Pyroptosis in Pulpitis

Introduction

Dental pulp is a kind of distinct connective tissue located in the central part of teeth, which is wrapped by dental hard tissues and consists of cells, fibers, lymphatic vessels, blood vessels and nerves. The pulp and dentin are jointly known as the pulpo-dentinal complex, because of they both develop from the dental papilla, and have closely relationships in anatomy and histophysiology. As the pulpo-dentinal complex was infected or exposed to slight stimuli, dental pulp stem cells were found to differentiate into odontoblasts/odontoblast-like cells, which play a role in the formation of reparative dentin and protection of pulp tissue.

Pulpitis is a common inflammatory oral disease. Without timely intervention, pulpitis will seriously damage the function of teeth and affect the oral health of patients, thus causing time and economic losses. As a type of inflammatory cell death, pyroptosis is a key pathway in the development and progression of pulpitis. This review outlines the activation mechanism and potential role of cellular pyroptosis in the progression of pulpitis, as well as the interaction of pyroptosis with other cell death pathways, including apoptosis, autophagy, and necroptosis. This review is intended to enrich the mechanistic background of pulp inflammation and explore potential therapeutic strategies for preventing and treating pulpitis.

The Concept of Pyroptosis

In 1992, the characteristics of pyroptosis were described for the first time in Shigella flexneri infected macrophages: chromatin condensation, cytoplasmic vesiculation, cytoplasmic vacuolization, endoplasmic reticulum expansion, retention of organelle structure, and genomic DNA fragmentation. Pyroptosis had been mistaken as a kind of apoptosis for a time.¹ Apoptosis triggers programmed cell death by cleaving substrates through a caspase cascade reaction. In apoptosis, cells shrink and divide into apoptotic bodies, which are usually engulfed by surrounding macrophages, leading to cell death of a non-inflammatory character. Meanwhile, pyroptosis is an inflamed death that releases inflammatory factors of IL-1β and IL-18, removes intracellular pathogens and triggers an inflammatory response.² All these findings indicate that pyroptosis is different from apoptosis. In 2001, Brennan MA and Cookson BT formally brought out the notion of “pyroptosis”, which distinguished pyroptosis from apoptosis for the first time.³ Pyroptosis, also referred to as inflammatory necrosis, was first defined by the NCCD (The Nomenclature Committee on Cell Death) in 2018 as a specific type of regulated cell death that is dependent on the formation of plasma membrane pores by gasdermin family proteins.

Pyroptosis is classified into two distinct pathways, termed “canonical” and “non-canonical” on the basis of the underlying mechanism. The canonical pathway facilitates the recruitment and activation of caspase-1 by sensing pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs) and cytoplasmic disturbances through different inflammasomes. The currently recognized inflammasomes mainly include nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), NLRP1, NOD-like receptor family CARD domain-containing protein (NLRC4), absent in melanoma 2 (AIM2) and pyrin. Caspase-1 cleaves and activates IL-18, IL-1β and other executive proteins of pyroptosis including cleaved GSDMD with N-terminal sequence (GSDMD-N), which binds to the membrane and generate membrane pores, resulting in pyroptosis.⁴ In the non-canonical pathway, lipopolysaccharide (LPS) activates human-derived pro-caspase 4/5 or rat-derived pro-caspase 11, which then cleaves the domain of GSDMD-N, and induces pyroptosis.⁵ Recent studies have shown a new activation mechanism of incomplete pyroptosis. Fas associated with death domain (FADD) was found to play a role in maintaining intestinal inflammatory homeostasis by inhibiting caspase-8/GSDMD-dependent pyroptosis-like cell death in the downstream of ZBP1 and TNFR1.⁶ It has been shown that FADD was expressed in hDPSCs⁷ while its role in pulpitis through the alternative pathway of NLRP3/Caspase1 activation deserves further investigation. In caspase-1/11-deficient macrophages, NLRP3 inflammasome was capable of activating caspase-8 through apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and subsequently cleaved GSDME, at last the IL-1α was released instead of the classical pyroptotic factor IL-1β.⁸ Some chemotherapy drugs, such as adriamycin, actinomycin-D, bleomycin and topotecan, can switch apoptosis induced by TNF or chemotherapeutic drugs into pyroptosis by means of the activation of caspase-3 to cleave GSDME.⁹ In 2020, Shao Feng’s team proved that Granzyme A (GZMA) secreted by cytotoxic lymphocytes could specifically cleave GSDMB and release GSDMB-N fragments. Moreover, the liposome leakage assay confirmed that GSDMB cleaved by GZMA could cause cell perforation to induce pyroptosis.¹⁰

