Apoptosis signal-regulating kinase 1是七氟烷抑制小胶质细胞活化的关键分子靶点？

Stroke is a devastating clinical condition for which an effective neuroprotective treatment is currently unavailable. Microglial activation occurs in ischemic core and penumbra regions during acute and delayed phases after cerebral ischemia. Whereas microglial function may be beneficial to stroke recovery during late stage by removing cell debris and stimulating neurovascular remodeling, excessive early microglial activation (the first 3 days) after ischemia is widely believed to exacerbate brain damage. Mounting evidence shows that overactivated microglia release cytotoxic molecules including NO, ROS, and proinflammatory cytokines, which augment neuronal death in sequel. In addition, the cytokines produced by damaged brain cells after the initial ischemic injury or by activated peripheral inflammatory cells could further activate microglia, thus triggering a vicious cycle that can propel neuronal death. However, the intracellular signaling mechanism underlying microglial activation after ischemia is poorly understood. Among the potential signaling molecules are NF-κB which is an important nuclear transcription factor for regulating expression of genes involved in innate and inflammatory responses. However, little is known about the signaling pathway that links NF-κB activation in microglia in ischemic models.We have reported preconditioned with sevoflurane can significantly inhibit the activation of microglia sparked by cerebral ischemia/reperfusion injury in rats, and robustly supress the activation of NFκB as well as expression of pro-inflammatory cytokines. However, few studies focus on the influence of sevoflurane on the cerebral inflammation, not mention to the role of microglia and the involved signaling pathway. Based on the existing evidence, we hypothesized that - sevoflurane can directly inhibit activation of microglia induced by LPS, which is related to ASK1 and its downstream NFκB signaling pathway. In the present study, the impacts of sevoflurane on the activation of microglia induced by LPS are explored in vitro. To study microglia-neuron interactions, we have successfully established a neuron-microglia co-culture model in preliminary studies.This study is the first one to concentrate on the effects of sevoflurane on activation of microglia stimulated with LPS and the potential molecular target-ASK1.

近年研究表明, 在脑缺血损伤急性期及慢性期，小胶质细胞在脑缺血的核心区及半影区被激活。活化的小胶质细胞对于慢性期中风的恢复是有益的，但在缺血早期小胶质细胞的过度激活却加重了缺血性脑损伤。Apoptosis signal-regulating kinase 1 (ASK1)不仅通过线粒体途径触发神经元的凋亡，而且是小胶质细胞活化信号途径的必需部分。ASK1对神经元及小胶质细胞的这种双重作用预示了它可能是缺血性脑损伤的重要分子靶点。我们前期的研究发现，七氟烷预处理能够抑制脑缺血损伤后早期小胶质细胞的活化，减少COX-2、iNOS等炎症因子表达，并且降低其上游转录因子NF-κB的活化，但NF-κB上游信号通路仍未明了。因此我们推测，脑缺血损伤小胶质细胞活化与ASK1有关，并且七氟烷预处通过影响ASK1/p38信号通路抑制脑缺血损伤后早期小胶质细胞的活化而发挥其神经保护作用。

小胶质细胞／巨噬细胞有两种极化细胞形态——M1与M2。“经典激活”M1的小胶质细胞/巨噬细胞能够产生并且释放炎性细胞因子如IL-1β、TNFα、IFN-γ等加重神经元的损伤。与之相反，“可选择性激活”M2的小胶质细胞/巨噬细胞则释放促进神经元修复的神经营养因子，并且通过抑制局部炎症从而发挥神经保护作用。去乙酰化酶3（Histone Deacetylase 3，HDAC3）在刺激因素的作用下在核及胞浆内迁移,使组蛋白C末端的赖氨酸去乙酰化而使DNA更加紧密地包裹于组蛋白周围，抑制转录因子与其调节区域结合从而阻断基因转录。除此之外，HDAC3乙酰化NFκB的p65亚单位能够使其与DNA结合能力极低、促进p65亚单位与DNA分离并且迁移出核，从而关闭NFκB基因转录、抑制其下游炎症因子的产生。简言之，HDAC3的活化能够促进NFκB的转录活性，前者或许是后者上游信号分子。我们首先利用体外原代小胶质细胞OGD后给予七氟烷后处理，证明了七氟烷后处理能够促使M0向具有抗炎症利修复的M2表型变化、而同时抑制M0向促炎症的M1表型转化，然后利用神经元OGD之后给予七氟烷干预的CM（conditioned medium，CM）作用于神经元细胞-小胶质细胞共培养体系，阐明了七氟烷后处理对小胶质细胞极化状态的影响对OGD之后的神经元损伤具有保护作用。最后在诱导极化的M1/M2小胶质细胞中发现氟烷后处理抑制M1小胶质细胞中HDAC3与NFκB的入核活化，同时也抑制了二者之间相互作用；而与之相反的是七氟烷干预能够促进M2小胶质细胞中HDAC3的活化，并且同样也影响了二者之间的相互作用。这些结果表明七氟烷后处理能够影响小胶质细胞的极化状态，从而对OGD损伤的神经元产生保护作用，并且HDAC3与NFκB在七氟烷后处理对小胶质细胞极化影响方面发挥了重要作用。在体实验方面，我们发现七氟烷干预能够通过抑制局灶性缺血损伤后脑组织内caspase3、AIF及cytochrome C的活化、增加抗凋亡因子Bcl-2、Bcl-xl的表达、减少促凋亡因子Bax、Bid的水平、抑制上游JNK及p53通路激活而发挥其抗凋亡作用。这些研究成果为深入阐明七氟烷神经保护作用的分子机制提供了理论基础与研究方向，尤其在探讨七氟烷后处理对小胶质细胞极化状态及HDACs的影响方面目前还未有相类似的研究发现。