The Role of NLRP3 Inflammasome in Cerebrovascular Diseases Pathology and Possible Therapeutic Targets

Pathology of the Ischaemic Core: Oxidative Stress and Excitatory Toxicity

In the ischaemic core, the decreased blood flow leads to insufficient oxygen and glucose supply in the brain tissue, triggering a series of cascading reactions, including sodium-potassium pump dysfunction, extensive depolarisation of neuronal cell membranes, activation of glutamate and voltage-gated calcium channels, the influx of calcium ions in neurons, and release of glutamate into the synaptic cleft (Zhang et al., 2006; Rashidian et al., 2007; Alishahi et al., 2019). Intracellular calcium overload promotes nitric oxide and ROS production, mitochondrial dysfunction, endoplasmic reticulum stress responses, activation of calpain proteases, and ultimately neuronal death (Rashidian et al., 2007; Hossmann, 2009; Ramos-Cabrer et al., 2011). Oxidative stress plays an important role in neuronal injury following cerebral ischaemia. Several factors can lead to oxidative stress during cerebral ischaemia, including intracellular calcium, zinc, extracellular glutamate elevation, anaerobic glycolysis, and mitochondrial dysfunction (Hossmann, 2009; Alishahi et al., 2019). Oxidative stress can mediate neuronal death by destroying mitochondria, the endoplasmic reticulum, and cellular components, promoting calcium overload, excitotoxicity, reperfusion injury, and inflammation (Hossmann, 2009; Lakhan et al., 2009; Alishahi et al., 2019).

Glutamate is an excitatory neurotransmitter. Following the release of glutamate into the synaptic cleft, the presynaptic neuronal reuptake of glutamate occurs. Glutamate reuptake is an energy-intensive process, and energy consumption leads to severe disruption of glutamate transporters, causing excitotoxic effects that trigger neuronal death (Castillo et al., 2016; Perez-Mato et al., 2019). Glutamate can also overstimulate glutamate receptors, promote calcium ion influx, and induce mitochondrial ROS production (Castillo et al., 2016; Alishahi et al., 2019; Perez-Mato et al., 2019).

Ischaemic Penumbra—The Region Whose Progression Is Affected by the Ischaemic Core

Penumbra formation is influenced by several factors, including excitotoxicity, peri-infarct depolarisations, spreading depression, lactacidosis, microcirculatory disturbances, and flow-metabolism uncoupling (Back and Schuler, 2004; Nedergaard and Dirnagl, 2005; Lakhan et al., 2009). In the ischaemic core, glutamate can mediate peri-infarct depolarisations or spreading depression, cause acidosis, increase energy demand and neurotransmitter leakage, affect the survival of penumbral cells, and gradually expand the infarct size (Ramos-Cabrer et al., 2011). The ischaemic core can also promote penumbral cell death through numerous mechanisms, including oxidative stress, apoptosis, inflammatory cytokine release, adhesion molecule expression, and matrix metalloproteinase production (Ramos-Cabrer et al., 2011). Penumbral tissues can tolerate several hours of hypoperfusion, and neurological function can be fully restored with timely restoration of blood flow (Lakhan et al., 2009); Therefore it is crucial to restore blood flow before irreversible damage occurs to penumbral tissues.

Post-Ischaemic Inflammation—A Double-Edged Sword

Post-ischaemic inflammation is a dynamic process involving various inflammatory cells, chemokines, and proteases, which play an important role during the course of cerebral ischaemia. Post-ischaemic inflammation is a double-edged sword, which not only aggravates cerebral oedema in the early stage of ischaemia but also promotes tissue recovery during the recovery stage (Zheng and Yenari, 2004; Shichita et al., 2012). During the early stages of ischaemia, excitotoxic events and cytotoxic substances appear to be involved in the induction of an in situ post-ischaemic inflammatory reaction (Zheng and Yenari, 2004). In situ inflammation induces the expression of adhesion molecules on endothelial cells of the blood-brain barrier, which further promotes the migration and adhesion of white blood cells from the peripheral blood into brain tissues (Lakhan et al., 2009). Increased expression of chemokines is also involved in the aggregation of inflammatory cells (Lakhan et al., 2009). The aggregation and adhesion of inflammatory cells can damage the integrity of the blood-brain barrier endothelial cells and microvessels. Furthermore, proinflammatory cells can increase the blood-brain barrier permeability directly through endothelial cells (Lakhan et al., 2009; Shichita et al., 2012). Increased matrix metalloproteinases reportedly impair the blood-brain barrier integrity after stroke and are associated with vasogenic oedema (Lakhan et al., 2009). During the recovery period, inflammatory cells infiltrating the brain tissue can remove necrotic cells and tissue fragments, promoting tissue repair and regeneration (Shichita et al., 2014).

Pathology of Early Intracerebral Haemorrhage: Toxicity of Blood, Oxidative Stress, Brain Edema and Mass Effect

Primary intracerebral haemorrhage occurs in the absence of vascular malformation or brain parenchymal lesions, blood vessel rupture, blood into the brain parenchyma, and may spread to the ventricular system; simultaneously, blood components such as leucocytes, haemoglobin, thrombin, plasmin, complement components, plasma, and fibrin degradation products enter the brain parenchyma (Bai et al., 2020). A few minutes after intracerebral haemorrhage occurrence, toxic components in the blood, such as blood-derived coagulation factors, complement components, immunoglobulins, and other bioactive molecules, present a potent toxic effect on brain cells and promote inflammation and oxidation (Aronowski and Zhao, 2011). After 24 h, red blood cell lysis produces cytotoxic haemoglobin, haem, and iron, which also induce intense oxidative damage to the brain cells (Aronowski and Zhao, 2011). In the early stage of intracerebral haemorrhage, serum protein deposition can lead to cytotoxic oedema, whereas in the late stage, cerebral oedema is mainly vasogenic (Badjatia and Rosand, 2005). The mass effect of haematoma and secondary edema can lead to tissue compression and displacement. Compression of adjacent blood vessels results in local hypoperfusion of brain tissues (Kingman et al., 1987), resulting in pathological changes similar to those in the penumbra area of cerebral infarction.