Pyroptotic Proteins Expressed in Inflamed Dental Pulp Tissues

Studies have confirmed that the expressions of NLRP3,¹¹ AIM2¹² and NLRP6¹³ inflammasome are upregulated in inflamed pulp tissues. However, the expressions of NLRP1 and NLRC4 have no significant differences between healthy and inflamed pulp tissues.¹⁴ NLRP3 was highly expressed in pulp tissues with irreversible pulpitis compared to normal pulp or tissues with reversible pulpitis. In inflamed pulp tissues, the gene expression of caspase-1 and IL-1β were also upregulated comparing with normal pulp.¹¹ Normally, AIM2 protein was expressed only in the cytoplasm of odontoblast, while it was significantly expressed in both inflamed odontoblasts and fibroblasts. Studies have shown that interferon γ and cytoplasmic DNA activated AIM2 inflammasome in HDPCs, and the inhibition of AIM2 downregulated the expression of cleaved caspase-1 and suppressed the release of IL-1β.¹² Persistent development of pulpitis may lead to periapical inflammation. The levels of NLRP3, AIM2 and ASC were significantly upregulated in apical lesions compared to healthy tissues. Also, NLRP3 and AIM2 were significantly overexpressed in inflammatory cells of periapical lesions, including neutrophils, monocytes, macrophages, and plasma cells.¹⁵

Immunohistochemical staining analysis of human samples confirmed that NLRP6 was expressed in both normal and inflamed pulp tissues. Meanwhile, in LPS-pretreated DPSCs, the expression of NLRP6 was found to increase in a time-dependent manner and IL-1β was significantly decreased after NLRP6 was silenced.¹⁶ ELISA assay also revealed that the level of NLRP6/caspase-4 inflammasome was up-regulated in inflamed human pulp tissues.¹³ These results indicate that NLRP6 is significantly involved in the LPS-induced inflammatory immune response of DPSCs, yet the specific mechanism needs to be further investigated.

The Activation of Pyroptosis in Dental Pulp Cells

A variety of pathogen recognition receptors (PRRs) that detect PAMPs and DAMPs are expressed in odontoblasts, fibroblasts, and DPSCs, especially Toll-like (TLR) and NOD-like receptorsin (NLR).^17–19 These PRRs regulate the immune defense and pathological processes of dental pulp tissues by inducing the inflammasome, mitochondrial dysfunction, and other mechanisms upon activation.

The Bacteria and Their Metabolites

Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) have significant roles in the progression of dental pulp infection^20,21 and apical abscesses.²² In HDPCs, LPS from Pg upregulates the levels of NLRP6, CASP4 and CASP1 in a type I interferon-dependent way.¹³ It has also been shown that with ATP induction, Pg remarkably enhances the level of IL-1β in HDPCs by NLRP3 upregulation and CARD16 downregulation. Fn could increase the expression of precursor and mature IL-1β in HDPCs without upregulating the levels of AIM2 and NLRP3 inflammasomes. Therefore, it was deduced that Fn may not regulate the inflammatory reactions of HDPCs via inflammasomes. Notably, simultaneous stimulation of Pg and Fn may also upregulate AIM2 and lead to a dysregulation of IL-1β.²³

