Ischemic stroke is a common, severe, disabling health-care problem (Wafa et al., 2020), and contributes to a high mortality rate and disability rate (Cao et al., 2023). Therefore, developing new brain protectants for ischemic stroke is in urgent.

Post-stroke injury is responsible for the reduction of viable neurons in the peri-infarct area (Wang et al., 2020b). Microglia-mediated neuroinflammation is an important cause of secondary brain injury (Benarroch, 2013; Han et al., 2021), and influences the permeability of blood brain barrier (BBB) (Lou et al., 2016). Microglia in the peri-infarct area would reach the peak amounts two to three days after the ischemic injury and linger for weeks afterward. At the onset of the ischemia event, the cerebral microenvironment would soon be changed and activate microglia (Zhou et al., 2023) through NOD-like receptor (NLR) (Luo et al., 2022), Toll-like receptors (TLR) (Ling et al., 2021), and C-type lectin receptors (Ye et al., 2020). Additionally, TLR4/NF-κB pathway is a classic pathway, which is not only directly responsible for immune response in ischemic stroke, but also one of the upstream regulation pathways of NLRP3 (Liu et al., 2020).

NLRP3 inflammasome is also considered as the therapeutic target in various inflammatory diseases (Coll et al., 2022; Mangan et al., 2018), which is an important immune effector could be triggered by infection and tissue injury (Huang et al., 2021). Activated NLRP3 can form a large multiprotein signaling platform, including oligomerized ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) and activated caspase1 (Christgen et al., 2020). Subsequently, cleaved caspase1 contributes to the production of IL-1β, IL-18, and induces the next coming inflammatory effect (Heneka et al., 2014; Mangan et al., 2018), which could aggravate acute damage in the peri-infarct area (Franke et al., 2021; Palomino-Antolin et al., 2022). Hence, inhibiting NLRP3 is protective to the infarcted penumbra (Ismael et al., 2018).

Echinatin, a chalcone separated from licorice (Fu et al., 2013), is recently discovered to be protective in DSS-induced colitis and could suppress NLRP3 inflammasome activation by binding to heat-shock protein 90 (HSP90) (Xu et al., 2021). Besides, as a flavonoid of phenolic compounds, echinatin also have the effect of anti-inflammation and antioxidation (Li et al., 2019; Liang et al., 2018). Meanwhile, as a chalcone, echinatin has low molecular weight, and easy to optimize its lipophilicity (logP) with appropriate substituents which allows it to have better BBB permeability than other chemical compounds (Thapa et al., 2021). In previous studies, echinatin was reported to be capable of reducing the production of interlukin-6 (IL-6), prostaglandin E2 (PGE2), reactive oxygen species (ROS) and nitric oxide (NO) in LPS-induced RAW 264.7 cells (Fu et al., 2013). However, the effect of echinatin on ischemic stroke or microglia is still unclear.

The transient Middle Cerebral Artery Occlusion (tMCAO) model is the most common animal model for simulating ischemic stroke (Franke et al., 2021). We measured the infarct zone, cerebral edema, BBB permeability, and motor impairment to determine the extent of neurological injury in a murine model of tMCAO. Moreover, the underlying mechanisms of echinatin were investigated in primary microglia following the stimulation of lipopolysaccharide (LPS) or LPS + adenosine triphosphate (ATP) challenge.