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Inhibition Effect of Justicia procumbens L. on Melanogenesis in B16F10 Melanoma Cells

B16F10 흑색종 세포의 멜라닌 생성에 대한 쥐꼬리망초의 억제 효과

  • 유정연 (Yu Jeong Yeon, 강릉원주대학교 일반대학원)

초록/요약 도움말

In this study, we investigated the effects of Justicia procumbens L. on melanogenesis and the mechanism of these effects using a B16F10 mouse melanoma model. Justicia procumbens L. is known to be rich in antioxidant compounds. It is also known for its efficacy in treating various ailments such as common cold, hepatitis, myalgia, gout, and arthritis. In this study, we investigated the inhibitory effect of Justicia procumbens L. on melanin production and its basic mechanism in B16F10 melanoma cells. We measured cell viability, reactive oxygen scavenging ability, intracellular melanin content, and intracellular/extracellular active tyrosinase levels in B16F10 cells induced by α-MSH. In addition, we investigated the effect on the expression of melanogenesis-related genes and proteins and the activation of PKA/CREB, AKT/GSK3β signaling pathways. The results showed that Justicia procumbens L. had no cytotoxicity in α-MSH-induced B16F10 cells and the reactive oxygen scavenging ability increased in a concentration-dependent manner. It also significantly reduced tyrosinase activity and melanin content in the cells in a concentration-dependent manner. Justicia procumbens L. suppressed major melaninproducing proteins such as microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related proteins (TRP-1, 2). Justicia procumbens L. also inhibited the phosphorylation of PKA/CREB proteins and increased the phosphorylation of AKT/GSK3β proteins, thereby reducing the expression levels of MITF and its downstream transcription factors. These results suggest that Justicia procumbens L. can effectively inhibit melanin synthesis in B16F10 melanoma cells and, by down-regulating the PKA/CREB signaling pathway and up-regulating the AKT/GSK3β signaling pathway, it can be considered as a skin lightening agent that can inhibit excessive pigment deposition and melanin production in the skin.

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목차 도움말

Ⅰ. Introduction 12

Ⅱ. Materials and Methods 16
2.1. Materials 16
2.2. Cell lines and cell culture 16
2.3. Cell proliferation assay 17
2.4. Colony assay 17
2.5. Apoptosis assay 18
2.6. DPPH radical scavenging activity test 18
2.7. Intracellular radical Measurement (H2DCF-DA) 19
2.8. In situ intracellular Tyrosinase activity (L-DOPA staining) 19
2.9. Intracellular Tyrosinase assay 20
2.10. Intracellular Melanin content 20
2.11. Protein analysis by Western blot 21
2.12. Statistical analysis 21

Ⅲ. Results 23
3.1. Effect of Justicia procumbens L. on cell viability in B16F10 melanoma cells or B16F10 melanoma cells induced by α-MSH 23
3.2. Antioxidant properties of Justicia procumbens L. extract 29
3.3. Effects of Justicia procumbens L. on intracellular tyrosinase inhibitory activity in B16F10 melanoma cells or B16F10 melanoma cells induced by α-MSH 34
3.4. Effects of Justicia procumbens L. on intracellular melanin synthesis in B16F10 melanoma cells or B16F10 melanoma cells induced by α-MSH 38
3.5. Effects of Justicia procumbens L. on the expression levels of melanogenesis-related proteins (MITF, Tyrosinase, TRP-1, TRP-2) in B16F10 melanoma cells induced by α-MSH 43
3.6. Effects of Justicia procumbens L. on melanogenesis-related pathways 47
3.6.1. Effect Justicia procumbens L. on the AKT/GSK3β pathway in B16F10 cells induced by α-MSH 47
3.6.2. Effect Justicia procumbens L. on the PKA/CREB pathway in B16F10 cells induced by α-MSH 48
3.6.3. Effect Justicia procumbens L. on the MAPK pathway in B16F10 cells induced by α-MSH 48
3.7. Effect of Justicia procumbens L. on the signalling pathway of B16F10 cells induced by a specific inhibitor of this pathway 55

Ⅳ. Conclusion 60

Ⅴ. References 65

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