Fucoidan from Fucus vesiculosus prevents the loss of dopaminergic neurons by alleviating mitochondrial dysfunction through targeting ATP5F1a

Carbohydrate Polymers 303 (2023) 120470

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Fucoidan from Fucus vesiculosus prevents the loss of dopaminergic neurons
by alleviating mitochondrial dysfunction through targeting ATP5F1a
Meimei Xing a, 1, Guoyun Li b, c, 1, Yang Liu a, 1, Luyao Yang b, Youjiao Zhang a, Yuruo Zhang a,
Jianhua Ding d, Ming Lu d, *, Guangli Yu b, c, **, Gang Hu a, d, ***
a

Department of Pharmacology, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing, 210023, Jiangsu, China
Key Laboratory of Marine Drugs of Ministry of Education, Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, School of Medicine and
Pharmacy, Ocean University of China, Qingdao 266003, China
c
Laboratory for Marine Drugs and Bioproducts, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China
d
Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu 211116, China
b

A R T I C L E I N F O

A B S T R A C T

Keywords:
Fucoidan
Neuroprotection
Parkinson's disease
Mitochondrial dysfunction

ATP5F1a

Parkinson's disease is a neurodegenerative disease that is characterized by the loss of dopaminergic neurons.
Fucoidan, which has emerged as a neuroprotective agent, is a marine-origin sulfated polysaccharide enriched in
brown algae and sea cucumbers. However, variations in structural characteristics exist among fucoidans derived
from different sources, resulting in a wide spectrum of biological effects. It is urgent to find the fucoidan with the
strongest neuroprotective effect, and the mechanism needs to be further explored. We isolated and purified four
different fucoidan species with different chemical structures and found that Type II fucoidan from Fucus ves­
iculosus (FvF) significantly improved mitochondrial dysfunction, prevented neuronal apoptosis, reduced dopa­
minergic neuron loss, and improved motor deficits in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)induced PD mouse model. Further mechanistic investigation revealed that the ATP5F1a protein is a key target
responsible for alleviating mitochondrial dysfunction of FvF to exert neuroprotective effects. This study high­
lights the favorable properties of FvF for neuroprotection, making FvF a promising candidate for the treatment of
PD.

1. Introduction
Parkinson's disease (PD) is a degenerative disease of the nervous
system that is characterized by the loss of dopaminergic (DA) neurons in
the substantia nigra (Grayson, 2016; Simon, Tanner, & Brundin, 2020).
The cause of PD can be related to multiple factors, including aging,
genetics, and environmental factors. The major motor symptoms of PD
include bradykinesia, rigidity, and resting tremors (Ascherio &

Schwarzschild, 2016; Xu, Fu, & Le, 2019). PD is also associated with
many nonmotor symptoms, including olfactory impairment, cognitive
impairment, psychiatric symptoms, and autonomic dysfunction. All of
these symptoms add up to overall disability (Schapira, Chaudhuri, &
Jenner, 2017). The existing drugs to treat PD, such as levodopa and
carbidopa, only focus on increasing the concentration of dopamine in
the brain to relieve motor symptoms (Vijiaratnam, Simuni, Bandmann,
Morris, & Foltynie, 2021). These drugs do not provide neuroprotection

Abbreviations: MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; HpF, Holothuria polii; LjF, Laminaria japonica; AnF, Ascophyllum nodosum; FvF, Fucus ves­
iculosus; PD, Parkinson's disease; MPP+, 1-methyl-4-phenylpyridine; DHE, dihydroethidium; TH, tyrosine hydroxylase; SNpc, substantia nigra pars compacta; DA,
dopamine; DOPAC, dihydroxy-phenylacetic acid; 5-HT, 5-hydroxytryptamine; LC-MS/MS, liquid chromatography tandem mass spectrometry; KEGG, Kyoto Ency­
clopedia of Genes and Genomes; ROS, reactive oxygen species; MAO-B, monoamine oxidase B; DAT, dopamine transporter; Oli A, oligomycin A.
* Correspondence to: M. Lu, Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, 101 Longmian Avenue,
Nanjing, Jiangsu 211116, China.
** Correspondence to: G. Yu, Laboratory of Marine Drugs of Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road,
Shandong, Qingdao 266003, China.
*** Correspondence to: G. Hu, Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, 101 Longmian Avenue,
Nanjing, Jiangsu 211166, China.
E-mail addresses: (M. Lu), (G. Yu), (G. Hu).
1
These authors contributed equally to this work.
/>Received 1 October 2022; Received in revised form 11 December 2022; Accepted 12 December 2022
Available online 15 December 2022
0144-8617/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( />

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Carbohydrate Polymers 303 (2023) 120470

or prevent or delay the degeneration of DA neurons. They also have
serious adverse effects, including nausea and vomiting, upright hypo­
tension, sedation, confusion, sleep disturbances, hallucinations, dyski­
nesia, chorea, and progressive dystonia. There is an urgent need to
develop drugs with neuroprotective effects and few side effects to treat
PD.
Although the underlying pathology of PD is still not well understood,
oxidative stress-induced mitochondrial dysfunction is thought to be an

