Is there any association between the presence of biomarkers and apical periodontitis? A systematic review.

This systematic review aimed to verify whether there is evidence of an association between apical periodontitis and the presence of systemic biomarkers. This study adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses - PRISMA. For this, the acronym PECO was used; population (P) of adult humans exposed (E) to the presence of apical periodontitis, compared (C) to adult humans without apical periodontitis, and the outcome (O) of the presence of biomarkers was observed. The articles were searched in PubMed, Scopus, Web of Science, LILACS, Cochrane Library, OpenGray, and Google Scholar grey databases. Subsequently, studies were excluded based on title, abstract, and full article reading, following the eligibility criteria. The methodological quality of the selected studies was evaluated using the Newcastle-Ottawa qualifier. After exclusion, 656 studies were identified, resulting in 17 final articles that were divided into case-control, cross-sectional, and cohort studies. Eight studies were considered to have a low risk of bias, one had a medium risk of bias, and eight had a high risk of bias. In addition, 12 articles evaluated biomarkers in blood plasma, four evaluated them in saliva, and only one evaluated them in gingival crevicular fluid. The results of these studies indicated an association between apical periodontitis and the systemic presence of biomarkers. These markers are mainly related to inflammation, such as interleukins IL-1, IL-2, and IL-6, oxidative markers, such as nitric oxide and superoxide anions, and immunoglobulins IgG and IgM. Systematic review registration: https://www.crd.york.ac.uk/prospero/, identifier (CRD42023493959).

Unveiling the oral-gut connection: chronic apical periodontitis accelerates atherosclerosis via gut microbiota dysbiosis and altered metabolites in apoE-/- Mice on a high-fat diet.

The aim of this study was to explore the impact of chronic apical periodontitis (CAP) on atherosclerosis in apoE-/- mice fed high-fat diet (HFD). This investigation focused on the gut microbiota, metabolites, and intestinal barrier function to uncover potential links between oral health and cardiovascular disease (CVD). In this study, CAP was shown to exacerbate atherosclerosis in HFD-fed apoE-/- mice, as evidenced by the increase in plaque size and volume in the aortic walls observed via Oil Red O staining. 16S rRNA sequencing revealed significant alterations in the gut microbiota, with harmful bacterial species thriving while beneficial species declining. Metabolomic profiling indicated disruptions in lipid metabolism and primary bile acid synthesis, leading to elevated levels of taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), and tauroursodeoxycholic acid (TDCA). These metabolic shifts may contribute to atherosclerosis development. Furthermore, impaired intestinal barrier function, characterized by reduced mucin expression and disrupted tight junction proteins, was observed. The increased intestinal permeability observed was positively correlated with the severity of atherosclerotic lesions, highlighting the importance of the intestinal barrier in cardiovascular health. In conclusion, this research underscores the intricate interplay among oral health, gut microbiota composition, metabolite profiles, and CVD incidence. These findings emphasize the importance of maintaining good oral hygiene as a potential preventive measure against cardiovascular issues, as well as the need for further investigations into the intricate mechanisms linking oral health, gut microbiota, and metabolic pathways in CVD development.

The importance of mechanosensitive cell mediated prostaglandin and nitric oxide synthesis in the pathogenesis of apical periodontitis: comparative with chronic periodontitis.

