Department of Dermatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People's Republic of China.
Department of Dermatology, Children's Hospital of Soochow University, Suzhou, Jiangsu, People's Republic of China.
Drug Des Devel Ther. 2021 Nov 30;15:4837-4848. doi: 10.2147/DDDT.S336066. eCollection 2021.
Oxidative stress is considered a major determinant in the pathogenesis of vitiligo. Methylcobalamin (MeCbl) is an activated form of vitamin B12 that regulates inflammatory factors, counters oxidative stress, and reduces apoptosis in many disease models. However, the specific mechanism of MeCbl repigmentation against vitiligo is unknown. In this study, we explored the effect of MeCbl on melanocytes following hydrogen peroxide (HO)-induced oxidative stress.
We established an oxidative stress model using the immortalized human normal melanocyte cell line PIG1. We used a Cell Counting Kit-8 (CCK-8) to detect drug cytotoxicity, and we measured the melanin content of cells using the NaOH method. Intracellular oxidative damage was assessed by flow cytometry and antioxidant enzyme detection kits. In addition, we assessed the presence of apoptosis by flow cytometry and Western blots. We explored the underlying mechanisms of MeCbl during oxidative stress in melanocytes by analyzing the results of experiments based on real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and laser scanning confocal immunofluorescence microscopy. Finally, we repeated the experiments after applying an inhibitor to block the Nrf2 pathway.
We found that MeCbl treatment enhanced cell viability, increased melanin content, reduced intracellular reactive oxygen species (ROS) accumulation, increased the activities of antioxidant enzyme superoxide dismutase (SOD) and catalase (CAT), reduced melanocyte apoptosis, and up-regulated the expression of the Nrf2/HO-1 pathway. Moreover, the protective effects of MeCbl were significantly weakened after inhibiting the Nrf2/HO-1 pathway.
Our results indicate that MeCbl attenuated the HO-induced oxidative stress in melanocytes by activating the Nrf2/HO-1 pathway, this suggests that MeCbl may be an effective treatment against vitiligo.
氧化应激被认为是白癜风发病机制的主要决定因素。甲钴胺（MeCbl）是维生素 B12 的一种活性形式，可调节炎症因子，对抗氧化应激，并减少许多疾病模型中的细胞凋亡。然而，MeCbl 使白癜风复色的具体机制尚不清楚。在这项研究中，我们探讨了 MeCbl 对过氧化氢（HO）诱导的氧化应激后黑素细胞的影响。
我们使用永生化的正常人黑素细胞系 PIG1 建立了氧化应激模型。我们使用细胞计数试剂盒-8（CCK-8）检测药物细胞毒性，并使用 NaOH 法测量细胞中的黑色素含量。通过流式细胞术和抗氧化酶检测试剂盒评估细胞内氧化损伤。此外，我们通过流式细胞术和 Western blot 评估细胞凋亡的存在。我们通过基于实时定量聚合酶链反应（RT-qPCR）、Western blot 和激光扫描共聚焦免疫荧光显微镜的实验结果分析，探讨了 MeCbl 在黑素细胞氧化应激中的潜在机制。最后，我们在应用抑制剂阻断 Nrf2 通路后重复了实验。
我们发现 MeCbl 处理可增强细胞活力，增加黑色素含量，减少细胞内活性氧（ROS）积累，增加抗氧化酶超氧化物歧化酶（SOD）和过氧化氢酶（CAT）的活性，减少黑素细胞凋亡，并上调 Nrf2/HO-1 通路的表达。此外，抑制 Nrf2/HO-1 通路后，MeCbl 的保护作用明显减弱。
我们的结果表明，MeCbl 通过激活 Nrf2/HO-1 通路减轻了 HO 诱导的黑素细胞氧化应激，这表明 MeCbl 可能是治疗白癜风的有效方法。