important cause of DA neuron loss in PD mice (Ascherio & Schwarzs­
child, 2016; Jankovic & Tan, 2020). Thus, antioxidants are often studied
as potential compounds for the protective effects of PD (Schapira &
Jenner, 2011). The great potential of marine-derived glycans to treat
neurodegenerative diseases has emerged because marine natural prod­
ucts are rich in the biological activity of antioxidants (Karthikeyan,
Joseph, & Nair, 2022). For example, GV-971, a compound derived from
acidic oligosaccharides in brown algae (Wang et al., 2019), was the first
drug developed in China to treat Alzheimer's disease. Therefore, we have
been focusing on finding a potential marine-derived compound as a
possible treatment for PD.
Fucoidans are sulfated polysaccharides rich in L-fucose, usually
found in brown algae and sea cucumbers. Fucoidan normally has two
types of backbones: type (I) encompasses repeated (1→3)-L-fucopyr­
anose, and type (II) encompasses alternating and repeated (1→3)- and
(1→4)-L-fucopyranose (Usoltseva et al., 2019; Usoltseva et al., 2021).
Owing to its beneficial biological activities, fucoidan is studied exten­
sively as a multifunctional and nontoxic polysaccharide (Zhang et al.,
2020). A variety of classic bioactivities have been reported for fucoidan,
including antitumor, antibacterial, antiviral, antioxidant, anticoagulant,
antiobesity, and immune-modulating properties. In addition, fucoidan
has also been shown to play a role in neuroprotection (Kim et al., 2019;
Wang et al., 2021). However, variations in structural characteristics
exist among different species of seaweeds or sea cucumbers (Li, Lu, Wei,
& Zhao, 2008). The molecular weight also varies between different
species (Fitton, Stringer, & Karpiniec, 2015), and the relationship be­
tween molecular weight and bioactivity is not clear (Yang et al., 2018).
Previous studies showed that fucoidan from Laminaria japonica pro­
tected dopaminergic neurons from rotenone-induced PD in rats and
reduced behavioral deficits and increased striatal dopamine (Luo et al.,

2009; Zhang et al., 2018). Sulfated fucoidan isolated from Saccharina
japonica showed better neuroprotective activity than low molecular
weight fucoidan (Liu, Wang, Zhang, & Zhang, 2018). The bioactivities of
fucoidan from Fucus vesiculosus gradually depolymerized fractions were
also proven to decrease with decreasing molecular weight (Han, Lee, &
Lee, 2019; Lahrsen, Schoenfeld, & Alban, 2018). However, He et al.
showed that low-molecular-weight fucoidan reduces mitochondrial
dysfunction in aged mice compared to higher molecular weight de­
rivatives (Wang, Zhu, & He, 2016). These results confirmed that diverse
sources of fucoidans result in diverse structures and a broad spectrum of
bioactivities, indicating that the structure-effect relationship deserves
further study.
Target fishing technology is a method combining active small
molecule probes and pull-down technology, which can accurately detect
protein targets of small molecule compounds. Tu et al. identified precise
targets of small molecule compounds in neuroinflammation, diabetes
and fatty liver using this method, which illustrates the feasibility of
targets for fishing applications in the mechanistic study of carbohydratebased pharmaceutical molecules (Dai et al., 2022; Ma et al., 2022; Yang
et al., 2021; Zheng et al., 2022).
In this study, we isolated and purified four kinds of fucoidans with
different chemical structures from Holothuria polii (HpF), Laminaria
japonica (LjF), Ascophyllum nodosum (AnF) and Fucus vesiculosus (FvF).
We found that type II fucoidan (FvF) had the best neuroprotective effect
in the MPTP-PD mouse model. Finally, we identified ATP5F1a as the
target of FvF to protect DA neurons by improving mitochondrial func­
tion, suggesting that FvF may be a pluripotent and promising drug for
PD therapy.

2. Materials and methods
2.1. Materials

Holothuria polii (HpF), Laminaria japonica (LjF), Ascophyllum nodosum
(AnF) and Fucus vesiculosus (FvF) were provided by the Key Laboratory
of Glycoscience and Glycotechnology, School of Medicine and Phar­
macy, Ocean University of China. Oligomycin A (Selleck, #S1478);
MPTP (Selleck, #S4732); 1-methyl-4-phenylpyridine (MPP+) iodide
(Sigma, #D048); anti-tyrosine hydroxylase (TH) antibody (Sigma,
#T1299); Hoechst 33342 (Sigma, #B2261); JC-1 (Invitrogen, #T3168);
MitoSOX (Invitrogen, #M36008); streptavidin agarose resin (Invi­
trogen, #20347); Anti-MAP2 antibody (Santa Cruz, #Sc-32791);
Annexin V/PI (Vazyme Biotech Co., Ltd., #A21-02); and dihydrothi­
dium (Beyotime Biotechnology; #M36008).
2.2. Preparation and characterization of fucoidans from different sources
Crude polysaccharides were extracted from the body wall of the sea
cucumber Holothuria polii or seaweeds by methods reported previously
(Li et al., 2020; Shan et al., 2016) and then separated by a Q Sepharose
Fast Flow anion-exchange column to obtain pure high-sulfated fucoi­
dans. The chemical composition and physical properties of fucoidans
were analyzed. The monosaccharide composition was determined by
using the 1-phenyl-3-methyl-5-pyrazolone (PMP)-HPLC method. The
composition and sulfate content were determined by the BaCl2-Gelatin
method. Purity and relative molecular weight (Mw) were determined by
gel filtration columns (Shodex OHpak SB-804 HQ and SB802.5 HQ)
connected to an HPLC system. In addition, we also carried out 1H NMR
at 298 K on an Agilent DD2 500 MHz to assign the structural type of
fucoidans. Table S1 shows the structural information and molecular
mass of the four kinds of fucoidan.
2.3. Animals
Three-month-old male C57BL/6 mice were purchased from Nanjing
Medical University Animal Centre. Mice were housed in SPF grade
laboratories and were allowed to drink and feed ad libitum. All exper­

iments were carried out in accordance with the Guide for the Care and
Use of Laboratory Animals. All animal experiments in the present study
were approved by the Ethical Committee of Nanjing Medical University
(Permission No. 1903038).
2.4. Cell culture
Culture of primary midbrain and cortical neurons was performed
according to previous studies (Han et al., 2018). Pregnant C57BL/6 fe­
male mice were rapidly cervically dislocated and disinfected with
iodophor and 75 % medical alcohol. The midbrain or cortical tissue of
fetal mice was carefully dissected, and the meninges and blood vessels
were removed. The midbrain or cortical tissues were next digested by
trypsin. Subsequently, the cells were incubated in neurobasal medium
containing 2 % B27 (Gibco, 17504044).
SH-SY5Y cells were purchased from the Cell Bank of the Chinese
Academy of Sciences and cultured in 10 % FBS in a constant temperature
incubator.
2.5. Cell treatment
Neurons were administered FvF (10 μM) and MPP+ (10 μM) for 48 h.
SH-SY5Y cells were administered FvF (5, 10 and 25 μM) and MPP+ (500
μM) for 48 h.
2.6. CCK-8 assay
Neurons or SHSY5Y cells were seeded in a 96-well plate and then
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pretreated with LjF, HpF, AnF and FvF (5 μM) for 24 h and treated with

