运动调控BDNF-ERK-CREB-GAD65/67途径减轻脊髓损伤后痉挛的机制研究

Spasticity is one of the major complications following spinal cord injury (SCI) which would possibly devastate patients’ quality of life. Although exercise rehabilitation has been proved to ameliorate spasticity, the underlying mechanisms remain unclear. Recent studies have revealed that the decrease in the synthesis of GAD65/67 (synthetase of inhibitory transmission-GABA) following SCI promotes the occurrence of spasticity. According to our previous studies, the incidence and tension of spasticity were reduced by exercise rehabilitation through enhanced level of BDNF/TrkB, CREB, and GAD67. However, the mitigation was restrained by blocking BDNF/TrkB. Hence, we presume that exercise rehabilitation might regulate the BDNF-ERK-CREB-GAD65/67 pathway, and thus promote the synthesis of GABA which in turn relieve the rate and tension of spasticity. To clarify this hypothesis, experiments will be designed to demonstrate the regulatory impact of Ras/ERK, and CREB on GAD65/67 expression in SCI rat model. In addition, the mechanism will be investigated to figure out the transcriptional regulation of CREB on GAD65/67 in vitro. Finally, clinical research will be carried out to explore the probability of the amount of BDNF and CREB in blood as indicators of prognoses of spasticity. Generally, the project aims to clarify the mechanisms of improvement in spasticity after SCI through BDNF-ERK-CREB-GAD65/67 and guide the theoretical formulation of clinical exercise rehabilitation program for SCI patients.

脊髓损伤（SCI）后痉挛会严重影响生活质量，运动训练可有效缓解痉挛，但机制尚未阐明。既往研究表明，SCI后GAD65/67（抑制性神经递质GABA的合成酶）合成减少，促使了痉挛的产生。我们前期研究发现，运动训练可增加BDNF/TrkB、CREB、GAD67的合成，减轻SCI后痉挛；阻断BDNF/TrkB后，上述作用则被抑制。因此，我们推测运动训练可能调控BDNF-ERK-CREB-GAD65/67途径，增加GABA合成，减轻SCI后痉挛。为证实此假说，我们在动物水平验证Ras/ERK、CREB对GAD65/67表达的影响；在细胞水平验证CREB对GAD65/67转录的调控；在临床层面探究运动强度与SCI患者血液BDNF和CREB水平及痉挛预后的相关性。本研究将阐明BDNF-ERK-CREB-GAD65/67途径在运动训练改善SCI后痉挛中的作用，为SCI运动训练方案的制定提供理论依据。

脊髓损伤（SCI）后痉挛状态会严重影响生活质量，运动训练可有效缓解痉挛，但机制不清。既往研究表明，SCI后谷氨酸脱羧酶-65/67（GAD-65/67）合成减少，促使了痉挛的产生。运动训练可增加SCI大鼠受损远端脊髓内脑源性神经营养因子（BDNF）、酪氨酸受体激酶 B（TrkB）、钾氯协同转运蛋白2（KCC2）和GAD-65/67的合成，改善痉挛状态。本项目进一步探讨了运动训练是否能够通过调控BDNF-ERK-CREB-GAD65/67途径，增加GABA合成，减轻SCI后痉挛。项目主要研究内容包括：①运动训练对SCI大鼠痉挛、痛觉阈值及损伤远端脊髓内GAD-65/67和KCC2表达的影响。②运动训练对SCI大鼠脊髓内BDNF、TrkB合成的影响；③阻断BDNF-TrkB信号通路后，运动训练对SCI大鼠损伤远端脊髓神经元活性、突触可塑性、神经环路重建、痉挛程度、神经病理性疼痛、CREB/p-CREB、GAD-65/67和KCC2表达的影响；④阻断Ras/Erk后，运动训练对GAD-65/67和GABAB受体合成的影响；⑤探讨CREB对GAD-65/67基因转录的调控作用；⑥运动训练对SCI患者痉挛状态和血清BDNF/CREB含量的影响。结果：①运动训练可减轻SCI大鼠的痉挛状态，提高痛觉阈值，增加脊髓内GAD-65/67和KCC2的合成；②运动训练可以增加SCI大鼠脊髓内BDNF和TrkB的合成；③阻断BDNF-TrkB通路后，运动训练对SCI大鼠损伤运动功能恢复、远端脊髓神经元活性、突触可塑性、神经环路重建、痉挛程度、神经病理性疼痛、CREB/p-CREB、GAD-65/67和KCC2表达的促进/改善作用被抑制；④阻断Ras/Erk后，运动训练对GAD-65/67和GABAB受体合成的促进作用被抑制；⑤CREB是调控GAD-65/67基因转录的关键因子，且BDNF含量的增加可增强CREB对GAD-65/67基因转录的促进作用；⑥运动训练可能改善SCI患者的痉挛状态，其机制可能与血清BDNF/CREB含量增多有关。本研究结果提示，运动训练可通过BDNF-ERK-CREB-GAD65/67途径促进SCI大鼠的运动功能恢复、减轻痉挛状态、提高痛觉阈值、增加受损远端脊髓内神经元的活性、增强突触可塑性，并增加远端脊髓内KCC2和抑制性神经递质GABA的合成。