Melatonin inhibits UVB-induced melanin synthesis by regulating p53-mediated premature senescence in HaCaT cells / 中华放射医学与防护杂志

Objective:To explore the mechanism and regulatory effects of melatonin on UVB-induced melanin synthesis in human immortalized keratinocytes (HaCaT), so as to provide a theoretical basis for the skin protection of melatonin.Methods:HaCaT cells were pretreated with 10 -5 mol/L melatonin and then irradiated with 80 mJ/cm 2UVB. The melanin content was detected by NaOH assay, the proportion of premature senescence cells was detected by β-galactosidase staining kit, and the protein expression levels of both p53 and tyrosinase (TYR) were detected by Western blot at 72 h after UVB exposure. After 12 h pretreatment of ATM/ATR inhibitor, p53 inhibitor and melatonin, the proportion of premature senescence and the change of melanin content in HaCaT cells were detected at 72 h after 80 mJ/cm 2 UVB irradiation. Results:Melatonin inhibited UVB-induced increases of melanin content ( t=56.65, 13.39, P<0.05) and TYR expression ( t=16.46, P<0.05) in HaCaT cells. Melatonin alleviated UVB-induced premature senescence ( t=7.139, P<0.05) and inhibited UVB-induced increase of p53 expression ( t=19.08, P<0.05) in HaCaT cells. In addition, ATM/ATR inhibitor, p53 inhibitor and melatonin all inhibited UVB-induced increase of melanin content in HaCaT cells. Conclusions:Melatonin inhibits TYR-mediated melanin synthesis by regulating p53-related premature senescence in HaCaT cells after UVB irradiation.

Melanin synthesis and regulation in vivo and commonly used melanin inhibitors from natural products and traditional Chinese medicine / 中国中药杂志

Melanin is an important factor affecting human skin color. This study defines its synthetic pathways and regulatory pathways have the practical significance for the treatment of diseases caused by pigmentation problems, such as chloasma. Besides, it also provides a theoretical basis for screening out melanin inhibitors and developing whitening and freckle products. At present, the melanin inhibitors used in whitening and freckle products mainly come from natural products and traditional Chinese medicine. This article first introduces the melanin biosynthesis pathway with tyrosinase as the core, defines the synthesis, transport and catalytic activity of tyrosinase as the three key factors affecting melanin synthesis, and then reviews two common types of melanin inhibitors, namely tyrosinase synthesis inhibitors and tyrosinase inhibitors derived from natural products and traditional Chinese medicine. This article provides guidance for the development of new melanin inhibitors, and puts forward the idea for combining and synergizing inhibitors according to different mechanisms, in order to develop new whitening formulas.

The effects of green tea (Camellia sinensis) flower extracton melanin synthesis in B16-F10 melanoma cells / 대한수의학회지

The present study observed the effects of a green tea (Camellia sinensis) flower extract (GTFE) on melanin synthesis in B16-F10 melanoma cells. GTFE exhibited antioxidant activity on 2,2-diphenyl-1-picrylhydrazyl and inhibited mushroom tyrosinase activity in a dose-dependent manner. Furthermore, GTFE significantly diminished α-melanocyte stimulating hormone (α-MSH) stimulated cellular melanin content and tyrosinase activity throughout the concentration range evaluated. Based on RNA sequencing analysis, differential gene expression patterns observed in α-MSH stimulated B16-F10 melanoma cells were normalized by the addition of GTFE. In particular, the expression levels of melanoregulin and tyrosinase genes which are key regulating genes in melanin synthesis were up-regulated by 3.5 and 3 fold respectively by α-MSH, and were normalized to control levels by the addition of GTFE. The results suggest that GTFE inhibits melanin synthesis in α-MSH stimulated B16-F10 melanoma cells by normalizing expression of genes that are essential for melanin synthesis. Overall, the results suggest that GTFE could be applied in the development of a whitening agent for the treatment of dermal hyperpigmentation.

