Study of the Cytotoxic and Wound Healing Activity of Polysaccharides from Bovistella utriformis

Abstract

Bovistella utriformis (Bull.) Demoulin & Rebriev is a cosmopolitan puffball mushroom traditionally recognized for its anti-inflammatory and wound-healing properties. This study aimed to evaluate the cytotoxic profile, regenerative potential, and molecular effects of polysaccharides extracted from B. utriformis (PsBU) using complementary in vitro and in vivo approaches. Thermogravimetric analysis revealed good thermal stability up to 300 °C with 26.91% residual mass at 800 °C. Human HaCaT keratinocytes were used to assess cell viability, proliferation, and cell-cycle distribution through MTT assays and flow cytometry, while wound-healing activity was examined using in vitro scratch assays. PsBU exhibited no cytotoxic effects across concentrations from 19.53 to 10,000 µg mL⁻¹, with cell viability remaining above 68%. At 5000 and 10,000 µg mL⁻¹, viability increased to 130% and 126%, respectively. The optimal in vitro wound-healing effect was observed at 500 µg mL⁻¹, achieving 40% wound closure at 32 h. Label-free quantitative proteomic analysis by UHPLC-HRMS identified 83 differentially expressed proteins, including upregulation of tissue repair-related factors such as plasminogen and FHL2, alongside modulation of cell-cycle regulation and mRNA transport pathways. In vivo zebrafish caudal fin regeneration assays demonstrated maximal regenerative activity at 200 µg mL⁻¹ (p < 0.001 vs. control). Overall, these findings demonstrate that B. utriformis polysaccharides are safe bioactive compounds that promote key biological processes involved in tissue regeneration, supporting their potential application as natural wound-healing agents.

1. Introduction

The kingdom Fungi is one of the most diverse, encompassing approximately 150,000 described species [1], although this number could be significantly higher. Present in various habitats, including extreme environments such as Antarctica and the Chernobyl exclusion zone [2,3,4], they serve as key decomposers, driving nutrient turnover and contributing to biogeochemical cycles [5]. In addition to their ecological importance, they have been used for centuries for their nutritional and therapeutic properties, offering nutritional value comparable to that of animal proteins [6,7].

Bovistella utriformis (Bull.) Demoulin & Rebriev (Basidiomycota, Agaricales, Agaricaceae) is a cosmopolitan puffball mushroom traditionally recognized for its anti-inflammatory and wound-healing properties [8]. Its polysaccharides show promising potential in modern medicine for their immunomodulatory properties and ability to promote tissue regeneration [9], particularly in wound healing and oncology contexts. Previous studies have demonstrated that these polysaccharides possess antioxidant, antimicrobial, and anticancer effects [10], which supports their application as therapeutic agents.

Polysaccharides from medicinal mushrooms have been shown to modulate cell-cycle pathways, cell migration, and the innate immune response [11,12]. Much of this research has focused on cancer models, where these compounds can inhibit tumor cell proliferation by regulating key cell cycle checkpoints, inducing apoptosis through mitochondrial pathways and caspase activation, and preventing cancer cell migration [13,14]. These polysaccharides also act as biological response modifiers, stimulating immune cells such as macrophages, dendritic cells, and T cells through receptors including toll-like receptors (TLRs) and dectin-1, leading to enhanced production of pro-inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6) and nitric oxide [15,16,17].

Importantly, the same biological activities observed in cancer contexts, controlled cell proliferation, enhanced cell migration, and immune modulation, are also fundamental to wound healing. During tissue repair, keratinocyte proliferation and migration must be precisely coordinated to achieve effective re-epithelialization, while immune cells orchestrate inflammation resolution and extracellular matrix remodeling [18]. Therefore, polysaccharides capable of modulating these interconnected pathways represent promising candidates for regenerative medicine applications. However, a critical distinction must be made: while tumor cells exhibit uncontrolled proliferation that should be inhibited, wound healing requires regulated proliferation that supports tissue regeneration without inducing harmful hyperproliferation or genomic instability. Thus, evaluating the effects of polysaccharides on cell cycle progression in healthy keratinocytes is essential to assess both their safety and therapeutic potential for skin repair.

To elucidate the molecular mechanisms underlying the biological effects of bioactive compounds, proteomic approaches have proven valuable for identifying differentially expressed proteins and modulated cellular pathways. For instance, proteomic analysis of mushroom polysaccharide-treated cells has successfully revealed key proteins and interaction networks involved in cell cycle regulation, signaling pathways, and cellular responses [19]. Label-free quantitative proteomics, in particular, enables unbiased detection of protein abundance changes in response to treatment, providing insights into cytoskeletal dynamics, migration, and other cellular processes relevant to tissue regeneration [20,21]. Therefore, combining proteomic analysis with functional assays offers a comprehensive strategy to assess both the efficacy and molecular mechanisms of potential therapeutic agents.

In our previous study [10], we characterized the structural and biochemical properties of PsBU, demonstrating that these polysaccharides are predominantly composed of glucose (>80%), with smaller amounts of galactose and mannose, and possess thermal stability suitable for pharmaceutical applications. FTIR spectroscopy confirmed the presence of characteristic polysaccharide functional groups with β-glycosidic linkages. PsBU exhibited significant antioxidant activity in ABTS and DPPH assays, antihyperglycemic potential in vitro and in vivo, and selectively promoted HaCaT keratinocyte proliferation without inducing cytotoxicity, indicating a regenerative response. Building on these findings, we hypothesized that PsBU could exert wound-healing activity through coordinated modulation of cell proliferation and migration pathways. However, the molecular mechanisms underlying these effects remain poorly understood, and no previous studies have combined proteomic analysis with both in vitro and in vivo regenerative models to comprehensively evaluate the therapeutic potential of B. utriformis polysaccharides.

Accordingly, the present study combines cell cycle analysis, proteomic profiling, in vitro scratch assays, and in vivo zebrafish fin regeneration assays to elucidate the molecular mechanisms and wound-healing potential of PsBU. This integrated approach aims to identify key proteins and pathways modulated by PsBU treatment, assess its safety in healthy keratinocytes, and evaluate its therapeutic efficacy in tissue repair models.