MPP+ (500 μM) for another 24 h. Then, CCK-8 was added to each well
for 4 h. Finally, the absorbance was detected at 450 nm.

surfaced, 50 cm high and 1 cm diameter iron rod. The time required
for the mouse to reach the bottom of the floor (T-TLA) and the time
required for the mouse to be fully head down (T-turn) were recorded.
Motor function for all mice was trained during the lights-on cycle from
9:00 to 14:00. All tests were similar to those of previous research (Yun
et al., 2018).

2.7. LDH assay
Culture media was collected according to the manufacturer's in­
structions to assess LDH levels using an assay kit (Nanjing Jiancheng
Bioengineering Institute).

2.14. Brain sample collection
Mice were rapidly anesthetized after behavioral testing. The
midbrain and stratum tissues of whole brains were quickly dissected,
and all samples were stored at − 80 ◦ C. The isolated brains were fixed in
4 % paraformaldehyde (Aladdin, #F111941) for 2 days and dehydrated
for 2 days using 20 % and 30 % sucrose (Saint-Bio, #T16373). Next, the
brains were embedded in OCT (Tissue-Tek) and sliced into 25 μm slices
using a freezing microtome (Leica CM1950, Nussloch, Germany). Slices
in 50 % glycerol PBS solution could be stored at − 80 ◦ C for long-term
storage (Han et al., 2021).

2.8. Hoechst staining
Fluorescence microscopy (Olympus, Tokyo, Japan) was used to
observe cells that had been stained with Hoechst 33342 for 10 min.
2.9. Quantitative real-time PCR

TRIzol reagent (Invitrogen, #10296028) was used to extract total
RNA from the midbrains and neurons. A NanoDrop 5500 (Thermo) was
used to perform total RNA quantification analysis. Reverse transcription
was carried out using Hiscript III RT SuperMix for qPCR (Vazyme,
#R323-01). SYBR Green Master Mix (Roche, #04913914001) was used
to determine the relative expression of mRNAs. Table S2 shows the
primer sequences used for real-time qPCR.

2.15. Neurotransmitter measurement using HPLC
Each striatum was weighed and then homogenized in ice-cold buffer
comprising 0.01 % DHBA. Following that, centrifugation was performed
(15,000 ×g, 30 min, 4 ◦ C), and collection of the supernatant was used for
determination of monoamine neurotransmitters.

2.10. Flow cytometry analysis

2.16. Immunofluorescence and immunohistochemistry

According to the manufacturer's instructions, Annexin V/propidium
iodide (PI) staining (Vazyme Biotech, China) was used to assess cellular
apoptosis using flow cytometry (Qiao et al., 2017). In brief, neurons
were washed in ice-cold PBS, resuspended in binding buffer, and incu­
bated with PI and Annexin V-fluorescein isothiocyanate for 10 min. A
flow cytometry instrument was immediately used to perform a flow
cytometric analysis (Millipore, USA).

For immunofluorescence staining, the neurons were incubated in
PBS buffer with 0.2 % Triton X-100 (Sigma, #T9284) for 20 min before
being cultured with 5 % BSA (Yi Fei Biotechnology, #YB0006-100) for 2
h. Next, the neurons were incubated with anti-MAP2 antibodies (Santa

Cruz, #SC-32791; 1:500) at 4 ◦ C for 24 h. Furthermore, the neurons
were washed and incubated with Goat anti-mouse IgG (Alexa Fluor 555conjugated Invitrogen, A21422; 1:700) for 2 h. Finally, the neurons were
photographed by fluorescence microscopy (Olympus, Tokyo, Japan).
For immunohistochemistry staining, slides encompassing the entire
SNpc were incubated in PBS buffer with 0.2 % Triton X-100 (Sigma,
#T9284) for 20 min before being cultured with 5 % BSA for 2 h. The
slides were then incubated at 4 ◦ C overnight with the primary antibodies
anti-TH (Sigma, #T1299; 1:500), washed, and incubated with secondary
antibody (Thermo, #SA5-10275, 1:500) for 2 h at room temperature,
and then the slides were stained with DAB (Shanghai Gene Company,
#GK500705) for 30 s and washed three times. Finally, slides were
visualized and photographed under a fluorescence microscope
(Olympus, Tokyo, Japan). The immunostaining signals were analyzed
quantitatively by using Micro bright field Stereo-Investigator software
(Stereo Investigator software; Micro bright field) (Han et al., 2021).

2.11. ROS detection in intracellular and mitochondrial environments
Dihydroethidium (DHE) was used to measure intracellular ROS. In
brief, SH-SY5Y cells were treated with 10 μM FvF and 500 μM MPP+ for
24 h. Next, the cells were incubated with 2 μM DHE for 30 min in a 37 ◦ C
thermostatic incubator. Finally, the cells were washed three times with
PBS, and the fluorescence intensity was observed by flow cytometry
(Millipore, USA). MitoSOX was used to measure mitochondrial ROS in
SH-SY5Y cells. In brief, cells were incubated with 5 μM MitoSOX for 30
min in a 37 ◦ C thermostatic incubator and washed three times with PBS.
Finally, the fluorescence intensity was measured by fluorescence mi­
croscopy (Olympus, Tokyo, Japan).
2.12. Administration of FvF in MPTP-induced PD mouse models

2.17. Target fishing

In total, thirty four-month-old male C57BL/6 mice weighing 22–28 g
were separated into 5 groups (n = 6 in each group): control, MPTP (20
mg/kg), FvF (40 mg/kg), MPTP + FvF (10 mg/kg), and MPTP + FvF (40
mg/kg). FvF was administered daily starting one day before MPTP in­
jection. Then, both MPTP and FvF were administered together daily for
5 days. FvF was then provided for another two days. Mice in the control
group were injected with an equal volume of saline.