Secondary Brain Injuries: Toxic Effects of Heme Metabolites and Thrombin

Secondary brain injuries include inflammatory responses, oxidative stress, cytotoxicity and excitotoxicity (Zhu, Wang, et al., 2019). Haem in erythrocytes is released into the brain tissue and metabolised by haem oxygenases. Metabolite iron can result in neuronal loss around blood clots through oxidative damage (Egashira et al., 2015; Dang et al., 2017). Thrombin produced immediately after intracerebral haemorrhage is not only involved in the hemostasis cascade reaction but also the formation of brain oedema. High-dose thrombin can induce inflammation, excitotoxicity, apoptosis, complement system activation, mesenchymal cell proliferation, scar tissue formation, and increase the blood-brain barrier permeability, while extremely low-dose thrombin has a protective effect (Lee et al., 1997; Xi et al., 2006; Zhu, Wang, et al., 2019). Damage to brain tissue by the thrombin-activated complement system is associated with the formation of membrane attack complexes and typical inflammatory responses (Xi et al., 2006). After intracerebral haemorrhage, microglial activation induces an inflammatory response by releasing matrix metalloproteinases, ROS, active nitrogen, inflammatory cytokines, and chemokines, inducing extensive neuronal death (Bai et al., 2020).

Pathology of Early Subarachnoid Hemorrhage—A Vicious Circle Formed by Increased Intracranial Pressure, Hypoperfusion, Cerebral Edema, and Neuroinflammation

SAH is a condition in which blood enters the subarachnoid space of the brain and spinal cord. Blood release, acute reactive congestion, and acute vasospasm during the first 72 h after a SAH can cause early brain damage including increased intracranial pressure, cerebral edema, neuroinflammation, oxidative stress, cell death, neuronal dysfunction, blood-brain barrier disruption, microvascular changes, and transient global cerebral ischaemia (Cahill et al., 2006; Zheng and Wong, 2017; Pang et al., 2019); these pathological changes are mutually promoting. The blood entering the subarachnoid space, together with vasoparalysis and obstruction of cerebrospinal fluid drainage, contribute to increasing the intracranial pressure. Increased intracranial pressure, cerebral vasospasm, and inflammatory cascades lead to whole-brain hypoperfusion (Cahill et al., 2006; Tso and Macdonald, 2014). NLR correlates with cerebral perfusion in the early phase of SAH (Wu et al., 2019). After SAH occurs, microthrombosis formed by platelet aggregation can also aggravate local cerebral hypoperfusion (Tso and Macdonald, 2014; Pang et al., 2019). Whole-brain hypoperfusion can cause a series of pathological changes, including neuronal cell and vascular endothelial cell death, sodium-potassium pump dysfunction, activation of inflammation and apoptosis cascade, oxidative stress response, and matrix metalloproteinase-9 activation, all of which can induce the destruction of the blood-brain barrier and lead to brain oedema (Cahill et al., 2006; Tso and Macdonald, 2014; Pang et al., 2016; Pang et al., 2019). Brain oedema further increases intracranial pressure, forming a vicious circle. Neuroinflammation plays an important role in early brain injury. Following the occurrence of SAH, microglia are activated and release inflammatory factors, which further activate glial cells and aggravate brain injury. Whole-brain hypoperfusion, oxidative stress, and blood-brain barrier destruction also increase neuroinflammatory responses (Pang et al., 2019).

Delayed Brain Injury: Cerebral Vasospasm, Inflammatory Cascade, Epilepsy, Rebleeding, and Hydrocephalus

SAH can result in delayed brain injury, including cerebral ischaemia associated with cerebral vasospasm, seizures, rebleeding, and hydrocephalus (Welty and Horner, 1990; Miller et al., 2014). The pathology of delayed cerebral ischaemia includes angiographic vasospasm, arteriole contraction, microthrombosis, cortical diffusion depolarisation, and self-regulation dysfunction (Macdonald, 2014). These factors cause cerebral vasospasm, which in turn causes delayed cerebral ischaemia (Macdonald, 2014; Veldeman et al., 2020). Inflammation plays a vital role in both early and delayed brain injury. Degradation products of red blood cells, such as haemoglobin, methaemoglobin, oxyhaemoglobin haem, and haem, increase oxidative stress in brain tissues, triggering an inflammatory cascade (Welty and Horner, 1990; Zheng and Wong, 2017). Inflammatory cascades, neuronal excitotoxicity, and blood-brain barrier disruption can lower the seizure threshold and increase susceptibility to epilepsy (Wang, Liang, et al., 2021). Rebleeding after SAH is associated with hypertension. Some studies have observed that larger aneurysms are a greater risk of rebleeding (van Donkelaar et al., 2015). However, few reports have found that the rebleeding rate of small, ruptured aneurysms is not lower than that of large, ruptured aneurysms (Zheng et al., 2019). Hydrocephalus after SAH is associated with inflammation, apoptosis, autophagy, oxidative stress and blood-brain barrier destruction (Chen, Luo, et al., 2017).

In general, cerebrovascular diseases are accompanied by oxidative stress. Oxidative stress is one of the main activators of NLRP3 inflammasome activation. Therefore, the generation of oxidative stress may be the main mechanism of activation of NLRP3 inflammasome in cerebrovascular diseases. Finally, activation of NLRP3 inflammasomes aggravates cerebrovascular diseases through multiple pathways.

NLRP3 Inflammasome Is Closely Related to the Development of Atherosclerosis

Cholesterol crystals and oxidised low-density lipoprotein (ox-LDL) act as stimuli, activate the NLRP3 inflammasome and the oxidative stress response and cause an inflammatory response, promoting atherosclerosis lesion development (Freigang et al., 2011; Li et al., 2014; Paramel Varghese et al., 2016; Abdul-Muneer et al., 2017; Liu, Zeng, et al., 2018). In atherosclerotic plaques, mRNA expression levels of P2X7R, NLRP3 inflammasome, ASC, caspase-1, IL-1β, and IL-18 are significantly increased when compared with those in normal arteries (Xiao et al., 2013; Peng et al., 2015; Paramel Varghese et al., 2016). Numerous studies have shown that enlarged atherosclerotic lesions are accompanied by the activation of the NLRP3 inflammasome (Yamaguchi et al., 2015; Tumurkhuu et al., 2016; Couchie et al., 2017; Tumurkhuu et al., 2018; Wu et al., 2018), and suppression of the NLRP3 inflammasome and ROS attenuates atherosclerotic plaques (Leng et al., 2016). In Nlrp^-/- mice, atherosclerosis stimuli such as cholesterol crystals and ATP were unable to induce IL-1β release; accordingly, atherosclerotic plaques were alleviated (Abderrazak et al., 2015; Deng et al., 2015; Bode et al., 2016; He, Wang, et al., 2016; Leng et al., 2016; Zhou et al., 2016; Fuster et al., 2017; Ma et al., 2018; Zhang, Han, et al., 2018; Zhang, Liu, et al., 2018). These results indicate that the NLRP3 inflammasome-mediated inflammatory response accelerates the formation and development of atherosclerosis. Herein, we explore the role of the NLRP3 inflammasome in atherosclerosis.

