Inhibiting Nav1.7 channels in pulpitis: An in vivo study on neuronal hyperexcitability.

Pulpitis constitutes a significant challenge in clinical management due to its impact on peripheral nerve tissue and the persistence of chronic pain. Despite its clinical importance, the correlation between neuronal activity and the expression of voltage-gated sodium channel 1.7 (Nav1.7) in the trigeminal ganglion (TG) during pulpitis is less investigated. The aim of this study was to examine the relationship between experimentally induced pulpitis and Nav1.7 expression in the TG and to investigate the potential of selective Nav1.7 modulation to attenuate TG abnormal activity associated with pulpitis. Acute pulpitis was induced at the maxillary molar (M1) using allyl isothiocyanate (AITC). The mice were divided into three groups: control, pulpitis model, and pulpitis model treated with ProTx-II, a selective Nav1.7 channel inhibitor. After three days following the surgery, we conducted a recording and comparative analysis of the neural activity of the TG utilizing in vivo optical imaging. Then immunohistochemistry and Western blot were performed to assess changes in the expression levels of extracellular signal-regulated kinase (ERK), c-Fos, collapsin response mediator protein-2 (CRMP2), and Nav1.7 channels. The optical imaging result showed significant neurological excitation in pulpitis TGs. Nav1.7 expressions exhibited upregulation, accompanied by signaling molecular changes suggestive of inflammation and neuroplasticity. In addition, inhibition of Nav1.7 led to reduced neural activity and subsequent decreases in ERK, c-Fos, and CRMP2 levels. These findings suggest the potential for targeting overexpressed Nav1.7 channels to alleviate pain associated with pulpitis, providing practical pain management strategies.

Macrophage M2 polarization promotes pulpal inflammation resolution during orthodontic tooth movement.

Mechanical force induces hypoxia in the pulpal area by compressing the apical blood vessels of the pulp, triggering pulpal inflammation during orthodontic tooth movement. However, this inflammation tends to be restorable. Macrophages are recognized as pivotal immunoreactive cells in the dental pulp. Whether they are involved in the resolution of pulpal inflammation in orthodontic teeth remains unclear. In this study, we investigated macrophage polarization and its effects during orthodontic tooth movement. It was demonstrated that macrophages within the dental pulp polarized to M2 type and actively participated in the process of pulpal inflammation resolution. Inflammatory reactions were generated and vascularization occurred in the pulp during orthodontic tooth movement. Macrophages in orthodontic pulp show a tendency to polarize towards M2 type as a result of pulpal hypoxia. Furthermore, by blocking M2 polarization, we found that macrophage M2 polarization inhibits dental pulp-secreting inflammatory factors and enhances VEGF production. In conclusion, our findings suggest that macrophages promote pulpal inflammation resolution by enhancing M2 polarization and maintaining dental health during orthodontic tooth movement.

Unveiling Therapeutic Potential: Targeting Fusobacterium nucleatum's Lipopolysaccharide Biosynthesis for Endodontic Infections-An In Silico Screening Study.

Complex microbial communities have been reported to be involved in endodontic infections. The microorganisms invade the dental pulp leading to pulpitis and initiating pulp inflammation. Fusobacterium nucleatum is a dominant bacterium implicated in both primary and secondary endodontic infections. Drugs targeting the molecular machinery of F. nucleatum will minimize pulp infection. LpxA and LpxD are early acyltransferases involved in the formation of lipid A, a major component of bacterial membranes. The identification of leads which exhibit preference towards successive enzymes in a single pathway can also prevent the development of bacterial resistance. A stringent screening strategy utilizing physicochemical and pharmacokinetic parameters along with a virtual screening approach identified two compounds, Lomefloxacin and Enoxacin, with good binding affinity towards the early acyltransferases LpxA and LpxD. Lomefloxacin and Enoxacin, members of the fluoroquinolone antibiotic class, exhibit wide-ranging activity against diverse bacterial strains. Nevertheless, their effectiveness in the context of endodontic treatment requires further investigation. This study explored the potential of Lomefloxacin and Enoxacin to manage endodontic infections via computational analysis. Moreover, the compounds identified herein serve as a foundation for devising novel combinatorial libraries with enhanced efficacy for endodontic therapeutic strategies.

Study on pulp metabolism of patients with pulpitis using ultra-performance liquid chromatography coupled with Orbitrap mass spectrometry.