2. Results

2.1. Thermal Gravimetric Analysis (TGA) of PsBU

The thermal stability of the PsBU was evaluated by TGA and DTG analysis (Figure 1). The TGA curve shows two main weight loss events. The first stage occurs at approximately 166.5 °C, corresponding to a minor mass loss due to moisture evaporation, as evidenced by the DTG peak. This initial loss reflects the intrinsic water content of the sample rather than decomposition of the polysaccharide structure. The second, more significant degradation occurs around 299.5 °C, which is associated with the breakdown of the polysaccharide backbone. After heating to 800 °C, the residual mass was 26.91%, indicating that a substantial fraction of the sample is thermally stable inorganic residue, likely corresponding to mineral components. Overall, the TGA demonstrates that PsBU exhibits good thermal stability up to approximately 300 °C.

2.2. MTT Cytotoxicity Assay

The cytotoxicity and potential proliferative effects of PsBU were evaluated on human keratinocytes (HaCaT) using the MTT assay (Figure 2). Cells were exposed to a range of concentrations from 19.53 to 10,000 µg mL⁻¹ for 72 h. No cytotoxic effects were observed across the tested concentrations, with cell viability consistently remaining above 68%. In fact, a concentration-dependent proliferative effect was noted at higher doses, particularly at 5000 µg mL⁻¹ and 10,000 µg mL⁻¹, where cell viability increased to 130% and 126%, respectively, compared to untreated controls. The IC₅₀ value could not be determined because the extract did not reduce viability below 50% at any concentration, indicating its safety for keratinocytes. The minimum effective concentration tested was 19.53 µg mL⁻¹, while the maximum was 10,000 µg mL⁻¹. These observations suggest that the extract may promote keratinocyte proliferation, supporting its potential use in skin regeneration and wound healing applications. Importantly, the observed maximal proliferation did not lead to abnormal or excessive cell growth under the tested conditions, indicating that PsBU promotes regenerative proliferation without inducing harmful hyperproliferation.

2.3. Proteomic Study of the Effect of Polysaccharides

Label-free quantitative proteomic analysis identified a total of 1829 proteins with a false discovery rate (FDR) < 1%. Among these, 83 proteins showed statistically significant differences in abundance between PsBU-treated and control HaCaT cells, including 42 proteins with increased abundance and 41 proteins with decreased abundance (Supplementary Table S1). Figure 3 provides a global overview of the magnitude and statistical significance of abundance changes induced by PsBU. Proteins located far to the left or right show the largest decreases or increases in abundance, respectively, whereas proteins higher on the plot are supported by stronger statistical evidence. Importantly, the plot shows that the differentially abundant proteins are not restricted to a single extreme outlier region but are distributed across both directions of change, indicating a balanced proteomic remodeling rather than an artefact driven by one or a few proteins. The selected thresholds (p < 0.05 and |log₂ ratio| ≥ 1) define the subset of proteins used for the functional interpretation described below.

A summary of selected differentially abundant proteins highlighted in this section, including their main functions and relevance to wound healing-related processes, is presented in

Table 1. Selected differentially abundant proteins in PsBU-treated HaCaT cells and their relevance to wound healing–related processes. Upward and downward arrows indicate increased or decreased protein abundance, respectively, in PsBU-treated cells compared with untreated controls. Proteins were selected from the quantitative proteomic dataset (Supplementary Table S1) as representative markers of pathways relevant to keratinocyte migration, cytoskeletal remodeling, RNA processing, nucleocytoplasmic transport, extracellular matrix remodeling, and broader cellular remodeling associated with wound closure.

Protein (Full Name)	Gene	Main Function (Brief)	Wound Healing-Related Process	Change
Nucleoporin NDC1	NDC1	Nuclear pore complex component; nuclear envelope anchoring	Nucleocytoplasmic transport supporting stress/adaptation programs	↑
Mitotic checkpoint serine/threonine-protein kinase BUB1B	BUB1B	Spindle checkpoint control; mitotic fidelity	Cell-cycle control influencing re-epithelialization balance	↑
Kinesin-like protein KIFC1	KIFC1	Microtubule-based motor; spindle/transport dynamics	Cytoskeleton and intracellular transport	↑
Zinc finger RNA-binding protein	ZFR	RNA binding; post-transcriptional regulation	mRNA metabolism affecting migration/repair programs	↑
Vasodilator-stimulated phosphoprotein	VASP	Actin polymerization and focal adhesion dynamics	Keratinocyte migration and wound closure	↑
Serine/arginine-rich splicing factor 3	SRSF3	Pre-mRNA splicing regulation	RNA processing linked to repair-associated gene expression	↑
Plasminogen	PLG	Precursor of plasmin; fibrinolysis/ECM remodeling	Matrix remodeling and re-epithelialization	↑
Four-and-a-half LIM domain protein 2	FHL2	Cytoskeletal adaptor; mechanotransduction	Migration/adhesion remodeling during wound closure	↑
Myosin regulatory light chain 12B	MYL12B	Actomyosin contractility regulation	Cytoskeletal tension and migration dynamics	↓
60S ribosomal protein L19	RPL19	Ribosomal large subunit component	Translation-related processes (general cellular remodeling)	↓

↑ Indicates upregulated proteins; ↓ indicates downregulated proteins in PsBU-treated cells compared to untreated controls.

.

Principal component analysis (PCA) provided a global overview of sample similarity across all quantified proteins and revealed a clear separation between PsBU-treated and control samples along PC1, which explained more than 40% of the total variance, indicating that treatment accounted for a major fraction of the proteomic variability and produced a consistent global shift in the proteomic profile (Figure 4A). Consistently, unsupervised hierarchical clustering based on normalized protein abundances grouped samples according to treatment condition and displayed coherent abundance patterns within each group, further supporting the reproducibility of the PsBU-associated proteomic signature across biological replicates (Figure 4B).

