Sirt1 通过分子伴侣介导的自噬调控脑损伤后星形胶质细胞活化的机制

Over activated astrocytes directly or indirectly cause neuronal death and exhibit neuronal regeneration after traumatic brain injury. Sirt1 is considered to be one of the important molecules to protect neurons after injury, but the mechanism of its protective effect on neuronal regeneration has not been clarified. Our previous study reported that up-regulation of Sirt1 expression could attenuate the astrocytes activation after traumatic brain injury. In addition，Sirt1 has been identified to play an important role in the process of LC3 deacetylation, which can effectively protect the injured peripheral nerve tissue, but whether it is involved in the other two autophagy has not been clearly reported. Autophagy occurred in neurons showing neuroprotective effect and also participated in the astrocyte activation after injury. The result of Co-Immunoprecipitation (Co-IP) shows that Sirt1 and DNAJB1, a molecular chaperon-mediated autophagy associated protein, bind directly in astrocytes in vitro. We propose that the mechanism of Sirt1 attenuating the astrocyte activation after brain injury may be related to the interaction with DNAJB1 and the regulation of molecular chaperone mediated autophagy. Therefore, we will observed the expression of molecular chaperon-mediated autophagy associated proteins and the activation of astrocytes via up-regulating Sirt1 expression and silencing DNAJB1 in vivo and in vitro, to explore the relationship between Sirt1 and DNAJB1 involved in molecular chaperon-mediated autophagy and astrocyte activation and discuss the neuroprotective mechanism of Sirt1. This study will provide a solid theoretical basis for neuronal rehabilitation after traumatic brain injury.

创伤性脑损伤后星形胶质细胞过度活化可引起神经元死亡而阻碍神经修复，Sirt1被认为是损伤后保护神经元的重要分子之一，但具体作用机制尚未明确。前期工作中发现脑损伤后上调Sirt1表达可减弱星形胶质细胞活化。另外，Sirt1可通过去乙酰化LC3参与巨自噬过程，但其是否参与其它两种自噬尚未有明确报道。自噬的发生可有效的保护损伤周边的神经组织，且参与星形胶质细胞的活化过程。预实验显示星形胶质细胞中Sirt1和分子伴侣介导的自噬相关蛋白DNAJB1直接结合。由此我们设想：Sirt1减弱星形胶质细胞活化的作用机制，可能与调控分子伴侣介导的自噬有关。因此，我们通过体内外上调Sirt1的表达并沉默DNAJB1，观察分子伴侣介导的自噬相关信号蛋白的表达和星形胶质细胞的活化情况，探究Sirt1和DNAJB1之间通过何种作用关系参与分子伴侣介导的自噬从而影响星形胶质细胞的活化，从中探讨Sirt1的神经保护机制。

我们的前期研究发现Sirt1可以通过改善创伤性脑损伤后的星形胶质细胞活化进而促进神经保护作用，但其机制尚未完全了解。相关文献报道，脑损伤后，分子伴侣介导的自噬在受损的神经组织中上调，并发挥神经保护作用。由此我们推测Sirt1与CMA活性相关的潜在机制，可能是TBI治疗的新靶点。 .本项目研究的体内模型采用闭合性颅脑损伤模型，体外模型应用IL-1β刺激原代脑皮质星形胶质细胞来模拟创伤性脑损伤诱导的星形胶质细胞活化。通过慢病毒过表达Sirt1或敲低Dnajb1。多种行为测试评估动物神经功能，其中包括mNSS，滚轮测试，握力测试和热痛测试。采用免疫荧光染色来评估组织学和功能损伤。通过Nissl染色评估组织缺损情况。定量PCR和蛋白质印迹检测并分析基因和蛋白质表达。Co-IP用于检测Sirt1和Dnajb1两种蛋白之间的相互作用。ChIP和双荧光素酶报告基因检测转录激活效应。研究结果显示， 过表达Sirt1改善了脑损伤后神经功能缺损，减少了组织损失，并减弱了星形胶质细胞活化，而DNAjb1-shRNA给药则逆转了这一作用；接着，体内动物实验和体外细胞原代培养星形胶质细胞实验均发现脑损伤或IL-1β刺激后，分子伴侣介导的自噬活性上调；Dnajb1敲低后，分子伴侣介导的自噬受到抑制。而且，过表达Sirt1可以上调Dnajb1表达并促进分子伴侣介导的自噬。在机制方面，Co-IP结果显示Sirt1可以与Dnajb1相互作用并调节Dnajb1的去乙酰化和去泛素化。总而言之，本研究结果明确了Sirt1通过调节分子伴侣介导的自噬相关蛋白Dnajb1的去乙酰化和去泛素化调控分子伴侣介导的自噬的活性，进而改善脑损伤导致的星形胶质细胞活化。