Comparing with LPS, lipoteichoic acid (LTA) treated HDPCs not only significantly up-regulated the expression of NLRP3 and IL-1β, but also provoked more ASC specks formation, suggesting LTA may be the first signal of inflammasome activation.²⁴ For HDPCs pre-treated with LPS of Pg, LTA can promote caspase-1 maturation as well as the release of IL-18 and IL-1β via activating NLRP6/caspase-4 inflammasome. NLRP6 or caspase-4 knockdown effectively inhibited the inflammasome activation triggered by cytoplasmic LTA, suggesting that NLRP6/caspase-4 inflammasome may involve in inflammation and immune defense of pulp.¹³ Enterococcus faecalis-derived lipoteichoic acid (Ef. LTA) and short-chain fatty acids consist of acetate, propionate, and butyrate had been found in inflammatory apical lesions. Furthermore, the combination of butyrate and Ef. LTA were confirmed to significantly activate pyroptosis via K⁺ efflux and NF-κB pathway without LDH releasing in human acute monocytic leukemia cell line (THP-1). In addition, caspase-4 and caspase-1 inhibitors reduced the maturation and release of IL-1β, suggesting that both canonical and non-canonical activation contributed to the secretion of IL-1β. In rat model of periapical periodontitis treated with butyrate and Ef. LTA, the levels of IL-1β and cleaved caspase-1 were substantially increased around the apical tissues, moreover, the pulp was severely necrosis comparing with other groups.²⁵ It has also been shown that THP-1 treated with LPS from Porphyromonas gingivalis, resulting in an increased expression of IL-1β and caspase-1 and activation of NLRP3 and AIM2 in the cells.¹⁵ LPS combines with Nigerian bacteriocin, poly (dA: dT) or LPS transfection by lipofectamine 2000 were separately used to induce pyroptosis of periodontal ligament fibroblasts (PDLFs) and dental pulp cells (DPCs). The results suggested that agonists of AIM2 and NLRP3 inflammasome cannot induce typical pyroptosis in PDLFs and DPCs. In contrast, after the LPS was transfected into cells by Lipofectamine 2000, cytoplasmic LPS induced significant pyroptosis and expression of cleaved-gasdermin D in PDLFs and DPCs.²⁶ Above results led to the conclusion that PDLFs and DPCs may be much more sensitive to non-canonical pyroptosis elicited by cytoplasmic LPS compared to the canonical pathway.

In dermal fibroblasts stimulated with LPS or pre-treated with TNF-α/IL-1β, the expression of AIM2 inflammasome and caspase-1 was synergistically upregulated, and this effect was further amplified by co-culture with DPSCs. Notably, co-cultured fibroblasts exhibited an augmented inflammatory response, characterized by increased release of IL-1β, IL-6, and fibronectin. These findings suggest that DPSCs may influence the fibrosis by modulating the AIM2 inflammasome pathway to regulate caspase-1 activation and inflammatory cytokine secretion, while balancing fibrotic progression by suppressing collagen deposition and promoting extracellular matrix degradation. Thus, AIM2 may be a key target for DPSCs to mediate the balance between inflammation and fibrosis.²⁷

NLRP6 was functionally expressed in inflamed periapical tissues and human periodontal membrane cells (HPDLCs). Meanwhile, NLRP6 has been shown to exert anti-inflammatory effects in apical periodontitis tissues and the LPS-induced inflammatory response of HPDLCs, which significantly reduced IL-6 and TNF-α by inhibiting the NF-κB and ERK signaling pathways.²⁸ NLRP6 may be a potential target for future apical periodontitis therapy in the future.

