A role for the calcium-sensing receptor in the expression of inflammatory mediators in LPS-treated human dental pulp cells.

The aim of this study is to investigate the role of calcium-sensing receptor (CaSR) in the expression of inflammatory mediators of lipopolysaccharide (LPS)-treated human dental pulp cells (hDPCs). The expression profile of CaSR in LPS-simulated hDPCs was detected using immunofluorescence, real time quantitative PCR (RT-qPCR), and Western blot analyses. Then, its regulatory effects on the expression of specific inflammatory mediators such as interleukin (IL)-1ß, IL-6, cyclooxygenase 2 (COX2)-derived prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, and IL-10 were determined by RT-qPCR and enzyme-linked immunosorbent assay (ELISA). LPS significantly downregulated the gene expression of CaSR, but upregulated its protein expression level in hDPCs. Treatments by CaSR agonist R568 or its antagonist Calhex231, and their combinations with protein kinase B (AKT) inhibitor LY294002 showed obvious effects on the expression of selected inflammatory mediators in a time-dependent manner. Meanwhile, an opposite direction was found between the action of R568 and Calhex231, as well as the expression of the pro- (IL-1ß, IL-6, COX2-derived PGE2, and TNF-α) and anti-inflammatory (IL-10) mediators. The results provide the first evidence that CaSR-phosphatidylinositol-3 kinase (PI3K)-AKT-signaling pathway is involved in the release of inflammatory mediators in LPS-treated hDPCs, suggesting that the activation or blockade of CaSR may provide a novel therapeutic strategy for the treatment of pulp inflammatory diseases.

Differential Protein Expression in Human Dental Pulp: Comparison of Healthy, Inflamed, and Traumatic Pulp.

Trauma or injury to the dental pulp causes inflammation. This study compared the proteome of healthy pulp with inflamed pulp and traumatic pulp to identify the differentially expressed proteins in the diseased state. Five participants were grouped based on the pulpal status of the teeth: healthy, inflamed, or traumatic pulp. Pulp was extirpated and stored immediately in liquid nitrogen. Pulp tissues were subjected to 2-dimensional gel electrophoresis, and spot selection was performed. The selected spots were analyzed using liquid chromatography-tandem mass spectrometry and identified by correlating mass spectra to the proteomic databases. Fifteen spots showed increased expression in the inflamed and traumatic pulp. Annexin V, type II keratin, and hemoglobin levels were increased two-fold in the inflamed and traumatic pulp group and annexin V, mutant beta-actin, and hemoglobin were increased by ten-fold in the inflamed or traumatic pulp group, compared to levels in the healthy pulp group. Annexin V constituted two out of fifteen protein spots, and seemed to play a critical role in inhibiting inflammation and promoting the immune reaction. Further studies on this protein concerning its role in pulp repair are necessary to elucidate the underlying mechanisms.

The effect of DNA methylation on the miRNA expression pattern in lipopolysaccharide-induced inflammatory responses in human dental pulp cells.

Endodontic infection is a widespread oral problem. DNA methylation is a key epigenetic modification that plays important roles in various inflammatory responses, but its role in dental pulp inflammation is poorly understood. In this study, we assessed the expression of DNA methyltransferases (DNMTs) in human dental pulp cells (hDPCs) during lipopolysaccharide (LPS)-induced inflammation and found that DNMT3B mRNA expression was reduced and DNMT1 mRNA and protein levels decreased significantly. Pretreatment with the DNMT inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) significantly enhanced the expression of the inflammatory cytokines IL-6 and IL-8 in LPS-stimulated hDPCs, indicating that DNA methylation may play a role in hDPC inflammation. Studies have reported that some microRNAs (miRNAs) are involved in dental pulp infection. DNA methylation can modulate the inflammatory response by regulating miRNA expression, but this phenomenon has not yet been reported in pulp inflammation. The present study used next-generation sequencing to examine the effect of 5-Aza-CdR on the miRNA expression profile of LPS-treated hDPCs, and the results showed that 5-Aza-CdR pretreatment changed the miRNA expression pattern in hDPCs during inflammation. Among the changed miRNAs, miR-146a-5p, which is a pulp inflammation-related miRNA, demonstrated the most noticeably altered expression. miR-146a-5p could be induced by LPS in hDPCs, and 5-Aza-CdR preincubation or DNMT1 knockdown markedly increased its expression level. However, no significant difference was found in the methylation pattern of the MIR146A promoter with 5-Aza-CdR pretreatment or DNMT1 knockdown in LPS-stimulated hDPCs. These results indicate that DNA methylation may regulate the LPS-induced inflammatory response by changing the miRNA expression in hDPCs.

