Molecular Characteristics of Cell Pyroptosis and Its Inhibitors: A Review of Activation, Regulation, and Inhibitors

Pyroptosis is an active and ordered form of programmed cell death. The signaling pathways of pyroptosis are mainly divided into canonical pathways mediated by caspase-1 and noncanonical pathways mediated by caspase-11. Cell pyroptosis is characterized by the activation of inflammatory caspases (mainly caspase-1, 4, 5, 11) and cleavage of various members of the Gasdermin family to form membrane perforation components, leading to cell membrane rupture, inflammatory mediators release, and cell death. Moderate pyroptosis is an innate immune response that fights against infection and plays an important role in the occurrence and development of the normal function of the immune system. However, excessive pyroptosis occurs and leads to immune disorders in many pathological conditions. Based on canonical pathways, research on pyroptosis regulation has demonstrated several pyroptotic inhibitors, including small-molecule drugs, natural products, and formulations of traditional Chinese medicines. In this paper, we review the characteristics and molecular mechanisms of pyroptosis, summarize inhibitors of pyroptosis, and propound that herbal medicines should be a focus on the research and development for pyroptosis blockers.


Introduction
The modes of cell death include programmed and non-programmed cell death. Programmed cell death refers to the programmed process of cell death in order to maintain the stability of the internal environment after cells receive a certain signal or are stimulated by certain factors. Apoptosis, autophagy, programmed necrosis, and pyroptosis are the manifestations of programmed death. Thereinto, pyroptosis is a newly discovered form of programmed cell death, which was firstly discovered by Zychlinsky et al. in macrophages infected with Shigella flexneri, because its morphological characteristics were significantly different from apoptosis [1]. Later, Cookson et al. found that it is dependent on the activity of caspase-1 and different from caspase-3-activity-dependent apoptosis, defined this form of cell death as caspase-1-dependent cell death for the first time, and proposed the concept of pyroptosis for the first time [2]. Due to the dependency on inflammatory caspase-1, pyroptosis primarily refers to inflammatory cell death, which is obviously different from apoptosis and necrosis. Meanwhile, with the deepening of research on pyroptosis, it is found that pyroptosis plays an important role in the occurrence and development of many diseases.
Pyroptosis is an important innate immune response that is critical in fighting infection. It is characterized by the expansion of cells until the cell membrane ruptures, resulting in the release of intracellular contents and activating the body's strong inflammatory response. IL-1β and IL-18 released by pyroptosis cells are endogenous immune factors that cause fever, stimulate the activation of immune cells, and promote lymphocyte proliferation Figure 1. Domain structure of mouse (caspases-1, -11) and human (caspases-1, -4, -5) inflammatory caspases. A CDL connects N-terminal CARD to the protease domain that is composed of a large subunit (p20) and a small subunit (p10) separated by an IDL. Each caspase contains some autocleavage sites (black site) within the linker sequences. The catalytic cysteine (red site) is located within the large protease subunit, while the dimerization interface is within the small subunit.
Caspase-11 is essentially an endogenous receptor of LPS. For caspase-11 activation, LPS directly binds to the CARD domain of caspase-11 via its lipid A tail [48]. In the case of Gram-negative bacterial infections, the hexacylated lipid A moieties of LPS bind directly to the caspase-11 CARD domain, resulting in oligomerization and adjacent induced activation of caspase-11, which may be facilitated due to the polymeric tendencies of LPS. Cytoplasmic pentacylated and hexacylated LPS could induce caspase-11 activation. However, this was not observed for tetracylated LPS [49]. The main effects of caspase-11 are associated with non-canonical pyroptosis [50]. During non-canonical pyroptosis, caspase-11 directly cleaves GSDMD as non-canonical inflammasome [51]. Domain structure of mouse (caspases-1, -11) and human (caspases-1, -4, -5) inflammatory caspases. A CDL connects N-terminal CARD to the protease domain that is composed of a large subunit (p20) and a small subunit (p10) separated by an IDL. Each caspase contains some autocleavage sites (black site) within the linker sequences. The catalytic cysteine (red site) is located within the large protease subunit, while the dimerization interface is within the small subunit.
Caspase-11 is essentially an endogenous receptor of LPS. For caspase-11 activation, LPS directly binds to the CARD domain of caspase-11 via its lipid A tail [48]. In the case of Gram-negative bacterial infections, the hexacylated lipid A moieties of LPS bind directly to the caspase-11 CARD domain, resulting in oligomerization and adjacent induced activation of caspase-11, which may be facilitated due to the polymeric tendencies of LPS. Cytoplasmic pentacylated and hexacylated LPS could induce caspase-11 activation. However, this was not observed for tetracylated LPS [49]. The main effects of caspase-11 are associated with non-canonical pyroptosis [50]. During non-canonical pyroptosis, caspase-11 directly cleaves GSDMD as non-canonical inflammasome [51].
In the noncanonical pathway of pyroptosis, the complex of LPS-pro-caspase-11 was recognized as a noncanonical inflammasome, and LPS directly activates caspase-11, which is independent to TLR4 signaling pathway [77]. When caspase-11 mediates noncanonical pyroptosis, it leads to cleavage of GSDMD, and GSDMD-N executes pyroptosis to induce the release of cell contents. However, noncanonical pyroptosis still requires the help of the NLRP3 inflammasome that activates caspase-1 to induce maturation of pro-IL-1β and pro-IL-18 [77]. In the canonical pathway of pyroptosis, when cytosolic pathogen recognition receptors (PRRs), NLRP1b, NLRP3, NLRC4, AIM2, or Pyrin are stimulated by the corresponding PAMPs and DAMPs, these proteins recruit ASC and pro-caspase-1 to assemble into inflammasomes. The NLRP3 inflammasome is described in detail, and its assembly and activation process is divided into two steps. Firstly, pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) are recognized by Toll-like receptor 4 (TLR4) and activate the nuclear factor kappa-B (NF-κB) pathway, leading to increased transcription of NLRP3, pro-caspase-1, pro-IL-1β, and pro-IL-18 [74]. Secondly, under the further stimulation of immune and inflammatory molecules, the NLRP3 protein is oligomerized and assembled with ASC and pro-caspase-1 to form the NLRP3 inflammasome [75]. Formation of NLRP3 inflammasome results in cleavage of pro-caspase-1 to form the active form of caspase-1 and promotes cleavage of pro-IL-1β and pro-IL-18 to form IL-1β and IL-18 mature bodies, leading to a cascade of immune or inflammatory responses [76].
In the noncanonical pathway of pyroptosis, the complex of LPS-pro-caspase-11 was recognized as a noncanonical inflammasome, and LPS directly activates caspase-11, which is independent to TLR4 signaling pathway [77]. When caspase-11 mediates noncanonical pyroptosis, it leads to cleavage of GSDMD, and GSDMD-N executes pyroptosis to induce the release of cell contents. However, noncanonical pyroptosis still requires the help of the NLRP3 inflammasome that activates caspase-1 to induce maturation of pro-IL-1β and pro-IL-18 [77].

