人参皂苷作用靶点BB型肌酸激酶在其抗应激致大鼠抑郁样行为中的作用研究

By means of pull-down technique, we previously found that brain-type creatine kinase (BCK) might be a target of ginsenosides in brain tissues, which has direct interaction with metabolites of ginsenosides in vivo. First, this project plans to detect the affinity parameters between protopanaxidiol (a representative of ginsenoside metabolites) and BCK via multiple biophysical techniques (i.e. biolayer interferometry), and analyze the binding sites via molecular docking software to confirm the target of ginsenosides in brain tissues from multiple perspectives. Second, the project intends to confirm that the downregulation of hippocampal BCK acitivity is the crutial phathological mechanism of chronic stress-induced depression-like behavior in rats by using chronic unpredictable stress model, BCK RNAi and BCK overexpression schemes. Finally, the project intends to investigate the effects of ginsenosides on BCK activities in vivo and in vitro, and determine whether ginsenosides can block the chronic stress-induced downregulation of BCK activities, impairment of hippocampal structural plasticity, and depression-like behavior by using chronic unpredictable stress model. Meanwhile, we will determine whether the benefical effects of ginsenosides depend on BCK in the hippocampus by the knockdown of BCK using RNAi technology. In conclusion, this project will contribute to the ‘effect-mechanism-target’ evidence chain construction for anti-depression activities of ginsenosides, provide a strong evidence for ginsenosides to prevent and treat depression in clinic, and promote the clinical application of ginseng in the field of psychiatry and neurology.

课题组前期采用亲和垂钓技术发现肌酸激酶可能是人参皂苷脑内作用靶点之一，其与原形人参皂苷的体内代谢产物人参次皂苷和苷元有直接的相互作用。本项目拟以与肌酸激酶亲和力最强的苷元原人参二醇为代表，采用生物膜层干涉等生物物理技术测定其与BB型（脑型）肌酸激酶的亲和常数，并利用分子对接软件分析、确定结合位点，从多角度进行靶点验证；其次，采用慢性应激致大鼠抑郁模型、肌酸激酶RNAi以及肌酸激酶过表达等方案，验证肌酸激酶活性下降是长期应激致大鼠抑郁样行为的病理生理机制。最后，体内外观察人参皂苷对BB型肌酸激酶活性的影响，并采用慢性应激致大鼠抑郁模型结合肌酸激酶RNAi病毒质粒，从正反两个方面阐释人参皂苷通过激活BB型肌酸激酶的活性保护海马结构可塑性、发挥抗抑郁作用的机理。本项目通过完善人参皂苷抗抑郁“药效-机理-靶点”证据链，为临床应用人参防治抑郁症提供实验依据。

亲和垂钓技术发现：BB型肌酸激酶（CKBB）可能是人参皂苷脑内作用靶点之一，其与原形人参皂苷的体内代谢产物人参次皂苷和苷元有直接的相互作用。生物膜层干涉（BLI）技术检测表明：人参皂苷苷元原人参二醇与CKBB直接相互作用最强。随后，两者之间的相互作用得到了BLI动力学、Drug Affinity Responsive Target Stability (DARTS)、Cellular Thermal Shift Assay (CETSA)、等温滴定量热技术（ITC）、分子对接以及氨基酸定点突变等技术的多维验证。接着，项目采用慢性应激致大鼠抑郁模型、腺病毒敲减CKBB以及CKBB抑制剂等方案，验证了CKBB活性下降是长期应激致大鼠抑郁样行为的病理生理机制。最后，项目从体内和体外两个角度观察了原人参二醇对CKBB活性的影响，并从正反两个方面阐释了原人参二醇可通过激活CKBB的活性保护海马结构可塑性、发挥抗抑郁作用的机理。本项目完善了人参皂苷抗抑郁的“药效-机理-靶点”证据链，为临床应用人参防治情志障碍提供了实验依据，并解读了人参安神定魄传统功效的科学内涵。