Pulp Metabolic Pathways and Its by Products

The studies on NLRP3/caspase-1 inflammasome showed that ATP triggers K⁺ efflux and induces the formation of pannexin-1 channels by activating P2X7 receptors on cell membrane in human dental pulp fibroblasts (HDPFs). As a result, the extracellular LPS transfers into the cytoplasm and then activates NLRP3 inflammasome. Moreover, the intracellular microenvironment with low K⁺ concentration induces the release of reactive oxygen species (ROS), which can also activate NLRP3.¹¹ In a word, the priming signal is LPS upregulates the expression of NLRP3 and IL-1β by TLR4/MyD88/NF-κB pathway, while the activation signal is ATP induced ROS production, which can regulate IL-1β secretion and activation of caspase-1 and NLRP3 inflammasomes by LPS and ATP.²⁹

Dimethyl fumarate (DMF) is a cyto-permeable fumaric acid derivative. Studies have confirmed that DMF attenuates pyroptotic cell death in LPS-transfected PDLFs and DPCs by inhibiting GSDMD-N expression and cell localization. As DMF can prevent LPS-induced non-canonical pyroptosis of PDLFs and DPCs, the therapy strategies of pulpitis based on DMF may be potential research interests.²⁶

Lu Zhang et al showed that LPS induced mitochondrial damage in mDPC6T cells, and promoted the translocation of BAX from cytoplasm to mitochondrial membrane. Subsequently, the mitochondrial DNA (mtDNA) triggered the pyroptosis of mDPC6T cells and increased the release of CXCL10 and IFN-β. After overexpression of mtDNA in cells with GSDMD knockdown, the STING pathway was substantially activated, the levels of cleaved-GSDMD, CXCL10 and IFN-β were upregulated, resulting in severe inflammation of odontoblasts.³⁰ These results suggest that cytoplasmic mtDNA may be implicated in regulating odontoblast inflammation in vitro through GSDMD mediated pyroptosis (Figure 1).

Figure 1 Pyroptosis in dental pulp tissues. Pyroptosis has been demonstrated to occur in HDPCs, HDPFs and mDPC6T. Early and moderate pyroptosis in favor of rapid response to bacterial infection, whereas dysregulation of the inflammatory response induced by hyperactivated pyroptosis may be associated with the irreversible pulpitis. Figure 1 was independently designed by the authors using Microsoft PowerPoint 2019, with all elements created using native software features.

Studies showed that mitochondrial oxidative stress can activate NLRP3 inflammasome and promote pyroptosis in an odontoblast-like cell line named mDPC6T cells. After co-cultured with mDPC6T, mouse bone marrow mesenchymal stem cells (mBMSCs) were able to inhibit mitochondrial oxidative stress and pyroptosis by selectively transferring mitochondria to mDPC6T that pre-treated with LPS and ATP. Further studies revealed that tunnelling nanotubes (TNT) played a role in mitochondrial transferring between mBMSCs and mDPC6T. Furthermore, the pyroptotic mDPC6T secreted TNF-α and promoted the formation of TNT in mBMSCs via NF-κB signaling. This positive feedback leaded to increased mitochondrial translocation, thereby reducing the pyroptosis initiated by mitochondrial dysfunction and NLRP3 inflammasome in mDPC6T cells³¹ (Figure 2).

Figure 2 Intercellular mitochondrial transfer alleviates pyroptosis in dental pulp. The pyroptotic mDPC6T secreted TNF-α and promoted the formation of tunnelling nanotubes (TNT) in mBMSCs via NF-κB signaling, thereby reducing the pyroptosis initiated by mitochondrial dysfunction and NLRP3 inflammasome in mDPC6T cells. Created in BioRender. Zhang, X. (2025) https://BioRender.com/0dn3cjf.

Other Small Molecules and Exogenous Compounds

In 2021, Wenkai Jiang’s team identified NLRP3 as a straight target of miR-223 in HDPFs by dual‑luciferase reporter assay. In HDPFs pre-treated with ATP and LPS, the overexpression of NLRP3 promoted the expression of caspase-1 p20 and the secretion of mature IL-18 and IL-1β. Otherwise, knockdown or inhibitors of miR-223 facilitated the activation of NLRP3/caspase-1 pathway.³² Furthermore, as a co-negative regulatory factor, miR-22 was also found to be correlated with the regulation of the secretion of pro-inflammatory cytokines IL-18 and IL-1β by targeting HIF-1α and NLRP3/caspase-1 inflammasome pathway.³³

