黄荆木脂素VB1调控ROS-MAPK信号通路影响胶原重构在皮肤光老化过程中的作用及机制研究

Mechanistic study of Purified vitexin compound 1 (VB1) affecting collagen reconstruction in dermis through ROS-MAPK pathway during photoaging. Collagen reconstruction mediated by MMPs is the most significant change in the dermis during the process of skin photoaging, Oxidative stress reaction activating MAPK signaling pathways (ROS-MAPK pathway) is one of the most important mechanism of photoaging. In our previous study of screening lots of small molecular substances in murine models of photoaging, Purified vitexin compound 1 (VB1), a novel antioxidant，was proved to exhibit an optimized effect of anti-photoaging. A series of molecular and cytological experiments subsequently indicated that VB1 may take effect through ROS-MAPK pathway. Co-immunoprecipitation and mass spectrometry analysis showed that MAPK1, a key signal molecular of MAPK, is the target of VB1. We speculated that besides inhibiting the production of ROS, VB1 may also directly regulate MAPK signal pathway by combining with MAPK1 to suppress the process of photoaging. In this study, we will first observe the effects of VB1 on UVA-induced collagen reconstruction and the activation of ROS-MAPK pathway, in human fibroblasts, then determine the target site where VB1 combining with MAPK1 by segmented expression vectors. After that, we will further investigate if the combination of VB1 with MAPK1 affecting the activation of MAPK pathway and then inhibiting collagen reconstruct to reverse the process of photoaging using lentivirus vector technology. Finally, we will confirm these mechanisms mentioned above in the murine models in vivo. The aim of our study is to provide theoretical and experimental evidences for the development of VB1 to be a novel drug of anti-photoaging.

光老化过程中真皮最显著的改变为MMPs介导的胶原降解。氧化应激激活MAPK信号通路（ROS-MAPK信号通路）是皮肤光老化最重要的机制之一。我们前期筛选出一种新型抗氧化物－黄荆木脂素VB1，能有效缓解小鼠皮肤光老化外观，并抑制ROS-MAPK途径激活，并进一步鉴定出MAPK关键信号分子MAPK1是木脂素的靶向蛋白。由此推测：VB1除抑制ROS产生，还可与MAPK1直接结合，调节MAPK信号途径，影响光老化过程。在本研究中，我们将以人成纤维细胞为研究对象，观察VB1作用下，UVA诱导人成纤维细胞衰老引起胶原重构及ROS-MAPK通路活化情况的变化，同时利用分段表达载体技术，明确VB1与MAPK1结合的靶位点，并进一步探讨VB1与MAPK1相互结合在逆转光老化过程中MAPK途径激活及胶原重构中的作用，最后利用小鼠光老化皮肤模型在体内验证该作用，为将VB1开发为新型抗衰老药物提供理论和实验依据。

研究背景: .氧化应激激活MAPK信号通路(RO S-MAPK信号通路)是皮肤光老化最重要的机制之一。若能有效抑制氧化应激反应及MAPK信号通路相关蛋白激活，可能会延缓皮肤细胞衰老，我们前期筛选出一种新型抗氧化物-黄荆木脂素VB1，能有效改善小鼠皮肤光老化外观，但其具体的抗衰老机制尚不明确，本研究旨在明确VB1对ROS-MAPK通路活化的影响及其可能的作用机制。.研究内容及重要结果: .我们研究证明VB1对于UVA诱导的皮肤成纤维细胞光 老化进程及其胶原重塑起到一定的保护作用，VB1对UVA诱导下MAPK、EGFR及AKT的信号通路活化均有抑制作用，其中对MAPK的抑制作用最明显，对p38 MAPK、JNK和ERK1/2及下游转录因子AP-1、ELK1的活化均有强抑制作用，且存在量效关系，推测其抗衰老机制主要通过抑制MAPK途径活化起作用。通过质谱分析及IP技术，进一步鉴定出MAPK关键信号分子MAPK1是木脂素的靶向蛋白。通过计算机分子模拟系统，进一步明确E58和R65可能是VB1与MAPK1结合的靶位点，最后利用小鼠光老化皮肤模型在体内验证VB1的抗衰老作用。.科学意义:.整个研究证明VB1除抑制ROS产生，还可与MAPK1直接结合，调节MAPK信号途径，影响光老化过程，并首次确定VB1 与MAPK1 的结合位点。VB1抗衰老机制和作用靶点的明确，将作为候选化合物有望开发为新型抗衰老药物。