The concept of pyroptosis was first proposed by Cookson and Brennan in 2001 (11). The authors described pyroptosis as a caspase-1-mediated, proinflammatory form of programmed cell death observed in Salmonella typhimurium-infected macrophages. This process is characterized by membrane rupture and cellular swelling, which facilitates the release of intracellular contents, leading to a marked inflammatory response. Morphologically and mechanistically, pyroptosis is distinct from apoptosis and necrosis (12). While apoptosis is characterized by cell shrinkage and non-inflammatory clearance, and necrosis is generally an uncontrolled lytic process, pyroptosis is a form of programmed lytic death defined by cellular swelling, membrane rupture and the robust release of pro-inflammatory cytokines. The mechanistic foundation of pyroptosis dates back to 1992, where Zychlinsky et al (13) reported an unconventional form of caspase-1-dependent cell death in a Shigella infection model; these findings established the groundwork for subsequent studies.

Further investigations revealed that pyroptosis is not limited to the caspase-1 pathway. In mice, caspase-11 can directly bind lipopolysaccharide (LPS) to induce pyroptosis. The human homologs, caspase-4 and caspase-5, function similarly, collectively constituting the non-canonical pathway (14). In 2015, Shi et al (15) and Kayagaki et al (16) independently identified GSDMD as the key substrate of inflammatory caspases. Upon cleavage, the N-terminal domain (NTD) of GSDMD inserts into the plasma membrane; this process forms large non-selective pores that drive cellular content release and robust inflammatory responses. This breakthrough established the core execution mechanism of pyroptosis and represented a major advancement in the field (15,16).

Subsequent research has elucidated the crosstalk between pyroptosis and other cell death pathways. In 2017, Wang et al (17) demonstrated that caspase-3 can cleave GSDME, thereby converting apoptosis into pyroptosis. As summarized in the 2018 recommendations of the Nomenclature Committee on Cell Death, pyroptosis, which can also be initiated by caspase-8 via GSDMD cleavage, was redefined as a GSDM-dependent form of programmed cell death (18). This pathway is typically triggered by the activation of inflammatory caspases. The GSDM family comprises GSDMA, GSDMB, GSDMC, GSDMD, GSDME and GSDMF; among these members, GSDMD is the most extensively characterized (19).

The canonical caspase-1-dependent pyroptotic pathway is driven by the activation of inflammasomes. Inflammasomes are cytosolic multiprotein complexes, which are composed of pattern recognition receptors (PRRs), the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and pro-caspase-1. These complexes detect pathogen-associated molecular patterns and host-derived danger-associated molecular patterns (DAMPs) (20). Common PRRs include members of the NOD-like receptor (NLR) family, such as NLRP1, NLRP3 and NLRC4, and other sensors include AIM2-like receptors and pyrin. Among these, the NLRP3 inflammasome is the most extensively characterized (21). NLRP3 activation typically follows a two-step model: First, a priming signal involving microbial or endogenous factors activates NF-κB signaling to upregulate NLRP3 and pro-IL-1β; second, an activation signal is triggered by cellular events, such as potassium efflux, mitochondrial dysfunction or lysosomal rupture (22). Upon activation, NLRP3 recruits ASC and pro-caspase-1 to orchestrate inflammasome assembly. Pro-caspase-1 then undergoes autocatalytic cleavage to generate active caspase-1 (23), which subsequently cleaves pro-IL-1β and pro-IL-18 into their mature forms. These cytokines are then released through membrane pores to propagate the inflammatory response. Simultaneously, caspase-1 cleaves GSDMD to release its NTD. This domain oligomerizes within the plasma membrane to form pores, thereby disrupting osmotic balance and ultimately precipitating cell rupture and pyroptosis (24).

The non-canonical pyroptotic pathway operates independently of canonical inflammasomes (25). In humans, this pathway is mediated by caspase-4 and caspase-5, whereas caspase-11 serves as the mediator in mice. In this pathway, LPS directly binds the caspase recruitment domain (CARD) of caspase-4/5/11. This binding event leads to their activation, and activated caspases subsequently cleave GSDMD. This cleavage releases the pore-forming NTD, which executes pyroptosis (26). Notably, caspase-4/5/11 do not directly process pro-IL-1β or pro-IL-18, but instead, GSDMD-mediated pore formation promotes potassium efflux. This ion shift secondarily activates the NLRP3 inflammasome and caspase-1, thereby driving the maturation and secretion of IL-1β and IL-18 (27).

