The risk of developing neurodegenerative diseases increases with age(Reference Hou, Dan and Babbar1). More than one in ten (10·9 %) people age 65 and older have Alzheimer’s disease, and the prevalence increases to approximately one-third of those age 85 and older(Reference Dement2). Likewise, the risk of Parkinson’s disease increases with age, and the WHO estimates that the prevalence of Parkinson’s disease has doubled over the past two decades(Reference Willis, Roberts and Beck3,4) . Considering the percentage of the global population age 65 and older is growing faster than any other age group, the burden of these age-related neurodegenerative diseases will intensify(5), especially as widely consumed dietary factors such as fructose, alcohol and high-fat diets have been implicated in promoting oxidative stress, lipid peroxidation, mitochondrial dysfunction and possibly ageing(Reference Alwahsh and Gebhardt6–Reference Song, Akbar and Abdelmegeed8). Therefore, nonpharmacological targets for prevention are warranted. In particular, a neuroprotective dietary strategy that can be implemented in daily life may be ideal. An increasing number of studies have demonstrated the protective effects of brown seaweed for brain health(Reference Ferdous, Sarihi and Ellis9,Reference Park, Crowe-White and Ciesla10) . A diet rich in brown seaweed is inversely associated with cerebrovascular disease among centenarians(Reference Willcox, Willcox and Shimajiri11,Reference Willcox, Scapagnini and Willcox12) . Seaweed contains bioactive compounds with antioxidant and anti-inflammatory effects that could mediate ageing processes(Reference Ferdous, Sarihi and Ellis9,Reference Peng, Yuan and Wu13) . Notably, brown seaweed is rich in fucoxanthin, a carotenoid pigment that is a strong antioxidant with the ability to scavenge reactive oxygen species (ROS) including hydroxyl radical, peroxyl radical and hypochlorous acid(Reference Sachindra, Sato and Maeda14–Reference Takashima, Shichiri and Hagihara16).
Fucoxanthin is reported to exhibit neuroprotective effects against neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Studies have shown that treatment with fucoxanthin prevented amyloid-β (Aβ)-induced oxidative stress and apoptosis in vitro (Reference Lee, Youn and Yoon17), and oral supplementation with fucoxanthin in mice improved cognitive behaviours against Aβ challenges(Reference Xiang, Liu and Lin18). In addition, fucoxanthin protected 6-hydroxydopamine (OHDA)-challenged PC12 cells by regulating the nuclear factor erythroid 2-related factor 2 pathway via binding to the hydrophobic regions of Keap1(Reference Wu, Han and Liu19). A 6-OHDA-induced mouse Parkinson’s disease model also demonstrated that oral supplementation with fucoxanthin-enhanced motor function(Reference Liu, Lu and Tang20). Despite these encouraging results that have demonstrated protective effects of fucoxanthin in neurodegenerative disease models, the neurodegenerative processes that underlie these diseases begin decades before clinical symptoms appear(Reference Wilson, Cookson and Van Den Bosch21). Currently, the role of fucoxanthin in the non-pathological ageing brain is less known. In this study, we hypothesised that treatment with fucoxanthin prevents ageing-associated oxidative stress and thus supports natural brain ageing. We tested the role of fucoxanthin against ROS and ageing in vitro. Additionally, this is the first test to examine whether orally administered fucoxanthin is found in the brain and exhibits a direct antioxidant effect in healthy middle-aged rats, and we further explored genes that are sensitive to fucoxanthin treatment.
Materials and methods
Culture of primary neurons
Primary rat hippocampal neurons were prepared from rat feti (Sprague-Dawley, day 18 of gestation; Envigo) as described previously(Reference Park, Licznerski and Alavian22–Reference Park, Licznerski and Mnatsakanyan25). Briefly, neurons (0·3 × 106 cells/35 mm plate) were seeded on poly-L-lysine-coated plates and grown in neurobasal medium supplemented with B-27, glutamine and antibiotics (Invitrogen Gibco Life Technologies) for 3 or 6 weeks in vitro. Fucoxanthin: Neurons were either treated with fucoxanthin (100 nM, Cayman Chemical) or a vehicle (DMSO). Hydrogen peroxide treatment: 25 µM of hydrogen peroxide (Sigma-Aldrich) were freshly prepared in sterile PBS and added to the cell culture medium. The vehicle control for hydrogen peroxide experiments was sterile PBS. All protocols were approved by the Institutional Animal Care Committee (IACUC) of University of Alabama, Tuscaloosa, AL (23-11-7123).
