Fucoxanthin protects placenta-derived human mesenchymal stem cells against oxidative stress-induced apoptosis by modulating genes involved in DNA damage repair, ER stress response, p53-induced apoptosis

Abstract

Human mesenchymal stem cells (hMSCs) hold significant promise in regenerative medicine due to their ability to reduce inflammation and promote tissue repair. However, their therapeutic potential is often compromised by their high susceptibility to apoptosis under oxidative stress, prevalent in the microenvironment of the target tissues. Our previous study showed that fucoxanthin, a carotenoid derived from brown algae, can improve the viability of placenta-derived mesenchymal stem cells (PL-MSCs) by reducing intracellular ROS levels through the activation of the PI3K/Akt/Nrf-2 signaling pathway. In this study, we further investigate the mechanisms underlying the protective effect of fucoxanthin against oxidative stress-induced apoptosis in PL-MSCs, using an in vitro model. PL-MSCs were cultured with 750 µM H₂O₂ to induce oxidative stress and treated with various concentrations of fucoxanthin for 48 h. The effect of fucoxanthin on PL-MSC apoptosis under oxidative stress conditions was determined using CCK-8, Annexin V/DRAQ7™ apoptosis assays, as well as the expression of apoptosis-related genes and proteins. The effect of fucoxanthin on the transcriptome of PL-MSCs under oxidative stress conditions was also assessed by high-throughput Nanostring analysis. The results showed that fucoxanthin significantly decreased the apoptosis of PL-MSCs under oxidative stress in a dose-dependent manner by reducing the expression of pro-apoptotic proteins and inhibiting their activation, while increasing the expression of anti-apoptotic proteins in these cells. Furthermore, fucoxanthin also downregulates the expression of genes associated with the endoplasmic reticulum stress, p53-induced apoptosis, while increasing the expression of genes involved in the regulation of the cell cycle, DNA damage repair, cytokine signaling, nucleotide synthesis, PI3K/mTOR pathway and AMPK pathway in PL-MSCs under oxidative stress conditions. Taken together, the findings provide compelling evidence that fucoxanthin protects PL-MSCs against oxidative stress-induced apoptosis by modulating the expression of various genes involved in DNA damage repair, ER stress response, p53-induced apoptosis in these cells. This suggests that fucoxanthin could be used in combination with other agents to increase the therapeutic potential of MSCs by improving their viability under conditions of oxidative stress in the target tissue microenvironment.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-025-04629-3.

Background

Human mesenchymal stem cells (hMSCs) could potentially be used in cell-based therapies due to their ability to home in on specific tissues and release various beneficial factors to reduce inflammation and promote tissue repair [1, 2]. However, there is evidence indicating that most MSCs undergo apoptosis shortly after being introduced into the target tissues [3, 4]. The poor viability of grafted MSCs is largely due to oxidative stress caused by an excessive amount of reactive oxygen species (ROS) in target tissues [5]. Oxidative stress has been shown to inhibit proliferation, increase senescence, and induce apoptosis in transplanted MSCs by damaging their cellular components [6].

Fucoxanthin, a marine carotenoid derived from edible brown algae, is known for its strong antioxidant properties that provide various health benefits, including anti-obesity [7], anti-cancer [8] and anti-inflammation [9] effects. Previous studies indicate that fucoxanthin significantly increases glutathione levels, reduces ROS, inhibits DNA damage, restores mitochondrial membrane potential and suppresses apoptosis in human keratinocytes [10, 11], retinal pigment epithelial cells [12, 13] and neurons [14] under oxidative stress conditions. Furthermore, our previous study found that fucoxanthin also protects human placenta-derived mesenchymal stem cells (PL-MSCs) from oxidative stress by reducing intracellular ROS production and improving their viability by activating the PI3K/Akt/Nrf-2 signaling pathway [15].

However, the effect of fucoxanthin on the prevention of MSC apoptosis under oxidative stress conditions has not yet been determined. Therefore, in this study, we further investigate the mechanisms underlying the protective effect of fucoxanthin against oxidative stress-induced apoptosis in PL-MSCs. We use PL-MSCs, which can be harvested in large numbers by a non-invasive procedure, and have a higher proliferative and engraftment capacity than bone marrow-derived mesenchymal stem cells (BM-MSCs) [16, 17], as a primary cell type for our investigation.

To study the effects of fucoxanthin on PL-MSC apoptosis under oxidative stress conditions, the percentages of MSC apoptosis, the expression of genes related to apoptosis, and the levels of apoptotic regulator proteins after fucoxanthin treatment were determined. The effect of fucoxanthin on the transcriptome of PL-MSCs under oxidative stress conditions was also studied by high-throughput Nanostring analysis.

Materials and methods

Ethical approval and consent

All experimental procedures were conducted in accordance with the Declaration of Helsinki and the Belmont Report. This study received approval from the Human Research Ethics Committee of Thammasat University (Medicine) under the title ‘The effects of fucoxanthin on the proliferation and osteogenic differentiation of human mesenchymal stem cells.’ (Approval number: 024/2021) on 4 February 2021. All samples were collected from donors who gave their written informed consent.

Isolation and culture of placental-derived human mesenchymal stem cells

Placental tissues were collected from five healthy volunteers after normal deliveries at the Thammasat University Hospital. The tissues were cut into small pieces and digested by incubation with 0.5% trypsin-EDTA (Gibco, USA) at 37 °C for 2 h. After washing twice with phosphate-buffered saline (PBS), cells were cultured in a complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS; Gibco, USA), 1% penicillin/streptomycin, and 1% glutamax (Gibco, USA), at 37 °C in a humidified atmosphere with 5% CO₂. The medium was replaced twice a week. When cultures reached 80–90% confluence, cells were trypsinized with 0.25% (w/v) trypsin/EDTA (Gibco, USA). Cells from passages 3 to 6 were used for all experiments.

Characterization of PL-MSCs

Immunophenotyping

5 × 10⁵ PL-MSCs were incubated with 5 µL fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated antibodies against human leukocyte antigen – DR isotype (HLA-DR) (Bio Legend, USA), CD34 (Bio Legend, USA), CD45 (Bio Legend, USA), CD73 (Bio Legend, USA), CD90 (BD Biosciences, USA) and CD105 (BD Biosciences, USA) for 30 min at 4 °C in the dark. Subsequently, the cells were fixed with 1% paraformaldehyde in PBS and analyzed using flow cytometry (DxFLEX flow cytometer, Beckman Coulter, USA) with CytExpert software (DxFLEX flow cytometer, Beckman Coulter, USA).

Trilineage differentiation assays

For osteogenic differentiation, PL-MSCs were cultured in complete DMEM in 6-well plates (Corning, USA) at a density of 4.5 × 10³ cells/cm² overnight. Subsequently, the medium was replaced with osteogenic differentiation medium, a complete DMEM supplemented with 100 nM dexamethasone and 50 µg/mL ascorbic acid (Sigma-Aldrich, USA). On day 7, 10 µM β-glycerophosphate (Sigma-Aldrich, USA) was added to the osteogenic differentiation medium and the cells were cultured for an additional 3 weeks, with medium replacements every 3–4 days. On day 28, cells were fixed, stained with 40 mM Alizarin red S (Sigma-Aldrich, USA) for 30 min at room temperature, and observed by light microscopy (Nikon Eclipse Ts2R, Japan).