Effects of broadband ultraviolet B on non-canonical Wnt pathways in human epidermal melanocytes / 中华皮肤科杂志

Objective To investigate the effects of broadband ultraviolet B (BB-UVB) on the proliferation of, tyrosinase activity and melanogenesis in melanocytes.Methods Melanocytes isolated from human foreskin were subjected to primary culture.Some cultured primary melanocytes were irradiated with BB-UVB at 10, 20, 30, 40, 50, 100, 200 and 300 mJ/cm2.Then, CCK-8 assay was performed to evaluate the proliferative activity of melanocytes, dopa oxidation assay to estimate the activity of tyrosinase, and sodium hydroxide (NaOH)-lysis method was used to determine melanin content.Real-time fluorescence-based quantitative PCR was conducted to measure the mRNA expressions of genes involved in non-canonical Wnt pathways in melanocytes after irradiation with BB-UVB at 30, 50 and 100 mJ/cm2.Western blot was carried out to determine the expressions of proteins involved in non-canonical Wnt pathways in melanocytes before and after irradiation with BB-UVB of 100 mJ/cm2.The melanocytes receiving no treatment served as the control group.Statistical analysis was carried out by one-way analysis of variance followed by least significant difference (LSD)-t test for multiple group comparisons and by the independent sample t test for two-group comparisons.Results After irradiation with BB-UVB at 10-300 mJ/cm2, the proliferative activity of melanocytes was gradually reduced compared with the control group (all P ＜ 0.05), and the survival rate of melanocytes was less than 50％ when the irradiation dose of BB-UVB was higher than 100 mJ/cm2.Furthermore, tyrosinase activity gradually increased in melanocytes after irradiation with BB-UVB at 10-100 mJ/cm2 compared with the control group, and the increase was statistically significant at the radiation dose of 100 mJ/cm2 (P ＜ 0.05).Compared with the control group, the WIF-1 mRNA expression level decreased, while c-Jun N-terminal kinase (JNK), microphthalmia-associated transcription factor (MITF), Ras-related C3 botulinum toxin substrate 1 (RAC 1) and tyrosinase (TYR) mRNA expression levels increased in melanocytes after irradiation with BB-UVB at 30, 50 and 100 mJ/cm2 (all P ＜ 0.05);the WNT5A mRNA expression significantly decreased in melanocytes irradiated with 30 and 50 mJ/cm2 BB-UVB, but increased in those irradiated with 100 mJ/cm2 BB-UVB (all P ＜ 0.05).The radiation with 100 mJ/cm2 BB-UVB significantly decreased the expression of WIF-1 protein, but enhanced the expressions of WNT5A, JNK, MITF, RAC1 and TYR proteins in melanocytes compared with the control group (all P ＜ 0.05).Conclusions BB-UVB can decelerate the proliferation of, elevate tyrosinase activity and melanin level in, melanocytes.The WIF-1 gene may inhibit melanogenesis, and the decrease in its expression may promote melanogenesis by activating the JNK/MITF/TYR pathway through the combined effects of proteins involved in non-canonical Wnt pathways.

Study on the Whitening activity and pathway of red eucommia herbs / 中国生化药物杂志

Objective To determine the impact of PEE with different ethanol concentration extract on the activity of tyrosinase in B16 melanoma and to explore the feasibility of PEE used as skin whitening agent make use of a model organism .Methods 30%, 50%, 70%, 95%, different concentration of ethanol reflux got four kinds of PEE alcohol extract, respectively.And selected the most active inhibition to mushroom tyrosinase ( PEE30、PEE50、PEE70、PEE95).The effect of extracts from the selected on B16 cell model was determined by MTS, NaOH splitting decomposition, L-DOPA oxidation progress.The change in protein expression level after ethanol extract of PEE was determined by Western bolt.Results The capacity of inhibition to mushroom tyrosinase in each concentration of PEE extraction, but PEE70 had the most active inhibition.When challenged with B16 cell model, PEE70 showed the capacity of decrease in tyrosinase activities and melanin synthesis (P<0.01) and had a does dependence ;it also decreased Tyrosinase and TRP-1 expression compared with control B16 cells.Conclusion These data support the idea that PEE can restrain melanogenesis, through its inhibitory effect on the activity of tyrosinase.

