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AIM: Apical periodontitis is an inflammatory disorder triggered by an immune response to bacterial infection, leading to the periapical tissue damage and alveolar resorption. However, the underlying mechanisms driving this process remain elusive, due to the complex and interconnected immune microenvironment within the local lesion site. In this study, the influence of Nlrp3 inflammasome-mediated immune response on the apical periodontitis was investigated. METHODOLOGY: RNA sequencing, immunohistochemistry and ELISA assay were performed to investigate the activation of Nlrp3 inflammasome signalling pathways in the human periapical tissues, including radicular cysts, periapical granulomas and healthy oral mucosa. A mouse model of apical periodontitis was established to study the role of Nlrp3 knockout in periapical bone resorption and Treg cell stability, and the underlying mechanism was explored through in vitro experiments. In vivo Treg cell adoptive transfer was performed to investigate the effects of Treg cells on the progression of apical periodontitis. RESULTS: Our findings find that the hyperactivated Nlrp3 inflammasome is present in human periapical lesions and plays a vital role in the immune-related periapical bone loss. Using a mouse model of apical periodontitis, we observe that Nlrp3 deficiency is resistant to bone resorption. This protection was accompanied by elevated generation and infiltration of local Treg cells that displayed a notable ability to suppress RANKL-dependent osteoclast differentiation. In terms of the mechanism of action, Nlrp3 deficiency directly inhibits the osteoclast differentiation and bone loss through JNK/MAPK and NF-κB pathways. In addition, Nlrp3 induces pyroptosis in the stem cells from apical papilla (SCAPs), and the subsequent release of cytokines affects the stability of Treg cell in periapical lesions, leading indirectly to enhanced bone resorption. In turn, adoptive transfer of both Nlrp3-deficient and wild-type Treg cells effectively prevent the bone erosion during apical periodontitis. CONCLUSIONS: Together, our data identify that the Nlrp3 inflammasome modulates the Treg cell stability and osteoclastogenesis in the periapical inflammatory microenvironment, thus determining the progression of bone erosion.
Assuntos
Modelos Animais de Doenças , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Periodontite Periapical , Linfócitos T Reguladores , Animais , Humanos , Camundongos , Perda do Osso Alveolar/imunologia , Perda do Osso Alveolar/metabolismo , Inflamassomos/metabolismo , Inflamassomos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Granuloma Periapical/imunologia , Periodontite Periapical/imunologia , Periodontite Periapical/metabolismo , Cisto Radicular/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , MasculinoRESUMO
INTRODUCTION: Bone loss is strongly associated with the immunologic milieu in apical periodontitis (AP). Tertiary lymphoid structures (TLSs) are organized lymphoid cell aggregates that form in nonlymphoid tissues under persistent inflammatory circumstances. To date, there has been no relevant report of TLSs in periapical lesions. This work aimed to investigate the formation and potential function of TLSs in AP. METHODS: Tissues from human apical lesions (n = 61) and healthy oral mucosa (n = 5) were collected. Immunohistochemistry and multiplex immunofluorescence were used to detect the formation of TLSs. Correlation analyses were performed between clinical variables and TLSs. In addition, immunohistochemistry was used to evaluate the expression of interleukin-1 beta, interleukin-6, receptor activator of nuclear factor kappa-B ligand, and macrophage subsets in the apical lesions. RESULTS: Periapical granulomas (n = 24) and cysts (n = 37) were identified by histologic evaluation. TLSs, composed of B-cell and T-cell clusters, developed in periapical granulomas and radicular cysts. The CXC-chemokine ligand 13, its receptor CXC-chemokine receptor 5, follicular dendritic cells, and high endothelial venules were localized in TLSs. The quantity and size of TLSs were positively associated with bone loss in AP. Moreover, proinflammatory cytokines and macrophage subsets were also substantially elevated in TLS regions of apical lesions. CONCLUSIONS: The formation of TLSs in periapical granulomas and cysts was closely associated with persistent immune responses and bone loss in apical lesions. TLSs provide an updated insight into the complicated immune response process in AP.
