Targeting pyroptosis as a preventive and therapeutic approach for stroke

Abstract

Stroke has caused tremendous social stress worldwide, yet despite decades of research and development of new stroke drugs, most have failed and rt-PA (Recombinant tissue plasminogen activator) is still the accepted treatment for ischemic stroke. the complexity of the stroke mechanism has led to unsatisfactory efficacy of most drugs in clinical trials, indicating that there are still many gaps in our understanding of stroke. Pyroptosis is a programmed cell death (PCD) with inflammatory properties and are thought to be closely associated with stroke. Pyroptosis is regulated by the GSDMD of the gasdermin family, which when cleaved by Caspase-1/Caspase-11 into N-GSDMD with pore-forming activity can bind to the plasma membrane to form small 10-20 nm pores, which would allow the release of inflammatory factors IL-18 and IL-1β before cell rupture, greatly exacerbating the inflammatory response. The pyroptosis occurs mainly in the border zone of cerebral infarction, and glial cells, neuronal cells and brain microvascular endothelial cells (BMECs) all undergo pyroptosis after stroke, which largely exacerbates the breakdown of the blood-brain barrier (BBB) and thus aggravates brain injury. Therefore, pyroptosis may be a good direction for the treatment of stroke. In this review, we focus on the latest mechanisms of action of pyroptosis and the process by which pyroptosis regulates stroke development. We also suggest potential therapeutic stroke drugs that target the pyroptosis pathway, providing additional therapeutic strategies for the clinical management of stroke. The role of pyroptosis after stroke. After stroke, microglia first rush to the damaged area and polarize into M1 and M2 types. Under the influence of various stimuli, microglia undergo pyroptosis, release pro-inflammatory factors, and are converted to the M1 type; astrocytes and neuronal cells also undergo pyroptosis under the stimulation of various pro-inflammatory factors, leading to astrocyte death due to increased osmotic pressure in the membrane, resulting in water absorption and swelling until rupture. BMECs, the main structural component of the BBB, also undergo pyroptosis when stimulated by pro-inflammatory factors released from microglia and astrocytes, leading to the destruction of the structural integrity of the BBB, ultimately causing more severe brain damage. In addition, GSDMD in neutrophils mainly mediate the release of NETs rather than pyroptosis, which also aggravates brain injury. IL-10=interleukin-10; TGF-β = transforming growth factor-β; IL-18=interleukin-18; IL-1β = interleukin-1β; TNF-α = tumor necrosis factor-α; iNOS=induced nitrogen monoxide synthase; MMPs=Matrix metalloproteinases; GSDMD = gasdermin D; BMECs=brain microvascular endothelial cells; BBB = blood-brain barrier.

The role of pyroptosis after stroke. After stroke, microglia first rush to the damaged area and polarize into M1 and M2 types. Under the influence of various stimuli, microglia undergo pyroptosis, release pro-inflammatory factors, and are converted to the M1 type; astrocytes and neuronal cells also undergo pyroptosis under the stimulation of various pro-inflammatory factors, leading to astrocyte death due to increased osmotic pressure in the membrane, resulting in water absorption and swelling until rupture. BMECs, the main structural component of the BBB, also undergo pyroptosis when stimulated by pro-inflammatory factors released from microglia and astrocytes, leading to the destruction of the structural integrity of the BBB, ultimately causing more severe brain damage. In addition, GSDMD in neutrophils mainly mediate the release of NETs rather than pyroptosis, which also aggravates brain injury. IL-10=interleukin-10; TGF-β = transforming growth factor-β; IL-18=interleukin-18; IL-1β = interleukin-1β; TNF-α = tumor necrosis factor-α; iNOS=induced nitrogen monoxide synthase; MMPs=Matrix metalloproteinases; GSDMD = gasdermin D; BMECs=brain microvascular endothelial cells; BBB = blood-brain barrier.

Fig. 1. Mechanism of cell death induced by pyroptosis.

In the classical inflammatory pathway, DAMPs and PAMPs increase the activation of NLRP3 inflammasomes, thereby promoting Caspase-1 cleavage of GSDMD and the pro-inflammatory factors IL-18 and IL-1β, causing pyroptosis; in the non-classical inflammatory pathway, LPS directly induces Caspase-4/5/11 to cleave GSDMD, thereby promoting pyroptosis. In addition to GSDMD, GSDMA/B/C/E can also induce pyroptosis and are not functionally significantly different in that they are cleaved by SpeB (secreted by GAS), Granzyme A, Caspase-8, and Caspase-3, respectively. DAMPs damage-associated molecular pattern molecules; TLR4 toll-like receptor 4, NF-κB nuclear factor kappa-B, NLRP3 NOD-like receptor thermal protein domain associated protein 3, ASC apoptosis-associated speck-like protein containing CARD, IL-18 interleukin-18, IL-1β interleukin-1β, dsDNA double-stranded DNA, LPS lipopolysaccharides, GSDMA/B/C/D/E gasdermin A/B/C/D/E, INF-γ interferon-γ, GAS group A Streptococcus, SpeB streptococcal pyrogenic exotoxin B.

