This study aimed to investigate the effects of Ganoderma sichuanense subcritical extract (GSE) on factors related to skin barrier improvement. Subcritical water extraction was employed as an environmentally friendly method capable of extracting both polar and moderately non-polar bioactive compounds. Cytotoxicity analysis demonstrated that GSE was non-toxic at concentrations up to 3%. Gene and protein expression levels were evaluated using quantitative real-time PCR and western blotting in HaCaT cells. GSE did not significantly affect TRPV1 expression, suggesting that it may not primarily act through inflammatory pathways. In contrast, GSE significantly increased the expression of transglutaminase 1 (TGM1) and filaggrin (FLG), indicating enhanced epidermal barrier function. Furthermore, GSE significantly reduced the expression of MMP1 and MMP3 while increasing COL1A1 expression, suggesting inhibition of collagen degradation and promotion of collagen synthesis. These findings demonstrate that GSE exerts dual protective effects on both epidermal and dermal skin barrier components and may serve as a promising functional ingredient for cosmetic and skin health applications.

Abstract
1. Background
2. Materials and Methods
    2.1. Specimen extraction
    2.2. Cell viability
    2.3. Gene expression
    2.4. Protein expression
    2.5. Statistical processing
3. Results and Discussion
    3.1. Effects on cell viability
    3.2. Effects on inflammatory responserelatedmarker (TRPV1)
    3.3. Effects on epidermal barrier-relatedfactors (TGM1 and FLG)
    3.4. Effects on ECM degradation-relatedfactors (MMP1, MMP3, MMP9)
    3.5. Effects on collagen synthesis (COL1A1)
4. Conclusion
References

• Hea-Jin Lee(Department of Beauty Design, Daejeon University (Lecturer)) | 이해진 (대전대학교 화장품학과, 겸임교수)
• Mi-Hye Lim(Department of Cosmetic, Daejeon University (Professor)) | 임미혜 (대전대학교 화장품학과, 교수) Corresponding author