Sargassum fusiforme fucoidan modifies the gut microbiota during alleviation of streptozotocin-induced hyperglycemia in mice

Sargassum fusiforme fucoidan modifies the gut microbiota during alleviation of streptozotocin-induced hyperglycemia in mice

Accepted Manuscript Sargassum fusiforme fucoidan modifies the gut microbiota during alleviation of streptozotocin-induced hyperglycemia in mice Yang ...

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Accepted Manuscript Sargassum fusiforme fucoidan modifies the gut microbiota during alleviation of streptozotocin-induced hyperglycemia in mice

Yang Cheng, Luthuli Sibusiso, Lingfeng Hou, Huijing Jiang, Peichao Chen, Xu Zhang, Mingjiang Wu, Haibin Tong PII: DOI: Reference:

S0141-8130(18)37075-2 https://doi.org/10.1016/j.ijbiomac.2019.04.040 BIOMAC 12116

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

18 December 2018 27 March 2019 7 April 2019

Please cite this article as: Y. Cheng, L. Sibusiso, L. Hou, et al., Sargassum fusiforme fucoidan modifies the gut microbiota during alleviation of streptozotocin-induced hyperglycemia in mice, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.04.040

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ACCEPTED MANUSCRIPT Research Article Sargassum fusiforme fucoidan modifies the gut microbiota during alleviation of

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streptozotocin-induced hyperglycemia in mice

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Yang Cheng 1, Luthuli Sibusiso 1, Lingfeng Hou, Huijing Jiang, Peichao Chen, Xu

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Zhang, Mingjiang Wu *, Haibin Tong *

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College of Life and Environmental Science, Wenzhou University, Wenzhou 325035,

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China;

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*Correspondence: [email protected] (M.W.); [email protected] (H.T.); Tel.: +

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Contributed equally to this work.

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1

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86-577-86689078

ACCEPTED MANUSCRIPT Abstract: Diabetes is a complicated endocrine and metabolic disorder, which has become an epidemic health issue worldwide. Fucoidan is extensively distributed in the brown algae and several marine invertebrates exhibiting diverse biological activities. In the present study, the physicochemical property of Sargassum fusiforme

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fucoidan (SFF) and its effects on streptozotocin (STZ)-induced diabetic mice and gut

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microbiota were investigated. Diabetes mice not only showed abnormal blood glucose,

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but also accompanied by multiple symptoms, such as gradual emaciation, decreased body weight, increased food and water intake. Compared with diabetic mice after

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6-week treatment, administration of SFF significantly decreased the fasting blood

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glucose, diet and water intake. Furthermore, SFF attenuated the pathological change in the heart and liver, improved the liver function, and suppressed oxidative stress in

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STZ-induced diabetic mice. Simultaneously, SFF significantly altered the gut

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microbiota in the faeces of diabetic mice, decreased the relative abundances of the diabetes-related intestinal bacteria, which is a potential mechanism for relieving the

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symptoms of diabetes. Therefore, SFF might be considered as one of the promising

future.

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complementary and alternative medicines for the management of diabetes mellitus in

Keywords: Sargassum fusiforme; Gut microbiota; Diabetes

ACCEPTED MANUSCRIPT 1. Introduction Sargassum fusiforme, an edible brown alga that belongs to the Sargassaceae family, is extensively distributed in eastern Asian countries, particularly China, Japan, and South Korea [1]. S. fusiforme is a popular functional seaweed which can

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prolong life, also has been developed as a traditional Chinese medicine for thousands

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of years to treat patients with scrofula, oedema, indigestion, and stagnation of Qi

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[2,3]. Polysaccharides, which account to 40 to 80% of dry defatted seaweed biomass, have been considered as one of the most important bioactive macromolecules in S.

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fusiforme [4]. Modern pharmacological studies have demonstrated that S. fusiforme

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polysaccharides possess multiple medicinally effects, such as anti-tumor, anti-hyperlipidemia, and immunomodulatory, and antioxidant activity [5-7].