Neurons were collected and homogenized in 1 % NP-40 lysis buffer
(GenSar, #E124-01), and the extracted protein was measured with a
BCA protein quantification kit (KeyGen BioTech, #KGP903). The pro­
tein was incubated with 10 μM FvF-biotin for 12 h at 4 ◦ C and incubated
with streptavidin agarose resin for 6 h at 4 ◦ C. Next, the protein was
centrifuged for 5 min at 3000 rpm. The precipitate was washed 3 times,
added to 25 μL 5× loading buffer and boiled for 5 min. Finally, the
protein was removed by 10 % SDS-PAGE and then analyzed by highperformance liquid chromatography tandem mass spectrometry (LCMS/MS). The potential target information of FvF is shown in Table S3.

2.13. Behavior analysis
Behavioral tests were performed after the last administration of
MPTP to mice (Sampson et al., 2016; Yun et al., 2018). For the opened
field test, the crawling track and distance of mice in the activity box of
50 cm × 50 cm × 42 cm in 5 min were logged. For the rotarod test, the
Rotarod Analysis System recorded the time of latency to fall at 20 rpm
for 300 s. For the pole test, mice were placed on the top of a rough-

2.18. Docking studies
The RCSB Protein Data Bank was used to obtain the threedimensional structure of ATP5F1a (PDB ID: 1BMF). The docking
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Carbohydrate Polymers 303 (2023) 120470

application was AutoDock Vina. PyMOL version Open-Source 1.6.x was
used to make the graphics.

variance, or one-way analysis of variance (ANOVA) followed by Tukey's
post hoc test was used to determine the significance of differences.
Differences were considered significant at P < 0.05.

2.19. Knockdown of ATP5F1a

3. Results

ATP5F1a siRNA was synthesized by Sangon Biotech. Negative con­
trol siRNA and ATP5F1a siRNA were also transiently transfected into
neurons or SH-SY5Y cells by the utilization of Lipofectamine 3000
(Invitrogen, #L3000-15) in agreement with the manufacturer's
protocols.

3.1. Neuroprotective activity screening of carbohydrate compounds of
marine origin
Based on previous work in our laboratory (Cui et al., 2018; Jiao, Yu,
Zhang, & Ewart, 2011; Li et al., 2018; Wang et al., 2012), we constructed
a library of marine carbohydrates. From this library, forty-two marineorigin carbohydrates belonging to 7 categories (fucosylated chondroitin
sulfate, alginate derivative, fucoidan, chitosan derivative, agar deriva­
tive, homoxylan and marine glycosaminoglycans) were selected to

2.20. Statistical analysis
GraphPad Prism 9.0 was used to analyze the results. The data are
presented as the mean ± SD. Student's t-test, two-way analysis of

Fig. 1. Neuroprotective activity of carbohydrate compounds of marine origin. (A) Cell viability of SH-SY5Y cells after treatment with 42 carbohydrate compounds at
a concentration of 10 μM. (B) Cell viability of SH-SY5Y cells treated with MPP+ (500 μM) and carbohydrate compounds. (C) 1H NMR spectra of four kinds of fucoidan.
(D) Structure of four types of fucoidan. (E) Cell viability of neurons treated with HpF, LjF, AnF, and FvF for 48 h. (F) LDH release was detected after treatment with
HpF, LjF, AnF, and FvF. (G) Effect of HpF, LjF, AnF, and FvF on neuronal viability with MPP+ stimulation. (H) Effect of HpF, LjF, AnF, and FvF on LDH release with
MPP+ stimulation. (I–J) Immunostaining for MAP2 after MPP+ and fucoidan treatment. Red color defines neuronal axons, showing the protection of neurons from
MPP+ by AnF and FvF. Scale bar is 20 μm. Data are analyzed as the means ± SD using one-way ANOVA and then combined with Dunnett's test to assess the dif­
ferences between groups. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. MPP+ group.
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evaluate their neuroprotective effect. Marine carbohydrates are soluble
in water and insoluble in organic solvents. Therefore, all marine car­
bohydrates were dissolved in PBS, and PBS was used as a negative
control in the cell experiments. Each carbohydrate compound was
incubated with SH-SY5Y cells at a concentration of 10 μM for 24 h, and
then cell viability of each group was detected by the CCK-8 method
(Fig. 1A). Subsequently, thirty-six compounds with cell viability >90 %
was exposed to 500 μM MPP+ for another 24 h (Fig. 1B). In MPP+induced SH-SY5Y cells, we noticed that one compound, fucoidan from
Fucus vesiculosus (FvF), markedly improved the cell viability. Fucoidan
includes a large family with different chemical structures, and exists in a
variety of marine organisms, which may result in a wide spectrum of
bioactivity. To investigate the potential protective effect of different

fucoidans, we then isolated and purified fucoidans from three other
sources: Holothuria polii (HpF), Laminaria japonica (LjF) and Ascophyllum
nodosum (AnF) (Li et al., 2020; Shan et al., 2016). All structural infor­
mation of four fucoidans is shown in Fig. 1C–D. All of these were highly
sulfated (35.6 %– 40.5 %) fucans with trace amounts of galactose or

xylose residues. As shown in Fig. 1C, strong signals at about 1.1–1.4 ppm
in the 1H NMR spectra of HpF, LjF, AnF and FvF can be readily assigned
to the methyl protons of fucose residues (CH3). In addition, the chemical
shifts of the envelope of anomeric signals at 4.9–5.6 ppm are consistent
with the presence of α-L-fucopyranosyl units. Differently, the spectra of
HpF and LjF show only one broad peak at 5.31 ppm in their anomeric
regions, which suggest the anomeric proton signals of 3-linked α-Lfucose. A feature at around 5.10 ppm can be tentatively assigned to their
H1 of non-sulfated fucose residues according to literature comparison.
The spectra of AnF and FvF show two broad peaks at 5.27 ppm, 5.04
ppm and 5.35 ppm, 5.13 ppm respectively in their anomeric regions,
which suggest the anomeric proton signals of both 3-linked and 4-linked
α-L-fucose. In conclusion, HpF and LjF were classified as Type I fucoidan,
with the main chain composed of 1,3-linked α-L-fucopyranose residues.
AnF and FvF was classified as Type II fucoidan, which encompassed
alternating and repeated (1→3)- and (1→4)-L-fucopyranose.
To further evaluate the potential protective effect of fucoidans, CCK8 and LDH assays were used. We first demonstrated that the four types of