OBJECTIVES: Mechano-sensitive odontoblast cells, which sense mechanical loading and various stresses in the tooth structure, synthesize early signaling molecules such as prostaglandin E2 (PGE2) and nitric oxide (NO) as an adaptive response. It is thought that these synthesized molecules can be used for the diagnosis and treatment of periodontal and periapical diseases. The aim of this study was to investigate the relationship between the severity of apical periodontitis (AP) and chronic periodontitis (CP) and serum (s) TNF-α, IL-10, PGE2 and NO levels, as well as PGE2 and NO levels in gingival crevicular fluid (GCF) samples. MATERIALS & METHODS: A total of 185 subjects were divided into three categories: AP group (n = 85), CP group (n = 50) and healthy control group (n = 50). The AP group was divided into 3 subgroups according to abscess scoring (AS-PAI 1, 2 and 3) based on the periapical index. The CP group was divided into 4 subgroups according to the periodontitis staging system (PSS1, 2,3 and 4). After recording the demographic and clinical characteristics of all participants, serum (s) and gingival crevicular fluid (GCF) samples were taken. TNF-α, IL-10, PGE2 and NO levels were measured in these samples. RESULTS: Unlike serum measurements (sTNF-α, sIL-10, sNO and sPGE2), GCF-NO and GCF-PGE levels of the AP group were significantly higher than the control group in relation to abscess formation (54.4 ± 56.3 vs. 22.5 ± 12.6 µmol/mL, p < 0.001 and 100 ± 98 vs. 41 ± 28 ng/L, p < 0.001, respectively). Confirming this, the GCF-NO and GCF-PGE levels of the AS-PAI 1 group, in which abscesses have not yet formed, were found to be lower than those in AS-PAI 2 and 3, which are characterized by abscess formation [(16.7(3.7-117.8), 32.9(11.8-212.8) and 36.9(4.3-251.6) µmol/mL, p = 0,0131; 46.0(31.4-120.0), 69.6(40.3-424.2) and 74.4(32.1-471.0) ng/L, p = 0,0020, respectively]. Consistent with the increase in PSS, the levels of sTNF [29.8 (8.2-105.5) vs. 16.7(6.3-37.9) pg/mL, p < 0.001], sIL-10 [542(106-1326) vs. 190(69-411) pg/mL, p < 0.001], sNO [182.1(36.3-437) vs. 57.0(15.9-196) µmol/mL, p < 0.001], sPGE2 [344(82-1298) vs. 100(35-1178) ng/L, p < 0.001], GCF-NO [58.9 ± 33.6 vs. 22.5 ± 12.6 ng/L, p < 0.001] and GCF-PGE2 [ 99(37-365) vs. 30(13-119), p < 0.001] in the CP group were higher than the control group. Comparison ROC analysis revealed that the GCF-PGE2 test had the best diagnostic value for both AP and CP (sensitivity: 94.1 and 88.0; specificity: 64.0 and 78.0, respectively; p < 0.001). CONCLUSIONS: GCF-PE2 and GCF-NO have high diagnostic value in the determination of AP and CP, and can be selected as targets to guide treatment. In addition, the measurements of PGE2 and NO in GCF can be used as an important predictor of pulpal necrosis leading to abscess in patients with AP. CLINICAL RELEVANCE: In this article, it is reported that syntheses of early signaling molecules such as PGE2 and NO can be used for the diagnosis and treatment target of periapical and periodontal infections.

Enterococcus faecalis Extracellular Vesicles Promote Apical Periodontitis.

Enterococcus faecalis is an important contributor to the persistence of chronic apical periodontitis. However, the mechanism by which E. faecalis infection in the root canals and dentinal tubules affects periapical tissue remains unclear. Bacterial extracellular vesicles (EVs) act as natural carriers of microbe-associated molecular patterns (MAMPs) and have recently attracted considerable attention. In this study, we investigated the role of EVs derived from E. faecalis in the pathogenesis of apical periodontitis. We observed that E. faecalis EVs can induce inflammatory bone destruction in the periapical areas of mice. Double-labeling immunofluorescence indicated that M1 macrophage infiltration was increased by E. faecalis EVs in apical lesions. Moreover, in vitro experiments demonstrated the internalization of E. faecalis EVs into macrophages. Macrophages tended to polarize toward the M1 profile after treatment with E. faecalis EVs. Pattern recognition receptors (PRRs) can recognize MAMPs of bacterial EVs and, in turn, trigger inflammatory responses. Thus, we performed further mechanistic exploration, which showed that E. faecalis EVs considerably increased the expression of NOD2, a cytoplasmic PRR, and that inhibition of NOD2 markedly reduced macrophage M1 polarization induced by E. faecalis EVs. RIPK2 ubiquitination is a major downstream of NOD2. We also observed increased RIPK2 ubiquitination in macrophages treated with E. faecalis EVs, and E. faecalis EV-induced macrophage M1 polarization was notably alleviated by the RIPK2 ubiquitination inhibitor. Our study revealed the potential for EVs to be considered a virulence factor of E. faecalis and found that E. faecalis EVs can promote macrophage M1 polarization via NOD2/RIPK2 signaling. To our knowledge, this is the first report to investigate apical periodontitis development from the perspective of bacterial vesicles and demonstrate the role and mechanism of E. faecalis EVs in macrophage polarization. This study expands our understanding of the pathogenic mechanism of E. faecalis and provides novel insights into the pathogenesis of apical periodontitis.

Experimental apical periodontitis alters salivary biochemical composition and induces local redox state disturbances in the salivary glands of male rats.