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The polysaccharides in brown seaweeds mainly contain alginate, laminaran and

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fucoidan [8]. However, present studies on S. fusiforme polysaccharides reported the bio-activities using a mixture of polysaccharides without separating those

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compounds [3]. Recently, fucoidan has been extensively studied because of their

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diverse beneficial functions to human health [9,10]. Fucoidan is a family of sulfated homo- and heteropolysaccharides, extensively distributed in the brown algae and several marine invertebrates [11,12]. Fucoidan usually contains a high percentage of fucose and galactose residues, as well as variable levels of a range of other neutral and acidic monosaccharides, including mannose, glucose, xylose, and glucuronic and galacuronicacids [13,14]. Fucoidan from brown seaweeds may have -(1→ 3)-backbones or repeating disaccharide units of -(1→3)-and -(1→4)-linked fucose

ACCEPTED MANUSCRIPT residues with O-2 branches depending on brown algal species [12,14]. Due to the structure of the main chain, fucoidan can be sulfonated at O-4, O-2, or at both positions of the fucose units [14]. The newest global estimate from the International Diabetes Federation is that in

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221 countries there were 425 million people with diabetes mellitus (1 in 11 adults) in

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2017; and it was predicted that the number of diabetes patients aged 20-79 years will

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increase to 629 million or to 693 million among 18-99 years by 2045 [15]. With the escalating incidence, pharmacotherapy involving synthetic insulin and anti-diabetic

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agents is clinically administered for diabetic therapy, but these are deficient in

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having multiple dosage regimens, side effects, toxicity, and high costs [16]. Therefore, it is essential to screen novel and effective medicines for the management

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of diabetes mellitus. Recent studies have demonstrated that the modulation of the gut

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microbiota may be one of the potential mechanisms contributing to the anti-diabetic effects of natural polysaccharides from seaweeds [17,18]. Fucoidan, as

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macromolecular carbohydrate, is indigestible and fermentative in the colon.

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Fortunately, it has been widely reported that fucoidan possesses anti-diabetic activities through distinct mechanisms, such as inhibiting α-glucosidase activity, improving β-cell dysfunction, ameliorating glucose metabolism in liver and muscle tissues, enhancing insulin sensitivity [19-22]. However, whether the fucoidan from S. fusiforme is able to alleviate diabetes by the modulation of gut microbiota in STZ-induced hyperglycemic mice is still unknown. In the present study, the anti-diabetic effects and the modulation on gut microbiota of SFF were evaluated by

ACCEPTED MANUSCRIPT STZ-induced diabetic mice.

2. Materials and methods 2.1. Materials and chemicals

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S. fusiforme was collected in October 2017 from the East China Sea on the coast

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of Dongtou District, Wenzhou City (Zhejiang Province, China). The voucher

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specimen (No. 2017-0195) was identified by Prof. Mingjiang Wu and deposited at the College of Life and Environmental Science, Wenzhou University, Wenzhou, China

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(Supplemental Figure S1). STZ was purchased from Sigma-Aldrich Co. (St. Louis,

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MO, USA). Standard monosaccharides [including arabinose (Ara), rhamnose (Rha), xylose (Xyl), fucose (fuc), mannose (Man), galactose (Gal), glucose (Glc), glucuronic

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acid (GlcA), and galacturonic acid (GalA)] were purchased from Aladdin Reagent Int

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(Shanghai, China). All other chemical reagents used were analytical grade. 2.2. Extraction of S. fusiforme fucoidan

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The procedure for the fucoidan extraction from S. fusiforme is illustrated in Figure

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1. Briefly, S. fusiforme was dried to constant weight at 60℃, and then the dried alga was ground into powder and defatted with 80% ethanol for 12 h to degrease. The fucoidan from defatted algal powder was extracted by soaking 0.01 M HCl (1:20, w/v), stirred for 6 h and filter through a mesh nylon sieve. Then, the filtrate was added with 4 M CaCl2, which can react with alginate to form a precipitate of calcium alginate. After incubated for 30 min, the mixture solution was re-filtered to remove the precipitate. Filtrate was diluted using distillated water until the final CaCl2

ACCEPTED MANUSCRIPT concentration of 2 M, centrifuged at 15000 rpm for 15 min, and then added with 3 M CaCl2 and then centrifuged at a speed of 15000 rpm for another 15 min. Supernatant was dialyzed (MWCO 1000 Da) against deionized water for 48 h. The sample was then concentrated and precipitated with four volumes of 95% ethanol, kept at 4℃ for

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and lyophilized to yield S. fusiforme fucoidan, coded as SFF.

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S. fusiforme powder

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Dried alga of S. fusiforme Ground

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12 h. The precipitate was collected by centrifugation. Then, the sample was collected

Defatted with 80% ethanol

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Dry defatted powder

Extracted (soaked in 0.01 M HCl)

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Filtrate

4M CaCl2

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Filtrate

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Residue

Residue

Diluted to a final CaCl2 concentration of 2M

Supernatant

Precipitate

3M CaCl2 Supernatant

Precipitate

Dialyzed, precipitated with ethanol S. fusiforme fucoidan Figure 1. The procedure of fucoidan extraction from S. fusiforme

2.3. Physicochemical characterization of SFF

ACCEPTED MANUSCRIPT Total carbohydrate content of SFF was quantified according to Dubois methods using L-fucose as the standard [23]. The sulfate group was quantified based on the BaCl2-gelatin method using K2SO4 as the standard [24]. Protein content was determined by the Bradford’s method [25]. Fourier transform-infrared spectroscopy

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(FT-IR) was recorded on a BRUKER Tensor 27 FT-IR spectrometer in the range of

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500-4000 cm-1, using KBr-disk method. Molecular weight was determined by HPLC.

