高迁移率族蛋白1在蛛网膜下腔出血后脑损伤中作用机制的实验研究

The brain injury after subarachnoid hemorrhage(SAH) usually brings poor outcomes to the patients, while the mechanisms have not been well elucidated. We previously found an up-regulation of Toll-like receptor 4(TLR4) and high-mobility group box 1(HMGB-1) in the cerebral cortex after SAH. Moreover, we found that inflammatory response could be induced and amplified following recombinant HMGB-1(rHMGB-1) addition in cultured neurons. Based on the latest research in this field, we raised a hypothesis that HMGB-1, a DNA-binding protein, was a prototypical member of damage-associated molecular pattern(DAMP) family and could be passively released from the hemotocyte lysis to initiate the inflammation through TLR. The inflammatory mediators then acted on the neuronal cells to make them actively release HMGB-1 continuously, manifesting as double phases. HMGB-1 might be able to induce and amplify sterile inflammation, and thus be the key factor of brain injury after SAH. In this study, we are planning to apply specific inhibitors, rHMGB-1, RNA interference and gene overexpression aiming at HMGB-1 to investigate the mechanisms of HMGB-1 in brain injury after SAH, which will be validated through DAMP-induced inflammation both in vivo and in vitro. From this research, we expect to better understand the relationship between DAMP-induced inflammation and brain injury, thereby providing a novel therapeutic target and establishing a rationale for brain injury after SAH.

蛛网膜下腔出血（SAH）后脑损伤严重影响患者预后，但目前对其病理分子机制认识甚微。我们前期研究发现SAH后脑内Toll样受体4（TLR4）及高迁移率族蛋白1（HMGB-1）表达增高，神经元中施加外源性重组HMGB-1可诱发炎性反应。结合国际最新研究，我们推测作为DNA结合蛋白的HMGB-1，是损伤相关分子模式（DAMP）的重要炎性介质，最初由血细胞裂解被动释放，通过TLR启动炎性反应，刺激神经细胞损伤后再次主动释放，呈"双相"效应，级联放大DAMP型炎性反应，即SAH后脑损伤的核心因素。本课题拟通过体内、体外实验，针对HMGB-1，运用特异性抑制剂、重组HMGB-1、RNA干扰及基因过表达技术，立足DAMP分子机制，探讨HMGB-1在SAH后脑损伤中的具体作用机制。通过此项研究，有助于深入理解SAH后DAMP型炎性反应与脑损伤的关系，为SAH后脑损伤的临床治疗提供新的靶点，并奠定理论基础。

蛛网膜下腔出血(SAH)是一种高病死率和病残率的脑血管疾病，研究表明脑内无菌性炎性反应是SAH后早期脑损伤的重要机制之一，但究竟是何种分子启动了SAH后的炎性反应仍不得而知。本项目深入探讨了内源性危险因子高迁移率族蛋白1（HMGB1）对SAH后启动炎性反应的作用及其相关调控机制。我们分别通过体内、体外实验建立SAH模型，使用Western blot、real-time PCR及免疫荧光等实验方法，研究发现在体内实验中SAH早期HMGB1即被释放至细胞外，同时伴随其mRNA及蛋白表达水平上调，且绝大部分释放出的HMGB1来自神经元细胞；在体外实验中，我们通过神经元混合胶质细胞培养，发现神经元释放的HMGB1能够发挥促炎作用。在调控机制方面，本项目发现TLR4/NF-κB信号通路对HMGB1诱发的炎性反应起着非常重要的作用。同时我们使用HMGB1特异性抑制剂甘草酸铵盐（GA），进一步发现抑制HMGB1可减轻SAH后炎性反应及神经细胞凋亡，同时改善神经功能。综上所述，本项目明确了SAH后HMGB1作为损伤因子的释放模式及对炎性反应的启动作用机制，因此HMGB1可能成为SAH后早期脑损伤的新的防治靶点。