The Therapeutic Effect of Porcine Placenta Extract for Improvement Sequelae of Burn

PURPOSE: Burn injury cause pruritis, pain, psychological and functional sequelae. The one of burn injury sequelae is the hypertrophic scar. It is difficult to control devastating fibrotic condition for hypertrophic scar. The objective of this study was to investigated the therapeutic effect on burn hypertrophic scar and wound healing for sequelae of burn injury by Porcine placenta extract (PPE). METHODS: To investigate the effect of PPE, we performed in vitro cell cytotoxity test (MTT assay), antioxidant activity assay (SOD like activity), melanin content assay, cell migration asssay and RT-PCR. RESULTS: As a result of cell cytotoxity test (MTT assay), PPE showed above 80% cell viability. From Antioxidant activity assay (SOD like activity), this effect was similar to vitamin C. In the melanin content assay, melanin synthesis was inhibited 23% on PPE treatment than control. PPE enhanced cell migration on human fibroblast and decreased the expression of hypertropic scar related gene (a-SMA and P311). CONCLUSION: Our data showed anti-oxidant effect, diminution of melanin and decrease of the expression of hypertropic scar related gene on the treatment of PPE. These results may provide the insight into the potential use of porcine placenta extract as support to control skin fibrosis related to burn hypertrophic scar and alternative medicine for burn sequelae.

Induction of Melanogenesis by Rapamycin in Human MNT-1 Melanoma Cells

BACKGROUND: Melanogenesis is one of the characteristic parameters of differentiation in melanocytes and melanoma cells. Specific inhibitors of phosphatidylinositol 3-kinase (PI3K), such as wortmannin and LY294002, stimulate melanin production in mouse and in human melanoma cells, suggesting that PI3K and mammalian target of rapamycin (mTOR) might be involved in the regulation of melanogenesis. OBJECTIVE: The involvement of the mTOR pathway in regulating melanogenesis was examined using human MNT-1 melanoma cells, and the effects of the potent inhibitor of mTOR, rapamycin, in the presence or absence of alpha-melanocyte-stimulating hormone (alpha-MSH) were evaluated. METHODS: In cells treated with rapamycin, cell viability, melanin content, and tyrosinase (TYR) activity were measured and compared with untreated controls. Protein levels of TYR, tyrosinase-related protein (TYRP)-1, TYRP-2, and microphthalmia-associated transcription factor (MITF) were also analyzed by Western blot. RESULTS: In rapamycin-treated cells, the melanin content increased concomitantly with an elevation in TYR activity, which plays a major role in melanogenesis. There was also an up-regulation of TYR, TYRP-1, and MITF proteins. Combined treatment with rapamycin or wortmannin and alpha-MSH increased melanogenesis more strongly than alpha-MSH alone. CONCLUSION: Rapamycin-induced melanin formation may be mediated through the up-regulation of TYR protein and activity. Furthermore, rapamycin and wortmannin, inhibitors of mTOR and PI3K, respectively, have co-stimulatory effects with alpha-MSH in enhancing melanogenesis in melanocyte cells.

The Evaluation of Skin Safety and Skin Cell Toxicity for Scutellaria baicalensis Georgi Extract according to Extraction Conditions / 대한피부과학회지

BACKGROUND: Scutellaria baicalensis Georgi extract is used as a traditional herbal medicine. The efficacy of Scutellaria baicalensis Georgi extract is known for antioxidative activity, antiinflammation effect, antibacterial effect, inhibitory effect of melanin synthesis, sun protection effect, antiallergy effect, and etc. OBJECTIVE: We confirmed the cell viability or inhibitory effect of melanin synthesis in HaCaT (human keratinocyte cell line) and B16F10 (murine melanoma cell line) cells and the skin safety test through a clinical test (dermal irritation study) for Scutellaria baicalensis Georgi extract, according to the extraction methods. METHODS: We checked the cell viability, using MTT assay and inhibitory effect of melanin synthesis in B16F10 cells or HaCaT cells for thirty one Scutellaria baicalensis Georgi extract, according to the extraction methods. Then, we evaluated the skin safety for selected eight Scutellaria baicalensis Georgi extract through a primary dermal irritation test. RESULTS: Among the thirty one Scutellaria baicalensis Georgi extracts, according to the extraction methods, we selected eight Scutellaria baicalensis Georgi extracts that were not detected with cell toxicity in HaCaT cells and B16F10 cells, and could have inhibited the melanin synthesis in B16F10 cells. The selected eight Scutellaria baicalensis Georgi extracts identified the skin safety through a primary dermal irritation test. CONCLUSION: We expect clinical trials for whitening efficacy based on inhibitory effect of melanin synthesis and human skin safety for Scutellaria baicalensis Georgi extracts.