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4 November 2026

27 Pages

Characterization and Evaluation of the Neuroprotective Potential of Peptides Derived from Holothuria scabra Inner Wall Proteins: An In Vitro Study

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1
Chulabhorn International College of Medicine, Thammasat University, Rangsit Campus, Pathumthani 12120, Thailand
2
Research Unit in Innovative Marine Biotechnology and Natural Bio-Resources for Sustainable Health and Wellness, Thammasat University, Pathumthani 12120, Thailand
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Department of Biochemistry and Genetic Engineering, Faculty of Medicine, Kasetsart University, Bangkok 10900, Thailand
4
Division of Health and Applied Sciences, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand
Mar. Drugs2026, 24(11), 396;https://doi.org/10.3390/md24110396
This article belongs to the Section Synthesis and Medicinal Chemistry of Marine Natural Products
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Abstract

Neurodegenerative diseases are characterized by progressive neuronal dysfunction driven by interconnected mechanisms, including oxidative stress, apoptosis, and impaired protein homeostasis. Marine-derived proteins and peptides represent promising sources of multifunctional neuroprotective agents; however, the neuroprotective potential of gastrointestinal digestion-derived peptides from Holothuria scabra remains poorly characterized. This study investigated the neuroprotective effects of a gastrointestinal digest of H. scabra inner-wall proteins (IWD), a mixture of digestion-derived peptides, in an OA-induced SH-SY5Y cell model. The original protein extract (IW) was characterized by LC–MS/MS and PANTHER, while IWD-derived peptides were profiled by LC–MS/MS and evaluated using in silico bioactivity and ADMET prediction. In vitro, IWD pretreatment significantly attenuated OA-induced neuronal injury by reducing intracellular ROS accumulation while preserving cell viability and neuronal morphology; 5 μg/mL showed the strongest protective effect. Proteomic analysis identified STMN1 as a major discriminator associated with neuronal structural adaptation, while RPS27A and RPLP2 were associated with translational regulation. HSPB1, HSP90AB1, and PARK7 emerged as additional candidate proteins related to cellular stress responses and homeostasis. Integrated proteomic and network analyses further highlighted proteins associated with ribosomal function, RNA processing, and proteostasis. These findings suggest that IWD-associated neuroprotection involves coordinated changes in neuronal structure and cellular maintenance pathways, supporting further validation of H. scabra-derived peptides as potential neuroprotective candidates.

1. Introduction

Neurodegenerative diseases are a group of progressive disorders characterized by the gradual loss of neuronal structure and function within the central nervous system. Major neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease, and amyotrophic lateral sclerosis (ALS), represent an increasing global health burden due to the rapid transition to an aging society. Despite differences in clinical manifestations, these disorders share several common pathological mechanisms, including oxidative stress, mitochondrial dysfunction, neuroinflammation, impaired proteostasis, excitotoxicity, and abnormal protein aggregation, ultimately leading to neuronal dysfunction and cell death [1].
Among these mechanisms, oxidative stress is considered a central driver of neurodegeneration. Neurons are particularly susceptible to oxidative damage because of their high metabolic demand, elevated oxygen consumption, and limited antioxidant capacity. Excessive generation of reactive oxygen species (ROS) disrupts redox homeostasis. This promotes oxidative damage to proteins, lipids, and nucleic acids, leading to mitochondrial impairment, dysregulated signaling, and neuronal apoptosis [2]. Oxidative stress also contributes to abnormal protein aggregation and defective protein clearance, which are key pathological features observed in AD and PD. Although current therapies, such as acetylcholinesterase inhibitors and NMDA receptor antagonists for AD, provide symptomatic benefits, they do not effectively prevent or reverse disease progression [3]. Therefore, the development of multi-target therapeutic strategies that protect neurons and modulate disease-related pathways remains an important unmet need.
Natural products have emerged as promising sources of neuroprotective agents because of their ability to regulate multiple biological pathways simultaneously. In particular, marine organisms represent an underexplored reservoir of bioactive compounds with diverse pharmacological activities, including antioxidant, anti-inflammatory, and neuroprotective effects. Among marine resources, sea cucumbers (class Holothuroidea) have attracted increasing attention owing to their nutritional value and rich composition of bioactive molecules [4].
Holothuria scabra (sandfish) is an economically and nutritionally important sea cucumber species and contains a high proportion of protein, accounting for approximately 40–70% of its dry weight [5,6]. This abundant protein content, together with its diverse amino acid composition, makes H. scabra a promising source of bioactive peptides. Marine-derived peptides exhibit a broad range of biological activities, including antioxidant, anti-inflammatory, antimicrobial, antihypertensive, and cytoprotective effects [7]. Increasing evidence further indicates that marine peptides may exert neuroprotective effects by attenuating oxidative stress and inflammation, modulating apoptotic signaling, and maintaining neuronal homeostasis. In addition, sea cucumber-derived oligopeptides improved spatial learning and memory in vivo and reduced lipid and protein oxidation while enhancing antioxidant enzyme activities, including SOD and GSH-Px, in vitro [8].
Despite this potential, research on H. scabra has predominantly focused on secondary metabolites, particularly triterpene glycosides, polysaccharides, and other low-molecular-weight compounds [9,10]. In contrast, the biological functions of H. scabra proteins and their derived peptides, particularly in neuronal systems, remain comparatively underexplored. Previous studies have demonstrated antioxidant, anti-inflammatory, immunomodulatory, wound-healing, and cytoprotective activities of proteins or protein-derived fractions from H. scabra [11,12,13]. This suggests that its proteins may provide a valuable source of multifunctional bioactive peptides. However, whether these proteins or their digestion-derived peptides can protect neuronal cells against neurodegenerative stress remains largely unknown.
Importantly, dietary proteins undergo extensive hydrolysis by gastrointestinal enzymes following consumption, generating peptide fragments that ultimately determine which bioactive sequences become available for absorption. Therefore, evaluating the biological properties of gastrointestinal-digested H. scabra proteins is essential for establishing their physiological relevance and identifying peptides with potential neuroprotective activity. Investigating these digestion-derived peptides may provide a more realistic basis for assessing the potential of H. scabra protein as a functional food or nutraceutical ingredient for neuroprotection [14,15].
Therefore, the present study aimed to investigate the neuroprotective effects of gastrointestinal-digested proteins derived from the inner wall of H. scabra (IWD) using an okadaic acid (OA)-induced neurotoxicity model in SH-SY5Y neuronal cells. Since OA-induced inhibition of protein phosphatase 2A (PP2A) reproduces several pathological features of Alzheimer’s disease, including oxidative stress, tau hyperphosphorylation, and neuronal apoptosis, this model provides a relevant platform for evaluating neuroprotective agents. In addition to assessing cell viability and intracellular reactive oxygen species (ROS) production, quantitative proteomic analysis was performed to elucidate the molecular mechanisms underlying the neuroprotective effects of the digested proteins. Moreover, the gastrointestinal-generated peptides were characterized by LC-MS/MS and subsequently evaluated using in silico, ADMET, and blood–brain barrier (BBB) permeability prediction tools to identify peptide candidates with neuroprotective potential. This integrated experimental and computational approach identifies molecular changes associated with the protective effects of IWD and provides a basis for further investigation of H. scabra-derived peptides as potential sources of in vitro neuroprotective activity.