OBJECTIVES: The objective was to evaluate the effects of experimental apical periodontitis on the inflammatory, functional, biochemical, and redox parameters of the parotid and submandibular glands in rats. MATERIALS AND METHODS: Twenty 12-week-old male Wistar rats were randomly divided into two groups (n = 10): a control group and apical periodontitis group. After 28 days, the saliva was collected for salivary flow rate and biochemistry composition. Both glands were sampled for quantification of the tumor necrosis factor-alpha (TNF-α) and biochemical analyses of redox state. RESULTS: TNF-α concentrations were higher in both salivary glands adjacent to the periapical lesions in animals with apical periodontitis and also compared to the control group. The apical periodontitis group increased the salivary amylase, chloride, potassium, calcium, and phosphate. The total oxidant capacity increased in the parotid gland adjacent to the periapical lesions in the same rat and compared to the control group. Conversely, the total antioxidant capacity of the parotid glands on both sides in the apical periodontitis group was lower than that in the control group. Furthermore, glutathione peroxidase activity increased in the submandibular gland adjacent to the apical periodontitis group compared to the control group. CONCLUSIONS: Experimental apical periodontitis alters salivary biochemical composition, in addition to increasing inflammatory marker and inducing local disturbances in the redox state in the parotid and submandibular glands of male rats. CLINICAL RELEVANCE: Apical periodontitis could exacerbate the decline in oral health by triggering dysfunction in the salivary glands.

STING inhibition alleviates bone resorption in apical periodontitis.

AIM: The goal of this study was to investigate the potential effects of an immunotherapeutic drug targeting STING to suppress the overreactive innate immune response and relieve the bone defect in apical periodontitis. METHODOLOGY: We established an apical periodontitis mouse model in Sting-/- and WT mice in vivo. The progression of apical periodontitis was analysed by micro-CT analysis and H&E staining. The expression level and localization of STING in F4/80+ cells were identified by IHC and immunofluorescence staining. RANKL in periapical tissues was tested by IHC staining. TRAP staining was used to detect osteoclasts. To clarify the effect of STING inhibitor C-176 as an immunotherapeutic drug, mice with apical periodontitis were treated with C-176 and the bone loss was identified by H&E, TRAP, RANKL staining and micro-CT. Bone marrow-derived macrophages (BMMs) were isolated from Sting-/- and WT mice and induced to osteoclasts in a lipopolysaccharide (LPS)-induced inflammatory environment in vitro. Moreover, WT BMMs were treated with C-176 to determine the effect on osteoclast differentiation by TRAP staining. The expression levels of osteoclast-related genes were tested using qRT-PCR. RESULTS: Compared to WT mice, the bone resorption and inflammatory cell infiltration were reduced in exposed Sting-/- mice. In the exposed WT group, STING was activated mainly in F4/80+ macrophages. Histological staining revealed the less osteoclasts and lower expression of osteoclast-related factor RANKL in Sting-/- mice. The treatment of the STING inhibitor C-176 in an apical periodontitis mice model alleviated inflammation progression and bone loss, similar to the effect observed in Sting-/- mice. Expression of RANKL and osteoclast number in periapical tissues were also decreased after C-176 administration. In vitro, TRAP staining showed fewer positive cells and qRT-PCR reflected decreased expression of osteoclastic marker, Src and Acp5 were detected during osteoclastic differentiation in Sting-/- and C-176 treated BMMs. CONCLUSIONS: STING was activated and was proven to be a positive factor in bone loss and osteoclastogenesis in apical periodontitis. The STING inhibitor C-176 administration could alleviate the bone loss via modulating local immune response, which provided immunotherapy to the treatment of apical periodontitis.

Ferroptosis of macrophages facilitates bone loss in apical periodontitis via NRF2/FSP1/ROS pathway.

Apical periodontitis (AP) is an infectious disease that causes periapical tissue inflammation and bone destruction. Ferroptosis, a novel type of regulated cell death, is closely associated with inflammatory diseases and the regulation of bone homeostasis. However, the exact involvement of ferroptosis in the bone loss of AP is not fully understood. In this study, human periapical tissues were collected, and a mouse model was established to investigate the role of ferroptosis in AP. Colocalization staining revealed that ferroptosis in macrophages contributes to the inflammatory bone loss associated with AP. A cell model was constructed using RAW 264.7 cells stimulated with LPS to further explore the mechanism underlying ferroptosis in macrophages upon inflammatory conditions, which exhibited ferroptotic characteristics. Moreover, downregulation of NRF2 was observed in ferroptotic macrophages, while overexpression of NRF2 upregulated the level of FSP1, leading to a reduction in reactive oxygen species (ROS) in macrophages. Additionally, ferroptotic macrophages released TNF-α, which activated the p38 MAPK signaling pathway and further increased ROS accumulation in macrophages. In vitro co-culture experiments demonstrated that the osteogenic ability of mouse bone marrow stromal cells (BMSCs) was suppressed with the stimulation of TNF-α from ferroptotic macrophages. These findings suggest that the TNF-α autocrine-paracrine loop in ferroptotic macrophages can inhibit osteogenesis in BMSCs through the NRF2/FSP1/ROS signaling pathway, leading to bone loss in AP. This study highlights the potential therapeutic value of targeting ferroptosis in the treatment of inflammatory bone diseases.

