Pyroptosis

1 Mechanism Overview

Pyroptosis is a form of programmed inflammatory cell death mediated by inflammatory caspases and gasdermin family proteins, and it is primarily activated through two pathways: the canonical pathway and the non‑canonical pathway. In the canonical pyroptosis pathway, bacterial, viral, and damage‑associated signals induce the assembly of inflammasomes (such as NLRP3, AIM2, and NLRC4), leading to the activation of Caspase‑1. In the non‑canonical pathway, cytosolic lipopolysaccharide (LPS) directly activates human Caspase‑4/5 or mouse Caspase‑11. Activated inflammatory caspases subsequently cleave gasdermin D (GSDMD), releasing the pore‑forming N‑terminal domain of GSDMD (GSDMD‑N). GSDMD‑N oligomerizes on the plasma membrane to form transmembrane pores, resulting in ionic imbalance, cellular swelling, and loss of membrane integrity, ultimately causing cell lysis. Meanwhile, GSDMD‑mediated membrane pore formation facilitates the release of leaderless inflammatory cytokines, including IL‑1β and IL‑18, further amplifying local inflammatory responses and immune cell recruitment. In addition, Caspase‑8, Caspase‑3, and other pathogen‑associated proteases can also induce pyroptosis‑like cell death through cleavage of gasdermin family members. Overall, the core molecular axis of pyroptosis can be summarized as: Inflammatory signal activation → Inflammasome assembly → Caspase activation → GSDMD cleavage → Membrane pore formation → Inflammatory cytokine release and cell lysis Pyroptosis plays essential roles in antimicrobial immunity, inflammatory diseases, and tissue injury by regulating immune responses and inflammation.

Pyroptosis pathway schematic. Extracellular pathogen‑associated molecular patterns (PAMPs) or damage‑associated molecular patterns (DAMPs) activate inflammasomes (such as NLRP3, AIM2, and NLRC4), leading to the activation of Caspase‑1. Meanwhile, cytosolic LPS activates Caspase‑4/5 in humans or Caspase‑11 in mice through the non‑canonical pyroptosis pathway. Activated inflammatory caspases cleave gasdermin D (GSDMD), releasing the pore‑forming N‑terminal domain of GSDMD (GSDMD‑N). GSDMD‑N subsequently oligomerizes on the plasma membrane to form membrane pores, resulting in cell swelling, membrane rupture, and the release of inflammatory cytokines such as IL‑1β and IL‑18, ultimately leading to inflammatory cell death.

2 Target List

Mechanism Module	Target / Detection Marker	Full Name	Biological Function / Mechanistic Significance	Recommended Detection Method
Inflammasome Assembly	NLRP3	NOD‑like receptor family pyrin domain‑containing 3	Senses danger signals such as ROS, K⁺ efflux, and crystals to form the NLRP3 inflammasome	WB / IF / qPCR
	AIM2	Absent in melanoma 2	Recognizes cytosolic DNA and activates inflammasome signaling	WB / IF
	NLRC4	NOD‑like receptor family CARD domain‑containing 4	Detects bacterial flagellin and type III secretion system components	WB
	ASC/PYCARD	Apoptosis‑associated speck‑like protein containing a CARD	Inflammasome adaptor protein linking inflammasome sensors with Caspase‑1	WB / IF
Canonical Pyroptosis Activation	Caspase‑1	Cysteine‑dependent aspartate‑directed protease 1 (Caspase‑1)	Inflammatory caspase that cleaves GSDMD and promotes maturation of IL‑1β and IL‑18	Cleaved‑Caspase‑1 p20 detection (WB)
	Caspase‑1 p20	Activated Caspase‑1 p20 fragment	Active form of Caspase‑1 indicating inflammasome activation	WB
Non‑canonical Pyroptosis Pathway	Caspase‑4 (human)	Cysteine‑dependent aspartate‑directed protease 4	Senses cytosolic LPS and induces GSDMD cleavage	WB
	Caspase‑5 (human)	Cysteine‑dependent aspartate‑directed protease 5	Functions similarly to Caspase‑4 in human non‑canonical pyroptosis	WB
	Caspase‑11 (mouse)	Cysteine‑dependent aspartate‑directed protease 11	Key mediator of LPS‑induced pyroptosis in mouse models	WB
Core Execution Phase	GSDMD	Gasdermin D	Major executioner protein of pyroptosis	WB
	GSDMD‑N	N‑terminal domain of Gasdermin D	Pore‑forming domain generated by caspase‑mediated cleavage; forms membrane pores	WB (priority detection)
Inflammatory Cytokine Release	IL‑1β	Interleukin‑1 beta	Major inflammatory cytokine released during pyroptosis	ELISA / WB
	Cleaved IL‑1β	Mature active interleukin‑1 beta	Determines functional activation of inflammasome signaling	WB / ELISA
	IL‑18	Interleukin‑18	Promotes NK cell activation and IFN‑γ responses	ELISA
Membrane Rupture / Cell Death Validation	LDH release	Lactate dehydrogenase release	Indicates plasma membrane integrity loss during pyroptosis	LDH assay
	PI uptake	Propidium iodide uptake	Indicates increased membrane permeability caused by pore formation	Flow cytometry / fluorescence staining
	SYTOX Green	SYTOX Green nucleic acid dye	Enters cells with damaged membranes and labels DNA	IF / Flow cytometry
Auxiliary Regulatory Pathways	Caspase‑8	Cysteine‑dependent aspartate‑directed protease 8	Can induce GSDMD/GSDME cleavage under specific inflammatory conditions	WB
	Caspase‑3	Cysteine‑dependent aspartate‑directed protease 3	Can cleave gasdermin family proteins and induce pyroptosis‑like cell death	WB
	GSDME	Gasdermin E	Cleaved by Caspase‑3 to generate membrane pore‑forming fragments	WB

3 Application Scheme

Analysis Purpose	Recommended Markers
Determination of pyroptosis initiation	NLRP3 / AIM2 / NLRC4
Inflammasome activation	ASC, Caspase‑1 p20
Core execution	GSDMD‑N
Membrane pore formation	GSDMD cleavage
Inflammatory cytokine release	IL‑1β, IL‑18
Non‑canonical pathway activation	Caspase‑4/5 (human), Caspase‑11 (mouse)
Plasma membrane rupture	LDH release, PI uptake
Exclusion of apoptosis	Cleaved‑Caspase‑3, PARP
Exclusion of necroptosis	p‑RIPK3, p‑MLKL

4 References

1. Devant P, Kagan JC. Molecular mechanisms of gasdermin D pore‑forming activity. Nat Immunol. 2023;24(7):1064‑1075. doi:10.1038/s41590‑023‑01526‑w.
2. Chen KW, Broz P. Gasdermins as evolutionarily conserved executors of inflammation and cell death. Nat Cell Biol. 2024;26:1394‑1406. doi:10.1038/s41556‑024‑01474‑z.
3. Zhang N, Zhang J, Yang Y, et al. A palmitoylation–depalmitoylation relay spatiotemporally controls GSDMD activation in pyroptosis. Nat Cell Biol. 2024;26:757‑769. doi:10.1038/s41556‑024‑01397‑9.
4. Pyroptosis inhibiting nanobodies block Gasdermin D pore formation. Nat Commun. 2023;14:7923. doi:10.1038/s41467‑023‑43707‑z.
5. Zhou B, Jiang ZH, Dai MR, et al. Full‑length GSDME mediates pyroptosis independent from cleavage. Nat Cell Biol. 2024;26:1545‑1557.