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SFF was dissolved in distilled water, applied to a Waters 1525 HPLC system equipped with a TSK-GEL G5000 PWXL column (7.8 × 300 mm, TOSOH, Japan),

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eluted with 0.1 M Na2SO4 solution and detected by a Waters 2424 Refractive Index

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Detector. Dextran standards with different molecular weights (2000 kDa, 150 kDa, 41.1 kDa, 21.4 kDa, 7.1 kDa, 4.6 kDa and 180 Da) were to establish a standard curve

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using linear regression.

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The monosaccharide composition of SFF was determined by HPLC with a 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization method [26] with

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some modification. Briefly, SFF (4 mg) was hydrolyzed with 2M trifluoroacetic acid

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(TFA) at 120°C for 3 h in a sealed tube. After removed excess acid by repeated co-distillations with methanol, 100 μL of 0.3 M NaOH and 100 μL of 0.5 M PMP solution were added into the reaction mixture at 70 °C for 30 min. HCl solution was added to neutralize the reaction mixture, and then equal volume of chloroform was added and shaken. After centrifugation, the aqueous phase was filtered for HPLC analysis with a Hypersil ODS-2 column (5 μm, 4.6 mm × 250 mm) detected at the wavelength of 254 nm. The mobile phases were 0.05 M phosphate buffer solution (pH

ACCEPTED MANUSCRIPT 6.8) and acetonitrile (83 : 17, v/v), at a flow rate of 0.8 mL min−1. The composition and the content of monosaccharides were determined by the retention time and the peak area, in comparison with monosaccharide standards (see Supplemental Figure 2).

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2.4. Experimental animals

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Male ICR mice weighing 20 ± 2 g, purchased from Laboratory Animal Center at

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Wenzhou Medical University (Certificate no. SYXK Z2015-0009), were used for the experiment. The mice were kept under standardized conditions at a temperature of

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22-24 °C, and 50 ± 5% humidity with a 12 h light/dark cycle, and they had free access

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to standard diet and water ad libitum. All experimental protocols were formulated according to the guidelines prescribed by Wenzhou University Animal Care and Use

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Committee. They were allowed to acclimatize for one week before the experiments

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started.

2.5. STZ-induced diabetic mice and experimental design

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The STZ-induced diabetic mouse model was established as described [27] with

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some modification. Briefly, the freshly prepared solution of STZ (40 mg/kg) in 0.1 M citrate buffer (pH 4.2) was intraperitoneal injected (i.p.) to the overnight fasted mice for continuous 5 days. The fasting blood glucose (FBG) level was determined at the 7 days after STZ-injection. The mice with the levels of FBG ranging from 11.1 to 25 mmol/L were considered as diabetic mice for further study. Twenty STZ-induced diabetic mice were randomly divided into the diabetic control (DC) group and SFF-treatment (SFF) group together with ten normal mice in normal

ACCEPTED MANUSCRIPT control (NC) group. Treatments were administered intragastrically once a day for 6 weeks as follows: NC group (n=10): normal mice treated with distilled water DC group (n=10): diabetic mice with distilled water

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SFF group (n=10): diabetic mice with 100 mg/kg of SFF

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The consumption of diet and water was recorded daily, and FBG was monitored

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weekly throughout the whole experimental period. At the end of the experiment (day 42), the faece of each mouse was collected and every three or four samples in the

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same group were pooled to minimize individual variation, then preserved at -80 °C

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for microbiota analysis [28]. Blood samples were collected from the mice fasting overnight by eyeball extirpating and centrifuged to collect serum, and stored at -80 °C

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for further assay. Subsequently, the mice were sacrificed by neck dislocation, the

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hearts and livers were harvested and snap-frozen in liquid nitrogen, and then stored at -80 °C.

