Inhibiting NLRP3 inflammasome signaling pathway promotes neurological recovery following hypoxic-ischemic brain damage by increasing p97-mediated surface GluA1-containing AMPA receptors

Background The nucleotide-binding oligomeric domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is believed to be a key mediator of neuroinflammation and subsequent secondary brain injury induced by ischemic stroke. However, the role and underlying mechanism of the NLRP3 inflammasome in neonates with hypoxic-ischemic encephalopathy (HIE) are still unclear. Methods The protein expressions of the NLRP3 inflammasome including NLRP3, cysteinyl aspartate specific proteinase-1 (caspase-1) and interleukin-1β (IL-1β), the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid receptor (AMPAR) subunit, and the ATPase valosin-containing protein (VCP/p97), were determined by Western blotting. The interaction between p97 and AMPA glutamate receptor 1 (GluA1) was determined by co-immunoprecipitation. The histopathological level of hypoxic-ischemic brain damage (HIBD) was determined by triphenyltetrazolium chloride (TTC) staining. Polymerase chain reaction (PCR) and Western blotting were used to confirm the genotype of the knockout mice. Motor functions, including myodynamia and coordination, were evaluated by using grasping and rotarod tests. Hippocampus-dependent spatial cognitive function was measured by using the Morris-water maze (MWM). Results We reported that the NLRP3 inflammasome signaling pathway, such as NLRP3, caspase-1 and IL-1β, was activated in rats with HIBD and oxygen-glucose deprivation (OGD)-treated cultured primary neurons. Further studies showed that the protein level of the AMPAR GluA1 subunit on the hippocampal postsynaptic membrane was significantly decreased in rats with HIBD, and it could be restored to control levels after treatment with the specific caspase-1 inhibitor AC-YVAD-CMK. Similarly, in vitro studies showed that OGD reduced GluA1 protein levels on the plasma membrane in cultured primary neurons, whereas AC-YVAD-CMK treatment restored this reduction. Importantly, we showed that OGD treatment obviously enhanced the interaction between p97 and GluA1, while AC-YVAD-CMK treatment promoted the dissociation of p97 from the GluA1 complex and consequently facilitated the localization of GluA1 on the plasma membrane of cultured primary neurons. Finally, we reported that the deficits in motor function, learning and memory in animals with HIBD, were ameliorated by pharmacological intervention or genetic ablation of caspase-1. Conclusion Inhibiting the NLRP3 inflammasome signaling pathway promotes neurological recovery in animals with HIBD by increasing p97-mediated surface GluA1 expression, thereby providing new insight into HIE therapy. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-023-04452-5.