The effects of dimethyl fumarate on cytoplasmic LPS-induced noncanonical pyroptosis in periodontal ligament fibroblasts and dental pulp cells.

AIM: Pyroptosis is a type of inflammatory cell death and is related to pulpitis and apical periodontitis. In this study, the aim was to investigate how periodontal ligament fibroblasts (PDLFs) and dental pulp cells (DPCs) respond to pyroptotic stimuli and explore whether dimethyl fumarate (DMF) could block pyroptosis in PDLFs and DPCs. METHODOLOGY: Three methods (stimulation with lipopolysaccharide [LPS] plus nigericin, poly(dA:dT) transfection and LPS transfection) were used to induce pyroptosis in PDLFs and DPCs, two types of fibroblasts related to pulpitis and apical periodontitis. THP-1 cell was used as a positive control. Afterwards, PDLFs and DPCs were treated with or without DMF before inducing pyroptosis to examine the inhibitory effect of DMF. Pyroptotic cell death was measured by lactic dehydrogenase (LDH) release assays, cell viability assays, propidium iodide (PI) staining and flow cytometry. The expression levels of cleaved gasdermin D N-terminal (GSDMD NT), caspase-1 p20, caspase-4 p31 and cleaved PARP were examined by immunoblotting. Immunofluorescence analysis was used to detect the cellular distribution of GSDMD NT. RESULTS: Periodontal ligament fibroblasts and DPCs were more sensitive to cytoplasmic LPS-induced noncanonical pyroptosis than to canonical pyroptosis induced by stimulation with LPS priming plus nigericin or by poly(dA:dT) transfection. In addition, treatment with DMF attenuated cytoplasmic LPS-induced pyroptotic cell death in PDLFs and DPCs. Mechanistically, it was shown that the expression and plasma membrane translocation of GSDMD NT were inhibited in DMF-treated PDLFs and DPCs. CONCLUSIONS: This study indicates that PDLFs and DPCs are more sensitive to cytoplasmic LPS-induced noncanonical pyroptosis and that DMF treatment blocks pyroptosis in LPS-transfected PDLFs and DPCs by targeting GSDMD, suggesting DMF might be a promising drug for the management of pulpitis and apical periodontitis.

Creating an atlas of the bone microenvironment during oral inflammatory-related bone disease using single-cell profiling.

Oral inflammatory diseases such as apical periodontitis are common bacterial infectious diseases that may affect the periapical alveolar bone tissues. A protective process occurs simultaneously with the inflammatory tissue destruction, in which mesenchymal stem cells (MSCs) play a primary role. However, a systematic and precise description of the cellular and molecular composition of the microenvironment of bone affected by inflammation is lacking. In this study, we created a single-cell atlas of cell populations that compose alveolar bone in healthy and inflammatory disease states. We investigated changes in expression frequency and patterns related to apical periodontitis, as well as the interactions between MSCs and immunocytes. Our results highlight an enhanced self-supporting network and osteogenic potential within MSCs during apical periodontitis-associated inflammation. MSCs not only differentiated toward osteoblast lineage cells but also expressed higher levels of osteogenic-related markers, including Sparc and Col1a1. This was confirmed by lineage tracing in transgenic mouse models and human samples from oral inflammatory-related alveolar bone lesions. In summary, the current study provides an in-depth description of the microenvironment of MSCs and immunocytes in both healthy and disease states. We also identified key apical periodontitis-associated MSC subclusters and their biomarkers, which could further our understanding of the protective process and the underlying mechanisms of oral inflammatory-related bone disease. Taken together, these results enhance our understanding of heterogeneity and cellular interactions of alveolar bone cells under pathogenic and inflammatory conditions. We provide these data as a tool for investigators not only to better appreciate the repertoire of progenitors that are stress responsive but importantly to help design new therapeutic targets to restore bone lesions caused by apical periodontitis and other inflammatory-related bone diseases.

The expression of cyclic GMP-AMP synthase in human apical periodontitis: A laboratory investigation.