2. Results

2.1. Functional Annotation of Proteins Identified in the H. scabra Inner Wall Protein Extract (IW) Using the PANTHER Database

Proteins identified in the IW fraction were functionally annotated using the PANTHER database based on molecular function, protein class, and associated signaling pathways (Figure 1). At the molecular function level, binding proteins predominated, followed by proteins involved in catalytic activity and structural molecule activity, with smaller proportions assigned to antioxidant, ATP-dependent, adaptor, regulatory, and cytoskeletal motor activities. Protein class analysis similarly showed predominance of metabolite interconversion enzymes, cytoskeletal proteins, protein-binding modulators, and transfer/carrier proteins, together with chaperones, translational, structural, and protein-modifying proteins.
Figure 1. Protein profile and classification of H. scabra protein extraction using LC-MS/MS and pathway enrichment analysis, respectively.
Pathway analysis identified proteins associated with diverse signaling pathways, including interleukin, integrin, inflammatory/cytokine, EGF, FGF, angiogenesis, Rho GTPase, and axon guidance pathways. Notably, pathways related to neuronal regulation, including dopamine receptor, metabotropic glutamate receptor, JAK/STAT, TGF-β, PI3K, VEGF, and Alzheimer disease–presenilin signaling, were also represented. Overall, the PANTHER analysis revealed a functionally diverse protein repertoire in the H. scabra IW extract, encompassing proteins involved in metabolism, structural maintenance, signaling, protein homeostasis, and neuronal regulation.

2.2. Bioactivity Prediction of Selected Peptides Identified from IWD

The IW protein extract was subjected to gastrointestinal digestion, and the resulting peptide mixture (IWD) was profiled by LC–MS/MS. A total of 162 unmodified peptide sequences were identified and subsequently screened using PEP-ADMET based on predicted BBB permeability, non-toxicity, and moderate Caco-2 permeability. Of these, 31 peptides met all selection criteria and were further analyzed using the BIOPEP-UWM database to predict their potential biological activities (Table 1). BIOPEP analysis predicted diverse biological activities among the selected peptides (Figure 2). The most abundant predicted activities were angiotensin-converting enzyme inhibitor (ACE inhibitor) (n = 32), dipeptidyl peptidase IV (DPP-IV) inhibitory (n = 31), antioxidative (n = 17), and neprilysin inhibitory (n = 13) activities. Additional predicted functions included glutamate carboxypeptidase II inhibitory, antiamnestic, antithrombotic, hypouricemic, α-glucosidase inhibitory, hypotensive, and neuroprotective activities.
Table 1. LC–MS/MS-identified peptides selected based on favorable ADMET properties, including blood–brain barrier permeability, moderate Caco-2 permeability, and non-toxicity.
Figure 2. Predicted bioactivity profiles of selected peptides identified from IWD based on BIOPEP-UWM analysis.
Among these, several predicted activities are associated with mechanisms implicated in neurodegeneration. Antioxidative peptides may reduce oxidative stress, while neuroprotective and antiamnestic peptides may help preserve neuronal function. In addition, peptides predicted to inhibit glutamate carboxypeptidase II and neprilysin may modulate glutamatergic signaling and neuropeptide metabolism, respectively. Although ACE- and DPP-IV-inhibitory peptides were the most abundant, these activities have also been associated with reduced oxidative stress, neuroinflammation, and improved neuronal homeostasis. Thus, the combination of predicted BBB permeability, non-toxicity, moderate intestinal absorption, and multiple neurorelevant bioactivities supports the potential of the IWD peptide mixture as a neuroprotective candidate. These findings provided the rationale for subsequent evaluation of the peptide fraction as a pretreatment against okadaic acid-induced neurotoxicity in SH-SY5Y cells.

2.3. Cell Viability Assay of IWD and Okadaic Acid

The cytotoxic effects of IWD on SH-SY5Y cells were evaluated after 24 and 48 h of treatment at concentrations ranging from 1 to 100 µg/mL. As shown in Figure 3A, cell viability was assessed using the MTT assay and expressed as a percentage relative to the untreated control. At 24 h, treatment with the highest concentration (100 µg/mL) significantly reduced cell viability to approximately 10–15% compared to the control group (p < 0.0001). In contrast, lower concentrations (1–25 µg/mL) did not exhibit cytotoxic effects and maintained cell viability close to or above 90%, with some concentrations (5–10 µg/mL) showing slightly enhanced viability compared to the control. At 48 h, a similar dose-dependent cytotoxic pattern was observed. Thus, IWD exhibited dose-dependent cytotoxicity, and concentrations of 1, 2.5, 5, 10, and 25 µg/mL were considered non-cytotoxic and were therefore selected for subsequent neuroprotective studies.
Figure 3. Cell viability of SH-SY5Y cells after being treated with various concentrations of inner wall of H. scabra protein digest (IWD) (A) and okadaic acid (OA) (B). Data are presented as means ± SD (n = 3, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared with the control group).
Moreover, in the okadaic acid (OA) cytotoxicity assay, concentrations of 100, 80, and 40 nM significantly reduced cell viability by more than 80% compared to the control group (p < 0.0001) at both 24 and 48 h. In contrast, treatment with 20 nM OA for 24 h resulted in approximately 50% cell viability relative to control. Therefore, this concentration was selected as the optimal condition for inducing neuronal cytotoxicity in subsequent assays (Figure 3B).

2.4. Neuroprotection Assay

The neuroprotective effect of IWD was evaluated against 20 nM OA-induced cytotoxicity in SH-SY5Y cells. As shown in Figure 4A, OA treatment significantly reduced cell viability to approximately 60% of the control (p < 0.05), indicating successful induction of neuronal toxicity. Pretreatment with IWD markedly attenuated OA-induced cytotoxicity in a concentration-dependent manner. Among the tested concentrations, 5 µg/mL exhibited the strongest protective effect, restoring cell viability to approximately 80%, which was significantly higher than the OA-treated group (p < 0.01). Similarly, 10 µg/mL also showed a significant improvement in cell viability (75–78%, p < 0.05). Higher concentration (25 µg/mL) demonstrated only a moderate protective effect, while lower concentrations (1 and 2.5 µg/mL) showed minimal protection.
Figure 4. Neuroprotective effect of IWD against okadaic acid (OA) induced neuronal cytotoxicity in SH-SY5Y cells. (A) Protective effects of various concentrations of IWD treatment on cell viability without and with OA. (B) Effect of various concentrations of the treatment on SH-SY5Y cell morphology (10×). Data are presented as means ± SD (n = 3; * p < 0.05 and ** p < 0.01 compared with the 20 nM OA group). The scale bar indicates 100 µm.
Morphological observations further supported these findings. Untreated control cells displayed normal neuronal morphology with elongated processes and intact cell bodies. In contrast, OA-treated cells exhibited cell shrinkage, rounding, and reduced neurite outgrowth, characteristic of neuronal damage. Pretreatment with IWD, particularly at 5 µg/mL, was associated with improved cellular morphology, increased cell density, and partial recovery of neurite structures. Overall, these findings suggest that IWD attenuated OA-induced cellular injury, with 5 µg/mL producing the greatest observed morphological improvement under the experimental conditions tested (Figure 4B).