NLRP3 Inflammasome Increases the Susceptibility of Macrophages to Lipid Deposition and Promotes Macrophage Recruitment and Transfer Into Foam Cells

Macrophage recruitment and transfer into foam cells is the key process involved in the development of atherosclerosis, and high-density lipoproteins (HDL) play a protective role in atherosclerosis by suppressing inflammatory monocyte cell recruitment and IL-1β secretion (Thacker et al., 2016). CD68-positive macrophages are reportedly located in atherosclerotic lesions with NLRP3 inflammasome expression (Paramel Varghese et al., 2016). NLRP3 inflammasome activation accelerates neutrophil and macrophage recruitment and neutrophil extracellular trap formation, increases the susceptibility of macrophages to lipid deposition, and promotes foam cell formation (Figure 2), while Nlrp3^-/- reduces the atherosclerotic lesion size, prevents plaque progression and reduces macrophage infiltration (Li et al., 2014; Zheng et al., 2014; Leng et al., 2016; Tumurkhuu et al., 2016; Wang et al., 2017; Wang, Wu, et al., 2018; Westerterp et al., 2018; Yan et al., 2018). Furthermore, inhibition of the NLRP3 inflammasome plays an anti-inflammatory role by promoting M1 macrophage transfer into the M2 phenotype (Abderrazak et al., 2015). The downstream factor, IL-1β, of the NLRP3 inflammasome promotes foam cell formation. Ox-LDL upregulates the expression of pro-IL-1β and ROS induced by phagocytosis of ox-LDL through the cathepsin B pathway to activate the NLRP3 inflammasome, induce macrophages to secrete IL-1β, and promote macrophage transfer into foam cells during atherosclerosis (Figure 2) (Jiang et al., 2012; Ding et al., 2014). IL-1β inhibits cholesterol efflux through negative feedback, resulting in the accumulation of intracellular cholesterol and foam cell formation (Tumurkhuu et al., 2018).

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Figure 2.

Main Mechanism of NLRP3 Inflammasome in Development of Atherosclerosis. The phagocytosis of ox-LDL generates ROS through the cathepsin B pathway to activate the NLRP3 inflammasome. Activation of the NLRP3 inflammasome accelerates neutrophil and macrophage recruitment, increases the susceptibility of macrophages to lipid deposition, promotes foam cell formation, induces macrophages secreting IL-1β, and impairs plaque stability. Rupture of atherosclerotic plaques can cause stroke. Ox-LDL also upregulates the expression of the pro-IL-1β. IL-1β inhibits cholesterol efflux through a negative feedback, results in accumulation of intracellular cholesterol and foam cell formation. CD36 converts intracellular soluble ligands into crystals or fibrils, while UDCA increases cholesterol solubility, decreases cholesterol crystals-depositions, and inhibits NLRP3 inflammasome dependent inflammation. HDL play a protective role in atherosclerosis by suppressing monocyte cell recruitment and IL-1β secretion. Abbreviations: ox-LDL: oxidized low-density lipoprotein, ROS: reactive oxygen species, UDCA: ursodeoxycholic acid, HDL: high-density lipoproteins.

NLRP3 Inflammasome Influences Cholesterol Solubility and Disrupts Atherosclerotic Plaque Stability

In atherosclerotic mice, cholesterol crystals that accumulate in atherosclerotic plaques activate the NLRP3 inflammasome in phagocytes. Thus, decreasing cholesterol crystal deposition in atherosclerotic plaques by employing ursodeoxycholic acid (UDCA) could increase cholesterol solubility in macrophages, impair NLRP3 inflammasome-dependent inflammation, and diminish atherosclerotic plaque development (Figure 2) (Bode et al., 2016). In atherosclerosis, an increase in the pattern recognition receptors CD36 is accompanied by NLRP3 inflammasome activation (Du et al., 2018). Studies have reported that CD36 converts intracellular soluble ligands into crystals or fibrils (Duewell et al., 2010; Sheedy et al., 2013), and targeting CD36 downregulated serum IL-1β concentrations and suppressed cholesterol crystal accumulation in plaques (Sheedy et al., 2013). NLRP3 inflammasome activation also disrupts plaque stability, whereas atherosclerotic plaque rupture leads to stroke (Hutcheson et al., 2014). Studies have observed that inhibition of the NLRP3 inflammasome enhances atherosclerotic plaque stability, reduces plaque volume and maximal stenosis, as well as the average plaque size of atherosclerotic lesions, preventing plaque progression (Zheng et al., 2014; Leng et al., 2016; van der Heijden et al., 2017; Ma et al., 2018). It can be seen that the improvement of atherosclerosis induced by inhibition of NLRP3 inflammasome is also beneficial for cerebrovascular diseases.