Fig. 2. Types of pyroptotic cell death after stroke.

After stroke, dormant microglia first rush to the damaged area and polarize into M1 and M2 types to exert pro-inflammatory and anti-inflammatory effects, respectively. However, under various stressful stimuli, microglia undergo pyroptosis and are converted to the M1 type, aggravating brain damage. Pro-inflammatory factors stimulate astrocytes to undergo pyroptosis, leading to increased membrane osmotic pressure and consequent water absorption and swelling, ultimately causing massive brain edema after stroke. Neuronal cells are more likely to undergo pyroptosis after losing the protection of astrocytes, resulting in irreversible brain damage. Pro-inflammatory substances released by microglia and astrocytes, among others, contribute to the pyroptosis of BMECs, accelerating the disruption of the BBB. In addition, GSDMD may exert other unknown effects in neutrophils; it seems to promote the release of NETs to induce secondary phagocytosis rather than directly triggering neutrophil rupture and death. IL-10 interleukin-10, TGF-β transforming growth factor-β, IL-18 interleukin-18, IL-1β interleukin-1β, GSDMD gasdermin D, dsDNA double-stranded DNA, TNF-α tumor necrosis factor-α, iNOS induced nitrogen monoxide synthase, PN-OP protein nanoparticle-induced osmotic pressure, AIM2 absent in melanoma 2, TLR4 toll-like receptor 4, CCR5 CC-chemokine receptor 5, CXCR4 chemokine (C-X-C motif) receptor 4, NLRP1 NOD-like receptor thermal protein domain associated protein 1, NLRP3 NOD-like receptor thermal protein domain associated protein 3, BBB blood-brain barrier, MMP-9 matrix metalloproteinase-9, BMECs brain microvascular endothelial cells, ELANE neutrophil elastase, NETs neutrophil extracellular traps.

Fig. 3. Crosstalk associations between the three types of PCD after stroke.

After stroke, TLR4/DR on the cell membrane receives the corresponding inflammatory stimulus and triggers pyroptosis, apoptosis, or necroptosis. Caspase-8 is a key protein regulating several modes of PCD death; normal expression of Caspase-8 enzyme activity favors the induction of apoptosis, diminished Caspase-8 activity helps induce necroptosis, whereas inactive Caspase-8 can promote ASC-procaspase-1 binding to induce pyroptotic cell death. The PANoptosome, a key complex triggering PAN-optosis, is mainly activated by IAV invasion, in which ZBP1 and RIPK3 act as sensors and Caspase-6 enhances their sensitivity; the reduction of TAK1 after stroke also promotes PANoptosome formation, triggering PANoptosis and aggravating brain injury. TLR4 toll-like receptor 4, DR death receptor, RIPK1 receptor interacting serine threonine kinase 1, RIPK3 receptor interacting serine threonine kinase 3, MLKL mixed-lineage kinase domain-like protein, ZBP1 Z-DNA binding protein 1, NLRP3 NOD-like receptor thermal protein domain associated protein 3, ASC apoptosis-associated speck-like protein containing CARD, NF-κB nuclear factor kappa-B, GSDMD gasdermin D, IAV influenza A virus, PANoptosis pyroptosis + apoptosis+necroptosis, TAK1 TGF beta-activated kinase 1.

Fig. 4. Potential drugs for stroke—targeting pyroptosis.

Pinocembrin and berberine ameliorate neuroinflammation and pyroptosis after stroke by inhibiting the NF-κB pathway activation, thereby reducing the activation of NLRP3 inflammasomes. MCC950, OLT1177, RRx-001, and CY-09 reduce post-stroke pyroptosis by inhibiting NLRP3 inflammasome activation, whereas lycorine and taraxasterol act by affecting the structural domain of ASC, indirectly blocking NLRP3 inflammasome activation. VX765 and nitrosonisoldipine improve post-stroke neuroinflammation and pyroptosis by inhibiting Caspase-1 and Caspase-4/5/11, respectively. DMF and NSA reduce pyroptotic cell death by targeting Caspase-1-mediated processing of GSDMD at Cys191/Cys192. Disulfiram reduces pyroptosis by directly affecting the pore-forming activity of N-GSDMD. JMZ trimetazidine, OLT007 dapansutrile, NSA necrosulfonamide, DMF dimethyl fumarate, LPS lipopolysaccharides, NLRP3 NOD-like receptor thermal protein domain associated protein 3, ASC apoptosis-associated speck-like protein containing CARD, GSDMD gasdermin D.