For adipogenic differentiation, PL-MSCs were cultured in adipogenic differentiation medium, a complete DMEM supplemented with 100 µM indomethacin, 25 mM glucose, 1 µM dexamethasone, and 1 µg/mL insulin (all purchased from Sigma-Aldrich, USA) for 28 days. On day 28 of culture, cells were fixed with 37% formalin vapor for 20 min, stained with 0.5% Oil red O (Sigma-Aldrich, USA) in 6% isopropanol for 20 min at room temperature and observed under an inverted light microscope.

For chondrogenic differentiation, PL-MSCs were cultured in a 96-well U-bottom plate (Jet Biofil, China) containing complete DMEM at a density of 3 × 10⁴ cells/cm² overnight to generate spheroids. Subsequently, the medium was replaced with MSCgo^™ Chondrogenic XF medium (Sartorius, Germany); spheroids were cultured for an additional 3 weeks. At the end of the culture, spheroids were fixed with 10% formalin for 30 min, stained with 1% Alcian blue (HiMedia, India) in 0.1 M HCl at room temperature overnight, in the dark, washed twice with 0.1 M HCl and observed by stereoscopic microscopy. In all differentiation experiments, PL-MSCs cultured in complete DMEM served as controls.

CCK-8 cell viability assay

Cytotoxicity was determined using the cell counting kit-8 (CCK-8) assay (Dojindo Laboratories, Japan). Briefly, PL-MSCs were seeded in a 96-well plate (Corning, USA) at a density of 3 × 10⁴ cells/cm². Cells were then treated with fucoxanthin (Sigma-Aldrich, USA) at final concentrations of 1, 2, 3, 4, 5, 10, and 20 µM for 48 h. At 24 and 48 h after treatment, 10 µL of CCK-8 solution was added to each well and cells were incubated at 37 °C for 4 h. After incubation, absorbance was measured at 450 nm using an Agilent BioTek Synergy H1 Multimode Reader; analysed with Agilent BioTek Gen5 software version 3.14 (BioTek Instruments Inc., USA). Cell viability was presented as percentage of control, calculated using the following equation.

H₂O₂-induced oxidative stress

To induce oxidative stress, PL-MSCs were seeded in a 96-well plate (Corning, USA) at a density of 3 × 10⁴ cells/cm² and treated with 50-1500 µM H₂O₂ (Sigma-Aldrich; Merck Millipore, Germany) in a complete DMEM at 37 °C for 24 h. Cell viability was assessed using a CCK-8 assay as described above.

MTT assay

To assess the effects of fucoxanthin on PL-MSCs under acute oxidative stress to mimic the environment in damaged tissues, PL-MSCs were seeded in 96-well plates (Corning, USA) at 3 × 10⁴ cells/cm² and treated with 750 µM H₂O₂ and fucoxanthin (1–5 µM) for 24 and 48 h. Untreated PL-MSCs cultured in complete DMEM served as controls. Cell viability was assessed using the 3- (4, 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, USA). The absorbance was measured at 570 nm using a Synergy HT Multi-Detection Microplate Reader and Agilent BioTek Gen6 software (BioTek Instruments Inc., USA). Cell viability was calculated as shown in the equation:

Apoptosis assay

The percentage of cell apoptosis was measured using the Annexin V-FITC / DRAQ7™ apoptosis assay (Abcam, USA) according to the manufacturer’s protocol. Briefly, PL-MSCs were seeded in a 6-well plate (Corning, USA) at a density of 1.5 × 10⁴ cells/cm² and co-treated with 750 µM H₂O₂ and 1–5 µM fucoxanthin for 24 h. PL-MSCs treated with H₂O₂ without fucoxanthin supplementation for 24 h served as controls. After treatment, cells were harvested, labeled with 5 µM Annexin V-FITC and DRAQ7™ in 200 µL cold labeling buffer for 15 min, protected from light, and, apoptosis was determined using BD FACSMelody™ flow cytometer (BD Biosciences, USA) followed by analysis with FlowJo™ software (BD Biosciences, USA). Total percentage of apoptosis profiles was determined as follows: Live (-) Annexin V-FITC; (-) Draq7, Early apoptosis (+) Annexin V-FITC; (-) Draq7, Late apoptosis (+) Annexin V-FITC; (+) Draq7. Dead (-) Annexin V-FITC; (+) Draq7.

Gene expression analysis by quantitative real‑time polymerase chain reaction (qRT-PCR)

To investigate the expression levels of genes related to apoptosis in PL-MSCs under oxidative stress conditions, PL-MSCs were treated with 750 µM H₂O₂ and 1–5 µM fucoxanthin for 24 h at 37 °C. Total RNA was isolated using the RNeasy^® Mini Kit (Qiagen, Germany) and 1 µg of RNA was reverse transcribed using a High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems; Thermo Fisher Scientific, USA) to generate cDNA according to the manufacturer’s instructions. qRT-PCR was performed for 40 cycles using PowerTrack™ SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, USA). and CT values determined using QuantStudio™ 5 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, USA). All reactions were carried out in triplicate (n = 3) and analysed using Design & Analysis 2 (DA2) software version 2.6.0 (Applied Biosystems; Thermo Fisher Scientific, USA). The expression levels of the target genes were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and presented as relative fold change compared to untreated controls using the 2^−ΔΔCt method.

Protein isolation and Western blot analysis

The levels of proteins associated with the intrinsic and extrinsic apoptotic pathway in PL-MSCs under oxidative stress conditions were determined by Western blot analysis. Total protein was isolated using cold RIPA buffer (Sigma-Aldrich, USA) containing protease inhibitor cocktail (Cell Signaling Technology, USA). Total protein concentrations were determined using Pierce^® BCA Protein Assay Kit (Thermo Fisher Scientific, USA). 20 µg total protein containing Laemmli sample buffer supplemented with 2-mercaptoethanol (1.43 M) was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, USA) and transferred to a nitrocellulose membrane (0.45 μm pore-size; Bio-Rad, USA) using a mini trans-blot electrophoretic transfer cell (Bio-Rad, USA). Subsequently, the membranes were blocked with 1X Pierce™ Clear Milk Blocking Buffer in Tris-buffered saline with 0.1% Tween 20 (TBST) for 2 h at room temperature. Then, the membranes were incubated overnight at 4 °C with gentle agitation in the following primary antibodies: rabbit anti-human caspase 3, rabbit anti-human cleaved-caspase 3, rabbit anti-human caspase 7, rabbit anti-human cleaved-caspase 7, rabbit anti-human caspase 8, rabbit anti-human cleaved-caspase 8, mouse anti-human caspase 9, rabbit anti-human cleaved-caspase 9, rabbit anti-human PARP, rabbit anti-human cleaved-PARP, rabbit anti-human DR4, rabbit anti-human FADD, rabbit anti-human phospho-FADD, rabbit anti-human β-actin, (all from Cell Signaling Technology, USA; diluted 1:1000 in blocking buffer). Following primary antibody incubation, membranes were washed with 0.05% TBST and incubated at room temperature with either goat anti-rabbit or horse anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (1:2000) (Cell Signaling Technology, USA) in 0.05% TBST for 1 h, gently rocked. Following secondary antibody incubation, membranes were washed again in 0.05% TBST and protein expression was determined using enhanced chemiluminescence (ECL) with Clarity™ Western ECL Substrate (Bio-Rad, USA), detected using a ChemiDoc™ MP Imaging System (Bio-Rad, USA). Membranes were then stripped with stripping buffer (0.5 M Tris HCl, pH 6.8; 2% SDS; 100 mM 2-mercaptoethanol) at 60 °C for 1 h. Protein signal intensity was quantified using NIH ImageJ software and expressed as a fold-change relative to β-actin.