Caspase-3 and caspase-8 are traditionally regarded as key executioners of apoptosis; however, recent evidence implicates them in pyroptosis via the cleavage of specific GSDM family members (28). In cells with high GSDME expression, caspase-3 cleaves the GSDME linker region, which releases the pore-forming NTD and shifts the mode of death from apoptosis to pyroptosis (29). Notably, GSDME expression levels dictate the cell death outcome, where high expression favors caspase-3-mediated pyroptosis and low expression favors apoptosis (30). Additionally, during Yersinia infection, the effector YopJ inhibits TAK1 signaling, which subsequently activates caspase-8. Activated caspase-8 then cleaves both GSDMD and GSDME. This process liberates their NTDs to form membrane pores and trigger pyroptosis (31). This pathway demonstrates that caspase-8 can initiate pyroptosis independently of canonical inflammasomes.

Gzms are the key effector molecules of cytotoxic T lymphocytes and natural killer cells. They enter target cells via perforin-mediated pores to induce cell death (32). Studies have elucidated their direct role in pyroptosis regulation; GzmB can induce pyroptosis indirectly through caspase-3 activation. Furthermore, it directly cleaves GSDME at the same site to release the pore-forming NTD and drive cell lysis (33). In addition, GzmA was found to specifically cleave GSDMB at Lys229/Lys244. This cleavage liberates the NTD to form pores, establishing a caspase-independent pyroptotic pathway. Notably, GSDMB is constitutively expressed in epithelial cells and certain brain tissues, and its expression is markedly upregulated within inflammatory microenvironments (34). These findings provide new mechanistic insights and a theoretical basis for targeting pyroptosis in cerebral IR injury. Fig. 1 illustrates the key molecules and pathways involved in pyroptosis.

Moreover, the mitochondrial-GSDMD axis functions as a pivotal amplifier of neuroinflammation during cerebral IR (35). Specifically, the N-terminal fragment of GSDMD (GSDMD-N) facilitates mitochondrial membrane permeabilization, triggering the release of mitochondrial DNA (mtDNA) into the cytosol (36). This translocation subsequently engages the cyclin GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, markedly exacerbating secondary neuroinjury and inflammatory cascades following ROSC.

To ensure scientific rigor and distinguish between pro-inflammatory signaling and terminal cell lysis, the following three-stage experimental validation framework is proposed.

Focuses on transcriptional readiness with key readouts of NF-κB nuclear translocation; mRNA and protein induction of NLRP3 and pro-IL-1β.

Focuses on proteolytic processing with key readouts: ASC speck formation; detection of cleaved caspase-1 (p20); and quantification of mature IL-1β/IL-18 secretion.

Focuses on membrane perforation and cytolysis with key readouts; Identification of GSDMD-N or GSDME-N fragments; LDH release assays; and propidium iodide or ethidium homodimer staining.

While Stage 2 markers indicate an active inflammatory response, terminal pyroptosis must be confirmed by Stage 3 markers (GSDMD cleavage and membrane rupture), which represent the definitive hallmarks of lytic cell death.

Neurons are the most vulnerable cell type during cerebral IR injury. Their heightened sensitivity to ischemia-hypoxia largely determines the severity of neurological deficits (37). Pyroptosis has emerged as a distinct form of inflammatory programmed cell death, which amplifies neuroinflammation through inflammasome activation and cytokine release; this mechanism has been validated across multiple neurological disorders, including Alzheimer's disease, Parkinson's disease and traumatic brain injury (38). At the molecular level, NLRP3 inflammasome activation triggers caspase-1 activation and GSDMD cleavage. These events lead to pore formation, neuronal swelling and lysis. Such findings are well-demonstrated in both cerebral IR injury and OGD/R models (39). Moreover, genetic deletion or pharmacological inhibition of NLRP3, caspase-1 or GSDMD markedly reduces neuronal death and attenuates inflammation. These interventions also improve motor and behavioral outcomes, supporting pyroptosis as a potential neuroprotective target (40). Notably, neuronal pyroptosis is not restricted to ischemic injury; it has also been implicated in Parkinson's disease and Alzheimer's disease (AD), suggesting it may represent a common neuronal response to diverse insults (41). In summary, elucidating the molecular mechanisms of neuronal pyroptosis in cerebral IR injury deepens the understanding of pathology. It also provides novel avenues for neuroprotective intervention (42).

