The NLRP3 inflammasome drives inflammation in ischemia/reperfusion injury after transient middle cerebral artery occlusion in mice
The NLRP3 inflammasome drives inflammation in ischemia/reperfusion injury after transient middle cerebral artery occlusion in mice
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DOI：
10.1016/j.bbi.2020.12.009
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发表时间：
2021-02-16
影响因子：
15.1
通讯作者：
Schuhmann, Michael K.
中科院分区：
文献类型：
--
作者：
Franke, Maximilian;Bieber, Michael;Schuhmann, Michael K.
目的：脑缺血可引起严重的神经炎性反应，但其分子机制尚不清楚。炎性小体(NLRP1、NLRP3、NLRC4、AIM2)是细胞内的多蛋白复合体，可诱导多种促炎细胞因子和趋化因子，从而引导炎症反应。方法：采用C57BL/6小鼠短暂性大脑中动脉闭塞(TMCAO)60min，再灌注3、7、23h的动物模型，观察NLRP3在脑缺血再灌注损伤中的作用。半定量实时定量聚合酶链式反应和Western Blot分析结合免疫细胞化学进行蛋白定位，评价炎性小体在缺血侧的表达模式。最后，动物在tMCAO再通前或再通后直接用萝卜硫素、Genipin、MCC950或赋形剂进行治疗。结果：TMCAO后1d，缺血侧脑内NLRP3基因相对表达水平升高20~30倍，神经元NLRP3表达增强。相应地，NLRP3调节剂、Bruton‘s酪氨酸激酶(BTK)和NLRP3诱导的细胞因子IL-1β的基因表达水平显著上升。其他炎性小体的改变较轻或不明显。在tMCAO前后应用覆盖所有炎症体的炎性小体抑制剂或特别是NLRP3可显著缩小脑梗塞体积，并伴随着caspase 1活性降低的明显证据。这种减轻中风的效果与缺血侧免疫细胞的减少和血脑屏障完整性的保护相一致。结论：我们的数据表明在神经元中诱导NLRP3炎性小体可以促进急性缺血性卒中的神经炎症。早期阻断NLRP3通过减轻炎症和稳定血脑屏障来保护I/R损伤。
Purpose: Cerebral ischemia induces a profound neuro-inflammatory response, but the underlying molecular mechanisms are poorly understood. Inflammasomes (NLRP1, NLRP3, NLRC4, AIM2) are intracellular multi-protein complexes which can induce sets of pro-inflammatory cyto- and chemokines, and thereby guide inflammation. We, here, assessed the functional role of NLRP3 in ischemia/reperfusion (I/R) injury in a mouse model of transient cerebral ischemia.Methods: Ischemic stroke was induced in C57Bl/6 mice by 60 min transient middle cerebral artery occlusion (tMCAO) and 3, 7 or 23 h of reperfusion, a paradigm of I/R injury. The expression patterns of inflammasomes in the ischemic hemispheres were evaluated by semiquantitative real-time PCR and Western Blot analysis accompanied by protein localization using immunocytochemistry. Finally, animals were treated with the inflammasome inhibitors Sulforaphane, Genipin, MCC950 or vehicle, directly before or upon recanalization after tMCAO. Stroke outcome was assessed, including infarct size and functional deficits, local inflammatory response, neuronal survival as well as blood-brain barrier function on day 1 after tMCAO.Results: After tMCAO the relative gene expression levels of NLRP3 increased 20-30x within 1 day in the ischemic hemisphere which translated into an increased expression of NLRP3 in neurons. Accordingly, the gene expression levels of the NLRP3-modulator, Bruton's Tyrosine Kinase (BTK), and the NLRP3-inducible cytokine IL-1 beta significantly rose. Lesser or non-significant changes were seen for the other inflammasomes. Application of inflammasome inhibitors covering all inflammasomes or specifically NLRP3 significantly reduced infarct volumes when given before or after tMCAO and was accompanied by clear evidence for reduced activation of caspase 1. This stroke attenuating effect coincided with less immune cell infiltration in the ischemic hemisphere and preservation of the blood-brain barrier integrity.Conclusions: Our data show that induction of the NLRP3 inflammasome in neurons drives neuroinflammation in acute ischemic stroke. Early blockade of NLRP3 protects from I/R injury by mitigating inflammation and stabilizing the blood-brain barrier.