Nanostring^® nCounter analysis

The effect of fucoxanthin on the transcriptome of PL-MSCs under oxidative stress conditions was investigated by NanoString^® nCounter Technology (NanoString Technologies, Seattle, WA, USA). The PL-MSCs were treated with 750 µM H₂O₂ and 5 µM fucoxanthin for 24 h. Total RNA was isolated using the PureLink™ RNA Mini Kit (Ambion, USA). 100 ng of RNA were hybridized with nCounter Reporter and Capture probes at 65 °C overnight. After hybridization, samples were processed using the nCounter Prep Station to purify and immobilize the target/probe complex onto the cartridge. The nCounter digital analyzer was then used to perform a high-density scan to measure the abundance of target RNA molecules in each sample. Multiplexed gene expression analysis of 768 genes in 34 annotated pathways that are essential for biosynthesis, nutrient capture, anabolic metabolism, catabolic metabolism, cell stress, metabolic signaling, and transcriptional regulation was performed by nSolver software, version 4.0 (NanoString Technologies, Seattle, WA, USA). The mRNA copy numbers were standardized using the geometric mean of 20 housekeeping genes for baseline normalization. A threshold count value of 50 was applied as the baseline subtraction parameter and the changes in gene expression levels were calculated by comparing the fucoxanthin-treated samples with the untreated controls (PL-MSCs treated with 750 µM H₂O₂ without fucoxanthin supplementation).

The differential expression of genes (DEGs) was represented as volcano plots, showing individual genes with -log10 (p-value) plotted against log2 fold change relative to a pairwise comparison between (A) PL-MSCs treated with H₂O₂ and untreated control and (B) PL-MSCs treated with a combination of H₂O₂ and fucoxanthin and PL-MSCs treated with H₂O₂ alone. The Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO) enrichment, and protein-protein interaction (PPI) analysis were performed through the String database using ShinyGO V0.82 [18] to assess functional and pathway enrichment of the DEGs. A Venn diagram visualizing overlapping DEG sets was generated using Venny2.1 (https://bioinfogp.cnb.csic.es/tools/venny/). Transcription factor (TF) regulatory networks were predicted using TRRUST version 2 (https://www.grnpedia.org/trrust/) [19] and validated using STRING version 12.0 (https://string-db.org/) to map protein associations. Statistical significance for enriched terms, pathways, and interactions was defined as p < 0.05.

Statistical analysis

Data were presented as the mean ± standard error of the mean (SEM). Significant differences between groups were determined by paired t-test, using SPSS software version 25 (SPSS, Inc.). A p-value of less than 0.05 was considered statistically significant.

Results

Characterization of PL-MSCs

Mesenchymal stem cells derived from the human placenta exhibited spindle-shaped morphology (Fig. 1A) expressed typical MSC surface markers at very high percentages, CD73 (99.04 ± 1.20%), CD90 (98.71 ± 0.52%), and CD105 (99.44 ± 0.50%), and did not express hematopoietic stem cell surface markers CD34, CD45 and HLA-DR (Fig. 1B). These PL-MSCs also have trilineage differentiation capacity toward osteocytes (Fig. 1C), adipocytes (Fig. 1D) and chondrocytes (Fig. 1E) after being induced by appropriate culture conditions, as demonstrated by Alizarin red S staining, Oil-red O staining and Alcian blue staining, respectively.

Fig. 1.

Characteristics of MSCs derived from human placentas (PL-MSCs). A Morphology of PL-MSCs cultured in DMEM supplemented with 10% FBS at day 2 after initial seeding, passage 0, passage 3, and passage 10. Cells retained a spindle-shaped morphology across passages, with no signs of replicative senescence. B Flow cytometry confirmed that PL-MSCs were positive for MSC surface markers (CD73, CD90, CD105) and negative for hematopoietic lineage markers (CD34, CD45, HLA-DR). C Osteogenic differentiation demonstrated by Alizarin Red S staining (orange-red calcium deposits) after 28 days of induction. D Adipogenic differentiation revealed by Oil Red O staining (red lipid droplets) in cytoplasmic vacuoles. E Chondrogenic differentiation is shown by Alcian Blue staining (blue proteoglycan-rich matrix). n = 5, Images (A), (C), and (E) were taken at 10X, 10X, and 20X, respectively; scale bar = 100 μm. Image D was taken at 40X; scale bar = 50 μm

Cytotoxic effect of fucoxanthin on PL-MSCs

We first determined the cytotoxic effects of fucoxanthin on PL-MSCs using the CCK-8 assay. Treatment of PL-MSCs with fucoxanthin at concentrations of up to 5 µM increased their viability at 24 and 48 h compared to untreated control, while 10 and 20 µM fucoxanthin significantly decreased the viability of PL-MSCs at 24 and 48 h compared to untreated control (Fig. 2A). The half-maximal inhibitory concentration (IC₅₀) of fucoxanthin in PL-MSCs was 14.39 ± 4.0 µM after 24 h and 7.81 ± 0.2 µM after 48 h of treatment. Therefore, a fucoxanthin concentration up to 5 µM was chosen for subsequent experiments to avoid any cytotoxic effects.

Fig. 2.

Cytotoxicity of fucoxanthin and H2O2 on PL-MSCs. (A) The CCK-8 assay showed the viability of PL-MSCs treated with increasing concentrations of fucoxanthin (1, 2, 3, 4, 5, 10, and 20 μM) for 24 and 48 h. (B) The viability of PL-MSCs treated with H₂O₂ (ranging from 50-1500 μM) for 24 h was evaluated using the CCK-8 assay. (C) The viability of PL-MSCs after 24 h of treatment with 750 μM H₂O₂ and fucoxanthin (1-5 μM). (D) The viability of PL-MSCs after 48 h of treatment with 750 μM H2O2 and fucoxanthin (1-5 μM). (E) Flow cytometry scatter plots categorizing live (Annexin V⁻/DRAQ7^TM−, Q4), early apoptotic (Annexin V⁺/DRAQ7^TM−, Q3), and late apoptotic (Annexin V⁺/DRAQ7^TM+, Q2) in PL-MSCs following 24-h treatment with 750 μM H₂O₂ and fucoxanthin (1–5 μM). (F) Quantification of early and late apoptotic PL-MSCs under H₂O₂ and fucoxanthin treatment. Data are presented as mean ± SEM (n = 4). Statistical significance was assessed using the paired t-test. *p ≤ 0.05 compared to the control (PL-MSCs cultured in complete DMEM), while #p ≤ 0.05 compared to H₂O₂-treated PL-MSCs without fucoxanthin.

Fucoxanthin decreases PL-MSC apoptosis under oxidative stress conditions

To establish an in vitro model of oxidative stress, the PL-MSCs were treated with H₂O₂ at concentrations ranging from 50 to 1500 µM for 24 h. The results of the CCK-8 assay showed that H₂O₂ decreased the viability of PL-MSCs in a dose-dependent manner (Fig. 2B) and H₂O₂ at a concentration of 750 µM, which reduces the viability of PL-MSCs to 75%, was chosen for subsequent experiments.

