基于下丘脑弓状核Fam111a介导LepRb-STAT3信号通路调控摄食神经元探讨肝郁脾虚证“食欲减退”的生物学基础

The feeding neurons in hypothalamic arcuate nucleus is closely related to the appetite loss of liver stagnation and spleen deficiency syndrome, but it is still unclear which mechanism regulates the feeding neurons and causes appetite loss in liver stagnation and spleen deficiency syndrome. We used the whole genome DNA methylation and transcriptome sequencing to screen the target molecule Fam111a in the feeding center-hypothalamic arcuate nucleus, which regulates appetite and promotes hypothalamic neurogenesis, and increases hypothalamic neurons to LepRb-STAT3. The sensitivity of the signaling pathway is thus associated with up-regulation of the anorexia neuron POMC. Therefore, we propose a hypothesis: the biological basis of appetite loss in liver stagnation and spleen deficiency syndrome is related to the regulation of the feeding neurons by the Fam111a-mediated LepRb-STAT3 signaling pathway in the hypothalamic arcuate nucleus. This project intends to replicate the rat model of liver stagnation and spleen deficiency in vivo, establish a hypothalamic neurogenesis cell model in vitro, and use Xiaoyao San to test the syndrome model, use overexpression and silence hypothalamic Fam111a, immunofluorescence double label, western blot and RT-qPCR, to research the biological basis of appetite loss in liver stagnation and spleen deficiency syndrome from Fam111a-mediated LepRb-STAT3 signaling pathway in the regulation of feeding neurons. This is of great significance for revealing the scientific connotation of appetite loss in liver stagnation and spleen deficiency syndrome.

下丘脑弓状核摄食神经元与肝郁脾虚证食欲减退密切相关，但何种机制调控摄食神经元引起食欲减退，仍不清楚。申请人前期用全基因组DNA甲基化和转录组测序，在肝郁脾虚证摄食中枢—下丘脑弓状核筛选到重要分子Fam111a，其抑制食欲可能与促进下丘脑神经再生，继而激活LepRb-STAT3信号通路，从而上调抑食欲神经元POMC有关。因此，我们提出假说：肝郁脾虚证食欲减退的生物学基础与下丘脑弓状核Fam111a介导LepRb-STAT3信号通路调控摄食神经元有关。本项目拟通过体内复制肝郁脾虚证模型、体外建立下丘脑神经再生模型、逍遥散以方测证，运用过表达和沉默下丘脑Fam111a、免疫荧光双标、免疫印迹、RT-qPCR等方法，从动物和细胞两个水平、正反两个方面探究Fam111a介导LepRb-STAT3信号通路调控摄食神经元与肝郁脾虚证食欲减退相关。这对于揭示肝郁脾虚证食欲减退科学内涵有重要意义。

下丘脑弓状核摄食神经元与肝郁脾虚证食欲减退密切相关，但何种机制调控摄食神经元引起食欲减退，仍不清楚。申请人前期用全基因组DNA甲基化和转录组测序，在肝郁脾虚证摄食中枢—下丘脑弓状核筛选到重要分子Fam111a，其抑制食欲可能与促进下丘脑神经再生，继而激活LepRb-STAT3信号通路，从而上调抑食欲神经元POMC有关。因此，本项目拟通过复制肝郁脾虚证模型及运用逍遥散以方测证，运用免疫荧光双标、免疫印迹、RT-qPCR等方法，探究Fam111a介导LepRb-STAT3信号通路调控摄食神经元与肝郁脾虚证食欲减退相关。通过研究本项目首先构建稳定的肝郁脾虚型抑郁症的病证结合大鼠模型，并完善模型评价手段；基于以上模型采用全基因组DNA甲基化测序联合转录组测序筛选下丘脑弓状核“食欲减退”关键分子Fam111a；明确下丘脑弓状核Fam111a的细胞定位，预测其与“摄食”的关系及调控机制；通过体内外下丘脑弓状核过表达Fam111a，明确其在肝郁脾虚证“食欲减退”中的关键作用，并初步揭示其调控肝郁脾虚证“食欲减退”的内在机制；逍遥散改善肝郁脾虚证“食欲减退”与下丘脑弓状核Fam111a及其调控的LepRb-STAT3信号及AdipoR1/AMPK/ACC通路有关；最后筛选能够促进下丘脑弓状核神经干细胞增殖、分化的逍遥散活性化合物，为明确逍遥散内发挥改善肝郁脾虚证“食欲减退”关键物质基础奠定前期。以上，对于揭示肝郁脾虚证食欲减退及逍遥散作用机制科学内涵有重要意义。