HMGB1/TLR4通路调节血脊髓屏障功能对大鼠脊髓损伤后脊髓离子性与血管源性水肿的作用及其分子机制

After spinal cord injury (SCI), the spinal cord edema could lead to secondary spinal cord injury. HMGB1 is a mediator of inflammatory responses and plays a critical role after SCI. The study, completed by us, has proved the promoting role of HMGB1/TLR4 on the spinal cord astrocyte swelling (cytotoxic edema) in rats after SCI. Therefore we make the hypothesis that HMGB1/TLR4 regulating the function and permeability of blood–spinal cord barrier (BSCB) plays an important role on the spinal cord ionogenic edema and vasogenic edema, which result in the influx of liquid from circulation system into spinal cord tissue after SCI. The hypothesis was supported by our pre-experiment, in which inhibiting HMGB1/TLR4 decreased BSCB permeability and reduced spinal cord edema in rats after SCI. In further research, we will use rat spinal cord injury model, and oxygen-glucose deprivation model of rat spinal astrocytes and rat microvascular endothelial cells to evaluate the effects of HMGB1/TLR4 regulating BSCB on the spinal cord ionogenic edema and vasogenic edema after SCI and its molecular mechanism by histomorphology, cell morphology, and molecular biology techniques. This study will further reveal the mechanism of spinal cord edema after SCI, and will explore original targets to reduce spinal cord edema after SCI.

脊髓损伤(SCI)后，脊髓水肿是其重要的继发性损害。SCI后细胞外HMGB1增多，发挥炎症因子作用。我们已结题项目证明HMGB1/TLR4通路对大鼠SCI后以星形胶质细胞肿胀为主的脊髓细胞性水肿发挥促进作用。我们推测，HMGB1/TLR4通路可能参与调节血脊髓屏障（BSCB）的功能与通透性、对真正造成SCI后水进入脊髓实质的离子性与血管源性水肿发挥重要调节作用。预实验结果初步证实了这一推测，抑制HMGB1/TLR4能降低大鼠SCI后BSCB通透性、减轻脊髓水肿。为进一步深入研究，我们拟制备大鼠脊髓损伤模型以及体外脊髓星形胶质细胞和微血管内皮细胞氧糖剥夺模型，应用组织细胞形态学及分子生物学技术，从整体和细胞两个层次，观察HMGB1/TLR4通路调节BSCB功能对大鼠SCI后脊髓离子性与血管源性水肿的作用及分子机制。本研究将进一步揭示SCI后脊髓水肿形成机制，为减轻SCI后脊髓水肿提供新靶点。

在星形胶质细胞和内皮细胞中，通过Na+-K+-Cl-共转运体-1(NKCC1)和钠/氢交换体-1(NHE1)的钠/水运输对脊髓损伤(SCI)后的细胞毒性和离子水肿至关重要。脊髓损伤后血管源性水肿主要与血脊髓屏障(BSCB)的破坏以及液体和蛋白质的外渗有关。高迁移率族蛋白B1 (HMGB1)作为脊髓损伤后炎症反应的介质促进脊髓水肿。在本研究中，我们探讨了HMGB1/toll样受体4(TLR4)通路在大鼠SCI后脊髓离子型水肿和血管源性水肿中的作用及HMGB1/TLR4通路调控BSCB功能的潜在机制。采用重物坠落法建立成年雌性大鼠SCI模型，并采用大鼠脊髓星形胶质细胞和微血管内皮细胞建立体外氧糖剥夺/复氧(OGD/R)模型。研究表明，抑制HMGB1可降低SCI大鼠脊髓、培养的脊髓星形胶质细胞和培养的微血管内皮细胞NKCC1和NHE1的表达。HMGB1对NKCC1和NHE1表达的影响至少部分是通过激活TLR4/白细胞介素-1受体结构域接头诱导干扰素-β(TRIF)-核因子-κB(NF-κB)信号通路介导的。抑制NKCC1或NHE1可降低SCI后大鼠脊髓含水量，增加OGD/R后培养星形胶质细胞培养液Na+浓度，减少星形胶质细胞体积和AQP4表达。此外，抑制HMGB1可降低SCI后大鼠BSCB的通透性，增加脊髓内claudin-5、occludin和ZO-1的表达，从而降低SCI后脊髓含水量，改善SCI后1至4周大鼠后肢神经的运动功能和电生理特征。对于体外培养的CNS微血管内皮细胞，抑制HMGB1可增加OGD/R后内皮细胞claudin-5、occludin和ZO-1的表达，降低内皮细胞通透性。这些作用是通过激活TLR4/TRIF/NF-κB信号通路介导的。综上所述，HMGB1在大鼠SCI后脊髓离子性水肿、血管源性水肿和BSCB的调节中发挥重要作用。HMGB1有望成为未来研究和治疗SCI后脊髓水肿的新靶点。