Treatment with 750 µM H₂O₂ for 24 h significantly reduced PL-MSC viability compared to untreated controls. However, treatment with increasing fucoxanthin concentrations (1–5 µM) progressively restored viability, with 2–5 µM fucoxanthin showing statistically significant recovery (p ≤ 0.05). Remarkably, 5 µM fucoxanthin fully restored viability to control levels (101.82% ± 3.71) (Fig. 2C). Similarly, after 48 h of H₂O₂ exposure, viability was significantly reduced but improved dose-dependently with fucoxanthin. A significant increase (p ≤ 0.05) occurred at 3–5 µM fucoxanthin, achieving 82.09% ± 4.47 viability relative to controls (Fig. 2D). Antioxidant effects of fucoxanthin were further confirmed by reduced intracellular ROS levels in H₂O₂-treated PL-MSCs, as measured by DCFH-DA fluorescence (Supplementary Fig. S1).

To determine the cytoprotective effects of fucoxanthin against oxidative stress-induced apoptosis in PL-MSC, PL-MSCs were co-treated with 750 µM H₂O₂ and 1–5 µM fucoxanthin for 24 h. The percentage of apoptosis was determined by the Annexin V-FITC and DRAQ7™ apoptosis assay. As expected, H₂O₂ treatment significantly increased the percentage of total apoptotic populations, early apoptotic (Annexin V⁺/ DRAQ7⁻), and late apoptotic (Annexin V⁺/ DRAQ7⁺) in PL-MSCs compared to the untreated control (Fig. 2E-F).

Fucoxanthin treatment significantly decreased PL-MSC apoptosis under oxidative stress conditions in a dose-dependent manner compared to control (PL-MSCs treated with 750 µM H₂O₂ without fucoxanthin supplementation) (Fig. 2E-F). Furthermore, 5 µM fucoxanthin treatment reduced PL-MSC apoptosis under the oxidative stress conditions compared to control (PL-MSCs without H₂O₂ treatment) (Fig. 2E-F).

Fucoxanthin decreased the expression of pro-apoptotic genes and increased the expression of anti-apoptotic genes in PL-MSCs under oxidative stress conditions

To determine the effects of fucoxanthin on the expression levels of genes related to apoptosis in PL-MSCs under oxidative stress conditions, PL-MSCs were co-treated with 750 µM H₂O₂ and 1–5 µM fucoxanthin for 24 h. Following treatments, expression levels of pro- and anti-apoptotic genes in PL-MSCs were determined by qRT-PCR. Consistent with its effect on PL-MSC apoptosis, H₂O₂ treatment significantly increased the expression levels of many pro-apoptotic genes in PL-MSCs including Caspase 3 (CASP3), Caspase 8 (CASP8), Apoptotic protease activating factor 1 (APAF-1), Cytochrome c (CYC C), BH3 interacting-domain death agonist (BID), BCL-2 associated agonist of cell death (BAD), BCL-2–associated X (BAX), Fas-associated death domain (FADD), Tumor necrosis factor receptor superfamily member 10 A (TNFRSF10A), and Tumor necrosis factor receptor superfamily member 10B (TNFRSF10B), while decreasing the expression levels of anti-apoptotic genes, B-cell lymphoma 2 (BCL-2) and Survivin (BIRC5) compared to the untreated controls (Fig. 3).

Fig. 3.

Effects of fucoxanthin on apoptotic gene expression in H₂O₂-treated PL-MSCs. Quantitative real-time RT-PCR analysis revealed the downregulation in the expression levels of pro-apoptotic genes, including CASP3 (A), CASP8 (B), APAF-1 (C), CYC C (D), BID (E), BAD (F), BAX (G), FADD (H), TNFRSF10A (I), and TNFRSF10B (J) in PL-MSCs after treatment with 750 µM H₂O₂ and fucoxanthin at 1–5 µM for 24 h. Upregulation in the expression levels of anti-apoptotic genes, BCL-2 (K), and BIRC5 (L) was detected under the same treatment conditions. Data are presented as mean ± SEM, with n = 3. Statistical significance was evaluated using a paired t-test, with ^#p ≤ 0.05 indicating significance compared to PL-MSCs cultured in complete DMEM and *p ≤ 0.05 indicating significance compared to H₂O₂-treated PL-MSCs without fucoxanthin

To ascertain the mechanistic basis of the positive effects of fucoxanthin on the survival of PL-MSCs, its effects on these key regulatory pro- and anti-apoptotic genes were also ascertained following H₂O₂ treatment. Fucoxanthin significantly decreased the expression levels of pro-apoptotic genes, CASP3, CASP8, APAF-1, CYC C, BID, BAD, BAX, FADD, TNFRSF10A and TNFRSF10B (Fig. 3A-J), while increasing the expression levels of anti-apoptotic genes, BCL-2 and BIRC5 (Fig. 3K-L) in PL-MSC compared to controls (PL-MSCs treated with 750 µM H₂O₂ without fucoxanthin supplementation) in a dose-dependent manner. These findings suggest that fucoxanthin reduces PL-MSC apoptosis under oxidative stress conditions by decreasing the expression of several pro-apoptotic genes and increasing the expression of anti-apoptotic genes.

Fucoxanthin decreases the expression and inhibits the activation of several proteins related to extrinsic and intrinsic apoptotic pathways in PL-MSCs under oxidative stress conditions

The levels of proteins involved in the extrinsic and intrinsic apoptotic pathways in PL-MSCs treated with 750 µM H₂O₂ with or without fucoxanthin supplementation were determined by Western blot analysis. Similar to its effect on pro-apoptotic gene expression, H₂O₂ treatment activated an extrinsic apoptotic pathway in PL-MSCs, as demonstrated by an increase in ratios of phosphorylated Fas-associated death domain (p-FADD) / total Fas-associated death domain (FADD) protein, and cleaved caspase 8/total caspase 8 protein, compared to untreated control.

Consistent with its positive effect on PL-MSC survival, fucoxanthin prevented the activation of the extrinsic apoptotic pathway in PL-MSCs under oxidative stress conditions by significantly decreasing p-FADD/total FADD, and cleaved caspase 8/total caspase 8 protein ratios in H₂O₂-treated PL-MSCs compared to controls (PL-MSCs treated with 750 µM H₂O₂) in a dose-dependent manner (Fig. 4A, C-E; Supplementary Fig. S2-S4). In addition to its inhibitory effect on the extrinsic apoptotic pathway, fucoxanthin also inhibited the activation of the intrinsic apoptotic pathway in PL-MSCs under oxidative stress conditions by significantly decreasing the cleaved caspase 9/total caspase 9 protein ratio in a dose-dependent manner (Fig. 4B, F; Supplementary Fig. S5) (Table 1).

Fig. 4.

Effects of fucoxanthin on proteins related to the extrinsic and intrinsic apoptotic pathway in H₂O₂-treated PL-MSCs. A Western blot analysis demonstrated DR4, p-FADD/FADD, and cleaved caspase 8/caspase 8 expression levels. B Western blot analysis demonstrated the expression levels of cleaved caspase 9/caspase 9, cleaved caspase-3/caspase 3, cleaved caspase 7/caspase 7, and cleaved PARP/PARP. The original blots are shown in Supplementary figures S2-S8. C–I The relative protein expression levels of DR4, p-FADD/FADD, cleaved caspase 8/caspase 8, cleaved caspase 9/caspase 9, cleaved caspase 3/caspase 3, cleaved caspase 7/caspase 7, and cleaved PARP/PARP in PL-MSCs after treatment with 750 µM H₂O₂ and fucoxanthin for 24 h. Data are presented as mean ± SEM, n = 3. A statistical significance of ^#p ≤ 0.01 indicates comparisons with control cultures of PL-MSCs in complete DMEM, while *p ≤ 0.05 indicates comparisons with H₂O₂-treated PL-MSCs without fucoxanthin.