Background
Hypoxic-ischemic encephalopathy (HIE) is the most prevalent type of asphyxia-induced neonatal brain damage during the perinatal period [1].The incidence of HIE ranges from 1 to 3 per 1000 live births in developed countries and approximately 20 per 1000 live births in developing countries [1][2][3].Nearly 25% of surviving children with HIE experience neurodevelopmental delays, neurological dysfunction and learning and memory impairments.Due to serious neurological sequelae, HIE can have a significant negative impact on surviving children and their families as well as society [4,5].Hypothermia, hyperbaric oxygen and systemic supportive care are the most common treatments for HIE, but their effectiveness is limited [6,7].Therefore, exploring the pathogenesis of HIE and searching for feasible interventions to prevent hypoxia-ischemia (HI)-induced brain dysfunction have become urgent needs.
Innate immunity is the first barrier of the immune system, plays a critical role in defending against and eliminating pathogens, and guides the body to mount an adaptive immune response [8].Pathogen-associated molecular patterns (PAMPs) are molecular structures encoded by pathogens that are shared among multiple related microorganisms.The function of innate immunity involves recognizing PAMPs through pattern recognition receptors (PRRs) [8,9].Inflammasomes, which are important components of innate immunity, are multiprotein complexes formed by PRRs.Among the NODlike receptor family, NLRP3 is the most extensively studied receptor [9][10][11].NLRP3 oligomerizes through the NACHT domain in response to diverse stimuli such as pathogens and stress, recruits apoptosis-related specific protein (ASC) through the pyrin domain, and leads to caspase-1 activation [12,13].Activated caspase-1 promotes the maturation of IL-1β and interleukin-18 (IL-18), further recruiting more inflammatory cells and amplifying the inflammatory response [14,15].Growing evidence has demonstrated that the NLRP3 inflammasome signaling pathway is significantly activated in brain injuries caused by HI [16,17].For example, HI promotes the expression of NLRP3, caspase-1, and IL-1β in patient or animal brain samples, as well as in vitro [18][19][20].Neuronal death and behavioral deficits can be prevented by suppressing the NLRP3 inflammasome pathway during stroke [18,19].However, whether the NLRP3 inflammasome signaling pathway is activated and its role in HIinduced brain dysfunction in the immature brain remains largely unknown.
As the basic structure of the nervous system, synapses play key roles in transmitting information via the release of neurotransmitters from the presynapse to the postsynapse [21].Synapses can be classified as excitatory and inhibitory based on the binding effects of neurotransmitters.Glutamate is an excitatory neurotransmitter that binds to excitatory receptors such as N-methyl-d-aspartate receptor (NMDAR), AMPAR, and kainate receptor (KAR) [22,23].Glutaminergic excitotoxicity is primarily mediated by NMDARs and AMPARs and plays a critical role in HI-induced cell death [3,24,25].HI Sensitivity varies among neurons in the affected region.Irreversible neuronal necrosis occurs in the central part of the ischemia, and neurons around the central part exhibit reversible delayed neuronal death, indicating the potential for intervening in hypoxic-ischemic brain damage (HIBD) [26].Glutaminergic excitotoxicity is characterized by elevated glutamate release and binding to postsynaptic NMDARs, resulting in excessive Ca 2+ and H 2 O influx, as well as cytotoxic edema and acute cell dissolution [3,27,28].Numerous studies have explored the mechanisms underlying NMDARs in animals with HIBD and found that NMDAR antagonists can successfully prevent HI-induced neuronal damage [29][30][31].However, NMDAR antagonists have a limited therapeutic window, and they impair normal NMDAR-mediated brain processes, such as neural circuit maturation, neuronal survival, and learning and memory [32][33][34].Therefore, NMDAR antagonists are not suitable for clinical use in human HIE patients.
Compared to the function and underlying mechanisms of NMDARs, the role of AMPARs in HI-induced brain damage remains largely unclear.Some reports suggest that elevated levels of glutamate can induce the phosphorylation of GluA2 subunits, leading to the endocytosis of GluA2-containing AMPARs.The absence of GluA2 subunits causes excessive influx of Ca 2+ and subsequent cell excitotoxicity and death [35,36].Therefore, the regulation of surface AMPARs may also have the potential to prevent cell death after HI.Indeed, a recent report shows that inhibiting caspase-1 modulates surface AMPAR expression and alleviates chronic social defeat stress, indicating that NLRP3 inflammasome signaling pathway blockers could be useful in the treatment of AMPAR-associated ischemic brain injury [37].However, the specific mechanism by which the NLRP3 inflammasome pathway affects surface AMPARs remains largely unknown.
In this study, we hypothesized that HI reduces surface AMPARs by activating the NLRP3 inflammasome pathway, thereby causing neural damage.To investigate this hypothesis, we performed a series of in vivo and in vitro biochemical and molecular experiments under ischemialike conditions.Additionally, multiple behavioral experiments were conducted in well-characterized HIBD models to examine the protective effects by inhibiting the NLRP3 inflammasome pathway on neurological function.
The rat HIBD model was established as described previously with some modifications [38][39][40].In brief, unsexed 7-day-old rats were randomly divided into 4 groups: sham, sham + vehicle, HIBD + vehicle, and HIBD + AC-YVAD-CMK.After the rats were anesthetized with isoflurane, the left common carotid artery was isolated and ligated, and a gelatin sponge was used to promote hemostasis.The rats with HIBD were allowed to recover for 2 h in their nests.Then they were placed in a hypoxic environment (8% O 2 + 92% N 2 ) at 37 °C for 2.5 h.In sham rats, the left common carotid arteries were only isolated but not ligated.After observing normal vital signs, the rats were returned to their nests.
The mouse HIBD model was established as previously described with some modifications [41].In brief, unsexed 10-day-old mice were randomly divided into 4 groups: WT-sham, WT-HIBD, NLRP3 −/− -sham or caspase-1 −/−sham, and NLRP3 −/− -HIBD or caspase-1 −/− -HIBD.After the mice were anesthetized with isoflurane, the left common carotid artery was isolated and ligated, and a gelatin sponge was used to promote hemostasis.The mice with HIBD were returned to their nests for 2 h.Due to the physiological and size differences between rats and mice, the mice were subjected to hypoxic conditions (8% O 2 + 92% N 2 ) at 37 °C for 50 min.In sham mice, the left common carotid arteries were only isolated but not ligated.After observing normal vital signs, all the mice were returned to their nests.
All animals were raised in the Animal Care Center of Children's Hospital of Chongqing Medical University, where they had unlimited access to food and water.The room was controlled on a 12-h light and dark cycle (8:00-20:00) and at a temperature of 21 °C.The use of animals and their suffering are kept to a minimum.Mortality due to surgery was approximately 2% among SD rats and WT mice and approximately 3% among NLRP3 −/− and caspase-1 −/− mice.The experiments were performed in a double-blind manner to ensure unbiased results.

