基于“诸痛痒疮，皆属于心”理论探讨镇心安神方调控特应性皮炎GRPR/MrgprA3/TRPs信号通路的研究

Atopic dermatitis (AD) is a common skin disease characterized by pruritus, chronically relapsing inflammatory and early occurrence. Currently, it is still lack of durgs which can cure or relief the disease. Seeking for the safe and effective treatment of AD has been a hot scientific issues in related fields. According to the theory of " All kinds of diseases with pain, itching and sore are exclusively related to the heart " referred in neijing, Formulation for Tranquilizing Heart (FTHM), as a new method, is used for the treatment of AD. In our previous research, FTHM was efficacious and safe for AD patients. We suppose that FTHM relieves pruritus by restraining GRPR/MrgprA3/TRPs signaling pathway. In this study, we intend to evaluate the pruritic behaviors in Nc/Nga mice, simultaneously, use immunofluorescence technique, ELESA, Western blot and Real-time PCR methods to test associated protein, molecular and mRNA expression of signaling pathway through vivo and vitro experiments. Through the above studies, we expect to reveal the molecular mechanism of FTHM relieving chronic itching of AD, and provide the scientific basis for the theory of "Treatment determination from heart and mind " in AD and other pruritic diseases.

特应性皮炎是临床上常见的慢性瘙痒性炎症性皮肤病，发病年龄低，病程长，目前尚缺乏治愈或长期缓解的治疗药物，探索安全有效的治疗方法及其作用机制一直是相关领域的热点科学问题。镇心安神方是基于《内经》“诸痛痒疮，皆属于心”理论指导下形成的治疗新思路，在前期应用本方治疗特应性皮炎取得可靠疗效基础上，提出“镇心安神方可能通过调控GRPR/MrgprA3/TRPs信号通路来阻断瘙痒信号传导”的假说。本研究拟以Nc/Nga小鼠为动物模型，通过行为学观察、体内及体外实验，采用免疫荧光、ELISA、Western bolt、Real-time PCR等检测技术，研究镇心安神方对通路相关细胞、蛋白、分子及基因表达的影响，探索镇心安神方缓解特应性皮炎慢性瘙痒的疗效及其分子机制，为“从心神论治”特应性皮炎及其它瘙痒性疾病提供科学依据。

特应性皮炎（atopic dermatitis, AD）是临床上常见的慢性、复发性、炎症性皮肤病，好发于儿童，目前全球约有2%～20%的人群受到本病的困扰。慢性瘙痒是AD的主要特征之一，成为影响AD患者生活、工作和学习重要的因素。因此探索AD慢性瘙痒的产生机制及寻找瘙痒相关靶向治疗策略具有重要的理论及现实意义。本研究基于《内经》“诸痛痒疮，皆属于心”理论，在前期应用镇心安神方（Zhen-Xin-An-Shen-Fang，ZXASF）治疗AD取得可靠疗效基础上，以现代医学的技术和方法为研究手段，从整体水平及细胞分子水平探讨ZXASF治疗AD瘙痒的作用靶点和分子机制。. 研究采用1-氯-2,4-二硝基苯（DNCB）致敏法构建AD样模型，观察治疗前后模型小鼠皮损评分和搔抓频率变化，HE染色观察皮损组织病理学变化，免疫组织化学法、蛋白免疫印迹法和实时荧光定量技术检测皮肤及脊髓组织中GRPR、TRPV1、TRPA1、MrgprA3蛋白及mRNA的表达。在体外实验中，采用辣椒素刺激原代背根神经节（DRG）细胞，采用CCK8法检测细胞增殖，流式细胞技术检测DRG细胞Ca2+内流，ELISA法检测PLC表达。结果显示ZXASF通过下调模型动物体内GRPR、TRPV1、TRPA1、MrgprA3等瘙痒相关受体的表达，减少DRG细胞Ca2+内流，抑制PLC信号通路的活化，抑制痒觉信号的传递，从而改善AD样皮损及瘙痒，以上结果初步阐明GRPR/MrgprA3/TRPs为ZXASF可能的作用靶点，为ZXASF治疗AD及瘙痒性皮肤病提供了科学依据。

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