Adult male Sprague-Dawley rats (weighing 230-280 g) were used in our study. Rat model of SAH was built in accordance with previous published method (22). Before SAH, anesthetization was administered with 1% sodium pentobarbital. A marked filament (4-0) was used to puncture the origin of the left middle cerebral artery. Sham-operated rats received the same treatment, but not with vessel perforation. In vivo experiments, rats were randomly allotted to sham group (n= 18), SAH + vehicle group (n= 22, 4 animals died), SAH + LDC7559 group (n= 60, 6 animals died), and SAH + LDC7559 + nigericin group (n=21, 3 animals died). Investigators blinded to the experimental groups performed experimental analyses.

Primary neurons and microglia were dissociated from rat pups (postnatal days 0-1) and cultured as previously described (23). Primary cortical neurons were seeded in poly-D-lysine-coated plates and maintained in neurobasal media containing B27 and glutamate. Primary microglia were cultured in Dulbecco’s modified Eagle Medium (DMEM, GIBCO). The co-culture system was supplemented with DMEM with 10% FBS. In vitro experiments, primary cortical neurons and microglia were exposed to 10 μM hemoglobin (Hb).

In vivo, LDC7559 (MedChemExpress) was diluted in 10% DMSO and 90% corn oil before use. LDC7559 (10, 20, or 30 mg/kg) or vehicle was treated at 2 h after SAH by intraperitoneally administration and then once a day. Nigericin (MedChemExpress, 2 μg) was prepared in 2 μl ethanol and physiologic saline and was intracerebroventricularly administered at 2 h before SAH. For experiments in vitro, three different doses of LDC7559 (5, 25, or 50 μM) was dissolved in culture medium. Nigericin (5 µM) was added into culture medium after Hb stimulation. The concentrations and administration routes of LDC7559 and nigericin were based on previous studies (21, 24).

A commercial cell counting kit-8 (CCK-8) assay was used to detect cell viability. According to the instructions (Beyotime Biotechnology, China), cells were incubated with CCK-8 reagent. The optical density was measured at 450 nm.

After rats were deeply anesthetized, the animals were transcardially perfused with phosphate buffer solution at room temperature. The fresh inferior basal temporal lobe was employed for western blotting and ELISA assay. For histological studies, brains were immersed in 4% paraformaldehyde overnight. After brain samples were transferred sequentially into different sucrose solution (15%, 25% and 35%), they were sectioned into 8-μm coronal sections. The brain sections were kept in refrigerater at -20°C.

After sample preparation, we determined the expressions of IL-1β, IL-6, and IL-18 using the respective commercial kits (Multi Sciences. China). The expressions of these cytokines were calculated based on the instructions.

After sample preparation, the same mass of sample was separated by SDS-PAGE gels. After they were transferred to PVDF membrane, the membranes were incubated with diluted specific antibodies overnight at 4°C. Then, they were incubated with appropriate secondary antibodies. Protein signals were analyzed with Image J software. Antibodies used for western blot can be seen in Supplementary Material .

Immunofluorescence staining was carried out by standard procedures (25, 26). Briefly, frozen brain sections and cells were fixed with 4% paraformaldehyde. After blocking, the slides were stained with primary antibodies and appropriate secondary antibodies. 4,6-diamidino- 2-phenylindole (DAPI) was employed to indicate the nuclei. A fluorescence microscope was employed for fluorescence detection. Antibodies used for immunofluorescence staining can be seen in Supplementary Material .

TUNEL was conducted in line with the manufacturer’s protocol (Beyotime Biotechnology). Frozen brain sections and cells were incubated with the NeuN antibody, and then with a TUNEL reaction solution. After washing, they were stained with DAPI. Brain sections and cells were observed with a fluorescence microscope.

The neurologic deficits of SAH rats were determined by using a previous published method (27). A lower score suggests more severe deficits. To further evaluate the motor deficits after SAH, the beam-balance score test was performed according to previous studies (6). Behavior functions were recorded in a blinded manner.

All data are expressed as mean ± SD. Comparisons between groups were determined by one-way analysis of variance with Bonferroni post hoc test. The GraphPad Prism 8 software was employed for all statistical analysis. Probability value <0.05 was considered significantly different.