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Granuloma Periapical , Periodontite Periapical , Cisto Radicular , Estruturas Linfoides Terciárias , Humanos , Granuloma Periapical/metabolismo , Ligantes , Cisto Radicular/metabolismoRESUMO
BACKGROUND: Occlusal trauma is an important factor promoting bone loss caused by periodontal diseases. Although there are reports of traumatic force promoting bone resorption in periodontal diseases, no studies examining the inhibition of bone formation by traumatic force and the underlying mechanism have been reported. The aim of this study was to investigate the mechanism whereby traumatic force inhibits bone formation. METHODS: MC3T3-E1 cells were induced to undergo osteogenic differentiation and subjected to cyclic uniaxial compressive stress with or without stimulation with Pg. LPS. The expression of osteoblast markers and the activation of IKK-NF-κB signaling were evaluated in vitro. Then, MC3T3-E1 cells were induced to undergo osteogenic differentiation and subjected to cyclic uniaxial compressive stress with or without IKK-2 Inhibitor VI. The expression of osteoblast markers was determined. Then, the classic Wnt signaling pathway (ß-catenin, Gsk3ß, p-Gsk3ß, and Dkk1) was further evaluated in vitro. Finally, occlusal trauma was induced in Wistar rats with or without the injection of IKK-2 Inhibitor VI, to evaluate changes in bone mass and IKK-NF-κB and Wnt/ß-catenin signaling in vivo. RESULTS: After stimulation with Pg. LPS and traumatic force, IKK-NF-κB signaling was significantly activated in vitro. The expression of osteoblast markers and the activity of alkaline phosphatase in MC3T3-E1 cells declined after traumatic force loading and were rescued when IKK-NF-κB signaling was blocked. Wnt/ß-catenin signaling was accordingly inhibited upon force loading, but this inhibition was reversed when IKK-NF-κB was antagonized in vitro. X-ray and Micro-CT analysis of the mandibles of the rats as well as HE and TRAP staining showed that bone loss induced by occlusal trauma declined after IKK-NF-κB was inhibited. The expression of p65 and IκBα was increased when occlusal trauma was induced in Wistar rats, whereas ß-catenin, OCN, and Runx2 levels were decreased. After blocking IKK-NF-κB, significant upregulation of ß-catenin, OCN, and Runx2 was observed in rats suffering from occlusal trauma. CONCLUSIONS: IKK-NF-κB signaling could be activated by traumatic force or occlusal trauma. Its activation promoted the degradation of ß-catenin, ultimately inhibiting osteogenic differentiation in vitro and bone formation in vivo.
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Oclusão Dentária Traumática , Osteogênese , Animais , Diferenciação Celular , NF-kappa B , Osteoblastos , Ratos , Ratos Wistar , Via de Sinalização Wnt , beta CateninaRESUMO
BACKGROUND AND OBJECTIVE: Diabetes influences the frequency and development of periodontitis. Inflammation of human periodontal ligament cells (HPDLCs) participates in this pathologic process. Hence, this study aims to explore whether advanced glycation end products (AGEs), by-products of diabetes, could exaggerate inflammation induced by muramyl dipeptide (MDP) in HPDLCs, and whether nucleotide-binding oligomerization domain-like receptors (NLRs) signaling pathway was involved. MATERIAL AND METHODS: Human periodontal ligament cells were pre-treated with 100 µg/mL AGEs for 24 hours and stimulated with 10 µg/mL MDP for 24 hours. IL-6, IL-1ß, and RAGE were detected, and the activation of NF-κB signaling pathway was observed. The expression of NLRs was evaluated with or without silencing RAGE or blocking NF-κB pathway under AGEs stimulation. Statistical analyses were performed by using independent sample t test. RESULTS: Advanced glycation end products induced significant increase of inflammatory cytokines in HPDLCs (P < 0.05). Results of western blot (WB) showed that after 45 minutes stimulation of AGEs, p-p65/p65 ratio peaked; AGEs promoted the expression of NLRP1, NLRP3, and apoptosis-associated speck-like protein containing a CARD (ASC). After silencing RAGE or blocking NF-κB pathway, the up-regulation of NLRs protein caused by AGEs was attenuated. Additionally, AGEs pre-treatment could enhance the inflammatory response of MDP and the expression of NLRs, which were demonstrated by more expression of IL-6, IL-1ß, NOD2, NLRP1, NLRP3, and ASC. CONCLUSION: Advanced glycation end products induced inflammatory response in HPDLCs via NLRP1-inflammasome and NLRP3-inflammasome activation in which NF-κB signal pathway was involved. Besides, AGEs promoted the inflammatory response of MDP via NOD2, NLRP1-inflammasome, and NLRP3-inflammasome.