Research progress on pyroptosis-mediated immune-inflammatory response in ischemic stroke and the role of natural plant components as regulator of pyroptosis: A review

This suggests that breviscapine may significantly improve the cognitive function of CCI rats and reduce the pathological damage of ischemic neurons, and its mechanism may be related to the inhibition of NLRP3 inflammasome activation and pyroptosis pathway in brain tissue [211].


  • This review summarized the latest molecular mechanism of pyroptosis on ischemic stroke.
  • This review summarizes the role of natural plant components as regulator of pyroptosis in ischemic stroke
  • Regulation of pyroptosis in ischemic stroke is proposed as a potential therapeutic strategy.


Ischemic stroke (IS) is one of the leading causes of death and disability. Its pathogenesis is not completely clear, and inflammatory cascade is one of its main pathological processes. The current clinical practice of IS is to restore the blood supply to the ischemic area after IS as soon as possible through thrombolytic therapy to protect the vitality and function of neurons. However, blood reperfusion further accelerates ischemic damage and cause ischemia-reperfusion injury. The pathological process of cerebral ischemia-reperfusion injury involves multiple mechanisms, and the exact mechanism has not been fully elucidated. Pyroptosis, a newly discovered form of inflammatory programmed cell death, plays an important role in the initiation and progression of inflammation. It is a pro-inflammatory programmed death mediated by caspase Caspase-1/4/5/11, which can lead to cell swelling and rupture, release inflammatory factors IL-1β and IL-18, and induce an inflammatory cascade. Recent studies have shown that pyroptosis and its mediated inflammatory response are important factors in aggravating ischemic brain injury, and inhibition of pyroptosis may alleviate the ischemic brain injury. Furthermore, studies have found that natural plant components may have a regulatory effect on pyroptosis. Therefore, this review not only summarizes the molecular mechanism of pyroptosis and its role in ischemic stroke, but also the role of natural plant components as regulator of pyroptosis, in order to provide reference information on pyroptosis for the treatment of IS in the future.


Pyroptosis; Ischemic stroke; Caspases; Natural plant components; Inflammasome

1. Introduction

With the aging of the world’s population, the incidence of cerebrovascular disease (such as stroke) has become the second largest in the world, and the threat to human beings is increasing [1]. Stroke includes ischemic stroke (IS) and hemorrhagic stroke, and up to 87% of strokes are ischemic [2]. IS is a common clinical emergency, and its incidence is increasing year by year. The current clinical practice of IS is to restore the blood supply to the ischemic area after IS as soon as possible through thrombolytic therapy to protect the vitality and function of neurons [3][4]. However, ischemia-reperfusion injury (IRI) after restoration of blood flow often causes more serious damage to brain tissue and nerve cells, which further becomes an important factor leading to poor prognosis and dysfunction of patients [5]. The worldwide epidemiological survey analyzed data from 1990 to 2019 in 204 countries and regions around the world. Their report states that stroke remains the second leading cause of death (11.6% of total deaths) and the third leading cause of death and disability [5.7% of total Disability adjusted life years (DALYs)], which is second only to neonatal disease (7.3% DALYs) and ischemic heart disease (7.2% DALYs), and the number of stroke patients is still increasing [6][7], especially in China. China has become the country with the highest risk of stroke in the world, with the risk of stroke among residents reaching 39.3%, and the risk of stroke among Chinese males is also the highest among males in the world, exceeding 41%; IS has risen from the third leading cause of death (as of 1990) to the first (as of 2017) among Chinese residents [8]. Current studies suggest that the pathological mechanisms of cerebral IRI (CIRI) are mainly oxidative stress injury, inflammatory injury, mitochondrial injury, autophagy, and apoptosis [9][10][11][12][13][14]. However, these are still insufficient to explain the pathological mechanism of CIRI. For example, neuronal death after CIRI is thought to be mainly caused by apoptosis, and the direction of neuronal cell protection is mainly anti-apoptosis. However, apoptosis is not directly related to inflammation [15][16], but there is a substantial the inflammatory response in CIRI [16]. Therefore, it is necessary to further clarify the pathophysiological mechanism of IS and further explore the etiology of CIRI.

Pyroptosis, also known as cell inflammatory necrosis, is a newly discovered and confirmed programmed cell death in recent years [17]. Pyroptosis is dependent on inflammatory caspases (mainly caspase-1, 4, 5, 11) and is accompanied by the release of a large number of pro-inflammatory factors. It is manifested by the continuous expansion of cells until the cell membrane ruptures, resulting in the release of the cellular contents and the activation of a strong inflammatory response [17][18]. The morphological characteristics, occurrence and regulatory mechanism of pyroptosis are different from other cell death methods such as apoptosis and necrosis [18]. Pyroptosis is widely involved in the occurrence and development of various diseases such as infectious diseases, nervous system-related diseases, atherosclerotic diseases and malignant tumors [19][20][21]. The latest studies show that pyroptosis and its mediated inflammatory response are involved in the pathological process of IS, and preventing the activation of pyroptosis is beneficial to inhibiting the inflammatory cascade and reducing ischemic brain injury [22][23]. Based on this, the study of pyroptosis is crucial for the treatment of IS; regulating pyroptosis may reduce the fatality rate of IS, improve the survival rate of neurons, and improve the symptoms of IS patients. Currently, drugs that exert neuroprotective effects by regulating the pyroptosis pathway are being developed. In particular, there are many studies on natural plant components regulating the pyroptosis of vascular neuronal units in IS. Our previous studies also showed that saponins [24][25][26] and multicomponent compounds [27][28] can regulate the biological process of pyroptosis in IS. Therefore, this review not only summarizes the molecular mechanism of pyroptosis and its role in ischemic stroke, but also the role of natural plant components as regulator of pyroptosis so as to provide lead compounds or natural plant components for future pyroptosis-related drug development.

2. Pyroptosis

2.1. The characteristics of pyroptosis

Pyroptosis is a form of programmed cell death dependent on pro-inflammatory caspases, which is characterized by the formation of transmembrane pores, swelling and rupture of cell membranes, and the release of pro-inflammatory contents [29]. In the process of apoptosis, cells have nuclear fragmentation and cell membrane integrity, which does not cause inflammatory response in surrounding tissues, while cells in the process of pyroptosis have a complete nuclear morphology and cell membrane rupture, which causes peripheral inflammation [30]. When pyroptosis occurs, the cell membrane will gradually rupture to form 1–2 nm cell membrane pores, and then the cell contents such as intracellular inflammatory factors and other substances are released outside the cell membrane. Membrane-penetrating dyes such as ethidium bromide can be used to observe whether cells undergo pyroptosis [31]. In addition, the occurrence of necrosis depends on the mixed series protein kinase-like domain protein (MLKL), which is selective for ions entering the cell, and the nuclear chromatin is flocculent or edge-clumped. Pyroptosis is dependent on the pore-forming protein GSDMD, a non-selective protein that causes nuclear pyknosis and DNA fragmentation [32]. There are similarities and differences among pyroptosis, apoptosis, necroptosis and ferroptosis, such as the cause, cell changes, etc. The details are shown in Table 1.

Table 1. Similarities and differences among pyroptosis, apoptosis and necroptosis.

Characteristics Signature components Changes in cells General cause Inhibitors Reference
Pyroptosis Pyroptosome, inflammasome Pore formation, cell swelling, plasma membrane rupture, chromatin condensation, DNA fragmentation but nuclear integrity, etc. DAMP, PAMP, infection GSDMD inhibitors [19][33][34][35][36]
Apoptosis Apoptosome Cell shrinks with intact membrane, plasma membrane blebbing, chromatin condensation, DNA fragmentation, etc. Gene regulation of physiological states Regulating caspase family, Bcl-2 family, p53, etc. [37][38][39]
Necroptosis Necrosome Cell swelling, plasma membrane rupture, organelle swelling, chromatin condensation, etc. Serious injury Nec-1, NSA, etc. [33][40][41][42][43]
Ferroptosis NRF2, GPX4, ACSL4, etc. Occurs mainly in mitochondria, with reduced mitochondrial cristae, condensed membranes, and ruptured outer membranes. Fe2 + overload and ROS GSH, deferoxamine, liproxstatin-1, ferrostatin-1, etc. [44][45][46][47]

2.2. The molecular mechanism of pyroptosis

The formation of inflammasome is the key substance for pyroptosis after injury. When injured, the body can activate the pattern signal related to pathogen-associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) [48]. When signal stimulation occurs, the intracellular promoter protein nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) can recruit a large amount of procaspase-1 using the inflammasome adaptor molecule ASC through oligomerization [49], the formation of inflammasome aggregated proteins. Procaspase-1 is the precursor of caspase-1, and normally procaspase-1 exists in the body in the form of zymogen. When stimulatory signals are delivered to procaspase-1, it generates P20 and P10 subunits by autohydrolysis. This subunit first forms a heterodimer that cannot function, and then aggregates to form a tetramer that promotes caspase-1 activity, leading to caspase-1 activation [50]. When caspase-1 is activated, the active caspase-1 is responsible for the formation of cell membrane pores and rapid lysis of the cell membrane to form an inflammatory response. Meanwhile, caspase-1 can also induce the precursors of IL-1β and IL-18, pro-IL-1β and pro-IL-18, to accelerate the maturation process. The mature IL-1β and IL-18 are released extracellularly, thereby recruiting more inflammasomes. After the inflammasome aggregates, the inflammatory response is further aggravated, and the tissue damage is aggravated. After IL-1β is released from cells, inflammatory factors spread to adjacent tissues along with the flow of lymph, further aggravating the inflammatory response. The secretion of a large amount of IL-18 causes Th1 and Th2 immune responses, and stimulates the immune response to play a role [51].

