白藜芦醇调控Nrf2-ARE信号通路提高光甘草定抑制黄褐斑生成的机制研究

Pigment melanin is formed by tyrosine which is catalyzed by tyrosinase inside the melanocytes, and chloama is induced by abnormal melanin. Tyrosinase inhibitor is a common treatment of chloama, but there are limitations with transdermal delivery system. The skin retention of these tyrosinase inhibitor can hardly reach the effective dose, so the effect are not good. We have found the synergistic effect on inhibiting the tyrosinase of potent antioxidants and tyrosinase inhibitors. It was proposed that the activated Nrf2-ARE pathway and the melanin can scavenge free radical, so the screening of external-use drugs for chloama can base on the two therapeutic targets above. B16 murine melanoma cells was chosen as the research object, the expression of Nrf2 was upregulated and downregulated in the B16 cells which was treated with UV, then the antioxidative activity、Biochemical index related to formation of melanin and the consumption of active constituent were determined. The expression level of genes and protein related to Nrf2 pathway and formation of melanin. Potential protein kinase was screened by protein phosphorylation analysis software. The protein kinase was upregulated and downregulated in B16 cells to verify the molecular mechanism that RES and GLA can enhance the effect of inhibiting tyrosinase by activating Nrf2-ARE pathway. This study provides a new thought for innovation and development of Chinese medicine to treat chloama.

基底层黑素细胞受酪氨酸酶（Tyr）作用催化酪氨酸生成黑素导致黄褐斑，目前对黄褐斑治疗主要停留在抑制Tyr活性上，但因经皮给药系统的局限性，活性成分在基底层滞留量很难达到其起效量，故疗效差。我们前期研究中发现强抗氧剂与强酪氨酸酶抑制剂复配具有协同抑制Tyr效果。我们提出Nrf2-ARE信号通路激活与黑素生成均可清除自由基，外用药物治疗黄褐斑时可同时针对以上两个作用靶点选择药物。本项目选择B16黑素瘤细胞为研究对象，构建Nrf2过表达和沉默的B16、UV刺激，测定药物协同作用后的抗氧化能力、黑素生成相关的指标及药物消耗，检测与Nrf2信号通路、黑素生成相关的基因和蛋白表达；蛋白磷酸化分析软件筛选出磷酸化Nrf2的潜在蛋白激酶，构建潜在蛋白激酶过表达和沉默的B16细胞，验证白藜芦醇和光甘草定激活Nrf2-ARE信号通路提高对Tyr抑制的分子机制，为治疗黄褐斑的创新中药研发提供思路。

UVB 照射诱导细胞产生大量的 ROS，产生氧化应激损伤，激活 Nrf2及其相应的二相蛋白 HO-1；在此同时，与黑色素生成相关的蛋白MITF、TYP、TRP1和TRP2也被激活。随着 UVB 照射剂量的增大，TYP 的表达升高，促进黑色素生成。在细胞内自我保护系统的共同启动下，产生的 ROS 会被机体逐渐清除而接近正常水平。Nrf2-ARE 信号通路的激活与黑色素的生成存在关联性。在机体产生 ROS 一定量的情况下，Nrf2 过表达，增加抗氧化能力，降低机体 ROS 的含量；HO-1的表达上调，启动抗氧化基因的转录；TYP 的表达下调，促进黑色素生成作用减弱，抑制黑色素的生成。抗氧化剂与酪氨酸酶抑制剂协同作用可显著提升强酪氨酸酶抑制剂的抑制酪氨酸酶活性。这种协同效应只有在以 L-DOPA 为底物时才能观察到。协同作用与化合物对酪氨酸酶的抑制方式无关。在选定的浓度范围内，RES 和 OXYR 摩尔比为1:9可作为最佳配比应用于后续研究。UVB 引起 PIG1 细胞氧化应激条件下，RES 与 OXYR组合物一方面通过调控 Nrf2 表达水平，从而调控 HO-1；另一方面抑制 MITP、从而下调 TYR、TRP1、TRP2等黑色素合成酶表达，最终协同抑制黑色素生成。Nrf2 过表达后，组合物这种抑制作用与 TYR 无关。说明抑制 MITF 蛋白表达水平抑制黑色素生成的作用，可能不是通过诱导 TYR 的调控来调节的，而是通过调控TRP1和TRP2 的表达来调节的。