TTC staining
Three days after HIBD surgery, rat brain tissues were collected for TTC staining.After being frozen at − 20 °C for 20 min, the brain was sectioned into 5 coronal slices: middle forebrain and optic chiasm, optic chiasm, funnel stalk, middle of the funnel stalk, and caudal pole of the posterior lobe.The sections were washed and stained with phosphate-buffered saline (PBS) and 2% TTC solution.During the staining process, the sections were flipped every 5 min.

OGD
To mimic immature cerebral ischemia in vitro, cortical/hippocampal neurons were exposed to OGD after 7 days of culture as previously described [39].Neurobasal feeding buffer was replaced with 199/Earle's balanced salt solution (EBSS).Then, the neurons were placed in a Thermo Scientific incubator for 1.5 h under hypoxic conditions (5% O 2 + 95% N 2 ).Then the 199/EBSS buffer was replaced with the previously saved neurobasal feeding buffer.The neurons were allowed to recover for 0 to 24 h in incubator with 5% CO 2 at 37 °C.

Western blotting
Total proteins were extracted in ice-cold NP-40 buffer (Beyotime, China) containing a protease inhibitor.Synaptic proteins were extracted as previously described [43,44], and membrane proteins were extracted by a Minute ™ Plasma Membrane Protein Isolation and Cell Fractionation Kit.The protein concentration was measured by a Pierce ™ BCA Protein Assay Kit.The aliquoted proteins were boiled with 5× sample buffer for 5 min at 95 °C.

Co-immunoprecipitation (Co-IP)
Cultured primary neurons were lysed in IP lysis buffer (containing a protease inhibitor).Protein A/G Magnetic Beads were used to separate the cell debris and the concentration was measured by a Pierce ™ BCA Protein Assay Kit.The lysates were aliquoted into two parts: in the input groups, the proteins, IP lysis buffer and 5× sample buffer were mixed, and in the IP groups, the proteins, IP lysis buffer and antibodies were mixed.The IP groups were incubated with anti-GluA1 or anti-VCP/p97 overnight at 4 °C.After the magnetic beads and the IP group samples were mixed for 2 h at 4 °C, IP lysis buffer and 5× sample buffer were added to the magnetic beads, and the target proteins were boiled for 5 min at 95 °C.

Grasping test
After 3 weeks of recovery from HIBD surgery, the rats/ mice were subjected to the grasping test.The grip of the forelimbs was tested by a Grip Strength Meter (Chatillon, USA).The animals were evaluated 5 times (10-min intervals).Myodynamia was calculated using the mean values.

