以痛温触觉障碍恢复为核心探究推拿促进SNI大鼠感觉功能障碍恢复的机理

As shown by research on the mechanism of early stage tuina treatment for peripheral nerve injury, Tuina can: ①improve the nociceptive and thermal behavioral indicators in peripheral nerve damaged rats ②increase neurotrophic factor expression ③protect neurons ④repair myelin sheath morphology ⑤prevent muscular atrophy. This research tentatively confirms Tuina’s scientific foundation for treatment of this condition. As further research of the mechanism of Tuina treatment and its effect on superficial sensation after peripheral nerve injury, this study will continue to use sciatic nerve injury as the model to study peripheral nerve injury, as well as utilizing an analog Tuina-technique simulator to simulate Tuina techniques while maintaining qualitative and quantitative controls. Intervention will be carried out on rats injured via clamping of their sciatic nerves. Behavioral and functional indicators will be used as indicators to evaluate treatment efficacy. Light and electron microscopy will be used to observe morphological changes in glial cells related to nociception, skin keratinocytes involved in thermal sensation, and merkel cells related to tactile sensation. Key regions along sensory pathways from the periphery to the spinal cord will be examined (skin --- dorsal root ganglion---ending in the dorsal horn). Immunohistochemical techniques and molecular biological methods will be utilized to test for nociceptive indicators (Iba1, GFAP, P2Y12, p38MAPK, NMDA receptor, JNK, et al), thermal sensation indicators (TRPV1, TRPM8, et al) and tactile indicators (Piezo2, et al). The eventual aim being to explore the possible mechanism of Tuina in the recovery of nociceptive, thermal, and mechanical sensations after peripheral nerve injury and to eventually answer the scientific question of how Tuina can treat pain, coldness and numbness.

前期研究主要结论如下：推拿可以①改善周围神经损伤大鼠痛温觉行为学指标；②促进NTFs表达；③保护神经元；④修复神经髓鞘；⑤防止肌肉萎缩；初步确立推拿治疗本病的科学性。为进一步研究推拿对周围神经损伤浅感觉恢复的影响及机理，本课题继续以坐骨神经损伤为例研究周围神经损伤，采用推拿手法模拟仪定性定量模拟手法，对SNI模型大鼠进行干预。将行为学作为疗效评价指标，采用光镜、电镜观察与痛觉相关神经胶质细胞；与温度觉相关角质细胞；与触觉相关梅克尔细胞的形态学变化，运用免疫组化及分子生物学技术，在外周至脊髓感觉通路上选择皮肤、背根节、脊髓背角为靶点，检测疼痛指标（Iba1、GFAP、P2Y12、p38MAPK、NMDA受体、JNK等）；温度觉指标（TRPV1、TRPM8等）；触觉指标（Piezo2等）；探寻推拿促进周围神经损伤痛、温、触觉恢复的可能机理，最终解释推拿能够治疗痛、凉、木这一科学问题。

背景：本课题前期研究得出推拿可以①改善周围神经神经损伤大鼠痛温觉行为学指标；②促进NTFs表达；③保护神经元；④修复神经髓鞘；⑤防止肌肉萎缩。.研究内容：本课题继续以坐骨神经损伤（sciatic nerve injury, SNI）为例研究周围神经损伤，采用推拿手法模拟仪定性定量模拟“三法三穴”推拿干预。通过行为学及形态学研究，得出推拿对感觉及运动功能恢复的表型证据；机理方面通过Western Blot、RT-PCR、免疫荧光、免疫组化等分子生物学方法，从痛觉、温度觉、细胞自噬、轴突再生等方面，系统阐述了“三法三穴”推拿干预促进周围神经损伤恢复的机制；通过RNA-Seq技术筛选出“三法三穴”推拿干预促进周围神经损伤恢复的关键基因及通路，为下一步继续研究奠定了基础。.重要结果及关键数据：行为学研究得出：推拿可以明显改善①冷感觉及热感觉异常，促进温度觉恢复；②病理性疼痛症状，促进痛觉恢复；③精细动作及后肢肌力，促进运动功能恢复。形态学研究得出：推拿可以①减少DRG尼氏体的崩解；②改善脊髓背角小胶质细胞活化状态；③促进神经损伤点细胞自噬过程；④促进脊髓腹角自噬体的生成；⑤缓解腓肠肌萎缩状态。分子生物学研究得出：推拿可以①调节TRPA1冷感觉通路中相关蛋白的表达，促进温度觉恢复；②下调促炎因子（IL-6、IL-1β）表达，上调抑制性神经递质（5-HT2A及GABA-B受体）的表达，发挥镇痛作用；③通过AMPK-mTOR信号通路调节细胞自噬，下调TGF-β1/Smad2通路蛋白表达，促进运动功能恢复；④通过调节髓鞘再生相关指标（NRG1、ErbB2）、轴突再生相关指标（NgR）的表达，促进神经修复。RNA测序研究得出：推拿可以改变神经损伤点、DRG、脊髓背角及腹角、腓肠肌的基因表达情况，从而促进周围神经损伤的修复。.科学意义：本研究①从痛觉、温度觉等角度对推拿促进周围神经损伤感觉恢复进行探究，为推拿良好的临床疗效提供科学支撑；②从基因层面全面分析推拿促进周围神经损伤的潜在机制，为进一步研究推拿起效机制提供切入点，为推拿临床治疗此类疾病提供理论依据。