Several proteins involved in cell cycle progression and mitotic processes displayed markedly increased abundance. Notably, the mitotic checkpoint kinase BUB1B exhibited a fold change (FC) of 6.25, while the kinesin-related motor protein KIFC1 showed an FC of 4.43. Cyclin K (CCNK) also presented increased abundance (FC = 2.21), consistent with alterations in cell cycle-associated protein networks.

Proteins associated with nucleocytoplasmic transport and nuclear pore organization were prominently affected. Nucleoporin NDC1 showed the highest increase in abundance among all identified proteins (FC = 11.72), while NUP85 exhibited an FC of 2.07. In contrast, NUP210 displayed a marked decrease in abundance (FC = 0.35), indicating differential modulation of nuclear pore complex components.

Several RNA-binding and mRNA-processing proteins showed increased abundance, including SRSF3 (FC = 2.86), U2AF1 (FC = 2.14), and ZFR (FC = 3.15), indicating coordinated changes in the abundance of proteins involved in RNA splicing and mRNA metabolism. STRING protein–protein interaction analysis revealed a significantly enriched interaction network (PPI enrichment p = 0.00885), indicating that the proteins increased in abundance after PsBU treatment are functionally connected beyond random expectation rather than representing an unrelated list. The network highlighted a coherent module dominated by factors involved in mRNA processing/export and nucleocytoplasmic transport, including nucleoporins, consistent with coordinated changes in RNA handling and cellular adaptation programs relevant to migration and repair (Figure 5).

Proteins linked to cytoskeletal dynamics and intracellular transport were also affected. Vasodilator-stimulated phosphoprotein (VASP), a key regulator of actin filament assembly and cell motility, showed increased abundance (FC = 2.87), while dynein light chain DYNLRB1 exhibited an FC of 2.04. Conversely, myosin regulatory light chain MYL12B showed decreased abundance (FC = 0.46).

Notably, proteins directly implicated in tissue repair and wound healing exhibited increased abundance following PsBU treatment. Plasminogen (PLG), involved in fibrin matrix remodeling, showed an FC of 2.82, while four-and-a-half LIM domain protein 2 (FHL2), associated with keratinocyte migration and wound repair, exhibited an FC of 2.23.

In contrast, several ribosomal and translation-related proteins showed decreased abundance, including RPL7 (FC = 0.46), RPL19 (FC = 0.27), RPS13 (FC = 0.47), and MRPS10 (FC = 0.25), indicating a reduction in ribosome-associated protein abundance in PsBU-treated cells.

Overall, the proteomic profiling indicates that PsBU treatment induces a consistent and reproducible global remodeling of the HaCaT proteome. Beyond individual protein changes, the enriched interaction patterns point to coordinated modulation of functionally connected modules, including nucleocytoplasmic transport and mRNA processing/export, together with proteins linked to cytoskeletal organization and cell motility. These proteomic signatures provide a supportive mechanistic context for the phenotypic effects described in the subsequent sections.

2.4. Cell Cycle Analysis by Flow Cytometry in Cell Line HACAT

In the negative control (C−), the Sub-G1 population was minimal (1.17%), indicating low levels of spontaneous apoptosis, while G0/G1 (14.22%) and G2/M (13.66%) phases were well represented and balanced, reflecting normal proliferative behavior under untreated conditions. As the S-phase was not gated separately, the reported percentages represent partial cell-cycle distributions and do not sum to 100%. In contrast, the positive control (C+) exhibited a marked increase in Sub-G1 cells (4.73%), indicative of apoptosis induction, alongside an accumulation in the G2/M phase (19.32%). Upon treatment with the PsBU extract at concentrations ranging from 250 to 1250 µg mL⁻¹, the Sub-G1 population remained low (1.49–1.96%), with no significant increase relative to the negative control. The G0/G1 phase distribution remained stable (~12–13%) across all tested doses. A slight decrease in the G2/M phase was observed at the highest concentration (from 11.34% to 9.50%), without evidence of G2/M accumulation or increased Sub-G1 population (

Table 2. Effects of PsBU Treatment on Cell Cycle Distribution and Apoptotic Sub-G1 Population in HaCaT Cells Assessed by Flow Cytometry.

Treatment	% P2 (Sub-G1)	% P3 (G0/G1)	% P4 (G2/M)
C− (Negative Control)	1.17	14.22	13.66
C+ (Positive Control) a	4.73	4.31	19.32
PsBU 250	1.60	12.94	11.34
PsBU 500	1.96	12.85	11.90
PsBU 750	1.49	12.47	11.39
PsBU 1250	1.69	11.89	9.50

^a 20 µM 2-methoxyestradiol. PsBU concentrations are expressed in µg mL⁻¹. C−: negative control (untreated cells); C+: positive control (20 µM 2-methoxyestradiol). Sub-G1 values < 2% indicate absence of apoptosis induction.

). Representative flow cytometry plots for the negative control (C−), positive control (C+), and PsBU-treated cells at 500 µg mL⁻¹ are shown in Figure 6 to illustrate the gating strategy and DNA content distribution. Complete flow cytometry datasets corresponding to all tested PsBU concentrations (250, 500, 750, and 1250 µg mL⁻¹), including FSC/SSC plots, PI-A histograms, and PI-W distributions, are provided in the Supplementary Material (S3) for detailed examination.

2.5. Scratch Wound Healing Assay

The wound closure ability of PsBU was assessed in HaCaT cells using a scratch assay over a period of 32 h. As shown in Figure 7, PsBU treatment modulated scratch closure compared to the untreated control. At 32 h, the control group exhibited a reduction in scratch width of 37%, while treatment with 250 µg mL⁻¹ and 750 µg mL⁻¹ PsBU resulted in 36% and 35% closure, respectively. Notably, treatment with 500 µg mL⁻¹ PsBU showed the highest healing activity, achieving a 40% reduction in scratch width. These results indicate an optimal pro-regenerative effect at 500 µg mL⁻¹, while lower and higher concentrations did not further enhance scratch closure compared to the control. Representative images of scratch progression at 0, 24, and 32 h under different treatment conditions are shown in Figure 8. Maximal closure is observed at 500 µg mL⁻¹ PsBU, which is significantly different from any other concentration (*: p < 0.05, **: p < 0.01; T-test). Collectively, these findings indicate that PsBU effectively promotes wound healing in vitro by accelerating keratinocyte migration and scratch closure at 500 µg mL⁻¹.