Canonical Pyroptosis Pathway
Canonical pyroptosis is mediated by caspase-1, mainly in macrophages, and its key steps are the recruitment and activation of caspase-1 ( Figure 3). Taking NLRP3 inflammasome as an example, when NLRP3 protein is stimulated by specific PAMPs and DAMPs, NLRP3 protein recruits ASC and pro-caspase-1 and assembles into NLRP3 inflammasome with the assistance of NIMA-related kinase 7 (NEK7) [78]. NLRP3 inflammasome assembly activates pro-caspase-1, and the activated caspase-1 can not only mediate the maturation and secretion of IL-1β and IL-18, but also directly cleave GSDMD to produce GSDMD-N [79]. Subsequently, GSDMD-N binds to phosphatidylinositol, phosphatidic acid, and phosphatidylserine on the inner surface of the membrane through membrane lipid interaction and forms oligomeric pores (GSDMS pore) with an inner diameter of 10~20 nm in the lipid bilayer. Then, LDH, IL-1β, and IL-18 are leaked out through the pores as well as other small cytosolic proteins and eventually cause pyroptosis [57]. Meanwhile, a large number of holes in the plasma membrane lead to the connection between the inner and outer membrane, forming a non-selective membrane channel, resulting in the efflux of K + ions, which imbalance the ion concentration on both sides of the plasma membrane and cause a large amount of water to enter the cell, causing cell swelling and the eventual death of cells [8,80]. Under normal conditions, K + efflux is generally considered to be both sufficient and necessary for NLRP3 inflammasome activation [81]. In addition, NLRC4, which is recognized by DAMPs or PAMPs, can directly activate caspase-1 to promote canonical pyroptosis [82].

