NLRP3 inflammasome deficiency attenuates cerebral ischemia-reperfusion injury by inhibiting ferroptosis

Abstract

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.

Keywords: Cerebral ischemia-reperfusion injury (CIRI); Ferroptosis; Inflammation; Keap1/Nrf2 signalling pathway; NLRP3.