2.6. Effects of PsBU on Caudal Fin Regeneration

In Figure 9, 72 hpa regenerated fin areas (µm²) are compared with respect to the concentrations of polysaccharides and control treatments. The highest significant difference (***, p < 0.001) was observed between the regenerated area at 200 µg mL⁻¹ and the untreated control. This indicates potent fin regeneration-promoting activity at this PsBU concentration. The promoting effect was maximal at 200 µg mL⁻¹ and decreased at both lower and higher concentrations (Figure 9).

Representative images of zebrafish caudal fins obtained during the regeneration assay are shown in Figure 10.

Our results strongly suggest that 200 µg mL⁻¹ PsBU in E3 medium behaves as an optimal dose for the promotion of fin regeneration in zebrafish larvae. Any other concentration shows a lower promotion of the regeneration effect.

3. Discussion

The thermal decomposition profile of PsBU reveals important insights into its structural integrity and potential applications. The initial weight loss observed at ~166.5 °C is primarily due to the evaporation of physically adsorbed water and low-molecular-weight volatiles. This type of event is characteristic of natural polysaccharides, as they possess hygroscopic properties due to abundant hydroxyl groups, which promote water retention through hydrogen bonding [21]. Such behavior is consistent with that of other fungal polysaccharides, in which the first thermal transition is typically associated with bound water loss rather than polymer decomposition [22].

The major degradation event detected at ~299.5 °C corresponds to the breakdown of the polysaccharide backbone. This stage involves cleavage of glycosidic linkages and depolymerization of the carbohydrate matrix, leading to rapid weight loss of volatile products such as CO and CO₂, and small organic compounds are released [23]. The relatively high onset of this degradation step indicates that the extract possesses good thermal stability compared to some plant-derived polysaccharides, which often degrade at lower temperatures (200–280 °C) [23]. Comparatively, this degradation onset temperature (~299.5 °C) matches other macromycete polysaccharides, such as β-glucans from Lentinula edodes [24] (shiitake, 285–305 °C) and polysaccharides from Ganoderma lucidum [25] (reishi, ~292 °C), confirming PsBU’s competitive thermal stability among macrofungal biopolymers. Such stability may be advantageous for potential biotechnological applications where moderate heating processes are required, including the formulation of biomaterials or nutraceuticals. The final residual mass of ~26.91% at 800 °C was observed.

In summary, the TGA profile indicates that PsBU maintains structural integrity up to ~300 °C, which supports its potential use in food, pharmaceutical, and biomedical applications where thermal processing is required.

The in vitro wound-healing assay performed on HaCaT keratinocytes demonstrated that PsBU significantly enhanced scratch wound closure compared with untreated cells, with the greatest effect observed at 500 µg mL⁻¹. After 24 h, cells treated with PsBU exhibited migratory activity, resulting in rapid coverage of the scratch width. This response reflects the ability of PsBU to stimulate keratinocyte proliferation and migration, both of which are critical steps in the early phase of wound healing [26].

At 32 h, following PBS washing to remove non-adherent cells, the closure process was dominated by firmly attached keratinocytes originating from the wound edges. The cells treated with 500 μg mL⁻¹ PsBU progressively migrated and bridged the scratch gap more quickly than control or other PsBU concentration treatments. This event that resembles re-epithelialization and regeneration has also been induced by polysaccharides from other origins [27]. The transition from proliferative coverage at 24 h to adhesive regenerative closure at 32 h highlights the coordinated effect of PsBU polysaccharides in promoting both cell expansion and tissue stabilization.

The significant increase in wound closure across all treated groups (p < 0.05), with a peak effect at 500 µg mL⁻¹, confirms that PsBU enhances skin cell regenerative capacity in a dose-responsive manner. These results are consistent with the wound-healing properties of β-glucan-rich polysaccharides from Ganoderma lucidum and Lentinus edodes [28,29]. However, while those studies primarily evaluated proliferation endpoints, our combined proteomic and functional approach reveals that PsBU specifically modulates proteins involved in both cell migration (VASP, FHL2) and matrix degradation (plasminogen), providing a more comprehensive molecular basis for the observed regenerative effects. Notably, the absence of apoptosis induction in our flow cytometry analysis further corroborates the regenerative effect, reinforcing the safety and therapeutic potential of PsBU.

To further elucidate the molecular mechanisms underlying these regenerative effects, a comparative proteomic analysis was performed to identify key proteins and pathways involved. The observed over-expression of BUB1 points to reinforced mitotic surveillance that prevents aneuploidy, in line with its established roles: loss of BUB1/BubR1 favours tumorigenesis, whereas elevated levels prolong healthy lifespan in murine models [30]. The pronounced down-regulation of several ribosomal proteins supports the notion of a global inhibition of protein synthesis, an antimitogenic mechanism similar to that caused by the ribosome-inactivating protein calcaelin isolated from this same fungus [31]. Concurrent changes in nucleoporins (NUP210, NUP85, NDC1) suggest altered mRNA export and nucleocytoplasmic trafficking, potentially impacting gene expression during the cellular response to treatment [32].

Meanwhile, the over-expression of VASP and plasminogen provides a molecular basis for the pro-healing effects observed: VASP enhances actin-cytoskeleton dynamics and accelerates the cell migration required for wound closure [33], whereas increased plasminogen facilitates fibrin-matrix remodelling and re-epithelialisation [34]. Notably, FHL2 is likewise up-regulated; this multifunctional protein is induced during wound repair, promotes the transition of keratinocytes to a contractile myofibroblast-like phenotype, and supports controlled cell proliferation [35].