2.2.1. Canonical pyroptosis pathway

Caspase-1 is a key protein in the canonical pyroptotic pathway, in which the inflammasome plays an important role in the activation of Caspase-1 [52]. The inflammasome is an important part of the innate immune system and is a multi-protein complex composed of sensor proteins, ASC and pro-Caspase-1. It exists in the cytoplasm of stimulated immune cells and can sense extracellular stimulatory signals [53][54]. Inflammasomes are divided into inflammasomes containing nucleotide-binding oligomerization domain-like receptors (NLRs), abstract in melanoma 2 (AIM2) inflammasomes and NLRC4 inflammasomes [55][56]. Purinergic 2 × 7 (P2X7) receptor is an ATP-gated transmembrane ion channel receptor expressed in microglia and is a key factor in inflammasome activation. Studies have shown that extracellular ATP can regulate K+ efflux by activating the P2X7 receptor, inducing the activation of the NLR family, pyrin domain-containing 3 (NLRP3) [57]. The NLRP3 inflammasome is currently the most studied. Activation of the NLRP3 inflammasome requires a two-step response: initiation of the response, microbial or endogenous factor nuclear factor-κB (NF-κB) into the nucleus, and upregulation of NLRP3 and pro-IL-1β expression [58][59]. When immune cells are stimulated by DAMPs, the sensor protein recruits pro-Caspase-1 through the ASC, which is then activated by autohydrolysis. Mature Caspase-1 processes downstream inactive pro-IL-1β and pro-IL-18 into active IL-1β and IL-18 [60]. Meanwhile, Caspase-1 cleaves the downstream GSDMD protein into GSDMD-N fragment and GSDMD-C fragment with pore-forming activity. The GSDMD-N fragment specifically recognizes and binds to the membrane lipids on the inner side of the cell membrane, causing changes in intracellular and extracellular osmotic pressure, which in turn leads to cell swelling and rupture and the release of inflammatory factors IL-1β and IL-18, inducing inflammatory response and cell pyroptosis [61].

2.2.2. Non-canonical pyroptosis pathway

Caspase family is mainly divided into two categories: apoptosis-related and inflammation-related according to different functions. Caspase-3/2/10 mainly mediate apoptosis, while caspase-1/4/5/11 are key mediators of inflammation and innate immune responses [62][63]. The non-canonical pyroptotic pathway is dependent on Caspase-4/5/11 activation, whereas the inflammasome is not essential for the maturation of IL-1β and IL-18 [64]. The non-classical pathway of pyroptosis is induced by lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria. LPS can directly bind to human Caspase-4/5 and mouse Caspase-11. These caspases act as both sensor proteins and effector molecules of LPS. The activated Caspase-4/5/11 directly cleave GSDMD and induce pyroptosis [65]. In addition, studies have found that Caspase-11 can activate the downstream gap junction channel protein Pannexin-1 to promote K+ efflux, and K+ efflux can activate the NLRP3 inflammasome, which in turn activates Caspase-1 and promotes the maturation of IL-1β and IL-18 [66].

2.2.3. The relationship between GSDMD and pyroptosis

GSDMD (Gsdermin D) is a protein composed of 484 amino acid residue fragments and is a member of the Gasdermin protein family. There are mainly 5 members in this family, they are: DFNA5, DFNB59, GSDMA, GSDMB, GSDMCD. The amino acid structures of these five proteins are highly similar, and these proteins contain two spatial domains, N-terminal and C-terminal [67]. GSDMD protein is a long-chain amino acid protein, and the structure between its N-terminal and C-terminal domains is relatively loose. GSDMD is typically cleaved at amino acid sequence 275, exposing the N-terminal and C-terminal domains. In mammalian cells, the simple N-terminal structure of GSDMD can regulate cell pyroptosis. Therefore, GSDMD often expresses the N-terminal domain but cannot express all GSDMD proteins [68][69]. However, studies have shown that when the N-terminal domain of GSDMD was introduced into E. coli for culture, strong toxic effects occurred. In contrast, intact GSDMD and the C-terminal domain of GSDMD showed relatively low toxicity [70]. The N-terminal structure of intracellular GSDMD will gradually transfer to the cell membrane with the invasion of damage factors, and on the cell membrane, where it specifically binds to phosphosarcosinase and phosphatidylserine on the cell membrane to produce biological effects [71]. Using equipment such as atomic force electron microscopy and cryo-electron microscopy, L. Sborigi observed that the N-terminal structure of GSDMD can be combined with biofilms to form a hollow ring-like polymer on the biofilm. This polymer induces the formation of biofilm pores, due to which GSDMD induces pyroptosis in cells when endogenous damage occurs. When external pathogens invade, they are able to adsorb on the cell surface of pathogens and protect the body from external harmful substances by lysing the cells [72]. GSDMD is a substance that specifically binds to caspase-1/4/5/11 proteins downstream of signal transduction [73]. Caspase-1 promotes the release of downstream IL-1β and IL-18 out of the cell membrane through inflammasome responses generated by complexes with various proteins. Activated caspase-1 cleaves GSDMD protein, converting GSDMD into active peptides. The decomposition products of GSDMD specifically bind to the cell membrane, and components such as extracellular water molecules enter the cell membrane through the pores, causing the cells to swell and eventually rupture [74].

2.3. Noncoding RNAs participate in the expression of pyroptotic genes in IS

In recent years, the regulatory mechanism at the gene level related to the occurrence of pyroptosis has also been fully developed. Modern molecular biology studies show that 98% of the genome is not involved in coding proteins. Long-chain coding RNAs (lncRNAs) are RNAs with no protein-coding function, so named because they are more than 200 nucleotides in length [75][76]. However, although lncRNA does not have the function of encoding protein, it is the most expressed gene in the body and the most conserved gene in transcription. It participates in important pathological and physiological processes in the body, especially in reperfusion injury-related diseases, and plays an important role [77]. Studies have shown that in microglia, the expression level of lncRNA-H19 is positively correlated with the duration of reperfusion [78]. Overexpression of LncRNA-H19 plays an inflammatory role when it promotes the expression of the downstream signaling molecule NLRP3/6 and initiates GSDMD [79][80][81]. lncRNA-H19 is able to activate members of the caspase protein family [82], leading to mitochondrial dysfunction, participating in the activation of inflammasomes, and causing neuronal damage [83]. In addition to the regulation of molecular structure, lncRNA-H19 can also recruit more transcription factors to regulate the mRNA transcription process [84]. In addition, lncRNA-H19 can also promote the nuclear transport process of transcription factors, thereby increasing the specific expression of more target genes, and generating an inflammatory cascade network when ischemia occurs [84]. Therefore, lncRNA-H19 is a strong danger signal when CIRI occurs, and inhibition of H19 may be a potential treatment for ischemia-reperfusion injury [84]. In AIM2 inflammasome-mediated pyroptosis capable of ischemia-reperfusion injury, the lincRNA MEG3/miR-485/AIM2 axis promotes pyroptosis by activating caspase1 signaling during CIRI, and thus this axis may be an effective therapeutic target for IS [84]. For the immune regulation of microglia, Wang et al. found that LncRNA-Fendrr protects the ubiquitination and degradation of NLRC4 protein through HERC2 and regulates microglial pyroptosis [85]. Zhang et al. found that the lncRNA NEAT1/miR-22–3p axis inhibited pyroptosis and attenuated CIRI injury [86]. In vitro oxygen-glucose deprivation (OGD) injury experiments after IS showed that OGD increased NOD-like receptor protein 3 (NLRP3) expression to induce pyroptotic death of NSCs, which was rescued by hyperbaric oxygen therapy. The upregulated lncRNA-H19 acts as a molecular sponge for miR-423–5p, targeting NLRP3 after OGD to induce neural stem cell (NSC) pyroptosis. Therefore, it was confirmed that hyperbaric oxygen therapy protects NSCs from pyroptosis by inhibiting the lncRNA-H19/miR-423–5p/NLRP3 axis [87]. miR-21 is a microRNA (miRNA) that is closely related to the occurrence of pyroptosis. Studies have shown that miR-21 can specifically regulate NLRP3 protein, activate NLRP3 protein, stimulate NLRP3 secretion, and express in macrophages through NF-κB signaling through the inflammatory feedback pathway [88]. In addition to activating NLRP3, miR-21 can also regulate the activation of downstream caspase-1, promote the secretion of IL-1β, and promote the occurrence of pyroptosis. When miR-21 is deficient, the expression of caspase-1 pathway regulated by NLRP3 is significantly reduced. Therefore, the expression level of miR-21 is also a key link in regulating the occurrence of pyroptosis [89][90]. Modern molecular biology studies have shown that miR-214–3p has a binding site for caspase-1, which can specifically bind to the occurrence of caspase-1, thereby regulating the inflammatory response and increasing the incidence of pyroptosis [91].