Noncanonical Pyroptosis Pathway
Numerous studies have found that caspase-1-independent pyroptosis pathway also exists in cells, which is named noncanonical pyroptosis and mediated by direct activation of caspase-4/5/11 under the action of LPS [83]. When Gram-negative bacteria infects mice,

Chemical Agents as Inhibitors of Pyroptosis
Small molecule drugs mainly refer to chemically synthesized drugs, usually organic compounds with a molecular weight of less than 1000. They are usually signal transduction inhibitors, which can specifically block signal transduction pathways, and their chemical properties determined that small-molecule drugs have good therapeutic properties and pharmacokinetic properties [96]. In recent years, the research on small-molecule drugs to inhibit pyroptosis has received more and more attention.

Structure Mechanism of Action IC 50 References
NSA Ac-yvad-cmk Prevents caspase-1 activation / [121] GSDMD is a key protein in pyroptosis. Screening and designing small molecule inhibitors specifically targeting GSDMD can prevent pyroptosis. As shown in Table 3, these are some GSDMD inhibitors described in literature. Necrosulfonamide (NSA) directly binds to GSDMD and inhibits the oligomerization of GSDMD-N, reducing the opening degree of cell membrane pores and the release of downstream inflammatory factors, but does not affect the expression of upstream NLRP3 and caspase-1 and the cleavage of GSDMD protein [122]. LDC7559 is a small molecule compound selected from the search for inhibitors of the special death form of human neutrophils, which blocks the toxicity of GSDMD-N and reduces inflammation [123]. Disulfiram, a drug used to treat alcohol addiction, can effectively inhibit the formation of GSDMD pores in human and mouse cells, thereby inhibiting pyroptosis [124]. It has been reported that fumarate, a tricarboxylic acid cycle intermediate, can acylate cysteine in GSDMD and prevent its interaction with cysteine proteases and subsequent processing activation, thus inhibiting the occurrence of pyroptosis [125]. Bay 11-7082 was previously identified as an NF-κB inhibitor, which can directly lead to covalent modification of the cysteine 191/192 residue of GSDMD, interfere with the formation of GSDMD pore and IL-1β secretion, and effectively inhibit pyroptosis [126].

Natural Product as Inhibitor of Pyroptosis
Natural products are metabolites produced by organisms in nature and have been an important source of new drug discovery. For the development of new drugs, research has Binds to GSDMD and inhibits the oligomerization of GSDMD-N / [122] LDC7559 Ac-yvad-cmk Prevents caspase-1 activation / [121] GSDMD is a key protein in pyroptosis. Screening and designing small molecule inhibitors specifically targeting GSDMD can prevent pyroptosis. As shown in Table 3, these are some GSDMD inhibitors described in literature. Necrosulfonamide (NSA) directly binds to GSDMD and inhibits the oligomerization of GSDMD-N, reducing the opening degree of cell membrane pores and the release of downstream inflammatory factors, but does not affect the expression of upstream NLRP3 and caspase-1 and the cleavage of GSDMD protein [122]. LDC7559 is a small molecule compound selected from the search for inhibitors of the special death form of human neutrophils, which blocks the toxicity of GSDMD-N and reduces inflammation [123]. Disulfiram, a drug used to treat alcohol addiction, can effectively inhibit the formation of GSDMD pores in human and mouse cells, thereby inhibiting pyroptosis [124]. It has been reported that fumarate, a tricarboxylic acid cycle intermediate, can acylate cysteine in GSDMD and prevent its interaction with cysteine proteases and subsequent processing activation, thus inhibiting the occurrence of pyroptosis [125]. Bay 11-7082 was previously identified as an NF-κB inhibitor, which can directly lead to covalent modification of the cysteine 191/192 residue of GSDMD, interfere with the formation of GSDMD pore and IL-1β secretion, and effectively inhibit pyroptosis [126].

