基于下丘脑NES1-OT-脑干POMC神经通路的肝郁脾虚证食欲不振的机理研究

The syndrome of stagnation of liver qi and spleen deficiency is one of common syndromes in clinic, the main symptoms of which is loss of appetite. It has been discovered that the activation of hypothalamic/nesfatin-1 (NES1)-oxytocin (OT)-brainstem/pro-opiomelanocortin (POMC) neuronal pathway could effectively regulate appetite, in which the suppressor peptide NES1 plays a pivotal role. Our former research indicated that syndrome of stagnation of liver qi and spleen deficiency showed increased NES1 levels, which were positively correlated with the reduction in food intake in rat model. Therefore, in this current study, we propose a hypothesis that the mechanism of loss of appetite of syndrome of stagnation of liver qi and spleen deficiency may be related to the activation of hypothalamic/NES1-OT-brainstem/POMC neuronal pathway. This study firstly develops the rat model of syndrome of stagnation of liver qi and spleen deficiency that will be effectively assessed with the effects of Xiaoyao Powder highly correlating the syndrome and the behavioral experiments. Then, three key nodes in this neural pathway related to appetite are respectively blocked with the NES1 antibody, OT receptor blocker and α-melanocyte stimulating hormone (α-MSH, a POMC derived peptides) receptor blocker via the intracerebroventricular microinjection, and meanwhile the methods including reverse transcription-quantitative polymerase chain reaction, in situ hybridization, immunohistochemistry, double immunofluorescence, electron microscopy are used to detect the expressions of related gene and protein in the neural pathway. Overall, the purpose of this study is to clarify the molecular mechanism of loss of appetite in syndrome of stagnation of liver qi and spleen deficiency based on the hypothalamic/NES1-OT-brainstem/POMC neuronal pathway. This study will be of great significance in understanding the biological basis of loss of appetite manifested in the syndrome of stagnation of liver qi and spleen deficiency.

肝郁脾虚证为临床常见证候，其主症之一为食欲不振。下丘脑nesfatin-1（NES1）-催产素（OT）-脑干前阿黑皮素原（POMC）神经通路在食欲调节过程中起重要作用，NES1为此通路的关键分子。课题组前期实验发现：该证候大鼠模型下丘脑NES1水平明显升高，与进食量的降低呈正相关。本项目提出假说：“肝郁脾虚证食欲不振的生物学基础与下丘脑NES1-OT-脑干POMC食欲神经通路的激活有关”。拟在复制该证候大鼠模型基础上，分别以NES1抗体、OT受体阻滞剂、POMC受体阻滞剂对此神经通路的三个关键节点进行阻断，以抑制该神经通路，采用动物行为学、脑室微量注射法、RT-qPCR、原位杂交、免疫组化、免疫荧光双标、电镜等技术方法，并通过逍遥散以方测证，研究基于下丘脑NES1-OT-脑干POMC食欲神经通路在肝郁脾虚证食欲不振形成的分子机制。这对于深刻理解肝郁脾虚证食欲不振的形成机理具有重要意义。

肝郁脾虚证为临床常见证候，其主症之一为食欲不振。下丘脑nesfatin-1（NES1）-催产素（OT）-前阿黑皮素原（POMC）神经通路在食欲调节过程中起重要作用，NES1为此通路的关键分子。课题组前期实验发现：肝郁脾虚证大鼠模型下丘脑NES1水平明显升高，与进食量的降低呈正相关。因此提出假说：肝郁脾虚证食欲不振的生物学基础与下丘脑NES1-OT-POMC食欲神经通路的激活有关。为证实以上假说，本研究运用21天慢性束缚应激法复制了肝郁脾虚证大鼠模型，对大鼠下丘脑及脑干NES1的基因及蛋白表达进行检测，明确肝郁脾虚证NES1水平的变化及其与食欲的相关性。然后使用脑室微量注射法阻断外周NES1对下丘脑相关神经元的激活。从食欲及体重变化、下丘脑NES1-OT-POMC食欲神经通路的结构、功能以及上、下游神经肽表达等方面进一步观察下丘脑NES1-OT-POMC食欲神经通路的激活对肝郁脾虚证食欲不振的影响及可能机制。同时给予逍遥散干预，观察逍遥散对下丘脑NES1-OT-POMC食欲神经通路及肝郁脾虚证食欲不振的影响。研究结果显示：肝郁脾虚证模型大鼠存在着外周血清NES1浓度的升高和下丘脑NES1-OT-POMC神经通路的过度激活，而脑干区域的NES1及POMC表达并无显著变化。模型大鼠的进食量和体重出现了明显的降低，并且伴随着抑郁样的情绪状态。逍遥散干预后相关指标的逆转说明其可能通过调节外周NES1信号以及下丘脑NES1-OT-POMC神经通路从而起到疏肝解郁、调节食欲的作用。以此为切入点揭示了肝郁脾虚证脑-肠轴食欲调节失衡的机制，以及中药复方逍遥散疏肝解郁、健脾调节食欲作用的靶点和机理。这一研究也为进一步阐明逍遥散的作用机理和探寻肝郁脾虚型抑郁症的治疗方法提供了新的思路。