亚硫酸氢钠穿心莲内酯抗紫外线所致皮肤光老化过程中对Nrf2与NF-κB平衡体系的调节机制研究

Skin photoaging is premature skin-aging exposure to ultraviolet radiation. How to prevent photoaging is a very concerned problem. Photoaging has two main features of inflammatory immune imbalance and oxidative stress, in which the crosstalk between Nrf2 and NF-κB pathways plays a crucial role in the skin photodamage process. Our previous study showed that Andrographolide sodium bisulfate (ASB) protected skin from photoaging while inhibited the expression of proinflammatory cytokines and oxidation products, which is often used in the treatment of various inflammatory diseases. Based on previous literature data and our preliminary experimental results, we propose a hypothesis that ASB exerts protective effect against photoaging by regulating the crosstalk between Nrf2 and NF-κB pathways. To support this novel hypothesis, we will explore the protective effect of ASB against UV-induced skin photoaging from animals to cells, from in vivo to in vitro. Molecular biology and biochemistry techniques (such as western blot, RT-PCR, flow cytometry and so on) will be applied to assess the in vivo and in vitro effects of ASB on the activities of the Nrf2 and NF-κB pathways. Furthermore, we will knockdown the Nrf2 and NF-κB p65 genes in the cell model of photoaging using RNA interference (RNAi), and assess whether ASB has protective effects against the inflammation, oxidative damage, and apoptosis in the model and regulates the crosstalk between Nrf2 and NF-κB pathways. Through this study lay the foundation for the material basis of anti-photoaging of ASB and other plant derived components. The completion of this study will also provide exploratory experience and guidance for the modernization of TCM ingredients and their derivatives in terms of anti-photoaging efficacy.

光老化是皮肤暴露于紫外线下导致的过早老化性改变，具有炎症免疫失衡和氧化应激反应两个主要特征，其中Nrf2/NF-κB通路平衡体系的失衡，在皮肤光损伤过程起重要作用。前期研究表明常用于治疗各种炎症性损伤疾病的亚硫酸氢钠穿心莲内酯(ASB)能抑制前炎症因子和氧化产物表达，有良好的抗光老化活性。结合文献与前期研究，本课题组提出“ASB通过调节Nrf2/NF-κB通路平衡体系抗紫外线所致光老化”的假说。本研究拟通过动物和细胞实验进一步评估ASB抗光老化活性，并利用RT-PCR等分子生物学技术揭示ASB对Nrf2/NF-κB平衡体系的影响；采用RNAi技术构建Nrf2与NF-κB p65基因共沉默细胞模型，分析ASB对该模型氧化损伤及炎症反应的保护作用及对Nrf2/NF-κB通路的调控机制。本研究能为ASB等植物源成分抗光老化作用机制提供依据，也可为中药活性成分及其衍生物抗光老化研究提供方法学借鉴。

亚硫酸氢钠穿心莲内酯（ASB）具有显著的抗炎抗氧化活性，可有效改善UV辐射诱导的皮肤光老化。药效评价实验结果显示ASB可显著改善光老化小鼠的皮肤宏观表现、皮肤弹性及皮肤厚度；组织病理学观察显示，ASB可显著降低紫外线福射诱导的表皮层异常增厚，抑制胶原纤维和弹性纤维的降解；同时，皮肤涂布给予ASB可显著升高抗氧化酶（CAT和SOD）的活力，降低MDA、MMP-1和MMP-3的含量；ASB可有效降低炎症因子TNF-α、IL-1β、IL-6和IL-10的过度表达。为进一步探明ASB治疗皮肤光老化的分子机制，我们构建了UV辐射诱导的HaCat细胞光老化模型，并结合分子生物学的方法与技术，对Nrf2和NF-κB信号通路参与调控的蛋白和基因的分子机制进行了深入研究。实验结果显示，ASB可通过调节Nrf2和NF-κB信号通路的交互作用发挥抗炎抗氧化治疗UV辐射诱导的皮肤光老化作用。在UV辐射诱导的HaCat细胞光老化模型中，发现ASB处理后能明显改善细胞凋亡和坏死现象；同时可减少UV辐射诱导的ROS的大量聚集，提高抗氧化基因GCLC和NQO1的表达，降低炎症因子IL-1β，IL-6 和TNF-α的含量；免疫荧光结果显示ASB处理后Nrf2蛋白的核转录效率明显上升，胞核中Nrf2蛋白含量显著增加，同时抑制了p65的核转录，细胞核中p65的蛋白含量明显降低；给予细胞ML385（Nrf2信号通路抑制剂）、BAY 11-7082（NF-κB 信号通路抑制剂）和ASB，检测Nrf2和NF-κB 信号通路相关蛋白和基因，结果显示keap1/Nrf2和NF-kB信号通路在UV辐照诱导的HaCat细胞中存在交互作用，ASB能通过调节keap1/Nrf2和NF-κB信号通路的平衡体系治疗UV辐照诱导的HaCat细胞光损伤。综合上述结果，本课题为探讨ASB治疗UV辐射诱导的光老化及其分子机制奠定了良好的工作基础。同时对于认识keap1/Nrf2和NF-κB信号通路平衡体系在治疗UV辐照诱导的光老化模型中的关键作用提供了重要的线索。