NLRP3 Inflammasome Is Associated with Increased Proinflammatory Cytokines and Inflammatory Infiltration

Ischaemia/reperfusion or oxygen-glucose deprivation/reoxygenation can induce the first-step signal of NLRP3 activation, resulting in increased proinflammatory cytokines and NLRP3 expression in injured tissues (Ito et al., 2015; Wang, Wang, et al., 2015; Chen, Dixon, et al., 2018; Lemarchand et al., 2019; Zhang, Zhao, et al., 2020). Reportedly, the NLRP3 inflammasome was first activated in microglia cells after brain ischaemia/reperfusion injury and then expressed in neurons and microvascular endothelial cells, but mainly in neurons (Gong et al., 2018). The NLRP3 inflammasome not only releases inflammatory cytokines such as IL-1β and IL-18, but also promotes microglial activation, infiltration of neutrophils, and the aggravates neurotoxicity of microglia in ischaemia/reperfusion (Figure 3) (Wang, Li, et al., 2015; Zhao et al., 2015; Li, Wang, et al., 2016; Lu et al., 2016; Qiu et al., 2016; Yu et al., 2017; Chen, Xu, et al., 2018; Liu, Cen, et al., 2018; Ma et al., 2019; Sha et al., 2019; She et al., 2019; Tang et al., 2019; Wang et al., 2019). A recent discovery has confirmed that Smad6 is a target for NLRP3 to interact with microglia cells in neonatal rat hypoxic-ischaemic encephalopathy model (Chen, Hu, et al., 2020). Chen et al. (2020)'s findings suggest that Smad6 may be a target for the treatment of nervous system diseases by intervening NLRP3 inflammasome (Chen, Hu, et al., 2020).

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Figure 3.

The Mechanism Underlying NLRP3 Inflammasome Aggravation of Cerebrovascular Diseases. NLRP3 inflammasomes are activated after brain injury. Activation of NLRP3 inflammasome aggravates brain edema by increasing inflammatory cell infiltration, destroying tight junction proteins, and enhancing permeability of brain microvessel endothelial cell. Inhibition of NLRP3 inflammasome with inhibitors or RNAi reduces proinflammatory cytokines levels, promotes the transformation of M1-type microglia cells to M2-type, inhibits the aggregation and activation of inflammatory cells, and reduces myeloperoxidase level. M2-type microglia have anti-inflammatory effects, and the level of anti-inflammatory cytokines is up-regulated after phenotypic transformation of microglia. Inhibition of NLRP3 inflammasome also increases tight junction proteins expression, reduces cerebral edema and blood-brain barrier permeability, reduces neuronal degeneration and apoptosis, and reduces microthrombosis. All of these lead to improvement of neurological functions.

NLRP3 Inflammasome Is Associated With Increased Blood–Brain Barrier Permeability and Brain Oedema

The blood-brain barrier is formed by specialised brain endothelial cells interconnected by tight junctions. Tight junctions in the blood-brain barrier are essential for maintaining the microenvironment (Sozen et al., 2009). Brain oedema mediated by the NLRP3 inflammasome is associated with increased blood-brain barrier permeability. Studies have shown that the NLRP3 inflammasome enhances the permeability of brain microvessel endothelial cells via IL-1β (Yang et al., 2014) and downregulates the expression of tight junction proteins, occludin and zona occludens-1, after ischaemia/reperfusion injury (Figure 4) (Cao et al., 2016; Qu et al., 2019). Occludin degradation renders the blood-brain barrier more vulnerable to reperfusion injury in vitro (Zhang, Li, et al., 2020). A recent study observed that the NLRP3 inflammasome influences the distribution of aquaporin-4 in the infarct area (Wang, Chen, et al., 2020). Similarly, diabetes reportedly stimulates NLRP3 inflammasome activation, disrupts the polarity of aquaporin-4 and increases the blood-brain barrier permeability after ischaemia (Ward et al., 2019). Moreover, NLRP3 inflammasome-mediated pyroptosis is related to brain oedema following ischaemia/reperfusion (An et al., 2019). The mechanisms underlying the association between pyroptosis and brain oedema warrant further study. In ischaemic tissues, recruited neutrophils can contribute to dramatic blood-brain barrier disruption and tissue damage, ultimately resulting in haemorrhagic transformation (Guo et al., 2018). NLRP3 inflammasome may disrupt the blood-brain barrier and promote brain oedema through various factors. Inhibition of NLRP3 inflammasome is expected to reduce the degradation of tight junction protein in blood-brain barrier and the permeability of blood-brain barrier, thus having a protective effect on cerebrovascular diseases.

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Figure 4.

Signalling Pathways of NLRP3 Inflammasome Activation in Cerebrovascular Diseases. Hypoxia and membrane damage induce mtROS production. ROS leads to NLRP3 inflammasome activation and IL-1β secretion through inducing the binding of TXNIP to NLRP3. ROS also activates NLRP3 inflammasome via NF-κB and apoptosis. The mitophagy/autophagy system removes mtROS and suppresses NLRP3 inflammasome activation. NF-κB regulates both pyroptosis and oxidative stress, it also influences macrophage differentiation into M1 or M2 cells. IFN-β inhibits NLRP3 in three pathways: (A) represses the activity of the NLRP3 inflammasome via the signal transducer and activator of transcription (STAT)1 transcription factor; (B) induces IL-10 in a STAT1-dependent manner, while IL-10 reduces pro-IL-1β production via STAT3 signalling; (C) suppresses ROS generation in antigen presenting cells. The opening of P2X7R allows potassium ions to efflux from cells and sodium and calcium ions to influx into cells. The NLRP3 inflammasome is activated when the intracellular potassium ion level is below the threshold of 90 Mm. ATP leads to neuroinflammation through activating the P2X7R/cryopyrin inflammasome axis. Ox-LDL upregulates the expression levels of P2X7R, NLRP3 inflammasome and IL-1β via phosphorylation of protein kinase R. Abbreviations: mtROS: mitochondrial ROS, ROS: reactive oxygen species, TXNIP: thioredoxin-interacting protein, IFN-β: interferon-β, STAT: signal transducer and activator of transcription, P2X7R: purinergic receptor-7.

