小胶质细胞TLR4-MyD88信号通路在异氟醚预处理保护脑缺血半暗带中的作用及机制研究

Cerebral ischemic penumbra has become the breakthrough point in the treatment of cerebral ischemic diseases, but how to protect it remains an urgent and unsolved issue as a result of its complexity of pathogenesis. Studies have shown that isoflurane preconditioning can protect cerebral ischemic prenumbra, but the exact mechanism is unclear. Our beforehand experiment results indicated that in ischemic penumbra, TLR4 mainly focused on microglia, and compared with the MCAO group, microglia activation remarkably weakened and the expression of HSP60, TLR4 along with MyD88 all significantly reduced in genes and protein level in the isoflurane preconditioning group. The above results strongly suggest that maybe isoflurane preconditioning protect cerebral penumbra by means of attenuating the activation of microglia, moreover HSP60 as well as TLR4-MyD88 signaling pathway are much more likely involved in that process. Based on the above-mentioned studies, the current project would further study the effect of isoflurane pretreatment against cerebral ischemic semi-dark band of microglia activity and HSP60 as well as the TLR4-MyD88 signaling pathway, and down-stream signaling molecules, and further discuss the molecular mechanisms of nerve cells apoptosis in penumbra in vitro, so as to clarify the key role of microglial TLR4-MyD88 signaling pathway in isoflurane preconditioning - induced cerebral ischemic penumbral protection and offer new theory along with experimental basis.

脑缺血半暗带已成为脑缺血疾病防治的突破口，由于其发病机理复杂，如何保护脑缺血半暗带目前仍为一个亟待解决的难题。研究显示异氟醚预处理对脑缺血半暗带有保护作用，但确切机制尚不清楚。我们的预实验结果表明，脑缺血半暗带中TLR4主要在活化的小胶质细胞上表达，且经异氟醚预处理后小胶质细胞活性明显减弱，HSP60、TLR4、MyD88在基因及蛋白水平表达均明显降低。上述结果强烈提示异氟醚预处理保护脑缺血半暗带可能与小胶质细胞的活性、HSP60及TLR4-MyD88信号通路有关。本项目拟在上述研究基础上，进一步观察异氟醚预处理对脑缺血半暗带小胶质细胞活性、HSP60、TLR4-MyD88信号通路的影响；并在细胞水平探讨脑缺血半暗带神经元凋亡的机制,以阐明小胶质细胞TLR4-MyD88信号通路在异氟醚预处理保护脑缺血半暗带作用中扮演重要角色，为异氟醚预处理和脑缺血保护研究提供新的理论和实验依据。

脑缺血半暗带已成为脑缺血疾病防治的突破口，由于其发病机理复杂，如何保护脑缺血半暗带目前仍为一个亟待解决的难题。研究显示异氟醚预处理对脑缺血半暗带有保护作用，但确切机制尚不清楚。本课题在预实验研究基础上，进一步观察了异氟醚预处理对脑缺血半暗带小胶质细胞活性、HSP60、TLR4-MyD88信号通路的影响；并在细胞水平探讨了脑缺血半暗带神经元凋亡的机制。结果通过体内实验证实2%异氟醚预处理能够降低神经功能缺损评分，减小脑梗死容积，减少脑缺血半暗带区神经细胞凋亡，减弱小胶质细胞活性，并且能够下调HSP60、TLR4、MyD88蛋白的表达，上调IκB-α蛋白的表达；通过体外实验证实2%异氟醚预处理和TLR4特异性抑制剂CLI-095均能下调TLR4, MyD88, IL-1β, TNF-α, Bax的表达,而上调IκB-α和Bcl-2蛋白的表达，并且均能减弱小胶质细胞活性以及改善脑缺血半暗带区神经细胞凋亡，而TLR4 DNA过表达却能逆转这种脑缺血半暗带神经保护效应。这些结果足以阐明异氟醚预处理可直接通过下调TLR4-MyD88信号通路的表达，从而抑制小胶质细胞活性，减轻神经炎性反应，最终起到脑缺血半暗带的神经保护效应。该研究为异氟醚预处理和围术期脑缺血保护研究提供了新的理论和实验依据。