慢性应激肝郁脾虚证大鼠模型下丘脑、胃基因表达谱及逍遥散干预机制的研究

As one of common patterns in clinic, although previous studies suggested that the syndrome of liver-qi staganation and spleen-qi deficiency has objective modern pathophysiological basis, its pathological mechanism remains unknown，and therefore the essence of this syndrome need further study. Xiao-Yao-San is widely used in clinic for various kinds of stress disease, but its mechanism is still not clear.In our study, after the rat model with liver-qi staganation and spleen-qi deficiency syndrome was induced by chronic immobilization stress and meanwhile evaluated, apply the technology of rat whole genome microarray to find out differential gene expression profilings of hypothalamus and stomach between the model group and the group treated with Xiao-Yao-San. And the relationship between the differentially expressed genes and the syndrome of liver-qi staganation and spleen-qi deficiency is analyzed by bioinformatic technology to reveal the commom mechanisms among the differentially expressed genes in hypothalamus and stomach and the emerge of the liver-qi staganation and spleen-qi deficiency syndrome and the regulatory effect of Xiao-Yao-San. Moreover, the results from the gene clips are validated from the histological and cytological level by Real-time PCR and in situ-hybridization, and the histological pathelogical lesion of the hypothalamus and stomach is also observed. Our study may reveal the biological mechanisms of the liver-qi staganation and spleen-qi deficiency syndrome, explain the mechanism and effect target on Xiao-Yao-San for prevention and treatment this syndrome as well as its effect in anti-stress damage. It may provide a scientific experimental evidence for the guidance of clinical practice and richment of the connotation of the Liver-Zang-Xiang Theroy.

肝郁脾虚证是临床上的常见证型，以往的研究表明肝郁脾虚证具有客观的现代病理生理学基础，但其本质规律尚不明确，仍需进一步深入研究。逍遥散广泛应用于临床各科疾病及应激性疾病，然而其作用机制仍不清楚。本项目通过慢性束缚应激方法制作肝郁脾虚证大鼠模型并对模型进行评价；应用大鼠全基因组表达谱芯片筛选模型大鼠及逍遥散干预大鼠动态演变过程中下丘脑、胃组织差异基因表达谱；利用生物信息学技术分析差异表达基因与肝郁脾虚证之间的关系，找出下丘脑、胃组织差异表达基因与肝郁脾虚证发生的共同作用机制及逍遥散的调节作用；同时运用Real-time PCR、原位杂交方法从组织和细胞水平对芯片结果进行验证并观察下丘脑、胃组织病理损害。通过研究从基因组学层面揭示肝郁脾虚证的现代生物学机制及逍遥散防治肝郁脾虚证及抗应激损伤的作用机制和靶点，为指导临床治疗及丰富中医肝藏象学说的内涵提供实验依据。

证候生物学基础及中药复方药效作用机制的发现和阐明，是中医理论现代化研究的主要领域。慢性应激是许多疾病发生和发展的重要因素，肝郁脾虚证是临床上常见的中医证型，涉及许多疾病，其病理机制与脑-肠轴功能紊乱密切相关，逍遥散是治疗肝郁脾虚证的经典名方，广泛应用于临床各科疾病及应激性疾病的治疗。本研究采用慢性束缚应激方法制作肝郁脾虚证大鼠模型并对模型进行评价，利用基因芯片筛选肝郁脾虚证大鼠及逍遥散干预大鼠下丘脑、胃组织差异基因表达谱，并对差异基因表达谱进行GO（Gene Ontology）功能分析、信号通路分析以及差异基因转录调控网络的构建，同时运用Real-time PCR、原位杂交方法对基因芯片结果进行验证。实验结果显示：①通过观测大鼠的宏观表征、行为学、食量、体重、血清D-木糖、CORT和血浆ACTH含量的变化以及逍遥散以方测证，明确制作的应激大鼠模型为肝郁脾虚证模型；②肝郁脾虚证大鼠下丘脑、胃组织存在众多差异表达基因，成功构建肝郁脾虚证大鼠及逍遥散干预大鼠下丘脑、胃组织差异基因表达谱；③下丘脑、胃组织差异表达基因参与了众多生物过程和信号通路的功能显著改变，涉及神经内分泌免疫系统等多系统功能的变化，脑肠轴上多种神经肽、激素、免疫调节因子及其受体出现了异常表达，如5-HT、CRF以及多巴胺系统均出现了异常变化；④肝郁脾虚证大鼠存在复杂的基因调控网络；⑤逍遥散对肝郁脾虚证大鼠显著改变的差异基因、生物过程、信号通路及基因调控网络均有调节作用，并找出了与逍遥散作用正相关最强的网络模块；⑥实时荧光定量PCR和原位杂交验证的差异表达基因和基因芯片结果基本吻合，证实了基因芯片数据结果的可靠性。总之，本次研究为今后从脑肠轴研究肝郁脾虚证的生物学基础提供了依据和思路，为指导临床治疗肝郁脾虚证、抗应激损伤及丰富中医肝藏象学说的内涵提供了科学的实验依据。