search
Contact Us
HOME

  • Crossref logo
  • Crossref Similarity Check logo
Journal Search Engine

to

Search Advanced Search Adode Reader(link)
Download PDF Export Citaion korean bibliography PMC previewer
ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.49 No.1 pp.25-36
DOI : https://doi.org/10.17779/KAOMP.2025.49.1.003

Inhibitory Effects on Melanogenesis by LED Light Irradiation and Bletillae Rhizoma Extract

Seojin Kim, Siyu Zhu, Okjoon Kim*
Department of Oral Pathology, School of Dentistry, Chonnam National University

† These authors contributed equally to this work.


* Correspondence: Ok Joon Kim, D.D.S, Ph.D., Department of Oral Pathology, School of Dentistry, Chonnam National University, Buk-Gu, Gwangju, 61186, Korea Tel: +82-62-530-4832 Email: js3894@jnu.ac.kr
January 20, 2025 January 21, 2025 February 14, 2025

Abstract


Skin color is primarily determined by the amount of melanin pigmentation in the skin. In recent years, cosmetic compositions have been developed to reduce the melanin pigmentation in the skin. Treatment of the skin with whitening agents, from the pharmacological and cosmetological views, should provide safety and efficacy without side effects. Currently used whitening agents which are mostly cause many harmful and cytotoxic effects. In view of the lack of safe whitening agents, this study has been conducted to find the stable and harmless compounds inhibiting melanogenesis. The use of light and Herbal extract for the purpose of skin whitening has been formally reported. So, this study is to investigate the effects of LED irradiation and Bletillae rhizoma (Br) extract on tyrosinase activity and melanin biosynthesis in B16F0 melanoma cells. Melanin biosynthesis was induced by α-MSH. Experimental group were conducted five groups; 1) Pre-LED group (LED light irradiation, followed by 1 μM α-MSH) 2) Post-LED group (α-MSH treat, followed by LED light irradiation) 3) Pre-Br extract group (Br extract treat, followed by 1 μM α-MSH) 4) Post-Br extract group (α-MSH treat, followed by 10 μg/ml Br extract) 5) LED and Br extract group. The melanin contents, tyrosinase activity, dendrite length of cells and expression of melanogenesis-related genes under LED light irradiation and Br extract treatment were investigated. Pre-635, Post-425 nm and Post-Br extract group were significantly reduced melanin contents and tyrosinase activity compared with other group. But both for LED irradiation and Br extract group, melanin contents and tyrosinase activity were increased. However, Pre-425 nm and Post-Br extract group could reduce the melanin contents, tyrosinase activity and dendrites length of cells. In addition, the expression of melanogenesis-related proteins, including microphthalmia associated transcription factor (MITF) and tyrosinase (TYR) is inhibited. The Pre-425 nm and Post-Br extract group activated Extracellular signal-regulated kinase (ERK) phosphorylation and involved in the inhibition of melanogenesis via stimulation of MITF degradation. These results indicate that the inhibitory effect of Pre-425nm irradiation and Post-Br extract on melanogenesis are derived from reduced TYR expression via the downregulation of MITF signaling, as well as acceleration of ERK phosphorylation. Thus, these results suggest that Pre-425nm irradiation and Post-Br extract could prevent and treat melanin hyperpigmentation or useful in whitening agents.


Melanogenesis , LED Light Irradiation , Bletillae Rhizoma Extract

LED 광 조사와 백급 추출물이 멜라닌 형성 억제에 미치는 영향

김서진, 주시유, 김옥준*
전남대학교 치의학전문대학원 구강병리학교실

초록

    Ⅰ. INTRODUCTION

    Skin color is primarily determined by the amount of melanin present in the skin. In recent years, cosmetic compositions have been developed to reduce the amount of melanin in the skin and therefore, whitening the skin1). Treatment of the skin with whitening agents, from the pharmacological and cosmetological views, should provide safety and efficacy without side effects2).

