栀子豉汤通过肠道菌群-肠-脑轴介导的抗抑郁作用机制及其药效物质基础研究

Clinical application of modern medicine indicated that ZHIZICHITANG had satisfactory effects on depression, but its therapeutic mechanism and the effective fractions are not clear. In our previous study, we found ZHIZICHITANG could shorten the immobility score of the rat in forced swimming test and tail suspension test, while it had no significant effect on germ-free animals. According to the results, we proposed that the main mechanism of ZHIZICHITANG for antidepressant effect might be the influence of intestinal microflora on brain gut axis. The project has been founded based on the completed National Natural Science Fund Project " Studies on characteristic fingerprint of the metabolism of iridoid glycosides in Gardenia jasminoides Ellis by multiple-dimensional chromatography-electrospray ionization mass spectrometry " and the technology system of separation, purification and structure identification of active components in the ZHIZICHITANG and the single herbs established in our previous work. Firstly, from the point of molecular microbial ecology and metabolomics, the effects of ZHIZICHITANG on the composition of intestinal microbiota will be studied and the bacteria related with antidepressant effect of ZHIZICHITANG will be founded. Secondly, small molecules participate the regulation of microbiota-gut-brain axis will be screened. Thirdly, the anti-depressive effect mechanism mediated by microbiota-gut-brain axis will be revealed. Thus, the anti-depressive effect fractions in Chinese herbal prescription "ZHIZICHITANG" will be founded. All these can supply reference for the screening of antidepressant for depression with intestinal microbiota as a new target.

现代中医临床应用表明栀子豉汤对于抑郁症具有良好的抗抑郁作用，但其作用机制及药效物质基础尚不明确。我们在前期研究中发现栀子豉汤能缩短大鼠强迫游泳和悬尾试验的不动时间，对胃肠道无菌群定植的无菌大鼠却无显著影响，提示栀子豉汤可能是通过肠道菌群-肠-脑轴介导其抗抑郁作用。本项目在已完成的国家自然科学基金项目技术成果及前期工作建立的栀子豉汤化学成分分离纯化及鉴定相关技术体系基础上，从分子微生物生态学代谢组学角度探讨栀子豉汤对肠道菌群微生物生态的影响，寻找与栀子豉汤抗抑郁作用相关菌群，筛选参与肠道菌群-肠-脑轴调节的活性小分子，从而揭示栀子豉汤通过肠道菌群-肠-脑轴介导的抗抑郁作用机制及其药效学物质基础，为将肠道菌群作为新靶点筛选抗抑郁中药复方提供思路和借鉴。

本课题以传统经方栀子豉汤为研究对象，通过建立慢性不可预见性轻度应激（CUMS）大鼠抑郁模型，发现栀子豉汤对抑郁大鼠行为及海马形态有明显的改善作用，表明栀子豉汤有明显的抗抑郁作用。采用基于16SrRNA高通量测序的方法，研究正常大鼠生理性肠道微生态和抑郁模型大鼠病理性肠道微生态的结构特点，比较栀子豉汤给药前后的菌群结构差异，发现在门和属水平上栀子豉汤均可改善由CUMS所致的盲肠内容物菌群的失调。进一步考察栀子豉汤提取物对脑-肠轴不同部位促炎细胞因子、脑源性神经营养因子（BDNF）以及5-羟色胺（5-HT）等物质的调控作用，发现栀子豉汤提取物可以降低CUMS大鼠不同部位中促炎细胞因子和5-HIAA的含量，升高其不同部位中BDNF、5-HT及5-羟色胺/5-羟吲哚乙酸（5-HT/5-HIAA）的含量。对栀子豉汤有效部位染料木苷等8种成分进行定量分析。比较栀子豉汤给正常和抑郁大鼠给药后栀子苷等5种环烯醚萜苷的药代动力学参数，发现CUMS大鼠药代动力学参数与正常组有显著差异，从药代动力学角度进一步佐证栀子豉汤通过肠道菌群-肠-脑轴介导抗抑郁作用的机制。采用代谢指纹图谱的方法比较栀子豉汤在正常大鼠和抑郁大鼠血浆及胃肠道的代谢差异，发现抑郁大鼠中栀子豉汤入血成分较正常大鼠多，而胃肠道代谢物较正常大鼠少。分别研究不同饲料对大豆异黄酮在大鼠体内的药代动力学的影响，建立了一种高灵敏度、高选择性的LC-MS/MS方法，同时检测大鼠血浆中的大豆苷、大豆苷元、黄豆黄苷、黄豆黄素、染料木苷、染料木素等6种成分，并成功测定了淡豆豉处理的大鼠进食常规含异黄酮和不含异黄酮的饲料后体内大豆异黄酮的动态变化水平，发现进食含异黄酮饲料的大鼠血浆中三种异黄酮苷含量低于无异黄酮饮食组，大豆苷元、黄豆黄素、染料木素的曲线下面积约为进食无异黄酮饲料大鼠体内的2-3倍。本研究完成了课题的预期目标，揭示了栀子豉汤通过肠道菌群-肠-脑轴产生抗抑郁作用的机制，为栀子豉汤临床用药提供实验基础和理论依据。