下丘脑弓状核羟色胺能神经元在瘙痒中枢敏化中的作用及其机制

Chronic obstinate pruritus is considered a histamine-independent itch, the main clinic characteristic of which is hyperknesis and alloknesis, and central sensitization is one of the main mechanisms. Hypothalamic arcuate nucleus is chemo-sensitive central nuclei and directly modulates energy metabolism. Serotonergic neurons in the hypothalamic arcuate nucleus synthesize serotonin (5-HT) by tryptophan hydroxylase (TPH). We hypothesize that after releasing from serotonergic neurons, 5-HT binds to its corresponding receptors in spinal dorsal horn, and mediates the facilitatory function of hypothalamus - brainstem - spinal cord to promote the maintenance of itch- related central sensitization. This project is to generate adult mutant mice that specific silence the expression of TPH and another type that specific knockout serotonergic neurons at the hypothalamic arcuate nucleus, mice that specific mark serotonergic neurons at the hypothalamic arcuate nucleus with green fluorescent protein and mice that specific down-regulate GRP receptors of serotonergic neurons at the hypothalamic arcuate nucleus. Using those mice models, the goal of our research is to understand the effect and mechanisms of serotonergic neurons at the hypothalamic arcuate nucleus modulating chronic itch-related central sensitization from animal behaviors, anatomy, electrophysiology and molecular level. We seek to demonstrate a novel theory in the mechanism of pathologic itch and provide a new rationale foundation for clinic treatment.

慢性顽固性瘙痒属于组胺非依赖性瘙痒，其主要临床特点是痒觉过敏和异常性瘙痒，中枢敏化是其主要机制之一。下丘脑弓状核是近年来倍受重视的直接调控能量代谢的化学敏感性中枢核团，推测弓状核内羟色胺能神经元通过色氨酸羟化酶（TPH）合成并释放5-羟色胺（5-HT），结合脊髓背角相应受体，介导下丘脑-脑干-脊髓下行易化作用，促进包括瘙痒在内的伤害性感受"中枢敏化"的维持，然而尚无相关研究报道。本研究拟通过建立成年小鼠下丘脑弓状核内特异沉默TPH 表达和羟色胺能神经元特异敲除两种模型、绿色荧光蛋白特异标记小鼠下丘脑弓状核内羟色胺能神经元的模型，以及特异下调小鼠下丘脑弓状核内羟色胺能神经元内GRP受体表达的模型，从动物行为学、解剖学、电生理学以及分子水平来研究下丘脑弓状核内羟色胺能神经元在慢性瘙痒中枢敏化中的作用及其机制，旨在病理性瘙痒的机理研究中有新的理论突破，并为临床治疗提供新的理论基础。

为了阐明下丘脑弓状核伤害性神经元在搔抓行为中的作用，本研究检测了弓状核神经元在后颈皮内注射compound 48/80所致瘙痒中的效应。在下丘脑弓状核注射对照病毒和TPH2小干扰RNA病毒后，将雄性C57BL/6J小鼠随机分为：对照组、compound 48/80组及TPH2-compound 48/80组。分别在小鼠接受颈背部皮内注射生理盐水（100µl）、compound 48/80 (100µg/100µl)后，立刻以5min为一个周期记录搔抓次数，记录30min。结果显示成功构建的小鼠TPH2 shRNA慢病毒载体能有效的抑制PC12细胞中TPH2的表达，立体定位下将其注射至瘙痒模型小鼠弓状核后，能抑制动物搔抓行为，同时下丘脑弓状核c-Fos表达减少，而小鼠体温、心率、体重等生理功能均未受到明显影响，由此证明下丘脑弓状核内注射TPH2 shRNA慢病毒可对瘙痒模型小鼠产生止痒效应，为顽固性瘙痒治疗提供新方法。