    Melanin plays a crucial role in the absorption of the free radicals generated within the cytoplasm and in shielding the host from various types of ionizing radiation, including UV light3). Melanin is formed by a process called melanogenesis through a combination of enzymatically catalyzed and chemical reactions4). Melanogenesis is initiated with tyrosine oxidation to dopaquinone catalyzed by the key enzyme, tyrosinase 5). After dopaquinone formation by tyrosinase, the compound is converted to dopa and dopachrome through auto-oxidation. Dopa is also the substrate of tyrosinase and oxidized to dopaquinone again by the enzyme. Therefore, tyrosinase-related protein 1 (TRP-1) and tyrosinase-related protein 2 (TRP-2) function in the biosynthesis of melanin downstream of tyrosinase (TYR). The expression of enzymes is strongly regulated by microphthalmia associated transcription factor (MITF)6). Then activates the gene expression of MITF via phosphorylation of the cAMP response element-binding protein (CREB)7). Finally, MITF efficiently activates the melanogenesis-related enzymes and stimulates melanogenesis8). Extracellular signal-regulated kinase (ERK) signaling is reportedly also involved in the regulation of melanogenesis via stimulation of MITF degradation9). ERK activation phosphorylates MITF, which is followed by MITF ubiquitination and proteasome-mediated degradation. Inhibition of the ERK pathway has been reported to enhance TYR promoter activity and increase melanogenesis10).

    Recently, LED light therapy using soothing the skin, reducing redness, inflammation, promote collagen production and increase the rate of skin cell renewal11). Also, research has been the LED irradiation inhibits Melanogenesis. For example LED at 830 and 850 nm inhibit melanin synthesis in vitro (Kim JM, et al, 2012), Blue light inhibits melanin synthesis in B16 melanoma cells and skin pigmentation induced by ultraviolet B in guinea-pigs (M.Ohara, et al, 2004), Blue light is phototoxic for B16F10 murine melanoma and bovine endothelial cell lines by direct cytocidal effect (Sparsa A, et al, 2010) and Effect of light-emitting diodes (LED) on migration of melanoblast cell line (Park SJ, et al, 2014). Although there were former studies on the inhibitory Melanogenesis, this research was performed in order to determine the detailed mechanism and site of action as a irradiation of LED.

    But LED irradiation can result different effects of the various skin conditions and intensity, and poor reproducibility. So, this study examined the effects of the LED light irradiation and herbal extract to supplement disadvantages of LED.

    In former study, Chinese herbal have been found to decrease melanin production and reduce melanogenesis. In our preliminary screening using melanin contents, Bletillae rhizoma (Br) extract was found that show decreased activity for melanin contents. Br, the roots of Bletillae striata, riches in phenanthrene derivates, blestiarenen A, B, C, batatasin III, polysaccharides, saponins, flavonoids, amino acids, and so on. In recent years, Br extract can promote the regeneration of the wound tissue through its anti-infection effect 12). There is research shows that Br is stable with respect to toxicity to the skin13). And as the efficacy of Br, it has been reported that the methanol extract inhibited melanin formation14). Thus, in this study investigated mechanisms for the inhibition of melanogenesis by LED light irradiation and Br extract.

    This study hypothesized that the LED light irradiation and Br extract that effectively inhibits melanogenesis and its molecular mechanisms effects on TYR, TRP-1, TRP-2, and MITF protein expression with intracellular signalling pathway using cultured B16F0 cells.

    Ⅱ. MATERIALS and METHODS

    1. Chemicals and reagents

    A-MSH, L-tyrosine, L-DOPA, mushroom tyrosinase, 3-(4,5-dimetylyhiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide were purchased from Sigma-Aldrich Aldrich (St. Louis, MO, USA). Penicillin/streptomycin and trypsin were purchased from Invitrogen (Carlsbad, CA, USA). Potassium dihydrogen phosphate and Triton-X were purchased from Merck (Frankfurt, Germany). Ultrapure water was obtained using a Milli-Q purification system from Millipore (Bedford, MA, USA). Sodium hydroxide and ethanol (analytical grade) were purchased from Biotec (Porto Alegre, RS, Brazil) and kojic acid from Galena (Campinas, SP, Brazil). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were obtained from Cultilab (Campinas, SP, Brazil).

    2. Cell culture

    Murine B16F0 melanoma cells from the Korean Cell Line Bank (KCLB, Seoul, Korea) were cultured in DMEM supplemented with 10% heat-in activated fetal bovine serum (FBS) and 1% penicillin/streptomycin (10,000 U/100 mg/mL) at 37 ℃ in a humidified atmosphere containing 5% CO2.

    3. Plant materials extracts

    Bletilla striata is 30–70 cm tall, including the inflorescence. Bletilla striata grows in grasses covered by pine forests, frequently forming patches connected by flattened corms and rhizomes. Br was purchased from Korean Plant Extract Bank. Br extract was prepared as follow. Br were extracted with 70% ethanol. The extract was filtered and concentrated by using the rotary evaporator. The extract was lyophilized by using freeze dryer and then with distilled water under reflux.