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2.6. Physiological and biochemical analysis

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The levels of FBG were determined by commercially available kits purchased from Jiancheng Bioengineering Research Institute (Nanjing, China). The enzyme activities of aspartate transaminase (AST), alanine transaminase (ALT), superoxide dismutase (SOD), and catalase (CAT) in the serum were determined by biochemical kits from Jiancheng Bioengineering Research Institute (Nanjing, China) following the manufacturer’s protocol. Lipid peroxidation was determined by measuring MDA concentration in the serum. The determination of MDA was performed according to

ACCEPTED MANUSCRIPT the instructions of MDA assay kit purchased from Jiancheng Bioengineering Research Institute (Nanjing, China). 2.7. Histological characterization by H&E staining Heart and liver samples were fixed in 10% formaldehyde overnight,

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paraffin-embedded, sectioned (4 μm thickness, 3-5 sections/specimen), and stained

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with hematoxylin and eosin (H&E) for histological analysis. Digital images of H&E

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stained sections were acquired with a Nikon Eclipse Ti light microscope at × 400 magnifications.

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2.8. Microbiota analysis by 16S rRNA amplicon sequencing

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Total fecal DNA was extracted using CTAB/SDS method. The V4 region of the 16S rRNA was amplified using the universal primers 515F and 806R. All PCR

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reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New

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England Biolabs). Then, the mixture PCR products were purified with GeneJETTM Gel Extraction Kit (Thermo Scientific). Sequencing libraries were generated using Ion

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Plus Fragment Library Kit 48 rxns (Thermo Scientific) following manufacturer's

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recommendations. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific). At last, the library was sequenced on an Ion S5TM XL platform (Thermo Scientific) and 400 bp/600 bp single-end reads were generated. Paired-end reads were merged using FLASH (V1.2.7), and the Raw fastq files were processed by QIIME (version 1.7.0). Sequence analysis was performed by Uparse software (v7.0.1001), and Sequences with ≥97% identity were assigned to the same Operational Taxonomic Units (OTUs). The principal component analysis

ACCEPTED MANUSCRIPT (PCA) was applied to quantify the compositional differences between microbial communities. The heatmap was implemented by using R packages heatmap (R3.1.0). Linear discriminant analysis (LDA) was carried out to determine the highly dimensional gut microbes and characteristics associated with diabetic mice and

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SFF-treated diabetic mice.

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2.9. Statistical analysis.

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Data were expressed as the means ± SEM (standard error of mean). Significant differences between two groups were analyzed using Student’s t test. Statistical

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comparisons between three groups were made by one-way ANOVA, followed by a

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Bonferroni post hoc test using GraphPad Prism 6 (San Diego, CA, USA). P values less than 0.05 were considered statistically significant.

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3. Results and discussion

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3.1. Physicochemical properties of SFF The yield of fucoidan extracted from S. fusiforme was 6.02%. The content of

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carbohydrate, sulfate group and protein, as well as average molecular weight and

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monosaccharide composition of SFF are summarized in Table 1. SFF has high sulfate content up to 14.55%, and the pH value of SFF dissolved in water is 5.45. The average molecular weight is 205.8 kDa calculated according to the calibration curve with standard dextrans. Fucose and galactose account for the highest proportion of monosaccharide composition in SFF. Due to the presence of glucose, it is speculated that SFF may contain laminaran. IR spectrum (Figure 2) of SFF revealed a typical major broad stretching peak at 3425 cm-1 for the hydroxyl group, indicating the

ACCEPTED MANUSCRIPT characteristics peak of the polysaccharide, and a weak band at 2918 cm-1 showing the C-H stretching vibration. We also found absorption at wavenumber of 1254 cm-1 that corresponds to S=O bonds. It was supported by the appearance of a peak at 827 cm-1 that corresponds to sulfate at equatorial position, assuming that sulfate group binds to

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the C-2 of fucose to form sulfate fucose. The band at 1632 cm-1 corresponded to C-O

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asymmetric stretching. The peak at 1425 cm-1 was due to the C–H variable angular

Table 1. Physicochemical properties of SFF Sulfate

(%)

(kDa)

(%)

68.33

205.8

14.55

Protein

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Mw

Molar ratios of monosaccharide

(%)

Man

Rha

Glc

Gal

Xyl

Fuc

4.13

4.45

3.34

5.44

20.83

3.70

55.67

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SFF

Carbohydrate

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and illustrated that there existed a pyranose ring.

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vibration. Peaks around 1053 cm-1 indicated C-O-C and C-O-H stretching vibration,

Figure 2. FT-IR spectrum of SFF. FT-IR was recorded on a BRUKER Tensor 27 FT-IR spectrometer in the range of 400-4000 cm-1, using KBr-disk method.

