脊髓水平HMGB1诱导自噬损伤活化小胶质细胞促进骨癌痛的机制研究

The activation of spinal microglia is one of the key factors for the development of bone cancer pain. Our preliminary study found that bone cancer pain led to autophagic dysfunction in spinal cord. Up-regulation of autophagy could significantly inhibit the activation of microglia and relieve hyperalgesia. HMGB1 in spinal cord plays an important role in the process of pain. We find that the inhibition of HMGB1 could significantly relieve the bone cancer pian in rats. HMGB1 has been shown to regulate the activity of microglia via autophagy in vitro. On this basis, we suppose that high level of HMGB1 in spinal cord may induce the activation of microglia through autophagy dysfunction, and eventually promote the development of bone cancer pain. The main research contents are shown as follows: 1. We will investigate the interation between HMGB1 and VPS34/Beclin-1 complex in microglia and show the regulation of autophagy by HMGB1 is relying on the bond of Beclin1. 2. We will investigate the effects of autophagy on microglia activity and NLRP3 inflammasome in vitro. 3. We will observe these changes in spinal cord. 4. We will investigate the effect of HMGB1, autophagy and NLRP3 on the pain behavior in rats. This study will help to clarify the molecular mechanism of microglia that involved in hyperalgesia, and may provide new theoretical basis for the therapies in bone caner pain.

脊髓小胶质细胞的活化是骨癌痛发生发展的关键因素之一，我们前期研究发现骨癌痛大鼠脊髓自噬功能紊乱，上调脊髓自噬水平明显抑制小胶质细胞的活化减轻痛觉过敏。脊髓水平高表达HMGB1在疼痛中发挥重要作用，我们抑制HMGB1能明显缓解大鼠骨癌痛。体外研究发现HMGB1可调节自噬影响小胶质细胞的活性。在此基础上我们提出新设想：脊髓水平高表达的HMGB1可介导自噬活化小胶质细胞促进骨癌痛的发展。研究内容主要包括：1、在细胞分子水平研究HMGB1调控VPS34/Beclin-1自噬复合体对小胶质细胞自噬形态及相关分子生物学的影响；2、在细胞分子水平研究调节自噬对小胶质细胞活性及NLRP3炎性小体的影响； 3、在骨癌痛大鼠脊髓水平验证上述影响的存在；4、分别对HMGB1、自噬、NLRP3干预，研究其对骨癌痛大鼠痛行为学的影响。这将有助于揭示小胶质细胞参与痛觉敏化的机制，为临床骨癌痛的治疗提供新的理论依据。

当前针对癌症患者疼痛的治疗仍主要基于经验性用药，这在一定程度上限制了对癌痛的诊治，因而亟需寻找新的有效治疗靶点。. 本项目前期实验发现骨癌痛大鼠脊髓自噬功能紊乱，鞘内注射自噬诱导剂雷帕霉素上调脊髓自噬水平，明显抑制小胶质细胞的活化减轻痛觉过敏，鞘内注射自噬抑制剂3-MA则加重此改变。在此基础上，我们证实骨癌痛大鼠脊髓水平高表达HMGB1，抑制HMGB1能够明显缓解大鼠骨癌痛。分子生物学和免疫荧光学实验显示高表达HMGB1能抑制Beclin1相关自噬体的形成，活化脊髓小胶质细胞，促进胶质细胞NLRP3炎性小体的明显增加，释放大量炎性因子。这将有助于揭示小胶质细胞参与痛觉敏化的机制，为临床骨癌痛的治疗提供新的理论依据，具有良好的科研意义和实际应用价值。