Reactive oxygen species measurement
2′,7′- dichlorodihydrofluorescein diacetate staining
Primary hippocampal neurons were treated with 10 µM of dichlorofluorescein (DCF) (Invitrogen) solution prepared in a light protected vessel, then incubated for 30 min at 37°C in the dark(Reference Duan, Gross and Sheu26) and processed as the manufacturer’s protocol. After incubation, neurons were carefully washed with pre-warmed Hanks’ Balanced Salt Solution (HBSS). Intracellular fluorescence was measured using a fluorescent microplate reader (CLARIOstar, BMG Labtech) at excitation and emission wavelengths of 470–15 and 515–20 nm, respectively.
mitoSOX staining
Production of mitochondrial ROS was analysed using MitoSOX Red (Invitrogen). The MitoSOX Red dye is oxidised by superoxide in the mitochondria, emitting red fluorescence. After treatment of neurons as described in relevant figure legends, 1·25 μM of MitoSOX was added to the cell culture medium. Cultures were incubated for 30 min at 37°C and washed twice with warm HBSS. Fluorescent images were taken with a Zeiss AxioVert.A1 microscope and analysed using AxioVision 4·9.
Viability measurement
Lactate dehydrogenase assay
The level of cytotoxicity in primary neurons was assayed by measuring leakage of lactate dehydrogenase (LDH) using an in vitro toxicology assay kit (Sigma-Aldrich) as previously described(Reference Park, Mnatsakanyan and Broman27). In brief, the culture media and lysed cells were collected after 24-h treatment. The LDH assay mixture was added to each sample. After 20-min incubation, the reaction was terminated by adding 1N HCl. LDH activity was spectrophotometrically measured with a Clariostar microplate reader (BMG Labtech) with absorbance set at 490 nm. Data were calculated by finding the activity of LDH which leaked into the medium from damaged cells/total LDH activity in the culture.
Propidium iodide (PI) staining
Dead cells were stained with PI as previously described(Reference Park, Licznerski and Alavian22,Reference Park, Licznerski and Mnatsakanyan25,Reference Alavian, Beutner and Lazrove28) . After treatment, 0·5 μM PI (Invitrogen) was added into the culture medium for 30 min at 37°C in the dark. Images were taken using a Zeiss AxioVert.A1 microscope using consistent exposure time. The number of PI positive neurons was analysed using AxioVision 4·9.
Experimental animals and study design
Male Sprague-Dawley rats (n 12, 17 months old, Envigo) were randomly divided into two groups using ‘sort by random’ tool in Excel: control (n 6) and supplemented (n 6) groups after a one-week acclimation period. To test the effect of dietary fucoxanthin in the brain, the supplemented group was orally gavaged with fucoxanthin (Algatechnologies Ltd) in olive oil at a dosage of 1 mg/kg body weight, 5 days per week for 4 weeks as described previously(Reference Zhang, Wang and Fan29). The control group was orally gavaged with olive oil with volume matching the mean volume of the supplemented group. After supplementation was completed, and tissue from control and supplemented animals were harvested. The exclusion criteria include animals with clinical and behavioural changes such as hunching, abnormal vocalisation, nosy breathing and hair loss. Animals were maintained under standard condition at 22 ± 2℃ with 12:12 dark: light cycles in the Animal Care Facility in the University of Alabama. The sample size was determined by a previous report with rats orally supplemented with fucoxanthin(Reference Zhang, Wu and Wen30). All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Alabama (20-07-3739).
Thiobarbituric acid reactive substance assay
The level of lipid oxidation was assessed by measuring malondialdehyde (MDA) using thiobarbituric acid reactive substance assay kit per the manufacturer’s protocol (Cayman ChemicalMI). In brief, the samples were homogenised in 1X radioimmunoprecipitation assay buffer containing protease inhibitors, and protein concentrations were determined using the bicinchoninic acid assay. Protein samples, sodium dodecyl sulphate and the colour reagent containing thiobarbituric acid, acetic acid and sodium hydroxide were mixed and boiled for 1 h. After 1 h, the samples were cooled in ice for 10 min followed by centrifugation. The MDA level was measured with a CLARIOstar microplate reader (BMG Labtech) with an excitation of 530 nm and an emission of 550 nm.