There was no significant difference in the average SAH grading scores ( Figure 1A ). The Modified Garcia test and beam-walking test were used to determine the effects of LDC7559 on short-term neurological impairments. As shown, SAH insults induced a significant neurological deterioration compared with the sham group. LDC7559 treatment dose-dependently improved neurological deficits and motor function compared with the vehicle-treated SAH group ( Figures 1B–E ). In addition, brain water content showed that LDC7559 treatment at both 20 mg/kg and 30 mg/kg significantly reduced brain edema as compared with the SAH group ( Figure 1F ). Based on these results, 20 mg/kg LDC7559 was adopted as the optimum dose. Nigericin, a potent NLRP3 activator, was further employed in this experiment. Our data further revealed that nigericin aggravated neurological impairments and brain edema after SAH and abated the cerebroprotective effects of LDC7559 against SAH ( Figures 1B–E ).

Microglial activation is essential to neuroinflammation after SAH. A previous study reported that LDC7559 can inhibit microglial proliferation and activation after TBI (21). In time, the pro-inflammatory cytokines release after TBI were decreased by LDC7559. We speculated that LDC7559 can also ameliorate microglial activation as well as the subsequent inflammatory response. The immunofluorescence staining results showed that microglial activation was significantly induced after SAH, which could be dose-dependently suppressed by LDC7559 ( Figures 2A, B ). In addition, SAH insults markedly triggered the levels of IL-1β, IL-6, and IL-18 in the brain samples, which could be decreased by LDC7559 ( Figures 2C–E ). In contrast, nigericin further aggravated microglial activation and abated the anti-inflammatory effects of LDC7559 against SAH ( Figures 2A–E ).

Evidence indicates that neuronal apoptosis and neuronal pyroptosis play determinant roles in the poor outcome after SAH. Pyroptosis is a recently discovered cell death form, which can be driven by inflammatory processes. Studies have demonstrated that pyroptosis is characterized by GSDMD-mediated. Inhibiting neuronal apoptosis and neuronal pyroptosis have been shown to improve neurological outcomes after SAH. LDC7559 is a newly selective GSDMD inhibitor. We then evaluated the influence of LDC7559 on neuronal apoptosis and pyroptosis. The data revealed that SAH significantly induced the number of GSDMD-positive and TUNEL-positive neurons, which could be markedly inhibited after LDC7559 ( Figures 3A–D ). However, the beneficial actions of LDC7559 on neuronal apoptosis and neuronal pyroptosis were abated by nigericin administration ( Figures 3A–D ).

NLRP3 inflammasome could trigger caspase-1 activation and subsequently induce IL-1β and IL-18 maturation. Additionally, activated caspase-1 exerts an important role in the intersection of the pyroptosis and the apoptosis pathways. Pyroptosis is characterized by GSDMD-mediated. Caspase-1 has been demonstrated not only to modulate GSDMD activation but also affect caspase-3 activation-mediated neuronal apoptosis. We further evaluated the effects of LDC7559 on NLRP3/caspase1/GSDMD signaling after SAH. It revealed that SAH significantly induced the activation of NLRP3/caspase1/GSDMD signaling. In contrast, LDC7559 treatment markedly inhibited the expressions of GSDMD and cleaved GSDMD after SAH ( Figures 4A–H ). However, LDC7559 did not significantly affect the levels of NLRP3 and caspase1. Nigericin was further used to activate NLRP3 inflammasome signaling. As expected, nigericin significantly induced NLRP3/caspase1/GSDMD signaling and abated the inhibitory effects of LDC7559 on GSDMD after SAH ( Figures 4A–H ).

We further verified the beneficial effects of LDC7559 on neuroinflammation and cell viability in vitro. A transwell co-culture system was established in this experiment. It showed that the neuronal cell viability was evidently decreased after SAH insults ( Figures 5A, B ). In addition, Hb stimulation significantly induced proinflammatory cytokines release, including IL-1β, IL-6, and IL-18 ( Figures 5C–E ). In contrast, LDC7559 dose-dependently inhibited the levels of proinflammatory cytokines and further improved cell viability. However, the beneficial effects of LDC7559 in vitro were abated by nigericin ( Figures 5A–E ).

Whether LDC7559 could inhibit neuronal apoptosis and pyroptosis in vitro remains unknown. We further evaluated the influence of LDC7559 on neuronal apoptosis and pyroptosis in vitro. It showed that the numbers of GSDMD-positive and TUNEL-positive neurons were significantly induced after Hb stimulation, whereas LDC7559 rescued the high levels of neuronal apoptosis and pyroptosis ( Figures 6A–D ). In contrast, NLRP3 inflammasome activator nigericin further aggravated neuronal apoptosis and pyroptosis and abated the beneficial effects of LDC7559 ( Figures 6A–D ). Overall, these findings indicated that LDC7559 exerted a protective effect on SAH-induced neuroinflammation and neuronal apoptosis and pyroptosis.