METTL3 regulates alternative splicing of MyD88 upon the lipopolysaccharide-induced inflammatory response in human dental pulp cells.

Dental pulp inflammation is a widespread public health problem caused by oral bacterial infections and can progress to pulp necrosis and periapical diseases. N6-methyladenosine (m6A) is a prevalent epitranscriptomic modification in mRNA. Previous studies have demonstrated that m6A methylation plays important roles in cell differentiation, embryonic development and stress responses. However, whether m6A modification affects dental pulp inflammation remains unknown. To address this issue, we investigated the expression of m6A and N6-adenosine methyltransferase (METTL3, METTL14) as well as demethylases (FTO, ALKBH5) and found that the levels of m6A and METTL3 were up-regulated in human dental pulp cells (HDPCs) stimulated by lipopolysaccharide (LPS). Furthermore, we knocked down METTL3 and demonstrated that METTL3 depletion decreased the expression of inflammatory cytokines and the phosphorylation of IKKα/ß, p65 and IκBα in the NF-κB signalling pathway as well as p38, ERK and JNK in the MAPK signalling pathway in LPS-induced HDPCs. The RNA sequencing analysis revealed that the vast number of genes affected by METTL3 depletion was associated with the inflammatory response. Previous research has shown that METTL3-dependent N6-adenosine methylation plays an important role in mRNA splicing. In this study, we found that METTL3 knockdown facilitated the expression of MyD88S, a splice variant of MyD88 that inhibits inflammatory cytokine production, suggesting that METTL3 might inhibit the LPS-induced inflammatory response of HDPCs by regulating alternative splicing of MyD88. These data shed light on new findings in epitranscriptomic regulation of the inflammatory response and open new avenues for research into the molecular mechanisms of dental pulp inflammation.

EZH2 regulates dental pulp inflammation by direct effect on inflammatory factors.

OBJECTIVE: Pulpitis is a multi-factorial disease that could be caused by complex interactions between genetics, epigenetics and environmental factors. We aimed to evaluate the role of Enhancer of Zeste Homolog 2 (EZH2) in the inflammatory response of human dental pulp cells (HDPCs) and dental pulp tissues. METHODS: The expressions of inflammatory cytokines in HDPCs treated by EZH2 complex or EZH2 siRNA with or without rhTNF-α were examined by quantitative real-time polymerase chain reaction (q-PCR). The levels of secreted inflammatory cytokines including IL-6, IL-8, IL-15, CCL2 and CXCL12 in culture supernatants were measured by Luminex assay. In rat pulpitis model, the effects of EZH2 on dental pulp tissues were verified by histology. We invested the mechanisms of the effect of EZH2 on the inflammatory factors by ChIP assay. RESULTS: EZH2 down-regulation inhibited the expression of inflammatory factors, including IL-6, IL-8, IL-15, CCL2 and CXCL12 in HDPCs. EZH2 complex promoted the expression and secretion of these inflammatory factors in HDPCs, while EZH2 silencing could attenuate the promotion of inflammatory factors that were induced by rhTNF-α. In pulpitis models of rats, EZH2 down-regulation inhibited the inflammatory process of dental pulp while EZH2 complex showed no significant facilitation of pulpal inflammation. In addition, EZH2 could bind on the promoters of IL-6, IL-8 and CCL2, but not IL-15 and CXCL12, to affect the transcription of these proinflammatory cytokines. CONCLUSIONS: In HDPCs, EZH2 could induce inflammation, while EZH2 down-regulation could attenuate the inflammatory responses. EZH2 plays an important role in this inflammatory process of dental pulp.