Natural Product as Inhibitor of Pyroptosis
Natural products are metabolites produced by organisms in nature and have been an important source of new drug discovery. For the development of new drugs, research has Blocks the toxicity of GSDMD-N / [123] Disulfiram GSDMD is a key protein in pyroptosis. Screening and designing small molecule inhibitors specifically targeting GSDMD can prevent pyroptosis. As shown in Table 3, these are some GSDMD inhibitors described in literature. Necrosulfonamide (NSA) directly binds to GSDMD and inhibits the oligomerization of GSDMD-N, reducing the opening degree of cell membrane pores and the release of downstream inflammatory factors, but does not affect the expression of upstream NLRP3 and caspase-1 and the cleavage of GSDMD protein [122]. LDC7559 is a small molecule compound selected from the search for inhibitors of the special death form of human neutrophils, which blocks the toxicity of GSDMD-N and reduces inflammation [123]. Disulfiram, a drug used to treat alcohol addiction, can effectively inhibit the formation of GSDMD pores in human and mouse cells, thereby inhibiting pyroptosis [124]. It has been reported that fumarate, a tricarboxylic acid cycle intermediate, can acylate cysteine in GSDMD and prevent its interaction with cysteine proteases and subsequent processing activation, thus inhibiting the occurrence of pyroptosis [125]. Bay 11-7082 was previously identified as an NF-κB inhibitor, which can directly lead to covalent modification of the cysteine 191/192 residue of GSDMD, interfere with the formation of GSDMD pore and IL-1β secretion, and effectively inhibit pyroptosis [126].

Natural Product as Inhibitor of Pyroptosis
Natural products are metabolites produced by organisms in nature and have been an important source of new drug discovery. For the development of new drugs, research has Inhibits the formation of GSDMD pores <1 M [124] Fumarate GSDMD is a key protein in pyroptosis. Screening and designing small molecule inhibitors specifically targeting GSDMD can prevent pyroptosis. As shown in Table 3, these are some GSDMD inhibitors described in literature. Necrosulfonamide (NSA) directly binds to GSDMD and inhibits the oligomerization of GSDMD-N, reducing the opening degree of cell membrane pores and the release of downstream inflammatory factors, but does not affect the expression of upstream NLRP3 and caspase-1 and the cleavage of GSDMD protein [122]. LDC7559 is a small molecule compound selected from the search for inhibitors of the special death form of human neutrophils, which blocks the toxicity of GSDMD-N and reduces inflammation [123]. Disulfiram, a drug used to treat alcohol addiction, can effectively inhibit the formation of GSDMD pores in human and mouse cells, thereby inhibiting pyroptosis [124]. It has been reported that fumarate, a tricarboxylic acid cycle intermediate, can acylate cysteine in GSDMD and prevent its interaction with cysteine proteases and subsequent processing activation, thus inhibiting the occurrence of pyroptosis [125]. Bay 11-7082 was previously identified as an NF-κB inhibitor, which can directly lead to covalent modification of the cysteine 191/192 residue of GSDMD, interfere with the formation of GSDMD pore and IL-1β secretion, and effectively inhibit pyroptosis [126].

Natural Product as Inhibitor of Pyroptosis
Natural products are metabolites produced by organisms in nature and have been an important source of new drug discovery. For the development of new drugs, research has Acylate cysteine in GSDMD and prevents its interaction with cysteine proteases and subsequent activation 0.003 µM [125] Bay11-7082 GSDMD is a key protein in pyroptosis. Screening and designing small molecule inhibitors specifically targeting GSDMD can prevent pyroptosis. As shown in Table 3, these are some GSDMD inhibitors described in literature. Necrosulfonamide (NSA) directly binds to GSDMD and inhibits the oligomerization of GSDMD-N, reducing the opening degree of cell membrane pores and the release of downstream inflammatory factors, but does not affect the expression of upstream NLRP3 and caspase-1 and the cleavage of GSDMD protein [122]. LDC7559 is a small molecule compound selected from the search for inhibitors of the special death form of human neutrophils, which blocks the toxicity of GSDMD-N and reduces inflammation [123]. Disulfiram, a drug used to treat alcohol addiction, can effectively inhibit the formation of GSDMD pores in human and mouse cells, thereby inhibiting pyroptosis [124]. It has been reported that fumarate, a tricarboxylic acid cycle intermediate, can acylate cysteine in GSDMD and prevent its interaction with cysteine proteases and subsequent processing activation, thus inhibiting the occurrence of pyroptosis [125]. Bay 11-7082 was previously identified as an NF-κB inhibitor, which can directly lead to covalent modification of the cysteine 191/192 residue of GSDMD, interfere with the formation of GSDMD pore and IL-1β secretion, and effectively inhibit pyroptosis [126].