Quantification of fucoxanthin in the brain
The brain extraction procedure was performed in accordance with a protocol adapted from Vishwanathan et al (Reference Vishwanathan, Neuringer and Snodderly31). Approximately 40 mg of brain tissue was homogenised with 60 μl saline and 0·1 ml ethanol in a 50 ml glass Dounce homogeniser. About 10 μl of internal standard (1 mg/ml Astaxanthin, Sigma) and 0·4 ml of ethanol were added to the homogenate and mixed well. The mixture was then incubated in a 70°C water bath for 2 min, followed by the addition of 25 % sodium ascorbate (0·1 ml) and 5 % sodium hydroxide (0·2 ml) and incubated again in a 60°C water bath for 20 min (saponification to remove esters, as carotenoids tend to be esterified with fatty acids)(Reference Toomey and McGraw32). Following incubation, 0·1 ml distilled water was added, and the mixture was cooled for 5 min. Subsequently, 1 ml of hexane was added, and the mixture was vortexed vigorously for 2 min. The mixture was then centrifuged at 1000 × g for 10 min at 4°C. The upper hexane layer was removed and evaporated to dryness under nitrogen. The extraction was repeated with 1 ml of hexane, and the mixture was processed as described above. The second hexane layer was combined with the first extract and evaporated to dryness under nitrogen. The dry residue was reconstituted in 20 μl of ethanol, vortexed, sonicated (30 s) and transferred to HPLC inserts. The inserts were centrifuged at 2000 × g for 3 min, and the pellet was discarded. The supernatant was then transferred to clean inserts, and 10 μl was injected into the HPLC-DAD-MS system comprising of C30 carotenoid column (3 μm, 150 × 4·6 mm, Acclaim, ThermoFisher)(Reference Waksmundzka-Hajnos and Sherma33) and DAD and MS detectors. Fucoxanthin was identified and quantified in the analysed samples by comparing their retention times, absorbance spectra and mass-to-charge ratio (m/z) to those of the external standards (Sigma Aldrich).
RNA extraction
Total RNA was isolated from brains of fucoxanthin-treated and control (vehicle) treated rats, and a NanoDrop ND-1000 (Thermo Fisher Scientific) was used to quantify RNA and evaluate quality. Standard denaturing agarose gel electrophoresis was used to assess RNA integrity.
Microarray analysis
Microarray analysis was performed to systematically profile expression changes in protein-coding mRNAs using the Arraystar Rat lncRNA Microarray v3.0 (Arraystar), which can detect 10 333 lncRNAs and 28 287 coding transcripts. The Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) was used to perform sample labelling and array hybridisation with minor modifications. Briefly, the Arraystar Flash RNA Labeling Kit (Arraystar) was used to amplify and transcribe each RNA sample into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilising a random priming method. The labelled cRNAs were purified using an RNeasy Mini Kit (Qiagen, Hilden, DE). A NanoDrop ND-1000 (Thermo Fisher Scientific) was used to measure the concentration and specific activity of the labelled cRNAs (pmol Cy3/μg cRNA). 1 μg of each labeled cRNA was fragmented by adding 5 μl 10× blocking agent and 1 μl of 25× fragmentation buffer. The mixture was then heated at 60°C for 30 min, and 25 μl 2× GE hybridisation buffer was then added to dilute the labelled cRNA. 50 μl of hybridisation solution was dispensed into the gasket slide and assembled to the lncRNA expression microarray slide. The slides were incubated for 17 h at 65°C in an Agilent Hybridisation Oven (Agilent Technology). The hybridised arrays were washed, fixed and scanned using the Agilent DNA Microarray Scanner, part number G2505C (Agilent Technology).
Bioinformatics analysis
Array images were analysed using Agilent Feature Extraction software, version 11.0.1.1 (Agilent Technology), and the GeneSpring GX v12·1 software package (Agilent Technology) was used for quantile normalisation and subsequent data processing. After quantile normalisation of the raw data, lncRNAs and mRNAs where at least four out of eight samples had present or marginal flags in ‘All Targets Value’ were chosen for further data analysis. Fold change filtering or volcano filtering was used to identify statistically significant and differentially expressed lncRNAs and mRNAs between the fucoxanthin and control (vehicle) samples. Hierarchical clustering was performed using the R software (R Core Team, R Foundation for Statistical Computing). Gene ontology (GO) analysis was performed using the topGO package in the R environment for statistical computing and graphics, and pathway analysis was calculated by Fisher’s exact test.
Statistical analysis
Data are reported as the mean (sd) of at least three independent samples. Differences in means were tested using one-way ANOVA with Tukey’s test or Student’s t test in GraphPad Prism software (San Diego, CA) and P < 0·05 was considered statistically significant. P values are provided in figure legends.