Taken together, these findings indicate that PsBU triggers a biphasic response: they transiently slow cell division, potentially preventing disordered proliferation or tumorigenesis, while simultaneously stimulating tissue-repair processes. Such dual action is characteristic of many medicinal-mushroom polysaccharides, which serve as biological modulators that suppress pathological cell growth while enhancing regeneration and innate immune responses [11,36]. In particular, previous studies have reported immune activation with increased pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in response to Trametes versicolor polysaccharides [19]. Such immunomodulatory effects could plausibly cooperate with the protein changes observed here to support wound repair and host defense; however, cytokine levels were not quantified in the present study. Thus, our results integrate experimental observations (enhanced keratinocyte migration and macrophage activation) with specific proteomic changes, providing a mechanistic explanation for the wound healing and immunomodulatory effects of PsBU.

These molecular and immunological findings are coherently supported by the cell cycle analysis in HaCaT keratinocytes exposed to PsBU. Flow cytometry of PI-stained cells showed that PsBU did not induce apoptosis, as indicated by a consistently low Sub-G1 fraction (1.4–1.9%) across all concentrations, comparable to untreated controls and consistent with basal levels observed in non-apoptotic cells (typically < 2%) [37]. The preservation of the G0/G1 fraction (~12–13%) and the absence of G2/M accumulation indicate that PsBU does not cause cell cycle arrest at either the checkpoint entry or the post-replication phase, supporting a profile compatible with ongoing proliferation rather than cytostatic stress.

These results align with previous studies reporting that mushroom-derived polysaccharides generally display low cytotoxicity toward normal mammalian cells while retaining robust bioactivity. For instance, mushroom beta-glucans from Ganoderma lucidum [38] maintained high viability in non-tumor epithelial cells at concentrations up to 0.5–1.0 mg mL⁻¹, with only minor reductions in cell number, supporting their biocompatibility with normal tissue. Similarly, polysaccharide-rich extracts or fractions from macrofungi are safe for human skin keratinocytes at concentrations in the 1–5 mg mL⁻¹ range, where they can significantly enhance cell metabolic activity and support regenerative responses rather than trigger apoptosis [39]. In this context, PsBU concentrations up to 1.25 mg mL⁻¹ maintained a Sub-G1 fraction below 2% and an unchanged G0/G1 population, indicating preserved cell viability and a non-cytotoxic, pro-regenerative profile.

The pro-proliferative and wound-healing properties observed in the scratch assay are also in line with independent reports on fungal polysaccharides and related bioactive fractions in HaCaT cells. Polysaccharide fractions from medicinal mushrooms such as Ganoderma lucidum oil or polysaccharide-enriched preparations have been shown to accelerate wound closure by promoting HaCaT proliferation and migration, in some cases via activation of signaling pathways such as TGF-β/SMAD that orchestrate the transition from inflammation to the proliferative phase of skin repair. The TGF-β/SMAD signaling pathway plays a central role in cutaneous wound healing by regulating keratinocyte behavior. In particular, TGF-β signaling promotes keratinocyte migration and re-epithelialization through modulation of cytoskeletal dynamics, cell–matrix interactions, and extracellular matrix remodeling, while limiting excessive proliferation [40]. In this context, the proteomic changes observed in the present study are consistent with molecular processes that support keratinocyte motility during wound closure. Likewise, mushroom-derived polysaccharide fractions from species such as Coriolus (Trametes versicolor) were reported to enhance HaCaT migration and re-epithelialization, achieving up to ~95% wound closure at optimized concentrations, compared with ~66% in untreated controls, highlighting the capacity of these glycoconjugates to stimulate keratinocyte-driven repair. The scratch assay results obtained for PsBU, together with the absence of apoptosis induction in flow cytometry, thus support the view that this extract promotes wound closure primarily through enhanced keratinocyte proliferation and migration rather than compensatory responses to cell death [41].

Taken together, the concordance between cell cycle homeostasis, low Sub-G1 levels, and improved scratch closure suggests that PsBU behaves as a safe, bioactive polysaccharide fraction that stimulates skin cell renewal and tissue repair. This profile mirrors the broader literature, where fungal polysaccharides are increasingly recognized as promising candidates for topical regenerative formulations due to their combination of biocompatibility, immunomodulatory activity, and ability to support keratinocyte proliferation and migration [42]. Within this framework, PsBU can be positioned as a mushroom-derived polysaccharide preparation with a safety and efficacy profile comparable to that of well-studied polysaccharides from Ganoderma lucidum, Pleurotus species, and other medicinal macrofungi, reinforcing its potential as a regenerative topical agent for skin repair applications [38,39,43].

These in vitro findings are further strengthened by the results obtained from our in vivo regeneration assay. Among several other assays, zebrafish fin regeneration is emerging as a very useful drug screening assay [44]. The fin fold of zebrafish larvae is a keratinocyte folded structure exclusively supported by collagen fibrils and scarce migrating fibroblasts [45]. Fin regeneration assays with zebrafish larvae have been used to test regenerative promotion activities of biomolecules [41,42]. Different molecules from medicinal plants, for instance, i.e., phenols, saponins, flavonoids, alkaloids, or others, have been satisfactorily tested in zebrafish fin regeneration [46]. Algal polysaccharides have also been extensively tested in this assay [47], which is emerging as an excellent anti-inflammatory, immunomodulatory, anti-oxidative, or pro-regenerative drug screening technique [46,47]. Even mushroom glucosamine hydrochloride has been tested in a zebrafish fin regeneration assay searching for anti-osteoporotic drugs [48].

Polysaccharides extracted from Spirulina maxima have been shown to promote wound healing and fin regeneration, activating gene transcription of several signaling and chemokine pathways [49]. Similar effects have been found using polysaccharides from Ulva rigida, the ulvans (preliminary results from our group), further supporting the existence of this property. Moreover, algal polysaccharides also show anti-inflammatory properties after a zebrafish fin cut assay, activating neutrophil migration and NO or ROS expression [50,51]. These results agree with our finding that 3-day treatment with 200 µg/mL PsBU activates fin fold regeneration in 72 hpf zebrafish larvae, a concentration lower than the optimal 500 µg/mL observed in keratinocyte scratch assays. This difference likely reflects enhanced bioavailability or metabolic processing in the whole-organism system compared to isolated cell cultures, as well as inherent differences in the regenerative mechanisms between zebrafish tissue regeneration and human keratinocyte wound healing. This is also consistent with the activation of pro-inflammatory cytokines, mRNA export, nucleocytoplasmic trafficking, and wound healing observed in our in vitro proteomics study of PsBU-treated HaCaT cells.