Table 1.

List of primers used for qRT-PCR

Gene	Forward primer	Reserve primer	Product size (bp)
CASP3	5’-GAGGCGGTTGTGGAAGTTAA-3’	5’-GGATGCCGTCTAGAGTCCTA-3’	252
CASP8	5’-GTGCCCAAATCAACAAGAGC-3’	5’-ATCAGACAGTATCCCCGAGG-3’	222
APAF-1	5’-CTTCTTCCAGTGTAAGGACAGT-3’	5’-CAGCCTGCCATTCCATGTAT-3’	157
CYC C	5’-GGGTGATGTTGAGAAAGGCA-3’	5’-TTGTTCTTATTGGCGGCTGT-3’	164
BID	5’-CTTCTTCCAGTGTAAGGACAGT-3’	5’-CAGCCTGCCATTCCATGTAT-3’	116
BAD	5’-TGAGTGACGAGTTTGTGGAC-3’	5’-GTGGAGTTTCGGGATGTGG-3’	184
BAX	5’-CAGGGTTTCATCCAGGATCG-3’	5’-AGAAAACATGTCAGCTGCCA-3’	157
FADD	5’-ACACCAAGATCGACAGCATC-3’	5’-ATGCGTCTGAGTTCCATGAC-3’	240
TNFRSF10A	5’-TCAGCTGCAACCATCAAACT-3’	5’-ACTCTCCCAAAGGGCTATGT-3’	73
TNFRSF10B	5’-TTGAGGACCACTTGTTGAGC-3’	5’-CTTCCAGTACCGGTCATGTG-3’	186
BCL-2	5’-GGATTCCTGCGGATTGACAT-3’	5’-GGCAACGATCCCATCAATCT-3’	156
BIRC5	5’-GCCATTAACCGCCAGATTTG-3’	5’-AGAAGAAACACTGGGCCAAG-3’	89
GAPDH	5’-CAATGACCCCTTCATTGACC-3’	5’-TTGATTTTGGAGGGATCTCG-3’	158

Consistent with these results, fucoxanthin also inhibited the activation of several executioner caspases in PL-MSCs under oxidative stress conditions, as demonstrated by a significant decrease in the ratios of cleaved caspase 3/total caspase 3, cleaved caspase 7/total caspase 7, and cleaved PARP/total PRAP proteins in H₂O₂-treated PL-MSCs compared to control (PL-MSCs treated with 750 µM H₂O₂) in a dose-dependent manner (Fig. 4B, G-I, Supplementary Fig. S6-S8). These findings suggest that fucoxanthin reduces PL-MSC apoptosis under oxidative stress conditions by decreasing the activation of proteins related to extrinsic and intrinsic apoptotic pathways.

Fucoxanthin increases PL-MSC viability under oxidative stress conditions by modulating the expression of genes involved in cell cycle regulation, DNA damage repair, ER stress response, P53-induced apoptosis

To study the effect of fucoxanthin on the global transcriptome of PL-MSCs under oxidative stress conditions, NanoString nCounter analysis was performed using the metabolic pathway panel to identify differentially expressed genes (DEGs). The results showed that fucoxanthin downregulated 23 genes mostly associated with apoptosis, unfolded protein response, the PI3K/Akt pathway and reactive oxygen response in the H₂O₂-treated PL-MSCs compared to controls (PL-MSCs treated with 750 µM H₂O₂) (Fig. 5A, B). Key downregulated genes included UPP1 (fold change [FC] = − 1.59, p = 0.002), PNP (FC = − 1.44, p = 0.029), and ERN1 (FC = − 1.39, p = 0.000), which are associated with ROS management and apoptosis (Fig. 5A, D, E; Table 2, Supplementary Tables 1 and 2). Conversely, 74 genes were upregulated, primarily involved in DNA repair and survival pathways. The top upregulated targets, such as BUB1 (FC = 3.10, p = 0.000), EME1 (FC = 2.99, p = 0.001), and BUB1B (FC = 2.81, p = 0.000), highlighted enhanced DNA damage repair and cell cycle regulation (Fig. 5A, D, E; Table 2 Supplementary Tables 1 and 2). These transcriptomic shifts suggest fucoxanthin mitigates oxidative stress by suppressing stress-response pathways while promoting genomic stability and survival.

Fig. 5.

NanoString analysis of differential gene expressions related to apoptosis and its associated pathways in PL-MSCs subjected to oxidative stress. A A heatmap displays the differentially expressed genes (DEGs) in PL-MSCs treated with 750 µM H₂O₂ and PL-MSCs treated with 750 µM H₂O₂ and 5 µM fucoxanthin. B Volcano plots highlight individual differentially expressed genes. The X-axis represents -log10 (p-value) while the Y-axis represents the log2 fold change between each pair of treatment groups. C A Venn diagram of DEGs shows the intersection of up-regulated and down-regulated genes in PL-MSCs treated with 750 µM H₂O₂ and 5 µM fucoxanthin, compared to those treated with 750 µM H₂O₂. D A bar plot representative of genes in PL-MSCs treated with 750 µM H₂O₂ and 5 µM fucoxanthin compared to those treated with 750 µM H₂O₂. The Y-axis represents the number of genes, while the X-axis represents the signaling pathway/function involved with apoptosis and its associated pathways. E Fold change and statistical significance (p-value) in gene expression of fucoxanthin-treated PL-MSCs compared to untreated PL-MSCs under oxidative stress.

Table 2.

Fold change of gene expression in fucoxanthin-treated PL-MSCs compared to untreated PL-MSCs under oxidative stress conditions

Upregulated Genes	Function/Pathway	Fold change	P value
BUB1	BUB1 mitotic checkpoint serine/threonine kinase	3.10	0.000
EME1	Essential meiotic structure-specific endonuclease 1	2.99	0.001
BUB1B	BUB1 Mitotic Checkpoint Serine/Threonine Kinase B	2.81	0.000
PCLAF	PCNA Clamp Associated Factor	2.56	0.000
BRCA2	BRCA2 DNA Repair Associated	2.43	0.003
RAD51AP1	RAD51 Associated Protein 1	2.29	0.000
RAD51	DNA repair protein RAD51 homolog 1	2.27	0.006
BRIP1	BRCA1 Interacting DNA Helicase 1	2.20	0.000
EXO1	Exonuclease 1	2.11	0.009
XRCC2	X-Ray Repair Cross-Complementing 2	1.98	0.000

Downregulated Genes	Function/Pathway	Fold change	P value
UPP1	Uridine phosphorylase 1	-1.59	0.002
PNP	Purine Nucleoside Phosphorylase	-1.44	0.029
ERN1	Endoplasmic reticulum to nucleus signaling 1	-1.39	0.000
FDXR	Ferredoxin Reductase	-1.33	0.009
PSAT1	Phosphoserine Aminotransferase 1	-1.07	0.046
ASNS	Asparagine Synthetase	-1.05	0.043
SLC3A2	Solute carrier family 3 member 2	-1.00	0.010
SQSTM1	Sequestosome 1	-0.95	0.000
TIGAR	TP53-inducible glycolysis and apoptosis regulator	-0.90	0.006
LY96	Lymphocyte Antigen 96	-0.86	0.015

The Venn diagram revealed distinct transcriptional responses between PL-MSCs subjected to oxidative stress (H₂O₂ treatment) and those co-treated with H₂O₂ and fucoxanthin. H₂O₂ treatment alone resulted in a downregulation of 75 genes associated with cell cycle progression and DNA repair pathways. In contrast, co-treatment with H₂O₂ and fucoxanthin led to an upregulation of 74 genes related to survival and DNA repair pathways. In particular, a significant overlap of genes whose expression was significantly altered between these two groups (59 genes, 65.6%) was observed. This result suggests that fucoxanthin reverses H₂O₂-induced suppression of critical pro-survival genes. In contrast to pro-survival genes, H₂O₂ upregulated 25 genes related to the stress response and the pro-apoptotic pathway. In which 14 of these genes (41.2%) were downregulated in PL-MSCs co-treated with H₂O₂ and fucoxanthin, indicated that fucoxanthin reverses H₂O₂-induced activation of stress response and pro-apoptotic pathway (Fig. 5C). Taken together, these findings suggest that fucoxanthin reduces oxidative damage by modulating the expression of several pro-survival, stress response and pro-apoptotic genes in PL-MSCs under oxidative stress conditions.

