Figure 1A illustrates the effects of Cyn on a mouse model of transient middle cerebral artery occlusion (tMCAO), which replicates cerebral I/R injury. The mice were assessed for their neurologic behavior based on their deficit scores after MCAO induction. A significantly higher deficit score was observed in MCAO mice compared to sham-operated control mice. Neurological deficit scores decreased significantly after treatment with Cyn (Figure 1B). Additionally, brain water content, which was elevated in MCAO mice, decreased significantly after Cyn treatment (Figure 1C). TTC staining was used to visualize the infarcted area, and the percentage of infarcted area in each brain section was calculated. Infarction on the ipsilateral side of the brain was not detected following the sham operation, whereas it was significantly increased following the MCAO. Treatment with Cyn inhibited tMCAO-induced infarction (Figure 1D). On the ipsilateral side of tMCAO, HE staining revealed neuronal loss in the cerebral cortex region. Cyn treatment mitigated this neuronal loss (Figure 1E).

Using SwissTargetPrediction, we identified Alox15 as having the highest betweenness centrality (Figure 2A). Subsequently, we conducted an analysis of the binding model between Cyn and the Alox15 protein using molecular docking technology. The result of 3D-molecular docking illustrated that Cyn can effectively bind to the residues Gly211 and His212 of the Alox15 protein, demonstrating a stabilizing binding conformation (Figure 2B). Bio-layer interferometry (BLI) results demonstrated a kinetic association constant (Kd) of 21.63 μM for Cyn binding to Alox15 (Figure 2C). Western blotting and immunofluorescence analyses indicated an upregulation of Alox15 expression following tMCAO, which was subsequently downregulated in the presence of Cyn, with a more pronounced downregulation observed at higher doses of Cyn compared to lower doses (Figures 2D–F).

Iba1 immunofluorescence staining showed that increased Iba1-positive microglia within the infarct region on the ipsilateral side of tMCAO, which decreased when treated with Cyn (Figure 3A). Cyn treatment in tMCAO mice decreased NLRP3-mediated inflammatory cytokine IL-1β and IL-18 release, as evidenced by ELISA tests, in the mice (Figures 3B,C). The levels of NLRP3, ASC, and cleaved caspase-1 were elevated following tMCAO, but decreased following Cyn treatment, However, full-length caspase-1 expression was not differentiated among the experimental groups (Figures 3D,E). These findings underscore the potential of Cyn in attenuating NLRP3 inflammasome-mediated inflammation subsequent to tMCAO.

She et al. reported that factors involved in ferroptosis regulation include lipid peroxidation, Fe²⁺ levels and GPX4 activity (18). Rats subjected to MCAO/R for an hour, reactive oxygen species, malondialdehyde, glutamate, and iron levels significantly increased, while glutathione levels decreased. After treatment with Cyn, these changes were reversed (Figures 4A–E). Moreover, The Western Blot analysis revealed a significant upregulation in the expression levels of COX2, Tfrc, and Acsl4 following tMCAO, with a subsequent decrease in protein expression levels after treatment with Cyn (Figures 4F,G).

We further investigated Cyn’s effects in vitro using BV-2 microglia cells with oxygen and glucose deprivation/reoxygenation (OGD/R) and varying concentrations of Cyn (25, 50 μM). ELISA analysis indicated an increase in the release of IL-1β and IL-18 mediated by NLRP3 inflammasome after OGD/R, which was attenuated by Cyn treatment (Figures 5A,B). Western blot analysis revealed that the expression levels of NLRP3, ASC, and caspase-1, key NLRP3 inflammasome proteins, were higher in OGD/R-treated cells but decreased in Cyn-treated cells. Full-length caspase-1 expression showed no significant difference (Figures 5C,D).

We observed that Cyn modulates ferroptosis post-OGD/R treatment by increasing ROS, MDA, glutamate, and iron levels, while reducing GSH levels, as evidenced by increased levels of ROS, MDA, glutamate, and iron. Remarkably, Cyn intervention reversed these alterations (Figures 6A–E). Correspondingly, Cyn downregulated COX2, Tfrc, and Acsl4 levels in OGD/R-exposed cells, consistent with our observations in mice (Figures 6F,G).

In this study, a total of 50 mice aged 10–12 weeks and weighing 30-35 g were obtained from Guizhou Medical University and housed under controlled conditions at 22°C and 45–55% humidity with a 12 h light/dark cycle and access to water and food ad libitum. Following a one-week acclimatization period, the mice were randomly assigned to one of four groups: (1) Sham, (2) MCAO, (3) MCAO + Cyn (L), (4) MCAO + Cyn (H). Consistent with prior studies (40, 41), embolization was performed on the right hemisphere of the brain using a 10 × microscope in a sterile environment. Induction and maintenance anesthesia were administered using 5% isoflurane and 2.5% halothane, respectively, prior to surgery (RWD Life Science Co, Shenzhen, China). The carotid sheath, common carotid artery, and internal carotid artery were exposed. Using a 0.26 mm nylon thread, a plug is inserted into the internal carotid artery through the common carotid artery and advanced into the intracranial space. After ligating and securing the plug, skin sutures are placed. Following a 150-min middle cerebral artery embolization procedure, the plug was retracted approximately 1 cm to restore blood flow. Mice in the sham group underwent identical surgical interventions without middle cerebral artery occlusion. Cyn (MCE, Wuhan, China) was administered intraperitoneally at doses of 10 mg/kg (low dose) or 20 mg/kg (high dose) 30 min before middle cerebral artery occlusion. For the MCAO group, saline was administered to mice. Animals displaying a 30% reduction in cerebral blood flow during MCAO, as well as those that expired during reperfusion, were excluded from subsequent experiments. An evaluation of neurological deficit scores was performed in mice after they had received 24 h of reperfusion. Afterward, mice were euthanized with 200 mg/kg pentobarbital, and brain tissues were collected.

