基于Nrf2-Keap1-ARE/NF-κB通路互作研究18β-甘草次酸壳聚糖微球抗皮肤光老化的作用机制

Skin photoaging (SP) is a premature skin-aging damage after repeated exposure to UV, mainly characterized by oxidative stress and inflammatory reaction, in which the interaction between Nrf2-Keap1-ARE and NF-κB pathways plays a crucial role. Our previous work indicated that compared with oral ingestion, percutaneous administration of 18-β glycyrrhetinic acid (GA) could effectively inhibit SP in mice through suppressing the over-expressions of proinflammatory cytokines and oxidation products. Based on previous literature data and our preliminary experimental results, we proposed a hypothesis that percutaneous administration of GA might exhibit anti-SP activity by regulating the interaction between Nrf2-Keap1-ARE and NF-κB pathways. However, as for GA, the barrier function of stratum corneum was observed remarkable, along with short skin adhesion time and low cumulative percutaneous absorption. Hence, this project proposes to employ natural polymer chitosan (CS) to prepare GA-loaded nano-microspheres, with the aim to improve the skin-adhesive capacity and controlled-release performance of GA, which may contribute to lower the barrier function of stratum corneum, and finally enhances the skin absorption and activity of GA. And then, the mice model of SP will be applied to verify this hypothesis; meanwhile, RNA interference (RNAi) and the Nrf2 and NF-κB p65 genes knockout cells model of photoaging will also be employed to reversely authenticate the hypothesis. This study will provide promising information for the development of GA as a highly effective novel anti-SP agent.

皮肤光老化（SP）是皮肤反复暴露于UV下导致的过早老化性损伤，氧化应激和炎症反应是其主要特征，其中Nrf2-Keap1-ARE/NF-κB通路交互作用的失衡在该损伤中起关键作用。前期研究表明，相对口服而言，涂布给予18β-甘草次酸（GA）可明显抑制皮肤促炎因子及氧化产物的异常表达，呈良好的抗SP活性。结合前期研究与文献报道，我们提出以下假说：GA经皮给药可通过对Nrf2-Keap1-ARE/NF-κB通路交互作用的调控而发挥抗SP作用。此外，GA皮肤角质层屏障作用明显且附着时间短，经皮渗入量较低。故本课题拟采用壳聚糖制备GA的纳米载药微球，赋予GA皮肤粘附及缓释性并降低角质层屏障作用以提升其对皮肤的渗入，协同放大GA药效，进而利用SP整体动物模型验证上述假说，同时采用RNAi技术构建Nrf2与NF-κB p65基因共沉默SP细胞模型，反向验证该假说，为GA高效抗SP新药的研发提供理论依据。

项目背景.相对口服而言，涂布给予18β-甘草次酸（GA）可明显抑制皮肤促炎因子及氧化产物的异常表达，呈良好的抗皮肤光老化（SP）活性，前期研究与文献报道提示GA经皮给药可能通过对Nrf2-Keap1-ARE/NF-κB通路交互作用的调控而发挥抗SP作用，机制有待阐明。同时，GA皮肤角质层屏障作用明显且附着时间短，经皮渗入量较低，提高GA的经皮渗透量及其在皮肤中的滞留时间有待解决。..研究内容.1..18β甘草次酸纳米晶的制备、表征及抗炎活性；.2. 两亲性壳聚糖（ACS）的制备及表征；.3. 两亲性壳聚糖/18β甘草次酸纳米晶复合物的制备、细胞毒性及体外.皮肤渗透研究；.4. 两亲性壳聚糖/18β甘草次酸纳米晶复合物抗皮肤光老化药效研究；.5. 两亲性壳聚糖/18β甘草次酸纳米晶复合物抗皮肤光老化作用研究。..研究结果.1. 18β甘草次酸纳米晶体（NGA）：所制NGA混悬液稳定且带负电荷（-45.9 ± 1.3），粒径分布均匀，平均粒径为 288.6 ± 7.3 nm，其溶解性、经皮渗透性能及抗炎活性均明显提升。.2. 两亲性壳聚糖（ACS）：所制ACS在水和pH 7.2 PBS中的溶解度由水不溶变为易水溶，具有良好的血液相容性且当其浓度在1 mg/mL以下时对HaCaT无细胞毒性。.3. 两亲性壳聚糖/18β甘草次酸纳米晶复合物（ANGA）：ACS与NGA的质量比为1∶10时，能得到稳定的ANGA分散体系，且体系带正电荷，可使其在皮肤表面负电荷条件下因静电吸引而粘附性更强；同时，其透皮累积渗透率可达到 75.3%，比累积渗透率为56.5%的NGA提高了18.8%。.4. ANGA抗皮肤光老化作用研究：ANGA可通过抑制NF-κB核因子的转录，以降低小鼠皮肤促炎细胞因子的表达，同时增强激活核因子Nrf2的表达，大量表达下游抗氧化酶，两通路协同作用，最终影响光老化的进程，表现出更加显著的抗光老化效力。..科学意义.研究有助阐明18β-甘草次酸治疗皮肤光老化（SP）的机制，为探讨中药抗SP的机理研究提供新的研究思路。同时，两亲性壳聚糖/18β甘草次酸纳米晶复合物（ANGA）的构建，可为新型高效抗SP新药的研发提供新的视角和理论依据。