2.5. Measurement of Intercellular ROS Level

The effect of IWD on intracellular reactive oxygen species (ROS) generation was evaluated in SH-SY5Y cells using the DCFH-DA assay, followed by flow cytometry and fluorescence microscopy analysis (Figure 5). As shown in Figure 5A, treatment with 20 nM okadaic acid (OA) significantly increased intracellular ROS levels to approximately 180–200% relative to the control group (### p < 0.01), indicating pronounced oxidative stress. Pretreatment with IWD significantly attenuated OA-induced ROS production. Specifically, treatment with 10 µg/mL reduced ROS levels to approximately 150%, while 5 µg/mL further decreased ROS levels to approximately 130%, both showing significant reductions compared to the OA-treated group (p < 0.01).
Figure 5. (A) Effects of OA on intracellular ROS levels. Data presented as means ± SD (n = 3; ** p < 0.01 and ### p < 0.01 compared with the 20 nM OA group. (B–D) show fluorescent intensity and (E–G) fluorescence of intracellular ROS generation under a fluorescence microscope of control, 20 nM OA-induced toxicity, and 5 µg/mL of IWD with 20 nM OA-induced toxicity, respectively. The scale bar indicates 50 µm of cell morphology (20×).
Flow cytometry histograms (Figure 5B–D) further confirmed these findings. The OA-treated group exhibited a rightward shift in fluorescence intensity, indicating increased DCF fluorescence and elevated ROS production compared to the untreated control. In contrast, cells pretreated with IWD (5 µg/mL) showed a noticeable leftward shift, reflecting a reduction in intracellular ROS levels toward baseline. Consistent with these results, fluorescence microscopy images (Figure 5E–G) demonstrated increased green fluorescence intensity in OA-treated cells, indicative of elevated ROS accumulation. Pretreatment with IWD markedly reduced fluorescence intensity, particularly at 5 µg/mL, suggesting effective suppression of oxidative stress. These results indicate that IWD attenuates OA-induced oxidative stress in SH-SY5Y cells, possibly by modulating cellular redox defenses.

2.6. Comparative Proteomic Profiling Demonstrates That Pretreatment with IWD Induces Adaptive Remodeling of SH-SY5Y Cells Exposed to Okadaic Acid

To investigate global proteomic responses associated with neuroprotection, we performed comparative proteomic analysis among untreated control cells (CT), okadaic acid-induced neurotoxic cells (OA), and cells pretreated with 5 µg/mL IWD before OA exposure (IWD + OA).
Differential protein profiling revealed both partial restoration and induction of distinct proteomic responses following pretreatment. A total of 714 proteins were commonly identified across all groups, indicating preservation of a core proteome independent of treatment. Notably, 154 proteins were shared between control and IWD + OA, whereas 300 proteins overlapped between OA and IWD + OA, suggesting that pretreatment did not fully restore the proteome to the control state but induced an adaptive proteomic profile (Figure 6A). Three-dimensional principal component analysis (PCA) showed clear separation among control, OA, and IWD + OA groups, with PC1 explaining 61.2% of the total variance (Figure 6B). The IWD + OA group formed a distinct cluster positioned between the CT and OA groups, indicating an overall proteomic profile that differed from both groups. Consistent with this observation, heatmap analysis revealed distinct protein-abundance patterns across the three groups (Figure 6C).
Figure 6. (A) Venn diagram showing the distribution and overlap of identified proteins among the control (CT), okadaic acid-treated (OA), and IWD pretreatment followed by OA exposure (IWD + OA) groups. (B) Three-dimensional principal component analysis (3D-PCA) plot based on global protein expression profiles across the three groups with four biological replicates per group. (C) Heatmap showing the expression patterns of the 714 proteins commonly identified across all treatment groups. (D) Top 20 proteins ranked by variable importance in projection (VIP) score contributing to group discrimination.
Partial least squares discriminant analysis (PLS-DA) was further performed to investigate proteomic differences among the three groups. A five-component model was evaluated, with component 3 yielding the highest cross-validated Q2 value among the components tested. Together, the PCA, heatmap, and PLS-DA results indicate group-associated differences in the overall proteomic profiles. However, these multivariate analyses alone do not establish that IWD reversed OA-induced protein alterations or restored the proteome to its basal state.
Variable importance in projection (VIP) analysis identified Stathmin (STMN1), large ribosomal subunit protein P2 (RLA2), heat shock protein HSP90-beta (HSP90B), tropomyosin alpha-3 chain (TPM3), alpha-enolase (ENO1), and alpha-2-HS-glycoprotein chain A (AHSG) as major contributors to group discrimination (Figure 6D). These proteins are associated with cytoskeletal organization, protein homeostasis, and cellular stress responses, supporting the neuroprotective role of IWD against OA-induced toxicity.