ACCEPTED MANUSCRIPT 3.2. Effects of SFF on FBG, diet and water intake in STZ-induced diabetic mice STZ is a chemotherapeutic and anti-biotic medicine. In clinical practice, STZ is still used to treat endocrine neoplasms. Apart from that, STZ induces an inflammatory response in islet β cells that produce insulin resulting in absolute insulin deficiency

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[29,30]. Further, polyphagia, polydipsia, hyperglycemia, and reduced body weight

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were observed in STZ-induced diabetic animals. Therefore, STZ-induced diabetes is

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an accepted experimental model of type 1 diabetes mellitus [31]. Diabetes was characterized by increased FBG level, which is the fastest and simplest test used to

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diagnose diabetes. The FBG was monitored weekly during 6-week treatment. As

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shown in Figure 3A, the FBG of diabetic mice (DC and SFF groups) were significantly higher than the non-diabetic mice (NC group) at week 0 (1 week after

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diabetes induction). After the intervention started, the FBG of SFF group exhibited a

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gradually decreasing trend, and significantly decreased compared with the DC group at week 5 and 6. DC group showed the highest FBG (17.33 ± 2.27 mmol/L) at week 6,

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but FBG was decreased by 21.1% in SFF group. DC group always showed gradual

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emaciation, decreased body weight, increased diet and water intake compared with NC group. As shown in Figure 3B and C, both of the diet and water intakes were elevated significantly in DC group and SFF group compared with NC group. The diet and water intake showed a gradual decline in SFF group during 6-week experimental period, especially the diet intake. The diet and water intake in SFF group were markedly decreased compared with DC group at week 6. The water intake in DC group still had an upward trend, which also indicated the deterioration of the disease

ACCEPTED MANUSCRIPT state. However, there was no significant difference (p > 0.05) in the body weight between DC and SFF groups during the experimental period (see Supplemental Figure 3). These results revealed that SFF can effectively alleviate the pathological

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symptoms in STZ-induced hyperglycemic mice.

Figure 3. Effects of SFF on FBG, diet and water intake in STZ-induced diabetic mice. FBG (A) was monitored weekly, the consumption of diet (B) and water (C) was converted to weekly consumption. Data are presented as mean ± SEM (n = 10). *p < 0.05; **p < 0.01, compared to DC

ACCEPTED MANUSCRIPT group.

3.3. Effects of SFF on the histological change and oxidative stress in diabetic mice Diabetes causes various complications, which have become the major cause of mortality in the diabetic population. Several studies have shown that hyperglycemia

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as an independent risk factor directly causes cardiac damage, leading to diabetic

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cardiomyopathy displaying a reduction in cardiac mass over time, myocardial

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hypertrophy, and interstitial and perivascular fibrosis at late phase [32,33]. Meanwhile, diabetes could increase the risk of chronic liver disease and the occurrence of

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hepatocellular carcinoma [34]. The histological slides of heart and liver were showed

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in Figure 4. The longitudinal sections of cardiac tissues from NC group showed well organized and regular cell distribution, and normal myocardium architecture with

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centrally placed nuclei and typical symmetric myofibrils. In contrast, the cardiac

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sections from DC group exhibited mild to moderate disorganization of the myofibrils with loss of striations, focal degenerating myocytes, and vascular dilatation and

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congestion, the loss of cardiac muscle cells would eventually lead to compromised

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cardiac function. SFF group was found to possess fewer severe histological changes in the cardiac tissues compared to DC group, such as the loss of striations and focal degenerating myocytes, a similar pattern as NC group could be found in SFF group. H&E-stained liver sections from NC group showing normal hepatic architecture, such as normal hepatic cells each with well-defined cytoplasm, prominent nucleus, nucleolus, and well brought out central vein. However, livers from DC group exhibited histological damage with increased infiltration of inflammatory cells in

ACCEPTED MANUSCRIPT pericentral areas and swollen hepatic cells. SFF treatment normalized the histopathological alterations that infiltrated leukocytes along with swelling hepatic cells were reduced in the liver of diabetic mice. AST and ALT are markers of hepatic function. As shown in Table 2, ALT and AST levels in the serum showed an increase

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in DC group for 1.6- and 1.9-fold, respectively, compared with that of NC group. The

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oral administration of SFF reduced the levels of ALT and AST by 40.2% and 22.2%,

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respectively, compared with DC group. These data indicated that SFF-treatment

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alleviated liver function in STZ-induced diabetic mice.