Odontoblast necrotic cell lysat (ONCL) produced by freezing-thawing cycles of odontoblast-like cells can induce migration and proliferation of HDPCs. NLRP3 inflammasome was significantly found to be activated in THP-1 cells previously treated with ONCL, as well as leading to the inflammatory cytokine release including IL-8, IL-6, IL-1β, TNF-α, CCL2, and IFN-γ. These results proved that direct damage to odontoblasts triggered a severe inflammatory response. Notably, NLRP3 inflammasome activation products, especially IL-1β, were observed to effectively stimulate the mineralization and odontogenic differentiation of HDPCs. This was demonstrated by the upregulation of osteogenic/odontogenic-related genes, including DSP, RUNX-2, DMP-1, and Secreted Phosphoprotein 1 (SPP1), as well as an increase in alkaline phosphatase activity.³⁴ The shortcoming of this study is that the ONCL-induced inflammatory responses were verified only in THP-1 cells but not in HDPCs.

Triethyleneglycol dimethacrylate (TEGDMA) is a commonly used dental copolymer monomer,³⁵ which can diffuse into the dental pulp through dentinal tubules under high concentration environment. In human peripheral blood mononuclear cells, TEGDMA was shown to upregulate the pyroptotic factors IL-18 and IL-1β by promoting the formation of NLRP3 inflammasome.³⁶ Human primary dental pulp stem cells (hpDPSCs) and human immortalized dental pulp stem cells (hiDPSCs) were cultured with TEGDMA monomers and concurrently treated with N-acetyl cysteine (NAC). As a result, NAC rescued the damage on viability and proliferation of hpDPSCs and hiDPSCs induced by TEGDMA. Besides, NAC also decreased the expression level of NLRP3 inflammasome, COX2, and IL-8 in hpDPSCs and hiDPSCs.³⁷ All these suggest that NAC could be used to alleviate the toxic damage of TEGDMA on dental pulp cells.

The expression of GSDME in normal tissues of OSCC patients is significantly higher than that in carcinoma tissues, and cisplatin could cause chemotherapy-associated toxicities by inducing caspase-3 maturation and GSDME cleavage, which triggered the pyroptosis and the release of IL-1β and TNF-α.³⁸ Also, GSDMB is one of the key differentially expressed genes related to tumor mutational load in OSCC and may be a reliable biomarker for predicting the prognosis of OSCC patients.³⁹ However, the functions of GSDME and GSDMB in dental pulp inflammation and their interactions with other gasdermin family members still need to be further studied.

Crosstalk Between Pyroptosis with Other RCD in Pulpitis

As we learn more about RCD, it has been found that different RCD signaling pathways are interconnected. Thus, crosstalk between pyroptosis and other RCDs like apoptosis, autophagy and necroptosis are of great importance for cell death and the progression of dental pulp inflammation.

Apart from caspase-8 has crucial impacts on apoptosis,⁴⁰ it has also been proved to activate inflammasome and regulate pyroptosis.^41,42 In mouse macrophage infected by Yersinia outer protein J, TAK1 inhibition activated caspase-8, which subsequently cleaved GSDME and GSDMD, resulting in pyroptosis.⁴² Besides, the metabolite α-ketoglutaric acid (α-KG) was also able to trigger pyroptosis through mediating the caspase-8-dependent cleavage of GSDMC.⁴³

It has been found that Caspase-3 triggered the molecular transition from apoptosis to pyroptosis by specifically cleaving the Asp270 site of GSDME. In the GSDME-deficient model, apoptotic stimuli induced only apoptotic body formation, whereas wild-type cells shifted to secondary necrosis or pyroptosis via the caspase-3/GSDME axis.⁴⁴ In the presence of GSDMD, caspase-1 triggered pyroptosis by cleavage of GSDMD. Whereas in GSDMD-deficient cells, caspase-1 could induce apoptosis by directly cleavage and activation of Caspase-3/7,⁴⁵ or by activation of the Bid-caspase-9-caspase-3 signaling axis.⁴⁶ Activated caspase-3/7 during apoptosis specifically cleaved the Asp87 site of GSDMD, generating an inactive p43 fragment, thus blocking GSDMD-mediated pyroptosis signaling.⁴⁵