Astrocytes are essential for maintaining ion homeostasis, neurotransmitter metabolism and BBB integrity. They provide notable neuronal support in the central nervous system. Consequently, astrocyte dysfunction or death is linked to multiple neurological disorders (43). During cerebral IR injury, astrocytic pyroptosis occurs via two primary pathways. These include inflammasome-mediated caspase-1-dependent GSDMD cleavage and caspase-4/5/11 signaling. Overexpression of CD73 inhibits astrocytic pyroptosis via the adenosine A2B receptor/NF-κB signaling axis; this modulation effectively mitigates brain injury (44). Following pyroptosis, astrocytes release pro-inflammatory cytokines such as IL-1β and IL-18. This release exacerbates local inflammation and neuronal damage. In AD brains, astrocytic and neuronal pyroptosis have been linked to amyloid-β deposition and neurodegeneration (45). Specifically, amyloid-β aggregates act as DAMPs that trigger NLRP3 inflammasome activation; in turn, the resulting pyroptosis and chronic neuroinflammation can further promote amyloid-β production and aggregation, creating a feed-forward pathological loop. Furthermore, astrocytic inflammasome activation induces cell death and disrupts hippocampal synaptic plasticity. It also impairs glutamate homeostasis through IL-1 and IL-18 signaling, thereby aggravating excitotoxicity (46). Astrocytic pyroptosis is associated with early neurological deficits following brain injury. Conversely, TNF-stimulated gene-6 alleviates this process by suppressing NLRC4 inflammasome activation. This reduction in pyroptosis alleviates brain edema and improves neurological function (47). Astrocytic pyroptosis is broadly involved in diverse neurological diseases; therefore, targeting inflammasome, caspase or GSDM pathways offers therapeutic promise. However, challenges remain in achieving selectivity and clinical translation (48).

Microglia are the primary immune effector cells of the central nervous system, where they activate rapidly in response to cerebral IR injury. In this context, microglia mediate inflammation and exacerbate neuronal damage. Furthermore, their activation can impede long-term recovery (49). Beyond conventional immune activation, microglia can undergo pyroptosis. This process amplifies neuroinflammation through pore formation and cytokine release. The interaction between interferon-inducible protein 204 and SUMO-specific protease 7 activates STING signaling. This pathway induces both pyroptosis and mitochondrial dysfunction. Notably, silencing these factors alleviates neurological deficits (50). Mechanistically, microglial pyroptosis is largely dependent on the NLRP3/caspase-1/GSDMD axis, and ASC specks further amplify NLRP3 activity to promote pyroptosis. This process accelerates α-synuclein aggregation and neuronal degeneration, suggesting relevance to diverse neurodegenerative diseases (51). The pyroptotic release of cytosolic contents and inflammatory mediators exacerbates neuroinflammation and secondary injury. cGAS-STING activation drives NLRP3-mediated microglial pyroptosis, which exacerbates pathology and motor deficits. Conversely, pharmacological or genetic inhibition of this axis markedly alleviates tissue damage (52). In cerebral IR models, delayed administration [24 h post-transient middle cerebral artery occlusion (tMCAO)] of the selective NLRP3 inhibitor MCC950 still reduces infarct size and improves neurological outcomes. Although MCC950 is not cell-type specific, its neuroprotective effects in cerebral IR models are largely characterized by the suppression of microglial-mediated neuroinflammation and cytokine release (53). In summary, microglial pyroptosis represents a notable driver of cerebral IR injury and a promising therapeutic target; however, its complex regulation and clinical feasibility warrant further investigation (54).

Disruption of the BBB is a central pathological event in cerebral IR injury; this breach further exacerbates neuronal damage (55). As an inflammasome-dependent death pathway, pyroptosis aggravates BBB permeability via GSDM-mediated pore formation (56). The canonical NLRP3 inflammasome pathway is a validated driver of IR pathology; caspase-1 activation and IL-1β release promote inflammatory signaling. These events directly injure endothelial cells and compromise BBB integrity (57). Simultaneously, the non-canonical pathway contributes to this dysfunction; caspase-11-mediated GSDMD activation induces endothelial rupture and the release of pro-inflammatory cytokines. This process spreads inflammation and exacerbates BBB impairment (58), and oxidative stress further amplifies BBB disruption during pyroptosis. It induces lipid peroxidation and mitochondrial dysfunction, which enhance inflammatory signaling and membrane injury (59). Astrocytes also actively participate in this process; astrocytic secretion of CXCL10 promotes endothelial pyroptosis via the CXCR3/cGAS/AIM2 pathway. This mechanism leads to BBB breakdown, intensifying neuroinflammation and aggravating brain injury (60). Together, these findings demonstrate that pyroptosis contributes to BBB disruption through multiple mechanisms. These insights highlight pyroptosis as a notable therapeutic target in cerebral IR injury.