Results
Treatment with fucoxanthin prevents oxidative stress-mediated neuronal death
To test neuroprotective properties of fucoxanthin against oxidative stress, we quantified LDH release (Figure 1(a)) in primary hippocampal neurons 24 h after treatment with hydrogen peroxide (25 µM) and fucoxanthin (0, 10, 100 and 1000 nM). We have previously shown that 25 µM hydrogen peroxide exhibits a moderate cytotoxicity that is sensitive to carotenoid treatment(Reference Park, Stumpf and Broman34). Consistently, 25 µM hydrogen peroxide increased LDH release in primary hippocampal neurons (control 1·00 (sd 0·01) v. hydrogen peroxide 1·37 (sd 0·1), P < 0·0001), whereas treatment with 100 and 1000 nM fucoxanthin prevented the effect of hydrogen peroxide (100 nM fucoxanthin 1·23 (sd 0·05) v. hydrogen peroxide 1·37 (sd 0·1), P = 0·002; 1000 nM fucoxanthin 1·14 (sd 0·08) v. hydrogen peroxide 1·37 (sd 0·1), P = 0·01). We further measured mitochondrial superoxide using mitoSOX staining (Figure 1(b)). Primary hippocampal neurons treated with hydrogen peroxide significantly increased mitoSOX fluorescence (control 1·00 (sd 0·51) v. hydrogen peroxide 1·59 (sd 0·93), P < 0·0001). However, co-treatment with 100 nM fucoxanthin attenuated hydrogen peroxide-induced mitoSOX signal (hydrogen peroxide 1·59 (sd 0·93) v. fucoxanthin cotreatment 1·15 (sd 0·65), P = 0·0001). Additionally, 100 nM fucoxanthin lowered PI-positive cells (Figure 1(c)), indicating a decreased late apoptotic and necrotic population, against hydrogen peroxide challenge (control 1·60 (sd 2·44) v. hydrogen peroxide 6·33 (sd 7·68), P = 0·0004; hydrogen peroxide 6·33 (sd 7·68) v. fucoxanthin cotreatment 2·83 (sd 3·4), P = 0·02).
Fucoxanthin attenuates hydrogen peroxide-induced oxidative stress in primary hippocampal neurons. Primary hippocampal neurons were treated with fucoxanthin (10, 100 and 1000 nM) and hydrogen peroxide (25 μM) for 24 h. Quantified cytotoxicity was measured by lactate dehydrogenase (LDH) release (a), n 4 from three independent cultures. Primary hippocampal neurons were treated with hydrogen peroxide (25 µM) or a combination of hydrogen peroxide and fucoxanthin (100 nM) for 6 h. Mitochondrial superoxide was measured by mitoSOX staining (b), n 90 micrographs per group. Neurons were treated with fucoxanthin (100 nM), hydrogen peroxide (25 µM) or a combination of both for 24 h. Cell death was measured by quantifying PI-positive cells (c), n 30 micrographs per group. Scale bar = 50 μm. Red: PI; blue: 4′,6-diamidino-2-phenylindole (DAPI). *P < 0·05, **P < 0·01 and ***P < 0·001, one-way ANOVA with a Tukey’s post hoc analysis. PI, propidium iodide.
Treatment with fucoxanthin protects primary neurons against ageing in vitro
To test whether in vitro ageing changes intracellular oxidative stress level, we labelled 3 and 6 weeks old primary hippocampal neurons with DCF (Figure 2(a)). Six-week-old primary hippocampal neurons significantly increased DCF fluorescent intensity compared to the 3 weeks old group indicating increased intracellular ROS (3-week 1·00 (sd 0·09) v. 6-week 1·34 (sd 0·17), P = 0·01). Although fucoxanthin did not show any significance in the 3-week-old group, treatment with fucoxanthin significantly prevented mitochondrial superoxide production (6-week without fucoxanthin 1·59 (sd 0·63) v. 6-week with fucoxanthin 1·28 (sd 0·58), P = 0·02, Figure 2(b)) and PI-positive dead cells in the 6 weeks group (6-week without fucoxanthin 6·20 (sd 3·53) v. 6-week with fucoxanthin 4·30 (sd 2·35), P = 0·02, Figure 2(c)), suggesting neuroprotective effect during in vitro ageing.