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Produtos Finais de Glicação Avançada/farmacologia , Inflamassomos/metabolismo , Inflamação/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ligamento Periodontal/citologia , Antígenos de Neoplasias/metabolismo , Células Cultivadas , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas NLR , Transdução de SinaisRESUMO
INTRODUCTION: In regenerative endodontics, irrigation is an important step to ensure the success of treatment. EDTA as a common irrigant has been recommended in the American Associations of Endodontists guidelines. It has been suggested that EDTA-treated dentin slices could increase the attachment, differentiation, and migration of dental pulp stem cells. However, no information is available about the effect of EDTA on the migration of dental pulp cells (DPCs). The aim of this study was to explore how EDTA affects the migration of DPCs. METHODS: Cells were obtained from human premolars or third molars, and cell counting kit-8 was used to evaluate the influence of EDTA on cell proliferation at various concentrations and time points. Real-time polymerase chain reaction was used to detect the messenger RNA expression levels of transforming growth factor beta (TGF-ß) and chemokine receptor 4 (CXCR4). Protein expression was tested by the enzyme-linked immunosorbent assay and Western blot, respectively. In addition, the transwell migration assay was performed to investigate the role of EDTA pretreatment in stromal cell-derived factor 1α (SDF-1α)-induced DPC migration. RESULTS: Stimulation with 12% EDTA enhanced SDF-1α-induced migration of DPCs. Both expressions of TGF-ß1 and CXCR4 were increased by 12% EDTA in a time-dependent manner. After silencing CXCR4, EDTA-enhanced migration was decreased. Furthermore, the transcriptional regulation of CXCR4 by EDTA was found to be mediated by TGF-ß1/ERK1/2 and TGF-ß1/Smad2/3 signal pathways. CONCLUSIONS: Our results showed that 12% EDTA could promote SDF-1α-induced migration of DPCs by up-regulating CXCR4 expression in which TGF-ß1 signal pathways were involved.
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Quelantes de Cálcio , Movimento Celular , Quimiocina CXCL12 , Polpa Dentária , Ácido Edético , Receptores CXCR4 , Células Estromais , Quelantes de Cálcio/farmacologia , Movimento Celular/efeitos dos fármacos , Ácido Edético/farmacologia , Humanos , Receptores CXCR4/efeitos dos fármacos , Receptores CXCR4/metabolismo , Transdução de Sinais , Células Estromais/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate the role of the histone 3 lysine 27 trimethylation (H3K27me3) demethylase Jumonji domain-containing protein 3 (Jmjd3) in the epigenetic regulation of the inflammatory response in human periodontal ligament cells (HPDLs). DESIGN: HPDLs were stimulated with lipopolysaccharide from E. coli. The expression of Jmjd3 in HPDLs was examined by quantitative real-time polymerase chain reaction (Q-PCR), Western Blot and immunofluorescent staining. Potential target genes were selected by silencing Jmjd3 and were confirmed by Chromatin Immunoprecipitation (ChIP). RESULTS: Q-PCR, Western Blot and immunofluorescent staining revealed that the expression of Jmjd3 was increased in inflamed HPDLs. Knockdown of Jmjd3 led to the suppression of inflammation-induced up-regulation of interleukin-6 and interleukin-12. Moreover, ChIP assays demonstrated that Jmjd3 was recruited to the promoters of interleukin-6 and interleukin-12b and this recruitment was associated with decreased levels of trimethylated histone 3 lysine 27 (H3K27). CONCLUSIONS: It was concluded that Jmjd3 regulated the activation of interleukin-6 and interleukin-12b in the early inflammatory response of HPDLs via demethylation of H3K27me3 at promoters. This molecular event may play an important role in the regulation of the inflammatory response in HPDLs.