Cellular heterogeneity within the neurovascular unit. A key distinction exists within the neurovascular unit, while neurons are the primary victims of terminal lytic death, microglia and astrocytes function as ‘inflammatory amplifiers’. This glial activation sustains the neuroimmune response and exacerbates BBB instability, creating a feed-forward cycle of secondary neuroinjury.

Future research should shift toward the development of cell-specific modulators. The ultimate aim is to selectively inhibit neuronal loss without compromising the essential trophic and phagocytic support provided by glial cells during the recovery phase.

Oxidative stress is a key driver of cell death and tissue damage in cerebral IR injury. Together with mitochondrial dysfunction and excitotoxicity, it forms the pathological basis of injury in cerebral IR (61). In cerebral IR models, reperfusion is characterized by a sharp rise in reactive oxygen species (ROS) levels. These levels positively associate with infarct volume and neurological deficits. Notably, inhibition of NADPH oxidase 2 effectively reduces ROS production and tissue injury (62). Mechanistically, ROS promote NLRP3 inflammasome assembly by inducing the dissociation of thioredoxin-interacting protein from thioredoxin, which subsequently binds to the leucine-rich repeat domain of NLRP3 to trigger its activation, and caspase-1 activation; this cascade leads to GSDMD cleavage and pyroptosis (63). Conversely, ROS scavengers or inflammasome inhibitors can partially reverse this injury. Beyond direct effects, oxidative stress indirectly facilitates inflammasome activation via mitochondrial dysfunction and calcium dysregulation. These interactions form complex signaling networks that represent potential therapeutic targets (64). In animal IR models, nanomaterial-based antioxidant delivery effectively scavenges ROS. This intervention suppresses the NLRP3/caspase-1/GSDMD pathway, reducing neuronal pyroptosis and alleviating brain damage (65). These findings support antioxidant interventions as viable neuroprotective strategies. Collectively, oxidative stress drives neuronal pyroptosis via both direct and indirect mechanisms (66).

Calcium homeostasis is essential for neuronal survival and function. Its disruption triggers cell death via mechanisms such as endoplasmic reticulum stress and mitochondrial dysfunction (67). In cerebral IR injury, excessive glutamate receptor activation and abnormal calcium channel opening contribute to Ca²⁺ overload; the reversal of Na⁺/Ca²⁺ exchange also plays a role. These events collectively drive excitotoxicity and neuronal injury (68). Calcium overload activates the calcium-dependent cysteine protease calpain, which subsequently liberates caspase-1 from the cytoskeleton to promote its activation. This process leads to GSDMD cleavage and the formation of pore-forming N-terminal fragments that drive canonical pyroptosis (69). Aberrant activation of the endoplasmic reticulum Ca²⁺ channel inositol 1,4,5-triphosphate receptor 1 has been shown to enhance Ca²⁺ efflux and induce pyroptosis via the NLRP3/caspase-1 pathway (70). In cerebral IR models, xanthine has been identified as a key metabolite; it triggers endothelial Ca²⁺ overload and induces GSDME-dependent pyroptosis, thereby exacerbating BBB disruption and brain injury (71). Thus, calcium dyshomeostasis acts as both a potent driver of pyroptosis and a central pathological mechanism of IR injury. Elucidating the cross-talk between Ca²⁺ imbalance and pyroptosis will provide opportunities for identifying novel therapeutic targets (72).

Mitochondrial dysfunction during cerebral IR leads to marked energy failure, and also serves as an inflammatory signaling hub that drives pyroptosis. In this context, the NLRP3 inflammasome acts as a notable bridge in stroke-associated inflammation (73). Mitochondria-derived danger signals include excessive mitochondrial ROS, oxidized mtDNA leakage and abnormal mitochondrial permeability transition pore opening. These signals induce or amplify NLRP3 inflammasome activation to promote pyroptotic cascades (74). Previous evidence demonstrates that GSDMD forms pores in both the plasma and mitochondrial membranes; this process facilitates mtDNA release and activates the cGAS-STING pathway. This mechanism establishes a positive feedback loop between inflammation and pyroptosis, thereby exacerbating tissue damage (75). In cerebral IR models, hyperactivation of dynamin-related protein 1 (Drp1) induces mitochondrial dysfunction and GSDMD-mediated pore formation. This aggravates neuronal pyroptosis and tissue injury. Conversely, Apelin receptor early endogenous ligand mitigates this process by suppressing Drp1 hyperactivation and NLRP3 signaling (76). Furthermore, enhancing PTEN-induced putative kinase 1 (PINK1)/Parkin-dependent mitophagy markedly inhibits pyroptosis. This suggests that the coupling of mitochondrial quality control and pyroptosis represents a conserved regulatory axis (77). Overall, targeting mitochondrial function effectively suppresses inflammation and pyroptosis, underscoring its central role in the pathophysiology of cerebral IR injury (78).