Figure 4. SFF attenuated pathological change in the heart and liver of STZ-induced diabetic

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mice. Heart and liver samples were fixed in 10% formaldehyde, paraffin-embedded, sectioned, and stained with hematoxylin and eosin for histological analysis. Digital images of H&E stained sections were acquired with a Nikon Eclipse Ti light microscope at × 400 magnifications (scale bar, 10 μm). Table 2. Effect of SFF on liver function and oxidative stress in diabetic mice ALT

AST

SOD

CAT

MDA

(IU/L)

(IU/L)

(U/mL)

(U/L)

(nmol/mL)

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NC

49.67 ± 1.18

49.00 ± 1.00

191.10 ± 16.14

59.10 ± 9.65

0.39 ± 0.01

DC

68.49 ± 13.67

92.74 ± 11.34

155.68 ± 22.55

41.74 ± 10.60

0.83 ± 0.18

SFF

48.09 ± 4.60*

72.19 ± 19.75

171.49 ± 34.16

73.70 ± 10.58*

0.66 ± 0.09*

Data are expressed as means ± SEM (n = 10). ‘*’ and ‘**’ indicate significantly different from the

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DC group at the p < 0.05 and p < 0.01 levels, respectively.

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Diabetes mellitus is typically correlated with increased free radicals or impaired

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antioxidant defense system [35]. For diabetic individuals, hyperglycemia and incorrect protein glycation may damage the endogenous antioxidant enzyme system,

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such as SOD and CAT [36]. Thus, the improving antioxidant defense system indicates

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the alleviation from pathological status of diabetes. The antioxidant enzymes and MDA levels in the serum were summarized in Table 2. The levels of SOD and CAT

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were decreased in DC group, while MDA was increased compared with that of NC

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group, which was in coincidence with accumulated evidences that STZ injection caused a great loss in the antioxidant capacity in STZ-induced diabetic mice [37].

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Furthermore, SOD and CAT of SFF group were increased by 9.2% and 43.4%,

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respectively, compared with DC group. Meanwhile, SFF could cause the decrease of MDA in diabetic mice. It is widely accepted that oxidative stress is an important contributor to diabetes mellitus. Some of the consequences of oxidative stress are the major cause for β-cell dysfunction, pancreas injury, and impaired insulin secretion, ultimately leading to diabetes [38,39]. In addition, both of clinical and experimental data suggest an inverse association between insufficient insulin secretion and ROS levels. Oxidative stress

ACCEPTED MANUSCRIPT can be reduced by natural polysaccharides via activating antioxidant enzymes; therefore, the antioxidant activity might be of one of the mechanism for the alleviation of diabetes. Overall, our present results indicated that SFF, to a certain degree, could suppress oxidative stress by improving the activities of antioxidant enzymes,

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3.4. Gut microbiota modification in diabetic mice by SFF

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providing a potential mechanism for relieving the symptoms of diabetes.

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Although the knowledge of gut microbiota is limited, the view about their mediation and maintenance roles in host health is highly agreeable. Many important

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metabolic disorders, such as diabetes, are known to be partially caused by the

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imbalance of interactions between host and gut microbiota [40,41]. The gut microbiota in individuals with preclinical type 1 diabetes mellitus (T1DM) is

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characterized by reduced bacterial and functional diversity and low community

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stability [42,43]. In a case-control study that included 16 children with T1DM and 16 healthy children, gut microbial composition showed marked differences between the

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healthy children and the children with T1DM [44]. At the phylum level, the

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abundance of Actinobacteria and Firmicutes, and the Bacteroidetes/Firmicutes ratio were all lower in the children with T1DM than the healthy children. At the genus level, the healthy children had greater numbers of Lactobacillus, Bifidobacterium, Blautia coccoides, Eubacterium rectale, and Prevotella in the gut, whereas children with T1DM contained greater numbers of Clostridium, Bacteroides, and Veillonella. Similarly, Patterson et al. observed a dramatic impact of T1DM development on the intestinal microbiota was apparent post-STZ injection and for up to 5 weeks [45].

ACCEPTED MANUSCRIPT T1DM mice induced by STZ injection was associated with a shift in the Bacteroidetes/Firmicutes ratio, while at the genus level, increased proportions of Lactobacillus and Bifidobacterium, and reduced in microbial diversity. Furthermore, the aberrant microbiota that develops in the ileum in STZ-induced diabetic rats and

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the characteristic upsurge of the Gram-negative Klebsiella could therefore be directly

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associated with the inflammation and the development of the pathological

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microenvironment, leading to both diabetes mellitus and diabetic complications [46]. Therefore, rebuilding the balance of gut microbiota has become an effective treatment

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strategy against diabetes.