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Inflamação/metabolismo , Histona Desmetilases com o Domínio Jumonji/fisiologia , Ligamento Periodontal/citologia , Animais , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Ensaio de Imunoadsorção Enzimática , Epigênese Genética , Imunofluorescência , Humanos , Imuno-Histoquímica , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos , Masculino , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Regulação para CimaRESUMO
OBJECTIVE: Alveolar bone loss is a common outcome of chronic apical periodontitis. In this study, we investigated the involvement of the Dickkopf-1-Wnt/ß-catenin signaling pathway in the attenuation of osteogenic differentiation induced by Escherichia coli lipopolysaccharide, and we evaluated the use of Dickkopf-1 inhibitor and Dickkopf-1 recombinant protein to reverse bone loss in different phases of osteogenic differentiation. METHODS: MC3T3-E1 cells grown in osteogenic medium were treated with Escherichia coli lipopolysaccharide for 24h during osteogenic induction on days 0, 1, 7, 14 and 21. Dickkopf-1 siRNA was added on days 0 and 1, and Dickkopf-1 recombinant was added on days 7, 14, and 21. Quantitative real-time PCR, Western blotting and alkaline phosphatase activity assays were performed to measure osteogenic marker expression and Wnt/ß-catenin signaling. A rat apical periodontitis model was used to further evaluate the function of Dickkopf-1 in relation to bone loss. RESULTS: MC3T3-E1 cells treated with Escherichia coli lipopolysaccharide showed decreased mRNA expression of osteogenic markers. Wnt/ß-catenin signaling was also inhibited, and Dickkopf-1 showed corresponding variations as quantified by Western blotting. Using Dickkopf-1 inhibitor or Dickkopf-1 recombinant protein at different phases of osteogenic differentiation in vitro partially reversed the decrease in osteogenic marker expression. The rat apical periodontitis model indicated that the Dickkopf-1 inhibitor could restore bone loss in the periapical area in vivo. CONCLUSIONS: Dickkopf-1 may play a key regulatory role in determining the outcome for bone in inflammatory environments, and modulating the Wnt/ß-catenin signaling pathway via Dickkopf-1 inhibitor or recombinant protein may provide a potential therapeutic option to prevent bone destruction in endodontic disease.
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Perda do Osso Alveolar/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Periodontite Periapical/metabolismo , Via de Sinalização Wnt/fisiologia , Fosfatase Alcalina/metabolismo , Perda do Osso Alveolar/etiologia , Animais , Western Blotting , Células Cultivadas , Lipopolissacarídeos , Osteogênese/fisiologia , Periodontite Periapical/complicações , Distribuição Aleatória , Ratos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
INTRODUCTION: MicroRNA-335-5p has been reported to regulate osteogenic and chondrogenic differentiations of mesenchymal stem cells. The aim of this study was to explore the function and regulation mechanism of miR-335-5p in apical periodontitis (AP). METHODS: Total RNAs were extracted from human periodontal ligament fibroblasts (HPDLFs), 10 AP tissues, and 6 healthy periodontal ligament tissues using lysis buffer. Gene expression was detected using real-time polymerase chain reaction. The Dual Luciferase Assay (Promega, Madison, WI) was used to test miR-335-5p directly targeted urokinase-type plasminogen activator receptor (uPAR) and the receptor activator of nuclear factor kappa-B ligand (RANKL). Western Blot was used to detect protein expressions of RANKL, uPAR, and the fragile X-related 1 gene (FXR1). The enzyme-linked immunosorbent assay was used to detect the secretions of interleukin 6, tumor necrosis factor alpha, and RANKL. Data were analyzed using the Student t test. RESULTS: miR-335-5p acted as a positive mediator in HPDLF inflammation (P < .05). Two targets of miR-335-5p, uPAR and RANKL, were identified. Interestingly, uPAR was repressed by miR-335-5p at the basal level, but it can be relieved from miR-335-5p-mediated repression, which is called derepression, when HPDLFs were subjected to lipopolysaccharide stimulation. miR-335-5p promoted RANKL in HPDLFs regardless of whether or not it was under inflammatory conditions (P < .05). We proved FXR1 was responsible for the derepression of uPAR from miR-335-5p (P < .01). Both FXR1 and uPAR were positive mediators in HPDLF inflammation (P < .05). miR-335-5p, uPAR, RANKL, and FXR1 had the same expression profiles in HPDLF inflammation and AP tissues (P < .05). CONCLUSIONS: Our data showed that miR-335-5p may play dual roles in AP, and it might be considered as a target for therapeutic potency in clinical applications.
