栀子豉汤多成分多靶点神经保护作用机制及其药效物质基础研究

Psychological sub-health has become one of the main types of sub-health with the clinical manifestations of insomnia, anxiety and depression. Clinical application of modern medicine indicated that ZHIZICHITANG had satisfactory effects on psychological sub-health, but its therapeutic mechanism and the effective fractions were not clear. In our previous study, we found ZHIZICHITANG could promote the regeneration and proliferation of neuron cells. According to the results, we proposed that neural protective effects of ZHIZICHITANG may be the main mechanism for its adjusting and improving effection on psychological sub-health. The project had 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, an in vitro pharmacodynamic model was established with damaged PC12 cells induced by Cort and Glu. Secondly, the metabolic activity of the cells and related gene transcription and translation levels were revealed through the change of concentration level of endogenous compounds and degradation products inside and outside cells. Thirdly, the multi-target neural protective mechanism of multi-components in Chinese herbal prescription "ZHIZICHITANG" was revealed from the point of cell metabolome and the molecular biology, respectively. Thus, the effective fractions which can improve and adjust the psychological sub health in Chinese herbal prescription "ZHIZICHITANG" were founded.

心理性亚健康已成为亚健康的主要类型之一，主要临床表现为失眠、焦虑、抑郁等。现代中医临床应用表明栀子豉汤对于心理性亚健康具有良好的调节和改善作用，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可以促进神经元样细胞再生和增殖，提出栀子豉汤保护神经细胞作用可能是其调节和改善心理性亚健康状态的主要作用机制。本项目在已完成的国家自然科学基金项目“基于多维色谱-质谱联用技术的栀子总环烯醚萜苷代谢指纹特征研究”技术成果及建立的栀子豉汤复方及单味药材中化学成分分离、纯化与结构鉴定的技术体系基础上，以Cort和Glu损伤的PC12细胞作为体外药效学模型，从内源性化合物和细胞内外活性物质降解产物的浓度变化角度去揭示细胞的代谢活动以及相关基因的转录和翻译水平，分别从细胞代谢组学和分子生物学角度揭示栀子豉汤复方多成分多靶点神经保护作用机制，确定栀子豉汤改善和调节心理性亚健康状态的药效学物质基础。

栀子豉汤对于焦虑，抑郁等慢性应激造成的心理性亚健康疾病的治疗已达数千年之久，然而其药效物质基础与配伍作用机制至今尚不明确。我们建立了栀子豉汤配伍前后化学成分的定性鉴别方法，归纳总结了栀子豉汤中67种化合物，栀子中58种化合物以及淡豆豉中16种化合物。建立了同时测定栀子豉汤中17种指标性成分的LC-MS方法，考察栀子豉汤配伍比例对主要活性成分溶出变化的影响，发现栀子与淡豆豉的配伍比例为1:1时，异黄酮类和环烯醚萜活性成分溶出为最佳。建立谷氨酸损伤PC12细胞模型，比较了栀子豉汤复方和单方神经保护作用区别，结果表明栀子豉汤的神经保护作用要优于单方，栀子豉汤复方能显著增加PC12细胞存活率，显著降低LDH含量、ROS含量、细胞凋亡率，同时显著提升GR和GSH活性水平。进一步考察栀子豉汤对单胺类神经递质和凋亡相关蛋白影响，结果表明栀子豉汤可显著提高谷氨酸损伤的PC12细胞内单胺递质水平，并且显著增加CREB、p-CREB和Bcl-2蛋白的表达水平，降低Bax蛋白的表达水平。建立了基于GC-MS法的细胞代谢组学研究方法，验证了方法的稳定可靠性，并成功应用于栀子豉汤神经保护细胞代谢组学研究，从细胞代谢角度评价了栀子豉汤的神经保护作用效果，发现了与神经损伤和神经保护相关的40个生物标志物以及13条代谢通路，栀子豉汤发挥药效的机制与其调节改善了细胞内紊乱的能量代谢，氨基酸代谢和脂质代谢有关。应用谷氨酸诱导的PC12细胞损伤模型筛选了栀子豉汤中起到神经保护作用的化学成分，并从药理和代谢两方面探究了其发挥神经保护作用机制，结果表明，栀子苷，对香豆酸和山栀苷甲酯是栀子豉汤发挥神经保护作用的物质基础，并且其神经保护与抗细胞凋亡，维持细胞内氧化还原平衡以及改善细胞内紊乱氨基酸代谢有关。以上研究结果不仅揭示栀子豉汤复方多成分多靶点神经保护作用机制，而且为栀子豉汤的进一步开发提供了科学依据。