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

<i>Bovistella utriformis</i> (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 <i>B. utriformis</i> (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 (<i>p</i> < 0.001 vs. control). Overall, these findings demonstrate that <i>B. utriformis</i> polysaccharides are safe bioactive compounds that promote key biological processes involved in tissue regeneration, supporting their potential application as natural wound-healing agents.