    4. Light source and irradiation

    The source of light for irradiation was a continuous-wave LED (U-JIN LED, Korea) emitting at a wavelength of red LED peak at 635 nm and blue LED peak at 425 nm. Manufactured power density of 5 mW on the sample surface was used as the light source. about The manufactured LED irradiation tool kit was built in 5% CO₂ humidified chamber at 37 ℃. To examine the effect of LED irradiation, each group was cultured with or without irradiation for 1 hr.

    5. Cell viability assay

    The B16F0 cell line was purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and cultured in DMEM supplemented with 10% FBS, and penicillin/streptomycin at 37℃ in a humid atmosphere of 5% CO2. Cells suspended in the culture medium containing 10% FBS were split into flat-bottomed 96-well plate and after the cells were attached to the plate, they were treated with various concentrations (1-500 μM) of Br extract for 24 hrs. The assay for cell viability was based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehydrogenase in viable cells to produce a purple formazan product indicating the level of cell respiration. The cells were seeded at 1×105 cells per well with 100 μl of the medium in 96-well plates and incubated with chemicals for 1 day at 37°C. After incubation for 1 day, the medium was removed, and the cells were incubated in phosphate-buffered saline containing 30 μl of at 37°C for 3 hr.

    The formazan product was dissolved in 50 μl of dimethyl sulfoxide (Calbiochem, USA). The optical density was measured at 570 nm using a colorimetric microplate reader (BioTek, Winooski, USA). Cell viability was quantified as a fold as compared to the untreated control.

    6. Determination of melanin biosynthesis in B16F0 cells

    After 48 hrs from seeding, the cells were washed with phosphate buffer and then treated with 1 μM α-MSH plus cells at various concentrations for 48 hrs before melanin contents evaluation. melanin content was measured using a slightly modified version of the method developed by Tsuboi et al.15). Briefly, cells were treated with Br extract and/or LED light irradiation at the indicated concentrations in the presence or absence of α-MSH for 24 hrs in FBS free DMEM. After treatment, the supernatant was transferred to a fresh tube and read directly at 450 nm with an ELISA plate reader. Next, the cells were harvested and solubilized in 2 N NaOH at 80℃ for 2 hrs then centrifuged for 10 min at full speed. The optical density (OD) of the sample was then measured at 450 nm. The total melanin content was calculated as the sum of the melanin content from the supernatant and its cell pellet for each sample.

    7. Intracellular TYR activity assay

    The TYR activity was determined with respect to its DOPA oxidase activity using the method described by Takahashi et al.16) with slight modifications. Briefly, B16F0 cells were seeded on a 6-well plate (1.2 × 10⁶ cells per well) and cultured with different concentrations of Br extract (10 μM) in the presence of α-MSH. After 24 hrs, the cells were washed twice with cold phosphate-buffered saline (PBS) and lysed with 0.1 M sodium phosphate buffer (pH 6.8) containing 1% Triton X-100 and a protease inhibitor cocktail (Sigma, St. Louis, MO). The cells were disrupted by freeze thawing, and the lysates were clarified by centrifugation at 10,000 rpm for 10 min. After quantification of the protein levels by BCA protein assay (Pierce, Rockford, IL) and adjustment of the concentration with lysis buffer, 100 μl of each lysate was placed in a well of a 96-well plate and 100 μl of substrate solution (1 mM L-DOPA in PBS, pH 6.8) was added. After incubation for 30 min at 37℃, TYR activity was analyzed spectrophotometrically by following the oxidation of DOPA to dopachrome at 450 nm using an ELISA plate reader.

    8. Melanocyte dendrite quantification

    Images of melanocytes after a 48 hrs exposure to DMEM were acquired with a microscope equipped with phase contrast objectives. Images were imported into software Image J for analysis. Blinded evaluators analyzed 20x fields per condition, melanocytes were seeded into in 6-well plates at 6x105 cells per well. Dendrites length is measured from the nuclear of the cell to dendrites (Fig. 1). Arrow indicate the cells having abundantly branched dendrites. Dendrites length and number per cell were counted manually; results are expressed as percent of cells with greater than 20 dendrites and average number of dendrites per cell.