Fig. 2. FvF attenuated MPP+-induced cytotoxicity in SH-SY5Y cells and neurons. (A–B) Cell viability and LDH release after SH-SY5Y cells were treated with FvF (5,
10, 25 and 100 μM) for 24 h. (C–D) Cell viability and LDH release after SH-SY5Y cells were treated with FvF (5, 10 and 25 μM) and MPP+ (500 μM) for 24 h. (E–F)
Apoptosis by Annexin V-FITC/PI and quantitative analysis in neurons treated with FvF (10 μM) and MPP+ (500 μM) for 24 h. (G–H) Representative images of Hoechst
staining; the scale bar is 20 μm. (H) Quantitative analysis of Hoechst-positive neurons. (I–J) Representative images of MAP2 and (J) the average neurite length; the
scale bar is 20 μm. (K–L) Representative images of TH, and (L) the average neurite length; the scale bar is 40 μm. Data are analyzed as the means ± SD using one-way
ANOVA and then combined with Dunnett's test to assess the differences between groups. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Control group; *P < 0.05, **P <
0.01, ***P < 0.001 vs. MPP+ group.

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fucoidans have no significant cytotoxicity (Fig. 1E–F). In primary
neuron culture, cell viability was greatly reduced with damage from
MPP+, but with the two type-II fucoidans AnF and FvF, cell viability
decreased less, showing the neuroprotective effect of these two fucoi­
dans (Fig. 1G). LDH release was greatly increased with damage from
MPP+ but with the two type-II fucoidans AnF and FvF (Fig. 1H).
Microscopic observation confirmed that with 10 μM MPP+ stimulation
for 48 h, few neurons remained, which decreased in size, and cell pro­
trusions were also shortened. The AnF- and FvF-treated cells, on the
other hand, did not decrease in number and size as much as the un­
treated culture (Fig. S1). Fig. 1I–J shows the overall survival of neurons
by immunostaining with the pan-neuronal marker MAP2 and further
confirmed that AnF and FvF treatment could protect neurons from being
damaged by MPP+. Moreover, the neuroprotective effect of FvF was
superior to that of AnF. In previous studies, MAO-B inhibitor selegiline

was used as a positive tool drug in cell and animal models (Anastassova
et al., 2021; Zhang et al., 2022). However, the protection mechanism of
selegiline is not the same as that of the experimental compounds in our
study, so we did not include a positive control in our following experi­
ments (Fig. S2).
3.2. FvF attenuated MPP+-induced cytotoxicity in SH-SY5Y cells and
primary neurons

To verify the cytotoxic effects of FvF on SH-SY5Y cells and neurons,
cell viability was assessed with CCK-8 and LDH assays. FvF (5, 10, 25
and 100 μM, for 24 h) had no significant cytotoxicity in SH-SY5Y cells
(Fig. 2A–B). In the MPP+-induced SH-SY5Y cell model, 5, 10 and 25 μM
FvF markedly enhanced the viability of SH-SY5Y cells (Fig. 2C–D). A
concentration of 10 μM was chosen to investigate the mechanism behind

Fig. 3. Neuroprotective effects of FvF in the MPTP-induced PD mouse model. (A) Schematic diagram of the experimental design. FvF (10/40 mg/kg) or vehicle
(saline) was administered intraperitoneally for 7 consecutive days beginning on day − 1, and mice received MPTP (20 mg/kg) or vehicle (saline) each day for 5 days
before tissues were taken for molecular analysis on Day 9 following the behavior test. Behavior tests, such as the OFT (B–C), latency to fall (D), T-turns (E) and T-TLA
(F), were conducted. (G) Immunostaining of TH-positive neurons in the SNpc. Scale bars is 40 μm. (H) Stereological counts of TH-positive neurons in G. (I) Con­
centrations of DA, DOPAC and 5-HT in the striatum of mice were measured using HPLC. Data are analyzed as the means ± SD using one-way ANOVA and then
combined with Dunnett's test to assess the differences between groups. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Control group; *P < 0.05, **P < 0.01, ***P < 0.001
vs. MPTP group.
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the effects of FvF. As shown in Fig. S3, MPP+ (10 μM, 48 h) induced
morphological damage in neurons, and FvF (10 μM) significantly
attenuated morphological damage. Furthermore, MPP+ triggered cell
death, as demonstrated by an increase in the proportion of Annexin Vand PI-stained cells, which was protected by FvF (Fig. 2E–F). According
to the Hoechst staining data, FvF (10 μM) dramatically decreased the
proportion of apoptotic cells (Fig. 2G–H). Immunostaining with MAP2
was used to examine the survival of non-DA neurons. As shown in
Fig. 2I–J, non-DA neurons were much more susceptible to MPP+induced axon loss, and FvF (10 μM) therapy significantly reduced the
effect of MPP+. Additionally, FvF (10 μM) notably reversed MPP+induced damage in dopamine neurons (Fig. 2K–L). These findings sug­

gest that FvF (10 μM) can reverse the detrimental effects of MPP+ on
neurons.

