Saccharomycopsis ferment filtrate (SFF), mainly used in skin care products, has been reported to inhibit inflammatory nitric oxide production and prevent epidermal damage. However, the effects of SFF on epidermal kera...Saccharomycopsis ferment filtrate (SFF), mainly used in skin care products, has been reported to inhibit inflammatory nitric oxide production and prevent epidermal damage. However, the effects of SFF on epidermal keratinocytes have not yet been explored. We investigated the effects of SFF on skin barrier function using human primary epidermal keratinocytes. Cell viability was determined by MTT assay. The mRNA and protein expression levels of tight junction proteins (claudin-1, -3, -4, occludin, ZO-1) were analyzed by RT-PCR and Western blotting, respectively. The effect of SFF on the barrier formation of epidermal keratinocytes was measured by transepithelial electrical resistance (TER). Rescue of cell death from H2O2 treatment was evaluated by annexin V staining. SFF, at concentrations that did not cause significant change of cell viability, induced dose-dependent cell-cell adhesion and formation of an organized monolayer structure. Pretreatment of keratinocytes with EGTA, a Ca2+ chelator, did not inhibit the cell-cell adhesion of keratinocytes by SFF, indicating a Ca2+-independent mechanism. The mRNA and protein levels of claudin-1 in keratinocytes were up-regulated by SFF treatment in a dose-dependent manner. The expressions of other tight junctions (TJs) including claudin-3 & 4, occludin and ZO-1 were also similarly increased in SFF-treated epidermal keratinocytes. The promoting effect of SFF on the barrier function of epidermal keratinocytes was further confirmed by the increased TER value in SFF-treated epidermal keratinocytes. Annexin V staining confirmed that SFF markedly decreased the number of dead cells resulted from H2O2 injury. Taken together, our results provided the first evidence that SFF enhanced keratinocytes barrier function by increasing the expression of TJs and TER.展开更多
Melanin, synthesized by melanocyte, is transferred to neighboring keratinocyte and finally accumulates in perinuclear site. Except functioning as an internal sunscreen to protect from UV damage, the potential effect o...Melanin, synthesized by melanocyte, is transferred to neighboring keratinocyte and finally accumulates in perinuclear site. Except functioning as an internal sunscreen to protect from UV damage, the potential effect of melanin on modulating the bioactivity of keratinocyte has not yet been fully investigated. In this study, we added melanin directly to the culture of human epidermal keratinocytes and the uptake of melanin was found to be dose- and time-dependent as determined by spectrophotometric method. The uptaken melanin accumulated perinuclearly in keratinocytes that is similar to the pattern observed in human solar lentigo tissue by microscopic examination. Pretreatment of keratinocytes with either niacinamide or trypsin inhibitor reduced the uptake of melanin dose-dependently, indicating a PAR-2-dependent pathway involved. Melanin uptake by keratinocytes inhibited cell proliferation as demonstrated both by the decrease of cell number and nuclear Ki-67 expression. Inhibited Ki-67 expression in melanin-containing keratinocyte was also found in human lentigo tissue. The cell cycle arrested at G1 phase in melanin-uptaken keratinocytes was confirmed by flow cytometric method. The protein expressions of cyclin-dependent kinase 1 (CDK1), CDK2, cyclin E, cyclin A and cyclin B were significantly reduced by melanin treatment. Microarray analysis, RT/real-time PCR and western blot demonstrated the inhibited expression of DKK1, a protein known to reduce skin pigmentation, in melanin-uptaken keratinocytes. Together, the direct incubation of keratinocyte with melanin might serve as a useful model to study the potential mechanisms involved in melanin uptake and pigmentation process.展开更多
文摘Saccharomycopsis ferment filtrate (SFF), mainly used in skin care products, has been reported to inhibit inflammatory nitric oxide production and prevent epidermal damage. However, the effects of SFF on epidermal keratinocytes have not yet been explored. We investigated the effects of SFF on skin barrier function using human primary epidermal keratinocytes. Cell viability was determined by MTT assay. The mRNA and protein expression levels of tight junction proteins (claudin-1, -3, -4, occludin, ZO-1) were analyzed by RT-PCR and Western blotting, respectively. The effect of SFF on the barrier formation of epidermal keratinocytes was measured by transepithelial electrical resistance (TER). Rescue of cell death from H2O2 treatment was evaluated by annexin V staining. SFF, at concentrations that did not cause significant change of cell viability, induced dose-dependent cell-cell adhesion and formation of an organized monolayer structure. Pretreatment of keratinocytes with EGTA, a Ca2+ chelator, did not inhibit the cell-cell adhesion of keratinocytes by SFF, indicating a Ca2+-independent mechanism. The mRNA and protein levels of claudin-1 in keratinocytes were up-regulated by SFF treatment in a dose-dependent manner. The expressions of other tight junctions (TJs) including claudin-3 & 4, occludin and ZO-1 were also similarly increased in SFF-treated epidermal keratinocytes. The promoting effect of SFF on the barrier function of epidermal keratinocytes was further confirmed by the increased TER value in SFF-treated epidermal keratinocytes. Annexin V staining confirmed that SFF markedly decreased the number of dead cells resulted from H2O2 injury. Taken together, our results provided the first evidence that SFF enhanced keratinocytes barrier function by increasing the expression of TJs and TER.
文摘Melanin, synthesized by melanocyte, is transferred to neighboring keratinocyte and finally accumulates in perinuclear site. Except functioning as an internal sunscreen to protect from UV damage, the potential effect of melanin on modulating the bioactivity of keratinocyte has not yet been fully investigated. In this study, we added melanin directly to the culture of human epidermal keratinocytes and the uptake of melanin was found to be dose- and time-dependent as determined by spectrophotometric method. The uptaken melanin accumulated perinuclearly in keratinocytes that is similar to the pattern observed in human solar lentigo tissue by microscopic examination. Pretreatment of keratinocytes with either niacinamide or trypsin inhibitor reduced the uptake of melanin dose-dependently, indicating a PAR-2-dependent pathway involved. Melanin uptake by keratinocytes inhibited cell proliferation as demonstrated both by the decrease of cell number and nuclear Ki-67 expression. Inhibited Ki-67 expression in melanin-containing keratinocyte was also found in human lentigo tissue. The cell cycle arrested at G1 phase in melanin-uptaken keratinocytes was confirmed by flow cytometric method. The protein expressions of cyclin-dependent kinase 1 (CDK1), CDK2, cyclin E, cyclin A and cyclin B were significantly reduced by melanin treatment. Microarray analysis, RT/real-time PCR and western blot demonstrated the inhibited expression of DKK1, a protein known to reduce skin pigmentation, in melanin-uptaken keratinocytes. Together, the direct incubation of keratinocyte with melanin might serve as a useful model to study the potential mechanisms involved in melanin uptake and pigmentation process.