Rotarod test
Twenty-four hours after the grasping test, the rats/mice were subjected to the rotarod test.The rats/mice were pretrained for 2 rounds at constant speeds of 10 rpm and 20 rpm (3 min for rats, 5 min for mice).The rats were trained for 10 rounds at a constant speed from 10 to 55 rpm (5 rpm increases, 20-min intervals).The mice were trained for 8 rounds at the constant speed from 5 to 40 rpm (5 rpm increases, 20-min intervals).

Morris water maze (MWM) test
Four weeks after HIBD surgery, the MWM test was performed to evaluate spatial cognitive function as described previously [39,44].Prior to spatial training, nontoxic paint was used to darken the pool water (25 ± 1 °C).The animals were permitted to swim freely for 60 or 120 s to adapt to the testing conditions.The spatial learning task was performed in the next 5 days.During spatial learning, a platform was hidden 1 cm under the surface in the target quadrant.The animals were trained to find the hidden platform in four trials per day.Rats and mice that failed to find the hidden platform within 60 and 120 s were directed to stay on the platform for 20 s before being returned to their holding cage.Spatial memory was measured 24 h after the last training trial, and the hidden platform was removed.The Any-maze Tracking System (Stoelting, USA) was used to record and analyze latencies and swimming distances.

Statistical analysis
All data are presented as the mean ± SEM.Escape latency during MWM training and the time spent on the rotarod during the rotarod test were analyzed by Two-way ANOVA.All the other data were analyzed by one-way ANOVA.Statistical significance was set at p < 0.05.

Pharmacological inhibition of caspase-1 reduces the interaction between p97 and GluA1
Our recent study has reported that the ATPase valosincontaining protein (VCP/p97) specifically interacts with GluA1 and regulates GluA1-homo AMPAR formation and trafficking [45].We next wanted to determine the role of p97 in the NLRP3 inflammasome-induced reduction in synaptic GluA1 under OGD conditions.The results showed that OGD had no effect on p97 expression in total cell lysates (OGD-3 h: 90.2 ± 6.9%, p = 0.413 vs. control; OGD-6 h: 103.3 ± 10.7%, p = 0.781 vs. control; OGD-9 h: 101.5 ± 5.8%, p = 0.902 vs. control; OGD-12 h: 107.1 ± 10.5%, p = 0.552 vs. control; OGD-24 h: 114.4 ± 10.5%, p = 0.233 vs. control; n = 5; Fig. 5a).Consistent with our recent report [45], we found that p97 co-immunoprecipitated with GluA1 (n = 3; Fig. 5b), but not GluA2 (Additional file 1: Fig. S2).To further confirm the finding that p97 interacted with GluA1, we used a GluA1 antibody to precipitate p97 and obtained a similar result that GluA1 was able to co-immunoprecipitate with p97 (n = 3; Fig. 5c).More importantly, the interaction between p97 and GluA1 was increased in cultured neurons subjected to OGD, and AC-YVAD-CMK treatment dramatically reduced this interaction (Fig. 5b, c).Collectively, these data suggest that OGD-induced NLRP3 inflammasome activation promotes the formation of the p97 and GluA1 complex.Inhibiting caspase-1 with Fig. 4 Inhibiting caspase-1 prevents the OGD-induced decrease in GluA1 expression in the plasma membrane.a Western blotting was used to detect the expression of cytoplasmic AMPARs in cultured neurons following OGD.b, c The expression of GluA1 (b), but not GluA2 (c), was notably decreased in the total cell lysates of cultured neurons following OGD.d Western blotting was used to detect the expression of AMPARs in the plasma membrane of cultured neurons following OGD.e, f The expression of GluA1 (e) and GluA2 (f) were notably decreased in the plasma membrane of cultured neurons following OGD.g The expression of AMPARs in the plasma membrane of cultured neurons treated with AC-YVAD-CMK (5 µM) 1 h before OGD was detected by Western blotting.h, i The expression of GluA1 (h), but not GluA2 (i), was restored in the plasma membrane of cultured neurons after AC-YVAD-CMK treatment.The data were presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 Fig. 5 Inhibiting caspase-1 promotes the dissociation of p97 from GluA1 in cultured primary neurons subjected to OGD. a The expression of p97 in cultured primary neurons remained unchanged following OGD.b, c Co-IP assays were performed 6 h after OGD in cultured neurons.The interaction between p97 and GluA1 was enhanced in neurons subjected to OGD compared to control neurons.AC-YVAD-CMK led to the dissociation of p97 from GluA1.The data were presented as the mean ± SEM. *p < 0.05, ***p < 0.001 AC-YVAD-CMK may increase GluA1 trafficking from the intracellular reserve pool to the membrane by dissociating p97 from the GluA1 complex.