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We assessed the effects of SFF on gut microbiota in diabetic mice by performing 16S rRNA-based amplicon sequencing of fecal samples, and hypothesized that

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alleviating effects are modulated via changes in the gut microbiota. The principal

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component analysis (PCA) visualized the relative similarity of gut community composition and illustrated that each group of experimental mice had its own unique

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structure of gut microbiota (Supplementary Figure 4), indicating the modulatory

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effects of SFF on gut microbiota of diabetic mice. The microbial composition analysis indicated that the gut microbiota at phylum level included three major phyla: Firmicutes, Bacteroidetes and Proteobacteria (Figure 5A). Firmicutes and Bacteroidetes were the most abundant phyla in all groups. While a reduced relative abundance of Bacteroidetes and an increased relative abundance of Firmicutes and Proteobacteria in DC group were detected comparing with NC group. Although the difference did not reach a significant level, a mild increase in Bacteroidetes (Figure

ACCEPTED MANUSCRIPT 5B) combined with a decrease in Firmicutes (Figure 5C) was observed after SFF administration. Also, a higher ratio of Bacteroidetes to Firmicutes in SFF group compared with DC group was found (Figure 5D). Many indigestible dietary fibers, such as fucoidan, inulin, pectin, arabinoxylan oligosaccharides, and arabinogalactan,

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play crucial roles in the fermentation of gut microbiota in the colon [47-49].

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Bacteroidetes is one of the major bacterial phyla, which has been reported to be

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responsible for the fermentation of complex indigestible polysaccharides [50]. Growing evidence indicated that dietary fucoidans could increase the abundance of

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Bacteroidetes [51,52]. An in vitro study also reported that a significant increase in

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beneficial Bacteroidetes combined with a significant decline in harmful Firmicutes was found in Sargassum thunbergii polysaccharide fermented by human fecal

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inoculums [53]. In addition, the declined ratio of the Bacteroidetes/Firmicutes is an

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indicator of microbial imbalance, associated with the development of diabetes [54]. Therefore, increasing the ratio of Bacteroidetes/Firmicutes is of great importance for

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the intervention of diabetes. In accordance with previous studies, our results

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demonstrate that SFF treatment can effectively increase Bacteroidetes and decrease Firmicutes, along with an increased ratio of Bacteroidetes to Firmicutes, during alleviation of hyperglycemia in mice.

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ACCEPTED MANUSCRIPT

Figure 5. SFF remodels the gut microbiota at the phylum level. Relative abundance of gut

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microbiota at the phylum level (A). Relative abundance of Bacteroidetes (B) and Firmicutes (C).

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Ratio of Bacteroidetes to Firmicutes in the fecal of mice from DC and SFF group (D).

To identify the specific bacterial taxa in each group, the composition of the

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microbiota from NC, DC and SFF-treated groups were compared by linear

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discriminant analysis (LDA). The LDA scores (Figure 6) were higher than 3 indicating a higher relative community abundance in the corresponding group than in the other two groups. The composition of intestinal flora among three groups showed distinct variations. SFF showed selective enrichment in 6 communities including: family of Aerococcaceae, genus of Lachnoclostridium and Aerococcus, species of Tidjanibacter massiliensis, Streptococcus danieliae, and Aerococcus viridans. Diabetes-related

bacteria,

including

Oscillibacter,

Ruminococcaceae,

ACCEPTED MANUSCRIPT Peptostreptococcaceae, and Peptococcaceae, had an increased abundance in DC group. For example, the Oscillibacter genus, which is known to be associated with increased gut permeability and inflammation [55], were positively associated with the

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occurrence and development of diabetes.

Figure 6. Linear discriminant analysis (LDA) score for taxa differing between treatment groups. When LDA score threshold >3 were listed, indicating a higher relative abundance in the corresponding group than in the other two groups. Blue bars represent taxa significantly increased in the SFF group. Green bars represent taxa significantly increased in NC group. Red bars

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represent taxa significantly increased in the DC group.

Figure 7. Genus-based comparison and microbial similarities. Heatmap represents clustering of bacterial communities with their relative abundances at the genus level. The bacterial communities or OTUs were clustered phylogenetically by neighbour-joining method and the libraries were clustered by profile pattern by Unifrac analysis. Relative abundances (log values) of microbial genus are displayed in a red-to-blue color code (high to low abundance).

The relative abundances of the intestinal bacteria at genus level among all groups

ACCEPTED MANUSCRIPT were further analyzed by clustering as a heatmap (Figure 7; Supplementary Figure 4), where red color code indicates a higher abundance of the microbiota. At genus level, 32 intestinal bacteria were exhibited different between NC group and DC group. Compared with NC group, the relative abundances of Harryflintia, Desulfovibrio, Faecalibaculum,

Anaerotruncus,

Blautia,

Ruminiclostridium,

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Bacteroides,

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Mucispirillum, Parabacteroides, Oscillibacter, Bilophila, Butyricimonas, Candidatus,

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Romboutsia, Dubosiella, et al., were increased in DC group, while these intestinal bacteria abundance was decreased in the SFF group. Additionally, SFF treatment

Roseburia,

Erysipelatoclostridium,

Aerococcus,

Rikenella,

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Millionella,

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increased the relative abundances of Alloprevotella. Alistipes, Odoribacter,

Lachnoclostridium, Acetatifactor, et al.