NLRP3 Inflammasome Regulates Immune Cells and Cytokines

Microglial activation is critical for intracranial haemorrhage-induced secondary brain injury (Li, Wang, et al., 2018). Activated microglia release cytokines in response to haemorrhagic stroke and ultimately lead to neuroinflammation and brain injury. Disturbing the co-localisation of the NLRP3 inflammasome with microglial cells improved neurological functions (Yang, Sun, et al., 2018), and NLRP3 inflammasome expression increased in microglia in a time-dependent manner; NLRP3 inflammasome increases gradually and peaks at day 5 after intracranial haemorrhage (Yao et al., 2017; Miao et al., 2020). Suppression of the NLRP3 inflammasome is followed by inhibition of microglial accumulation and activation, as well as amelioration of neurological dysfunction post intracranial hemorrhage (Figure 3) (Zhao, Pan, et al., 2017; Li, Wang, et al., 2018; Wang, Nowrangi, et al., 2018). Furthermore, selective inhibition of caspase-1 could promote the polarisation of M1-type microglia into M2-type around the haematoma and ameliorate neurological dysfunction of limb movement (Figure 3) (Lin et al., 2018). The role of NLRP3 inflammasome in intracerebral haemorrhage is not only related to microglia, but also related to the release of inflammatory factors.

P2X7R plays a damaging role in intracranial haemorrhage through NLRP3 inflammasome-dependent neutrophil infiltration and IL-1β and IL-18 release (Feng et al., 2015). Reportedly, the suppression of the NLRP3 inflammasome downregulates levels of myeloperoxidase, a heme-containing peroxidase expressed primarily in neutrophils and monocytes, and inhibits recruitment and infiltration of leucocytes and neutrophils (Ma et al., 2014; Guo et al., 2018; Wang, Nowrangi, et al., 2018). Furthermore, inhibition of NLRP3 inflammasome attenuates neurological deficits, downregulates cytokine levels, including IL-1β, IL-6, TNF-α and lactate-dehydrogenase (Yang et al., 2015; Cheng et al., 2017; Zeng et al., 2017; Li, Wang, et al., 2018; Lin et al., 2018; Ren et al., 2018; Xu, Shen, et al., 2019). The proinflammatory effect of NLRP3 inflammasome may be related to neuroinflammation in intracerebral haemorrhage. Inhibition of NLRP3 inflammasome is beneficial to reduce neuroinflammation and the subsequent pathological changes caused by neuroinflammation.

NLRP3 Inflammasome Is Involved in Blood–Brain Barrier Disruption

NLRP3 inflammasome expression is accompanied by blood-brain barrier component destruction in delayed recombinant tissue-type plasminogen activator-induced haemorrhagic transformation, while NLRP3 shRNA decreases haemorrhage score and haemorrhage volume during haemorrhagic transformation (Guo et al., 2018). Neutrophil recruitment may be the mechanism underlying of NLRP3 knockdown-attenuated haemorrhagic transformation and motor deficits (Ma et al., 2014). IL-1β injected into the brain parenchyma increased the blood-brain barrier permeability. The IL-1β-induced increase in blood-brain barrier permeability was neutrophil-dependent (Blamire et al., 2000). In intracranial haemorrhage, accumulating evidence shares a similar conclusion that inhibition of the NLRP3 inflammasome maintains the blood-brain barrier integrity, attenuates brain oedema and alleviates neurobehavioral disorders in intracranial haemorrhage (Ma et al., 2014; Feng et al., 2015; Cheng et al., 2017; Yao et al., 2017; Zeng et al., 2017; Li, Wang, et al., 2018; Ren et al., 2018; Yang, Sun, et al., 2018; Xu, Shen, et al., 2019; Miao et al., 2020). Yang et al. (2015) reported that miR-223 inhibited NLRP3 expression, consequently relieving brain oedema via 3′ UTR sites on NLRP3 mRNA in intracranial hemorrhage (Yang et al., 2015). The destruction of the blood-brain barrier by NLRP3 inflammasome in intracerebral haemorrhage may be related to inflammatory infiltration. Inhibition of NLRP3 inflammasome is helpful to reduce cerebral edema after intracerebral haemorrhage.

NLRP3 Inflammasome Is Associated With Neuronal Degeneration and Apoptosis

Intracranial haemorrhage is associated with the activation of different cell death pathways. These cell death pathways lead to the removal of inactivated and damaged cells, resulting in neuronal cell damage (Lin et al., 2018). A previous study observed that inhibition of NLRP3 inflammasome assembly reduces neuronal cell degeneration and ameliorates histological damage (Cheng et al., 2017; Zeng et al., 2017; Li, Wang, et al., 2018), as well as decreases apoptosis in intracranial haemorrhage (Figure 3) (Lin et al., 2018; Xu, Shen, et al., 2019).

NLRP3 Inflammasome Is Involved in Inflammatory Infiltrates, Autophagy, and Apoptosis

In SAH, inhibition of the NLRP3 inflammasome upregulates the rate of survival and surviving neurons (Dong et al., 2016; Cao et al., 2017). Suppression of NLRP3 reportedly decreases neutrophil infiltration, microglial activation, and production of proinflammatory factors (Figure 3) (Yin et al., 2018). The NLRP3 inflammasome may also be associated with autophagy and apoptosis. Li et al. (2017) reported that NLRP3 inhibition increased autophagy marker beclin-1 expression (Li et al., 2017), and caspase-1 inhibition reduced neural necrotic cell death in SAH (Li et al., 2017). In another study, Li et al. (2016) observed that minocycline inhibited NLRP3 inflammasome activation and repressed neural apoptosis induced by P53-associated apoptotic proteins in early brain injury following SAH (Li, Chen, et al., 2016).

Blocking of NLRP3 Inflammasome Improves the Blood–Brain Barrier Permeability and Relieves Brain Oedema

NLRP3 inflammasomes are associated with disruption of the blood-brain barrier in SAH. ATP activates the NLRP3 inflammasome through the P2X7R/NLRP3 inflammasome axis, exacerbating neurological deficits and brain oedema following SAH (Chen et al., 2013). Inhibition of the NLRP3 inflammasome improved the blood-brain barrier permeability, reduced the brain water content, and exerted neuroprotective effects after SAH (Figure 3) (Li, Chen, et al., 2016; Li et al., 2017; Liu et al., 2017; Zhou et al., 2017). Additional studies have observed that NLRP3 inflammasome inhibition is accompanied by increased tight junction protein expression (Dong et al., 2016; Yin et al., 2018). The effect of NLRP3 on the blood-brain barrier may be attributed to the regulation of IL-1β-induced matrix metalloproteinase-9 expression, which opens the blood-brain barrier by degrading tight junction proteins (Sozen et al., 2009).

