基于靶标网络-印迹敲除技术研究西红花活性分子群的抗抑郁协同作用

Saffron is an effect spice used for depression treatment in clinical but the active material basis and mechanism are still unknown. Based on results of our previous researches, we bring forward a hypothesis that active compounds in saffron with anti-inflammatory activity are effective to depression treatment and the synergistic effect of these compounds will enhance efficacy. In order to verify this hypothesis, the network pharmacology of saffron will be firstly studied and the information of active compounds and action mechanisms will be got. According to the above information, characteristic components from saffron would been isolated by preparation HPLC, then magnetic molecularly imprinted polymer will been synthesized and used to selectively extract the characteristic compounds in saffron. After the characteristic compounds in saffron extraction are knocked out, the antidepressant activity of saffron extraction will been evaluated by chronic unpredictable mild stress (CUMS) model. In this project, the change of antidepressant activity of saffron extraction after and before knockout characteristic compounds in saffron will been as antidepressant activity of characteristic compounds. According to the antidepressant activity of characteristic compounds, the material basis will been elucidated. Then ,the targets of FABP7、MAP2K1、RXRB will been detected by Western blot and Immunofluorescence assay in order to study the antidepressant mechanism of saffron. The research idea reflects the overall and collaboration effect of traditional Chinese medicine and the results will be useful to clarify material basis of saffron used to depression treatment.

西红花治疗抑郁症临床疗效明确但物质基础和作用机制不明，不利于开发抗抑郁新药。本项目在前期研究结果的基础上，提出了“具有抑制免疫炎症反应的西红花活性分子群通过协同作用可显著性增强其抗抑郁活性”的假说。为此，本项目首先构建了西红花靶标网络，预测其抑制免疫炎症反应的作用靶点和活性分子群；接着采用磁性分子印迹技术选择性敲除靶标网络预测的西红花提取物中的某一活性成分，人为切割该成分与其它成分之间的协同作用，再采用大鼠CUMS抑郁模型测定敲除前后西红花提取物的抗抑郁活性变化值，以活性变化值作为该协同作用对西红花整体抗抑郁活性的贡献值，并以此依次确定西红花抗抑郁的主要活性分子群。最后采用Western blot和免疫荧光法检测FABP7、MAP2K1、RXRB等可能作用靶点，初步探讨西红花抗抑郁的作用机理。该项目体现了整体性和协同性的研究思路，也为阐明西红花活性分子群和作用机制提供科学依据。

临床实验研究表明，西红花具有良好的抗抑郁活性，其抗抑郁作用可能与其抗炎活性有关。本项目首先通过网络药理学的研究方法，发现西红花抗抑郁的潜在活性成分包括西红花酸、西红花苦苷等10种成分；其关键作用靶点为MAP2K1, MAPK1, HRAS, PIK3R1, ALB 和AKT1等；其作用的信号通路主要余免疫系统和信号转导等通路相关；接着采用机械化学固相提取、大孔吸附树脂法、分子印迹技术、制备高效液相色谱法等方法，制备了西红花苷类、西红花苷-1、西红花酸、西红花苦苷和敲除了西红花苷-1及西红花苦苷的西红花提取物；通过脂多糖诱导的BV2小胶质细胞模型，发现西红花酸是西红花中的主要抗炎活性成分之一；最后，采用抑郁症模型小鼠评价了西红花酸抗抑郁活性及其作用机制，认为西红花酸能显著性改善慢性束缚应激抑郁小鼠的抑郁样行为，能显著降低MKP-1、proBDNF、丙氨酸转氨酶、天冬氨酸转氨酶的升高，提高血清DA和CREB水平；组织病理学分析表明，西红花酸通过保护神经细胞抑制束缚应激小鼠的海马损伤。免疫荧光和WB分析显示，西红花酸能够升高海马区ERK1/2和CREB的表达，降低MKP-1和proBDNF的表达。同时，西红花酸组能够改善肠道生态系统。上述研究结果说明西红花酸可能通过调节MKP-1-ERK1/2-CREB信号通路和改善肠道菌群发挥神经保护及改善应激引起的大脑损伤。此外，基于网络药理学的研究结果，经过实验验证，发现西红花酸能增强PI3K、AKT、GSK3β和mTOR的磷酸化水平，减少血清炎症因子的含量，降低海马组织中炎症因子的蛋白表达，起到快速抗抑郁作用。