Pretreatment of keratinocytes with Galactomyces ferment filtrate (GFF) promotes stress resiliency in an in vitro oxidative stress model.

Abstract

Objective: Extrinsic, lifestyle stressors can increase oxidative stress and induce premature aging characteristics of the skin. We studied the effects of oxidative stress at the biomolecular level to gain insights into effective stress-resiliency strategies for skincare. We also investigated if Galactomyces ferment filtrate (GFF) pretreatment could enhance stress resiliency in keratinocytes in vitro.

Methods: In vitro single cell Raman spectroscopy was performed to identify an oxidative stress phenotype in keratinocytes exposed to hydrogen peroxide (H₂O₂) stress. The gene expression and functional consequences of acute stress were also determined by performing RNA sequencing, mitochondrial metabolism assays, and proliferation assays 24 h after H₂O₂ stress exposure. All experiments were performed with and without GFF pretreatment.

Results: Raman spectra revealed a novel stress phenotype which includes the formation of cysteic acid with increasing doses of H₂O₂, likely from the breaking of disulfide bonds in keratins. GFF pretreatment significantly mitigated this stress phenotype; GFF-pretreated cells were 11.9 times more likely to exhibit healthy spectra compared to control cells when exposed to high stress. RNA sequencing conducted 24 h after H₂O₂ exposure also demonstrated a stress-related phenotype characterized by enhanced regulation of apoptosis and reduced keratinocyte differentiation. Conversely, GFF pretreatment increased multiple pathways of skin development at baseline and prevented the stress-induced dysregulation of gene expression. Keratinocytes pretreated with GFF also had increased stress resiliency in mitochondrial stress and wound healing assays.

Conclusion: Our data establishes that oxidative stress induces a distinctive biomolecular stress phenotype in keratinocytes. GFF pretreatment enhances the resilience of keratinocytes, as demonstrated by increased expression across various gene pathways related to keratinocyte differentiation, cell-cell junctions, and lipid metabolism. Following oxidative stress, GFF-pretreated cells exhibited substantial protection from the stress-induced biomolecular alterations identified in Raman spectra. Furthermore, GFF pretreatment effectively mitigated the downstream consequences of stress, supporting maintenance of keratinocyte gene-expression programs, mitochondrial activity, and wound-closure capacity after oxidative stress.