The molecular mechanism of pyroptosis was summarized in Fig. 1.

Fig. 1

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Fig. 1. The molecular mechanism of pyroptosis (The mechanisms of pyroptosis include the canonical inflammasome pathway and the non-canonical inflammasome pathway. The canonical inflammasome pathway is triggered by DAMPs or PAMPs. The non-canonical inflammasome pathway is triggered by LPS of extracellular Gram-negative bacteria or death stimulation. After triggering, pyroptosis is triggered through a series of intermolecular interactions. TLR: Toll-like receptor; GSDM: Gsdermin; NLRP3: nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3; DAMP: damage associated molecular pattern; PAMP: pathogen-associated molecular pattern).

2.4. The relationship between pyroptosis and other cellular damage

There are many mechanisms involved in the occurrence of cerebral ischemia-reperfusion injury. The known mechanisms are related to various mechanisms such as inflammatory response, calcium channel disorder, mitochondrial function impairment, and autophagy. Pyroptosis is involved in all aspects of cerebral ischemia-reperfusion injury and is closely related to other injuries.

The occurrence of pyroptosis and apoptosis both belong to a way of cell death, but they are fundamentally different. Apoptosis is a physiological way of cell death, while pyroptosis is often associated with pathological cell death after body injury [92]. Autophagy is a kind of autophagy phenomenon that widely exists in eukaryotic cells. Autophagy is generally divided into three types, namely chaperone-mediated autophagy, microautophagy, and macroautophagy. At present, there are many studies on macroautophagy [93]. Autophagy is an indispensable part of the normal metabolic process of cells. Inhibition of autophagy can lead to the accumulation of harmful substances in tissues, organs or cells. Excessive activation of autophagy will destroy important intracellular organelles and essential proteins, triggering autophagic apoptosis [94].

Pyroptosis and autophagy are inextricably linked. Under normal circumstances, the occurrence of autophagy and pyroptosis in vivo is in a state of dynamic equilibrium. When the body mounts an inflammatory response to external stimuli, the balance between autophagy and pyroptosis is disrupted [95]. For example, NF-κB is a signal transduction pathway that participates in the signal transduction of many signal pathways to achieve signal transduction. The study found that in IS mice with knockout of the NF-κB gene, mTOR activity was inhibited and autophagy levels were enhanced. The signaling activation pathway of NF-κB is a large number of stimuli to activate NF-κB-induced kinases, which activate IκKα, IκKB and IκKγ trimers, resulting in the phosphorylation and degradation of IκB, and finally activate the NF-κB signaling pathway. After activation, NF-κB enters the cell and specifically binds to its corresponding DNA receptor, thereby stimulating the transcription of inflammatory factors and promoting the massive expression of inflammatory factors TNF-α, IL-1β and IL-6. Studies have shown that the NF-κB signaling pathway is involved in regulating the activation of the pre- and post-transcriptional levels of the key pyroptotic protein NLRP3 to induce pyroptosis. The abundantly expressed inflammatory factors may induce an inflammatory cascade in the body, induce pyroptosis, and further expand the inflammatory response, thereby aggravating brain damage [96][97][98].

2.5. Mechanisms of pyroptosis and inflammation involved in IS

Inflammatory cascades exist in various stages of IS [99]. In the early stages of IS, blood flow in the brain slows and neutrophils adhere to the endothelial cells of ischemic vessels, and an initial acute inflammatory response begins [100]. With the occurrence of reperfusion after cerebral ischemia, exogenous inflammatory factors, inflammatory cells and inflammatory factors pass through the blood-brain barrier, resulting in the secondary effect of ischemia after reperfusion, and leading to the release of a large number of oxidation factors and free radicals. At this time, the microglia in the brain tissue are gradually activated in large quantities and produce more inflammatory mediators. This type of inflammatory mediators can over-activate endothelial cells in the brain tissue and produce a large amount of tissue factor to accumulate the toxicity of amino acids. This further aggravates the release of oxidative factors, free radicals and carbon monoxide, and activates inflammatory response-related signaling pathways such as NF-κB, Toll-like receptors, and Nod-like receptors (NLRs) [101][102]. For example, NLRs, studies have shown that there are 23 family members of NLRs, which are mainly expressed in the cell cytoplasm and play a key role in the innate immune response of the body. Members of the NLR family, Nalp1, Nalp3, Nalp5, and Ipaf, are able to activate caspase-1 activation through the adaptor protein ASC, initiate a cascade of inflammatory responses, and mediate pyroptosis [103][104]. NLRP3 is an important component of inflammatory factors. NLRP3 contains three domains: PYD, NACHT, and LRR, which can be represented by LRR-NACHT-PYD: PYD-CARD: CARD-CARDC a spase domain. It responds to various signals of endogenous damage, so the activation of the NLRP3 inflammasome is considered to be the main type of pyroptosis [105][106][107].

Inflammasomes are protein complexes assembled after the body receives signals of infection or cell damage, and serve as a platform for the recruitment and activation of pro-caspase-1. The promoter protein NLRP3 can recruit large amounts of procaspase-1 through oligomerization using the inflammasome adaptor molecule ASC [108]. Procaspase-1 is the precursor of caspase-1, and active caspase-1 is responsible for rapidly lysing cells to activate and release extracellular IL-1β and IL-18, which further aggravates the inflammatory response. Therefore, the occurrence of pyroptosis is closely related to the appearance of inflammatory response and the formation of inflammasomes. The occurrence of cerebral ischemia-reperfusion injury is more closely related to the interaction between pyroptosis and inflammatory response [109][110]. Especially in the central nerve system, astrocytes induce the activation and proliferation of microglia and produce a large number of inflammatory mediators. These inflammatory mediators can activate endothelial cells to produce a variety of tissue factors, increase the toxicity of excitatory amino acids, and promote the release of nitric oxide and oxygen free radicals. The above substances further lead to the activation of multiple inflammatory signal transduction pathways such as NF-κB and JNK2/STAT3, and promote the assembly of inflammasomes such as NLRP1 and NLRP3. Activated caspase-1 induces pyroptosis of cells, expands the inflammatory response, and aggravates cerebral ischemia-reperfusion injury [111][112]. Some studies have found that compared with wild-type mice, the levels of AIM2 and IL-1β in the brain of AIM2 knockout mice after cerebral ischemia-reperfusion were significantly decreased, the infarct volume was significantly reduced, and the neurological function scores were also significantly improved. Inhibition of AIM2 inflammasome activation can inhibit the occurrence of pyroptosis to a certain extent, thereby reducing ischemic brain injury [113]. Therefore, pyroptosis plays an important role in the process of cerebral ischemia-reperfusion injury. The NLRP3 inflammasome is a key protein in the pyroptotic pathway, and inhibiting its expression can simultaneously inhibit the expression of downstream pyroptotic pathway-related proteins, limit the inflammatory response and alleviate cerebral ischemia-reperfusion injury.

2.6. Pyroptosis and mitochondrial damage mediate oxidative stress injury after IS

Mitochondria are mainly involved in the aerobic respiration of cells and are the place where aerobic respiration occurs. Mitochondria provides an ionized basis for normal signal transmission between cells and signal transduction of nerve cells [114]. Mitochondrial damage is mostly caused by abnormal mitochondrial metabolism and oxidative damage [115]. Studies have shown that cerebral ischemia-reperfusion injury is closely related to mitochondrial dysfunction [116]. Oxidative stress, as the key to the occurrence of oxidative damage, is closely related to pyroptosis [117][118]. Oxidative stress is more likely to be expressed in brain tissue injury, especially in cerebral ischemia and reperfusion injury after cerebral ischemia [119]. When the body is attacked by external harmful substances, the cells can secrete a large amount of ROS and activated carbon, which disrupts the balance between oxidation and anti-oxidation, resulting in a series of oxidative damage reactions [120]. Most of ROS are secreted by mitochondria [121]. When stimulated by external stimuli or endogenous damage occurs, the body stimulates the binding of nucleotides to NLRP3 by inducing high levels of ROS, activating the inflammasome protein complex. When ROS is overactivated, the normal order of signaling pathways in the body is disrupted, resulting in the destruction of genetic material, proteins, and various organelles (including mitochondria) in cells. When various organelles such as mitochondria in cells are damaged, the cells have abnormal nutrition and metabolism, and ROS can further aggravate the damage of the vascular endothelium and induce the disturbance of the microcirculation in the brain. The permeability disorder of the blood-brain barrier in the brain induces the overexpression of cell adhesion molecules, thereby aggravating brain injury [122]. Meanwhile, oxidative stress causes cells to gradually exhibit a cell death mode characterized by increased cell membrane permeability and the release of cell contents – pyroptosis [120]. Therefore, both pyroptosis and mitochondrial damage are involved in the pathological process of cerebral ischemia-reperfusion injury. It has been reported that the mitochondrial motility-related protein Drp1 is a key protein necessary for mitochondria to maintain normal physiological functions [123]. When the expression of Drp1 is increased, it can inhibit the mitochondrial fission process and reduce the level of pyroptosis, thereby slowing down the occurrence of damage [124]. When the body receives a variety of endogenous and exogenous damage stimuli, the regulatory process of signaling pathways in the body changes. It is manifested as decreased secretion of Drp1, mitochondrial damage, mitochondrial dysfunction, and increased apoptosis and pyroptosis, leading to various diseases in the body [125]. Recent studies show mitochondrial dysfunction induces NLRP3 inflammasome activation during cerebral ischemia/reperfusion injury [126]. Mitochondrial uncoupling protein 2 (UCP2) deficiency exacerbates brain injury after CIRI, and new studies show that UCP2 deficiency enhances NLRP3 inflammasome activation after hyperglycemia-induced CIRI exacerbation in vitro and in vivo. UCP2 may be a potential therapeutic target for hyperglycemia-induced worsening of CIRI. UCP2 deficiency also enhanced NLRP3 inflammasome activation and ROS production in neurons in vitro and in vivo [127]Adiponectin is an adipose-derived hormone with broad antioxidant and anti-inflammatory effects. Adiponectin peptide attenuates oxidative stress and NLRP3 inflammasome activation after cerebral ischemia-reperfusion injury by regulating AMPK/GSK-3β [128]. Therefore, in IS, there is a close relationship between the occurrence of pyroptosis and mitochondrial damage, and there is a close interaction between the two.