GSDMD-mediated feed-forward loops. Evidence identifies mitochondrial GSDMD pores as a critical amplifier. These pores allow mtDNA leakage, which directly amplifies intracellular inflammatory signaling beyond the initial insult.

Identifying the ‘point of no return’. Mapping the precise spatiotemporal sequence of these drivers is vital. Determining the transition from reversible stress to irreversible lysis will help identify the optimal therapeutic window for interventions.

Neuroimmune crosstalk and regeneration: Beyond localized cell damage, pyroptotic DAMPs (such as HMGB1) facilitate T-cell infiltration into the brain parenchyma. This sustained neuroimmune crosstalk may create a hostile microenvironment that markedly hinders long-term axonal regeneration.

Validation of biofluid biomarkers: A primary goal for clinical translation is the systematic validation of biofluid markers, specifically GSDMD-N and IL-18. Establishing the prognostic value of these markers in serum or cerebrospinal fluid is essential for the real-time monitoring of patients following cardiac arrest.

In cerebral IR injury, pyroptosis and ferroptosis are primary pathological forms of programmed cell death. These pathways independently regulate neuronal damage but also interact synergistically, and this crosstalk further exacerbates tissue injury (92). Mechanistically, iron overload and lipid peroxidation directly activate the NLRP3 inflammasome. This activation triggers caspase-1-mediated GSDMD cleavage and pyroptosis (93). Conversely, GSDMD pore formation facilitates Ca²⁺ influx and ROS accumulation. These events amplify lipid peroxidation and enhance ferroptotic susceptibility, thereby establishing a vicious cycle (94). In cerebral IR models, inhibiting ferroptosis suppresses lipid peroxidation via the SLC7A11/GPX4/ACSL4 axis. This inhibition also downregulates the caspase-1/GSDMD pathway, thereby attenuating pyroptosis and improving neurological outcomes (95). Moreover, NLRP3-deficient mice exhibit smaller infarct volumes and improved neurological scores. These benefits are attributed to both pyroptosis suppression and GPX4 upregulation. Reduced iron deposition and diminished lipid peroxidation collectively mitigate ferroptosis in these models (96). Autophagy, particularly ferritinophagy, has emerged as a notable node linking ferroptosis and pyroptosis by integrating iron homeostasis and inflammasome activity (97). These insights suggest that combined targeting of ferroptosis and pyroptosis may provide a promising therapeutic strategy for cerebral IR injury.

Autophagy is an essential degradative pathway which maintains cellular homeostasis by clearing misfolded proteins and damaged organelles to prevent the accumulation of cytotoxic factors (98). In cerebral IR injury, autophagy exerts a dual effect on pyroptosis. Moderate autophagy activation alleviates mitochondrial dysfunction and oxidative stress; this suppresses excessive inflammasome activation, thereby reducing pyroptosis and conferring neuroprotection (99). For instance, an Astragalus-Angelica herbal formulation activates autophagy via the AMPK/mTOR pathway. This activation suppresses NLRP3-dependent pyroptosis and attenuates cerebral IR injury (100). By contrast, excessive or sustained autophagy can promote GSDMD cleavage and pro-inflammatory cytokine release, which amplifies pyroptosis (101). In such settings, autophagy inhibition mitigates inflammasome-mediated damage and improves neurological function. Thus, autophagy exerts context-dependent effects, acting as either protective or detrimental. This paradox highlights its complexity as a therapeutic target. It underscores the need to dissect the spatiotemporal dynamics of autophagy-pyroptosis crosstalk across diverse cell types and disease stages.

