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目的:探讨姜黄素激活Nrf2对人黑素细胞免受H_2O_2诱导氧化应激损伤的预保护作用。方法:利用不同浓度的H_2O_2(50、100、200μmol/L)处理人黑素细胞24h,MTT检测细胞活性,筛选H_2O_2体外诱导人黑素细胞氧化应激的最适浓度;进一步使用10μmol/L的姜黄素预处理细胞2h后,使用最适浓度H_2O_2刺激24h。MTT检测细胞活性,DCFH-DA流式检测细胞内活性氧(ROS)含量,Real-time PCR测定Nrf2、Ho-1、Sod1、Cat的mRNA表达水平,Western blot检测Nrf2蛋白表达。结果:100μmol/L的H_2O_2处理人黑素细胞24h后细胞活性显著降低,10μmol/L的姜黄素预处理2h后,人黑素细胞活性显著增强,流式检测结果显示细胞内ROS含量显著降低,Western blot检测结果显示Nrf2蛋白表达增加,Real-time PCR结果提示Nrf2、Ho-1、Sod1、Cat的mRNA表达水平显著增强。结论:姜黄素保护人黑素细胞免受H_2O_2诱导氧化应激损伤的过程中激活了Nrf2信号通路,提示Nrf2是白癜风氧化应激致病机制中的关键分子,为临床治疗白癜风提供了一种思路。
Objective: To investigate the pre-protection effect of curcumin on activation of Nrf2 against oxidative stress induced by H_2O_2 in human melanocytes. Methods: Human melanocytes were treated with different concentrations of H 2 O 2 (50,100 and 200μmol / L) for 24 h. MTT assay was used to determine the optimal concentration of H 2 O 2 for inducing oxidative stress in human melanocytes. 10μmol / L Curcumin pretreatment cells 2h, the optimum concentration of H_2O_2 stimulation 24h. Cell viability was measured by MTT assay. ROS level was detected by DCFH-DA flow cytometry. Nrf2, Ho-1, Sod1 and Cat mRNA were detected by Real-time PCR. Western blot was used to detect Nrf2 protein expression. Results: After treated with 100μmol / L H 2 O 2 for 24 h, the viability of human melanocytes decreased significantly. After pretreatment with 10 μmol / L curcumin for 2 h, the activity of human melanocytes increased significantly. The results of flow cytometry showed that the intracellular ROS content was significantly decreased, The results of Western blot showed that the expression of Nrf2 protein was increased. Real-time PCR results showed that the mRNA expression of Nrf2, Ho-1, Sod1 and Cat were significantly increased. CONCLUSION: Curcumin protects human melanocytes from H 2 O 2 -induced oxidative stress-induced activation of Nrf2 signaling pathway, suggesting that Nrf2 is a key molecule in the pathogenesis of vitiligo oxidative stress and provides a new idea for the clinical treatment of vitiligo .