Furthermore, extracted polysaccharides from algae also reduce uncontrolled cell proliferation of tumors in culture and affect zebrafish larva growth during development [52,53,54,55,56,57]. This further agrees with the conclusion of reinforced mitotic surveillance and tumorigenesis prevention drawn from our proteomics results. Finally, the relevant involvement of cell migration, apoptosis and extracellular matrix remodelling during fin regeneration of zebrafish larvae [58,59,60] corroborates the molecular conclusions of PsBU-stimulation of keratinocyte migration, controlled apoptosis or matrix remodelling in this study.

The regenerative activity of PsBU is thus consistent with previous findings for other fungal polysaccharides. β-glucans from Ganoderma lucidum similarly promoted tissue repair in zebrafish wound models [29], while polysaccharides from Trametes versicolor accelerated fin regeneration through activation of macrophage-mediated immune responses [37]. Future studies must be focused on deciphering the exact molecular pathways involved in these promotion effects in zebrafish to facilitate future drug screening of mushroom biomass. Future studies should focus on deciphering the molecular pathways underlying these regenerative effects in zebrafish, including direct assessment of immunomodulatory mediators (e.g., TNF-α, IL-1β, IL-6), to facilitate future drug screening of mushroom biomass.

4. Materials and Methods

4.1. Biological Material

The biological material and polysaccharide extraction procedures in this study are identical to those described in Maaloul et al. (2025) [10]. Fruiting bodies of B. utriformis were collected in the Magallanes Region, southern Chile (54°04′00.8″ S, 68°57′27.0″ W), between January and April 2022, as part of systematic field sampling campaigns conducted across south Patagonia. Collected specimens were immediately frozen, then lyophilized and stored under controlled conditions before transport for polysaccharide extraction and biological evaluation. The polysaccharide extraction was carried out following the method described by Abdala et al. (2010) [61], as previously applied in Maaloul et al. (2025) [10], yielding the polysaccharide fraction from Bovistella utriformis (syn. Calvatia utriformis) PsBU. Briefly, the dried fungal biomass was treated three times with absolute ethanol to remove pigments, then resuspended in distilled water (1:10 w/v) and boiled for 1 h with continuous stirring. After centrifugation, phenolic compounds were removed using PVPP, and polysaccharides were precipitated with cold absolute ethanol. The precipitate was collected by centrifugation, frozen at −80 °C, and lyophilized. PsBU was prepared as previously described [10] to ensure methodological consistency and comparability of results.

4.2. Thermal Stability Analysis of Polysaccharide PsBU

The thermal stability of the PsBU was evaluated using a thermogravimetric analyzer (TGA 209 IRIS, Netzsch, Selb, Bavaria, Germany) at the Thermal Analysis Laboratory. Approximately 5–10 mg of the sample was weighed, and the mass of the empty alumina crucible was recorded before analysis. The crucible containing the sample was carefully placed on the thermocouple of the balance system. Measurements were carried out under a controlled nitrogen atmosphere, following a heating program from 30 to 550 °C at a rate of 10 °C/min. The experiment lasted approximately 60 min. Thermogravimetric analysis was used to determine the onset decomposition temperature, maximum decomposition temperature, and residual mass of the polysaccharide sample.

4.3. Cell Culture

In this investigation, HaCaT cells (ATCC, Manassas, VA, USA) were employed and routinely cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Biowest, Nuaillé, France), supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 5 mL penicillin-streptomycin, and 2.5 mL amphotericin B. Incubation was performed at 37 °C in a humidified environment with 5% CO₂. Cell harvest occurred at 80% confluency [10].

4.4. MTT Assay

For the MTT assay, cells were exposed to sample concentrations ranging from 20 mg mL⁻¹, followed by serial dilutions down to approximately 20 µg mL⁻¹ to cover a broad range of doses and identify the concentrations inducing maximal biological responses. Following this, the diluted samples were then mixed 1:1 with the cell suspension and plated into 96-well microplates, followed by incubation for 72 h at 37 °C in a humidified atmosphere with 5% CO₂. HaCaT cells were seeded at a concentration of 2.5 × 10⁵ cells mL⁻¹, and blank controls (wells without cells) as well as negative controls (non-treated cells) were included in each plate. Cell proliferation was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Sigma-Aldrich, St. Louis, MO, USA) [10].

In summary, an MTT stock solution (5 mg mL⁻¹ in phosphate-buffered saline) was prepared and subsequently diluted 1:10 in DMEM. A volume of 100 µL of this working solution (0.5 mg mL⁻¹) was then added to each well, and the plates were incubated at 37 °C for 4 h. Metabolically viable cells reduced the yellow MTT tetrazolium salt to form insoluble purple formazan crystals. These crystals were dissolved with acid-isopropanol (150 µL of 0.04 N HCl2-propanol) and measured spectrophotometrically at 550 nm using a MicroPlate Reader 2001 (Whittaker Bioproducts, Palatine, IL, USA). Cell viability was expressed as the mean percentage of viable cells compared to untreated cells. All experiments were conducted in quadruplicate.

4.5. Cell Cycle Analysis by Flow Cytometry

The cell-cycle distribution of HaCaT keratinocytes was evaluated by flow cytometry [62]. Briefly, 5 × 10⁵ cells were seeded into 6-well plates, with a final working volume of 1.5 mL per well, and maintained at 37 °C in a humidified incubator with 5% CO₂ until sub-confluence was reached. Once the appropriate density was achieved, the cells were detached, centrifuged, and reseeded in fresh 6-well plates for treatment with different concentrations of PsBU, previously dissolved in complete culture medium. Four PsBU concentrations were selected for the assay (250, 500, 750, 1250 µg mL⁻¹). A 20 µM solution of 2-methoxyestradiol (Sigma-Aldrich, M6383) served as the positive control.