Apoptosis and pyroptosis frequently coexist and interact during cerebral IR injury, and heir molecular signatures are defined by distinct caspase family members (102). Specifically, caspase-3 and caspase-9 drive classical apoptosis, whereas caspase-1, −4/5 and −11 mediate pyroptosis through GSDMD or GSDME cleavage. Notably, caspase-3-mediated cleavage of GSDME can convert apoptosis into secondary pyroptosis, a lytic transition where cells initially undergoing programmed apoptosis switch to a pro-inflammatory phenotype due to GSDME-mediated membrane pore formation (103). Caspase-8 exhibits dual functions; it triggers apoptosis through canonical pathways while also directly cleaving GSDMD or promoting inflammasome activation. This signaling bifurcation has been validated in TNF-α-induced cell death and infection responses (104). Mitochondrial pathways further integrate apoptosis and pyroptosis; BAX/BAK-mediated mitochondrial outer membrane permeabilization induces Cytochrome c release to drive apoptosis. Simultaneously, this process releases mtDNA to activate the NLRP3 inflammasome and promote pyroptosis (105). Regulatory molecules such as cellular FLICE-like inhibitory protein (c-FLIP), inhibitors of apoptosis proteins and receptor-interacting serine/threonine-protein kinase 1 (RIPK1) act as pivotal nodes in this crosstalk. For example, c-FLIPL deficiency facilitates complex II formation and promotes LPS-induced pyroptosis; this underscores its role in dual-pathway regulation (106). Based on these insights, pharmacological inhibition of caspases or GSDMs has been proposed to attenuate both apoptosis- and pyroptosis-related injury. However, efficacy remains context-dependent, warranting further refinement using multi-omics approaches (107).

Both pyroptosis and necroptosis are pro-inflammatory death pathways that often coexist in cerebral IR injury. These processes are tightly interconnected through the PANoptosome complex (108). Key molecular convergence points include RIPK1, RIPK3, MLKL, caspase-8 and GSDMD, which collectively determine cell fate. RIPK1 and RIPK3 activate MLKL to initiate necroptosis; simultaneously, caspase-8 cleaves GSDMD to induce pyroptosis (109). MLKL-mediated K⁺ efflux has been shown to activate the NLRP3 inflammasome, which induces GSDMD pore formation and couples necroptosis with pyroptosis (110). Caspase-8 further promotes IL-1β maturation, linking the two pathways in inflammatory cell death. The emerging concept of PANoptosis (pyroptosis, apoptosis, and necroptosis) emphasizes that these pathways are co-regulated within the PANoptosome complex (111). Consequently, the inhibition of a single pathway may be compensated by alternative mechanisms. In cerebral IR models, pyroptosis and necroptosis jointly exacerbate sterile inflammation and neuronal injury. Conversely, neural stem cell transplantation mitigates these effects by downregulating GSDMD and MLKL expression. This intervention ultimately improves functional recovery (112,113). These findings highlight the therapeutic potential of simultaneously targeting pyroptotic and necroptotic pathways.

Autophagy as a cellular rheostat: Autophagy acts as a complex rheostat in the context of ischemic injury. While physiological autophagy confers neuroprotection by degrading inflammasomes, excessive ‘autophagic stress’ can facilitate GSDMD cleavage and accelerate lytic cell death.

Toward multi-node therapeutic inhibition: Future therapies should transition toward ‘multi-node’ inhibition strategies. Targeting the shared molecular hubs of the PANoptosome may offer improved neuroprotection compared with inhibiting a single death pathway alone, particularly given the compensatory mechanisms inherent in programmed cell death.

The NLRP3 inflammasome is a central sensor in the pyroptotic pathway. It recognizes intracellular danger signals to mediate downstream caspase-1 activation and GSDMD cleavage. These events ultimately lead to plasma membrane pore formation and pro-inflammatory cytokine release (114). In a mouse cardiac arrest/cardiopulmonary resuscitation (CA/CPR) model, selective inhibition of NLRP3 using MCC950 administered post-ischemia notably reduces NLRP3/caspase-1 activation to improve survival and neurological deficit scores (115). Similarly, in a mouse model of tMCAO, β-caryophyllene administered post-ischemia alleviates white matter injury, and improves motor and cognitive functions by suppressing NLRP3-mediated pyroptosis (116). In a rat CA/CPR model, chrysophanol administered post-ischemia also mitigates cerebral IR injury via the inhibition of the TRAF6/NLRP3 signaling pathway (117). In a mouse tMCAO model, pre-ischemic inhibition of the lipocalin-2/24p3R/NLRP3 axis attenuates astrocyte pyroptosis and neuroinflammation by reducing GSDMD-N and IL-1β levels (118). Collectively, these findings establish NLRP3 as a pivotal mediator of ischemic brain injury; however, its activation mechanisms remain complex, with oxidative stress serving as a primary driver.