2.7. Oxidative stress/nitrosative stress mediates pyroptosis after IS

Oxidative/nitrosative stress and neuroinflammation are key pathological processes of cerebral ischemia-reperfusion injury, mediating neuronal damage, blood-brain barrier damage and hemorrhagic transformation during ischemic stroke [129][130]. Nitric oxide (NO) and peroxynitrite (ONOO-) are typical RNSs in CIRI [130][131]. There are 3 isomers of nitric oxide synthaseendothelial nitric oxide synthase (eNOS) produces low NO concentration and has physiological functions; while neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) produce high NO concentrations, which induces inflammation and increases blood-brain barrier permeability [132]. The overproduction of ROS/RNS creates a stressful microenvironment and leads to a series of cellular signaling cascades, leading to inflammation, hyperpermeability of the blood-brain barrier, brain edema and neuronal cell death [133][134]. Peroxynitrite mediates DNA strand breaks and activates the ribozyme poly(ADP-ribose) synthase (PARS), also known as poly(ADP-ribose) polymerase (PARP) or poly(ADPribose) transferase (pADPRT). ONOO- can directly activate PARP [135], and in vivo and in vitro experiments have demonstrated the role of ONOO-/PARP signaling pathway in ischemic brain injury. In vivo experiments showed that NOS-deficient mice displayed less PARP activation in an animal model of ischemic stroke. In vitro experiments showed that ONOO-donors, but not NO-donors, significantly induced PARP activation in cultured C6 glioma cellsGene disruption or silencing of PARP significantly reduces cerebral infarct size, alleviates neurotoxicity, protects neurovascular units and improves neurological outcomes [136][137][138]. Studies have found that ROS/RNS may be an activator of the inflammasome during CIRI [139]. The inflammasome is a multi-protein complex in the cytoplasm of microglia, of which NOD-like receptor 3 (NLRP3) is the most widely studied one. NLRP3 triggers and activates caspase-1, activates IL-1β and IL-18 and releases them into the extracellular space, promoting the development of inflammation [140]. In vivo experiments showed that the cerebral infarct size and BBB damage in NLPR3 knockout mice were lower than those in wild-type mice. Further experiments showed that NLRP3 could mediate the release of IL-1β and increase the permeability of cerebral microvascular endothelial cells [141]. Nuclear factor E2-related factor 2 (Nrf2) regulates cellular antioxidant responses, and Nrf2 inhibits ROS-mediated NLRP3 production in BV2 microglia under conditions of oxygen and glucose deprivation [142]. It was found that oxidative/nitrosative stress induces the formation of peroxynitrite, which may be a key trigger of caspase1/inflammasome activation [143]. Therefore, ROS/RNS-mediated inflammasome may be a potential therapeutic target for ischemic brain injury.

In addition, ROS/RNS mediates the activation of Toll-like receptors. Current studies have shown that Toll-like receptors (TLRs) have been widely studied as an innate immune receptor, and TLR4/2 have been studied more in cerebral ischemic injuryTLR4 can mediate the expression of IL-1β, MMP-9, iNOS and COX-2, and aggravate oxidative stress-induced brain damage [144]. The application of the TLR4 inhibitor E5564 showed that it can play a neuroprotective role by inhibiting the activation of microglia and the production of ROS [145]. After IS, the infarct size of TLR4-deficient mice was significantly smaller than that of wild-type mice [146]. TLR4 is also involved in post-IS neurogenesisPositron emission tomography studies have shown that TLR4-deficient mice have enhanced neurogenesis and suppressed inflammatory responses in IS [147]. In addition, in vivo experimental studies have shown that inhibiting the TLR2/4/NF-κB signaling pathway has a certain effect on regulating oxidative stress, inflammatory response, and protecting ischemic brain tissue [148]. Therefore, TLR4/2 may be a therapeutic target for ischemic brain injury.

3. The relationship between pyroptosis and IS

The pathological mechanisms of cerebral ischemia and post-ischemia-reperfusion injury are complex. The mechanisms involved in this process include inflammation, oxidative stress, autophagy, mitochondrial dysfunction, calcium overload, and programmed cell death [149][150][151]. A number of studies have shown that programmed cell death plays an important role in the pathological process of cerebral ischemia and ischemia-reperfusion injury, and is intertwined and closely related to the above mechanisms, especially pyroptosis [152][153]. Programmed cell death is the main event of ischemic stroke, involving neurons, microglia, astrocytes, vascular endothelial cells, etc., such as apoptosis [154], autophagy [155], programmed necrosis or necroptosis [156]ferroptosis [157], and pyroptosis [158]. Especially in pyroptosis, the expression of pyroptosis-related proteins such as NLRP1, ASC, Caspase-1, and GSDMD is increased in a rat model of cerebral ischemia, and inhibition of NLRP1 can alleviate inflammation and cerebral ischemic injury. In addition, in the event of chronic cerebral ischemia, cells promote inflammatory responses by releasing signals such as DAMPs and PAMPs, and trigger a series of complex molecular responses. At this time, intracranial neuroimmune inflammatory cells, such as microglia and astrocytes, appear to proliferate and activate, which further damages the neurovascular unit [159][160][161].

3.1. Pyroptosis and neurons

After IS, necrosis occurs in the central ischemic area in a short time, and dead cells release danger signals such as HMGB1 proteinheat shock proteinperoxidase family protein, etc. These danger signal molecules bind to pattern recognition receptors, form inflammasomes, initiate innate immune responses, and cause neuronal death [162][163]. Studies have confirmed that cerebral ischemia can lead to the high expression of NLRP1 and NLRP3 in ischemic brain tissue and neurons, and the activation of NLRP1 mainly exists in neurons [164]. The use of Caspase-1 inhibitors or immunoglobulin preparations can attenuate the expression of NLRP1 and NLRP3 in primary cortical neurons and reduce the size of cerebral infarction, and the mechanism may be related to the inhibition of NF-κB and MAPK pathway activation [164]. In the IS mice Li et al. [165] found that on the 3rd day after cerebral ischemia, ultrastructural damage of neuronal plasma, nuclear and mitochondrial membranes occurred, and the expressions of Caspase-1, GSDMD and IL-1β were significantly increased. The Caspase-1 inhibitor Vx765 can inhibit pyroptosis, promote the survival of neurons in ischemic areas, and improve brain dysfunction in mice. Liang et al. [166] found that long non-coding RNA maternally expressed gene 3 (MEG3) promotes pyroptosis and inflammatory responses by activating the AIM2/Caspase-1 pathway, resulting in cerebral ischemia-reperfusion injury. Knockout of MEG3 gene can inhibit the expression of AIM2, Caspase-1, GSDMD and other proteins, and alleviate ischemic brain injury. This suggests that MEG3 may be an effective therapeutic target for ischemic stroke.