Following the addition of treatments, the cultures were incubated for 16 h under standard conditions. Cells were then collected, centrifuged, washed with PBS, and fixed in 70% ethanol for 1 h at −20 °C. After fixation, the pellets were washed twice with PBS and resuspended in a staining solution containing propidium iodide (40 µg mL⁻¹; Sigma-Aldrich, P4864) and RNase A (0.1 mg mL⁻¹; Sigma-Aldrich, R6513) in PBS. Samples were incubated for 30 min at 37 °C in the dark.

Flow cytometry data were acquired on a FACS VERSE^TM flow cytometer (BD Biosciences, San Jose, CA, USA), recording 10,000 events per sample. Debris was excluded based on forward scatter (FSC-A) versus side scatter (SSC-A) parameters, and singlet cells were discriminated to exclude doublets or aggregates using propidium iodide (PI) pulse geometry (PI-A versus PI-W). Cell-cycle analysis was performed using PI staining detected in the PE/PI channel (filter 586/42, 560LP), and the distribution of cells across Sub-G1, G0/G1, and G2/M phases was quantified on the singlet-gated population using DNA content histograms with BD FACSuite software v1.0.6.

4.6. Proteomic Analysis (UHPLC-HRMS for Differential Protein Expression in Treated HaCaT Cells)

A proteomic analysis using ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) was performed on HaCaT cells to evaluate the effects of polysaccharides derived from PsBU on global protein expression profiles.

4.6.1. Cell Treatment and Protein Extraction

PsBU polysaccharides were dissolved in sterile distilled water to prepare stock solutions, sterilized by filtration (0.22 µm), and diluted to the required final concentrations in serum-free DMEM immediately before cell treatment. For proteomic profiling, HaCaT cells were exposed to PsBU under the same serum-free conditions used for the scratch assay and incubated for 24 h prior to protein extraction.

Proteomic analyses were performed using three independent biological replicates per condition (untreated controls and PsBU-treated HaCaT cells), each corresponding to an independent cell culture and protein extraction. No technical replicates at the UHPLC–HRMS level were included. HaCaT cells were incubated for 24 h under two experimental conditions: untreated controls and cells treated with 500 µg mL⁻¹ of PsBU.

Following treatment, cells were washed with ice-cold phosphate-buffered saline and lysed in RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) supplemented with Pierce Universal Nuclease (Thermo Fisher Scientific, Waltham, MA, USA). Lysates were subsequently sonicated to ensure complete protein release.

Protein precipitation was performed using the Clean-Up Kit (GE Healthcare, München, Germany), and pellets were resuspended in Milli-Q water. Protein concentration was quantified using the bicinchoninic acid (BCA) assay, and all samples were normalized to a final concentration of 1 µg mL⁻¹.

Protein samples were then subjected to in-gel enzymatic digestion. Briefly, the protein solution was immobilized within a polyacrylamide gel matrix, reduced with dithiothreitol, and alkylated using iodoacetamide. Proteolytic digestion was carried out overnight using trypsin (Pierce Trypsin Protease, Thermo Fisher Scientific, Waltham, MA, USA). The resulting peptides were extracted from the gel and purified using C18 ZipTip columns (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s protocol.

Peptide concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and all samples were normalized before UHPLC-HRMS injection.

4.6.2. Liquid Chromatography–High-Resolution Mass Spectrometry

The Ultra-High-Performance Liquid Chromatography coupled to High-Resolution Mass Spectrometry (UHPLC-HRMS) analysis was carried out as described elsewhere (Rojas-Velis et al., 2025) [63]. Briefly, peptide samples were injected into an Easy-nLC 1200 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

Peptides were initially loaded onto a trap column and subsequently separated on a 50 cm analytical column using a 180 min linear gradient at a flow rate of 300 nLmin⁻¹. Mass spectrometric data were acquired in positive electrospray ionization mode using a data-dependent acquisition (DDA) method to obtain the corresponding MS/MS spectra.

4.6.3. Data Analysis

The acquired raw data were analyzed using the Proteome Discoverer^TM 2.5 platform (Thermo Fisher Scientific, Waltham, MA, USA). MS/MS spectra were identified using the Sequest HT^® search engine against the Homo sapiens Swiss-Prot subset of the UniProt database. Protein identifications were validated using the Percolator^® algorithm with a strict false discovery rate (FDR) threshold of 1%.

Label-free quantification was performed using the Minora Feature Detector within Proteome Discoverer^TM 2.5, with abundances determined from precursor ion intensities. Normalization across samples was conducted based on total peptide content.

Protein abundance ratios obtained from three independent biological replicates per condition were evaluated using a one-way analysis of variance (ANOVA) applied to the whole protein dataset. Differentially expressed proteins were defined as those with p < 0.05. To classify proteins as over- or under-expressed following treatment, log₂-transformed abundance ratios were used: values > 1 indicated upregulation, whereas values < −1 indicated downregulation.

Bioinformatic characterization of significantly deregulated proteins was performed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) (https://www.string-db.org, accessed on 31 May 2023) to assess protein–protein interaction networks. Functional pathway enrichment was evaluated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/pathway.html, accessed on 31 May 2023).