Gene Ontology (GO) analysis was performed to determine the biological functions of DEGs between H₂O₂-treated PL-MSCs and untreated control (Fig. 6A) and between PL-MSCs treated with H₂O₂ together with fucoxanthin and PL-MSCs treated with H₂O₂ alone (Fig. 6B). The results show that H₂O₂ treatment downregulated the expression of genes involved in the cell cycle regulation, DNA metabolism, DNA damage repair, and chromosomal organization, while upregulating the expression of genes involved in the oxidative stress response and apoptosis in PL-MSCs (Fig. 6A).

Fig. 6.

The Gene Ontology (GO) enrichment analysis for the top 10 ranked GO enrichments across three functional classifications: Biological Process, Cellular Component, and Molecular Function, based on the DEGs. A The GO term was significantly associated with DEGs in PL-MSCs treated with 750 µM H₂O₂, compared to the untreated control. B The GO term for PL-MSCs treated with both 750 µM H₂O₂ and 5 µM fucoxanthin, relative to the baseline of H₂O₂-treated PL-MSCs. The X-axis represents -Log(p-value) of GO enrichment, which indicates the number of genes involved. The Y-axis represents the GO terms. Significantly upregulated DEGs are shown in orange, while significantly downregulated DEGs are shown in blue

Consistent with its survival-promoting effect on PL-MSCs under oxidative stress conditions, fucoxanthin increases the expression levels of genes involved in the cell cycle regulation, DNA damage repair, cytokine signaling, nucleotide synthesis, PI3K/mTOR pathway and AMPK pathways in PL-MSCs that were initially down-regulated by the H₂O₂ treatment, while downregulating the expression levels of genes involved in apoptosis, and ER stress response (Fig. 6B).

Consistent with the GO analysis, the analyses of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway also showed that H₂O₂ treatment significantly downregulated the expression levels of genes involved in the regulation of the cell cycle, DNA damage repair, cytokine signaling, nucleotide synthesis, PI3K/mTOR pathway and AMPK pathways in PL-MSCs (Fig. 7A) and that treatment with fucoxanthin increased the expression levels of genes involved in the regulation of the cell cycle, DNA damage repair, cytokine signaling, nucleotide synthesis, PI3K/mTOR pathway and AMPK pathways in PL-MSCs that were initially downregulated by H₂O₂ treatment (Fig. 7B).

Fig. 7.

The KEGG analysis of PL-MSCs under oxidative stress conditions was conducted using the Shiny GO web application. A KEGG term for significant DEGs in PL-MSCs treated with 750 µM H₂O₂ compared to the untreated control. B KEGG term in PL-MSCs treated with 750 µM H₂O₂ and 5 µM fucoxanthin, compared to the baseline H₂O₂-treated PL-MSCs. The X-axis represents the number of genes, while the Y-axis represents the KEGG pathway term. Significantly upregulated DEGs are shown in blue, while downregulated DEGs are displayed in orange

The PPI network analysis further showed that the top 20 downregulated DEGs in H₂O₂-treated PL-MSCs have 139 interaction partners, which were enriched in pathways related to DNA replication initiation, PI3K/Akt and DNA damage repair (Fig. 8A), while the top 20 upregulated DEGs in H₂O₂-treated PL-MSCs have 68 interaction partners enriched in oxygen stress response and apoptosis pathways (Fig. 8B). Consistent its positive effect on PL-MSC survival, fucoxanthin upregulated genes enriched in pathways related to DNA replication initiation, PI3K/Akt activation and DNA damage repair (Fig. 8C) and downregulated genes enriched in pathways related to ROS detoxification and apoptosis (Fig. 8D). The transcription factor (TF) analysis showed that the p53 (TP53, p < 0.0001), E2F transcription factor 1 (E2F1, p < 0.0001), and E2F transcription factor 4 (E2F4, p < 0.0001) were significantly associated with oxidative stress responses in PL-MSCs (Fig. 8E–F). These TFs, whose expressions were enriched in both (1) H₂O₂-treated PL-MSCs compared to untreated controls and (2) H₂O₂ and fucoxanthin-treated PL-MSCs compared to the H₂O₂ only group, play an important role in the regulation of cell cycle progression, DNA replication, and cell growth (Table 3). The consistent prominence of tumor suppressor TP53 and cell cycle regulators, E2F1/E2F4, underscores their important role in balancing stress adaptation and survival pathways in PL-MSCs under oxidative stress conditions.

Fig. 8.

Protein-protein interaction (PPI) networks were analyzed using STRING to identify functional clusters of differentially expressed genes (DEGs). A Upregulated protein cluster in PL-MSCs treated with 750 µM H₂O₂ vs. untreated controls, linked to oxidative stress adaptation. B Downregulated protein cluster in H₂O₂-treated PL-MSCs vs. controls, associated with suppressed metabolic pathways. C Upregulated protein cluster in H₂O₂ and 5 µM fucoxanthin-treated PL-MSCs vs. H₂O₂-only, enriched in DNA repair and survival pathways. D Downregulated protein cluster in H₂O₂ and fucoxanthin vs. H₂O₂-only, highlighting reduced apoptosis signaling. E Predicted transcription factors (TFs) for DEGs in H₂O₂-treated PL-MSCs vs. controls, dominated by stress-responsive regulators (e.g., TP53, E2F1). F TF network for DEGs in H₂O₂ and fucoxanthin vs. H₂O₂-only, revealing fucoxanthin-driven modulation of cell cycle and survival TFs

Table 3.