Rapamycin and 3-methyladenine (3-MA) can be utilized for activation and inhibition of autophagy, respectively. Among dental pulp cells, LPS activated pyroptosis via the pNF-κB/IκBα signaling pathway, which could be inhibited through the autophagy induced by rapamycin. In contrast, 3-MA promoted LPS-induced activation of caspase-1 and IL-1β.⁴⁷

Recent years, the interactive connection that existed between relationship between NLRP3 inflammasome and autophagy have been extensively proved. Autophagy was able to exert a negative effect on the NLRP3 inflammasome by eliminating related components, cytokines, and endogenous activators, including damaged mitochondria.⁴⁸ Autophagic mechanisms also contributed to the unconventional release of IL-1β, thereby modulating the inflammatory response.^49,50 Excessive inflammation can lead to tissue damage and the onset of inflammatory diseases. Conversely, autophagy may attenuate the inflammatory response by removing NLRP3 inflammasome activators and other inflammatory elements.

Meanwhile, crosstalk seemed to exist between the NLRP3 inflammasome and necroptosis. Phosphorylated mixed lineage kinase-domain like (MLKL) oligomers generated by necroptosis in small intestinal epithelial cells translocated and which caused the secretion of ATP into the extracellular matrix. In neighboring cells, such as immune and epithelial cells, ATP triggered the formation of the NLRP3 inflammasome, facilitating the maturation of IL-1β.⁵¹ Mitochondrial damage triggered by the MLKL/p-MLKL translocation in necroptosis of epithelial cells promoted ROS production, which subsequently promoted NLRP3 inflammasome and IL-1β, ultimately promoted Pseudomonas aeruginosa-mediated inflammatory responses in the lungs⁵² (Figure 3).

Figure 3 Functional overlap of apoptotic, autophagic, and necroptotic proteins with pyroptotic proteins. (A) Autophagy and pyroptosis. (B) Necroptosis and pyroptosis. (C) Apoptosis and pyroptosis. Created in BioRender. Tu, D. (2025) https://BioRender.com/gmxfo36.

Recent studies have provided substantial evidences supporting the interaction between the different regulated cell death pathways. However, the crosstalk of pyroptosis and NLRP3 inflammasome with other regulated cell deaths have not been directly demonstrated in pulpitis or dental pulp cells, which still need to be investigated in depth.

Prospects of Pyroptosis in the Treatment of Pulpitis

Currently, therapeutic strategies against pyroptosis primarily involve inhibiting inflammasome formation, preventing the activation of cysteine proteases, and antagonizing the function of gasdermin proteins.

Pyroptosis causes perforation of cell membrane, leading to the release of cell contents and inflammatory cytokines especially IL-1β and IL-18, thereby activating a severe inflammatory response. There is evidence that that activation of the NLRP3 inflammasome promoted inflammation and neuropathic pain. By establishing an experimental pulpitis model in rats, a study found that the decrease in pain threshold after pulp exposure was linked to the elevated expression of P2X7, caspase-1, NLRP3, IL-1β, and IL-18 in the trigeminal ganglion and dorsal horn of the medulla. Additionally, injection of P2X7 inhibitor in rats with pulpitis intraperitoneally elevated the pain threshold and suppressed the expression of NLRP3, IL-18 and IL-1β. Treating microglial cell lines to model the in vitro inflammatory microenvironment, the P2X7 inhibitor markedly diminished the expression of NLRP3, IL-1β, IL-18, and caspase-1 in microglial cells exposed to 10 μg/mL LPS.⁵³ This finding indicated that targeting the P2X7/NLRP3/caspase-1 pathway could be a promising therapeutic strategy for relieving pulp inflammatory pain.