AIM: As a key DNA sensor, cyclic GMP-AMP synthase (cGAS) has emerged as a major mediator of innate immunity and inflammation. Human apical periodontitis has yet to be studied for the presence of cGAS. This investigation was conducted to determine if cGAS is involved in the pathological process of human apical periodontitis. METHODOLOGY: Sixty four human periapical lesions, comprising 20 periapical granulomas and 44 radicular cysts, were employed in this investigation. Healthy gingiva (n = 6), dental pulp (n = 3), and apical papilla (n = 3) were used as control samples. The expression of cGAS in the periapical tissues was discovered using immunohistochemical staining. mRNA-Sequencing and qRT-PCR were utilized to determine the differentially expressed genes (DEGs) associated with DNA-sensing signalling in periapical lesions compared to the healthy control. Immunofluorescence labelling was used to identify the co-expression of cGAS, interleukin-1ß, and interleukin-18. RESULTS: A significantly greater expression level of cGAS was discovered in the periapical lesions, with no significant difference between radicular cysts and periapical granulomas. mRNA-Sequencing analysis and qRT-PCR identified differentially expressed mRNA, such as cGAS and its downstream DEGs, between periapical lesions and healthy control tissues. Immunofluorescence labelling further revealed that cGAS, interleukin-1, and interleukin-18 were co-localized. CONCLUSIONS: These observations imply that along with the synthesis of interleukin-1 and interleukin-18, cGAS may be involved in the aetiology of apical periodontitis.

Systemic inhibition of 5-lipoxygenase by MK-886 exacerbates apical periodontitis bone loss in a mouse model.

BACKGROUND: To investigate if 5-LO selective inhibitor (MK-886) could be used for systemic treatment of experimentally induced apical periodontitis in a mouse model. METHODS: Twenty-four C57BL/6 mice were used. After coronal opening, a solution containing Escherichia coli LPS (1.0 µg/µL) was inoculated into the root canals of the lower and upper right first molars (n = 72 teeth). After 30 days apical periodontitis was established, and the animals were treated with MK-886 (5 mg/kg), a 5-LO inhibitor, for 7 and 14 days. The tissues were removed for histopathological and histometric analyses, evaluation of osteoclast number and gene expression for receptor activator of nuclear factor kappa-B (Tnfrsf11a), receptor activator of nuclear factor kappa-B ligand (Tnfsf11), osteoprotegerin (Tnfrsf11b), tartrate-resistant acid phosphatase (Acp5), matrix metalloproteinase-9 (Mmp9), cathepsin K (Ctsk) and calcitonin receptor (Calcr). Statistical data analysis was performed using Kruskal Wallis followed by Dunn's tests (α = 0.05). RESULTS: Administration of MK-886 for 7 days exerted no effect on apical periodontitis progression compared to LPS inoculation without treatment (p = 0.3549), while treatment for 14 days exacerbated bone loss (p < 0.0001). Administration of MK-886 enhanced osteoclastogenesis signaling and osteoclast formation within 7 days (p = 0.0005), but exerted no effect at 14 days (p > 0.9999). After 7 days of treatment, MK-886 induced mRNA expression for Acp5 (p = 0.0001), Calcr (p = 0.0003), Mmp9 (p = 0.0005) and Ctsk (p = 0.0008), however no effect in those gene expression was observed after 14 days (p > 0.05). CONCLUSION: Systemic treatment with MK-886 exacerbated LPS-induced apical periodontitis in a mouse model.

OPN N-glycosylation promoted bone destruction.

OBJECTIVES: Exploring the role of OPN N-glycosylation in osteoblasts and osteoclasts. METHODS: Immunohistochemistry was used to detect the expression of OPN in mice with apical periodontitis. The asparagine at position 79 of the OPN protein was mutated to glutamine, and the above plasmids were transfected into osteoblasts and osteoclasts. The effect of OPN N-glycosylation on proliferation of osteoblasts and osteoclasts was detected by CCK8 assays. Western blotting was used to detect the expression of OPN N-glycosylation on osteoclasts and osteoblasts. Detection of N-glycosylation of OPN activated the NF-κB signaling pathway to regulate osteoblasts and osteoclasts. RESULTS: OPN increased the expression in a mice model of apical periodontitis. The expression curve of OPN resembled a reverse V shape. The OPN N-glycosylation site was identified as 79 by MS. N-glycosylation of OPN promoted the proliferation of osteoclasts. But the N79 glycosylation site of mutant OPN could not increase the proliferation of osteoblasts. OPN N-glycosylation modulated the expression of osteoclast- and osteoblast-associated factors through the NF-κB signaling pathway. N-glycosylation of OPN promoted nuclear translocation of NF-κB in osteoclasts and osteoblasts. CONCLUSIONS: The N-glycosylation site of OPN is 79. N-glycosylation of OPN played an important role in the biological function of OPN protein.

