加减青娥方基于雌激素受体调节ERK1/2-Nrf2-ARE信号通路抗皮肤光老化研究

The ERK1/2-Nrf2-ARE signaling pathway mediated antioxidant effect based on estrogen receptor activation is the key point to prevent skin aging. The Modified Qing E Formula is a prescription with the addition of the root of red-rooted salvia and subtraction of walnut and garlic based on the ancient Qing-er pill. It has been proved to exert estrogen-like activity and can suppress skin aging, but the exact mechanism is unknown. In this study, UPLC/DAD-QtofMS and Nrf2-ARE Dual-Luciferase Reporter Assay System were used to identify the antioxidant substance of MQEF through activation of ER in the first place. Human keratinocytes (HaCaT cells) after UVB irradiation and UV-induced skin aging mice were chosen as models of the research. RNA interference technique, laser confocal, FCM flow cytometry, RT-PCR and western blot are used to detect the effects of Modified Qing E Formula on suppressing photoaging by activating ERK1/2-Nrf2-ARE signaling pathway through Estrogen receptor, in order to provide the experimental basis for the scientific connotation of “invigorating kidney qi and promoting blood circulation to remove blood stasis” of traditional Chinese medicine in the treatment of skin aging, and provide experimental basis for the development of phytoestrogens drugs for skin anti-photoaging.

基于雌激素受体（ER）活化ERK1/2-Nrf2-ARE信号通路介导的抗氧化损伤是抗皮肤光老化的关键环节。加减青娥方（MQEF）是在经典方剂青娥丸基础上减掉胡桃仁、大蒜加上丹参化裁而来，前期研究证实其具有雌激素样活性，改善皮肤衰老，但具体机制不明。本研究首先应用UPLC/DAD-QtofMS及Nrf2-ARE双荧光素酶报告基因系统明确MQEF通过ER发挥抗氧化作用药效物质基础。建立UVB损伤HaCaT细胞和UV致皮肤衰老小鼠模型，应用ER阻断剂及基因沉默技术，采用激光共聚焦、流式细胞术、RT-PCR和免疫印迹等方法，从体外活性筛选、细胞和整体动物水平阐明MQEF基于ER调节ERK1/2-Nrf2-ARE信号通路发挥抗皮肤光老化的作用机制。本研究为“补益肾气，活血化瘀”方剂MQEF治疗皮肤衰老的现代科学内涵提供实验依据，并为开发植物雌激素类抗皮肤光老化创新中药奠定实验基础。

利用制备型HPLC，优化分离条件后按照保留时间制备加减青娥方标准组分得11份标准组分。成功构建了Nrf2-ARE通路的报告基因筛选系统，对加减青娥方的11份标准组分及全方进行了ARE转录活性的筛选，发现全方及标准组分2、3、4、5、6、7、8、9均具有良好的ARE转录活性。采用UPLC/DAD-QtofMS分析保留各标准组分的DAD-MS信息，鉴定各标准组分中的化合物结构。对鉴定得到的化合物及加减青娥方全方进行UVB辐射HaCaT细胞光损伤的保护作用研究，发现全方及鉴定化合物，能显著增强UVB辐射HaCaT细胞的细胞活力，减少细胞中ROS水平，减轻DNA损伤，降低其凋亡；预给予雌激素受体抑制剂后，鉴定化合物的上述作用减弱。生化试剂盒检测显示光损伤细胞给予全方及鉴定化合物后，细胞内MDA含量显著降低，细胞SOD、CAT、GSH-Px的酶活力显著增强，初步阐明了加减青娥方全方及鉴定化合物对HaCaT细胞经由ERK1/2-Nrf2-ARE信号通路发挥保护作用的调控机制。在以UV辐照的ICR小鼠为研究对象的动物实验中，得到了相同的结论。本研究为“补益肾气，活血化瘀”方剂加减青娥方治疗皮肤衰老的现代科学内涵提供实验依据，并为开发植物雌激素类抗皮肤光老化创新中药奠定实验基础。