Top 10 predicted key transcription factors (TFs) in PL-MSCs treated with 750 µM H₂O₂ compared to those treated with 750 µM H₂O₂ and 5 µM Fucoxanthin

Key TFs	Role/Associated Pathway	P value	FDR
PL-MSCs treated with 750 µM H2O2 vs. untreated control
TP53	Tumor protein p53	1.82e-13	9.66e-12
E2F1	E2F transcription factor 1	1.25e-10	3.32e-09
JUN	Jun proto-oncogene	9.61e-08	1.63e-06
TFDP1	Transcription factor Dp-1	1.52e-07	1.63e-06
ESR1	Estrogen receptor 1	1.53e-07	1.63e-06
CREBBP	CREB binding protein	1.84e-07	1.63e-06
MYC	v-myc myelocytomatosis viral oncogene homolog	1.01e-06	7.67e-06
SP1	Sp1 transcription factor	1.23e-06	8.18e-06
STAT1	Signal transducer and activator of transcription 1, 91 kDa	5.53e-06	3.22e-05
E2F4	E2F transcription factor 4, p107/p130-binding	6.08e-06	3.22e-05
PL-MSCs treated with 750 µM H2O2 and 5 µM fucoxanthin vs. H2O2-treated PL-MSCs
TP53	Tumor protein p53	2.08e-14	7.48e-13
E2F1	E2F transcription factor 1	2.35e-11	4.24e-10
E2F4	E2F transcription factor 4, p107/p130-binding	3.33e-06	4e-05
CREBBP	CREB binding protein	4.73e-06	4.25e-05
MYC	v-myc myelocytomatosis viral oncogene homolog	6.34e-06	4.57e-05
TFDP1	Transcription factor Dp-1	1.07e-05	6.43e-05
MEN1	Multiple endocrine neoplasia I	1.47e-05	7.55e-05
SP1	Sp1 transcription factor	5.6e-05	0.000252
EP300	E1A binding protein p300	0.000123	0.000493
CHD8	Chromodomain helicase DNA-binding protein 8	0.000204	0.000611

Taken together, these results suggest that fucoxanthin increases PL-MSC viability under oxidative stress conditions by modulating the expression of various genes involved in cell cycle regulation, DNA damage repair, ER stress response, p53-induced apoptosis.

Discussion

Human mesenchymal stem cells hold significant promise in regenerative medicine due to their ability to home in specific tissues and release various beneficial factors to reduce inflammation and promote tissue repair [20–22]. Although BM-MSCs are the most extensively studied [23, 24], several limitations hinder their clinical uses, especially limited availability, invasive harvesting procedures and a decrease in proliferation and differentiation capacity after culture expansion [25, 26]. Therefore, alternative sources of MSC, such as PL-MSCs, which have greater expansion potential than BM-MSCs and are easier to obtain by non-invasive procedures [27–29], are considered more suitable for research and clinical use [30, 31]. Consistent with previous reports, the characteristics of PL-MSCs established in this study met all the criteria for defining mesenchymal stromal cells recommended by the International Society for Cell & Gene Therapy [32], including adherence, expression of MSC surface markers, and trilineage differentiation capacity.

Regardless of the source of MSCs, previous studies showed that the viability of MSCs at the transplanted sites is compromised by oxidative stress and hypoxic microenvironment in targeted tissues [5, 33, 34]. Therefore, it is essential to develop strategies to increase MSC survival under oxidative stress conditions to improve their therapeutic potential for regenerative medicine applications.

The unique chemical structure of fucoxanthin, including its allenic bond and oxygen-rich functional groups (e.g., epoxides, hydroxyl groups), enables potent free radical scavenging, directly neutralizing ROS generated under oxidative stress [35]. This aligns with our data showing reduced intracellular ROS levels in H₂O₂-treated PL-MSCs co-treated with fucoxanthin. Recent studies showed that co-treatment and pre-treatment with the algal carotenoid fucoxanthin promotes cell survival under oxidative stress by reducing intracellular ROS and increasing the expression and activity of antioxidant enzymes in human hepatic cell lines [36], human retinal epithelial cells [12, 13], and human BM-MSCs [37]. Consistent with this, our previous study showed that fucoxanthin also increases the viability of PL-MSCs under oxidative stress conditions by increasing the activity of the antioxidant enzymes SOD and GSH, reducing intracellular ROS production, and activating PI3K/Akt/Nrf-2 signaling pathways [15]. Moreover, high‑throughput analysis revealed a dual role of fucoxanthin, which upregulates the survival pathway and downregulates apoptosis. Enhanced expression of DNA repair (BUB1, RAD51) and cell cycle genes (cyclin D1) promotes proliferation and genomic stability. Suppression of stress-response genes (UPP1, ERN1) reduces programmed cell death [15]. By concurrently scavenging ROS and activating repair/survival pathways, fucoxanthin creates a protective feedback loop. This dual action prevents oxidative damage from overwhelming cellular defenses, as evidenced by full viability restoration at 24 h and partial recovery at 48 h. Therefore, fucoxanthin enhances MSC viability through ROS neutralization, antioxidant pathway activation, and transcriptomic shifts favoring survival over apoptosis.

In this study, we focused on 24-h and 48-h time points to assess acute responses to oxidative stress and fucoxanthin intervention. The selection was guided by their clinical and mechanistic relevance to MSC therapies. In clinical settings, such as post-transplantation ischemia-reperfusion injury, MSCs encounter transient oxidative stress within these acute windows, making our experimental timeline reflective of real-world therapeutic scenarios. Furthermore, these timeframes provide mechanistic clarity: the 24-h period captures early apoptotic signaling and immediate stress responses, while the 48-h interval reveals cumulative damage and progressive repair processes, offering a holistic view of the cytoprotective effects of fucoxanthin. Our findings demonstrate that fucoxanthin restores PL-MSC viability in a time- and dose-dependent manner. At 24 h, 5 µM fucoxanthin fully rescued viability (101.82% ± 3.71), aligning with its potent antioxidant and anti-apoptotic properties observed in prior studies. By contrast, viability restoration at 48 h was partial (82.09% ± 4.47), suggesting that prolonged oxidative stress overwhelms reparative mechanisms despite treatment with fucoxanthin. This diminishing efficacy under chronic stress underscores the importance of early therapeutic administration in clinical contexts. The differential recovery rates may stem from the dual role of fucoxanthin, scavenging reactive oxygen species (ROS) to mitigate acute damage while simultaneously activating survival pathways like PI3K/Akt/Nrf-2, which may lose efficacy as oxidative insults persist [15].

While this study provides critical insights into the acute protective effects of fucoxanthin, longer-term observations (e.g., 72–96 h) were omitted to avoid confounding factors such as nutrient depletion or spontaneous differentiation in monolayer cultures. Future work employing 3D culture systems or in vivo models could better replicate the dynamic microenvironments MSCs encounter post-transplantation, enabling assessment of chronic stress adaptation and functional integration. Additionally, coupling viability assays with real-time metabolic profiling could clarify how fucoxanthin modulates energy utilization under oxidative duress. These findings position fucoxanthin as a promising adjuvant for enhancing MSC resilience in oxidative environments, particularly in acute settings. By bridging mechanistic insights with clinically relevant timeframes, this work lays a foundation for optimizing MSC-based therapies in regenerative medicine.

Additionally, we showed that fucoxanthin mitigated PL-MSC apoptosis under oxidative stress conditions by decreasing the expression and activation of pro-apoptotic proteins, including FADD, caspase 8, caspase 9, caspase 7, caspase 3, PARP, Bid, Bad, and Bax, while increasing the expression of anti-apoptotic proteins Bcl-2 and Survivin (Fig. 9). The results are consistent with previous studies showing that fucoxanthin down-regulated pro-apoptotic proteins Bax, caspase 9, caspase 3, and p53, and up-regulated anti-apoptotic proteins, Bcl-2, in human keratinocytes [10], human renal proximal tubular epithelial HK-2 cells [38] and human BM-MSCs [37]. Furthermore, fucoxanthin reduced ER stress and prevented amyloid β protein fragment-induced apoptosis in rat glial cells by reducing the levels of proteins associated with ER stress and apoptosis, such as CCAAT/enhancer-binding protein homologous protein (CHOP), caspase 12, caspase 3 and Bax [39].

Fig. 9.

