Talapphet Natchanok, Kim Moon-Moo
Department of Applied Chemistry, Dong-Eui University, Busan 614-714, Republic of Korea.
Food Technol Biotechnol. 2024 Mar;62(1):15-25. doi: 10.17113/ftb.62.01.24.8120.
Ageing is a biochemical, metabolic and genetic physiological phenomenon. The suppression of melanin biosynthesis, evident in the greying of the hair, is a hallmark of ageing resulting from translation failure, reduced enzyme activity and cellular senescence. Putrescine, the smallest member of the polyamine family and an organic chemical, is present in living mammalian cells and plays a crucial role in regulating skin melanogenesis. Therefore, the purpose of this study is to explore the effect of putrescine on the signalling pathways of melanogenesis in melanoma cells.
Melanin production capacity of putrescine was analysed using a tyrosinase activity assay. To assess the cell viability of B16F1 cells exposed to putrescine, a tetrazolium dye MTT assay was performed. The effect of putrescine on melanin synthesis in the presence of HO was evaluated using various assays in B16F1 cells. The effect of putrescine on melanin production in B16F1 cells was determined using a specific melanin production assay. Gene expression was analysed using real-time polymerase chain reaction (RT-PCR). Furthermore, the effect of putrescine on the expression of proteins related to melanin production in the cells treated with HO was analysed by immunofluorescence and Western blot analysis.
Putrescine increased tyrosinase activity and showed no cytotoxicity in B16F1 cells. In addition, putrescine effectively scavenged HO, as shown by the reduction of intracellular HO amounts in 2',7'-dichlorofluorescin diacetate analysis, and promoted melanin production in living cells. The stimulation of melanogenesis by putrescine was attributed to the increased expression of , , and genes. Immunofluorescence assays revealed that putrescine enhanced the expression of proteins associated with melanogenesis and upregulated TYR, TRP-1 and TRP-2 the microphthalmia-associated transcription factor (MITF) and increased the expression of methionine sulfoxide reductases A (MSRA) and B (MSRB) in the cells treated with HO, effectively promoting melanogenesis. These results suggest that putrescine can be used to stimulate melanin synthesis.
This is the first study to investigate the effect of putrescine on the signalling pathways of melanogenesis in B16F1 melanoma cells. The results confirm that putrescine can promote melanogenesis through the expression of TYR, TRP-1 and TRP-2 the MITF in cells treated with HO. Putrescine can be used exclusively as a cosmetic product to prevent premature greying of hair.
衰老乃一种生物化学、新陈代谢及遗传的生理现象。黑色素生物合成的抑制，在头发变白中表现明显，是由翻译失败、酶活性降低及细胞衰老导致的衰老标志。腐胺是多胺家族中最小的成员，属于有机化合物，存在于活体哺乳动物细胞中，在调节皮肤黑色素生成中起关键作用。因此，本研究旨在探讨腐胺对黑色素瘤细胞黑色素生成信号通路的影响。
采用酪氨酸酶活性测定法分析腐胺的黑色素生成能力。为评估暴露于腐胺的B16F1细胞的细胞活力，进行了四氮唑染料MTT测定。在B16F1细胞中，采用多种测定法评估腐胺在存在HO时对黑色素合成的影响。使用特定的黑色素生成测定法确定腐胺对B16F1细胞黑色素生成的影响。采用实时聚合酶链反应（RT-PCR）分析基因表达。此外，通过免疫荧光和蛋白质印迹分析，分析腐胺对用HO处理的细胞中与黑色素生成相关蛋白质表达量的影响。
腐胺增加了酪氨酸酶活性，且在B16F1细胞中未显示出细胞毒性。此外，如在2',7'-二氯荧光素二乙酸酯分析中细胞内HO量的减少所示，腐胺有效清除了HO，并促进了活细胞中的黑色素生成。腐胺对黑色素生成的刺激归因于 、 、 和 基因表达量的增加。免疫荧光测定显示，腐胺增强了与黑色素生成相关蛋白质的表达，并上调了小眼畸形相关转录因子（MITF）的TYR、TRP-1和TRP-2，且在用HO处理的细胞中增加了甲硫氨酸亚砜还原酶A（MSRA）和B（MSRB）的表达量，有效促进了黑色素生成。这些结果表明，腐胺可用于刺激黑色素合成。
这是第一项研究腐胺对B16F1黑色素瘤细胞黑色素生成信号通路影响的研究。结果证实，腐胺可通过在用HO处理的细胞中表达TYR、TRP-1和TRP-2以及MITF来促进黑色素生成。腐胺可专门用作防止头发过早变白的化妆品。