There are many NLRP3 inhibitors had been reported, such as MCC950,^24,54,55 CY-09,^56,57 and OLT1177.^58,59 As an alternative inhibitor of NLRP3 inflammasome, MCC950 was studied extensively. Among diabetic SD rats with periodontitis, a large number of pyroptotic macrophages were found in periodontal tissues, accompanying by a high expression of IL-1β. Local injection of MCC950 into gingiva inhibited the levels of GSDMD and IL-1β in periodontal tissues, which effectively attenuated alveolar bone resorption.⁵⁴ Meanwhile, other studies showed that MCC950 reduced LPS-induced pyroptosis of human gingival fibroblasts (HGFs) along with the level of caspase-1 p20 and IL-1β.⁵⁵ After DPCs along with THP-1 cells were co-cultured and subsequently exposed to LTA, MCC950 markedly downregulated the levels of IL-1β and IL-6, whereas it failed to affect IL-8 or MMP-9²⁴.

GSDMD inhibitors include necrosulfonamide (NSA),⁶⁰ disulfiram (DSF),⁶¹ GI-Y1,⁶² LDC7559,⁶³ fumarate⁶⁴ and Bay.11−7082⁶⁵ High concentrations of palmitic acid (PA) activated caspase-4 and GSDMD through NF-κB pathway in vitro, causing the pyroptosis of periodontal ligament cells (PDLCs). The NF-κB inhibitor SC75741 could reduce the level of IL-1β and partially inhibit the pyroptosis in PA-treated cells. Although the GSDMD inhibitors, neither NSA nor DSF had effects on the secretion of IL-1β, these two drugs significantly inhibited pyroptosis indeed.⁶⁶ GI-Y1 could bind to GSDMD-N and inhibit the oligomerization and membrane perforation activity of GSDMD-N by directly binding to Arg7 residues. Meanwhile, GI-Y1 prevented the binding of GSDMD-N to mitochondria and mitigated mitochondrial damage under hypoxia/reoxygenation (H/R) or ischemia/reperfusion (I/R) conditions.⁶² LDC7559 reduced the expression and cleavage of GSDMD in subarachnoid hemorrhage (SAH), but had no major effects on the expression of NLRP3.⁶³ In patients with periodontitis, the level of Synoviolin produced by peripheral blood mononuclear cells was downregulated, which resulted in less ubiquitination and higher levels of GSDMD proteins, increasing the activation of inflammasome.⁶⁷

VX-765,^68,69 Z-YVAD-FMK,⁷⁰ Ac-yvad-cmk,⁷¹ Boc-D-FMK⁷² and Ac-yvad-cmk⁷³ have been proved to be inhibitors of caspase-1. VX765 suppressed IL-1β, MCP-1, IL-6, and IL-8 through inhibiting caspase-1 in hPDLFs. In an experimental periapical inflammation on rat, VX765 was shown to increase the thickness of trabecular bone without altering the magnitude of periapical lesions. This suggests that caspase-1 inhibition could partially reduce bone loss.⁶⁸ In inflammatory gingival tissues from human and rat, the expressions of NLRP3, caspase-1, caspase-4/11, and IL-18 were increased, while the levels of E-cadherin were reduced. In comparison to the LPS-treated sample, the mRNA expression of E-cadherin was upregulated among oral mucosal epithelial cells treated with LPS and Z-YVAD-FMKGA, while the caspase-1, IL-18, and IL-1β were decreased. Therefore, Z-YVAD-FMKGA partly attenuated LPS-induced pyroptosis and reversed the changes in E-cadherin, which may influence epithelial junctions.⁷⁰

