1-磷酸鞘氨醇对ASD模型鼠背根神经节KCNQ钾通道活性的调控及机制研究

Autism Spectrum Disorders (ASD) is a severe neurodevelopmental disorder ,which begins at the stage of infancy and childhood, and they usually show obvious sensory abnormalities ,in which the abnormal pain sensitivity is paid increasing attention ,for which the mechanism is unknown. The current study suggests KCNQ potassium channels in dorsal root ganglion (DRG) neurons might be associated with this disease .Our early metabolism study found that sphingosine 1 phosphate (S1P) level ,which regulates the activity of ion channel in dorsal root ganglion neurons ,increased significantly in serum of children with ASD and correlated with its clinical phenotype .We speculate , the effection of abnormal level of S1P on KCNQ potassium channels in dorsal root ganglion neurons would mediate the formation of abnormal pain sensitivity in ASD. The proposed project will use C57BL/6 and BTBR rats (ASD model mouse) as the research objects, and change the level of S1P through the methods such as giving S1P and the inhibition of its receptor , from cellular 、molecular and organ levels ，by patch clamp、Western Blot and immunity fluorescence technique , to study effection of S1P on KCNQ potassium channels activity and regulatory mechanisms of S1P on KCNQ potassium channels is mediated by its receptors and the relevant signal transduction pathway.To provide the theoretical basis and new idea for clinical targeted intervention for ASD.

孤独症谱系障碍（ASD）是起始于婴幼儿时期的严重神经发育障碍性疾病，存在明显的多感官异常，其中痛觉异常备受关注，但机制不明。研究表明：背根神经节（DRG）KCNQ钾通道与ASD痛觉异常关系密切；本课题组前期代谢组学研究发现，ASD患儿血清中可调控背根神经节离子通道活性的1-磷酸鞘氨醇（S1P）明显升高，并与其临床表型相关。我们推测，S1P异常可能通过对背根神经节KCNQ钾通道活性的影响而导致ASD痛觉异常形成。本项目以C57BL/6小鼠和BTBR小鼠（ASD模型鼠）为研究对象，通过加入S1P及其受体阻断等方法改变DRG局部环境的S1P水平，应用膜片钳技术、免疫荧光和Western Blot技术，从细胞、分子和器官水平层面，探讨：S1P对DRG神经元KCNQ钾通道活性的作用；S1P经其受体及信号转导通路的介导调控KCNQ钾通道活动的机制。本课题将为ASD患者临床靶向干预提供新思路及理论依据。

孤独症谱系障碍（ASD）痛觉异常备受关注，背根神经节（DRG）KCNQ钾通道与ASD痛觉异常关系密切。本项目对142名ASD儿童和142名正常儿童的疼痛敏感特征进行调查，结果显示ASD儿童存在痛觉异常，并表现出对痛觉不敏感的特征。选取国际公认的BTBR鼠作为ASD模型鼠，采用热刺痛仪及Vonfrey纤维丝刺痛仪，检测其机械痛阈和热痛阈，结果显示BTBR的机械痛痛阈值和热痛阈值明显高于B6组，提示BTBR鼠痛觉敏感性与人群一样存在异常，对痛觉不敏感。检测BTBR鼠和对照鼠的血清和背根神经节中S1P、SPHK及KCNQ2/3/5水平，结果显示与对照组相比，BTBR鼠血清和DRG中S1P和SPHK水平显著升高(P<0.01)，KCNQ2/3/5水平表达降低(P<0.01)，同时给予SPHK阻断干预，BTBR小鼠的痛阈降低(P<0.05)，提示S1P调控KCNQ水平在ASD痛觉异常中发挥重要作用。通过对BTBR小鼠及对照鼠的S1PR检测，结果显示BTBR小鼠DRG中S1PR1、S1PR3的蛋白表达较B6组显著升高(P<0.01)；BTBR小鼠DRG中S1PR2水平较B6组没有明显差异，提示在BTBR鼠背根神经节中S1PR1/3可能发挥重要作用。通过S1PR1/3阻断干预前后，BTBR鼠痛觉行为对比分析，结果显示，VPC23019（S1PR1/3抑制剂）干预后痛阈降低(P<0.05)，提示S1PR1/3可能在ASD痛觉不敏感中发挥重要作用。给予SPHK和S1PR1/3阻断干预前后检测S1PR相关信号通路的表达变化，结果显示，BTBR鼠中ERK表达增高，干预后显著降低，提示MAPK信号通路参与S1P调控KCNQ通道的活性。分别给予S1PR1和3抑制剂（W146和CAY10444），采用免疫荧光技术，观察小鼠背根神经节KCNQ2/3的表达，结果显示干预后BTBR鼠的KCNQ2/3表达增加(P<0.01)。通过膜片钳技术，观察S1P抑制KCNQ通道活性，当分别加入S1PR1/3和ERK抑制剂，再加入S1P时发现，M电流没有变化，证实S1P由S1PR1/3受体介导通过激活MAPK信号通路调控KCNQ2/3通道活性。本项目从人群现象到动物模型的机制验证，通过体外和体内实验，综合阐明了S1P调控KCNQ通道活性与ASD痛觉异常的关联及可能机制，为临床靶向干预提供新思路。