BACKGROUND AND AIMS: Pulpitis, a pulp disease caused by caries, trauma, and other factors, has a high clinical incidence. This study focused on identifying possible metabolic biomarkers of pulpitis cases and analyzing the related metabolic pathways for providing a theoretical foundation to diagnose and prevent pulpitis. MATERIALS AND METHODS: Pulp samples from 20 pulpitis cases together with 20 normal participants were analyzed with a serum metabolomics approach using ultra-high-performance liquid chromatography (UPLC)/Orbitrap mass spectrometry. Moreover, this work carried out multivariate statistical analysis for screening potential biomarkers of pulpitis. RESULTS: Through biomarker analysis and identification, such as partial least squares discrimination analysis, orthogonal partial least squares discriminant analysis model establishment, correlation analysis, and biomarker pathway analysis, 40 biomarkers associated with 20 metabolic pathways were identified, including 20 upregulated and 20 downregulated metabolites. Those major biomarkers included oxoglutaric acid, inosine, citric acid, and PA(14:1(9Z)/PGD1). Among them, oxoglutaric acid and inosine were most significantly downregulated and had the highest correlation with pulpitis. Among these metabolic pathways, GABAergic synapse and alanine, aspartate, and glutamate metabolism were positively correlated with pulpitis. 4. CONCLUSIONS: These biomarkers as well as metabolic pathways may offer the theoretical foundation to understand pulpitis pathogenesis and develop preventive drugs.

TSG-6 Inhibits the NF-κB Signaling Pathway and Promotes the Odontogenic Differentiation of Dental Pulp Stem Cells via CD44 in an Inflammatory Environment.

In pulpitis, dentinal restorative processes are considerably associated with undifferentiated mesenchymal cells in the pulp. This study aimed to investigate strategies to improve the odonto/osteogenic differentiation of dental pulp stem cells (DPSCs) in an inflammatory environment. After pretreatment of DPSCs with 20 ng/mL tumor necrosis factor-induced protein-6 (TSG-6), DPSCs were cultured in an inflammation-inducing solution. Real-time polymerase chain reaction and Western blotting were performed to measure the expression levels of nuclear factor kappa B (NF-κB) and odonto/osteogenic differentiation markers, respectively. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays were used to assess cell proliferation and activity. Subcutaneous ectopic osteogenesis and mandibular bone cultures were performed to assess the effects of TSG-6 in vivo. The expression levels of odonto/osteogenic markers were higher in TSG-6-pre-treated DPSCs than nontreated DPSCs, whereas NF-κB-related proteins were lower after the induction of inflammation. An anti-CD44 antibody counteracted the rescue effect of TSG-6 on DPSC activity and mineralization in an inflammatory environment. Exogenous administration of TSG-6 enhanced the anti-inflammatory properties of DPSCs and partially restored their mineralization function by inhibiting NF-κB signaling. The mechanism of action of TSG-6 was attributed to its interaction with CD44. These findings reveal novel mechanisms by which DPSCs counter inflammation and provide a basis for the treatment of pulpitis.

Acute pulpitis promotes purinergic signaling to induce pain in rats via P38MAPK/NF-κB signaling pathway.

Toothache is one of the most common types of pain, but the mechanisms underlying pulpitis-induced pain remain unknown. The ionotropic purinergic receptor family (P2X) is reported to mediate nociception in the nervous system. This study aims to investigate the involvement of P2X3 in the sensitisation of the trigeminal ganglion (TG) and the inflammation caused by acute pulpitis. An acute tooth inflammation model was established by applying LPS to the pulp of SD rats. We found that the increased expression of P2X3 was induced by acute pulpitis. A selective P2X3 inhibitor (A-317491) reduced pain-like behavior in the maxillofacial region of rats and depressed the activation of neurons in the trigeminal ganglion induced by pulpitis. The upregulated MAPK signaling (p-p38, p-ERK1/2) expression in the ipsilateral TG induced by pulpitis could also be depressed by the application of the P2X3 inhibitor. Furthermore, the expression of markers of inflammatory processes, such as NF-κB, TNF-α and IL-1ß, could be induced by acute pulpitis and deduced by the intraperitoneal injection of P2X3 antagonists. Our findings demonstrate that purinergic P2X3 receptor signaling in TG neurons contributes to pulpitis-induced pain in rats and that P2X3 signaling may be a potential therapeutic target for tooth pain.