2.7. Pretreatment with IWD Induces a Distinct Protective Proteomic State

Differential protein expression analysis was performed to characterize proteomic alterations associated with OA-induced neuronal injury and the protective response induced by IWD pretreatment (IWD + OA).
In the OA versus control comparison, volcano plot analysis revealed substantial proteomic remodeling following OA exposure (Figure 7A). A total of 79 differentially expressed proteins (DEPs) were identified, comprising 48 upregulated and 31 downregulated proteins, indicating broad disruption of cellular homeostasis under neurotoxic conditions. Among the most significantly upregulated proteins were BAX (Bcl-2-associated X protein), KRT10 (keratin type I cytoskeletal 10), H4C4 (histone H4), and H2AJ (histone H2A.J). Increased abundance of BAX suggests activation of apoptosis-associated signaling, whereas altered expression of histone proteins may reflect changes in chromatin organization and stress-associated transcriptional regulation. Overall, these findings indicate that OA exposure induces coordinated alterations involving apoptotic signaling, intracellular organization, and nuclear regulatory mechanisms.
Figure 7. Differential proteomic analysis of SH-SY5Y cells under OA-induced neurotoxicity and IWD pretreatment. Volcano plots showing differentially expressed proteins (DEPs) in (A) OA-treated cells compared with the control group and (C) IWD pretreatment followed by OA exposure (IWD + OA) compared with OA treatment alone. Red, blue, and gray dots represent significantly upregulated proteins, significantly downregulated proteins, and proteins with no significant change in expression, respectively. The top 20 proteins ranked by the VIP score contributing to the discrimination between (B) OA-treated cells and the control group, and (D) IWD pretreatment followed by OA exposure (IWD + OA) and OA treatment alone, respectively.
Variable importance in projection (VIP) analysis identified proteins contributing most strongly to discrimination between OA-treated and control groups (Figure 7B). The highest-ranked protein was stathmin (STMN1), followed by molecular chaperones (HSPA8, HSPD1, and HSPB1) and proteins involved in translation and energy metabolism, including EEF1A1, PKM, PGAM1, ALDOA, and ENO1. Additional discriminative proteins included PARK7 (DJ-1), PRDX1, UCHL1, RPS27A, and TPM1. The predominance of metabolic enzymes, heat shock proteins, and oxidative stress-associated proteins suggests that OA exposure induces coordinated metabolic adaptation, proteostatic stress, and cytoskeletal reorganization. Notably, the enrichment of glycolytic proteins (PKM, PGAM1, ALDOA, and ENO1) indicates altered cellular energy regulation under neurotoxic conditions rather than maintenance of basal metabolism.
Additionally, comparison between IWD pretreatment (IWD + OA) and OA alone revealed a distinct proteomic response associated with adaptive remodeling rather than simple reversal of OA-induced changes (Figure 7C). A total of 129 DEPs were identified, including 47 upregulated and 82 downregulated proteins, demonstrating extensive reorganization of the proteomic landscape following pretreatment. Among significantly altered proteins, reduced abundance of translation-related proteins, including RLA2 (ribosomal protein P2) and RPS27A (ubiquitin-40S ribosomal protein S27a), suggests selective modulation of translational activity and protein turnover under stress conditions.
VIP analysis further identified proteins that contributed most strongly to the separation between the IWD + OA and OA groups (Figure 7D). The highest-ranking proteins included STMN1, RLA2, HSP90B, TPM3, ENO1, AHSG, TPI1, HSPB1, ATP5F1B, HNRNPA2B1, PARK7, ARHGDIA, SFPQ, and RPS27A. Functionally, these proteins are associated with cytoskeletal organization, mitochondrial energy production, RNA processing, translational regulation, and stress adaptation. The presence of RNA-binding and ribosome-associated proteins is consistent with enrichment analyses demonstrating regulation of RNA metabolism, translation, and proteostasis pathways following pretreatment.
In short, these findings suggest that OA-induced neurotoxicity is characterized by activation of apoptotic signaling, metabolic reprogramming, and proteostatic stress, whereas pretreatment with IWD promotes adaptive proteomic remodeling involving translational regulation, RNA-associated processes, and maintenance of cellular homeostasis, thereby potentially enhancing resistance to OA-mediated neuronal injury.

2.8. Functional Enrichment and Protein–Protein Interaction Analysis Reveal Cellular Processes Associated with Okadaic Acid-Induced Neurotoxicity

To investigate the biological significance of differentially expressed proteins, protein–protein interaction (PPI) and functional enrichment analyses were performed using Gene Ontology (GO), KEGG, and Reactome databases. In the OA versus control comparison, GO cellular component analysis showed enrichment of proteins localized to intracellular and membrane-bounded organelles, together with vesicles, extracellular region, extracellular space, and extracellular exosomes (Figure 8A), suggesting extensive remodeling of intracellular organization and extracellular communication following OA exposure. GO molecular function analysis further revealed enrichment of protein binding, RNA binding, nucleic acid binding, protein dimerization, and enzyme inhibitor activity (Figure 8B), indicating disruption of protein interaction networks and cellular homeostasis. Reactome analysis identified enrichment of pathways related to mitotic prophase, M phase, Senescence-Associated Secretory Phenotype (SASP), oxidative stress-induced senescence, and Spry regulation of FGF signaling (Figure 8C). Collectively, these findings suggest that OA-induced neurotoxicity is associated with intracellular dysfunction, altered molecular interactions, oxidative stress-associated senescence, and dysregulation of cell-cycle-related processes.
Figure 8. Functional enrichment analysis of differentially expressed proteins (DEPs) identified in OA-induced neurotoxicity compared with the control group using the STRING database v12.0. GO enrichment analysis was performed for (A) cellular component (CC) and (B) molecular function (MF) categories, followed by (C) Reactome pathway enrichment analysis.
Interestingly, enrichment analysis of IWD + OA vs. OA revealed a distinct functional profile associated with adaptive cellular responses. GO cellular component categories remained enriched in intracellular and extracellular structures but additionally showed enrichment of ribonucleoprotein complex, suggesting regulation of RNA-associated molecular assemblies and translational processes (Figure 9A). GO molecular function analysis demonstrated enrichment of RNA binding, mRNA binding, nucleic acid binding, protein binding, and structural constituent of ribosome (Figure 9B), indicating modulation of protein synthesis and post-transcriptional regulation following pretreatment. GO biological process analysis identified enrichment of cytoplasmic translation, peptide biosynthetic process, cellular nitrogen compound metabolic process, and macromolecule metabolic process (Figure 9C), suggesting maintenance of biosynthetic and metabolic capacity under OA-induced stress.
Figure 9. Functional enrichment analysis of differentially expressed proteins (DEPs) identified in IWD + OA compared with the OA alone group using the STRING database v12.0. GO enrichment analysis was performed for (A) cellular component (CC), (B) molecular function (MF), and (C) cellular process (CP) categories, followed by (D) KEGG and (E) Reactome pathway enrichment analysis.
KEGG analysis identified enrichment of pathways related to the spliceosome, ribosome, proteasome, pyruvate metabolism, and pathways commonly associated with neurodegenerative disorders, including Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (ALS) (Figure 9D). To clarify the molecular basis of the latter enrichments, the proteins mapped to these three pathways were examined (Table 2). Eight proteins were shared across all three pathways: PSMA3, PSMA6, PSMB6, PSMB4, BAX, UQCRH, ATP5PF, and TUBA1A. These proteins are associated with proteasomal protein degradation, mitochondrial energy metabolism, apoptosis, and cytoskeletal organization. The overlap suggests that these disease-annotated pathway enrichments may reflect changes in shared cellular processes related to proteostasis, energy metabolism, and cellular stress responses rather than disease-specific effects. Accordingly, these findings should be interpreted as pathway-level associations and do not establish that IWD directly targets or modulates Parkinson’s disease, Huntington’s disease, or ALS.
Table 2. Proteins mapped to KEGG pathways annotated as Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease in the IWD + OA group.
Reactome analysis identified enrichment of pathways associated with RNA metabolism, axon guidance, regulation of SLIT and ROBO expression, cellular responses to stress, eukaryotic translation termination, and apoptosis (Figure 9E). These findings indicate that proteins associated with RNA processing, neuronal guidance-related signaling, translational regulation, and cellular stress responses were represented among the differentially expressed proteins. However, pathway enrichment alone does not establish whether these processes were activated, inhibited, or functionally restored following IWD pretreatment.
Thus, comparative enrichment analysis indicates that OA exposure primarily induces proteomic changes associated with cellular stress, senescence, and regulatory disruption, whereas IWD promotes a distinct adaptive proteomic program characterized by RNA metabolism, translational regulation, proteostasis, and neuronal stress resilience.

