星形胶质细胞外泌体lnc-XR-380989增强神经元细胞膜Nav1.6表达在病理性疼痛中的作用

Exosomes are involved in the cell-to-cell communication and nervous system diseases. Our prior experiment showed that inhibition of exosome lnc-XR-380989 of astrocyte attenuated the mechanical allodynia induced by oxaliplatin and L5-ventral root transection (L5-VRT). It is well known that upregulation of Nav1.6 on neuronal membrane played a pivotal role in hyperalgesia. Furthermore, we found that inhibition of lnc-XR-380989 decreased the phosphorylation of STAT3 and Nav1.6 expression induced by oxaliplatin and L5-VRT. Moreover, application of KIF5B siRNA inhibited the upregulation of Nav1.6 on cell membrane mediated by lnc-XR-380989. All these suggested that exosome lnc-XR-380989 might increase the expression of Nav1.6 in cell plasma via activating STAT3, and upregulate Nav1.6 on cell membrane via enhancing KIF5B expression, thereby inducing hyperalgesia. The present project aims to investigate: I) The molecular mechanisms underlying lnc-XR-380989-mediated Nav1.6 upregulation via activating STAT3; II) The mechanisms underlying lnc-XR-380989-mediated transport of Nav1.6 to the membrane via upregulating KIF5B. Our study will provide novel common molecular targets for prevention and therapy of pathological pain.

外泌体作为细胞间通讯的重要媒介，参与神经系统疾病的发生。我们预实验首次显示：抑制星形胶质细胞外泌体lnc-XR-380989（lnc-XR）显著缓解奥沙利铂或腰5脊神经前根切除（L5-VRT）诱导的痛觉过敏。众所周知，神经元细胞膜上Nav1.6的表达增加是痛觉过敏的关键。预实验还发现：抑制lnc-XR或STAT3降低了奥沙利铂或L5-VRT诱导神经元胞浆Nav1.6表达；而抑制KIF5B减弱了lnc-XR介导的细胞膜Nav1.6上调。提示：外泌体lnc-XR，一方面可能通过激活STAT3上调胞浆Nav1.6表达；另一方面通过促进KIF5B表达，增加细胞膜上Nav1.6表达，最终介导痛觉过敏。本研究将探讨：1）lnc-XR激活STAT3介导神经元Nav1.6表达的分子机制；2）lnc-XR增强KIF5B表达介导Nav1.6转运至细胞膜的具体机制。本研究将为临床有效防治病理性疼痛提供新靶点。

CircRNA在神经系统中具有较高的丰度，提示其可能在神经系统疾病中发挥重要作用，但是否参与神经病理性疼痛的发生发展尚不清楚。 我们通过脊神经结扎（Spinal nerve ligation, SNL）建立神经病理性疼痛大鼠模型，采用circRNA 高通量测序发现脊髓背角circAnks1a的表达水平显著升高。我们还发现circAnks1a主要表达于脊髓背角神经元中，使用RNA干扰技术沉默circAnks1a 的表达可显著缓解SNL诱导的大鼠痛样行为，而使用腺相关病毒（Adeno-associated virus,AAV）在脊髓背角神经元中过表达circAnks1a则可以引起正常大鼠的痛觉阈值下降。此外，干预circAnks1a后，我们通过基因芯片发现脊髓背角中circAnks1a可以正性调控下游靶基因血管内皮生长因子 B（Vascular endothelial growth factor B, Vegfb）的表达，通过干预或过表达Vegfb以及借助Vegfbflox/flox 转基因小鼠进行功能验证后，我们证实了Vegfb在SNL模型中的促痛作用。分子机制上，我们发现circAnks1a可以与转录因子Y-box结合蛋白1（Y box binding protein 1, Ybx1）结合，促进Ybx1入核，进而增强其对Vegfb基因的转录调控。另外，circAnks1a可以作为分子海绵，吸附微小RNA-324-3p（miR 324-3p）并抑制其功能，进而增强Vegfb的翻译。 本研究为临床治疗神经病理性疼痛提供了新思路，也为解析环状RNA在神经系统中的表观遗传学机制打开了新视野。