Inhibition of NLRP3 Inflammasome Reduces Microthrombosis

A previous study reported that SAH induced microcirculatory dysfunction and microthrombosis in vivo (Friedrich et al., 2012). Microcirculatory dysfunction contributes to delayed cerebral ischaemia following SAH, and the number of microthrombi correlates with the number of apoptotic neuronal cells and prognosis of SAH (Sabri et al., 2012). Zuo et al. (2020) found that inhibition of the NLRP3 inflammasome reduced microthrombosis and improved neurobehavior following SAH (Figure 3) (Zuo et al., 2020). Currently, research on the NLRP3 inflammasome and microthrombosis remains scarce, and the study of microthrombosis is of great significance to improve the prognosis of SAH.

It appears that the NLRP3 inflammasome plays a crucial role in cerebrovascular diseases and atherosclerosis, and inhibition of NLRP3 inflammasome activation may have therapeutic significance in cerebrovascular diseases. Understanding the activation pathway of the NLRP3 inflammasome in cerebrovascular diseases can help assess suitable NLRP3 inflammasome inhibitors.

ROS/NF-κB/NLRP3 Inflammasome Pathway-Signalling Pathway Involved in the Priming

NF-κB is a heterodimeric protein involved in inflammatory responses (Sabir et al., 2017). Activation of NF-κB in brain injury plays an important role in the priming of the NLRP3 inflammasome, increasing the mRNA level of NLRP3 and creating conditions for NLRP3 inflammasome activation. Suppression of the NF-κB/NLRP3 inflammasome pathway plays a protective role in both haemorrhagic and ischaemic stroke (Li, Wang, et al., 2016; Shao et al., 2016; Zeng et al., 2017; Chen, Xu, et al., 2018; Fann et al., 2018; Li, Wang, et al., 2018; Liu, Cen, et al., 2018; Yin et al., 2018; Tang et al., 2019; Miao et al., 2020).

ROS induced by SAH and intracranial hemorrhage activates NF-κB, thus translocating the p65 subunit into the nucleus and increasing the mRNA levels of NLRP3 and proinflammatory cytokines (Figure 4) (Shao et al., 2016). Activation of the ROS/NF-κB/NLRP3 inflammasome led to early brain injury after SAH and intracranial hemorrhage (Shao et al., 2016), while inhibition of the ROS/NF-κB/NLRP3 inflammasome pathway improved neurological deficits following ischaemia/reperfusion injury (Zhang, Zhao, et al., 2020). Zeng et al. (2017) reported that nuclear factor E2-related factor-2 (Nrf2) is upstream of the ROS/NF-κB/NLRP3 inflammasome pathway or ROS/NLRP3 inflammasome pathway in intracranial haemorrhage (Zeng et al., 2017). Xu et al. (2018) observed that Nrf2 activation decreased ROS, inhibiting NLRP3 inflammasome activation in oxygen-glucose deprivation/reoxygenation-induced BV2 cells (Xu, Zhang, et al., 2018). However, the mechanism associating Nrf2 with the ROS/NF-κB/NLRP3 inflammasome pathway in cerebrovascular diseases remains to be elucidated. A previous report has observed Nrf2 activation attenuated oxidative stress and neuronal inflammation via the nicotinamide adenine dinucleotide phosphate oxidase 4/ROS/NF-κB pathway (Saha et al., 2020). Nrf2 negatively regulates the NF-κB signalling pathway via the following mechanisms: a) Nrf2 decreases intracellular ROS levels by upregulating antioxidant genes and mitophagy-activating genes, resulting in the inhibition of NF-κB activation (Liu, Zeng, et al., 2020). b) Nrf2 prevents IκB-α proteasomal degradation by increasing heme oxygenase-1 levels and inhibiting the nuclear translocation of NF-κB. c) Nrf2 inhibits NF-κB by competing with the transcription co-activator cyclic adenosine monophosphate response element-binding protein (Saha et al., 2020). Further studies need to assess whether the regulation of Nrf2 on NF-κB in cerebrovascular diseases is identical to the above-listed mechanisms.

Signalling Pathway Involved in the Activation

Cerebrovascular Disease Aggravation after NLRP3 Inflammasome Activation

Autophagy/Mitophagy

Autophagy maintains cell homeostasis by engulfing damaged and dysfunctional organelles. Autophagy dysfunction is associated with various diseases, including neurodegenerative diseases and cancer. In cerebrovascular diseases, the autophagy markers, including light chain 3-II/light chain 3-I, beclin-1, and autophagy-related gene 5, and the mitophagy marker Parkin, as well as phosphatase and tensin homolog-induced putative kinase 1 (PINK-1) expression, are negatively correlated with the NLRP3 levels (Cao et al., 2017; He et al., 2017; Li et al., 2017; Wang et al., 2019). Autophagy regulates the NLRP3 inflammasome through the following three mechanisms (Figure 4): Autophagy removes the intracellular activator of the NLRP3 inflammasome, such as mtROS, which is the primary source of mtROS, a major source of ROS (90%) (Figure 4). b) Autophagy removes components of the NLRP3 inflammasome. c) Autophagy removes intracellular pro-IL-1β (Zhou et al., 2011). Mitophagy is defined as autophagy that occurs in the mitochondria and can remove mtROS. Parkin/mitophagy plays a protective role in cerebral ischaemic injury and atherosclerosis by inhibiting the NLRP3 inflammasome (Ma et al., 2018; He et al., 2019). The mechanism underlying the Parkin/mitophagy pathway is relatively well established. PINK-1 protein assists the recruitment of Parkin into the dysfunctional mitochondria, and induces mitochondrial outer membrane protein ubiquitination; p62 recognises ubiquitin mitochondria and binds to light chain 3 to initiate mitophagy (He et al., 2019). However, few studies have assessed the mechanism of PINK-1 activation. Investigating the activation mechanism of PINK-1/Parkin is expected to provide a new method for treating cerebrovascular diseases.