2.9. Protein–Protein Interaction Analysis Identifies Translational and Proteostasis-Associated Hub Networks Following IWD Pretreatment

The resulting PPI network demonstrated extensive connectivity among differentially expressed proteins between IWD pretreatment (IWD + OA) and OA alone groups, indicating coordinated regulation rather than isolated protein alterations (Figure 10A). The PPI showed 112 nodes and 584 edges. Several functional clusters associated with protein synthesis, stress responses, RNA processing, cytoskeletal regulation, and protein homeostasis were identified.
Figure 10. (A) Protein–protein interaction (PPI) network of differentially expressed proteins (DEPs) in the IWD + OA group compared with the OA-only group. (B) The top 30 core targets identified from the PPI network using the maximal clique centrality (MCC) algorithm. Color intensity (orange to yellow) indicates the relative interaction strength of each target.
Hub analysis using the CytoHubba MCC (Maximal Clique Centrality) algorithm identified highly interconnected proteins within the PPI network (Figure 10B). Node color intensity (red to yellow) represents decreasing MCC scores, with darker colors indicating greater network centrality and potential biological importance rather than expression abundance. Several highly ranked hub proteins were identified, including ribosomal proteins (RPS3A, RPS13, RPS16, RPS18, RPS27A, RPL3, RPL23A, RPL27, RPL29, and RPLP2), together with RNA-processing regulators (SNU13, RBMX, HNRNPC, SRSF1, SF3B3, PTBP1, and SFPQ). Additional hub proteins involved in proteostasis and stress adaptation, including PSMA3, PSMA6, HSP90AB1, NACA, and FUS, were also identified.
The predominance of ribosomal and RNA-processing hubs suggests that pretreatment with IWD may reorganize translational and post-transcriptional regulatory networks under OA-induced stress conditions. This observation is consistent with enrichment analyses showing modulation of RNA metabolism, translation, and protein homeostasis pathways.