Berberine regulates bone metabolism in apical periodontitis by remodelling the extracellular matrix.

OBJECTIVES: The objectives of this study were to explore the role and related mechanism of berberine in repairing bone destruction in apical periodontics (AP). MATERIALS AND METHODS: AP was established in 14 of 21 male Wistar rats (four weeks of age; 70-80 g) for 3 weeks. The canals were cleaned and administered berberine (2 mg/ml; n = 7) or calcium hydroxide (100 mg/ml; control; n = 7), followed by glass ionomer cement sealing. After 3 weeks, specimen collection followed by micro-computed tomography (µ-CT) and histological staining was performed, including haematoxylin and eosin staining, Masson's trichrome staining, tartrate-resistant acid phosphatase staining, immunohistochemistry and immunofluorescence histochemistry. RESULTS: µ-CT showed that AP lesion volume reduced in the berberine group. Histopathology showed that berberine decreased the activity and number of osteoclasts but increased the expression of proteins related to osteoblast differentiation, including alkaline phosphatase and osterix. The immune cell, T cell, dendritic cell and macrophage counts were significantly decreased in the berberine group. In the berberine group, the expression of extracellular matrix-degraded proteases, metalloproteinases, was decreased; however, that of extracellular matrix-stable proteases, lysyl oxidases, was increased. CONCLUSIONS: Berberine controlled the inflammatory response and regulated bone metabolism in AP by reducing metalloproteinase expression and increasing lysyl oxidases expression.

Evaluation of Receptor Activator of Nuclear Factor Kappa B Ligand, Osteoprotegerin, Osteopontin, and Tumor Necrosis Factor Alpha on Chronic Apical Periodontitis in Smokers.

INTRODUCTION: Smoking can be considered a risk factor for chronic apical periodontitis (CAP). This study compared the immunoexpression of biomarkers receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), osteopontin (OPN), and tumor necrosis factor alpha (TNF-α) in CAP in smokers and nonsmokers. METHODS: Twelve smokers and 12 nonsmokers diagnosed with CAP and indicated for tooth extraction were selected. Exclusion factors were teeth with a diagnosis of root fracture, previous endodontic treatment, or endoperiodontal injury, in addition to individuals with systemic diseases, under 18 years of age, users of anti-inflammatory and/or antibiotics in the last 3 months, and drug users. Specimens were processed for histopathologic and immunohistochemical analysis. RESULTS: Qualitative analysis of RANKL expression showed 66.66% weak/moderate and 33.33% strong in smokers and 100% weak/moderate in nonsmokers. OPG and OPN expressions were 100% negative to focal in the smoker group and 50% negative to focal and 50% weak/moderate in the nonsmoker group. TNF-α was 25% negative to focal and 75% weak/moderate in the smoker group and 33.33% negative to focal and 66.66% weak/moderate in the nonsmoker group. Quantitative analysis of the data using the Mann-Whitney U test showed that there was a significant difference in the immunoexpression of RANKL (P < .05), OPG (P < .05), and OPN (P < .05), but there was no statistical difference in the immunoexpression of TNF-α (P > .05) between the 2 groups. CONCLUSIONS: These findings suggest that smoking is capable of altering the inflammatory response, influencing the evolution of CAP.

m6 A demethylase Fto regulates the TNF-α-induced inflammatory response in cementoblasts.

OBJECTIVE: Apical periodontitis is the most frequently occurring pathological lesion. Fat mass and obesity-associated protein (Fto) is the first identified RNA N6-methyladenosine demethylase. However, whether Fto regulates apical periodontitis remains unclear. This study aimed to explore the mechanisms of Fto in the tumor necrosis factor-α (TNF-α)-induced inflammatory response. MATERIALS AND METHODS: We established an apical periodontitis model. An immortalized cementoblast cell line (OCCM-30) cells were exposed to TNF-α. Fto, Il6, Mcp1, and Mmp9 expressions were assessed by qRT-PCR. We knocked down Fto using lentiviruses and detected TNF-α-induced inflammation-related gene expressions and mRNA stability. RESULTS: Mice with apical periodontitis showed downregulation of Fto expression. OCCM-30 cells exposed to TNF-α showed an upregulation of inflammation-related genes with a decrease in Fto. Furthermore, knockdown of Fto promoted the expressions of Il6, Mcp1, and Mmp9 in TNF-α-treated OCCM-30 cells as compared with negative control cells, whereas it did not affect the mRNA stability. Interestingly, Fto knockdown activated the p65, p38, and ERK1/2 pathways, and it slightly activated the JNK signaling pathway after TNF-α administration in OCCM-30 cells. CONCLUSION: A TNF-α-induced decrease in the expression of Fto might play a critical role in the inflammatory response in cementoblasts, and knockdown of Fto might upregulate the inflammatory response.