Oxidative stress is a primary driver of cerebral IR injury, and ROS are recognized as major triggers for NLRP3 activation (119). In a mouse distal middle cerebral artery occlusion model, administration of hydrogen-rich saline, a potent ROS scavenger, both pre- and post-ischemia effectively suppresses the ROS/NLRP3/caspase-1 pathway and reduces IL-1β levels, thereby improving neurological deficit scores (120). Additionally, in a mouse tMCAO model, OIP5 Antisense RNA 1 administered as a single dose post-ischemia promotes the degradation of thioredoxin-interacting protein, which inhibits oxidative stress-induced neuronal pyroptosis and alleviates cerebral IR injury (121). Likewise, methyl isoeugenol administered post-ischemia in a rat tMCAO model activates the nuclear factor erythroid 2-related factor 2 (Nrf2)/NQO1/HO-1 antioxidant pathway, which suppresses ROS-driven microglial pyroptosis and attenuates neuroinflammation (122). These findings underscore oxidative stress modulation as an important therapeutic strategy.

Calcium overload and mitochondrial dysfunction also trigger aberrant NLRP3 activation (123). Dexmedetomidine reduces microglial pyroptosis and neural injury in a rat permanent middle cerebral artery occlusion (pMCAO) model when administered via post-ischemic continuous infusion, a process mediated by the inhibition of the P2X7R/NLRP3/caspase-1 pathway (124). By suppressing Drp1-mediated mitochondrial fission, a single post-ischemic dose of FK866 in a rat CA/CPR model ameliorates mitochondrial dysfunction, thereby reducing both pyroptosis and neuroinflammation (125). Single post-ischemic administration of Apelin-13-loaded macrophage membrane-encapsulated nanoparticles in a mouse tMCAO model attenuates pyroptosis by enhancing Sirtuin 3 activity and suppressing NLRP3 assembly (126). These approaches highlight novel strategies targeting calcium overload and mitochondrial dysfunction.

Inflammatory propagation represents another notable pathological process mediated by NLRP3-dependent pyroptosis (127). Pre-ischemic administration of indobufen and aspirin, alone or in combination with clopidogrel or ticagrelor, attenuate pyroptosis in a rat tMCAO model by inhibiting NF-κB/NLRP3 signaling (128). Knockdown of maternally expressed gene 3 administered as a single pre-ischemic dose in a rat tMCAO model inhibits the microRNA (miR)-145-5p/TLR4/NLRP3 axis, thereby suppressing pyroptosis and conferring neuroprotection (129). In a rat tMCAO model, a single pre-ischemic dose of calycosin reduces pyroptosis by suppressing the HMGB1/NLRP3 signaling axis (130). Post-ischemic electroacupuncture (EA) at the ST36 acupoint in a rat CA/CPR model alleviates neuronal injury and inflammation by activating the α-7 nicotinic acetylcholine receptor, which effectively suppresses microglial pyroptosis (131). Furthermore, in a mouse tMCAO model, a single post-ischemic dose of the STING inhibitor C-176 reduces ischemic brain injury by disrupting the STING-NLRP3 interaction and suppressing microglial pyroptosis (132). Taken together, these findings confirm the central role of NLRP3 in pyroptosis induction and inflammatory amplification, underscoring its promise as a translatable therapeutic target.

Caspase-1 is a notable component of the inflammasome complex, which is responsible for the maturation of IL-1β and IL-18 and the initiation of pyroptosis (133). In a rat pMCAO model, post-ischemic treatment with the caspase-1 inhibitor VX-765 ameliorates BBB disruption and neuroinflammation by inhibiting caspase-1 and reducing pyroptosis, which ultimately leads to improved neurological outcomes (134). AIM2 inflammasome-driven caspase-1 activation exacerbates brain injury and cognitive impairment. Conversely, pre-ischemic treatment with the caspase-1 inhibitor Ac-YVAD-CMK in a mouse tMCAO model improves cognitive function by suppressing caspase-1-mediated pyroptosis (135). Additionally, miR-96-5p reduces cerebral IR injury in a mouse tMCAO model when administered as a single dose pre-ischemia primarily by downregulating caspase-1 and suppressing pyroptosis (136). Thus, targeting caspase-1 is a promising strategy to mitigate IR-induced pyroptosis and neural injury.

GSDMD is the primary executioner of pyroptosis, and cleavage of GSDMD liberates its NTD. This domain subsequently forms pores in the plasma membrane, ultimately leading to cell rupture (137). Administered post-ischemia in a mouse tMCAO model, the GSDMD inhibitor disulfiram reduces neutrophil infiltration and infarct size in cerebral IR injury (91). Similarly, in a mouse tMCAO model, genetic ablation (knockout) of GSDMD decreases microglial pyroptosis in cerebral IR, a reduction that markedly improves neurological function (138). Therefore, targeting the caspase-1/GSDMD axis enables multi-level intervention. This approach spans from inflammatory sensing to cellular rupture, providing a systematic framework for modulating pyroptosis (Table I) (91,115–118,120–122,124–126,128–132,134–136,138).