Pharmacological inhibition of caspase-1 alleviates behavioral deficits in rats with HIBD
A previous study has demonstrated that GluA1-homo AMPARs play critical roles in mediating long-term potentiation (LTP) and cognitive function [46].Our findings suggested that inhibiting caspase-1 could prevent the OGD-induced reduction in GluA1 in the plasma membrane (Fig. 4).Therefore, we conducted three different behavioral tests to evaluate the influence of AC-YVAD-CMK on HI-induced deficits in motor, learning and memory functions in rats with HIBD (Fig. 6a).
Taken together, the present data demonstrate that blocking the NLRP3 inflammasome pathway with the caspase-1 inhibitor AC-YVAD-CMK can prevent HIinduced motor and cognitive dysfunction.
Taken together, these findings demonstrate that blocking the NLRP3 inflammasome pathway via genetic ablation of NLRP3 or caspase-1 can alleviate HI-induced impairments in myodynamia, motor coordination, and spatial learning and memory.

Discussion
Brain injury caused by HI can persist for hours to weeks [49].Mitochondrial energy failure, excitotoxicity, inflammation, calcium overload and oxidative stress are the most extensively studied pathogenic mechanisms in HIBD [3,28].The early stage of cerebral ischemia is characterized by a decrease in cerebral blood flow, thereby reducing intracellular ATP levels and leading to cell membrane depolarization and excessive glutamate release.The overactivation of glutamate receptors eventually results in cell edema and dissolution [28,50].Few treatments can effectively minimize brain injury caused by the initial energy failure that lasts from a few minutes to hours [6,51,52].However, there is a potential damage period that usually occurs 1-6 h after HI that is between the first and second energy failures [52,53], indicating that targeted interventions could minimize excitotoxicity, inflammation, and oxidative stressrelated cell damage [3,53].A growing body of evidence has revealed that innate immunity is triggered a few minutes after HI, leading to the release of inflammatory cytokines including IL-1 and TNF-α by neurons, astrocytes, and microglia [54][55][56][57].Antagonists of the IL-1β receptor can alleviate inflammation-related brain injury caused by HI [58].Furthermore, previous studies have revealed time-dependent activation of the NLRP3 inflammasome pathway in ischemic stroke [18,19].In line with these reports, our results demonstrate that the NLRP3 inflammasome pathway, including caspase-1 and IL-1β, is time-dependently activated under ischemia-like conditions in vivo and in vitro (Figs. 1 and  2).Considering the pathological progression of HIBD, secondary energy failure occurs approximately 6 h after HI [3,52,53].Based on the inverted U-shape increase in the NLRP3 inflammasome proteins (Figs. 1 and 2), we hypothesize that inflammation may play a critical role primarily in the early phase of HIBD rather than in secondary energy failure stage.Since caspase-1 acts as a crucial mediator of the NLRP3 inflammasome pathway, we used a caspase-1 inhibitor to block the NLRP3 inflammasome pathway.Although there are various caspase-1 inhibitors, AC-YVAD-CMK was selected because of its superior specificity compared with other inhibitors such as VX-765 [59][60][61].Here, we reported that pharmacological intervention immediately after HIBD or genetic ablation of caspase-1 improved HI-induced myodynamia, motor coordination, learning, and memory impairment in animal models (Figs. 6 and 8).In addition, the neuroprotective effect of AC-YVAD-CMK was also evident at the histopathological level (Additional file 1: Fig. S3), which was consistent with the functional outcomes.Notably, although inhibiting the NLRP3 inflammasome pathway with AC-YVAD-CMK promoted neurological recovery following HIBD, its potential side effects need to be further explored in future studies.
