目的 基于药物亲和反应的靶点稳定性（drug affinity responsive target stability，DARTS）技术筛选京尼平龙胆双糖苷（genipin-1-β-D-gentiobioside，GG）脑内抗抑郁靶点，并探讨GG基于靶点发挥抗抑郁作用的潜在机制。方法 通过DARTS联合无标记液相色谱质谱联用（liquid chromatography-mass spectrometry，LC-MS）蛋白质组学筛选GG在脑组织内潜在蛋白质靶点，再将给药组特有的差异表达基因与抑郁症疾病靶点数据库取交集，共交集出1个潜在抗抑郁靶点蛋白激酶C相互作用蛋白1（protein interacting with C kinase 1，PICK1）。通过生物层干涉技术（biolayer interferometry，BLI）、分子对接和氨基酸定点突变对潜在靶标PICK1与GG之间的亲和力和结合位点进行分析。通过qRT-PCR和Western blotting考察GG对慢性不可预见性轻度应激（chronic unpredictable mild stress，CUMS）小鼠海马组织PICK1、谷氨酸受体A2（glutamate receptor A2，GluA2）和突触后致密蛋白95（postsynaptic density protein 95，PSD95）表达的影响，阐释GG基于靶点PICK1发挥抗抑郁作用的潜在机制。结果 基于DARTS联合无标记LC-MS蛋白质组学共获得29个潜在靶点，将29个潜在靶点与抑郁症靶点数据库交集获得1个GG在脑内的潜在抗抑郁靶标PICK1。BLI结果显示，GG与PICK1存在相互作用，亲和力（K_D）值为1.915×10⁻⁵ mol/L。分子对接结果显示，GG与PICK1存在相互作用，GLN91、LYS83、ILE37、SER36和ILE35与GG形成的氢键为主要作用力。氨基酸定点突变结果显示，突变SER36、GLN91、LYS83后GG与PICK1之间的亲和力变化显著，SER36、GLN91、LYS83为GG与PICK1结合的关键位点。qRT-PCR和Western blotting结果显示，GG显著下调抑郁模型小鼠海马组织中PICK1、GluA2和PSD95的mRNA和蛋白表达（P＜0.05、0.01）。结论 GG可能通过结合PICK1影响突触可塑性相关蛋白的表达水平，进而改善CUMS小鼠海马突触可塑性，发挥抗抑郁作用。

Objective To screen the anti-depressant targets of genipin-1-β-D-gentiopicide (GG) in brain by drug affinity responsive target stability (DARTS) technique, and explore the potential mechanism of GG exerting anti-depressant effect based on target. Methods DARTS combined with label-free liquid chromatography-mass spectrometry (LC-MS) proteomics were used to screen potential protein targets of GG in brain tissues, and the differentially expressed genes specific to drug groups were intersected with the depression disease target database to identify a potential anti-depression target protein kinase C interaction protein 1 (PICK1). Affinity and binding sites between the potential target PICK1 and GG were analyzed by biolayer interference (BLI), molecular docking and site-specific mutation of amino acids. The effect of GG on expressions of PICK1, glutamate receptor A2 (GluA2) and postsynaptic density protein 95 (PSD95) in hippocampus of mice with chronic unpredictable mild stress (CUMS) was investigated using qRT-PCR and Western blotting, and the potential mechanism of GG exerting anti-depressant effect based on target PICK1 was elucidated. Results Based on DARTS combined with label-free LC-MS proteomics, 29 potential targets were obtained, and a potential anti-depressive target PICK1 of GG was obtained by intersecting them with the depression target database. The BLI result showed that GG interacted with PICK1, with affinity (K_D) value of 1.915 × 10⁻⁵ mol/L. Molecular docking showed that PICK1 interacted with GG, and the hydrogen bond formed by GLN91, LYS83, ILE37, SER36 and ILE35 with GG was the main force. The results of amino acid site-directed mutagenesis showed that the affinity between GG and PICK1 significantly changed after mutations at positions SER36, GLN91 and LYS83. SER36, GLN91 and LYS83 were the key sites for the binding of GG to PICK1. qRT-PCR and Western blotting results showed that GG could significantly down-regulated the mRNA and protein expressions of PICK1, GluA2 and PSD95 in hippocampus of depression model mice (P < 0.05, 0.01). Conclusion GG may affect the expression levels of synaptic plasticity-related proteins by binding to PICK1, thereby improving the synaptic plasticity of hippocampus in CUMS mice and exerting an anti-depressant effect.

R285.5

国家自然科学基金项目（82204647，82404886，82574611）；南京中医药大学国自然青年基金经费配套项目（XPT82204647）