Fucoxanthin protects primary hippocampal neurons against in vitro ageing. Primary hippocampal neurons were grown in neurobasal media for 3 or 6 weeks. Intracellular hydrogen peroxide levels were assayed by measuring DCF (a), n 4 from three independent cultures. *P < 0·05, Student’s t test. Three or 6-week-old primary hippocampal neurons were treated with or without fucoxanthin (100 nM) for 24 h, and mitochondrial oxidative stress was measured by mitoSOX staining (b), n 45 micrographs per group. Scale bar = 50 μm. Red: mitoSOX. Cell death was measured by quantifying PI-positive cells (c), n 30 micrographs per group. Scale bar = 50 μm. Red: PI; blue: DAPI. *P < 0·05 and ****P < 0·0001, one-way ANOVA with a Tukey’s post hoc analysis. PI, propidium iodide.
Fucoxanthin is found in the brain after oral supplementation
Previous studies in animals that underwent oral supplementation with fucoxanthin were reported to demonstrate improved cognitive behaviours against Alzheimer’s disease and acute brain injury(Reference Xiang, Liu and Lin18,Reference Zhang, Lu and Tao35) . Similarly, intragastric administration of fucoxanthin alleviated behaviours associated with depression and ischaemia(Reference Jiang, Wang and Lin36,Reference Hu, Chen and Tian37) . However, it is still unclear whether orally administered fucoxanthin is delivered to the brain and exhibits a direct protective effect. In this study, male Sprague-Dawley rats were orally gavaged with 1 mg/kg fucoxanthin or the same volume of vehicle for 4 weeks. The concentration of fucoxanthin was determined based on the estimated habitual intake of fucoxanthin in Japanese women, which ranges from 0 to 5·66 mg/d, corresponding to approximately 0–0·12 mg/kg body weight(Reference Fukushima, Taguchi and Kishimoto38). The animal dose equivalence of which is around 0–0·7 mg/kg in rats(Reference Nair and Jacob39). Therefore, a dose of 1 mg/kg was chosen to ensure a physiologically relevant exposure. To evaluate the direct antioxidant effects of fucoxanthin, we measured MDA to determine the level of lipid peroxidation (Figure 3 (a) and (b)). MDA is a final product of oxidation of PUFA that are abundant in the brain. The MDA level in the fucoxanthin group significantly decreased, indicating that oral supplementation with fucoxanthin exhibits an antioxidant effect in blood (control 1·00 (sd 0·12) v. fucoxanthin 0·88 (sd 0·05), P = 0·04) and the brain (control 1·00 (sd 0·09) v. fucoxanthin 0·70 (sd 0·14), P = 0·02). Quantitative analysis showed that oral supplementation with fucoxanthin significantly increased the concentration of fucoxanthin in the brain compared with control groups (control 0·00 (sd 0·00) v. fucoxanthin 34·11 (sd 2·56), P < 0·0001, Figure 3(c)).
Fucoxanthin is found in the brain of rats after oral supplementation. Oral supplementation of fucoxanthin significantly decreases malondialdehyde levels in blood (a) and the brain (b) collected from rats (n 6). (c) Oral supplementation of fucoxanthin increased fucoxanthin in the brain (n 3). *P < 0·05 and ****P < 0·0001, Student’s t test.
Oral supplementation of fucoxanthin regulates gene expression in the brain
To determine whether oral administration of fucoxanthin resulted in changes in gene expression, we submitted whole brain tissue for rat lncRNA microarray (Arraystar) (heat map and hierarchical clustering shown in Figure 4(a)). Using this assay, we identified 5602 mRNAs that were significantly and differentially expressed (2666 upregulated, 2936 downregulated and 22252 unchanged) via volcano filtering with fold change cut-off of 2·0 and P value cut-off of 0·05 (Figure 4(b)). To determine the major biological processes, cellular compartments and molecular processes regulated by fucoxanthin administration, GO analysis using the topGO package was performed. The GO terms most highly enriched in upregulated genes were detection of stimulus (GO:0051606, biological processes), cell periphery (GO:0071944, cellular compartments) and olfactory receptor activity (GO:0004964, molecular processes). The ten most high upregulated GO terms are shown in Figure 5(a). The GO terms most highly enriched in downregulated genes were ion transport (GO:0006811, BP), extracellular space (GO:0005615, cellular compartments) and signalling receptor activator activity (GO:0030546, molecular processes). The ten most highly downregulated GO terms are shown in Figure 5(b).