3.2. Pyroptosis and astrocytes

Astrocytes are the most abundant glial cells in the central nervous system and are involved in the formation of the blood-brain barrier, regulating neuronal metabolism and stabilizing intercellular communication [167]. After IS, a prominent pathological change is reactive astrogliosis and glial scarring, which promote neuronal plasticity early after ischemic injury [168]. It is generally believed that when the hippocampus is ischemia and hypoxia, the antioxidant capacity of the hippocampus will be weakened, and a large number of inflammatory factors may be released, thereby aggravating the damage to the hippocampus [169]. Three (3) h after focal cerebral ischemia in SD rats, IL-1β-like immunoreactive positive cells appeared in the ischemic area, mainly inactivated astrocytes. Until 2 months after ischemia, the positive cells can still be detected in the ischemic hemisphere, especially around the necrotic foci, and the higher the degree of activation [170]. Activated astrocytes can induce the release of tumor necrosis factor, interleukin, growth factor and other inflammatory factors or neuronal toxic mediators, resulting in neuronal damage [171]. NLRP2 is mainly expressed in astrocytes, but hardly expressed in neurons and microglia. Moreover, the expression of NLRP2 was significantly up-regulated in the mouse cerebral ischemia model and after oxygen and glucose deprivation in astrocytes, and silencing NLRP2 could reduce the pyroptosis induced by oxygen and glucose deprivation [172]. Another study found that in an in vitro model of cerebral ischemia, oxygen-glucose deprivation led to an increase in NLRP3, ASC, Caspase-1, IL-1β, and IL-18, and a decrease in astrocyte survival. Hispidulin is a ketone compound isolated from Chinese herbal medicine. In vitro and in vivo experiments have shown that Hispidulin can inhibit NLRP3-mediated pyroptosis and exert a protective effect on the brain, and its mechanism is related to the activation of the AMPK/GSK-3β signaling pathway [173]. Meng et al. [174] found that the expression of NLRP6 reached a peak at 48 h after cerebral ischemia-reperfusion in rats. After oxygen and glucose deprivation in astrocytes, the production of NLRP6 and its activation products increases, and silencing NLRP6 can reduce ASC and Caspase-1, reduce the release of inflammatory factors, and increase neuronal activity [175]. Therefore, interventions targeting inflammasome activation in astrocytes may provide new ideas for the treatment of IS.

3.3. Pyroptosis and Microglia

Microglia are the innate immune cells of the central nervous system and the earliest activated cells after ischemic stroke [176]. In the acute phase of cerebral ischemia injury, microglia rapidly migrate to the lesion site and secrete inflammatory factors and cytotoxic substances, aggravating tissue damage [177]. In the chronic phase, microglia can produce anti-inflammatory cytokines and growth factors to promote tissue repair and remodeling [178]. Studies have found that microglial pyroptosis plays an important role in ischemic brain injury. NLRC4 was the first inflammasome to be significantly increased when microglia were deprived of oxygen and glucose for 3 h, while NLRP1, NLRP3 and AIM2 were not significantly increased until after 6 h of oxygen glucose deprivation. Silencing NLRC4 can reduce the production of GSDMD, IL-1β and IL-18, and inhibit microglial pyroptosis [179]. Xu et al. [180] reported that driver receptor 1 (TREM-1) expressed by myeloid cells in microglia after cerebral ischemia-reperfusion can activate the NLRP3/Caspase-1-mediated pyroptosis pathway and induce neuroinflammatory responses. Inhibition of TREM-1 reduces microglial pyroptosis and nerve damage. Li et al. [181] confirmed that the expression of cyclic guanosine monophosphate-adenosine synthase (cGAS) is up-regulated after cerebral ischemia and activates the AIM2 inflammasome to induce microglial pyroptosis. The cGAS antagonist A151 can inhibit AIM2 activation and microglial pyroptosis, significantly reduce cerebral infarct volume, and alleviate nerve damage. The above studies indicate that microglia pyroptosis and its mediated neuroinflammatory response may be an important mechanism leading to ischemic brain injury. Inhibiting the neurotoxic effects of microglia may be a new strategy for the treatment of ischemic stroke.

3.4. Pyroptosis and endothelial cells

Endothelial cells constitute the first barrier of the blood-brain barrier, providing scaffolds for claudinadhesion molecules and extracellular matrix [182]. During cerebral ischemia, immune-inflammatory response and oxidative stress can damage endothelial cells and disrupt the integrity of the blood-brain barrier, leading to vasogenic edema, hemorrhagic transformation, and increased mortality [183][184]. After cerebral ischemia, NLRP3 is up-regulated in neurons, microglia and vascular endothelial cells, and silencing NLRP3 gene can reduce the volume of cerebral infarction in mice with middle cerebral artery ischemia and reduce the permeability of blood-brain barrier [185]. Wang et al. [186] confirmed that cerebral ischemia can induce pyroptosis of microvascular endothelial cells and aggravate ischemia-reperfusion injury. They also found that activation of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) significantly reduced the expression of pyroptosis-related proteins and increased the expression of ZO-1 and Occludin proteins in brain microvascular endothelial cells, so as to protect the integrity of the blood-brain barrier. In addition, studies have found that the pyroptosis of cerebral microvascular endothelial cells may increase the possibility of hemorrhage after IS, leading to serious complications such as cerebral hemorrhage [187].

The association between cerebral ischemia and pyroptosis is summarized in Fig. 2.

Fig. 2

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Fig. 2. Summary of the mechanism of pyroptosis in vascular neuronal units after IS [Molecular mechanism of pyroptosis on vascular neuronal units (consists of microglia, astrocytes, neurons, vascular endothelial cells, pericytes) after IS. IRF: interferon regulatory factor; IFN: Interferon; CASP: Caspase; GPX4: Glutathione peroxidase 4].

4. Potential of pyroptosis inhibitors to treat IS

In recent years, the research of pyroptosis inhibitors in IS has received much attention. Liang et al. [188] found that the Caspase-1 inhibitor VX-765 can reduce Caspase-1, ASC, GSDMD and IL-1β, while up-regulating the levels of tight junction proteins and tissue inhibitors of metalloproteinases, protecting the integrity of the blood-brain barrier. In addition, VX-765 also promotes the transformation of microglia from M1 type to M2 type, reduce the inflammatory response mediated by microglia, and exert a neuroprotective effect [189][190]. MCC950 is a selective NLRP3 inhibitor. Studies have found that MCC950 can reduce the expression of NLRP3, Caspase-1, and IL-1β in the ischemic penumbra, and has a protective effect on focal cerebral ischemia in mice [191]. Studies have found that some microRNAs may inhibit the pyroptosis in cerebral ischemia model and play a neuroprotective role [192]. LP17 may inhibit myeloid cell trigger receptor-1, thereby inhibiting oxidative stress and pyroptosis, and reducing cerebral ischemia-induced neuronal damage [192]Histidine may inhibit NLRP3-mediated pyroptosis by regulating the adenylate-activated protein kinase/glycogen synthase kinase-3β signaling pathway, thereby exerting anti-CIRI neuroprotective effects [193]. Low-density lipoprotein receptors may inhibit NLRP3-mediated neuronal pyroptosis after CIRI [194]. The lipid alcohol-mediated peroxisome proliferator-activated receptor α-glutamate oxaloacetate transaminase 1 axis may inhibit the pyroptosis of endothelial cells after IS and improve ischemic brain injury [195]. Overexpression of CHRFAM7A may inhibit NLRP3/caspase-1 pathway-dependent microglial pyroptosis and attenuate CIRI [196]. TP53-induced glycolysis and apoptosis regulators may alleviate cerebral ischemia-induced microglial pyroptosis and ischemic brain injury [197]. In addition, exosomes derived from hypoxic bone marrow mesenchymal stem cells may modulate the microglial M1/M2 phenotype to alleviate CIRI-induced neuronal pyroptosis [198]. Hypoxia-preconditioned olfactory mucosa mesenchymal stem cells may inhibit the pyroptosis and apoptosis of microglia caused by cerebral ischemia by activating hypoxia-inducible factor-1α, thereby reducing CIRI [199].

As an executor of pyroptosis, GSDMD is an ideal molecular target for the treatment of IS. However, current research on GSDMD inhibitors is still in its infancy, and evidence of the efficacy of GSDMD inhibitors in the treatment of ischemic stroke is lacking. Recent studies have found that some natural plant components may treat IS by regulating the pyroptotic pathway.

5. Natural products as novel pyroptosis inhibitors and thus become potential candidates for the treatment of IS

Natural products have significant advantages in the prevention and treatment of cerebral infarction/cerebral ischemia-reperfusion due to their advantages of multi-component, multi-target, multi-channel and low toxicity, and have good application prospects. Currently, the research of natural products in the prevention and treatment of cerebral infarction/CIRI is gradually increasing, involving a variety of protective mechanisms. This section summarized the natural products, animal or cell models, administration methods, administration doses, treatment time, effects, and treatment mechanisms (signaling pathways) to provide theoretical support for the rapid search for natural plant components that are safe, effective, low toxicity, and regulate pyroptosis for the prevention and treatment of IS/CIRI.

5.1. Natural plant components

The main structure of natural plant components were shown in Fig. 3.

Fig. 3

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Fig. 3. The main structure of natural plant components.

5.1.1. Gastrodin (GAS)

GAS is a multifunctional compound derived from the herb, Gastrodia elata, with various pharmacological activities, such as antioxidant and anti-inflammatory activities. In addition, GAS has been shown to significantly reduce symptoms of cerebral ischemia-reperfusion by modulating pro-apoptotic factors including caspase-3 cleavage, IL-18, and IL-1β. Several studies have shown that GAS ameliorates cerebral ischemic injury and reduces the levels of reactive oxygen species and inflammatory cytokines in mice [200][201][202]. Lu et al. found that GAS could ameliorate pyroptosis associated with cardiac microvascular ischemia-reperfusion injury by targeting the NLRP3/caspase-1 axis [203]. GAS may significantly improve the neurological function score and reduce the size of cerebral infarction. Meanwhile, GAS inhibited pyroptosis by downregulating NLRP3, inflammatory factors (IL-1β, IL-18) and cleaved caspase-1. Furthermore, GAS attenuated ischemia-reperfusion-induced neuronal cell inflammation by regulating the lncRNA NEAT1/miR-22–3p axis. GAS significantly attenuated cerebral ischemia-reperfusion injury by regulating the lncRNA NEAT1/miR-22–3p axis [204]. Therefore, GAS may be used as a potential drug for the treatment of CIRI.

