Enamel Matrix Derivative Decreases Pyroptosis-Related Genes in Macrophages

Abstract

Background: Pyroptosis is a caspase-dependent catabolic process relevant to periodontal disorders for which inflammation is central to the pathophysiology of the disease. Although enamel matrix derivative (EMD) has been applied to support periodontal regeneration, its capacity to modulate the expression of pyroptosis-related genes remains unknown. Considering EMD has anti-inflammatory properties and pyroptosis is linked to the activation of the inflammasome in chronic periodontitis, the question arises whether EMD could reduce pyroptosis signalling. Methods: To answer this question, primary macrophages obtained from murine bone marrow and RAW 264.7 macrophages were primed with EMD before being challenged by lipopolysaccharide (LPS). Cells were then analysed for pyroptosis-signalling components by gene expression analyses, interleukin-1β (IL-1β) immunoassay, and the detection of caspase-1 (CAS1). The release of mitochondrial reactive oxygen species (ROS) was also detected. Results: We report here that EMD, like the inflammasome (NLRP3) and CAS1 specific inhibitors—MCC950 and Ac-YVAD-cmk, respectively—lowered the LPS-induced expression of NLRP3 in primary macrophages (EMD: p = 0.0232; MCC950: p = 0.0426; Ac-YVAD-cmk: p = 0.0317). EMD further reduced the LPS-induced expression of NLRP3 in RAW 264.7 cells (p = 0.0043). There was also a reduction in CAS1 and IL-1β in RAW 264.7 macrophages on the transcriptional level (p = 0.0598; p = 0.0283; respectively), in IL-1β protein release (p = 0.0313), and CAS1 activity. Consistently, EMD, like MCC950 and Ac-YVAD-cmk, diminished the ROS release in activated RAW 264.7 cells. In ST2 murine mesenchymal cells, EMD could not be tested because LPS, saliva, and IL-1β + TNF-α failed to provoke pyroptosis signalling. Conclusion: These findings suggest that EMD is capable of dampening the expression of pyroptosis-related genes in macrophages.

Enamel Matrix Derivative Suppresses Chemokine Expression in Oral Epithelial Cells

“…Based on a series of previous experiments indicating TGF-β activity of EMD [ 32 , 33 , 34 , 35 , 36 ], we tested the activation of canonical TGF-β signaling in HSC2 cells. In support of this assumption, EMD induced the phosphorylation of smad3 ( Figure 3 ) and the nuclear translocation of smad2/3 ( Figure 4 ).…”

“…Even though the underlying molecular and cellular mechanisms are not fully understood, the effects of EMD might involve the modulation of local inflammation [ 31 ]. This has prompted us to study the impact of EMD on macrophages in an LPS-induced inflammatory environment [ 32 , 33 ]. To this aim, EMD significantly attenuated the LPS-induced expression of lead cytokines in RAW 264.7 and primary macrophages, an activity that was attributed to TGF-β, a pleiotropic growth factor found in EMD [ 32 , 33 ].…”

“…This has prompted us to study the impact of EMD on macrophages in an LPS-induced inflammatory environment [ 32 , 33 ]. To this aim, EMD significantly attenuated the LPS-induced expression of lead cytokines in RAW 264.7 and primary macrophages, an activity that was attributed to TGF-β, a pleiotropic growth factor found in EMD [ 32 , 33 ]. The TGF-β activity was further attributed to mediating the effects of EMD on osteoclastogenesis [ 34 ], adipogenesis [ 35 ], and changes in the genetic signature of gingival fibroblasts [ 36 ].…”

Pyroptosis in inflammatory bone diseases: Molecular insights and targeting strategies

“…Direct inhibitors of the NLRP3 inflammasome include CY‐09, 128 glyburide, 93 icariin, 82 MCC950, 21,98,99 monomeric derivatives of paeoniflorin (MDP), 119 metformin, 129 cranberry proanthocyanidins (PACs), 130 and resveratrol 75 . Indirect NLRP3 inhibitors include A3AR activator (CF101), 90 ASIC‐1a inhibitors (amiloride, psalmotoxin‐1), 122 GRK2 phosphorylation inhibitor (MDP), 119 glycogen synthase kinase‐3β (GSK‐3β) inhibitor (GSKI), 131 NF‐κB inhibitors (Baeckein E, loganin, morroniside, PDTC, punicalagin), 21,132–135 Nrf2/HO‐1 activators (eldecalcitol, licochalcone A), 88,98 and ROS inhibitors (enamel matrix derivative, NAC, quercetin) 98,99,136,137 …”

“…75 Indirect NLRP3 inhibitors include A3AR activator (CF101), 90 ASIC-1a inhibitors (amiloride, psalmotoxin-1), 122 GRK2 phosphorylation inhibitor (MDP), 119 glycogen synthase kinase-3β (GSK-3β) inhibitor (GSKI), 131 NF-κB inhibitors (Baeckein E, loganin, morroniside, PDTC, punicalagin), 21,[132][133][134][135] Nrf2/HO-1 activators (eldecalcitol, licochalcone A), 88,98 and ROS inhibitors (enamel matrix derivative, NAC, quercetin). 98,99,136,137 5.2 | MicroRNAs (miRNAs) targeting strategies Epigenetic processes, including DNA methylation, histone modifications, and miRNAs, which causing the heritable silence of genes without alteration to their coding sequence. 138 Since epigenetics is crucial for NLRP3 inflammasome activation and may regulate GSDM levels, 139,140 it may be possible to treat inflammatory diseases by regulating the epigenetic process.…”