    9. Western blot analysis

    The medium was removed and washed twice with phosphate- buffered saline (pH 7.4), and the cell lysate was prepared in 200 ml of cold lysis buffer (1% NP-40, 50 mM Tris– HCl, pH 7.5, 150 mM NaCl, 0.02 % sodium azide, 150 mg/ml PMSF, 2mg/ml aprotinin, 20 mg/ml leupeptin, and 1 mg/ml pepstatin A). Approximately 30 mg of the cell lysate was separated in a 10 % sodiumdodecyl sulfate polyacrylamide gel and transferred onto a polyvinylidenedifluoride membrane (Amersham, NJ, USA). The membrane was blocked with a blocking solution containing 5 % skim milk in TBST (2.42 mg/l Tris–HCl,8 g/l NaCl, 0.1 % Tween 20, pH 7.6) for 30 min and rinsed briefly in TBST. The membrane was incubated overnight at 4 °C with the appropriate primary antibodies: anti-MITF (1:1000; Cell signaling UK), anti-TYR (1:1000; Cell signaling UK), anti-TRP1 (1:1000; Cell signaling UK), anti-TRP2 (1:1000; Cell signaling UK), anti-CREB (1:1000; Cell signaling UK), anti-p-CREB (1:1000; Cell signaling UK), anti-ERK (1:1000; Cell signaling UK), anti-p-ERK (1:1000; Cell signaling UK), and A mouse monoclonal IgG anti-beta actin antibody (1:2500; Cell signaling UK) was used as the control. Finally, the membrane was washed in TBST, and the immunore activity of the proteins was detected using an enhanced chemiluminescence detection kit (Amersham, USA). The levels were determined by densitometric analysis using Scion Image software (Scion Corp, Frederick, MD, USA).

    10. Reverse transcription polymerase chain reaction (RT-PCR)

    The total RNA was isolated from culture cells using RNA iso Plus reagent (Takara, Japan), according to the manufacturer’s instructions. The complementary DNA was synthesized from the total RNA using a Maxime RT Premix kit (Intron, Korea). After 5 min at 95°C, the reaction was carried out at 45°C for 1 hr, followed by enzyme inactivation at 95°C for 5 min. The B16F0 cells were stimulated with 100 nM of α-MSH in the presence or absence of Br. For analysis of the TYR, TRP-1, TRP-2 and MITF mRNA levels, total cellular RNA was prepared using TRI reagent (MRC Inc., Cincinnati, OH) following the manufacturer’s instructions.

    The reaction was cycled 40 times for TYR, TRP-1, TRP-2, and MITF for 30 s at 94℃, 30 s at 58℃, and 60 s at 72℃. The reaction for β-actin was cycled 30 times for 30 s at 9 4℃, 30 s at 55℃, and 60 s at 72℃ (Table. 1). Each reaction was performed in triplicate, and the levels of β-actin mRNA expression were calculated and normalized to the level of mRNA. The data were analyzed using Exicycler II™ software (Bioneer, CA, USA).

    Ⅲ. RESULTS

    1. Effect of α-MSH on melanin contents

    B16F0 cells were treated with various concentrations of α-melanocyte stimulating hormone (MSH) for 48 hrs. Melanogenesis was then determined by measuring intracellular melanin contents, which are shown as percentage values. The effects of α-MSH on melanin contents of B16F0 cells are shown in Fig. 2. α-MSH showed a melanogenesis enhancing effect in a concentration dependent manner, but it decreased at 10 μM concentration. Thus, α-MSH at concentration 1 μM was used for further study.

    2. Effect of LED light irradiation on melanin contents

    To investigate the effects of 635 and 425 nm light irradiation on B16F0 cells, B16F0 cells were irradiated with 635 and 425 nm LED wavelengths for 1 hr. As shown in Fig. 3, melanin contents in B16F0 cells were significantly reduced by LED irradiation at Pre-635 nm and Post-425 nm group compared with other group.

    3. Effect of Br extract on cell viability and melanin contents

    Before the effects of Br extract on melanin contents were examined, the cytotoxic effects of Br extract were determined by an MTT assay. When B16F0 cells were treated with Br extract at concentrations ranging from 1 to 500 μ g/ml for 1 hr. As shown in Fig. 4A, treatment with Br extract did not affect cell proliferation up to 10 μg/ml; however, at concentrations ranging from 50 to 500 μg/ml, strong cytotoxicity was observed. Thus, Br extract at concentration 10 μg/ml was used to determine its effect on melanogenesis in B16F0 cells, B16F0 cells were stimulated with α-MSH and subsequently treated with 1 μg/ml for 12 hrs. As shown in Fig. 4B, melanin contents in B16F0 cells were significantly reduced by Post-Br extract group compared with Pre-Br ex- tract group for 1 hr without influencing melanocyte viability.