Natural Product as Inhibitor of Pyroptosis
Natural products are metabolites produced by organisms in nature and have been an important source of new drug discovery. For the development of new drugs, research has

Herb Medicine as Inhibitor of Pyroptosis
Herbal medicine, especially traditional Chinese medicine, has become an important repository for new drug discovery. Some representative formulas of TCM have been found to play a role in pyroptosis regulation as shown in Table 5. For example, Baihu Guizhi Tang inhibits the activation of NLRP3 inflammasome and suppresses cell pyroptosis [153]. Daphnes Cortex inhibits macrophage pyroptosis by regulating TLR4/NF-κB/NLRP3 signaling pathway [154]. Kuijieling Tang reduces the mRNA levels of Caspase-1, ASC, IL-1β, and IL-18 and significantly reduces the protein expression of NLRP3, caspase-1, GSDMD-N, IL-1β, and IL-18 [155]. Shenling Baizhu San inhibits the production of pro-inflammatory factors IL-1β, IL-18, and TNF-a and reduces the mRNA expression levels of NLRP3, ASC, and GSDMD-N [156]. Yushi Anchang Fang can down-regulate the expressions of NLRP3, ASC, and caspase-1 to close to normal levels, thereby inhibiting pyroptosis [157]. Due to the flexible composition of TCM prescriptions, we just listed the above as some representative prescriptions studied in recent years. As shown in Table 5, TCM formulas often consist of two or more herbs, and effective components are also complex. However, the complexity of effective components also makes the characteristics of multi-target inhibition of pyroptosis of TCM formulas, which reflects the pan-inhibitory effect on pyroptosis. Therefore, we suggest that TCM should be considered as a candidate library for development of pyroptosis blockers.

Herb Medicine as Inhibitor of Pyroptosis
Herbal medicine, especially traditional Chinese medicine, has become an important repository for new drug discovery. Some representative formulas of TCM have been found to play a role in pyroptosis regulation as shown in Table 5. For example, Baihu Guizhi Tang inhibits the activation of NLRP3 inflammasome and suppresses cell pyroptosis [153]. Daphnes Cortex inhibits macrophage pyroptosis by regulating TLR4/NF-κB/NLRP3 signaling pathway [154]. Kuijieling Tang reduces the mRNA levels of Caspase-1, ASC, IL-1β, and IL-18 and significantly reduces the protein expression of NLRP3, caspase-1, GSDMD-N, IL-1β, and IL-18 [155]. Shenling Baizhu San inhibits the production of pro-inflammatory factors IL-1β, IL-18, and TNF-a and reduces the mRNA expression levels of NLRP3, ASC, and GSDMD-N [156]. Yushi Anchang Fang can down-regulate the expressions of NLRP3, ASC, and caspase-1 to close to normal levels, thereby inhibiting pyroptosis [157]. Due to the flexible composition of TCM prescriptions, we just listed the above as some representative prescriptions studied in recent years. As shown in Table 5, TCM formulas often consist of two or more herbs, and effective components are also complex. However, the complexity of effective components also makes the characteristics of multi-target inhibition of pyroptosis of TCM formulas, which reflects the pan-inhibitory effect on pyroptosis. Therefore, we suggest that TCM should be considered as a candidate library for development of pyroptosis blockers.

Herb Medicine as Inhibitor of Pyroptosis
Herbal medicine, especially traditional Chinese medicine, has become an important repository for new drug discovery. Some representative formulas of TCM have been found to play a role in pyroptosis regulation as shown in Table 5. For example, Baihu Guizhi Tang inhibits the activation of NLRP3 inflammasome and suppresses cell pyroptosis [153]. Daphnes Cortex inhibits macrophage pyroptosis by regulating TLR4/NF-κB/NLRP3 signaling pathway [154]. Kuijieling Tang reduces the mRNA levels of Caspase-1, ASC, IL-1β, and IL-18 and significantly reduces the protein expression of NLRP3, caspase-1, GSDMD-N, IL-1β, and IL-18 [155]. Shenling Baizhu San inhibits the production of pro-inflammatory factors IL-1β, IL-18, and TNF-a and reduces the mRNA expression levels of NLRP3, ASC, and GSDMD-N [156]. Yushi Anchang Fang can down-regulate the expressions of NLRP3, ASC, and caspase-1 to close to normal levels, thereby inhibiting pyroptosis [157]. Due to the flexible composition of TCM prescriptions, we just listed the above as some representative prescriptions studied in recent years. As shown in Table 5, TCM formulas often consist of two or more herbs, and effective components are also complex. However, the complexity of effective components also makes the characteristics of multi-target inhibition of pyroptosis of TCM formulas, which reflects the pan-inhibitory effect on pyroptosis. Therefore, we suggest that TCM should be considered as a candidate library for development of pyroptosis blockers.

