TRPC1参与糖尿病机械性痛敏的机制研究

Mechanical allodynia is one of the most common modalities of pain for diabetic patients, but the clinic treatments have been stranded because of the molecular mechanisms underlying the symptom are largely unknown. Transient receptor potential canonical 1 (TRPC1) is a one member of transient receptor potential family with mechanical sensitivity, our preliminary study showed that with the development of diabetic mechanical allodynia (DMA), the expression level of TRPC1 protein was significantly increased in large- and medium-sized dorsal root ganglion (DRG) neurons and afferent projection area of Aβ fibers in the spinal dorsal horn. Further pharmacology behavioral study revealed that inhibition of TRPC1 channel activity with non-specific and relatively specific blockers caused significant alleviation of DMA. These results strongly suggest that TRPC1 may play a pivotal role in the progression or the development of DMA. Thus, this project is aimed to investigate the molecular mechanism of TRPC1 in the involvement of DMA by conducting the following: (1) the spatio-temporal expression and function of TRPC1 along with the development of DMA, (2) the upstream activating mechanisms of TRPC1 in the DRG neurons in vivo, (3) the downstream signal transduction mechanisms of TRPC1 in the algesic process after activation. This project will pave the new avenue for clarifying the peripheral molecular mechanisms of DMA and provide the theoretical basis for the development of new analgesics in clinical treatment of chronic pain.

机械性痛是糖尿病患者常见的痛敏形式之一，但因其分子机制不清使临床治疗陷于困境。TRPC1是有机械敏感性的瞬时感受器电位基因家族成员，我们的预实验结果显示，随糖尿病机械性痛（diabetic mechanical allodynia, DMA）的发展，TRPC1在背根神经节大、中型神经元和脊髓背角Aβ纤维分布区的表达显著上调；阻滞TRPC1通道能显著缓解大鼠的机械性痛行为，提示TRPC1可能参与了DMA的发展和维持。因此，本项目拟聚焦于TRPC1参与DMA的分子机制，研究：①TRPC1随DMA发展的时空表达特点和功能；②背根神经节神经元中TRPC1的上游激活机制；③TRPC1激活后发挥促痛作用的下游信号转导机制。本项目将为揭示目前研究较薄弱的糖尿病机械性痛的外周分子机制开辟新思路，并为研发临床镇痛新药提供科学依据。

机械性刺激导致的机械性痛对糖尿病患者影响严重，因而聚焦于糖尿病机械性痛（DMA），探索并揭示该类疼痛形式的分子机制，将为临床开发治疗糖尿病性痛的新靶标提供科学依据。方法先应用免疫组化染色观察生理条件下TRPC1在背根神经节（DRG）和脊髓背角（SDH）中的表达特点；再采用雄性SD大鼠随机分为Vehicle组，DMA 7、14、21和28 d组，腹腔注射STZ建立糖尿病模型；继而应用测痛仪筛选DMA模型大鼠；分别取Vehicle组和DMA组大鼠的L4-5节段的DRG和SDH，应用RT-PCR、Western blot和免疫荧光化学染色，观察TRPC1的时空表达变化和TRPC1的神经生物学特性；应用行为药理学方法，观察干预TRPC1后对DMA大鼠痛行为的影响；钙成像和电生理方法观察DRG神经元中TRPC1通道的功能特点；最后观察TRPV4，TRPC6和TRPA1在DMA发展中的表达和功能特点。结果表明，与Vehicle组相比，在DRG和SDH中TRPC1的mRNA和蛋白质表达均呈增高趋势，主要表现为DMA 28 d组TRPC1表达明显升高；DRG神经元的TRPC1/NF200双标阳性细胞数显著增加；单次或多次给予SKF96365或TRPC1 AS ODN抑制TRPC1，均能有效缓解DMA大鼠痛行为，且减少了TRPC1在DRG神经元中的表达量；通过提高动作电位（AP）的频率以及自发AP的产生，可能是TRPC1参与DMA痛的机制。此外，DRG和SDH中TRPC6、TRPV4和TRPA1的mRNA和蛋白质表达分别在DMA7、14和21d组显著升高；在DMA14d给予TRPV4拮抗剂后，也能有效缓解DMA大鼠的痛行为。结论过表达的TRPC1参与了DMA的发展和维持，其机理与TRPC1在DRG大细胞中表达增多，进而影响神经元AP的频率和产生有关；在DMA发展过程中，机械性敏感性的TRPC6，TRPV4和TRPA1也以不同的作用方式，与TRPC1共同参与构成DMA的分子谱系。