Differentially expressed mRNAs between rats treated with fucoxanthin or vehicle. Hierarchical clustering (a) of differentially expressed mRNAs and volcano plots (b) of mRNA expression in the brain of rats. In this plot, genes with high relative expression are colored in red, while genes with low expression are coloured in green. Data were generated from four rats receiving fucoxanthin treatment (Fuco-1 through Fuco-4) and four rats receiving vehicle treatment (Con-1 through Con-4).
GO analysis of rats treated with fucoxanthin compared with vehicle. The ten most enriched GO categories containing mRNAs that are upregulated (a) and the ten most enriched GO categories containing mRNAs that are downregulated (b) by fucoxanthin treatment grouped by biological process, cellular compartment and molecular function. GO, gene ontology.
Pathway analysis of rats treated with fucoxanthin compared with vehicle
To determine biological pathways represented by differentially expressed mRNAs following fucoxanthin administration, pathway analysis based on the Kyoto Encyclopedia of Genes and Genomes database was performed. Compared with vehicle control, fucoxanthin administration resulted in twenty-two upregulated pathways and forty-eight downregulated pathways. Treatment with fucoxanthin highly upregulated olfactory transduction, Ca signalling pathway, focal adhesion and ECM–receptor interaction (Figure 6(a)), whereas cytokine–cytokine receptor interaction, inflammatory mediator regulation of TRP channels, protein digestion and absorption and neuroactive ligand–receptor interaction were highly downregulated (Figure 6(b)).
Pathway analysis of rats treated with fucoxanthin compared with vehicle. KEGG pathway analysis was used to identify the ten most enriched pathways containing mRNAs that are upregulated (a) and the ten most enriched pathways containing mRNAs that are downregulated (b) by fucoxanthin treatment. KEGG, Kyoto Encyclopedia of Genes and Genomes.
Included in the significantly downregulated pathways such as thyroid cancer (rno05216), pathways in cancer (rno05200), transcriptional misregulation in cancer (rno05202) and PI3K-Akt signaling pathway (rno04151) were numerous genes involved in cell death processes such as apoptosis, including Bak1 encoding Bak (2·90-fold downregulated) and Bcl2l11 encoding Bim (9·17-fold downregulated and among the 50 most downregulated genes, Table 1). Given the prior known neuroprotective properties of fucoxanthin, we also found it interesting that the cytokine–cytokine receptor interaction pathway (rno04060) included Tnfrsf 9 gene (2·11-fold downregulated), a deletion of which was recently associated with a juvenile-onset Parkinson’s disease phenotype(Reference Guler, Gul and Guler40). An expanded analysis of candidate genes associated with neurodegenerative diseases revealed downregulation of Snca encoding α-synuclein (3·16-fold downregulated), Park7 encoding DJ1 (2·01-fold downregulated), Aplp2 encoding amyloid precursor-like protein 2 (2·36-fold downregulated) and Apbdlip encoding amyloid beta precursor protein binding family B member 1 interacting protein (2·54-fold downregulated).
Top 50 differentially expressed mRNAs in fucoxanthin-treated rats v. vehicle-treated rats
FDR, false discovery rate.
Fold change is fucoxanthin v. vehicle (control).
Discussion
In this study, we showed neuroprotective roles of fucoxanthin against oxidative stress and in vitro ageing in primary hippocampal neurons. We further quantified fucoxanthin in the brain after oral supplementation and demonstrated its delivery to the brain. Fucoxanthin exhibited direct effects in the brain as an antioxidant and a gene regulator. We identified over 5000 genes that are sensitive to fucoxanthin treatment, including genes that are associated with neurodegeneration.
While the pharmacokinetics of fucoxanthin in the blood and other tissues, including liver, lung, heart and adipose tissue, have been reported in rodents(Reference Zhang, Wu and Wen30,Reference Hashimoto, Ozaki and Taminato41) , no prior studies have demonstrated the delivery of fucoxanthin to the brain. Our data are the first to provide a quantitative analysis of fucoxanthin in the brain after oral administration. Although a single administration may be inadequate for the distribution of fucoxanthin in tissue, as fucoxanthin can be converted to fucoxanthionol and amarouciaxanthin A via hydrolysis and dehydrogenation, respectively(Reference Asai, Sugawara and Ono42). Hashimoto et al. showed that fucoxanthin was detectable in various tissues after daily oral administration of 160 nM fucoxanthin for 1 week(Reference Hashimoto, Ozaki and Taminato41). We successfully detected fucoxanthin in the brain after the administration of fucoxanthin (1 mg/kg) for 4 weeks, and thus, habitual intake may favour the accumulation of fucoxanthin in the target tissues. Although we did not perform quantitative analysis on fucoxanthin’s metabolites in this study, the mechanisms of fucoxanthin and metabolites may be distinctive(Reference Pruccoli, Balducci and Pagliarani43). To understand the effect of the habitual intake of fucoxanthin, future quantitative analysis with metabolites following the prolonged administration of fucoxanthin can elucidate the metabolism, kinetics and bioavailability of fucoxanthin in the brain.
