基于MrgprA3-TRPA1/P2X3R通路探讨白鲜碱抑制非组胺依赖性痒的机制研究

Histamine-independent itch predominates in chronic pruritus, is a debilitating disorder that is refractory to conventional anti-histamine treatment, which is a lack of effective therapeutic drugs in the clinic. MrgprA3+ neurons are itch-specific neurons that play an important role in histamine-independent itch. We are first time discovered that dictamnine, the main active ingredient in cortex dictamni, has an inhibitory effect on chloroquine and dermatitis induced histamine-independent itch. The mechanism may be that dictamnine affects the function of MrgprA3+ neurons by regulating TRPA1 or P2X3R. However, whether the cross-talk of P2X3R and TRPA1 involved in the MrgprA3 itch signaling transduction remains unknown. This project uses transgenic mice to first investigate the inhibitory effect of dictamnine on acute and chronic histamine-independent itch and the expression of MrgprA3, TRPA1 and P2X3R in peripheral from the overall animal level. Secondly, the effects of dictamnine on the function of MrgprA3+ neurons and TRPA1, P2X3R were investigated by calcium imaging and whole-cell patch clamp recording technique. The above studies will help to deepen our understanding of histamine-independent itch, and clarify the role and mechanism of dictamnine in the treatment of histamine-independent itch, and provide a theoretical basis for the application of dictamnine in the treatment of histamine-independent itch.

非组胺依赖性痒在慢性瘙痒中占主导地位，对抗组胺药物治疗无效，临床尚缺乏有效治疗药物。MrgprA3+神经元是痒觉特异性神经元，在非组胺依赖性痒中起重要作用。我们发现中药白鲜皮中的活性成分白鲜碱对氯喹和皮炎引起的非组胺依赖性痒具有抑制作用。其机制可能是白鲜碱通过调控下游的TRPA1或P2X3R，影响MrgprA3+神经元功能。但是P2X3R和TRPA1在MrgprA3介导的痒中关系还有待探讨。本项目利用转基因小鼠，首先从整体动物水平探讨白鲜碱对急慢性非组胺依赖性痒的抑制作用及对MrgprA3、TRPA1、P2X3R在外周表达的影响。其次采用钙成像、膜片钳技术探讨白鲜碱对MrgprA3+神经元功能及TRPA1、P2X3R活性的影响。以上研究有助于加深我们对非组胺依赖性痒的认识，明确白鲜碱治疗非组胺依赖性痒的作用及机制，为白鲜碱应用于临床治疗非组胺依赖性痒提供理论依据。

慢性瘙痒是临床许多皮肤疾病的显著特征之一，而非组胺依赖性痒在慢性瘙痒中占主导地位。我们前期研究发现白鲜皮在临床炎症性皮肤疾病中使用频率较高，白鲜碱是白鲜皮的主要活性止痒成分之一，但白鲜碱的止痒机制并不清楚。本研究旨在从动物行为-分子-细胞水平探讨白鲜碱能否通过调控非组胺依赖性痒觉通路抑制特应性皮炎（Atopic dermatitis，AD）引起的慢性瘙痒，并明确其机制。实验结果表明：白鲜碱能够抑制氯喹引起的急性瘙痒和AD引起的慢性瘙痒，且对AD小鼠的皮损部位具有保护作用。在AD小鼠皮肤和背根神经节（Dorsal root ganglion，DRG）中，Mas相关G蛋白偶联受体A3（Mas-related G protein-coupled receptor A3，MrgprA3）、瞬时受体电位锚蛋白1（Transient receptor potential ankyrin 1，TRPA1）、P2X3R的表达明显上调，证实了非组胺依赖性痒觉通路在慢性瘙痒中的重要作用。在给予AD小鼠白鲜碱治疗后，白鲜碱能明显抑制MrgprA3、TRPA1、P2X3R的表达。钙成像和电生理实验则进一步明确了白鲜碱对MrgprA3阳性神经元兴奋性的抑制作用。上述研究结果综合表明：白鲜碱能够通过调控MrgprA3介导的非组胺依赖性痒觉通路抑制瘙痒，这为白鲜碱在慢性瘙痒中的临床应用提供了理论依据。