3.4. FvF is a neuroprotective agent associated with signaling pathways
involved in oxidative stress and mitochondrial function
Given that FvF exerted significant neuroprotective activity both in
vitro and in vivo, it is worthwhile to discover its macromolecular
binding partners and fully define the molecular mechanism. We syn­
thesized biotin probes to target the protein target of FvF that is
responsible for its neuroprotective impact. The workflow chart of pro­
tein target identification is shown in Fig. 4A. FvF was conjugated with
biotin and then enhanced on streptavidin beads. To identify proteins
targeted by FvF, the digested peptides were examined using liquid
chromatography tandem mass spectrometry (LC-MS/MS). Kyoto Ency­
clopedia of Genes and Genomes (KEGG) analysis using Metascape was
performed to identify the pathways associated with the 512 proteins
captured by FvF (Fig. 4B). Pathways associated with Parkinson's disease,
mitochondrial function, apoptosis and energy metabolism were found.
Strikingly, we found that FvF recovered the downregulation of gene
expression related to mitochondrial function (Mfn1, Mfn2, Drp1 and
Opa1), anti-apoptosis and anti-oxidative stress (Dj1, Nqo1, Sod1, and
Gpx3) in primary neurons and midbrains and lowered gene expression
levels related to oxidative stress and apoptosis. However, FvF had no
effects on genes associated with ER stress (Fig. 4C, D). In conclusion,
these results suggested that the PD-protective effect of FvF could be
mediated through signaling pathways related to oxidative stress and
mitochondrial function.

3.3. FvF exerted neuroprotective effects in the MPTP-induced PD model
To assess the neuroprotective effect of FvF in PD, the MPTP-induced

PD mouse model was used. We treated MPTP-PD mice with 10 and 40
mg/kg doses of FvF for 8 days to evaluate the neuroprotective effects of
FvF in vivo (Fig. 3A). The open field test, rotarod test, and pole test were
used to evaluate the motor and behavioral performance of the mice. As
shown in Fig. 3B–F, the impaired motor coordination and balance ability
were relieved by FvF. FvF treatment significantly slowed the loss of TH
markers in the substantia nigra pars compacta (SNpc) region of MPTPPD mice (Fig. 3G, H). The decrease in dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) content in the striatum was mitigated by
FvF but had no effect on the level of 5-hydroxytryptamine (5-HT)
(Fig. 3I). In conclusion, in MPTP-induced PD mice, FvF slowed the
progression of dopamine neuron loss, protected the function of the
nigral striatum, and alleviated motor dysfunction.

3.5. FvF rescued mitochondrial dysfunction in SH-SY5Y cells
Owing to our findings on the restoration in gene expression associ­
ated with oxidative stress and mitochondrial function following FvF
treatment, we next examined mitochondrial function using dihy­
droethidium and MitoSOX superoxide dye. Reactive oxygen species
(ROS) production was increased in MPP+-treated SH-SY5Y cells
compared to the control group but was recovered with the addition of

Fig. 4. FvF is a neuroprotective agent associated with signaling pathways involved in oxidative stress and mitochondrial function. (A) Schematic illustration of target
identification, biotin probe design and target fishing of FvF. (B) KEGG analysis of the 512 protein targets identified by LC-MS/MS revealed their association with
Parkinson's disease. (C) Gene heatmap of the midbrain in MPTP-induced PD mice by q-PCR analysis. (D) Gene heatmap of neurons in MPP+-induced neuronal injury
by q-PCR analysis.
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Carbohydrate Polymers 303 (2023) 120470

FvF (Fig. 5A–B). MitoSOX mitochondrial superoxide indicator was used
to detect mitochondrial ROS. Superoxide increased in mitochondria of
MPP+-treated SH-SY5Y cells as predicted, compared to the control
group, and FvF therapy restored this accumulation. ROS accumulation is
a result of mitochondrial dysfunction, and therefore the above results
suggested a rescue in mitochondria (Fig. 5C–D). Transmission electron
microscopy showed that MPP+ induced a decrease in mitochondrial
density and an increase in the number of abnormal mitochondria but
was recovered with the addition of FvF in SH-SY5Y cells (Fig. 5E–G).
These findings imply that FvF can protect SH-SY5Y cells against MPP+induced mitochondrial dysfunction.

at approximately 60, 110 and 190 kDa. Among the potential targets
shown in Table S3, the bands at approximately 110 or 190 kDa did not
have any matching results. The band at approximately 60 kDa matched
ATP5F1a after excluding other targets at similar molecular weights but
did not have biological functions related to the pathogenesis of PD
(Fig. S4C, Table S3). Next, we discovered the potential protein binding
with FvF by LC-MS/MS (Fig. 6A) and verified the target protein by
Western blotting (Fig. 6B). Then, we performed a molecular docking
(MD) analysis to investigate the binding site of the ATP5F1a/FvF com­
plex based on the published crystal structure of human ATP5F1a (PDB
ID: 1BMF). The corresponding docking conformation suggested that FvF
fits into the active pocket, forming multiple important interactions with
surrounding amino acid residues, including Asp252, Thr255, Asp312,
Lys316, Lys218, Thr19, Gly217, Glu371, and Gln215 (Fig. 6C).
To investigate whether the target of FvF, ATP5F1a, played a role in
the neuroprotective effects of MPP+ on neurological damage, we used
ATP5F1a siRNA to test the function of FvF on ATP5F1a (Fig. S5). The
knockdown of ATP5F1a stopped the anti-apoptosis effects that FvF

should exert on MPP+-treated primary neurons (Fig. 6D–F). FvF also lost
its potency to alleviate the production of mitochondrial ROS (Fig. 6G–H)

3.6. ATP5F1a was recognized as a potential target of FvF
To study which proteins FvF binds to exert its neuroprotective bio­
logical activity, we first evaluated the effects of FvF-biotin on neuron
survival. The results indicated that FvF-biotin (10 μM, 48 h) had no
significant cytotoxicity (Fig. S4A–B). The proteins labeled with biotin
probes were separated by SDS-PAGE and visualized by Coomassie bril­
liant blue. As shown in Fig. S4C, there were three distinct protein bands