    4. Effects of LED irradiation and Br extract on Melanin contents in pellets

    To determine the potential inhibitory effects on melanogenesis, the B16F0 cells were treated with or without α-MSH (1 μM), LED irradiation and Br extract (10 μg/ml). When the cell were simultaneously added with α-MSH, melanin contents were increased in terms of pellet color (Fig. 5). 635, 425 nm light irradiation and Br extract markedly regulated melanin contents in pellet. Pre-635, Post-425 nm and Post-Br extract group significantly reduced the melanin contents in pellet compared with those of other group. But both for LED irradiation and Br extract group, melanin contents in pellet was increased. Only Pre-425 nm and Post-Br extract group could reduce the melanin contents in pellets.

    5. Inhibitory effects of LED light irradiation and Br extract on Melanin contents and tyrosinase activity

    635 and 425 nm light irradiation and Br extract markedly regulated the melanin contents and tyrosinase activity. Pre-635 nm and Post-425 nm group were significantly reduced melanin contents and tyrosinase activity compared with other group. But both for LED irradiation and Br extract group, melanin contents and tyrosinase activity in cells was increased. Only Pre-425 nm and Post-Br extract group could reduce the melanin contents and tyrosinase activity in cells.

    6. Effect of LED light irradiation and Br extract on cellular morphology

    To examine the inhibitory effect of LED light irradiation and Br extract on cellular morphological changes during melanogenesis, branched dendrites in cells exposed to α -MSH were observed. Pre-635, Post-425 nm and Post-Br group were significantly reduced dendrites length compared with other group. But both for LED irradiation and Br extract group, dendrites length was increased. Only Pre-425 nm and Post-Br extract group could reduce the dendrites length in cells. (Fig. 7A). Average dendrites length were graphed (Fig. 7B).

    7. Effects of LED light irradiation and Br extract on melanogenic enzyme expression.

    To determine whether LED irradiation and Br extract influences the mRNA and melanogenic enzymes such as MITF, TYR, TRP-1, TRP-2, CREB, p-CREB, ERK and p-ERK, this study performed RT-PCR and Western blotting analysis. As shown in Fig. 8A, mRNA levels of MITF and TYR were inhibited by Pre-635, Post-425 nm and Post-Br groups. However, expression levels of MITF and TYR were increased by LED irradiation and Br extract group. Interestingly, only Pre-425 nm and Post-Br extract group inhibited the expression levels of MITF and TYR. But, there was no significant change in TRP-1 and TRP-2 expression level. Results of protein expression such as MITF, TYR, TRP-1 and TRP-2 were same with mRNA level (Fig. 8B). Just LED light irradiation groups seriously affected to regulate phosphor-CREB expression, but both for LED and Br extract group degraded MITF via ERK phosphorylation.

    Ⅳ. DISCUSSION

    Melanin is a pigment found in skin, eyes, hair and so on. It determines the skin color17). Also, it is responsible for absorbing UVA and UVB rays to protect the body from their harmful effects18). The synthesis of melanin is stimulated primarily by environment stimulation; ultraviolet (UV) irradiation and alpha-melanocyte stimulating hormone (α -MSH). α-MSH causes a marked increase in tyrosinase activity and a consequent increase in melanin synthesis19). In this study, the reason why selecting α-MSH as a stimulating material to produce melanin, it is stable with respect to toxicity to the cells and has been used it in many literature. As shown in Fig. 1, α-MSH showed a increase of melanogenesis in dose dependent manner, but it decreased at 10 μM concentration. Thus, 1 μM α-MSH was used for this study.

