以痒觉特异性MrgprA3+神经元为靶标探究经DRG细胞膜色谱筛选的苦参活性成分对顽固性瘙痒的作用机制

Intractable itch can cause serious damage to the physical and mental health of patients. Because of the questionable effectiveness or serious adverse reactions of the present antipruritic agents, we are in urgent need to develop high efficiency antipruritic drugs with clear mechanism and less side-effect. Radix Sophorae Flavescentis have exact effects to intractable itch, but its components are complex and the antipruritic mechanism is unknown. Under the guide of Traditional Chinese Medicine theories, this study will target the MrgprA3+ receptor, and use cell membrane chromatography to prepare dorsal root ganglion cell membrane chromatography,then to screen activity components of Radix Sophorae Flavescentis which may be combined with MrgprA3+ receptor by it. We will prepare the animal models of intractable itch induced by the square acid butyl ester,chloroquine and evaluate its pharmacological activities through the behaviors and immunology test. The next step we will copy the itching models with MrgprA3+ - EGFP(enhanced green fluorescent protein) transgenic mice, accurate positioning the itch sensation specific neurons of dermatitis combined with red fluorescence tracer injected in skin receptive field. Finally we record the effect of components of Radix Sophorae Flavescentis to the target cell respectively by electrophysiology in vitro and in vivo. All of these will help us clarify the peripheral antipruritic nerve mechanisms.We will using cell membrane chromatography to "fishing" active components of Radix Sophorae Flavescentis and targeting MrgprA3+ itch-specific neuron combined with the curative effect in vivo, target cells and specific molecular and its receptor to study the effective components and mechanisms. This provides a solid foundation for developing new antipruritic drugs. It's a novel strategy and method to the antipruritic research.

顽固性瘙痒严重危害患者身心健康，常用止痒药物大多疗效欠佳。苦参对顽固性瘙痒疗效确切，但其止痒成分及作用机制尚未明确。申请者在前期研究中发现其对组胺非依赖性的顽固性瘙痒有明显抑制作用。本课题拟在中医药理论指导下，以痒觉特异性MrgprA3+受体为靶标，以课题组成熟技术制备背根神经节细胞膜色谱筛选苦参中可能作用于MrgprA3+受体的活性成分，用方形酸二丁酯及氯喹制备顽固性瘙痒模型以评价其药效，再用MrgprA3+-EGFP转基因小鼠复制以上模型，结合红色荧光示踪剂准确定位痒觉特异性神经元，即能离体、在体可视化研究活性成分对此痒觉神经元靶细胞的影响。这种以细胞膜色谱 "垂钓"中药活性成分，在体可视化精确定位痒觉特异性神经元的方法从整体疗效、靶细胞、痒觉特异性神经元及其受体几个层次阐明其物质基础和作用机制，为中药抗顽固性瘙痒研究提供新的策略和方法，也为进一步开发抗组胺非依赖性瘙痒新药奠定基础。

瘙痒，尤其是顽固性瘙痒对患者生活质量危害甚大且无特效药。苦参是中医止痒名方中的常用药，对顽固性瘙痒有确切疗效，但是其抗瘙痒有效成分及作用机制尚不明确。. 首先，利用面颊和小腿两种动物行为学模型，采用方形酸二丁酯（SADBE）分别建立变应性接触性皮炎（ACD）与过敏性接触性皮炎（ICD），对两种皮炎模型小鼠的瘙痒和疼痛行为进行量化，结果表明ACD小鼠的痒觉和痛觉强度均要显著地高于ICD小鼠，这与ACD和ICD小鼠在炎症、免疫相关因子的差异性表达有关，并确定ACD小鼠模型更适合研究顽固性瘙痒；此外在ACD和ICD面颊模型中，雌性小鼠痛觉和痒觉强度均高于雄性小鼠，表明不同性别小鼠可能对SADBE的反应程度不同。与美国约翰霍普金斯大学屈林涛教授、耶鲁大学医学院Robert H. LaMotte教授合作，发现此ACD模型能显著增强MRGPRA3+和MRGPRD+神经元的兴奋性，并且CXCR3/CXCL10趋化因子受体信号及下游Cl-通道在顽固性瘙痒形成中起关键作用。. 随后，通过液相-质谱联用的方法对苦参提取液中21个成分进行鉴定，建立苦参的指纹图谱，并利用改良的细胞膜色谱法制备小鼠背根神经节（DRG）细胞膜固相色谱(CMIC)，筛选出了五种与DRG细胞特异性结合的苦参活性成分（毛蕊异黄酮、氧化苦参碱、氧化槐果碱、高丽槐素、槐果碱），并选取代表性成分氧化苦参碱（OMT）进行药效学评价。首次证实氧化苦参碱对组胺非依赖型瘙痒有显著的抑制作用，并揭示其主要通过直接作用于TRPA1通道而发挥止痒作用。此外，OMT对ACD小鼠顽固性瘙痒具有明确的止痒作用，其作用机制主要与改善炎症及恢复Th1/Th2与Th17/Treg免疫平衡有关。 . 综上，小鼠面颊/小腿模型更能准确反映动物痒和痛的感觉，可以作为一种新的动物模型应用于神经生物学领域；而由SADBE诱导的顽固性瘙痒模型可以用来评价中药抗顽固性瘙痒的药效；细胞膜固相色谱技术为其他中药的研究提供了新的策略和方法；苦参活性成分的筛选及药效评价能促进其在临床的进一步推广使用；也为进一步开发具有自主知识产权的抗组胺非依赖性瘙痒新药奠定基础，具有广阔的应用开发前景。