5.1.2. Tanshinone IIA

Tanshinone IIA is the main active component of Salvia miltiorrhiza. It is widely used in the treatment of cardiovascular and cerebrovascular diseases due to its anti-oxidative and circulation-improving effects. Cai et al. [205] found that tanshinone IIA effectively reduced the expression of IL-1β and IL-18 in the NLRP3 inflammatory signaling pathway, and its effect is dose-dependent. This suggests that tanshinone IIA inhibits the activation of NLRP3 signaling pathway, thereby reducing the damage of oxygen-glucose deprivation/reperfusion on BV-2 cells after stroke.

5.1.3. Leonurine

Leonurine in Leonuri herba improved the learning ability and memory ability of rats with chronic cerebral ischemia, and reduced the expression of NLRP3. It is speculated that the purpose of protecting nerves may be achieved through anti-inflammatory effect. Activation of NLRP3 promoted the conversion of Caspase-1 precursor to Caspase-1, and promoted the production of IL-1β and IL-18, thereby causing a series of inflammatory responses. Leonurine may reduce the inflammatory response in brain tissue, reduce the neurological deficit score by inhibiting the pyroptosis of cells, and has a good neuroprotective effect [206].

5.1.4. Paeoniflorin

Paeoniflorin comes from the main component of Paeoniae radix rubra. Paeoniflorin is the main active component of total glucosides of peony (TGP), which also has good anti-inflammatory and immunomodulatory activities [207]. TGP and paeoniflorin also have various pharmacological activities such as antispasmodicanalgesic, and vasodilation [208]. Zulifiya Ekemu et al. found that after acute cerebral ischemia-reperfusion, the activation of microglia and neuronal pyroptosis in the brain was obvious, the activation of NLRP3 was increased, and Caspase-1 and IL-1β were up-regulated [209]. Paeoniflorin significantly reduced neurological function scores, significantly reduced cerebral infarct size, significantly down-regulated Iba1 expression, and significantly improved neuronal status. In addition, the expressions of NLRP3, Caspase-1 and IL-1β were significantly decreased after paeoniflorin intervention. It is suggested that paeoniflorin may reduce the activation of microglia, inhibit neuron pyroptosis, and improve ACI-mediated neurological damage by inhibiting the activation of NLRP3 and Caspase-1 [209].

5.1.5. Butylphthalide

L-butylphthalide was first isolated from cress seeds, and later it could be synthesized artificially. Butylphthalide has a unique dual role, which can not only reconstruct microcirculation and increase ischemia reperfusion, thereby protecting the integrity of vascular structure, restoring vascular diameter, increasing blood flow in ischemic area and the number of surrounding microvessels, but also protecting mitochondria and reducing cell death, thereby protecting the integrity of mitochondrial structure, improving the activity of mitochondrial complex enzyme IV, improving the activity of mitochondrial ATP enzyme and maintaining the stability of mitochondrial membrane [210]. The latest study shows the butylphthalide may affect pyroptosis in CIRI rats in a dose-dependent manner through the NLRP3 inflammasome signaling pathway.

5.1.6. Breviscapine

Breviscapine is extracted from Erigeron breviscapus (Vant.) Hand. -Mazz. Breviscapine is a mixture of scutellarin mainly containing scutellarin with a small amount of scutellarin, which has the functions of dilating cerebral blood vessels, reducing cerebrovascular resistance, increasing cerebral blood flow, improving microcirculation, and resisting platelet aggregation. The latest research shows that breviscapine significantly inhibited the activation of NLRP3 inflammatory cells in the hippocampus of CCI rats, down-regulate the expression of Caspase 1, IL-6 and IL-1β protein, inhibit the activation of Caspase-3 protein, and inhibit neuronal apoptosis. This suggests that breviscapine may significantly improve the cognitive function of CCI rats and reduce the pathological damage of ischemic neurons, and its mechanism may be related to the inhibition of NLRP3 inflammasome activation and pyroptosis pathway in brain tissue [211].

5.1.7. Resveratrol

Resveratrol is a non-flavonoid polyphenolic compound. It was first isolated from the roots of Veratrum grandiflorum, and resveratrol has been found in more than 700 plants. Studies have shown that resveratrol plays an important role in regulating oxidative stress in cerebral infarction, inhibiting inflammation, and improving brain neuroprotection. Resveratrol regulates the pyroptosis of ischemia-reperfusion brain tissue mainly through the regulation of microglia NLRP3 inflammasome, Caspase-1 and ZO-1 [212].

5.1.8. Salvianolic acid (SAFI)

Recent studies have shown that SAFI significantly increased neurological deficit scores, reduce infarct volume, attenuate histological damage to cerebral cortex and neuronal apoptosis in MCAO/R model, increase neuronal viability and reduce neuronal apoptosis in OGD model. SAFI also remodeled the microglial polarization pattern from an M1-like phenotype to an M2-like phenotype and inhibited the activation of the NLRP3 inflammasome and the expression of NLRP3 inflammasome/pyroxia-related proteins in vitro and in vivo. This suggests that SAFI may exert neuroprotective effects by reducing neuronal apoptosis, shifting the microglial phenotype from M1 to M2, and inhibiting the NLRP3 inflammasome/pyroptotic axis in microglia [213].

5.1.9. Hispidulin

Hispidulin is a flavonoid compound with various pharmacological properties and is one of the main active components of many Chinese herbal medicines. Hispidulin has a wide range of pharmacological properties, including antioxidant, antifungalantineoplastic, antiosteoporotic, antiinflammatory and antimutagenic properties. The latest study showed that Hispidulin improved neurological symptoms in rats after IRI, while reducing infarct size and brain edema. Mechanistically, hispidulin exerts its neuroprotective effects in vivo and in vitro by inhibiting NLRP3-mediated pyroptosis by regulating the AMPK/GSK3β signaling pathway [214].

5.1.10. Astragaloside IV

Astragali Radix can supplement deficiency, strengthen the spleen and stomach, promote blood circulation and promote blood circulation, and can treat the symptoms of Qi deficiency and blood deficiency [215]Astragaloside IV is one of the main active components of Astragali Radix and the monomer component of Astragalus saponins. It is often used as a standard for testing the quality of Astragalus [216]. Astragaloside IV has a variety of biological activities, and the research scope involves multiple organs and tissues such as brain, liver, heart, lung, kidney, stomach, intestine, blood vessels, etc. [217][218][219] It has biological activities such as anti-inflammatory, anti-virus, anti-apoptosis, promoting cell proliferation, regulating rabbit disease, regulating blood sugar, slowing down aging, and inhibiting cancer [217][218][219]. Our previous study identified astragaloside IV as a potential neuroprotective agent that played an important role in the treatment of IS. For example, it can protect the blood-brain barrier, improve energy metabolism, inhibit nerve cell apoptosis, inhibit inflammatory response, oxidative stress, and exert neuroprotective effects, thereby improving cerebral ischemia [220][221][222]. In terms of pyroptosis, Tang et al. found that NLRP3 inflammasome was activated during cerebral ischemia-reperfusion in rats, and inhibiting NLRP3 inflammasome or inhibiting its downstream Caspase-1 could alleviate cerebral ischemia-reperfusion in rats damage [223]. Astragaloside IV can reduce the neurological deficit score, reduce the volume of cerebral infarction, and reduce the protein levels of NLRP3, Caspase-1, pro-IL-1β, IL-1β, pro-IL-18 and IL-18 in brain tissue, and inhibit the expression of phosphorylated NF-κB protein. This suggests that astragaloside IV has an anti-CIRI effect, and its mechanism may be related to the inhibition of NF-κB protein phosphorylation and the inhibition of NLRP3 inflammasome activation [223]. Li et al. also found that astragaloside IV can reduce hypoxia-ischemia-induced brain damage in neonatal rats, and inhibit the inflammatory response of hypoxic-ischemic brain tissue and HT22 hippocampal neurons in neonatal rats, which may be exerted by regulating MMP-9-mediated NLRP3/Caspase-1 signaling pathway [224].