If interventions fail during the potential damage period, excitotoxicity increases and becomes the dominant factor during secondary energy failure [62][63][64].Clinical evidence has shown that secondary energy failure can result in moderate to severe neonatal HIE, causing a number of major neurological sequelae, such as cerebral palsy [4,5,65].Excitotoxicity appears to play a critical role in the pathological process of HIBD [3,53], and AMPAR regulation has the potential to minimize ischemic brain injury [37].Additionally, the immature brain exhibits higher sensitivity to excitotoxic insults than the mature brain [66,67], highlighting the necessity of exploring the potential mechanisms of AMPARs in HIBD.Previous studies have primarily focused on the roles of GluA2-containing AMPARs in HIBD [35,[68][69][70].However, in the early stage of brain development, many synapses contain GluA2-lacking AMPARs, and the expression of GluA2 subunits gradually increases in rats until 14 days after birth [69,71,72].In contrast to GluA2, the GluA1 subunit is expressed predominantly at higher levels, especially during early brain development [71,73,74], indicating that GluA1 may be more important in HI-induced immature brain damage.Indeed, we found an obvious decrease in the synaptic levels of the GluA1 subunit, but not GluA2 in vivo (Fig. 3).In addition, cultured neurons exhibited low total and membrane GluA1 expression, which was similar to recent reports [75][76][77][78], whereas GluA2 was only decreased in the membrane (Fig. 4).Notably, several previous reports have shown that OGD increases GluA1 expression on the plasma membrane in cultured neurons and brain slices [79,80].In the current study, we selected 7-day cultured neurons to mimic immature brain injury during OGD, while previous studies used neurons cultured 14-17 days before OGD [79][80][81].Therefore, the developmental stage of the subjects and other variations in the OGD conditions may at least partially account for the reported inconsistencies.The reduction in GluA2 in the membrane may be caused by phosphorylation-mediated endocytosis [35,68,82], but the underlying mechanism by which GluA1 levels are reduced still requires further exploration.
It has been widely reported that neuroinflammation impairs brain function, especially learning and memory, by altering membrane expression of AMPARs [37,[83][84][85].However, whether activation of the NLRP3 inflammasome pathway contributes to the reduction in membrane GluA1 is not unclear.In this study, we used AC-YVAD-CMK to inhibit the NLRP3 inflammasome pathway in cultured primary neurons.As expected, AC-YVAD-CMK rescued the HI-induced reduction in GluA1 but not GluA2 in the plasma membrane (Fig. 4).VCP/ p97, which is an ATPase, is widely present in the cytoplasm.Together with a network of cofactors, p97 plays a critical role in various cellular processes that maintain genomic stability and proteostasis [86,87].Importantly, abnormal expression of p97 is associated with many diseases including neurodegenerative diseases, amyotrophic lateral sclerosis and cancers [86,88].Since p97 has been reported to be involved in synaptic plasticity by regulating the formation and trafficking of GluA1-homo AMPARs [45], which is important for learning and memory [43,46], p97 may play an essential role in HI-induced brain dysfunction by regulating the surface expression of the GluA1 subunit.Indeed, our findings demonstrate that the interaction between p97 and GluA1 is up-regulated after OGD (Fig. 5).AC-YVAD-CMK markedly promotes the dissociation of p97 from GluA1, resulting in the release of GluA1 subunits from the intracellular reserve pool and transport to the postsynaptic membrane under ischemia-like conditions (Fig. 5).Notably, we found that caspase-1 was involved in p97-mediated GluA1 subunit trafficking following HIBD, but the underlying mechanism remains unclear.Thus, future studies should focus on elucidating how NLRP3 inflammasome signaling pathway factors, such as caspase-1, affect p97-mediated GluA1 trafficking after HIBD, which may benefit the development of novel HIE therapies.