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Fibroblastos/metabolismo , MicroRNAs/metabolismo , Periodontite Periapical/metabolismo , Ligamento Periodontal/metabolismo , Ligante RANK/metabolismo , Reabsorção Óssea/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Inflamação/metabolismo , Interleucina-6/metabolismo , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Achieving a precise diagnosis of the pulp status of traumatized teeth is difficult. The time to interfere through endodontic treatment for these teeth is greatly dependent on the doctor's experience. A 24-year-old male patient suffered a traumatic injury to tooth #9 that resulted in an apical root horizontal fracture 3 days before he came to the hospital. The injured tooth showed no response to pulp sensitivity testing at the first visit and was discolored 1 week later. Tooth #9 was immobilized to its adjacent teeth for 4 weeks by a composite splint. The patient was asked to make regular follow-up appointments for 72 weeks. The changes in the pulp microcirculation of tooth #9 after the injury were recorded using laser Doppler flowmetry (LDF) and a tissue oxygen monitor (OXY). The LDF was used to directly measure the pulpal blood flow, and the OXY was used to detect the tissue oxygen saturation and total hemoglobin at each appointment. Tooth #9 survived a period of pulp infarct that started at week 4 and showed complete revascularization within 10 weeks. The color of the tooth returned to normal after revascularization. The tooth responded to sensitivity testing at week 58, and radiographic examinations confirmed its hard tissue was healing. Without other clinical signs besides tooth discoloration and an absence of response to pulp sensitivity testing, the complete self-repair of pulp can be expected in teeth with pulp infarct after a horizontal root fracture, and more than 1 year of follow-up is recommended. The use of LDF combined with OXY monitoring has great value in the timely and precise reflection of changes in pulp status after dental trauma.
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Polpa Dentária/irrigação sanguínea , Incisivo/lesões , Raiz Dentária/lesões , Humanos , Incisivo/diagnóstico por imagem , Infarto , Fluxometria por Laser-Doppler , Masculino , Maxila , Microcirculação , Neovascularização Fisiológica , Oximetria , Radiografia Dentária , Fatores de Tempo , Raiz Dentária/diagnóstico por imagem , Adulto JovemRESUMO
INTRODUCTION: microRNAs (miRNAs) have been identified to be closely related to inflammatory diseases. The aim of our study was to identify expression profiles of miRNAs associated with inflammation in apical periodontitis (AP) lesions and human periodontal ligament fibroblasts (HPDLFs) inflammation. METHODS: Total RNAs were extracted from 10 AP lesions, 6 control tissues, and HPDLFs using lysis buffer. Expressions of miRNAs (miR-29b, 106b, 125b, 143, 155, and 198) were detected by real-time polymerase chain reaction. The dual luciferase assay was used to test miR-155 directly targeted semaphorein3a (SEMA3A). Western Blot and the enzyme-linked immunosorbent assay were used to detect the protein expressions of SEMA3A and proinflammatory cytokines, respectively. All experiments were repeated at least 3 times. Data were analyzed using the independent sample t test. RESULTS: The previously mentioned miRNAs were all significantly up-regulated in AP lesions (P < .05), whereas they were not in HPDLFs inflammation, in which miR-29b, 106b, 125b, and 198 were down-regulated (P < .05) and miR-143 and 155 were unchanged (P > .05). Overexpression of miR-155 induced proinflammatory phenotype, and down-regulation reduced the other miRNAs in HPDLFs (P < .05). Moreover, miR-155 directly targeted SEMA3A, which was significantly up-regulated (increased) in acute HPDLFs inflammation and down-regulated (decreased) in AP lesions (P < .05). Knockdown of SEMA3A led to induction of the proinflammatory phenotype; down-regulation of miR-29b, 106b, 125b, 143, and 198 (P < .05); and, more importantly, up-regulation of miR-155 (P < .01). CONCLUSIONS: Our study showed that the expression profiles of inflammation-associated miRNAs associated with inflammation in AP lesions and HPDLFs inflammation were different. miR-155 may play a crucial role in apical periodontitis progression by directly inhibiting SEMA3A.
Assuntos
Fibroblastos/metabolismo , Inflamação/metabolismo , MicroRNAs/metabolismo , Ligamento Periodontal/metabolismo , Semaforina-3A/metabolismo , Células Cultivadas , China , Citocinas/metabolismo , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Humanos , Inflamação/genética , MicroRNAs/genética , Periodontite Periapical/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Semaforina-3A/genética , Regulação para CimaRESUMO
OBJECTIVE: To determine whether IKK-NF-κB is activated either directly by compressive mechanical stress or by proinflammatory cytokines produced by MC3T3-E1 cells under compressive stress loading. RESULTS: MC3T3-E1 cells subjected to cyclic uniaxial compressive stress showed increased expression of proinflammatory cytokines and activation of the IKK-NF-κB signaling pathway with nuclear translocation of p65. Following treatment with antibodies to neutralize the action of the proinflammatory cytokines, IL-1ß and IL-6, the activation of IKK-NF-κB signaling was notably inhibited in MC3T3-E1 cells subjected to force loading. CONCLUSION: IKK-NF-κB signaling in MC3T3-E1 cells may be activated by proinflammatory cytokines that are produced as a consequence of mechanical stress loading and not by direct compressive mechanical stress.