5.1.11. Panax notoginseng saponins (PNS)

Notoginseng Radix Et Rhizoma is the dried root and rhizome of Panax notoginsen (Burk.) F. H. Chen, which has pharmacological activity in blood, cardiovascular, nervous and immune systems [225][226]. PNS is its main active ingredient, containing a variety of monomeric saponins, and its preparations such as Xuesaitong injection are widely used in the prevention and treatment of cardiovascular and cerebrovascular diseases [227]. Our previous studies and existing studies have shown that PNS has various pharmacological effects in ischemic brain injury, such as antithrombotic, anti-inflammatory, antioxidant, inhibiting brain nerve cell apoptosis, and improving blood-brain barrier damage [228][229][230][231]. This suggests that PNS may play a protective role in cerebral ischemia through multiple targets. Recent studies have found that PNS can inhibit the activation of the NLRP3 inflammasome and selectively promote mitophagy during cerebral ischemia-reperfusion in rats. Inhibition of mitophagy could reverse the inhibitory effect of PNS on NLRP3 inflammasome, indicating that mitophagy mediates the inhibitory effect of PNS on NLRP3 inflammasome activation in cerebral ischemia-reperfusion. In addition, the levels of PINK1 and Parkin proteins in mitochondria increased during cerebral ischemia-reperfusion in rats, and PNS could further increase the levels of PINK1 and Parkin proteins in mitochondria of brain tissue. This suggests that PNS may promote mitophagy in cerebral ischemia-reperfusion through the PINK1/Parkin pathway [232]. In summary, PNS selectively promotes mitophagy through the PINK1/Parkin pathway, inhibits the activation of NLRP3 inflammasome in cerebral ischemia-reperfusion, and alleviates cerebral ischemia-reperfusion injury.

5.2. Natural plant extract

5.2.1. Taohong Siwu Decoction (THSWD)

THSWD is one of the classic prescriptions for promoting blood circulation and removing blood stasis, removing blood stasis and regenerating new blood created by Wu Qian’s “Golden Mirror of Medicine” in Qing Dynasty. Current pharmacological studies have confirmed that the main active ingredients of THSWD include ferulic acidsafflower yellow, and polysaccharides. Among them, ferulic acid has pharmacological effects such as inhibiting platelet aggregation, antithrombotic, anti-inflammatory and anti-oxidation. Safflower yellow has anti-cerebral ischemia, anti-myocardial ischemia, anti-thrombosis, antioxidant, anti-tumor and other effects. Zhou et al. found that THSWD improved the neurological deficit function, cerebral infarction volume and brain tissue morphology after cerebral ischemia-reperfusion injury, and significantly reduced the levels of DRP1, NLRP3, Caspase-1 and IL-1 β proteins [233]. It indicated that the therapeutic effect of THSWD on cerebral infarction was related to the inhibition of DRP1/NLRP3 pathway related to pyroptosis. Further research showed that the contents of IL-1β and IL-18 in the THSWD group were significantly decreased, and the levels of NLRP3, Caspase-1, Caspase-1 p10, ASC, TXNIP, and GSDMD were decreased. The detection of signaling pathways showed that THSWD significantly reduced the expression levels of HMGB1/RAGE, TLR4/NF-κB, p38 MAPK and JNK in the penumbra. In summary, THSWD reduced the level of inflammatory response in MCAO/R rats, inhibit the activation of NLRP3 inflammasome in MCAO/R rats, and down-regulate GSDMD. THSWD has the effect of inhibiting pyroptosis, which may be affected by inhibiting HMGB1/TLR4/NF-kB and MAPK signaling pathways [234].

5.2.2. Naoxinqing Capsules (NXQC)

The main ingredient of NXQC is the leaf of Diospyros kaki Thunb (persimmon leaf). Studies have shown that persimmon leaf extract contains flavonoids, organic acids and coumarin and other chemicals, which have the effects of anti-inflammatory, antioxidant, antihypertensive, blood lipid lowering, improving vascular smooth muscle function and hemodynamic function. The drug has been widely used in the treatment of cardiovascular and cerebrovascular diseases [235][236][237]. Existing studies have shown that NXQC can effectively improve cerebral arteriosclerosis and protect nerve damage caused by cerebral ischemia [238]. Min et al. found that NXQC can effectively improve the decline of learning ability after cerebral ischemia, and increase the activities of superoxide dismutase and lactate dehydrogenase, increase the content of GSH, and reduce the content of malondialdehyde. NXQC can also down-regulate ASC, NLRP3 and Caspase-1 proteins in hippocampus, and significantly reduce IL-18 and IL-1β contents. They also found that the platelet endothelial cell adhesion molecule-1 positive cells in gerbils were significantly increased after the intervention with NXQC, and the intercellular junctions were tight. It is suggested that NXQC can effectively protect the morphology of hippocampal CA1 region of gerbils, protect cerebrovascular function, and then inhibit cerebral ischemia-reperfusion injury [239].

5.2.3. Buyang Huanwu Decoction (BYHWD) and Its Modifications

BYHWD is a famous traditional Chinese medicine formula used to treat stroke. Its prescription was first recorded in Wang Qingren’s “Medical Classics Correction” in the Qing Dynasty [240][241]. It is an outstanding representative of the prescription for nourishing qi and activating blood. It has the functions of tonifying qi, promoting blood circulation and dredging collaterals. Its evidence-based medicine studies have shown good clinical effects [242][243]. Our previous study showed that BYHWD glycosides can improve neurological dysfunction, reduce neuronal damage, and inhibit neuronal pyroptosis. BYHWD glycosides are the active ingredients extracted from BYHWD, mainly including astragaloside IV, paeoniflorin and amygdalin. It is the main pharmacological active ingredient of tonifying kidney and activating blood, and can treat cerebral ischemic nerve injury [244]. Furthermore, we observed that BYHWD glycosides significantly inhibited the expression of NLRP3, ASC, pro-caspase-1, caspase-1 and IL-1β proteins of the NLRP3-mediated canonical pyroptosis pathway [209]. In summary, BYHWD glycosides exert neuroprotective effects by inhibiting neuronal pyroptosis after CIRI, which is closely related to the regulation of classical pyroptotic pathway by NLRP3. In addition, Longzhi Decoction is an empirical formula formed by BYHWD with leeches and Achyranthes sichuanensis. It has been used in the clinical treatment of acute stroke for many years, and the effect is very good [245]. Recent studies have shown that Longzhi Decoction has obvious effects on improving neurological symptoms in rats after CIRI, maintaining the state of nerve cells in brain tissue, and reducing the volume of cerebral infarction. In terms of intervening cell pyroptosis, Longzhi Decoction can improve the symptoms of rats after CIRI and protect nerve cell damage after injury by down-regulating the expression of Caspase-1 and IL-18 proteins [245].

5.2.4. Yiqi Huoxue Prescription (YQHXP)

YQHXP consists of Angelicae Sinensis Radix, Astragali Radix, Chuanxiong Rhizoma, Scorpio, Leonuri Herba, Acori Tatarinowii Rhizoma, Borneolum Syntheticum, and has achieved good clinical results [246]. Animal studies have shown that YQHXP improved neurological function scores and cerebral infarction rates in rats, and reduce the levels of IL-1β, TNF-α, and IL-18 in brain tissue, and the relative expression of P2RX7, Caspase-1, Caspase-11, and GSDMD in cerebral ischemic penumbra tissue. After YQHXP intervention, the inflammatory exudation and edema of brain tissue were significantly reduced, and the cell morphology and neuronal vacuolar degeneration were improved. In summary, YQHXP may reduce brain tissue inflammation in acute ischemic stroke rats by inhibiting pyroptosis [247].

5.2.5. Shen Nao Fu Yuan Decoction (SNFYD)

SNFYD is an effective prescription for ischemic stroke based on the theory of simultaneous treatment of kidney and brain. Its composition is: Astragali Radix 30 gRehmanniae Radix Praeparata 10 g, Corni Fructus 10 g, Dioscoreae Rhizoma 15 g, Rhodiolae Crenulatae Radix Et Rhizoma 20 gMoutan Cortex 10 g, Angelicae Sinensis Radix 10 g, Paeoniae Radix Rubra 10 gPheretima 10 g [248]. It is combined with a large number of qi-invigorating, essence-replenishing and kidney-invigorating medicines, which are combined with activating blood and dredging collaterals. Studies have shown that after OGD modeling, PC12 was significantly damaged, and various cytokines in the pyroptosis pathway were significantly increased [249]. After SNFYD-containing serum and INF39 intervened in damaged PC12 cells, the morphology and activity of PC12 cells were improved, and the activation of the NLRP3/Caspase-1 pyroptosis pathway was weakened accordingly. It indicated that the inhibition of this pathway was related to the improvement of cell state, and both SNFYD and INF39 could inhibit the activation of this pathway. It is suggested that SNFYD may reduce the inflammatory apoptosis of nerve cells by inhibiting NLRP3/Caspase-1 and downstream pyroptosis pathway, thereby protecting nerve tissue and achieving the purpose of treating cerebral infarction [250].

5.2.6. Other extracts

In addition, recent studies have shown that antithrombotic drugs such as heparin can improve lung injury by inhibiting pyroptosis. For example, Yang et al. found that heparin inhibits lung endothelial cell apoptosis by blocking hMGB1 LPS-induced caspase-11 activation, which may be a potential way to treat sepsis-induced lung injury [251]. Prathapan et al. found that tender coconut water has antioxidant and antithrombotic effects in an experimental myocardial infarction model [252], while young coconut juice can significantly reduce some of the pathologies associated with Alzheimer’s disease [253]. It is indicated that coconut water may be a potential inhibitor of pyroptosis, and its role in regulating pyroptosis in IS may be studied in the future.

