基于下丘脑弓状核促进与抑制摄食调控通路的肝郁脾虚证候形成的机理研究

Liver-depression and spleen-deficiency syndrome is the chief syndrome of sub-health, and the frequent syndrome of various clinical diseases. At present, experimental studies emphasis on emotional disorders and behaviour changes relating with the liver depression, while lack of studies on spleen-stomach malfunction manifested with food-intake and body-weight decreased, and loose stool..The modern medicine think that two types of neuropeptides related to appetite express in the arcuate nucleus(ARC) of hypothalamus, such as Neuropeptide Y(NPY)/agout-related protein(AgRP) and pro-opiomelanocortin(POMC)/cocaine- andamphetamine regulated transcript(CART) . Leptin derived from periphery traverse the blood brain barrier and arrive in ARC to combined with leptin receptor(ob-R), and act on NPY/AgRP and POMC/CART. Our early studies demonstrated that the expression of ob-R increased obviously and instability changes of NPY in ARC of rats with liver-depression and spleen-deficiency..Combined with the function of ARC in hypothalamus on regulation of appetite and energy metabolism and our early studies, in this study, we establish the rats models with liver-depression and spleen-deficiency syndrome adopt the method of chronic immobilization stress, and adopt the RNAi method to silent NPY mRNA in ARC avoid of the interference of NPY, and adopt molecular biology methods(such as, ELISA, RT-qPCR, ISH, etc.), to dynamic observe changes of POMC/CART nerve pathway related to appetite, for exploring the critical central nervous endocrine basis of spleen and stomach malfunction in liver-depression and spleen-deficiency syndrome, and clarifying the main functional target and mechanism of Xiaoyaosan(Formularies of the Bureau of People's Welfare Pharmacies) for smoothing liver to invigorating spleen in order to regulate appetite.

肝郁脾虚证是亚健康人群首要证候，也是临床多种疾病常见证候之一。目前实验重点集中于动物模型出现情绪障碍和行为改变等肝郁样表现的基础研究，而肝郁脾虚证机体出现食少、便溏、消瘦等脾胃运化功能失常的机制研究涉及不足。.结合现代医学关于下丘脑弓状核主要表达NPY/AgRP和POMC/CART两类食欲相关神经肽，外周Leptin结合ob-R作用于这两类食欲肽而发挥作用。前期研究肝郁脾虚模型大鼠下丘脑弓状核中ob-R明显升高而NPY变化不定，故认为POMC/CART为Leptin结合ob-R后调控的主要神经通路。本研究通过慢性束缚应激建立肝郁脾虚模型，以RNAi沉默弓状核中NPY表达以排除NPY的干扰，从基因与蛋白水平动态观测下丘脑ARC中POMC/CART摄食相关神经通路变化机制，探讨肝郁脾虚证脾胃运化功能失常的关键中枢神经内分泌基础，及逍遥散（《局方》）疏肝健脾以调节食欲的主要作用靶点和机理。

本课题组一直致力于肝郁脾虚证候机体出现食少、便溏、消瘦等脾胃运化功能失常的机制研究。前期研究认为，慢性应激肝郁脾虚证候机体出现食欲下降、体重减轻等表现，下丘脑弓状核（ARC）中瘦素受体（ob-R）的表达增加为其关键中枢神经内分泌基础之一；进入ARC中的Leptin与ob-R结合，引起模型大鼠体重和摄食量下降的中间环节并非主要通过抑制NPY神经通路，而阿黑皮素原（POMC）/可卡因-安非他明调节转录因子（CART）通路极有可能为其关键环节。.所以本研究首先构建了NPY的腺病毒pAd-NYi干扰质粒，以RNAi沉默ARC中NPY表达，排除NPY对ARC中Leptin结合ob-R后下行摄食调控通路的干扰作用。并以每天3h，连续21天束缚应激方法制备慢性应激肝郁脾虚大鼠模型。同时对模型大鼠ARC注射pAd-NYi，结合分子生物学方法观测各组大鼠ARC中NPY、Leptin、ob-R、POMC、CART、а-MSH蛋白与基因表达。.结果表明：（1）动物外观表征结合行为学、血清D-木糖含量变化，及逍遥散干预以方测证，综合表明每天3h，连续21天束缚应激可成功复制慢性应激肝郁脾虚大鼠模型。（2）模型大鼠ARC中ob-R表达明显减少，与POMC、CART、α-MSH变化一致，说明应激状态下血清中升高的Leptin进入ARC与ob-R结合，下行调节POMC/CART通路，是引起模型大鼠体重和摄食量下降的关键环节。pAd-NYi能明确干扰模型大鼠下丘脑ARC中NPY表达同时，也导致了ob-R的下降，但POMC、CART、α-MSH变化趋势并未受明显影响。再次表明，应激状态下ARC中Leptin与ob-R结合后，主要通过影响POMC/CART通路，导致机体出现摄食、体重下降等脾胃功能失常表现。（3）逍遥散可调节模型大鼠ARC中ob-R、POMC、CART、α-MSH基因与蛋白表达。而pAd-NYi干扰模型大鼠ARC中NPY表达后，逍遥散也对ob-R、POMC、CART、α-MSH起到了调节作用，更进一步反证了ARC 中POMC/CART，是逍遥散疏肝以健脾调节慢性应激肝郁脾虚机体脾胃功能失常的重要通路。.本研究结论：外周Leptin进入下丘脑ARC中结合ob-R，下行调节POMC/CART通路，是慢性应激肝郁脾虚机体出现脾胃功能失常关键环节，也是逍遥散疏肝以健脾，调节食欲的重要作用靶点。