In addition, AR-A014418,^74,75 enamel matrix derivative (EMD),⁷⁶ curcumin⁷⁷ have been shown to inhibit pyroptosis. AR-A014418, was shown to increase the migration and osteogenic differentiation of rat dental pulp stem cells (rDPSCs).⁷⁴ Besides, AR-A014418 effectively inhibited the NLRP3 inflammasome, cleaved caspase-1 as well as pro-inflammatory factors of TNF-α, IL-6, CXCL1, and IL-1β within a dose-dependent response on rDPSCs.⁷⁵ EMD have been proved to be used to facilitate periodontal regeneration. Primary macrophages extracted from mouse bone marrow along with RAW 264.7 were pre-treated with EMD before LPS stimulation, which resulted in a notable reduction in NLRP3. Simultaneously, the levels of caspase-1 and IL-1β were downregulated among RAW 264.7.⁷⁶ Curcumin exhibited anti-inflammatory properties by inhibiting the phosphorylation of NF-κB p65 and inactivating NLRP3 inflammasome in human dental pulp stem cells (hDPSCs). In inflammatory hDPSCs induced by LPS and ATP, curcumin not only markedly reduced the activation of NLRP3, ASC, caspase-1, and IL-1β, but also suppressed phosphorylation of p65. Meanwhile, downregulated pro-inflammatory factors of IL-6, IL-8, TNF-α and COX-2 associated with NF-κB pathway.⁷⁷

Also, the therapies targeting pyroptosis show equal potential in the treatment of apical periodontitis, a more severe progression of pulpitis. In an in vivo model of periapical inflammation induced with Enterococcus faecalis, the expression of NLRP3, caspase-1, and IL-1β proteins was significantly elevated in inflamed tissues. Whereas the NF-κB inhibitor BAY 11–7082 effectively blocked Enterococcus faecalis LTA-induced NLRP3 inflammasome activation and IL-1β release by inhibiting the NF-κB pathway, suggesting that this drug might be used in the treatment of apical periodontitis.⁷⁸ In mouse experimental apical periodontitis demonstrated that NLRP3 was significantly overexpressed in apical periodontitis tissues, and NLRP3 inhibitor MCC950 could significantly decrease the proportion of Th17 cells, increase the proportion of Treg cells, and decrease the serum levels of IL-1β, IL-6, and IL-17, which resulted in attenuation of periapical inflammation and bone resorption. However, the NLRP3 activator nigericin showed the opposite effect.⁷⁹

The pyroptosis inhibitor exhibits the potential to reduce pulp and periodontal tissue inflammation, inhibit bone resorption, alleviate pain, and promote restoration via the key processes such as interfering NLRP3 inflammasome activation, blocking GSDMD-mediated membrane perforation, inhibiting caspase-1 activity, and decreasing the release of inflammatory factors. Further validation of pyroptosis inhibitors’ safety and efficacy in humans is necessary for clinical translation in future.

Conclusion

Recent studies have shown that pyroptosis is critical to the progression of pulpitis and its complications. Within this review, effects and roles of pyroptosis in pulpitis are summarized. Inflammasome is a key participant in pyroptosis, while NLRP3, AIM2, and NLRP6 inflammasomes were all detected to be differentially characterized in inflamed dental pulp tissues (Table 1). In pulpitis, pyroptosis can be activated by multiple pathways, causing the cell membrane perforation and inducing the release of IL-1β and IL-18 as well as cellular contents to activate an intense inflammatory response, which may even lead to pain. Notably, the therapeutic strategies of pyroptosis have been shown to attenuate the inflammatory response of pulp cells (Table 2). In previous studies, it has been demonstrated that programmed cell death exists in pulpitis including apoptosis,^80,81 autophagy,^47,82 and necroptosis.⁸³ However, studies related to the mechanisms of programmed cell death and the crosstalk between different ways of programmed cell death under pulpitis are expected to be enriched and explored in depth.

Table 1 Pathogenic Factors of Pyroptosis Related to Pulpitis

Table 2 Inhibitors of Pyroptosis

Also, most studies have only relied on in-vitro models. It is a potential direction to build an in-vivo model of rat pulpitis and explore the effect of pyroptosis on the occurrence and progression of pulpitis in the future.