Adult male C57BL/6 mice (8-10 wk old, weighing 20–25 g) were obtained from the Animal Core Facility of Fujian Medical University. All experimental procedures were complied with the rules for animal research by Fujian Medical University. Briefly, mice were anesthetized with isoflurane. After the common, external and internal carotid arteries were exposure, a marked 6-0 filament was employed to puncture the origin of the left middle cerebral artery through the internal carotid artery (20). Animals in sham group received similar procedures without the artery puncture. The SAH severity grading score was recorded according to previous studies (21). Mice with a SAH grading score of less than 8 were excluded.

In the first experiment, mice were assigned to sham group (n = 6) and post-SAH (6 h, 12 h, 24 h, 48 h, 72 h) (n = 6 per group). Western blot and immunofluorescence staining were performed in the experiment. In the second experiment, mice were assigned to sham group, vehicle-treated SAH group, Scramble small interfering RNA (siRNA)-treated SAH group, and PHLDA1 siRNA-treated SAH group (n = 12 per group). Animals were sacrificed at 24 h or 72 h after SAH. Post-treatment assessments included neurobehavior tests, western blot, immunofluorescence staining, TUNEL staining, enzyme-linked immunosorbent assay (ELISA), and biochemical estimation. In the third experiment, mice were assigned to sham group, vehicle-treated SAH group, PHLDA1 siRNA-treated SAH, and PHLDA1 siRNA plus nigericin -treated SAH group (n = 12 per group). Post-treatment assessments included neurobehavior tests, western blot, immunofluorescence staining, TUNEL staining, ELISA, and biochemical estimation.

For PHLDA1 knockdown, a volume of 3μl PHLDA1 siRNA (Santa Cruz Technology) or scramble siRNA was dissolved in transfection solution and then injected into the lateral ventricles at 48 h before the construction of SAH model. Nigericin (MedChemExpress, 2 μg), a potent NLRP3 activator, was prepared in 2 μl ethanol and physiologic saline. Nigericin or vehicle was intracerebroventricularly administered at 2 h before SAH operation. The dose of nigericin and administration route were based on previous studies (22).

The modified Garcia scale test was used to evaluate neurological deficits as previously reported (23). Six measurements were included in this score system. The higher score suggested the better neurobehavioral outcomes. For motor function, the beam-walking score test was performed according to previous reports (24). The animals’ walking distance within 1 min were recorded. Neurobehavior tests were conducted in a blinded manner.

The supernatant of brain samples was collected. The levels of interleukin (IL)-1β, IL-6, IL-18, and IL-10 were detected by using commercially available kits (Multi Sciences). The detailed methods were conducted according to the manufacturer’s instructions.

The brain tissue and protein samples were prepared according to previous studies (25). Briefly, the protein samples were loaded onto SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk. After that, they were incubated with primary antibodies: PHLDA1 (1:1000, Abcam), NLRP3 (1:200, Santa Cruz Biotechnology), ASC (1:200, Santa Cruz Biotechnology), caspase-1 (1:200, Santa Cruz Biotechnology), cleaved caspase-1 (1:200, Santa Cruz Biotechnology), and β-actin (1:3000, Bioworld Technology) in a 4°C freezer. Then, membranes were incubated with corresponding secondary antibodies. ImageJ software was employed to measure relative intensity.

The detailed methods were performed according to previous studies (26). In brief, the frozen tissue sections were treated with Triton X-100 (0.3%) and then blocked with 5% goat serum. After that, sections were incubated with primary antibodies: PHLDA1 (Abcam), CD16/32 (BD Biosciences), CD206 (Invitrogen), NeuN (EMD Millipore), IL-1β (Santa Cruz Biotechnology), and Iba-1 (Santa Cruz Biotechnology) in a 4°C freezer. After that, they were incubated with corresponding secondary antibodies followed by using DAPI staining. The slices were then observed under a fluorescence microscope.

TUNEL staining was performed by using a commercially available kit (Beyotime Biotechnology). Brain sections were incubated with primary antibody against NeuN in a 4°C freezer. After that, the sections were incubated with TUNEL reaction mixture. The slides were then washed and counterstained with DAPI. The slices were observed under a fluorescence microscope.

Data are expressed as mean ± SD. Statistical analysis was conducted with Graph- Pad Prism 8 software. Statistical evaluation was performed using one-way ANOVA or two-way ANOVA with Tukey’s post hoc test. The significant P-value was < 0.05.

Mounting evidence has indicated that PHLDA1 and NLRP3 might interact with each other. PHLDA1 activation could induce NLRP3 inflammasome signaling. In this experiment, western blot (Figure 1A) was performed to investigate the protein expression of PHLDA1 and NLRP3. As shown in Figures 1B, C, the expression of PHLDA1 and NLRP3 markedly increased in the early period after SAH, and peaked at 24 h post-SAH (P < 0.05). In addition, double immunofluorescence staining indicated that the enhanced PHLDA1 and NLRP3 were mainly distributed in microglia after SAH (P < 0.05) (Figures 1D–G).