IFN-β/NLRP3 Inflammasome Pathway

IFN-β plays an anti-inflammatory role by inhibiting the NLRP3 inflammasome in experimental autoimmune encephalomyelitis (Inoue and Shinohara, 2013) and tuberculosis (Sabir et al., 2017). IFN-β also plays an anti-inflammatory role in cerebrovascular diseases. IFN-β attenuates tight junction protein degradation in brain endothelial cells and affords protection against ischaemic stroke (Kuo et al., 2020). In intracranial hemorrhage, elevated levels of IFN-β and signal transducer and activator of transcription (STAT) 1 are accompanied by reduced levels of NLRP3 inflammasomes, caspase-1, and IL-1β (Wang, Nowrangi, et al., 2018). However, the mechanism underlying IFN-β effects in cerebrovascular diseases have not been directly evaluated. IFN-β is thought to inhibit NLRP3 inflammasome-mediated inflammatory response via three possible pathways (Figure 4): a) It represses the activity of NLRP3 inflammasomes via the STAT1 transcription factor. b) IFN-β induces IL-10 in a STAT1-dependent manner, while IL-10 reduces pro-IL-1α and pro-IL-1β production via STAT3 signalling (Guarda et al., 2011; Sabir et al., 2017). c) IFN-β can inhibit the NLRP3 inflammasome by suppressing ROS generation in antigen-presenting cells (Inoue and Shinohara, 2013). Although these three pathways have not been assessed in cerebrovascular diseases, we postulate that they might be applicable in cerebrovascular disease as STAT-1 activation is associated with neuronal cell death (Jung et al., 2015; Xu et al., 2015), and inhibition of STAT1 or STAT3 plays a protective role in stroke (Jiang et al., 2013; Tian et al., 2018; Cai et al., 2019; Cheng et al., 2019; Liu, Ran, et al., 2019; Li, Lv, et al., 2020). It is worth exploring the interaction between IFN-β and NLRP3 inflammasomes in future investigations. Understanding the relationship between the IFN-β/NLRP3 inflammasome and cerebrovascular diseases is crucial for the treatment of stroke.

Sirtuin (SIRT)/NLRP3 Inflammasome Pathway

SIRT is a class of NAD⁺ dependent deacylases related to cell metabolism. SIRT activation has an inhibitory effect on the NLRP3 inflammasome. SIRT acts on NLRP3 inflammasomes via two strategies: a) oxidative stress, including SIRT1-AMPK-sterol regulatory element-binding protein/NLRP3 inflammasome (Li et al., 2013) and SIRT3-superoxide dismutase 2-mtROS/NLRP3 inflammasome in atherosclerosis (Chen, Zhu, et al., 2017). b) Apoptosis and autophagy, including SIRT1/autophagy/NLRP3 inflammasome in ischaemic stroke, the SIRT3/class O of forkhead box 3a/Parkin/NLRP3 inflammasome signalling pathway in atherosclerosis, and the APMK/SIRT1/NF-κB pathway in diabetic nephropathy (He et al., 2017; Ma et al., 2018; Li, Chen, et al., 2020). However, the mechanism of action of SIRT and NLRP3 inflammasome remains unclear and requires further study.

PI3K/AKT/NLRP3 Inflammasome Pathway

The role of the PI3K/AKT pathway in stroke is well-known. It regulates downstream anti-apoptotic molecules and plays a protective role in rats after ischaemic stroke (Li, Liu, et al., 2019; Lu et al., 2019; Meng et al., 2020; Wei et al., 2020; Yang et al., 2020). However, PI3K/AKT has not been associated with the NLRP3 inflammasome in cerebrovascular diseases. Wang et al. (2020) observed that the ROS/PI3K/AKT/NLRP3 inflammasome alleviates acute lung injury (Wang, Zhang, et al., 2020), suggesting that PI3K/AKT has an inhibitory effect on the NLRP3 inflammasome. However, Cruz et al. (2007) reported that ATP-mediated, ROS-dependent PI3K activation is involved in the activation of caspase-1 and processing of IL-1β and IL-18 (Cruz et al., 2007). The findings of Cruz et al. (2007) suggest that the PI3K/AKT pathway promotes NLRP3 inflammasome activation, and the difference results between Cruz et al. (2007) and Wang et al. (2020) may be attributed to differences in models employed (Cruz et al., 2007; Wang, Zhang, et al., 2020). Accordingly, the relationship between PI3K/AKT and NLRP3 inflammasomes needs to be further explored, especially in cerebrovascular diseases.

Notably, ROS and Nrf2 can simultaneously affect “priming” and “activation.” However, ROS plays a stimulatory role, while Nrf2 plays an inhibitory role. NF-κB activates “priming” of the NLRP3 inflammasome, while P2X7R activates “activation” of the NLRP3 inflammasome. Activation of NLRP3 inflammasomes mediates damage to the nervous system through pyroptosis and apoptosis. In contrast, autophagy/mitophagy inhibits NLRP3 inflammasome activation by clearing NLRP3 inflammasome activators. The effect of the PI3K/AKT pathway on inflammasomes remains controversial, and the inhibitory mechanism of IFN-β and SIRT on NLRP3 inflammasome in cerebrovascular diseases is also unclear, necessitating further investigations. The interaction mechanism between IFN-β and NLRP3 inflammasome has been proposed, but whether this mechanism is applicable to cerebrovascular diseases needs further study. Inhibition of ROS, NF-κB, and P2X7R mediated signalling pathways and activation of autophagy, mitochondrial autophagy, Nrf2, IFN-β, and SIRT mediated signalling pathways can inhibit the NLRP3 inflammasome and play a protective role in cerebrovascular diseases. However, as these signalling pathways are not exclusively involved in NLRP3 inflammasome activation, inhibiting these signalling pathways may simultaneously affect other responses. In other words, inhibitors that target these signalling pathways are not specific. So we are going to talk about specific inhibitors of NLRP3 inflammasome.