Herb Medicine as Inhibitor of Pyroptosis
Herbal medicine, especially traditional Chinese medicine, has become an important repository for new drug discovery. Some representative formulas of TCM have been found to play a role in pyroptosis regulation as shown in Table 5. For example, Baihu Guizhi Tang inhibits the activation of NLRP3 inflammasome and suppresses cell pyroptosis [153]. Daphnes Cortex inhibits macrophage pyroptosis by regulating TLR4/NF-κB/NLRP3 signaling pathway [154]. Kuijieling Tang reduces the mRNA levels of Caspase-1, ASC, IL-1β, and IL-18 and significantly reduces the protein expression of NLRP3, caspase-1, GSDMD-N, IL-1β, and IL-18 [155]. Shenling Baizhu San inhibits the production of pro-inflammatory factors IL-1β, IL-18, and TNF-a and reduces the mRNA expression levels of NLRP3, ASC, and GSDMD-N [156]. Yushi Anchang Fang can down-regulate the expressions of NLRP3, ASC, and caspase-1 to close to normal levels, thereby inhibiting pyroptosis [157]. Due to the flexible composition of TCM prescriptions, we just listed the above as some representative prescriptions studied in recent years. As shown in Table 5, TCM formulas often consist of two or more herbs, and effective components are also complex. However, the complexity of effective components also makes the characteristics of multi-target inhibition of pyroptosis of TCM formulas, which reflects the pan-inhibitory effect on pyroptosis. Therefore, we suggest that TCM should be considered as a candidate library for development of pyroptosis blockers. Table 5. TCM formulas as inhibitors of pyroptosis.

Consists of Herbs Mechanism of Action References
Inhibits the NLRP3/GSDMD signaling pathway Vegetables and fruits [152] Aesculin is the main component of ash bark, which promotes the phosphorylation of protein kinase B (Akt)/glycogen synthase kinase 3β (GSK3β), inhibits the phosphorylation of nuclear factor-κb (NF-κB), and significantly reduces the expression levels of NLRP3, caspase-1, GSDMD, and IL-1β, thereby inhibiting pyroptosis and alleviation of inflammatory symptoms [136]. Notoginsenoside R1 is the main component of panax notoginseng, which can inhibit the NF-κB signaling pathway, block the activation of NLRP3 inflammasome, prevent the cleavage of caspase-1 and IL-1β, reduce the expression of the N-terminal domain of GSDMDN, and improve the cell function of rat nucleus pulposus cells, thereby inhibiting pyroptosis [137]. Ginsenoside Rb1 is another important component of Panax notoginseng, which can regulate the nuclear transcription factor E2-related factor 2 (Nrf2)/antioxidant response element signaling pathway to reduce the pyroptosis by inhibiting calcium overload [138]. Quercetin is a natural flavonoid widely distributed in vegetables and fruits, which can inhibit LPS/ATP-induced NLRP3 inflammasome activation and reduce the cleavage of GSDMD protein and the secretion of IL-1β and IL-18 [139]. Resveratrol is a polyphenolic compound that occurs naturally in a variety of plants, especially red grape skins, which can inhibit the activation of NLRP3 inflammasome and down-regulate the expression of caspase-1, thereby reducing the cleavage of GSDMD protein and the release of IL-1β and IL-18, and finally significantly inhibiting LPS/ATP-induced pyroptosis of macrophages [140]. Alliin is an organic sulfur compound extracted from garlic, which can inhibit NLRP3 inflammasome activation and reduce the secretion of IL-1β and IL-18 in mouse macrophages induced by LPS [141]. Curcumin is a natural polyphenol extracted from the rhizome of Curcuma longa, which can inhibit the NLRP3 inflammasome-mediated pyroptosis by regulating TLR4/NF-κB signaling pathway in mouse microglia [142]. Luteolin is a natural flavonoid found in a variety of plants, which can inhibit NLRP3 inflammasome activation and reduce the expression of the N-terminal domain of GSDMD and IL-1β by inhibiting Nrf2 and NF-κB signaling pathways [143]. Salidroside is extracted from Rhodiola rosea, which can reduce the levels of IL-1β, IL-18, and GSDMD. It can inhibit the NLRP3/caspase-1/GSDMD axis-mediated pyroptosis [144]. Salvianolic acid is extracted from salviae miltiorrhizae, which can reduce the cleavage of GSDMD-FL to GSDMD-N and inhibit the NLRP3 inflammasome [145]. Oroxylin is extracted from oroxylum indicum, which can reduce the activation of NLRP3 inflammasome and caspase-1 and inhibit GSDMD-mediated pyroptosis and the release of IL-1β and IL-18 [146]. Glycyrrhizin is a triterpene compound isolated from licorice, which can inhibit pyroptosis by inhibiting caspase-1/GSDMD signaling pathway [147]. In addition, many natural plant components with antioxidant and anti-inflammatory effects can inhibit pyroptosis by inhibiting the NLRP3/GSDMD signaling pathway, including paeoniflorin extracted from paeonia lactiflora pall., berberine extracted from coptis chinensis, lycorine isolated from lycoris radiata, isoliquiritigenin isolated from licorice, and protocatechuic acid present in many vegetables and fruits [148][149][150][151][152].