    The removal of hyperpigmentation remains a challenge 20). The LED irradiation has been used in recent research to inhibit Melanogenesis. For example, LED irradiation at 830 and 850 nm inhibit melanin synthesis in vitro (Kim JM, et al, 2012)21). Blue light inhibits melanin synthesis in B16 melanoma cells and skin pigmentation induced by ultraviolet B in guinea-pigs (M.Ohara, et al, 2004)22) and is phototoxic for B16F10 murine melanoma and bovine endothelial cell lines by direct cytocidal effect (Sparsa A, et al, 2010)23). Pilot study has shown α-MSH treatment, followed by LED light irradiation having the effects of inhibiting the production of melanogenesis. So, various condition of LED irradiation on melanogenesis is needed. This study was conducted with two groups by LED irradiation conditions. 1) Pre-LED group (LED light irradiation treatment, followed by 1 μM α-MSH) 2) Post-LED group (α-MSH treatment, followed by LED light irradiation). The results of Post-LED group are generally consistent with other research. This study additionally found that in the case of 635 nm light irradiation, Pre-635 nm treatment is better than Post-635 nm treatment on melanogenesis inhibitory effects. In the case of 425 nm light irradiation, however, Post-425 nm treatment has a more significant melanogenesis inhibitory effects than those of Pre-425 nm treated group.

    In pilot study, chinese herbal extract have been used as whitening agents and showed inhibitory melanogenesis24). Especially the extract of Br has shown significantly decreased activity for melanin synthesis. Br extract are used as a coating agent and cosmetic additive. Br also can promote the regeneration of the wound tissue through its anti- infection effect25). Many author found that Br is stable with respect to toxicity to the skin26). And as the efficacy of Br, it has been reported that the methanol extract inhibited melanin formation27). Br extract treat is just as LED irradiation, divided into two group by Br extract conditions. 1) Pre-Br extract group (Br extract treatment, followed by α-MSH) 2) Post-Br extract group (α-MSH treatment, followed by Br extract treatment). Although both groups had melanogenesis inhibitory effects, Post-Br extract group showed obvious inhibitory effects compared with Pre-Br extract group.

    The principal aim of this study was to investigate the inhibitory effects of LED light irradiation and Br extract on melanogenesis. Unlike the former studies, it is the first research to study the inhibitory effects on melanogenesis by LED with herbal extract.

    The results of this study show that Pre-425 nm and Post-Br extract group most significant inhibited melanogenesis. It was confirmed that the pellet colors brightened, reduced melanin contents, tyrosinase activity and length of dendrites in cells. Also, those were activated ERK phosphorylation and involved in the inhibition of melanogenesis via stimulation of degraded MITF and TYR. Interestingly, Just LED light irradiation groups seriously affected to regulate phosphor-CREB expression. It is expected to get effect of whitened skin through phosphor-CREB regulation with LED light irradiation.

    Ⅴ. CONCLUSION

    The inhibitory effect of Pre-425 nm irradiation and Post-Br extract on melanogenesis are derived from reduced TYR expression via the downregulation of MITF signaling, as well as acceleration of ERK phosphorylation. Thus, these results suggest that Pre-425 nm irradiation and Post-Br extract could prevent and treat melanin hyperpigmentation or useful in whitening agents.

    AKNOWLEDGEMENTS

    This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government( MSIT) (No. 2022R1F1A1068614)

    Figure

    KAOMP-49-1-25_F1.gif

    Pigmentation in melanocytes is related to their proliferation as well as stimulation of melanocyte dendrites. Arrow indicate the cells having abundantly branched dendrites. Dendrites length is measured from the center of nuclear to dendrites.

    KAOMP-49-1-25_F2.gif

    Changes of melanin contents in B16F0 melanoma cells by treatment of α-MSH. Cells were treated for 48 hrs by α-MSH for indicated concentration and measured melanin contents. α-MSH showed a melanogenesis enhancing effect in a concentration dependent manner, but it decreased at 10 μM concentration. Data are presented as the mean of three independent experiments.

    KAOMP-49-1-25_F3.gif

    Effects of 635 and 425 nm on melanin contents were evaluated in B16F0 cells. melanin contents in B16F0 cells were significantly reduced by LED irradiation at Pre-635 nm and Post-425 nm group compared with other group. P-value vs. the α -MSH only : ** < 0.01, * < 0.1

    KAOMP-49-1-25_F4.gif

    Effects of Br extract on cell viability (A) and melanin contents (B). B16F0 cells were incubated for 1 hr in the presence of Br extract at different concentrations. Br extract at concentration 10 μg/ml was used to determine its effect on melanogenesis in B16F0 cells. melanin contents in B16F0 cells were significantly reduced by Post-Br extract group compared with Pre-Br extract group for 1 hr without influencing melanocyte viability. P-value vs. the α-MSH only : ***<0.001, ** < 0.01, * < 0.1

    KAOMP-49-1-25_F5.gif

    Effects on melanin production in B16F0 cells. Cells were treated with or without α-MSH, LED irradiation and Br extract. Both for LED irradiation and Br extract group, melanin contents in pellet was increased. However, Pre-425 nm and Post-Br extract group could reduce the melanin contents in pellets. Pellets were pictured after cell harvesting by centrifugation to compare with colors.

