Background

The role of ferroptosis in ischemic stroke has been hotly debated recently, but the mechanism is not clearly clarified. It has been reported that the NLRP3 inflammasome is essential for the progression of ischemic stroke. Whether the ferroptosis after ischemic stroke mediated by the activation of NLRP3 inflammasome is still not reported. In this study, we investigated the effect of NLRP3 deficiency on ferroptosis following cerebral ischemia-reperfusion injury (CIRI) in vivo and in vitro.

Materials

In vivo, we used C57BL/6J mice and NLRP3^-/- mice to establish a model of middle cerebral artery occlusion (MCAO). After 3 days of reperfusion, we assessed neurological function and then performed TTC staining to measure the infarct volume. Besides, we measured the expression of NLRP3 inflammasome-related proteins and the ferroptosis-inhibiting protein glutathione peroxidase 4 (GPX4) by western blotting (WB) and immunofluorescence (IF). Moreover, we evaluated the levels of ferroptosis-related factors (Fe²⁺, MDA and GSH) in the infarct area by using appropriate kits. Furthermore, we used WB to measure the expression of Kelch-like epichlorohydrin-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2), which participate in the progression of ischemic stroke. In vitro, we knocked down NLRP3 with small interfering RNAs (siRNAs) and established an oxygen glucose deprivation/Reperfusion (OGD/R) model in BV2 cells to simulate ischemic conditions. Next, we assessed the viability of BV2 cells by the Cell Counting Kit (CCK)-8 cytotoxicity assay. Moreover, we used WB to measure the expression of NLRP3, IL-1β, GPX4, Keap1 and Nrf2 proteins which are involved in CIRI.

Results

Three days after MCAO, the NLRP3^-/- mice exhibited smaller cerebral infarct volumes and lower neurological deficit scores. The expression of NLRP3 inflammasome-associated proteins (IL-18 and IL-1β) and Keap1/Nrf2 signaling pathway moleculars (Keap1 and Nrf2) in mice brain tissue and BV2 cells were inhibited by NLRP3 knockout/knockdown, while the expression of GPX4, one of the ferroptosis-related factors was increased. Furthermore, the contents of Fe²⁺ and MDA in the brain tissues of NLRP3^-/- mice were decreased, while the content of GSH were increased significantly.

Conclusion

Inhibition of the NLRP3 inflammasome alleviates CIRI by inhibiting ferroptosis and inflammation, possibly through a mechanism of the Keap1-Nrf2 pathway.

中文翻译：

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NLRP3 炎症小体缺陷通过抑制铁死亡减轻脑缺血再灌注损伤

背景

铁死亡在缺血性脑卒中中的作用近期备受争议，但机制尚不明确。据报道，NLRP3 炎症小体对于缺血性中风的进展至关重要。NLRP3 炎症小体的激活介导的缺血性卒中后铁死亡是否仍未见报道。在这项研究中，我们在体内和体外研究了 NLRP3 缺乏对脑缺血再灌注损伤 (CIRI) 后铁死亡的影响。

材料

在体内，我们使用 C57BL/6J 小鼠和 NLRP3 ^-/-小鼠建立大脑中动脉闭塞 (MCAO) 模型。再灌注 3 天后，我们评估神经功能，然后进行 TTC 染色以测量梗死体积。此外，我们通过蛋白质印迹 (WB) 和免疫荧光 (IF) 测量了 NLRP3 炎性体相关蛋白和铁死亡抑制蛋白谷胱甘肽过氧化物酶 4 (GPX4) 的表达。此外，我们评估了铁死亡相关因子（Fe ²⁺, MDA 和 GSH) 在梗死区使用适当的试剂盒。此外，我们使用 WB 来测量参与缺血性中风进展的 Kelch 样表氯醇相关蛋白 1 (Keap1) 和核因子红细胞 2 相关因子 2 (Nrf2) 的表达。在体外，我们用小干扰 RNA (siRNA) 敲低 NLRP3，并在 BV2 细胞中建立氧糖剥夺/再灌注 (OGD/R) 模型以模拟缺血情况。接下来，我们通过细胞计数试剂盒 (CCK)-8 细胞毒性测定评估了 BV2 细胞的活力。此外，我们使用 WB 来测量参与 CIRI 的 NLRP3、IL-1β、GPX4、Keap1 和 Nrf2 蛋白的表达。

结果

MCAO 三天后，NLRP3 ^-/-小鼠表现出较小的脑梗死体积和较低的神经功能缺损评分。小鼠脑组织和BV2细胞中NLRP3炎性体相关蛋白（IL-18和IL-1β）和Keap1/Nrf2信号通路分子（Keap1和Nrf2）的表达受到NLRP3敲除/敲除的抑制，而GPX4、一种与铁死亡相关的因素增加了。此外， NLRP3 ^-/-小鼠脑组织中Fe ²⁺和MDA含量降低，而GSH含量显着升高。

结论

抑制 NLRP3 炎性体可通过抑制铁死亡和炎症减轻 CIRI，这可能是通过 Keap1-Nrf2 通路的机制实现的。