Drugs That Target NEK7

NEK7 is a recently discovered protein that may be involved in NLRP3 inflammasome activation. The decreased expression of NEK7 can assist MCC950 in altering the active conformation of NLRP3; however, MCC950 may disrupt the interaction between NLRP3 and NEK7 (Tapia-Abellan et al., 2019). One of the components of ginsenoside, Rg3, is a specific inhibitor of the NLRP3 inflammasome. Although it does not affect the upstream regulation of the NLRP3 inflammasome and does not affect the NLRC4 or AIM2 inflammasome, Rg3 inhibited the interaction between NEK7 and NLRP3, further inhibited the interaction between NLRP3 and ASC, inhibited the oligomerisation of ASC, and ultimately blocked the NLRP3 inflammasome cascade (Shi et al., 2020). Inhibition of NLRP3 by Rg3 is independent of ATP and has inhibitory effects in vivo. Rg3 also inhibited the priming of NLRP3, but not as potently as the interaction of NEK7-NLRP3. Furthermore, Rg3 inhibited both potassium ion efflux-independent activation of the NLRP3 inflammasome and the potassium ion efflux-dependent interaction of NEK7-NLRP3 (Shi et al., 2020). The possibility that Rg3 inhibits potassium ion outflow cannot be excluded, warranting further investigations (Shi et al., 2020). As NEK7 is a newly discovered component of the NLRP3 inflammasome, limited studies on inhibitors targeting it are available, and exploration of NEK7 inhibitors can be undertaken in the future. Aalinkeel et al. (2018) used nanotechnology to increase the transport of Rg3 on blood-brain barrier, which had a protective effect on Alzheimer's disease (Aalinkeel et al., 2018). The application of nanotechnology to other NLRP3 inflammasome inhibitors is expected to boost the ability of drugs to cross the blood-brain barrier and thus treat neurological diseases.

Drugs That Target NLRP3

Targeting NLRP3 is the most investigated strategy, especially targeting the NACHT structure. The NACHT domain is vital for NLRP3 oligomerisation, a key step in the assembly of NLRP3 inflammasomes (Huang et al., 2018).

Drugs That Target ASC

ASC oligomerisation is an upstream event in inflammasome activation and is involved not only in the activation of NLRP3 but also in the activation of AIM2; hence, inhibitors targeting ASC also affect AIM2 inflammasomes. Furthermore, inhibition of ASC weakly inhibits NLRC4 inflammasomes (Franklin et al., 2014; Chen, Li, et al., 2020).

Drugs That Target Caspase-1

Currently, Z-YVAD-FMK is the most commonly employed irreversible caspase-1 inhibitor with cell permeability (Li, Yan, et al., 2018). It blocks necrosis and the release of IL-1β and IL-18 by inhibiting caspase-1 p20 cleavage (Li, Yan, et al., 2018; Gao et al., 2019; Nyiramana et al., 2020). Chow et al. (2020) observed that Z-YVAD-FMK had a poor inhibitory effect on human caspase-1 and reduced cell death to a certain extent, but failed to completely inhibit NLRP3 inflammasome-mediated cell death (Chow et al., 2020). Other molecules were also found to inhibit caspase-1, such as Z-VAD-FMK (Chow et al., 2020) and Q-VD-OPH (Altaee and Gibson, 2020; Butkevych et al., 2020), thereby inhibiting the downstream caspase-1 reaction.

X-linked inhibitor of apoptosis protein is considered a caspase-1 inhibitor; however, Gao et al. (2019) proposed that X-linked inhibitor of apoptosis protein may be located downstream of caspase-1 and participate in the regulation of cytokine secretion (Gao et al., 2019). They also found that blocking caspase-1 p20 cleavage was associated with the preservation of the X-linked inhibitor of apoptosis protein (Gao et al., 2019). Inflammasome-mediated IL-1β and IL-18 release also depended on the expression of the X-linked inhibitor of apoptosis protein (Gao et al., 2019). Reports regarding NEK7 and X-linked inhibitor of apoptosis protein suggest that some unknown molecules may be involved in activating the NLRP3 inflammasome. Furthermore, exploring the NLRP3 inflammasome structure could help increase targets for future NLRP3 inflammasome therapy.

To sum up, inhibitors that inhibit the second-step activation of the NLRP3 inflammasome, especially those targeting NLRP3, have been studied extensively. Currently, there are few inhibitors targeting the newly discovered structure NEK7. Nanotechnology has promoted the transport of NEK7 inhibitors across the blood-brain barrier, and the application of nanotechnology is expected to broaden the range of therapeutic drugs for cerebrovascular diseases. MCC950 is the most widely used NLRP3 inflammasome specific inhibitor. The interaction mechanism between MCC950 and NACHT has been well defined. In recent years, there has been new understanding of the interaction between MCC950 and NLRP3, for example, it also affects NEK7 and ASC, and may even affect the priming of NLRP3 inflammasome. Unfortunately, the clinical trial found that MCC950 had an effect on serum liver enzyme levels, so the clinical trial was not further developed. However, MCC950 is still widely used as a specific inhibitor of NLR3 inflammasome, and has been used to compare the efficacy against NLRP3. Based on MCC950, several inhibitors of NLRP3 inflammasome have been identified. In the study of MCC950, THP1 cells were found to be a reliable and simple model for the study of NLRP3 inflammasome specific inhibitors. The interaction mechanism of oridonin on NLRP3 is similar to that of MCC950 and also related to NACHT and NEK7. The effects of MCC950 and oridonin on NACHT and NEK7 suggest that there may be a relationship between NACHT and NEK7, which requires further exploration. Other small molecule compounds also target NACHT, such as CY-09. It has been proved in animal models that CY-09 has a protective effect on cerebrovascular diseases, but whether it can be applied in clinical practice still needs further study. OLT1177 is also a small molecule targeting NACHT, and whether it affects priming is controversial. Phase II clinical trials of OLT1177 are currently underway and have found that OLT1177 is safe orally. OLT1177 has been shown to protect against cardiac ischemia/reperfusion injury and central nervous system diseases in animal models. The effect of OLT1177 on central nervous system diseases suggests that OLT1177 can penetrate the blood-brain barrier. Therefore, OLT1177 is expected to be applied in the treatment of cerebrovascular diseases. At present, there is no study on OLT1177 and cerebrovascular diseases, so it is of great significance to further study the role of OLT1177 in cerebrovascular diseases. Tranilast was initially used as an anti-allergy treatment. In recent years, a study found that tranilast has a protective effect in animal models of cerebral ischemia, and more evidence is needed. ASC is involved not only in the activation of NLRP3 inflammasome, but also in the activation of AIM2 inflammasome, so inhibitors targeting ASC may inhibit both AIM2 and NLRP3. There are relatively few drugs targeting ASC and caspase-1.