesis (32). Nrf2 and Tfam mRNA expression was also downregulated in MIN6 cells following exposure to H₂O₂, and SFG supplementation upregulated their expression (P < 0.05) ( Figure 6A ). Western Blot revealed a similar pattern in the Sirt1, Pgc1-a, Nrf2, and tfam mRNA levels (P < 0.05) ( Figure 6B ).