Herb Medicine as Inhibitor of Pyroptosis
Herbal medicine, especially traditional Chinese medicine, has become an important repository for new drug discovery. Some representative formulas of TCM have been found to play a role in pyroptosis regulation as shown in Table 5. For example, Baihu Guizhi Tang inhibits the activation of NLRP3 inflammasome and suppresses cell pyroptosis [153]. Daphnes Cortex inhibits macrophage pyroptosis by regulating TLR4/NF-κB/NLRP3 signaling pathway [154]. Kuijieling Tang reduces the mRNA levels of Caspase-1, ASC, IL-1β, and IL-18 and significantly reduces the protein expression of NLRP3, caspase-1, GSDMD-N, IL-1β, and IL-18 [155]. Shenling Baizhu San inhibits the production of pro-inflammatory factors IL-1β, IL-18, and TNF-a and reduces the mRNA expression levels of NLRP3, ASC, and GSDMD-N [156]. Yushi Anchang Fang can down-regulate the expressions of NLRP3, ASC, and caspase-1 to close to normal levels, thereby inhibiting pyroptosis [157]. Due to the flexible composition of TCM prescriptions, we just listed the above as some representative prescriptions studied in recent years. As shown in Table 5, TCM formulas often consist of two or more herbs, and effective components are also complex. However, the complexity of effective components also makes the characteristics of multi-target inhibition of pyroptosis of TCM formulas, which reflects the pan-inhibitory effect on pyroptosis. Therefore, we suggest that TCM should be considered as a candidate library for development of pyroptosis blockers.

Conclusions and Future Perspectives
Pyroptosis is a newly discovered form of programmed death of inflammatory cells. The canonical pyroptosis pathway of pyroptosis is mediated by caspase-1, and the noncanonical pyroptosis pathway is mediated by caspase-4/5 and caspase-11. Pyroptosis is characterized by the activation of various caspases through inflammasomes, resulting in the cleavage and multimerization of various Gasdermin family members to perforate the cell membrane, which in turn causes cell death. It is different from apoptosis in that pyroptosis occurs more rapidly and is accompanied by the release of a large number of proinflammatory cytokines. At the same time, inflammatory cytokines recruit other inflammatory cells and expand the inflammatory response. Actually, pyroptosis is an innate immune response that fights against infection and plays an important role in the occurrence and development of the normal function of the immune system in the body. Therefore, the regulation of pyroptosis to keep it within a reasonable range is helpful to the treatment of diseases and the health of the body. However, excessive pyroptosis occurs and leads to immune disorders in many pathological conditions. It is crucial to follow the mechanisms of pyroptosis and understand the potential drugs that inhibit excessive pyroptosis and their mechanisms, so as to keep pyroptosis at a moderate level. Since pyroptosis is an active and orderly form of cell death regulated by intracellular signals, its program can be interfered with or reprogrammed, which provides a theoretical basis for regulating pyroptosis and, therefore, makes it possible to study drugs that regulate or block pyroptosis. While some small molecule compounds have been reported that inhibit pyroptosis, there is still a long time before clinical drugs are developed. By contrast, some natural products and herbal medicines have significant inhibitory effects on pyroptosis, which may become a new growth point in the development of pyroptosis inhibitory drugs, and most of them have been used clinically for a long time. Therefore, more attention should be paid to herbal medicines for research and development of pyroptosis blockers.