Beyond canonical pathways, modulating cell-specific functional states and signaling networks represents a promising therapeutic avenue. Microglia are the primary resident immune cells of the brain where they play central roles in cerebral IR injury, and their polarization state (M1 vs. M2) dictates the extent of neuronal damage (139). Intermittent θ-burst stimulation (iTBS), applied twice daily for 7 days post-ischemia in a mouse tMCAO model, reduces neuronal pyroptosis by modulating microglial M1/M2 polarization (140). Via activation of the HACE1/Nrf2 axis, ETS variant transcription factor 5 administered as a single pre-ischemic dose in a rat tMCAO model inhibits microglial M1 polarization and attenuates brain injury (141). Salvianolic acids for injection administered daily post-ischemia in a rat tMCAO model exert neuroprotective effects by promoting microglial M1-to-M2 polarization and inhibiting the NLRP3 inflammasome (142). Autophagy is a marked cellular self-clearance mechanism, and its role in pyroptosis is complex and often described as a double-edged sword. Both excessive and insufficient autophagic activity can exacerbate injury (143). When administered as a single post-ischemic dose in a rat tMCAO model, dexmedetomidine enhances mitochondrial autophagy via the PINK1/Parkin pathway, thereby reducing pyroptosis and protecting cerebral cells (144). Pre-ischemic combination therapy involving EA and tigustilide in a rat tMCAO model activates the PI3K/AKT/mTOR pathway, which inhibits autophagy and subsequently reduces pyroptosis (145). These studies suggest that regulating microglial activation and balancing autophagy could provide new strategies for treating cerebral IR injury.

Non-coding RNAs (ncRNAs) are established as marked regulators of pyroptosis (146). For example, targeting the DEAD-box RNA helicase 3X/NLRP3 pathway via a single pre-ischemic dose of miR-135a-5p inhibits pyroptosis in a mouse tMCAO model (147). In a mouse tMCAO model, a single dose of circular RNA Fndc3b administered 4 weeks pre-ischemia suppresses microglial/macrophage pyroptosis via the enolase 1/Kruppel-like factor 2 axis, thereby promoting neurological recovery (148). Similarly, in a diabetic mouse tMCAO model, knockdown of the long ncRNA Fendrr via adeno-associated virus administration 3 weeks pre-ischemia reduces microglial pyroptosis by inhibiting NLRC4 ubiquitination, thereby protecting against cerebral IR injury (149). These findings highlight ncRNAs as potential therapeutic modulators of pyroptosis and neuroprotection.

As advanced drug delivery platforms, exosomes offer promising strategies for the treatment of cerebral IR injury (150). Post-ischemic treatment with astrocyte-derived exosomal miR-378a-5p suppresses NLRP3 activation and alleviates neuronal pyroptosis in a rat tMCAO model (151). Furthermore, a single post-ischemic dose of bone marrow mesenchymal stem cell-derived exosomes in a rat tMCAO model attenuate brain injury by modulating microglial M1/M2 polarization and suppressing NLRP3 inflammasome activation (152). Notably, transferrin receptor-targeted nanomicelles delivering MCC950, administered as a single post-ischemic dose in a Wistar rat model of tMCAO, effectively inhibit NLRP3 activation and ameliorate cerebral IR injury (153). A cascade-type microglial pyroptosis inhibitor was developed to integrate nanotechnology with antioxidative activity; a single post-ischemic administration of this system in a rat tMCAO model regulates the ROS/NLRP3/pyroptosis axis to mitigate neuroinflammation (154). Table II summarizes these emerging biological regulators and delivery platforms, including iTBS, transcription factors, ncRNAs and exosome/nanotechnology-based systems, across various ischemia models (140–142,144,145,147–149,151–154). These studies underscore the therapeutic potential of exosome- and nanotechnology-based interventions in cerebral IR injury.

The success of clinical translation hinges on BBB permeability, optimal administration routes and precise dosing. Small-molecule inhibitors, characterized by their inherent lipophilicity, possess a distinct advantage in the acute CA/ROSC setting due to rapid BBB penetration (155). While nanotechnology and exosome-based platforms offer superior targeting specificity, they must navigate challenges regarding off-target toxicity and potential immunosuppressive risks (156). Biologics, conversely, are often constrained by their large molecular weight, making BBB crossing a notable hurdle (157). Given the narrow therapeutic window in CA/ROSC, optimizing delivery protocols during the hyper-acute phase is imperative to enhance overall pharmacological feasibility.