基于TLR4/NF-κB信号通路调控GLT-1的针刺治疗创伤性脑损伤的机制研究

Acupuncture has been widely used to treat traumatic brain injury (TBI), but the mechanism is still not clear. Previous study showed that acupuncture could relieve brain edema of rats with TBI, improve the neurological function and relieve brain injury. Glutamate excitatory toxicity is an important pathological mechanism of TBI, which is closely related to glutamate transporter 1 (GLT-1). Preliminary study showed that acupuncture may increase the mRNA expression of GLT-1, which suggested acupuncture may regulate the expression of GLT-1 to inhibit of glutamate excitotoxicity. The TLR4/NF-κB signal pathway was activated after TBI and NF-κB could inhibit the expression of GLT-1. So we suppose that there is a negative correlation between GLT-1 and TLR4/NF-κB signal pathway，acupuncture can inhibit TLR4/NF-κB signal pathway to strengthen the expression of GLT-1, meanwhile glutamate excitotoxicity, inflammation and apoptosis can be inhibited. To verify this hypothesis, this project intends to use technologies such as water maze test, Western Blot, Real-time PCR, In situ cell death detection (TUNEL) and TLR4 antagonist to explore the mechanism of acupuncture treatment of TBI from GLT-1 and TLR4/NF-κB pathway and their relationship, so as to provide scientific basis for clinical acupuncture treatment for TBI.

针刺已被广泛用来治疗创伤性脑损伤(TBI)，但起效机制尚不明确。前期动物研究证实了针刺对TBI有效：针刺可减轻脑水肿、神经功能缺损和脑损伤的程度。谷氨酸兴奋性毒性是TBI重要的病理机制，其发生和谷氨酸转运体1（GLT-1）密切相关，预实验显示针刺对GLT-1表达有增强趋势。跟据他人研究：GLT-1表达受NF-κB抑制，TLR4/NF-κB信号通路在TBI后被激活，由此推测GLT-1和TLR4/NF-κB信号通路呈负相关性，针刺可通过抑制TLR4/NF-κB信号通路，提高GLT-1表达，防止谷氨酸兴奋性毒性，抑制炎症反应和细胞凋亡，最终达到治疗TBI的效果。为验证此假说，拟利用水迷宫实验、Western Blot、Real-time PCR、细胞凋亡检测和TLR4拮抗剂技术，从GLT-1和TLR4/NF-κB通路及二者关系的角度探讨针刺治疗TBI的效应机制，为临床针刺治疗TBI提供科学依据。

临床上针刺已被广泛用于治疗创伤性脑损伤（TBI）导致的意识、语言、肢体障碍等多种症状并且疗效确切，但关于针刺的起效机制仍不明确。本研究根据以往研究基础和他人研究提出假说：针刺通过抑制TLR4/NF-κB信号通路和GLT-1治疗TBI。本实验以大鼠TBI模型为研究对象，利用行为学、HE染色、TUNEL法、Western Blot、Real-time PCR、ELISA技术，并结合TLR4拮抗剂TAK-242作为对照，观察针刺对TLR4/NF-κB信号通路相关指标、GLT-1、细胞凋亡、炎性反应、运动、感觉等神经功能损伤的影响。根据实验结果针刺可降低TLR4和NF-κB蛋白、mRNA的表达，减轻脑组织损伤程度，降低细胞凋亡指数，抑制炎性细胞因子TNF-α、IL-1β和IL-6的表达，改善神经功能评分和Rotarod test。说明针刺可抑制TBI后被激活的TLR4/NF-κB信号通路，减轻细胞凋亡和炎性反应，促进运动、感觉等神经功能的恢复，并且针刺对TLR4/NF-κB信号通路的抑制效果好于TAK-242。揭示了针刺通过抑制TLR4/NF-κB信号通路治疗TBI，这为针刺对TBI的有效性提供科学佐证。但本研究发现TBI并未影响GLT-1的表达，针刺TBI大鼠也未对GLT-1的表达产生影响，由此未能证实针刺可通过调节GLT-1的表达、减轻谷氨酸兴奋性毒性治疗TBI。目前对于TBI是否影响GLT-1也存有争议，推测TBI伤后GLT-1的表达差异可能和不同造模仪器和造模方式、损伤部位、损伤程度、观察时间点等因素有关。未来拟从这些因素进一步研究针刺对GLT-1的影响，或从其他路径研究针刺对谷氨酸兴奋性毒性的作用。