Role of ß-1,3-galactosyltransferase 2 in trigeminal neuronal sensitization induced by peripheral inflammation.

Glycosyltransferases are enzymes that catalyze the formation of a variety of glycoconjugates. Glycoconjugates play important roles in the nervous system. ß-1,3-Galactosyltransferase 2 (B3galt2) belongs to the family of ß-1,3-galactosyltransferase, which is one of the major types of glycosyltransferases. Dental pulp inflammation may cause neurophysiological alterations in the trigeminal ganglion (TG), and serve as a good model for investigating the peripheral inflammation and trigeminal neuronal sensitization. In the present study, we investigated the expression of B3galt2 in neuroinflammation using the dental pulp inflammatory model induced by lipopolysaccharide in rat. The expression of B3galt2 gene and protein were determined by reverse transcription PCR, immunohistochemistry and western blot analysis. ELISA assays were used to measure the levels of cytokines in the TG neurons. Toll-like receptor 4 (TLR4) and nuclear factor-κB (NFκB) were evaluated by immunohistochemistry and western blotting. Our results demonstrated that B3galt2 was expressed in the TG, and dental pulp inflammation up-regulated B3galt2 expression in the TG. B3galt2 gene knockdown reduced the secretion of TNFα and IL-6 in the TG neurons. The expression of TLR4 and NFκB in the TG was activated during the inflammation, but B3galt2 gene knockdown inhibited the expression of TLR4 and NFκB. These observations indicated that dental pulp inflammation could induce B3galt2 expression in TG, and that B3galt2 might play a regulatory role in TG neuronal sensitization. These findings suggest that B3galt2 may play an important role in trigeminal neuronal sensitization induced by peripheral inflammation via mediating TLR4/NFκB signaling pathway.

Epigenetic regulation in dental pulp inflammation.

Dental caries, trauma, and other possible factors could lead to injury of the dental pulp. Dental infection could result in immune and inflammatory responses mediated by molecular and cellular events and tissue breakdown. The inflammatory response of dental pulp could be regulated by genetic and epigenetic events. Epigenetic modifications play a fundamental role in gene expression. The epigenetic events might play critical roles in the inflammatory process of dental pulp injury. Major epigenetic events include methylation and acetylation of histones and regulatory factors, DNA methylation, and small non-coding RNAs. Infections and other environmental factors have profound effects on epigenetic modifications and trigger diseases. Despite growing evidences of literatures addressing the role of epigenetics in the field of medicine and biology, very little is known about the epigenetic pathways involved in dental pulp inflammation. This review summarized the current knowledge about epigenetic mechanisms during dental pulp inflammation. Progress in studies of epigenetic alterations during inflammatory response would provide opportunities for the development of efficient medications of epigenetic therapy for pulpitis.

CCL3 and CXCL12 production in vitro by dental pulp fibroblasts from permanent and deciduous teeth stimulated by Porphyromonas gingivalis LPS

ABSTRACT Objective: The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS). Material and Methods: Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0 – 10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results. Results: Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF. Conclusion: These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation. .

Immunohistochemical assessment of lymphangiogenesis in inflamed human dental pulp

The present study aimed to investigate the effect of inflammation on lymphangiogenesis in human dental pulp using monoclonal mouse anti-human D2-40 antibody. Thirty samples of healthy dental pulps and twenty-seven pulps with irreversible pulpitis were included in this study. All cases were immunohistochemically stained for D2-40. Positively stained lymphatic vessels were counted by a light microscopy in an area of 1 mm2. The mean number and standard deviation of lymphatic vessels positive for D2-40 in the group with inflammation (8.04 ± 2.8) was significantly higher (p< 0.0001) than the mean number in the group without inflammation (3.93 ± 1.11). The study established an increased number of lymphatic vessels in the inflamed human dental pulp, which suggests that inflammation contributes lymphangiogenesis. The new lymphatic vessels can accelerate the removal of interstitial fluid and thus limit the effects of increased tissue pressure on the connective tissue. This may have importance in the process of regeneration of dental pulp and conservative procedures such as pulpotomy.