Conclusions
In summary, our present data suggest that NLRP3 inflammasome pathway activation enhances the interaction between p97 and GluA1, thereby inducing a decrease in surface GluA1.Blocking the NLRP3 inflammasome pathway with the caspase-1 specific inhibitor AC-YVAD-CMK prevents motor and cognitive impairments in HIBD models.These findings establish a scientific foundation for the development of NLRP3 inflammasome pathway inhibitors, such as AC-YVAD-CMK, as potential therapeutic interventions to improve outcomes in HIBD patients.Further studies should explore the underlying mechanisms by which caspase-1 is involved in the formation of the p97-GluA1 complex.In addition, further investigation of the potential side effects and limitations of NLRP3 inflammasome pathway blockers is warranted to provide a basis for validating our findings in a clinical setting and exploring alternative approaches for HIE treatment.

Fig. 1 Fig. 2
Fig.1The NLRP3 inflammasome signaling pathway is activated in rats with HIBD. a The expression of NLRP3 inflammasome proteins in the brain tissues of rats with HIBD was detected by Western blotting.b The expression of NLRP3 showed an inverted U-shape and peaks at 6 h following HIBD.c-f The protein levels of clv-caspase-1 (d) and mature IL-1β (f), but not caspase-1 (c) and pre-IL-1β (e), were significantly increased in rats with HIBD compared with sham rats.The data were presented as the mean ± SEM. *p < 0.05, **p < 0.01

Fig. 6
Fig. 6 Pharmacological inhibition of caspase-1 by AC-YVAD-CMK prevents HIBD-induced behavioral deficits in rats.a The experimental protocols of HIBD establishment and AC-YVAD-CMK (1 mg/kg, i.p.) injections as well as behavioral tests.b AC-YVAD-CMK treatment increased holding power of right forelimb during the grasping test in rats with HIBD.c AC-YVAD-CMK treatment of rats with HIBD increased the time spent on the rod during the rotarod test.d-f AC-YVAD-CMK treatment alleviated HIBD-induced spatial learning and memory deficits in the MWM test.AC-YVAD-CMK treatment of rats with HIBD shortened the latency to find the hidden platform (d) during training, and increased the time spent in the target quadrant (e) and the number of entries into the platform zone (f) during memory retrieval.The data were presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7
Fig. 7 Genetic ablation of NLRP3 prevents HIBD-induced behavioral deficits in mice.a Experimental protocols for model establishment and behavioral tests.b Genetic ablation of NLRP3 (NLRP3 −/− ) improved HIBD-induced impairment of the holding power of the right forelimb during the grasping test.c The time spent on the rod was significantly longer in NLRP3 −/− mice following HIBD than in WT mice during the rotarod test.d-f Genetic ablation of NLRP3 (NLRP3 −/− ) improved HIBD-induced impairment of spatial learning and memory during the MWM test.HIBD shortened the latency of NLRP3 −/− mice to find the hidden platform during spatial learning (d), compared with WT mice.Genetic ablation of NLRP3 (NLRP3 −/− ) increased the time spent in the target quadrant (e) and the number of entries into the platform zone (f).The data were presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 8
Fig. 8 Genetic ablation of caspase-1 prevents HIBD-induced behavioral deficits in mice.a Experimental protocols for model establishment and behavioral tests.b Genetic ablation of caspase-1 (caspase-1 −/− ) improved HIBD-induced impairment of the holding power of the right forelimb during the grasping test.c The time spent on the rod was significantly longer in caspase-1 −/− mice following HIBD than in WT mice during the rotarod test.d-f Genetic ablation of caspase-1 (caspase-1 −/− ) improved HIBD-induced impairments in spatial learning and memory during the MWM test.In caspase-1 −/− mice, HIBD shortened the latency to find the hidden platform during spatial learning (d) compared with WT mice.Genetic ablation of caspase-1 (caspase-1 −/− ) increased the time spent in the target quadrant (e) and the number of entries into the platform zone (f).The data were presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001