4.7. In Vitro Wound-Healing Assay

The wound healing assay was performed to evaluate the in vitro regeneration of a wound created on a monolayer of HaCaT keratinocytes, assessing the potential of PsBU to accelerate wound closure. HaCaT cells were seeded in a 6-well plate (3 replicates for the control and 3 for each tested concentration) at a density of 2.5 × 10⁵ cells/mL in 2 mL of complete DMEM (4.5 gL⁻¹ glucose, 4 mM L-glutamine, 10% FBS, and 1% P/S) and incubated at 37 °C with 5% CO₂ for 24 h to allow monolayer confluence. After incubation, the medium was removed, and the wells were gently washed with PBS. A cross-shaped wound was created in each well using a sterile P100 pipette tip, guided by the plate lid to ensure straight lines. Wells were washed twice with 1 mL of PBS to remove detached cells. Control wells received 2 mL of serum-free DMEM, while treated wells received 2 mL of PsBU solutions at 250 µg mL⁻¹, 500 µg mL⁻¹, and 750 µg mL⁻¹, prepared in serum-free DMEM. Cell migration was evaluated by direct comparison between PsBU-treated cells and untreated control cells cultured under identical conditions. No additional positive migration control was included, as the aim of the assay was to assess the relative effect of PsBU treatment on wound closure under basal conditions. Images of the wounds were taken immediately (time 0) and at 4, 8, 24, and 32 h over the same position in the well using an inverted optical microscope (Eclipse Ti, Nikon, Tokyo, Japan) equipped with a digital camera (DS-Ri2, Nikon, Tokyo, Japan). Image analysis was performed using ImageJ software (version 1.54g) to quantify the non-healed wound area or width over time. Wound width was measured six times per photograph using an overlaid grid mesh. Percentage of wound closure was measured following the equation:

$$\mathbf{W}\mathbf{o}\mathbf{u}\mathbf{n}\mathbf{d} \mathbf{c}\mathbf{l}\mathbf{o}\mathbf{s}\mathbf{u}\mathbf{r}\mathbf{e}=\left(\mathbf{1}-{\displaystyle \frac{\mathbf{U}\mathbf{n}\mathbf{h}\mathbf{e}\mathbf{a}\mathbf{l}\mathbf{e}\mathbf{d} \mathbf{a}\mathbf{r}\mathbf{e}\mathbf{a} \mathbf{o}\mathbf{r} \mathbf{w}\mathbf{i}\mathbf{d}\mathbf{t}\mathbf{h} \mathbf{a}\mathbf{t} \mathbf{32} \mathbf{h}}{\mathbf{I}\mathbf{n}\mathbf{i}\mathbf{t}\mathbf{i}\mathbf{a}\mathbf{l} \mathbf{w}\mathbf{o}\mathbf{u}\mathbf{n}\mathbf{d} \mathbf{a}\mathbf{r}\mathbf{e}\mathbf{a} \mathbf{o}\mathbf{r} \mathbf{w}\mathbf{i}\mathbf{d}\mathbf{t}\mathbf{h} \mathbf{a}\mathbf{t} \mathbf{0} \mathbf{h}}}\right)\times \mathbf{100}\mathbf{\%}$$

4.8. Zebrafish Husbandry and Embryo Collection

The AB wild-type zebrafish (Danio rerio) adults have been bred and maintained at the fish facilities of the Centre of Experimentation and Animal Behaviour at the University of Malaga. They were reared at 28 ± 0.1 °C under a 12:12 h light:dark photoperiod, following standard procedures [51]. The zebrafish strain was initially obtained from the European Zebrafish Resource Centre (EZRC, Karlsruhe Institute of Technology, Karlsruhe, Germany).

Spawning pairs were transferred to breeding tanks the day before spawning. After spawning, fertilized eggs were collected, bleached, washed, and incubated at 28 ± 0.1 °C in a Petri dish containing embryo medium [64]. PsBU concentrations were chosen to minimize toxicity, as higher doses were lethal to the larvae. Multiple preliminary tests were performed to identify safe, non-toxic concentrations that allowed evaluation of regenerative effects independent of drug-induced mortality.

4.9. Chemical Exposure and Caudal Fin Regeneration Assay

Embryos at 72 h post fertilization (hpf) were placed into each well of a 96-well plate containing 300 µL per well of PsBU solutions prepared in E3 medium. E3 medium alone served as a negative control, and a 50 µg mL⁻¹ ulvan solution was used as a positive control [47]. Three replicates of this setup were performed per treatment.

PsBU exposure concentrations were 25, 50, 100, 150, 250, and 500 µg mL⁻¹. The toxicity profile in zebrafish larvae was determined as previously described [54]. At 72 hpf, hatched larvae were anesthetized with tricaine, and caudal fins were amputated just posterior to the notochord. Each larva was then transferred to a well that contained one of the above-mentioned PsBU concentrations or to either the negative or positive control solution. Images were obtained from the caudal fin of tricaine anesthetized larvae at 72 hpf (0 h post amputation, hpa) and 144 hpf (72 hpa).

4.10. Morphometry of Caudal Fin Regeneration

Images of caudal fin regenerates were obtained using a CytoSmart Lux3 FL microscope (Axion Biosystems, Atlanta, GA, USA) [65]. Fin areas were quantified using ImageJ (NIH, USA) [66]. The original fin area before amputation was also measured for comparison. Statistical significance between fin areas was calculated using the T-test of the Statgraphics Centurion 19 program (Statgraphics, Inc., The Plains, VA, USA).

4.11. Statistical Analysis

Statistical significance of in vivo studies, the caudal fin regeneration assay and the in vitro scratch assay was determined by one-way ANOVA or T-test using GraphPad Prism 8.0 or Statgraphics Centurion 19 programs. Data were presented as boxplots showing the mean ± SD. For graphical representation, a total of n = 15 larvae per concentration were measured, except for the 500 µg mL⁻¹ treatment, n = 3, due to mortality observed across three biological replicates.

5. Conclusions

In summary, polysaccharides extracted from B. utriformis demonstrated high thermal stability, excellent biocompatibility, and regenerative activity. In vitro, PsBU promoted HaCaT keratinocyte proliferation and migration, with maximal effects observed at 500 µg/mL, while zebrafish fin regeneration was effectively stimulated at 200 µg/mL. Proteomic analysis suggested modulation of pathways involved in cell cycle regulation, cytoskeletal dynamics, and tissue repair, providing a mechanistic basis for these effects. While these findings highlight the therapeutic potential of PsBU, it is important to note that in vitro and zebrafish results may not fully predict human outcomes. Additional in vivo studies in rats and fish have been conducted and will be published soon, offering further support for the safety and efficacy of PsBU in wound healing and regenerative applications.