3. Discussion

The present study demonstrates that the gastrointestinal digest of H. scabra inner-wall proteins (IWD) attenuates OA-induced neuronal injury in SH-SY5Y cells, with differential proteomic analysis identifying molecular changes associated with the observed protective effects [17,18]. The findings suggest that the protective effects of IWD are associated with changes in oxidative stress, protein homeostasis, translational regulation, and cellular stress responses, although the contributions of individual molecular targets and pathways remain to be established.
Functional characterization of the H. scabra inner wall extract (IW) revealed a complex composition enriched in proteins involved in molecular binding, catalytic activity, structural functions, and multiple signaling pathways associated with cellular regulation and neuronal homeostasis [19]. Binding proteins represented the dominant molecular function, suggesting substantial capacity for molecular interaction and signaling regulation, whereas catalytic and structural proteins may contribute to maintenance of cellular metabolism and structural integrity under stress conditions [19,20]. In addition, protein class analysis identified metabolite interconversion enzymes, cytoskeletal proteins, chaperones, translational proteins, and protein-binding modulators, indicating that the extract contains functionally diverse proteins capable of influencing multiple biological processes simultaneously [21,22].
Importantly, pathway annotation identified signaling pathways closely associated with neuronal survival and neurodegenerative regulation, including integrin, FGF, PI3K, JAK/STAT, TGF-β, axon guidance, dopamine receptor, glutamate receptor, and Alzheimer’s disease–presenilin pathways [23,24,25]. Many of these pathways are recognized for regulating neuronal plasticity, cellular adaptation, oxidative stress responses, and resistance to apoptosis. This broad functional profile suggests that H. scabra proteins may possess intrinsic biological potential to support neuronal resilience under pathological stress [26].
Following gastrointestinal digestion, these parent proteins were further processed into smaller peptide sequences that may possess biological activities distinct from those of the intact proteins. Importantly, 31 digested peptides (4–11 amino acids long) predicted to exhibit BBB permeability, non-toxicity, and moderate Caco-2 permeability were identified and subjected to BIOPEP analysis. These peptides exhibited diverse predicted activities, particularly ACE-inhibitory, DPP-IV-inhibitory, antioxidative, and neprilysin-inhibitory activities, as well as predicted neuroprotective and antiamnestic functions [27,28]. The presence of antioxidative and neurorelevant peptide activities provides a plausible basis for the biological effects observed following exposure to IWD and suggests that gastrointestinal digestion may generate peptide sequences capable of contributing to neuroprotection [29].
Consistent with this hypothesis, pretreatment with IWD significantly attenuated OA-induced neurotoxicity in SH-SY5Y cells. OA exposure reduced cell viability and induced characteristic neuronal injury, including cell shrinkage and loss of neurite structures, accompanied by marked intracellular ROS accumulation [30]. On the other hand, IWD pretreatment improved cell survival, preserved neuronal morphology, and significantly reduced intracellular ROS levels, with 5 µg/mL producing the strongest protective effect. The absence of additional benefit at higher concentrations suggests that neuroprotection occurs within an optimal biological response range rather than increasing proportionally with dose [31].
The reduction in ROS indicates that IWD treatment was associated with decreased oxidative stress, a process implicated in neurodegenerative progression and neuronal dysfunction. Excessive ROS disrupts protein folding, mitochondrial function, RNA integrity, and intracellular signaling, ultimately leading to loss of cellular homeostasis [32]. Suppression of oxidative stress may represent an early protective event that limits downstream molecular damage [33]. Although ROS levels were assessed by flow cytometry in living cells using equal cell numbers across samples, the inclusion of a positive antioxidant control, such as N-acetylcysteine (NAC), would have strengthened comparison with a reference antioxidant [34].
Proteomic analysis demonstrated that pretreatment with IWD did not simply restore OA-treated cells to the untreated state but induced a distinct adaptive proteomic response associated with neuroprotection. PCA and hierarchical clustering showed that the IWD + OA group exhibited a distinct proteomic profile, with its PCA position intermediate between those of the control and OA groups. This pattern suggests that IWD treatment was associated with changes in the OA-related proteomic profile, although the intermediate position alone does not establish reversal of OA-induced alterations or complete restoration toward the control state.
OA exposure induced extensive proteomic remodeling characterized by increased expression of proteins associated with apoptosis, cellular stress, and structural adaptation, including BAX (Bcl-2-associated X protein) [35,36]. Functional enrichment further revealed broad disruption of intracellular organization, with significant enrichment of proteins localized to intracellular organelles, membrane-bounded compartments, vesicles, and extracellular exosomes [30]. These findings suggest that OA toxicity affects not only intracellular homeostasis but also mechanisms involved in cellular communication and stress propagation. Concurrent enrichment of protein binding, RNA binding, nucleic acid binding, and protein dimerization indicates perturbation of molecular interaction networks essential for maintaining neuronal function [37]. Reactome analysis further identified enrichment of Mitotic Prophase, M Phase, Senescence-Associated Secretory Phenotype (SASP), and oxidative stress-induced senescence, supporting activation of oxidative stress-associated and cell-cycle-related responses that have been increasingly recognized as features of neuronal dysfunction and neurodegeneration [38]. These findings are consistent with the biological outcomes observed in this study, where OA markedly increased intracellular ROS and reduced neuronal viability.
Importantly, IWD pretreatment shifted the proteomic profile toward pathways related to translation, RNA metabolism, proteostasis, and stress adaptation, suggesting that neuroprotection resulted from enhanced cellular resilience rather than complete suppression of stress responses. STMN1 (stathmin-1) was identified as an important discriminatory protein in the multivariate analysis, as indicated by its high variable importance in projection (VIP) score. However, the VIP score reflects the protein’s contribution to group separation and does not independently establish the direction or statistical significance of its expression change. STMN1 is a microtubule-associated regulatory protein involved in microtubule dynamics and cytoskeletal remodeling. Therefore, changes in STMN1 abundance may be associated with alterations in neuronal structural regulation under OA-induced injury and following IWD pretreatment [39]. Nevertheless, the potential contribution of STMN1 to the observed morphological improvement remains to be established, and its role in the response to IWD should be interpreted in the context of the direct differential-expression comparison between the IWD + OA and OA groups [40,41,42]. Proteins involved in translational regulation, including RPLP2 (Large ribosomal subunit protein P2) and RPS27A (Ubiquitin-40S ribosomal protein S27a), were less abundant following IWD pretreatment than in OA-treated cells. Because ribosomal protein abundance does not directly reflect translation rate [43,44,45], and RPS27A additionally contributes to the cellular ubiquitin pool, these changes are interpreted as alterations in ribosome- and ubiquitin-associated proteostasis rather than as evidence of altered protein synthesis. The reduced abundance of the RNA-binding proteins HNRNPA2B1 (Heterogeneous nuclear ribonucleoproteins A2/B1) and SFPQ (Splicing factor, proline- and glutamine-rich), both of which have been implicated in neurodegeneration-associated RNA dysregulation [46,47,48], together with the enrichment of spliceosome and RNA metabolism terms (Figure 9D–E), suggests that IWD pretreatment may influence RNA-processing networks under OA-induced stress.
Proteostasis-related proteins show coordinated regulation, with decreased HSP90AB1 (Heat shock protein HSP 90-beta) suggesting reduced activation of stress-associated chaperone pathways linked to protein misfolding and neurodegeneration [49]. On the other hand, increased HSPB1 (Heat shock protein beta-1) may indicate activation of adaptive neuroprotective mechanisms that maintain protein homeostasis and improve cellular resistance to stress [50,51]. In parallel, PARK7 (DJ-1) has been implicated in cellular responses to oxidative stress [52,53], whereas ATP5F1B is a catalytic subunit of mitochondrial ATP synthase [54]. These proteins are biologically relevant to oxidative stress responses and mitochondrial energy metabolism, respectively. However, because PARK7 and ATP5F1B were not identified among the proteins with reported differential fold changes in the present analysis, their involvement in the protective effects of IWD cannot be established froµ the current proteomic data. The observed reduction in ROS accumulation and preservation of cell viability and neuronal morphology provide experimental evidence of the protective effects of IWD, whereas the specific contributions of PARK7, ATP5F1B, and related cellular processes remain to be investigated through targeted functional studies.
This interpretation was further supported by enrichment analysis. IWD pretreatment retained enrichment within intracellular and extracellular compartments but additionally promoted enrichment of ribonucleoprotein complexes, together with RNA binding, mRNA binding, and structural constituents of the ribosome, indicating active regulation of protein synthesis under stress conditions. Biological process analysis identified enrichment of cytoplasmic translation, peptide biosynthesis, and macromolecule metabolism, suggesting preservation of biosynthetic and metabolic capacity despite OA challenge. KEGG and Reactome analyses identified pathways related to RNA processing, translation, proteostasis, energy metabolism, cellular stress responses, axon guidance, and SLIT–ROBO signaling. These enrichments indicate proteomic changes associated with neuronal guidance and cellular maintenance but do not demonstrate pathway activation or functional restoration.
Notably, KEGG analysis identified pathways annotated as Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (ALS), with eight shared proteins: PSMA3, PSMA6, PSMB6, PSMB4, BAX, UQCRH, ATP5PF, and TUBA1A. These proteins are associated with proteasomal degradation, mitochondrial energy metabolism, apoptosis, and cytoskeletal organization. Their overlap suggests that these enrichments reflect shared cellular processes rather than disease-specific effects, providing context for the proteomic response to IWD pretreatment under OA exposure without establishing a direct relationship between IWD and these neurodegenerative diseases.
The PPI network analysis revealed highly interconnected hub proteins involved in ribosomal function, RNA processing, proteostasis, and cellular stress responses. The identification of ribosomal proteins, including RPS3A, RPS13, RPS16, RPS18, RPS27A, RPL3, RPL23A, RPL27, RPL29, and RPLP2, highlights the potential importance of translational regulation in the proteomic response to IWD pretreatment. In parallel, RNA-processing regulators, including SNU13, RBMX, HNRNPC, SRSF1, SF3B3, PTBP1, and SFPQ, suggest the involvement of RNA maturation and post-transcriptional regulation. Additional hubs, including PSMA3 and PSMA6, are associated with proteasomal protein degradation, while HSP90AB1, NACA, and FUS are involved in protein homeostasis and cellular stress responses. Collectively, these network findings suggest associations with translational, RNA-processing, and proteostasis-related processes in the cellular response to IWD pretreatment under OA-induced stress. However, MCC-based hub status reflects topological centrality within the STRING-derived network, which may be influenced by database connectivity and annotation biases, particularly for ribosomal and RNA-processing proteins. Therefore, hub status does not establish causal involvement in neuroprotection, and the functional contributions of these proteins require further validation.
Taken together, the proteomic, KEGG, and PPI analyses suggest that IWD pretreatment is associated with adaptive remodeling of protein synthesis, RNA processing, proteostasis, and cellular metabolism during OA-induced neuronal stress. Further functional studies are needed to establish whether these changes directly contribute to the observed neuroprotective effects.
Several proteins identified in this study may serve as candidate markers of neuroprotection. STMN1 is a protein highly expressed during the development of the central nervous system. The phosphorylation of STMN1 involves microtubule dynamics, so STMN1 plays a vital part in neurite outgrowth and synaptic plasticity [55]. It emerged as a major discriminator associated with neuronal structural adaptation, while RPS27A and RLA2 were associated with changes in ribosomal and translational processes. HSPB1, HSP90AB1, and PARK7 represent additional candidates related to stress adaptation and cellular homeostasis, whereas PSMA3 and PSMA6 were identified as highly interconnected PPI hubs and shared proteins in the three disease-annotated KEGG pathways. These proteins represent complementary candidates associated with cytoskeletal regulation, translational control, stress responses, and proteostasis. However, they should be considered candidate biomarkers rather than validated biomarkers, as their diagnostic or mechanistic value in IWD-associated neuroprotection requires further experimental validation. Furthermore, these findings suggest that IWD-associated neuroprotection involves coordinated regulation of neuronal structure and cellular homeostasis. However, the observed effects cannot be attributed to any individual peptide among the 31 candidates identified by LC–MS/MS and screened in silico; their individual neuroprotective activities and potential synergistic effects require further experimental validation.

