HMGB1调节AQP4表达在脊髓损伤后星形胶质细胞水肿中的作用及其机制研究

After spinal cord injury (SCI), the astrocyte edema could lead to secondary spinal cord injury. Studies have shown that HMGB1 promoted cerebral astrocytes edema via activation of AQP4 after traumatic brain injury, however there has been no report about the effects of HMGB1 activating AQP4 on spinal astrocytes edema after SCI. In our pre-experiment, serious spinal astrocytes edema and increased expression of HMGB1 and AQP4 on astrocytes were found at 24 h after oxygen-glucose deprivation. So we make the hypotheses that after SCI, HMGB1 activating AQP4 plays an important role in promoting the spinal astrocytes edema, and its mechanism of signal transduction pathway may be related to HMGB1 cell surface RAGE, TLR2, TLR4 receptors and their intracellular signaling pathways. To confirm this hypothesis, we will use oxygen-glucose deprivation model of spinal astrocytes and rat spinal cord injury model to evaluate the effects of HMGB1 activating AQP4 on the edema of astrocytes after spinal cord injury and its mechanism of signal transduction pathway by cell morphology, histomorphology, and molecular biology techniques. This study will further reveal the mechanism of astrocytes edema after SCI, and will explore original targets to reduce spinal astrocytes edema and secondary injury after SCI.

脊髓损伤(SCI)后，星形胶质细胞水肿会造成脊髓继发损害。据报道，脑损伤后HMGB1增多促进星形胶质细胞AQP4表达，导致其水肿；但SCI后HMGB1调节AQP4表达在脊髓星形胶质细胞水肿形成中的作用尚未见报道。预实验中，我们用氧糖剥夺后复氧模型模拟SCI后缺血再灌注状态，培养脊髓星形胶质细胞，发现细胞HMGB1增多，AQP4表达增强，细胞水肿加重。我们推测SCI后HMGB1调节AQP4表达在星形胶质细胞水肿形成中发挥重要作用，其机制可能涉及HMGB1的RAGE、TLR2、TLR4受体通路。为证实这一假说，我们拟制备脊髓星形胶质细胞氧糖剥夺模型与脊髓损伤模型，应用细胞组织形态学及分子生物学技术，从细胞和整体两个层次，观察HMGB1调节AQP4表达在SCI后星形胶质细胞水肿中的作用及其受体通路。本研究将进一步揭示SCI后星形胶质细胞水肿形成机制，为减轻SCI后细胞水肿及继发损伤提供新靶点。

高迁移率组蛋白1（HMGB1）是中枢神经系统的一种炎症因子，在脊髓损伤（SCI）后发挥重要的作用。脊髓水肿是SCI主要的继发性损害之一，它与SCI后患者神经功能缺失以及SCI治疗效果不佳有关；脊髓星形胶质细胞水肿是脊髓水肿最主要的组成部分。目前，对于SCI后脊髓水肿的有效治疗，特别是争对特定的与脊髓水肿相关的炎性分子介质的治疗方法仍较为缺乏。我们的研究旨在观察HMGB1对于SCI大鼠早期脊髓水肿、脊髓星形胶质细胞活化及水通道蛋白4（AQP4）表达的作用；体外试验中，探讨HMGB1对于氧糖剥夺/复氧（OGD/R）所致脊髓星形胶质细胞水肿及AQP4表达的作用，并探讨其具体细胞通路与分子机制。动物实验我们建立了成年Sprague-Dawley大鼠脊髓损伤模型，观察抑制HMGB1时，SCI大鼠早期脊髓水肿情况、脊髓星形胶质细胞活化（GFAP）及AQP4表达情况。体外试验中，我们培养新生Sprague-Dawley大鼠脊髓星形胶质细胞并建立OGD/R模型，研究抑制HMGB1对于OGD/R所致的脊髓星形胶质细胞肿胀、HMGB1与AQP4表达及HMGB 释放的影响；之后进一步研究了Toll样受体4（TLR4）/核转录因子-κB （NF-κB）信号通路以及白介素（IL-6）在这一过程中的作用。我们的研究结果表明，丙酮酸乙酯（EP）及甘草甜素（GL）能够有效抑制SCI大鼠脊髓HMGB1的表达；抑制HMGB1，能够改善SCI大鼠后肢运动功能、减轻其早期脊髓水肿、降低SCI相关的脊髓GFAP及AQP4表达、并降低脊髓TLR4/MyD88/NF-κB通路的活化。体外试验结果表明，HMGB1干扰及EP能够抑制大鼠脊髓星形胶质细胞HMGB1表达；抑制HMGB1减轻了OGD/R所致的脊髓星形胶质细胞肿胀、HMGB1与AQP4表达、以及HMGB1的释放；HMGB1对于OGD/R所致脊髓星形胶质细胞肿胀与AQP4表达的作用是通过TLR4/MyD88/NF-κB信号通路发挥效应；此外，OGD/R与HMGB1抑制均可引起脊髓星形胶质细胞IL-6的释放改变，IL-6参与脊髓星形胶质细胞AQP4表达的调节。总之，HMGB1在大鼠SCI后脊髓水肿及星形胶质细胞肿胀的发生发展过程中发挥关键作用，是我们进一步研究和治疗SCI后脊髓水肿和脊髓星形胶质细胞肿胀的一个新的重要的靶点。