In the Octet RED96 System (ForteBio), the bio-layer interferometry (BLI) assay was used to determine Cyn’s binding affinity to Alox15. Biotinylation was performed according to the manufacturer’s instructions on ReadTM Streptavidin Biosensors to immobilize Alox15 proteins. The association of immobilized protein with flowing corosolic acid was detected with various concentrations of Cyn in the mobile phase. A running buffer is the PBS puffer with 0.1% DMSO and 0.02% Tween-20 was used for all experiments. Data were analyzed using ForteBio Data Analysis 9.0.

Following anesthesia, the brains were extracted and cryopreserved at −20°C for 15 min. Subsequently, the cerebellum and olfactory bulb were excised, and the brain tissue was sectioned into five equal parts in the coronal plane, each approximately 2 mm thick. The sections were then immersed in 0.4% TTC (Sangon Biotech Co., Ltd., Shanghai, China) solution at 37°C in a light-protected water bath, with regular flipping every 5 min to ensure uniform staining. Non-infarcted regions appeared red, while infarcted areas exhibited a gray-white hue. Following staining, the TTC solution was decanted, and the brain tissue was fixed in 4% paraformaldehyde. After a period of 6 h, the caudal side of each brain slice was examined using the Image-Pro Plus 6.0 system. The infarct size and total area of each slice were quantified, and the percentage of infarct area relative to the entire brain area was computed.

After 24 h of reperfusion, the whole brain was harvested and weighed (wet weight). A second weight was obtained after drying the brain at 100°C. In order to determine the percentage of water in the brain, the formula is 100 × (wet weight - dry weight)/wet weight.

Protein expression levels were assessed using western blot analysis with antibodies against NLRP3 (Proteintech, No. 68102-1-Ig), ASC (Proteintech, No. 10500-1-AP), and COX2 (Proteintech, No. 66351-1-Ig), anti-caspase-1 (Proteintech, No. 22915-1-AP), anti- Alox15 (CST, No. 82129), Cleaved Caspase-1 (Bioss, No. bs-10743R), anti-Tfrc (Proteintech, No. 65236-1-Ig), anti-Acsl4 (Proteintech, No. 22401-1-AP) and anti-β-actin (Proteintech, No. 81115-1-RR).

ChemDraw was used to generate Cyn’s structure, while Protein Data Bank (PDB) was used to retrieve proteins’ crystal structures. Subsequently, both the obtained protein structures and the Cyn structure were inputted into Autodock. Prior to docking, the protein structures underwent preparation steps involving the removal of water molecules and addition of hydrogen atoms. The docking process was executed using an algorithm, and the highest scoring binding model was selected for visualization of the interactions between Alox15 and Cyn.

After the BV-2 microglia cells were suspended in a sugar-free and serum-free medium, they were incubated in an hypoxic environment containing 0.2% O₂ and 5% CO₂ for 3 h before being switched to a sugar-containing medium containing 10% fetal bovine serum, and further incubated at 37°C with 5% CO₂ for 24 h. Using standard culture conditions, control cells and cell models of cerebral ischemia–reperfusion injury were treated with various treatments according to experimental protocols.

Brain tissues were removed, mechanically homogenized under an ice water bath, centrifuged at 2500 r/min for 10 min, and the supernatant was taken for determination. The Malondialdehyde (MDA), Reactive oxygen species (ROS), Glutathione peroxidase 4 (GPXs), Glutathione (GSH) and Fe²⁺ assay kit (Solarbio, Beijing, China) instructions were followed.

Blood was collected from mice’s eye sockets overnight at 4°C then centrifuged at 3000 r/min for 5 min to separate the upper serum and lower serum. The ELISA kits were purchased from Absin (Shanghai) biotechnology company. The procedure that was followed was based on the instructions on the kit.

Rat brain tissue from the ischemia–reperfusion area or microglia proteins were obtained with the RIPA lysis buffer supplemented with protease and phosphatase inhibitors. The homogenates were then centrifuged at 12,000 rpm for 15 min at 4°C to remove debris. And then protein concentrations were determined using the BCA protein assay kit. Each sample of protein were isolated equally to 30 μg by electrophoresis and then transferred to PVDF membranes. The membranes were placed into primary antibodies against Alox15, NLRP3, ASC, Cleaved Caspase-1, Caspase-1, COX2, Trfc, Acsl4 and β-acting overnight at 4°C after being blocked with 5% non-fat dry milk in TBS-T at 36°C. Subsequently, the membranes were then washed with TBS-T and incubated with HRP-conjugated secondary antibodies for an hour at 36°C. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit and quantified by densitometry using ImageJ software.

Neurological function was assessed 24 h after reperfusion by an investigator blinded to the treatment groups. The scoring was based on a five-point scale, as described by longa et al. (42): 0, no neurological deficit (normal behavior); 1, failure to fully extend the contralateral forelimb; 2, circling to the contralateral side when held by the tail; 3, falling to the contralateral side; 4, no spontaneous walking with a depressed level of consciousness.

The data, with the exception of neurological deficit scores, are presented as mean ± standard deviation. Multivariate analysis was applied to assess the relationships between different variables, using techniques such as principal component analysis (PCA) to reduce dimensionality and identify the key patterns in the data ANOVAs and Tukey’s multiple comparison tests were used to assess the statistical significance of the differences between groups. GraphPad Prism 7.0 software was used to conduct all statistical analyses, and a significance level of 0.05 was set for all statistical analyses. Statistical analysis was conducted using Kruskal-Wallis and Dunn’s multiple comparison tests.