4. Materials and Methods

4.1. Extraction of the Inner Wall Protein of Holothuria Scabra (IW)

Holothuria scabra (150–200 g body weight) were obtained from a commercial farm in Krabi Province, Thailand, as previously described (Animal Care and Use Committee of Thammasat University, NRCT, Protocol No. 019/2561) [21]. Before tissue collection, the sea cucumbers were anesthetized by immersing them in seawater containing 0.125 mL/L clove oil for approximately 10–15 min. After anesthesia, the inner body wall tissue was collected and cut into small pieces to facilitate the extraction process and make it easier. The tissue samples were then homogenized using a lysis buffer (PH 7.2) consisting of 10 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, and 1 mM EDTA prepared in deionized water with 1 mM phenylmethylsulfonyl fluoride (PMSF) added to the buffer as a protease inhibitor. The homogenized tissue lysate was then centrifuged at 12,000× g for 30 min at 4 °C to obtain the soluble protein fraction [21]. Following centrifugation, the supernatant containing crude protein extracts was then filtered through a 0.45 µm syringe filter, lyophilized, and stored at −80 °C until further experimental use.

4.2. Protein Identification by LC-MS/MS

The protein extracts from the inner wall (IW) were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The crude protein was subjected to enzymatic digestion using modified trypsin (Promega Corporation, Madison, WI, USA) at a concentration of 20 ng/spot in 50% acetonitrile containing 10 mM ammonium bicarbonate. Digestion was performed at 37 °C for 3 h to generate peptide fragments suitable for mass spectrometric analysis. Following digestion, the supernatant containing peptides was collected and resuspended in 0.1% (v/v) formic acid. Peptide separation and analysis were carried out using the nano chromatography system (Dionex Ultimate 3000; Thermo Fisher Scientific, Waltham, MA, USA) coupled to an HCTultra PTM Discovery System equipped with electrospray ionization (ESI). Peptides were first separated by liquid chromatography and subsequently introduced into the mass spectrometer for mass-to-charge (m/z) analysis, and detection was performed using a TripleTOF 6600+ mass spectrometer (AB SCIEX, Framingham, MA, USA).
The resulting MS/MS spectra were processed using a local MASCOT server and searched against the Swiss-Prot and NCBI databases for protein identification [56]. Search parameters included the use of monoisotopic mass values, with methionine oxidation specified as a variable modification and carbamidomethylation of cysteine as a fixed modification. Trypsin was selected as the digestion enzyme, and up to one missed cleavage was allowed to improve peptide identification accuracy. Additionally, proteins identified from H. scabra extract were functionally classified using the PANTHER Classification System (http://www.pantherdb.org/; accessed on 10 April 2025) according to molecular function, protein class, and associated signaling pathways to characterize the biological composition of the extract.

4.3. Simulated Gastrointestinal Digestion of H. scabra Inner Wall (IW) Proteins

The protein extract from the inner wall of H. scabra was digested using an in vitro gastrointestinal digestion method [57,58]. As the protein extract contained no starch, the oral phase was omitted. Briefly, 5 mL of protein solution (50 mg/mL) was mixed with 3.9 mL of simulated gastric fluid (SGF), 5 μL of 0.3 M CaCl2, and pepsin (final activity 200 U/mL) from porcine gastric mucosa (P6887, Sigma-Aldrich, Saint Louis, MO, USA). The pH was adjusted to 3.0 with 6 N HCl, and the final volume was adjusted to 10 mL with distilled water. The mixture was incubated at 37 °C for 2 h with shaking at 150 rpm. For the intestinal phase, 7 mL of simulated intestinal fluid (SIF), 40 μL of 0.3 M CaCl2, and pancreatin (10 U/mL) from porcine pancreas (P1750, Sigma-Aldrich, Saint Louis, MO, USA) were added. The pH was adjusted to 7.0 with 0.1 N NaOH, and the final volume was adjusted to 20 mL with distilled water. The mixture was incubated at 37 °C for an additional 2 h with shaking at 150 rpm. Digestion was terminated by heating the mixture in boiling water for 5 min. After centrifugation, the supernatant was carefully collected and lyophilized. Accordingly, the inner-wall protein extract (IW) was subjected to gastrointestinal digestion to obtain a peptide-containing digest, designated IWD. This digest, comprising a mixture of digestion-derived peptides, was used as the test material in subsequent cell-based experiments. The peptide composition of IWD was characterized by LC–MS/MS, as described above.

4.4. Toxicity and Biological Activity Profile Analysis of IWD

The peptide sequences identified by LC–MS/MS from IWD were subjected to in silico pharmacokinetic and safety evaluation using pepADMET (https://pepadmet.ddai.tech/; accessed on 1 July 2026). The predicted parameters included blood–brain barrier (BBB) permeability, Caco-2 intestinal permeability, and toxicity. Only peptides predicted to be BBB-permeable, non-toxic, and to exhibit moderate or higher Caco-2 permeability were selected for subsequent bioactivity analysis [59]. The selected peptides were analyzed using the BIOPEP-UWM database (https://biochemia.uwm.edu.pl/biopep-uwm/; accessed on 4 July 2026) with the “Profiles of Potential Biological Activity” function to predict their putative biological activities based on the occurrence of known bioactive peptide motifs [60]. The predicted activities were summarized according to the frequency of bioactive fragments identified within the selected peptide sequences [61]. The Sankey diagram was visualized by Displayr (https://www.displayr.com/graph-makers/sankey-diagram/; accessed on 5 July 2016).

4.5. Cell Culture

SH-SY5Y human neuroblastoma cell line (CRL-2266; ATCC, Manassas, VA, USA) was cultured and maintained in High-glucose Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were incubated at 37 °C in a humidified incubator with 5% CO2.

4.6. Cytotoxicity Assay of IWD and Okadaic Acid in SH-SY5Y Cell Line

Cells were seeded in 96-well plates at a density of 2 × 104 cells/well in 100 µL of culture medium and incubated for 24 h to allow cell attachment. Subsequently, cells were treated with various concentrations of IWD peptide mixture (1, 2.5, 5, 10, 25, 50, 75, and 100 µg/mL) or okadaic acid (5, 10, 20, 40, 80, and 100 nM). All treatments were performed in a final volume of 100 µL per well and incubated for an additional 24 h under standard conditions [62]. Cell viability was determined using the MTT assay that reflects cellular metabolic activity and was used as an initial viability screen. Briefly, MTT solution was added to each well to a final concentration of 0.5 mg/mL, followed by incubation for 3–4 h to allow viable cells to convert MTT into insoluble formazan crystals. The crystals were then dissolved in DMSO, and absorbance was measured at 570 nm using a microplate reader. Cell viability was expressed as a percentage relative to the untreated control group. The concentrations of all treatments that did not have cytotoxicity were selected for subsequent experiments. All treatments were performed in triplicate to ensure reproducibility.

4.7. Evaluation of the Neuroprotective Effect of IWD Pretreatment

The potential protective effects of IWD against okadaic acid-induced oxidative damage were evaluated using SH-SY5Y cells. Non-cytotoxic concentrations of IWD were selected for the neuroprotective assay against okadaic acid-induced toxicity at a concentration that produced approximately 50% cell death. Cells were seeded in 96-well plates at a density of 2 × 104 cells/well in 100 µL of culture medium and incubated for 24 h to allow cell attachment. Subsequently, cells were pretreated with different concentrations of IWD for 24 h. After the pretreatment period, okadaic acid was added, and the cells were further incubated for 24 h. At the end of the treatment, cell viability was assessed using the MTT colorimetric assay. All treatments were performed in triplicate.