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Growing evidence shows that dietary fucoidan from seaweeds contribute to the

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prevention and treatment of diverse diseases, such as diabetes, obesity, insulin resistance and other metabolic syndrome. Shang et al. [56] reported that fucoidan

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from Laminaria japonica (FuL) and Ascophyllum nodosum (FuA) could profoundly

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improve glucose homeostasis, reduce systematic inflammation, and modify the gut microbiota in diabetic mice. The fucoidan extracted from Saccharina japonica exhibited a considerable hypoglycemic effects, possibly by stimulating pancreas to release insulin and/or by reducing insulin metabolism [57]. Yu et al. [19] demonstrated that fucoidan isolated from Sargassum hemiphyllum exhibits anti-diabetic effects mainly through attenuation of β-cell death, thereby elevating insulin synthesis by upregulating PDX-1 and GLP1-R via a Sirt-1-dependent manner.

ACCEPTED MANUSCRIPT Low molecular weight fucoidan produced from the seaweed Laminaria japonica could alleviate diabetic retinal neovascularization and damage, possibly through lowering HIF-1α and VEGF expressions [58]. The highly sulfated fucoidan derived from the Sporophyll of Undaria Pinnatifida showed strongly suppression of blood

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glucose, and promoted to improve insulin sensitivity in db/db diabetic mice [59].

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Therefore, the application of natural fucoidan for the treatment of diabetes, not only

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has stable efficacy with low side effects, but also effectively prevents diabetic complications.

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The selective modulations of gut microbial communities may be associated with

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the actions of orally administered SFF in relieving STZ-induced diabetes. Growing evidence indicates that microbiota-produced short-chain fatty acids (SCFAs) have a

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distinctive role in the regulation of glucose homeostasis [60,61]. While SFF-treatment

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increased the relative abundances of Alloprevotella, a (SCFAs)-producing bacteria, which mainly produces butyric acid and acetic acid by the fermentation of dietary

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fibers or indigestible carbohydrates. The abundances of some bacteria, such as

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Bilophila, Oscillibacter, and Mucispirillum, showed significant positive correlation with the development of diabetes [62,63], while SFF-treatment reduced their relative abundances in diabetic mice. Previous studies have indicated a structure-dependent effects of fucoidan on diverse bioactivities [8,64]. Fucoidan is a family of sulfated polysaccharides that differed in glycosidic linkage, molecular weight, monosaccharide composition and sulfate content, depending on the algal source and harvesting time [12,14]. The backbone of brown algae fucoidans mainly contain two types, the type I

ACCEPTED MANUSCRIPT chains are organized in repeating (1→3)-linked α-L-fucopyranose residues, whereas type II chains contain alternating (1→3)- and (1→4)-linked a-L-fucopyranose residues [14]. Fucoidans with different structural feature exhibited distinctive modulation in gut microbiota [28,52,56]. Therefore, it is reasonable that fucoidans

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with defined structural feature could be utilized by distinct bacteria in the gut,

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eventually leading to the diversity in gut microbiota composition. Further evidence is

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still needed, especially structural analysis and metabolomics should be applied to

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elucidate the structure-dependent modulations of gut microbiota by SFF.

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Conclusion

In the present study, the physicochemical characterization and anti-diabetic effects

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of SFF on STZ-induced diabetic mice were investigated. Then 16S rRNA sequencing

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and multivariate statistics were utilized to analyze the structure and relative abundance of gut microbiota in diabetic mice. Compared with DC group, the

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administration of SFF significantly decreased the levels of FBG, diet and water intake,

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alleviated the pathological change in heart and liver, and suppressed oxidative stress. Simultaneously, SFF changed the composition of gut microbiota in STZ-induced diabetic mice. This study reveals the potential mechanisms of SFF in improving gastrointestinal health, and eventually mediating their beneficial effects on diabetes. In summary, our results suggest that the SFF has great potential as a strategy of adjuvant therapy for diabetes.

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Conflict of Interest The authors declare that they have no conflict of interest. Acknowledgement

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This work was financially supported by the National Natural Science Foundation of

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China (41876197 and 81872952), the Natural Science Foundation of Zhejiang

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Province (LY18C020006 and LGN18C020004), the Scientific Foundation of Education Department of Zhejiang Province (Y201737374), the Funding for Xinmiao

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Talents Program of Zhejiang Province (2017R426018).

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