Fig. 5. FvF recovered mitochondrial dysfunction in SH-SY5Y cells. (A) ROS levels and (B) mean fluorescence intensity analysis of dihydroethidium by flow
cytometry. (C) Fluorescence intensity of cells stained with MitoSOX analyzed by fluorescence microscopy, and (D) mean fluorescence intensity analysis of MitoSOX.
Scale bar is 20 μm. (E) Mitochondrial morphology was observed via transmission electron microscopy, and (F–G) the mitochondrial density and abnormal mito­
chondria were analyzed. Data are analyzed as the means ± SD using one-way ANOVA and then combined with Dunnett's test to assess the differences between
groups. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. MPP+ group.
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Carbohydrate Polymers 303 (2023) 120470

Fig. 6. IP-LC/MS identified ATP5F1a as a target of FvF for alleviating mitochondrial dysfunction. (A) A representative peptide of ATP5F1a was identified by LC-MS/
MS. (B) Pull-down/Western blotting for target validation of ATP5F1a with biotin probes. (C) The hydrogen bonds formed between FvF and neighboring amino acid
residues in ATP5F1a. (D) Fluorescence microscopy of primary neurons labeled with Hoechst 33342. Red arrow: apoptotic cells. (E) Quantitative analysis of Hoechstpositive neurons. Scale bar is 20 μm. (F) Cell viability of SH-SY5Y cells after treatment with FvF, MPP+ and ATP5F1a siRNA. (G) Fluorescence images of cells stained
with MitoSOX and (H) mean fluorescence intensity analysis of MitoSOX. The scale bar is 20 μm. Data are analyzed as the means ± SD using one-way ANOVA and
then combined with Dunnett's test to assess the differences between groups. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Control group; *P < 0.05, **P < 0.01, ***P <
0.001 vs. MPP+ group.

induced by MPP+ in SH-SY5Y cells when ATP5F1a was knocked down.
Taken together, the neuroprotective effect of FvF was reversed by
ATP5F1a siRNA, which confirmed that FvF exerts its neuroprotective
effects through ATP5F1a.

to protect against DAergic neuron loss induced by MPTP (Fig. 7G–H).
Taken together, these results suggested that the pharmacological in­
hibitor of ATP5F1a oligomycin A reversed the neuroprotective effect in
the MPTP PD mouse model induced by FvF.

3.7. The neuroprotective effects of FvF could be attenuated by a
pharmacological inhibitor of ATP5F1a, oligomycin A

4. Discussion
Oxidative stress and mitochondrial dysfunction play an important
role in the etiology of Parkinson's disease (Dai et al., 2018; Trist, Hare, &
Double, 2019). Evidence previously found suggests that oxidative stress,
which occurs in the case of neurotoxicity, has been an important
mechanism of impaired mitochondria that drives degenerative
neurology, making it an important therapeutic target for PD (Mahoney´nchez et al., 2021). Marked oxidative stress, which is increased
Sa
mitochondrial ROS and intracellular ROS, was also found in neurons in
PD pathology (Burbulla et al., 2017; Han et al., 2021). 1-Methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a highly lipophilic mole­
cule that can rapidly penetrate the blood–brain barrier (Langston, 2017)

Oligomycin A is widely used as a pharmacological inhibitor of
ATP5F1a. After treatment with oligomycin A, motor performance
improved by FVF was significantly reduced. In open field tests, mice
treated with Oligomycin A plus FvF did not show significant improve­
ment in speed of movement compared to MPTP mice, as FvF only would

have (Fig. 7A–C). Similarly, in rotarod tests, FvF treatment increased the
time on the rod of MPTP mice, while FvF plus oligomycin A treatment
did not (Fig. 7D). The same result of the time of turning and climbing in
pole tests was also observed: Oligomycin A canceled the effect of
decreasing the time FvF had (Fig. 7E–F). Furthermore, FvF lost its ability
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Carbohydrate Polymers 303 (2023) 120470

Fig. 7. Neuroprotective effects of FvF could be attenuated by a pharmacological inhibitor of ATP5F1a, oligomycin A. FvF (40 mg/kg) or Oli (0.5 mg/kg) was
administered intraperitoneally for 7 consecutive days beginning on day − 1 and received MPTP (20 mg/kg) each day for 5 days before tissues were taken for mo­
lecular analysis on Day 9 following the behavior test. Representative image (A), average speed (B) and distance (C) of movement were recorded simultaneously
during the open field test. Latency to fall (D), T-turns (E), and T-TLA (F) were conducted. (G) Representative photomicrographs of TH staining in the SNpc. (H)
Unbiased stereological counts of TH. Scale bar is 40 μm. Data are analyzed as the means ± SD using one-way ANOVA and then combined with Dunnett's test to assess
the differences between groups. n = 6. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. MPTP group.

with type І Fucoidan (HpF and LjF) in the primary neuron (Fig. 1E–J).
And the differences in neuroprotective effects between type І and type II
Fucoidan may be probably due to the differences in link and sulfated
modification. For the first time, we found that the type II fucoidan FvF
had neuroprotective and anti-PD effects in both a primary neuronal
model and an MPTP-induced PD mouse model.
In view of the key role of mitochondrial dysfunction in the patho­
genesis of PD, we hypothesized that the neuroprotective effect of FvF is
mediated by the alleviation of mitochondrial dysfunction (Zhang et al.,
2018). Oxidative stress produced by reactive oxygen species (ROS) is a
major contributor to the pathogenesis of PD. Mitochondria are the main

source of ROS, and mitochondrial dysfunction increases the formation of
ROS. Increased oxidation products were consistently observed in the
SNpc of PD animal models (Zhang et al., 2018). In Alzheimer's diseaserelated studies, fucoidan reduced the production of ROS stimulated by
Aβ in Caenorhabditis elegans (C. elegans) (Jin et al., 2013; Liu et al., 2018;
Wang et al., 2021; Wang, Yi, & Zhao, 2018). In addition, fucoidan
reversed the reduction in intracellular superoxide dismutase (SOD) and
glutathione (GSH) induced by MPP+ in a dopaminergic nerve precursor
cell line (MN9D) (Liang et al., 2018). The results of our in vitro assays
also indicate that FvF prevents the generation of excessive mitochon­
drial ROS and recovers abnormalities in mitochondrial membrane po­
tential, indicating that FvF has a neuroprotective effect by improving
mitochondrial function.
Utilizing target fishing technology, we detected ATP5F1a as a po­
tential target of FvF. ATP5F1a is a mitochondrial complex V component
and is one of the subunits of the ATPase complex. ATP synthase (ATPase)
is responsible for the majority of ATP production(Zech et al., 2022), but
only a few studies have shown that ATPase plays a role in ROS

