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

2.1. The characteristics of pyroptosis

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].

2.3. 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.

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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 synthase: endothelial 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 cells. Gene 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 injury. TLR4 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 neurogenesis. Positron 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.

3.1. 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 protein, heat shock protein, peroxidase 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 claudin, adhesion 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.

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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].

5.1. Natural plant components

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

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

5.2. Natural plant extract