Macrophage M1 polarization mediated via the IL-6/STAT3 pathway contributes to apical periodontitis induced by Porphyromonas gingivalis.

OBJECTIVE: To investigate the involvement of IL-6/STAT3 signaling pathway activation in macrophage polarization and bone destruction related to apical periodontitis (AP) stimulated by Porphyromonas gingivalis. METHODOLOGY: Macrophage polarization, IL-6/STAT3 expression, and the presence of P. gingivalis were detected in human AP tissues via RT-qPCR, western blotting, and immunohistochemistry staining. Murine bone marrow derived macrophages were isolated and cultured with P. gingivalis W83 in vitro, and levels of macrophage IL-6 expression, STAT3 phosphorylation, and macrophage polarization with or without the selective STAT3 phosphorylation inhibitor Stattic (5 µM) were detected via ELISA, western blotting, RT-qPCR, and flow cytometry, respectively. P. gingivalis-induced murine AP models were constructed, and bone destruction and macrophage polarization in the apical region were evaluated. Transwell co-culture systems were used to investigate the effects of macrophages infected with P. gingivalis on osteogenesis and osteoclastogenesis. RESULTS: P. gingivalis was detected in human AP tissues that highly expressed IL-6/STAT3, and the M1 subtype of macrophages was more abundant in these tissues. P. gingivalis infection induced IL-6 expression, STAT3 phosphorylation, and M1 polarization of macrophages, while 5 µM of Stattic partially abolished these activation effects. Systemic STAT3 blockade via oral administration of Stattic at a dose of 25 mg kg-1 alleviated murine periapical bone resorption and apical infiltration of M1 macrophages induced by P. gingivalis infection in vivo. Furthermore, macrophages infected with P. gingivalis promoted bone destruction via secretion of IL-6, TNF-α, and RANKL, which hinder pre-osteoblast expression of Runx2 and accelerate pre-osteoclast expression of NFAT2. CONCLUSIONS: The activation of IL-6/STAT3 signaling pathway is involved in mediating macrophages M1 polarization in the P. gingivalis induced apical inflammatory context and may also be intimately involved in the bone loss caused by P. gingivalis infection, directing the M1 macrophage infiltration during the progression of AP.

Leukotriene B4 Loaded in Microspheres Inhibits Osteoclast Differentiation and Activation.

To investigate osteoclast formation in vivo and if leukotriene B4 (LTB4) loaded in microspheres (MS) could be used as a therapeutical strategy to promote a sustained delivery of the mediator and prevent osteoclast differentiation. Methods: In vivo, apical periodontitis was induced in mice to investigate osteoclast differentiation and signaling in absence of 5-lipoxygenase (5-LO). In vitro, LTB4-MS were prepared using an oil-in-water emulsion solvent extraction-evaporation process. Characterization and efficiency of LTB4 encapsulation were investigated. J774A.1 macrophages were cultured in the presence of monocyte colony-stimulating factor (M-CSF) and ligand for receptor activator of nuclear factor kappa B (RANKL) and then stimulated with LTB4-MS. Cytotoxicity, in vitro MS-LTB4 uptake, osteoclast formation and gene expression were measured. Results: We found that 5-LO negatively regulates osteoclastic formation in vivo during apical periodontitis development. In vitro, LTB4-MS were up-taken by macrophages and were not cytotoxic to the cells. LTB4-MS inhibited osteoclast formation and the synthesis of osteoclastogenic genes Acp5, Mmp9, Calcr and Ctsk. LTB4-MS inhibited differentiation of macrophages into an osteoclastic phenotype and cell activation under M-CSF and RANKL stimulus.

Role of NOD2 and hepcidin in inflammatory periapical periodontitis.