4.8. Intracellular ROS Production Assay

The intracellular production of reactive oxygen species (ROS) in SH-SY5Y cells was evaluated using the fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA). This probe is widely used for detecting oxidative stress within cells because it can diffuse into cells and be converted into a fluorescent compound upon oxidation by intracellular ROS. Cells were seeded in 6-well plates at a density of 5 × 106 cells/well and incubated for 24 h. After incubation, cells were treated in accordance with the experimental design to evaluate the neuroprotective effects of IWD. Following treatment, cells were incubated with DCFH-DA probe for 20 min at 37 °C. During this step, the non-fluorescent DCFH-DA diffuses into cells and is hydrolyzed by intracellular esterases to form DCFH, which is subsequently oxidized by reactive oxygen species (ROS) to generate the fluorescent compound dichlorofluorescein (DCF) [63,64]. Intracellular DCF fluorescence was measured using a DxFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) with CytExpert software (version 2.5; Beckman Coulter, Brea, CA, USA) for analysis. DCFH-DA was used as a general indicator of intracellular oxidative stress. DCF fluorescence reflects oxidation by multiple reactive species and peroxidase-dependent processes and does not identify a specific ROS.

4.9. Statistical Analysis

Data analysis was performed with GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA, USA). Results were presented as mean ± standard deviation (SD) based on three independent experiments. Differences between groups were analyzed with one-way ANOVA (three or more groups). p < 0.05 was considered statistically significant.

4.10. Proteomic Approach

Effects of IWD on tested cells were analyzed using the nano chromatography system (Thermo Dionex Ultimate 3000) equipped with an Acclaim PepMap RSLC C18 column (75 μm × 15 cm, 2 μm particle size, 100 Å pore size. The column temperature was maintained at 35 °C, and the flow rate was set to 300 nL/min. Mobile phase A consisted of 0.1% formic acid in 2% acetonitrile, while mobile phase B consisted of 0.1% formic acid in 80% acetonitrile. The total LC runtime was 155 min. Eluted peptides were analyzed using a TripleTOF 6600+ mass spectrometer (AB SCIEX, USA) equipped with an electrospray ionization (ESI) source. Mass spectrometric analysis was conducted in SWATH acquisition mode, in which precursor ions were sequentially isolated across defined mass windows and fragmented to generate comprehensive MS/MS spectra for all detectable peptides. This DIA strategy enables reproducible and accurate quantification across multiple samples. For quantitative analysis, each sample was analyzed in four technical replicates under identical experimental conditions to ensure reproducibility and reliability of the proteomic data.

4.11. Pathway and Network Analysis

Differentially expressed proteins (DEPs) identified from pairwise comparisons of the OA and control (CT) groups and the IWD + OA and OA groups were subjected to bioinformatics and functional enrichment analyses to investigate the molecular processes associated with OA-induced neurotoxicity and IWD pretreatment. Proteins were considered differentially expressed based on fold changes (FCs) > 1.5 or < 0.66 and p-values < 0.05 in the respective comparisons. Functional enrichment analyses were performed using MetaboAnalyst (https://www.metaboanalyst.ca/; accessed on 1 April 2026). Partial least squares discriminant analysis (PLS-DA) was performed using MetaboAnalyst to explore proteomic differences between the OA and IWD + OA groups. The number of components was selected based on five-fold cross-validation using Q2 as the performance measure, and a one-component model was used to calculate variable importance in projection (VIP) scores. The 20 proteins with the highest VIP scores (>1) were selected as candidate discriminant proteins. Given the limited sample size (four biological replicates per group), the VIP-based findings were interpreted as exploratory.
Protein–protein interaction (PPI) networks were constructed using the STRING database version 12.0 (https://string-db.org/; accessed on 17 April 2026) with a minimum required interaction score of 0.7 (high confidence). Gene Ontology (GO) enrichment, including cellular component (CC), molecular function (MF), and cellular process (CP), KEGG, and Reactome pathway analyses, were conducted to identify significantly represented biological functions and pathways. Enrichment significance was determined based on false discovery rate (FDR)-adjusted p-values, and pathways with FDR < 0.05 were considered statistically significant.
The generated interaction networks were imported into Cytoscape version 3.10.2 for network visualization and downstream analysis. To identify densely connected functional modules within the PPI network, Molecular Complex Detection (MCODE; version 2.0.3) was applied using the following parameters: degree cutoff = 2, node score cutoff = 0.2, K-core = 2, and maximum depth = 100. Hub proteins were subsequently identified using the CytoHubba plugin in Cytoscape based on the maximal clique centrality (MCC) algorithm, where proteins with higher MCC scores were considered key network regulators.

5. Conclusions

This study showed that gastrointestinal-digested Holothuria scabra inner wall proteins (IWD) attenuated OA-induced injury in SH-SY5Y cells, reducing intracellular ROS and preserving cell viability and neuronal morphology, with the strongest protective effect observed at 5 μg/mL. Peptidomic and in silico analyses identified candidate peptides with predicted bioactivities, while differential proteomics revealed treatment-associated changes in pathways related to translation, RNA metabolism, proteostasis, cellular stress responses, and energy metabolism. These findings demonstrate the in vitro protective activity of the IWD peptide mixture in this cellular model and provide a basis for further investigation. However, the contributions of individual peptides and the causal roles of the associated molecular changes remain to be established. Further validation in additional cellular and in vivo models is required to determine their relevance to neurodegenerative disease research.

Author Contributions

Conceptualization, S.N., N.K., and S.S.; methodology, S.N., S.S., and J.N.; software, S.N., S.S., and P.J.; validation, S.N., N.K., S.D. (Supawadee Duangprom), and S.S.; formal analysis, S.N., N.K., and J.N.; investigation, S.N. and S.S.; resources, N.K., S.D. (Supawadee Duangprom), and P.A.; data curation, S.N., N.K., P.S., J.S., and S.D. (Sakda Daduang); writing—original draft preparation, S.N.; writing—review and editing, N.K., S.N., and J.S.; visualization, S.N. and S.S.; supervision, N.K.; project administration, N.K.; funding acquisition, N.K. and S.D. (Supawadee Duangprom). All authors have read and agreed to the published version of the manuscript.

Funding

This research received financial support from the Thailand Science Research and Innovation Fundamental Fund, fiscal year 2025. This study was supported by the Thammasat Postdoctoral Fellowship.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in this article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors are especially grateful to the Research Unit in Innovative Marine Biotechnology and Natural Bio-resources for Sustainable Health and Wellness, Thammasat University, Thailand.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
IWDHolothuria scabra inner wall protein digest
OAOkadaic acid
CTUntreated control cells
DEPsDifferentially expressed proteins
PPIProtein–protein interaction
FCFold change

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