Although it is still unclear whether administration of fucoxanthin in a range similar to 1 mg/kg is neuroprotective in humans, a single oral administration of 31 mg fucoxanthin to humans with a mean body weight of 60 kg (which is approximately 0·5 mg/kg) demonstrated pharmacokinetic profiles in plasma(Reference Hashimoto, Ozaki and Mizuno44). In addition, oral administration of a high concentration of fucoxanthin (500 and 1000 mg/kg) in mice for 30 d did not alter mortality and the growth rate(Reference Beppu, Niwano and Tsukui45). Although clinical trials investigating fucoxanthin for neuroprotection are lacking, the few that have investigated fucoxanthin supplementation for obesity and other cardiometabolic disorders have not shown adverse effects(Reference Kumarasinghe and Gunathilaka46). Thus, a milligram range in concentrations may be feasible to investigate the translational aspects of neuroprotective effects of fucoxanthin in the future.
We selected middle-aged healthy rats to eliminate the effects of ageing-associated pathology, thus our in vivo data may not directly represent neuroprotective function of fucoxanthin for specific disease states. Rather, we investigated the role of fucoxanthin in natural ageing. We found that oral supplementation with fucoxanthin alters numerous genes that control biological processes, cellular components and molecular function. In particular, pathway analysis based upon the Kyoto Encyclopedia of Genes and Genomes database revealed that fucoxanthin inhibits signaling pathways responsible for inflammation, such as cytokine–cytokine receptor interaction and inflammatory mediator regulation of TRP channels. Unexpectedly, we also found that treatment with fucoxanthin upregulated 244 genes in olfactory transduction. The results of this pathway analysis are interesting because preclinical processes of neurodegeneration are known to occur decades before the diagnosis of neurodegenerative diseases, and inflammation and olfactory dysfunction are important predictors(Reference Lafaille-Magnan, Poirier and Etienne47,Reference Alcolea, Martinez-Lage and Sanchez-Juan48) and biomarkers of neurodegeneration(Reference Tian, Bilgel and Moghekar49,Reference Morgan, Touchard and Leckey50) . Approximately 90 % of individuals with early-stage Parkinson’s disease and 85 % of those with early-stage Alzheimer’s disease exhibit olfactory dysfunction(Reference Dan, Wechter and Gray51). When the Movement Disorder Society’s Research Criteria for Prodromal Parkinson’s Disease were updated in 2019, olfactory dysfunction was identified as a strong marker with a 6·4 likelihood ratio of risk(Reference Heinzel, Berg and Gasser52). Evidence suggests that accumulation of misfolded α-synuclein in the olfactory bulb precedes accumulation in other brain regions(Reference Saramago and Franceschi53).
Neuroprotective properties of fucoxanthin have been reported in models of neurodegenerative diseases. Treatment with fucoxanthin prevents 6-OHDA-mediated neurotoxicity both in vitro and in vivo (Reference Wu, Han and Liu19,Reference Liu, Lu and Tang20) . 6-OHDA is taken up by dopaminergic neurons via dopamine receptors and releases hydrogen peroxide; thus, it is commonly used to mimic oxidative stress in Parkinson’s disease pathology. Wu et al. showed that fucoxanthin directly binds to Keap1 and increases expression of the downstream antioxidant enzymes of the Keap1/nuclear factor erythroid 2-related factor 2-ARE pathway such as nicotinamide heme oxygenase-1, glutamate-cysteine ligase modifier subunit and glutamate cysteine ligase catalytic subunit(Reference Wu, Han and Liu19). Further, fucoxanthin has a concentration-dependent agonist effect on dopamine D3 and D4 receptors(Reference Paudel, Seong and Jung54). Previously, fucoxanthin was reported to prevent the loss of mitochondrial membrane potential against 6-OHDA and levodopa challenges(Reference Liu, Lu and Tang20). Although the exact mechanism is unknown, we found that fucoxanthin downregulated the Ntrk3 gene, which encodes tropomyosin receptor kinase C. Tropomyosin receptor kinase C has been shown to inhibit DJ1 degradation(Reference Kim, Lee and Park55), and thus, treatment with fucoxanthin may help enhance DJ1’s stability.