    KAOMP-49-1-25_F6.gif

    Inhibitory effects of LED light irradiation and Br extract on Melanin biosynthesis. LED 635 nm irradiation with Br extract regulated melanin contents (A) and tyrosinase activity (B). LED 425 nm irradiation with Br extract regulated melanin contents (C) and tyrosinase activity (D). Both for LED irradiation and Br extract group, melanin contents and tyrosinase activity in cells was increased. However, Pre-425 nm and Post-Br extract group could reduce the melanin contents and tyrosinase activity in cells. P-value vs. the α-MSH only : ** < 0.01, * < 0.1

    KAOMP-49-1-25_F7.gif

    Effect of LED irradiation and Br extract on B16F0 cells length. Both for LED irradiation and Br extract group, dendrites length was increased. However, Pre-425 nm and Post-Br extract group could reduce the length of dendrites in cells. (A); Phase contrast microscopic findings of B16F0 cells. The individual length of B16F0 cells was determined with NIH ImageJ software (n = 20). Average dendrite length (B).

    KAOMP-49-1-25_F8.gif

    Effects of LED light irradiation and Br extract on the expression of melanogenesis-related genes. Cells were treated with or without α-MSH, LED irradiation and Br extract. (A) Total RNA was extracted and analyzed by RT-PCR for the expression of MITF, TYR, TRP-1 and TRP-2. (B) Intracellular TYR, TRP-1, TRP-2, MITF, ERK, p-ERK, CREB and p-CREB levels were measured by Western blotting. Equal protein loading was confirmed by reaction with β-actin.