L-arginine is an organic compound that is present in protamine in large amounts [254]. It is a precursor for the synthesis of nitric oxide (NO), which protects the intact endothelium of blood vessels, acts as a vasodilator and an endogenous anti-atherosclerotic molecule [254][255]. Animal experiments show that L-arginine may have complex anticoagulation, anticoagulation and fibrinolysis effects [255][256]. In hypertensive rats, Cylwik et al. found that L-arginine could reduce blood pressure in rats, and long-term treatment shortened the euglobulin clot dissolution time and bleeding time, and inhibited collagen-induced platelet aggregation. It is suggested that L-arginine plays an antithrombotic effect in a hypertensive rat model of venous thrombosis in a complex manner [256]. A recent study showed that L-arginine may be a potential inhibitor of pyroptosis: Tanuseputero et al. suggested that L-arginine may partially inhibit the NLRP3 inflammasome to alleviate sepsis-induced acute kidney injury in mice [257]. In view of the vasodilatory, antithrombotic and inhibiting NLRP3 inflammasome activities of L-arginine, it is suggested that L-arginine may have the potential to increase blood flow in the ischemic area and inhibit pyroptosis in IS.

The summary of natural products regulating pyroptosis is showed in Table 2.

Table 2. The summary of natural compounds regulating pyroptosis.

Natural products Model/Disease Species Effects Reference
Gastrodin Middle Cerebral Artery Occlusion/Reperfusion (MCAO/R), Oxygen-glucose deprivation/reoxygenation (OGD/R) Rattus norvegicus and rimary cortical neurons from Rattus norvegicus Inhibits pyroptosis by downregulating NLRP3, inflammatory factors (IL-1β, IL-18) and cleaved caspase-1; regulates lncRNA NEAT1/miR-22–3p axis and lncRNA NEAT1/miR-22–3p axis [204]
Tanshinone IIA OGD/R BV2 cells (microglia from Mus musculus) Reduces the expression of IL-1β and IL-18 in the NLRP3 inflammatory signaling pathway [205]
Leonurine MCAO Rattus norvegicus Reduces the expression of NLRP3 [206]
Paeoniflorin MCAO Rattus norvegicus Inhibits NLRP3, Caspase-1 and IL-1β level [209]
Butylphthalide MCAO Rattus norvegicus Regulates NLRP3 inflammasome signaling pathway [210]
Breviscapine MCAO/R Rattus norvegicus Inhibits the activation of NLRP3 inflammatory cells in the hippocampus of CCI rats, down-regulates the expression of Caspase 1, IL-6 and IL-1β protein, inhibits the activation of Caspase-3 protein [211]
Resveratrol MCAO/R Rattus norvegicus Regulates microglia NLRP3 inflammasome, Caspase-1 and ZO-1 [212]
Salvianolic acid MCAO/R and OGD/R Rattus norvegicus and microglia from Rattus norvegicus Inhibites the activation of the NLRP3 inflammasome and the expression of NLRP3 inflammasome/pyroxia-related proteins [213]
Hispidulin MCAO Rattus norvegicus Inhibites NLRP3-mediated pyroptosis by regulating the AMPK/GSK3β signaling pathway [214]
Astragaloside IV MCAO/R Rattus norvegicus Inhibites the activation of NLRP3 inflammasome; reduces the protein levels of NLRP3, Caspase-1, pro-IL-1β, IL-1β, pro-IL-18 and IL-18 in brain tissue; inhibits the expression of phosphorylated NF-κB protein [223]
Panax notoginseng saponins MCAO/R Rattus norvegicus Regulates PINK1/Parkin pathway; inhibits the activation of NLRP3 inflammasome [232]
Taohong Siwu Decoction MCAO Rattus norvegicus Reduces the levels of DRP1, NLRP3, Caspase-1 and IL-1 β proteins [233]
Taohong Siwu Decoction MCAO/R Rattus norvegicus Inhibites HMGB1/TLR4/NF-kB and MAPK signaling pathways [234]
Naoxinqing Capsules MCAO/R Meriones unguiculatus Down-regulates ASC, NLRP3 and Caspase-1 proteins; Reduces IL-18 and IL-1β contents. [239]
Buyang Huanwu Decoction MCAO/R Rattus norvegicus Inhibits the expression of NLRP3, ASC, pro-caspase-1, caspase-1 and IL-1β proteins of the NLRP3-mediated canonical pyroptosis pathway [244]
Longzhi Decoction MCAO/R Rattus norvegicus Down-regulates the expression of Caspase-1 and IL-18 proteins [245]
Yiqi Huoxue Prescription MCAO Rattus norvegicus Reduces the levels of IL-1β, TNF-α, and IL-18 and the relative expression of P2RX7, Caspase-1, Caspase-11, and GSDMD [247]
Shen Nao Fu Yuan Decoction OGD PC12 cells from Rattus norvegicus Inhibites NLRP3/Caspase-1 [250]

6. Prospects

Our research team has long studied the programmed cell death mode of various groups of cells (such as neurons, microglia, and astrocytes) in the vascular neural unit after IS. Combined with other research reports, we found that various cell death modes such as apoptosis, ferroptosis, and autophagy occurred after IS, and the morphology of apoptosis and pyroptosis had certain similarities. Neuroinflammation after IS is the most important pathological process leading to brain injury, in which pyroptosis is an important part of neuroinflammation. This suggests that pyroptosis is abnormally important in neuroinflammation-related programmed cell death after cerebral ischemia. The related signaling pathways triggered by pyroptosis also intersect with other programmed cell death pathways, suggesting that drug development targeting pyroptosis is an important way to treat IS.

For the current study, there are still areas for improvement: (1) Due to the problems of normalization and unification in the animal model of the current study, the conclusions about pyroptosis in different cells of the nervous system are inconsistent. Therefore, in the future research on pyroptosis requires a more standardized and unified stable model in animal models of ischemic stroke, which can also determine the real effect of anti-pyroptosis drugs. (2) Future studies can verify the long-term brain-protective effects of natural compounds and the mechanisms regulating pyroptosis in a variety of rodent and large mammal stroke models. In the future, more attention should be paid to the pharmacokineticspharmacodynamics, and toxicological properties of natural compounds that currently modulate pyroptosis. (3) In addition, the synergistic effect of the combination of natural compounds in regulating pyroptosis in cerebral ischemic stroke and the neuroprotective effect of inhibiting neuroinflammation need to be explored in the future. (4) If all results are favorable, the next step is to conduct clinical trials of potential phytochemicals to investigate their neuroprotective effects on cerebral ischemia/stroke (For example, the team is currently conducting a clinical trial of Naotai Fang in the treatment of cerebral small vessel disease: ChiCTR1900024524).

7. Summary

Pyroptosis, as a pro-inflammatory programmed cell death, plays an important role in the pathological process of ischemic stroke by inducing cell death and neuroinflammation mainly through the classical pyroptotic pathway mediated by Caspase-1. The relationship between non-canonical pyroptotic pathways and IS remains to be elucidated. Other mechanisms for pyroptosis require further study in the future. At present, studies have confirmed that drugs targeting key proteins of the pyroptosis pathway can alleviate ischemic brain injury to a certain extent, but their research is mainly limited to cell and animal experiments, and there is a lack of clinical research evidence. Therefore, further clinical research to explore the regulatory mechanism of pyroptosis in IS is expected to provide novel therapeutic strategies and theoretical basis for the prevention and treatment of ischemic stroke. Regarding the regulation of pyroptosis by natural compounds, through our generalization, it can be found that natural plant compounds can regulate cerebral ischemia by regulating pyroptosis not only on a single component. Multicomponent natural plant compounds can also be seen to exhibit potential synergistic effects in modulating the inflammatory cascade triggered by pyroptosis. This inspires us to study the regulatory effects of various natural compounds on pyroptosis-mediated neuroinflammation caused by different signaling pathways in the future.

CRediT authorship contribution statement

Kailin Yang: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft; Liuting Zeng: Conceptualization, Methodology, Software, Formal analysis, Investigation, Writing – original draft; Jinsong Zeng: Methodology, Formal analysis, Investigation; Tingting Bao: Methodology, Formal analysis, Investigation, Writing – original draft; Xiao Yuan: Methodology, Formal analysis, Investigation; Shanshan Wang: Methodology, Formal analysis, Investigation; Wang Xiang: Methodology, Formal analysis, Investigation; Hao Xu: Methodology, Formal analysis, Investigation; Jinwen Ge: Conceptualization, Methodology, Formal analysis, Investigation, Writing – review & editing.

Conflict of Interest Statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


This work is supported by the National Natural Science Foundation of China (81774174), the National Key Research and Development Project of China (No. 2018YFC1704904), National Natural Science Foundation of Hunan Province, China (2020JJ5424 and 2020JJ5442), Hunan University of Chinese Medicine “Double First-Class” Discipline Open Fund Project of Integrated Traditional Chinese and Western Medicine (2020ZXYJH08 and 2020ZXYJH09), Hunan Provincial Department of Education Youth Fund Project (21B0386).

Availability of data and materials

The data used to support the findings of this study are included within the article.


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