Fucoxanthin has been tested in models of Alzheimer’s disease. Treatment with fucoxanthin was found to alleviate Aβ-mediated cytotoxicity in vitro and cognitive impairments in vivo (Reference Lee, Youn and Yoon17,Reference Xiang, Liu and Lin18,Reference Alghazwi, Smid and Musgrave56,Reference Lin, Yu and Zhao57) . Notably, fucoxanthin showed a concentration-dependent Bax/Bcl-2 ratio reduction and prevented the loss of mitochondrial membrane potential against Aβ challenge, indicating mitochondrial protection(Reference Lee, Youn and Yoon17). Fucoxanthin binds to Aβ directly via hydrophobic interactions and prevents Aβ oligomerisation(Reference Xiang, Liu and Lin18). Similarly, a molecular docking simulation study identified fucoxanthin’s inhibitory effects on Aβ aggregation by its interaction with 16–20, 24–28 and 33–37 residues of Aβ (Reference Lakey-Beitia, Kumar and Hegde58). In addition to functional inhibition, fucoxanthin may prevent the production of Aβ. In addition, as an antioxidant, fucoxanthin may prevent cleavage of APP to Aβ by blocking oxidative stress-mediated secretase activation.
Because our pathway analysis revealed that fucoxanthin downregulates fifty-nine genes that regulate cytokine–cytokine receptor interaction and twenty-eight genes that regulate inflammatory mediator regulation of TRP channels, it is not surprising to find that fucoxanthin’s anti-inflammatory effects have been reported in the literature. Microglial cells challenged with lipopolysaccharide increased mRNA and protein levels of pro-inflammatory cytokines such as TNF-α and IL-6, while treatment with fucoxanthin exhibited a concentration-dependent inhibitory effect. The authors further showed that fucoxanthin inhibits Akt/ nuclear factor-kappa B and mitogen-activated protein kinase/ activator protein-1 pathways(Reference Zhao, Kwon and Chun59). Similarly, treatment with fucoxanthin attenuated the production of pro-inflammatory cytokines against Aβ challenge in microglial cells(Reference Pangestuti, Vo and Ngo60).
Although our study identifies fucoxanthin-sensitive genes and pathways in the brain, some limitations exist. In this study, we used whole-brain samples for in vivo analysis; therefore, our findings are insufficient for explaining the heterogeneity of the brain. For example, because the olfactory transduction is the major pathway affected by fucoxanthin, future analysis of the olfactory bulb can help explain the region-specific role of fucoxanthin. Given that current studies primarily focus on the role of fucoxanthin in natural ageing, introducing neuropathology with functional and behavioural analysis in future projects can bridge the gap between healthy brain ageing and neurodegenerative disease and can help elucidate the therapeutic potential of fucoxanthin in the brain.
Conclusion
In summary, treatment with fucoxanthin protects neurons against ageing-associated damage in vitro. We also show that orally administered fucoxanthin is detected in the brain and exhibits an antioxidant effect. We demonstrated that oral supplementation with fucoxanthin changes genes that are associated with neuronal damage in middle-aged rats without a phenotype of neurodegenerative diseases. Combined with evidence from reported epidemiological studies with populations with frequent dietary consumption of brown seaweed(Reference Brown, Allsopp and Magee61), our data suggest that fucoxanthin is a potential dietary strategy to benefit natural brain ageing.
Acknowledgements
We would like to thank Algatechnologies Ltd (Israel) for donating fucoxanthin.
This study was supported by The University of Alabama Joint Institute Pandemic Pilot Project (H-A. P. and A. E.), and R. N. C. was supported by a contribution from the Dale/Roy family.
H-A. P. was involved in conceptualization, formal analysis, funding acquisition, investigation, resources, supervision, and writing; E. A. was involved in investigation and formal analysis; G. B. was involved in investigation and formal analysis; K. A. F. was involved in investigation and formal analysis; M. S. was involved in investigation and formal analysis; J. J. was involved in investigation and formal analysis; S. B. was involved in investigation and formal analysis; M.S. was involved in investigation and formal analysis; R. N. C. was involved in formal analysis, funding acquisition, investigation, and writing; L. C. was involved in formal analysis, investigation, resources, and writing; A. E. was involved in funding acquisition, supervision, and writing.
The authors declare no conflict of interest.
