脊髓损伤后HMGB1蛋白激活炎症因子的释放及作用机理研究

Recent studies demonstrated that the release of high-mobility group box 1 (HMGB1) stimulated the inflammatory reaction after brain ischemic injury. However, the role of HMGB1 as an inflammatory cytokine after spinal cord injury has not been studied in detail. In our previous study, HMGB1 expression in the injured spinal cord appeared at an earlier phase than that of TNF-α and IL-1β. HMGB1 and its receptors RAGE contributed to the inflammatory response after spinal cord injury. Recent studies confirmed that HMGB1 could induce tumor necrosis factor (TNF)-α and interleukin (IL)-1β synthesis and secretion in monocyte/macrophages via the activation of NF-κB pathway. Therefore, we speculate that HMGB1 is likely an upstream inflammatory cytokine involved in the regulation of TNF-α and IL-1β secretion after spinal cord injury. In the present study, we intend to perform in vivo and in vitro models of spinal cord injury, and administrate HMGB1 monoclonal antibody or HMGB1 small interfering RNAs (siRNA) to suppress HMGB1 expression after spinal cord injury. The protein and mRNA expression of HMGB1, TNF-α and IL-1β are supposed to be examined in the different time points after spinal cord injury using western blotting, real-time polymerase chain reaction (RT-PCR) and immunohistochemical analysis. The purpose of current study is to verify that HMGB1 can promote the release of TNF-α and IL-1β after spinal cord injury, and to investigate the possible mechanism of HMGB1 stimulating the release of inflammatory cytokines. This study will help elucidate the initial mechanisms of inflammatory response related to the injury event and its mediators, and confirm the importance of HMGB1 as a potential candidate in a specific upstream pathway that promotes inflammation after spinal cord injury.

HMGB1能够加重大脑缺血性损伤后炎症反应，但它在脊髓损伤后的确切作用尚不明确。我们前期研究发现：脊髓损伤后早期HMGB1的表达上调，其释放早于TNF-α、IL-1β，它与受体RAGE结合诱导损伤后炎症反应。既往研究证实，HMGB1通过激活NF-κB信号通路，诱导单核/巨噬细胞释放TNF-α、IL-1β。因此，我们推测HMGB1可能作为上游炎症因子激活脊髓损伤后TNF-α、IL-1β的释放。本研究拟制备脊髓损伤体内和体外模型，通过给予HMGB1单克隆抗体或HMGB1 siRNA抑制脊髓损伤后HMGB1的表达，利用Western Blot、免疫组化和RT-PCR检测各组标本不同时间点HMGB1、TNF-α、IL-1β的蛋白及mRNA表达变化，验证HMGB1能够促进脊髓损伤后TNF-α、IL-1β的表达，并探讨HMGB1诱导炎症因子释放的机制，从而确立HMGB1在脊髓损伤后炎症反应中的重要地位

高迁移率族蛋白B1(HMGB1)近年来被证实在许多疾病中作为上游炎症因子，在多种病理生理过程中发挥重要作用。HMGB1在脊髓损伤中的作用尚不明确。本研究通过体内、体外实验研究了大鼠脊髓损伤后HMGB1参与激活炎症反应、加重组织损伤的机制，以及抗HMGB1抗体对大鼠脊髓损伤的治疗作用。结果发现：（1）HMGB1在脊髓损伤后由细胞核迁移至胞质，随后释放入胞外；这一过程主要发生于神经元和小胶质细胞中。（2）原代培养的脊髓神经元损伤后，培养基上清HMGB1的表达在伤后24 h明显上升。（3）神经元损伤后24 h培养液可激活BV-2小胶质细胞释放TNF-α；抗HMGB1抗体可明显抑制TNF-α的表达。（4）重组HMGB1蛋白可促进BV-2小胶质细胞内TNF-α mRNA的表达上调，抗HMGB1抗体可以抑制HMGB1引起的炎症因子mRNA表达上调。（5）HMGB1可增强BV2小胶质细胞迁移的能力，抗HMGB1抗体可抑制HMGB1促进迁移的作用。（6）抗HMGB1抗体可以促进大鼠脊髓半切损伤后，患侧肢体运动功能的恢复。这些研究结果提示，脊髓损伤后释放的HMGB1蛋白能够作为上游炎症因子，增强小胶质细胞迁移的能力，刺激小胶质细胞表达TNF-α，可能在脊髓继发性损伤的发生发展中发挥着重要作用；抗HMGB1抗体对抑制脊髓损伤后的炎症反应、促进运动功能改善有潜在的作用。