A schematic diagram illustrates how fucoxanthin mitigates oxidative stress and enhances PL-MSC survival

In addition to its inhibitory effect on pro-apoptotic genes and proteins, our results showed that fucoxanthin also increased the expression levels of genes involved in the cell cycle regulation, DNA damage repair, cytokine signaling, nucleotide synthesis, PI3K/mTOR pathway and AMPK pathways in PL-MSCs that were down-regulated under oxidative stress condition. Furthermore, fucoxanthin also reduced the expression levels of genes involved in apoptosis, and the ER stress response in these PL-MSCs. This is consistent with previous studies showing that fucoxanthin increases cell viability by protecting cells against oxidative damage and decreasing the expression of genes associated with ER stress and apoptosis, including ERN1 (also known as IRE1), ATF4, HSF1 and HSPA4 [40–42]. In addition, our results suggest that fucoxanthin decreases PL-MSC apoptosis under oxidative stress conditions by downregulating genes, such as UPP1, SQSTM1, CTSL and PRKAB1, as shown through NanoString pathway analysis, which is consistent with previous studies showing that these genes can trigger apoptosis under certain circumstances through the activation of caspase 8 and the depletion of endogenous apoptosis inhibitors [43–45].

The Venn diagram provided valuable information on the effect of fucoxanthin in reducing oxidative stress-induced transcriptional dysregulation in PL-MSCs. The substantial overlap of 59 genes (65.6%) whose expression was downregulated by H₂O₂ treatment but upregulated by co-treatment of H₂O₂ and fucoxanthin suggests that fucoxanthin effectively restores the expression of genes involved in cellular homeostasis, antioxidant, and DNA repair processes. This effect aligns with the unique chemical structure of fucoxanthin, characterized by its abundant allenic C-H bonds and oxygen-containing groups, which facilitates the ROS scavenging and the modulation of redox signaling pathways. Furthermore, the partial suppression of H₂O₂-induced upregulation of stress-response genes (14 genes, 41.2%) in the co-treatment group suggests that fucoxanthin also reverses H₂O₂-induced activation of stress response and pro-apoptotic pathway. This finding is consistent with the fucoxanthin capacity to activate pro-survival signaling pathways, such as the PI3K/Akt/Nrf-2 axis observed in previous research [15].

Our findings are also consistent with previous studies that demonstrate the cytoprotective effects of fucoxanthin on hepatic and retinal cells by increasing the activity of antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH), and reducing stress-induced apoptosis in these cells [12, 13, 36]. The increased expression levels of BUB1, RAD51, and other DNA repair genes further support the role of fucoxanthin in promoting DNA repair and genomic stability under oxidative stress. This is crucial to improve the viability of MSCs during therapeutic applications [38]. However, it is essential to note that the restoration of H₂O₂-induced gene dysregulation, such as those related to apoptotic signaling, by fucoxanthin treatment is incomplete. This underscores the complexity of oxidative damage that might require a combination treatment to further enhance the viability of MSCs under severe oxidative stress conditions.

The results suggest that the TP53 and E2F1/2 networks could corroborate the ability of fucoxanthin to activate the PI3K/Akt/Nrf-2 pathways, enhance DNA repair by upregulating the expression of BUB1 and RAD51 genes, and suppress the expression of the pro-apoptotic CASP8 gene, to increase PL-MSC viability by balancing stress adaptation and survival. However, future research is required to investigate the long-term effect of fucoxanthin on MSC properties, using an in vivo model to confirm its therapeutic efficacy. Furthermore, additional proteomic and metabolomic analyzes should be performed to further elucidate the molecular mechanisms that contribute to the cytoprotective effects of fucoxanthin.

Conclusions

This study provides compelling evidence that fucoxanthin protects PL-MSCs against oxidative stress-induced apoptosis by modulating the expression of many genes involved in cell cycle regulation, DNA damage repair, ER stress response, p53-induced apoptosis in these cells (Fig. 9). We believe that fucoxanthin could be used in combination with other agents to increase the therapeutic potential of MSCs by improving their viability in the oxidative stress microenvironment of the target tissues.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

APAF-1

Apoptotic protease activating factor 1

BAD

BCL-2 associated agonist of cell death

BAX

BCL-2–associated X

BCL-2

B-cell lymphoma 2

BID

BH3 interacting-domain death agonist

BIRC5

Survivin

BM-MSCs

Bone marrow-derived mesenchymal stem cells

CASP3

Caspase 3

CASP8

Caspase 8

cDNA

Complementary deoxyribonucleic acid

CHOP

CCAAT/enhancer-binding protein homologous protein

CO₂

Carbon dioxide

CYC C

Cytochrome c

DEGs

Differential expression of genes

DMEM

Dulbecco’s modified Eagle’s medium

DNA

Deoxyribonucleic acid

ECL

Enhanced chemiluminescence

FADD

Fas-associated death domain

FBS

Fetal bovine serum

FITC

Fluorescein isothiocyanate

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

GO

Gene ontology

H₂O₂

Hydrogen peroxide

HLA-DR

Human leukocyte antigen – DR isotype

hMSCs

Human mesenchymal stem cells

HRP

Horseradish peroxidase

KEGG

Kyoto Encyclopedia of Genes and Genomes

OD

Optical density

PBS

Phosphate-buffered saline

PE

Phycoerythrin

p-FADD

Phosphorylated Fas-associated death domain

PI3K

Phosphoinositide 3-kinase

PL-MSCs

Placenta-derived mesenchymal stem cells

PPI

Protein-protein interaction

qRT-PCR

Quantitative reverse transcription polymerase chain reaction

ROS

Reactive oxygen species

SEM

Standard error of the mean

tBid

Truncated BH3-interacting domain death agonist

TBST

Tris-buffered saline with Tween 20 detergent

TNFRSF10A

Tumor necrosis factor receptor superfamily member 10A

TNFRSF10B

Tumor necrosis factor receptor superfamily member 10B

Author contributions

G.S. conducted the experiments and drafted the manuscript. C.T. contributed to the data interpretation. D.G., I.M. P.K. also assisted with data interpretation and revised the manuscript. L.P. was involved in conceptualization, provided resources, supervised the work, contributed to data interpretation and revised the manuscript. S.M. contributed to conceptualization, study design, resources, funding, supervision, data analysis and interpretation, and was responsible for drafting and completing the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the Thailand Science Research and Innovation Fundamental Fund for the fiscal year 2024, Thammasat University (Grant No. TUFF 52/2567), and the Royal Golden Jubilee (RGJ) Ph.D. program (Grant No. NRCT5-RGJ63008-098) through the National Research Council of Thailand (NRCT) and the Thailand Research Fund (TRF). Additionally, support was provided by the Faculty of Medicine, Thammasat University and the Center of Excellence in Stem Cell Research and Innovation at Thammasat University. The funding body had no role in the design of this study, the collection, analysis, or interpretation of the data, nor in the writing of the manuscript.

Data availability

The data that support the findings of this study are available in the supplementary material of this article.

Declarations

Ethics approval and consent to participate

All experimental procedures were conducted under the Declaration of Helsinki and the Belmont Report. This study received approval from the Human Research Ethics Committee of Thammasat University (Medicine) under the title “Approval title: The effects of fucoxanthin on the proliferation and osteogenic differentiation of human mesenchymal stem cells.” (Approval number: 024/2021, Date of approval: February 4, 2021). All samples were obtained from donors who provided written informed consent.

Consent for publication

Not applicable.

Competing Interests

The authors declare that they have no competing interests.