and then be metabolized into MPP+ by monoamine oxidase B (MAO-B).
MPP+ is selectively absorbed and accumulates in dopaminergic neurons
through the dopamine transporter (DAT). MPP+ can interfere with the
function of mitochondrial complex I, mediate a variety of harmful
oxidative stress responses, and lead to the death of dopamine neurons
(Dionísio, Amaral, & Rodrigues, 2021; Schildknecht, Di Monte, Pape,
Tieu, & Leist, 2017).
Among the two type-I fucoidans we tested, HpF has been shown to
have an anticoagulant effect (Li et al., 2020; Mansour et al., 2019).
However, there has been no report on its neuroprotective effects. Our
results further confirmed that HpF does not protect against neuronal
damage in primary neuron culture. LjF, on the other hand, has been

extensively studied for its endothelial protective, immune activating,
antiviral, antithrombotic, anti-inflammatory and antioxidant activities
(Ahmad et al., 2021; An et al., 2022; Chen et al., 2017; Kim et al., 2020;
Liang et al., 2018; Zhang et al., 2021; Zhao et al., 2016). It has also been
reported to protect dopaminergic neurons in a rotenone-induced rat
model of PD (Zhang et al., 2018). AnF has been reported to suppress
postprandial hyperglycemia, ameliorate atherosclerosis and alleviate
gut microbiota dysbiosis (Shan et al., 2020; Wang et al., 2020; Yin et al.,
2019). We observed similar neuroprotective effect of LiF and AnF in our
study. Previous studies on FvF have only reported on its bioactivities not
related to the nervous system, such as reduced HBV DNA, hBsAg, and
HBeAg levels in the blood (Li et al., 2017), a bacteriostatic effect (Ayr­
apetyan et al., 2021) and a protective role in DOX-induced acute car­
diotoxicity (Zhang et al., 2020). We explored the structure-activity
relationship between type І and type II Fucoidan. It was found that
type II Fucoidan (AnF and FvF) had superior effects in lowering the
release of lactate dehydrogenase, improving cell viability compared
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M. Xing et al.

production. Quintana-Cabrera et al. showed that the OPA1 gene could
decrease mitochondrial ROS accumulation, but ATPase activity is
required in this process (Quintana-Cabrera et al., 2021). ATP5F1a itself
has been linked to various human diseases, including cancer, amyo­
trophic lateral sclerosis and frontotemporal dementia (Chin et al., 2014;
Choi et al., 2019; Feichtinger et al., 2018). A growing number of sci­

entists are noticing the role of ATP5F1a in mitochondrial function and
metabolism (Chin et al., 2014; Goldberg et al., 2018; Quintana-Cabrera
et al., 2021; Xiao et al., 2021). Previous information shows that the loss
of ATP5F1a participates in neurotoxicity and has a significant impact on
amyotrophic lateral sclerosis (ALS). In addition, induction of ectopic
ATP5F1a expression in poly (GR)-expressing neurons or reduction in
poly (GR) levels in adult mice after they have been rescued from poly
(GR)-toxicity (Choi et al., 2019). In this study, our findings reveal that
ATP5F1a may play a significant role in the pleiotropic effects of FvF in
Parkinson's disease therapy. FvF may provide protection against PD by
targeting ATP5F1a and mitigating mitochondrial dysfunction. Oligo­
mycin A abolishes the neuroprotective effect of FvF in MPTP PD mice.
This is also proof that ATP5F1a played a role in ROS reduction. Our data
suggested that the neuroprotective effects of FvF may be mediated via
ATP5F1a.
The significance of our study is to enrich the anti-PD effects of
different sources of fucoidan based on previous studies and to confirm
the potential value of marine glycoconjugate fucoidan in the develop­
ment of anti-PD drugs. We used target fishing technology for the first
time to assess the potential targets of FvF and to elucidate the mecha­
nism of its neuroprotection. Although we elucidated the role of ATP5F1a
in FvF alleviating mitochondrial dysfunction and apoptosis in primary
neurons, there might be other targets in FvF against PD that still need to
be explored. Furthermore, the main role of ATP5F1a is to regulate ATP
production and participate in energy metabolism. Therefore, it is worth
exploring in depth the role of FvF in the energy metabolism of neuronal
cells.

Acknowledgments
This work was supported by the National Natural Science Foundation

of China (81991523, 81991522, 31971210), National Key Research and
Development Program of China (2021ZD0202901), Taishan Scholar
Climbing Project (TSPD20210304) and Key Scientific and Technological
Projects of Shandong Province (2021ZDSYS22, 2021KJ012).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.carbpol.2022.120470.
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5. Conclusion
For the first time, we found that type II fucoidan from Fucus ves­
iculosus (FvF) had neuroprotective and anti-PD effects in both primary
neurons and MPTP-induced PD mice. Furthermore, FvF protected
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CRediT authorship contribution statement
Meimei Xing and Yang Liu designed and performed the part of
pharmacological experiments, analyzed the data, and wrote the manu­
script. Guoyun Li and Luyao Yang designed and performed the part of
chemical experiments. Jianhua Ding provided technical support. You­
jiao Zhang and Yuruo Zhang contributed to the experiments. Gang Hu,

Guangli Yu, and Ming Lu conceived the study concept, and designed the
experiments. Gang Hu and Guangli Yu supervised the project. All the
authors have read and approved the final version of the manuscript.
Declaration of competing interest
The authors have no competing interests to declare that they are
relevant to the content of this article.
Data availability
Data will be made available on request.

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