    Table

    RT-PCR primer sequences for melanogenic-related genes and proteoglycans

    Reference

    1. Fitzpatrick TB: The validity and practicality of sun-reactive skin types I through VI. Arch. Dermatol 1988;124:869-871.
    2. Friedmann PS and Gilchrest BA: Ultravioletradiation directly induces pigment production by cultured human melanocytes. J Cell Physiol 1987;133:88-94.
    3. Chang TS: An updated review on tyrosinase inhibitors. Int J Mol Sci 2009;10:2440-2475.
    4. Bennett DC: Genetics, development, and malignancy of melanocytes. I nt Rev Cytol 1993;146:191-260.
    5. Halaban R, Patton RS, Cheng E, Svedine S, Trombetta ES, Wahl ML, Ariyan S, Hebert DN: Abnormal acidification of melanoma cells induces tyrosinase retention in the early secretory pathway. J Biol Chem 2002;277:14821-14828.
    6. Gaggioli C, Busca R, Abbe P, Ortonne JP, Ballotti R: Microphthalmia-associated transcription factor (MITF) is required but is not sufficient to induce the expression of melanogenic genes. Pigment Cell Res 2003;16:374-382.
    7. Garcia Borron JC, Sanchez-Laorden BL, Jimenez-Cervantes C: Melanocortin-1 receptorstructure and functional regulation. Pigment Cell Res 2005;18:393-410.
    8. Takeda K, Yasumoto K, Takada R, Takada S, Watanabe K, Udono T, Saito H, Takahashi K, Shibahara S: Induction of melanocyte-specific microphthalmiaassociated transcription factor by Wnt-3a. J Biol Chem 2000;275:14013-14016.
    9. Kim DS, Park SH, Kwon SB, Park ES. Huh CH, Youn SW, Park KC: Sphingosylphosphorylcholine-induced ERK activation inhibits melanin synthesis in human melanocytes. Pigment Cell Res 2006;19:146-153.
    10. Englaro W, Bertolotto C, Buscà R, Brunet A, Pages G, Ortonne JP, Ballotti R: Inhibition of themitogen-activated protein kinase pathway triggers B16 melanoma cell differentiation. J Biol Chem 1998;273:9966-9970.
    11. Vink E, Coorevits P, Caqnie B, De Muynck M, Vandestraeten G, Cmbier D: Evidene of changes in sural nerve conduction mediated by light emitting irradiation. Lasers in Medical Science 2005;20:35-40.
    12. Takagi S, Yamaki M, Inoue K: Antimicrobial agents from Bletilla striata. Phytochemistry 1983;22:1011-1015.
    13. Zhang W, Ma S, Gu G, Shi J, Zhao B: Studies on the toxicological assessment on skin safety of poly-saccaride gum of Bletillae striata (THUNB.) REICHB. Chinese Wild Plant Res. 2003;22:59-61.
    14. Yoon HJ, Yoon JW, Yoon SW, Ko WS, Woo WH: Inhibitory effect on melanogenesis of Rhizoma Bletillae. J Oriental Med Ophthamol Otolaryngol Dermatol 2003;16:100-111.
    15. Tsuboi T, Kondoh H, Hiratsuka J, Mishima Y: Enhanced melanogenesis induced by tyrosinase gene-transfer increases boron-uptake and killing effect of boron neutron capture therapy for amelanotic melanoma. Pigment Cell Res 1998;11:275-282.
    16. Takahashi H, Parsons PG: Rapid and reversible inhibition of tyrosinase activity by glucosidase inhibitors in human melanoma cells. J Invest Dermatol. 1992;98:481-487.
    17. Ito K, Yoshikawa M, Fujii T, Tabunoki H, Yokoyama T: Melanin pigmentation gives rise to black spots on the wings of the silkworm Bombyx mori. J Insect Physiol 2016;1910:30129.
    18. Slominski A, Tobin DJ, Sh ibahara S, Wortsman J: Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol. Rev. 2004;84:1155-1228.
    19. Hsu MY, Meier F, Herlyn M: Melanoma development and progression: a conspiracy between tumor and host. Differentiation 2002;70(9-10):522-536.
    20. Parvez S, Kang M, Chung HS, Cho C, Hong MC, Shin MK, Bae H: Survey and mechanism of skin depigmenting and lightening agents. Phytother 2006;20:921-934.
    21. Kim JM, Kim NH, Tian YS, Lee AY. Light-emitting diodes at 830 and 850 nm inhibits melanin synthesis in vitro. Acta Derm Venereol. 2012;92:675-680.
    22. Ohara M, Kobayashi M, Fujiwara H, Kitajima S, Mitsuoka C, Watanabe H: Blue light inhibits melanin synthesis in B16 melanoma cells and skin pigmentation induced by ultraviolet B in guinea-pigs. Photodermatol Photoimmunol Photomed 2004;20:86-92.
    23. Sparsa A, Faucher K, Sol V, Durox H, Boulinguez S, Doffoel-Hantz V, Calliste CA, Cook-Moreau J, Krausz P, Sturtz FG, Bedane C, Jauberteau-Marchan MO, Ratinaud MH, Bonnetblanc JM: Blue light is phototoxic for B16F10 murine melanoma and bovine endothelial cell lines by direct cytocidal effect. Anticancer Res 2010;30:143-147.
    24. Kong JM, Goh NK, Chia LS, Chia TF: Recent advances in tradit ional plant drugs and orchids. Acta Pharmacologica Sinica 2003;24:7-21.
    25. Dong L, Xia S, Luo Y, Diao H, Zhang J, Chen J, Zhang J: Targeting delivery oligonucleotide into macrophages by cationic polysaccharide from Bletilla striata successfully inhibited the expression of TNF-alpha. J Control Release 2009;134:214-220.
    26. Fen GS, Yan XQ, Wang LY: Chinese medicine rhizoma Bletillae used as an embolization material in animal experiments and for clinical application. J Tongji Med Univ 1987;7:219-225.
    27. Tsai KS, Lin TC, Wu MT, Shen JL, Mao MY, Chen HY, Chen YH, Chen WC: Irritant contact dermatitis risk of common topical traditional chinese medicines used for skin-lightening. Evid Based Complement Alternat Med 2014;2014:60-64.
    28. Chang Taek Oh Tae‐Rin Kwon Eun Ja Choi Soon Re Kim Joon Seok Seog Kyun Mun Kwang Ho Yoo Yeon Shik Choi Sun Young Choi Beom Joon Kim: Inhibitory effect of 660‐nm LED on melanin synthesis in in vitro and in vivo. Photodermatol Photoimmunol Photomed. 2017;33:49-57.
    Copyright © Korean Association of Oral and Maxillofacial Pathology. All rights reserved.
    오늘하루 팝업창 안보기 닫기