The immunological response occurring during periapical inflammation includes expression of nucleotide binding oligomerization domain containing 2 and hepcidin. Nucleotide binding oligomerization domain containing 2 deficiency increases infiltration of inflammatory cells close to alveolar bone. Hepcidin has an important role in iron metabolism affecting bone metabolism.We investigated the role of nucleotide binding oligomerization domain containing 2 and hepcidin in inflammatory periapical periodontitis. Periapical periodontitis was induced in rats and confirmed by micro-computed tomography. Nucleotide binding oligomerization domain 2 and hepcidin were evaluated through immunohistochemistry. Bioinformatics analysis was undertaken usingthe Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases. Micro-computer tomography revealed alveolar bone resorption in the periapical region and furcation area of mandibular molars in rats of the periapical periodontitis group. Immunohistochemistry showed increased expressionof nucleotide binding oligomerization domain containing 2 and hepcidin around root apices in rats of the periapical periodontitis group. Bioinformatics analysis of differentially expressed genes in inflamed and non-inflamed tissues revealed enrichment in the NOD-like receptor signaling pathway. Our data suggest that nucleotide binding oligomization domain contain2 and hepcidin have important roles in periapical periodontitis severity because they can reduce alveolar bone loss.They could elicit new perspectives for development of novel strategies for periapical periodontitis treatment.

Trigeminal neurons control immune-bone cell interaction and metabolism in apical periodontitis.

Apical periodontitis (AP) is an inflammatory disease occurring following tooth infection with distinct osteolytic activity. Despite increasing evidence that sensory neurons participate in regulation of non-neuronal cells, their role in the development of AP is largely unknown. We hypothesized that trigeminal ganglia (TG) Nav1.8+ nociceptors regulate bone metabolism changes in response to AP. A selective ablation of nociceptive neurons in Nav1.8Cre/Diphtheria toxin A (DTA)Lox mouse line was used to evaluate the development and progression of AP using murine model of infection-induced AP. Ablation of Nav1.8+ nociceptors had earlier progression of AP with larger osteolytic lesions. Immunohistochemical and RNAscope analyses demonstrated greater number of macrophages, T-cells, osteoclast and osteoblast precursors and an increased RANKL:OPG ratio at earlier time points among Nav1.8Cre/ DTALox mice. There was an increased expression of IL-1α and IL-6 within lesions of nociceptor-ablated mice. Further, co-culture experiments demonstrated that TG neurons promoted osteoblast mineralization and inhibited osteoclastic function. The findings suggest that TG Nav1.8+ neurons contribute to modulation of the AP development by delaying the influx of immune cells, promoting osteoblastic differentiation, and decreasing osteoclastic activities. This newly uncovered mechanism could become a therapeutic strategy for the treatment of AP and minimize the persistence of osteolytic lesions in refractory cases.

Expression of PINK1 and Parkin in human apical periodontitis.

AIM: PTEN-induced putative kinase 1 (PINK1) and Parkin E3 ubiquitin-protein ligase (Parkin) are critical for immune and inflammatory regulation in health and disease. PINK1 and Parkin have been confirmed to be involved in the progression of apical periodontitis by affecting mitophagy-related osteoblast apoptosis; however, the expression of PINK1 and Parkin in macrophages, one of the most important cells in apical periodontitis, remains unknown. This study aimed to investigate the expression of PINK1 and Parkin in human apical periodontitis lesions, as well as their possible localization in macrophages. METHODOLOGY: Thirty-seven human periapical tissues, including periapical granulomas (PGs, n = 12), radicular cysts (RCs, n = 11) and healthy gingival tissues (n = 14) were examined. The inflammatory infiltrates of lesions were evaluated by haematoxylin staining, and the expression of PINK1 and Parkin was detected by immunohistochemistry. Double immunofluorescence was used to explore the colocalization of microtubule-associated protein 1 light chain 3 (LC3) and TOMM20, as well as the localization of PINK1 and Parkin, in macrophages of human apical periodontitis lesions. The ultrastructural morphology of mitochondria in human apical periodontitis lesions was visualized by transmission electron microscopy (TEM). Data were analysed by one-way anova with Student-Newman-Keul's test and the Mann-Whitney test. p < .05 was considered statistically significant. RESULTS: Immunohistochemistry demonstrated a significantly higher expression of PINK1 and Parkin proteins in human apical periodontitis lesions than in healthy gingival tissues (p < .0001), but no significant difference was demonstrated between PGs and RCs (p > .05). The higher expression of LC3 and the presence of more LC3-TOMM20 double-positive cells were also observed in human apical periodontitis. Double-labelling analysis of PINK1, Parkin and LC3 with CD68 indicated that macrophage mitophagy might be present in the progression of human apical periodontitis. Finally, the results of TEM morphological analysis revealed the appearance of double-membraned mitophagosomes and vacuolated mitochondria in macrophage-like cells of apical periodontitis lesions. CONCLUSIONS: Our findings indicated that PINK1 and Parkin proteins were highly expressed in clinical apical periodontitis lesions.