Previous study has demonstrated that PHLDA1 activation could induce NLRP3 inflammasome signaling. We applied PHLDA1 siRNA to inhibit PHLDA1 expression and explore whether PHLDA1 deficiency could reduce NLRP3 inflammasome activation. As shown, western blot results (Figure 2A) showed that PHLDA1 siRNA significantly reduced PHLDA1 expression after SAH (P < 0.05). Moreover, the activated NLRP3 inflammasome signaling pathway was markedly suppressed by PHLDA1 siRNA (P < 0.05) (Figures 2A–F). Consistently, double immunofluorescence staining confirmed that PHLDA1 siRNA significantly decreased PHLDA1 and NLRP3 expression in microglia in the brain cortex after SAH (P < 0.05) (Figures 2G–J).

The anti-inflammatory effects of PHLDA1 blockade have been verified in other diseases models. We further investigated the influence of PHLDA1 deficiency on inflammatory response after SAH. By using ELISA kits, we found that SAH insults induced a significant increase in proinflammatory cytokines release, including IL-1β, IL-6, and IL-18 (P < 0.05) (Figures 3A–C). All these cytokines were decreased by PHLDA1 deficiency (P < 0.05). In addition, PHLDA1 deficiency significantly induced an increase in IL-10 expression after SAH (P < 0.05) (Figure 3D). Simultaneously, IL-1β immunofluorescence staining verified that PHLDA1 deficiency significantly decreased the enhanced levels of IL-1β in the brain cortex after SAH (P < 0.05) (Figures 3E, F).

Microglial polarization plays a key role in inflammatory response after SAH. Studies have proved that suppression of microglia M1 polarization and promotion of M2 microglia could effectively reduce acute brain injuries and improve neurological outcomes. Interestingly, PHLDA1 has been reported to modulate microglia M1/M2 polarization in other diseases. To determine whether PHLDA1 deficiency affects microglial polarization after SAH, double immunostaining was performed to examine the levels of M1 microglia and M2 microglia. It showed that the number of Iba1⁺/CD16/32⁺ cells was significantly increased after SAH, which could be decreased by PHLDA1 silencing (P < 0.05) (Figures 4A, C). In addition, we examined the expression of M2 microglia and revealed that the levels of Iba1⁺/CD206⁺ cells were markedly increased after PHLDA1 siRNA treatment (P < 0.05) (Figures 4B, D). These data suggested that PHLDA1 silencing could suppress M1 microglia and promote M2 microglia polarization.

Next, we explored whether PHLDA1 deficiency could exert cerebroprotective effects after SAH. TUNEL staining revealed that SAH insults significantly induced neuronal apoptosis (P < 0.05) (Figures 5A, B). Concomitant with the exacerbated neuronal death, the neurological outcomes of SAH was further aggravated (P < 0.05) (Figures 5C, D). In contrast, PHLDA1 siRNA treatment evidently reduced SAH-induced neuronal apoptosis (P < 0.05). Simultaneously, PHLDA1 deficiency showed better neurological outcomes after SAH insults (P < 0.05) (Figures 5A–D). These suggested that PHLDA1 deficiency could protect against SAH-induced EBI by its anti-inflammatory effects.

NLRP3 inflammasome plays a key role in microglial activation after SAH. Moreover, NLRP3 inflammasome activation could induce microglia M1 polarization and inhibit NLRP3 inflammasome could promote M2 microglia polarization. As mentioned above, PHLDA1 deficiency could markedly inhibit NLRP3 inflammasome activation after SAH. We further explored whether NLRP3 inflammasome activation by nigericin could abate the cerebroprotective effects of PHLDA1 deficiency. As expected, western blot results showed that nigericin administration eliminated the inhibitory effects of PHLDA1 deficiency on NLRP3 inflammasome activation (P < 0.05) (Figures 6A–E). Consistently, the immunofluorescence staining results verified that NLRP3 inflammasome staining was further induced after nigericin treatment (P < 0.05) (Figures 6F, G).

Next, we examined the effects of nigericin administration on microglial polarization after PHLDA1 siRNA treatment. As expected, the immunofluorescence staining results showed that NLRP3 inflammasome activation by nigericin abated the effects of PHLDA1 silencing on microglia M1/M2 polarization, as evidenced by the increased number of Iba1⁺/CD16/32⁺ cells and decreased number of Iba1⁺/CD206⁺ cells (P < 0.05) (Figures 7A–D). Moreover, the ELISA data indicated that nigericin further exacerbated proinflammatory cytokines release and decreased anti-inflammatory cytokines after SAH (P < 0.05) (Figures 7E–H). These suggested that NLRP3 inflammasome activation contributed to the modulation effects of PHLDA1 on microglial polarization after SAH.

We suspected that NLRP3 inflammasome activation by nigericin might reverse the beneficial effects of PHLDA1 deficiency on neuronal survival and neurological function. Consistent with the aggravated inflammatory insults, nigericin administration further significantly increased neuronal apoptosis and exacerbated neurological deficits and motor dysfunction (P < 0.05) (Figures 8A–D). Based on the findings above, PHLDA1 blockade could attenuate EBI after SAH by regulating microglia M1/M2 polarization via NLRP3 inflammasome signaling.