Osteopontin interacts with dendritic cells and macrophages in pulp inflammation: Comprehensive transcriptomic analysis and laboratory investigations.

AIM: To investigate novel diagnostic markers for pulpitis and validate by clinical samples from normal and inflamed pulp. To explore the relationship between diagnostic markers and immune cells or their phenotypes during pulp inflammation. METHODOLOGY: Two microarray datasets, GSE77459 and GSE92681, and identified differential expression genes were integrated. To understand immune features, gene functions, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Disease Ontology (DO) and ImmuneSigDB Gene Set Enrichment Analysis (GSEA) were analysed. For predictive purposes, machine learning techniques were applied to detect diagnostic markers. Immune infiltration in inflamed pulp was studied using CIBERSORT. The relationship between diagnostic markers and immune cells was investigated and validated their gene expression in clinical samples from the normal or inflamed pulp by qRT-PCR. Finally, the correlation between one marker, secreted phosphoprotein 1 (SPP1), encoding osteopontin (OPN), and dendritic cells (DCs)/macrophages was identified via HE staining and multiplex immunohistochemistry. An in vitro inflammatory dental pulp microenvironment model of THP-1 macrophages cocultured with dental pulp cells derived conditioned media (DPCs-CM) to investigate OPN production and macrophage phenotypes was established. RESULTS: Analysis revealed unique immunologic features in inflamed pulp. Three diagnostic markers for pulpitis: endothelin-1 (EDN1), SPP1, and purine nucleoside phosphorylase (PNP), and validated them using qRT-PCR were predicted. Multiplex immunohistochemistry demonstrated OPN co-localized with activated DCs and M2 macrophages during pulp inflammation. In vitro experiments showed that THP-1 macrophages produced the highest levels of OPN when stimulated with DPCs-CM derived from the 20 µg/mL LPS pre-conditioned group, suggesting an M2b-like phenotype by increasing surface marker CD86 and expression of IL6, TNFα, IL10, and CCL1 but not CCL17 and MerTK. Levels of CCL1 and IL10 elevated significantly in the macrophages' supernatant from the 20 µg/mL LPS pre-conditioned CM group. OPN was proven co-localizing with CD86 in the inflamed pulp by immunofluorescence. CONCLUSIONS: The current findings suggest that OPN can serve as a promising biomarker for pulpitis, correlated with DCs and macrophages. OPN+ macrophages in the inflamed pulp are associated with M2b-like phenotypes. These insights offer the potential for improved diagnosis and targeted therapy.

LIF Aggravates Pulpitis by Promoting Inflammatory Response in Macrophages.

Leukemia inhibitory factor (LIF) has been recognized as a novel inflammatory modulator in inflammation-associated diseases. This study aimed to investigate the modulation of LIF in dental pulp inflammation. Experimental pulpitis was established in wild-type (WT) and Lif-deficient (Lif-/-) mice. Histological and immunostaining analyses were conducted to assess the role of LIF in the progression of pulpitis. Mouse macrophage cell line (RAW264.7) was treated with LPS to simulate an inflammatory environment. Exogenous LIF was added to this system to examine its modulation in macrophage inflammatory response in vitro. Primary bone marrow-derived macrophages (BMDMs) from WT and Lif-/- mice were isolated and stimulated with LPS to confirm the effect of Lif deletion on macrophage inflammatory response. Supernatants from LIF and LPS-treated human dental pulp cells (hDPCs) were collected and added to macrophages. Macrophage chemotaxis was assessed using transwell assays. The results showed an increased expression of LIF and LIFR with the progression of pulpitis, and LIFR was highly expressed in macrophages. Lif deficiency alleviated experimental pulpitis with the reduction of pro-inflammatory cytokines and macrophage infiltration. Exogenous LIF promoted inflammatory response of LPS-induced macrophages through a STAT3/p65-dependent pathway. Consistently, Lif deletion inhibited macrophage inflammatory response in vitro. Supernatants of LIF-treated hDPCs enhanced macrophage migration in LPS-induced inflammatory environment. Our findings demonstrated that LIF aggravates pulpitis by promoting macrophage inflammatory response through a STAT3/p65-dependent pathway. Furthermore, LIF plays a crucial role in driving the recruitment of macrophages to inflamed pulp tissue by promoting chemokine secretion in DPCs.

Guanylate-binding protein 5-mediated cell-autonomous immunity suppresses inflammation in dental pulpitis: An in vitro study.

AIM: Guanylate-binding protein 5 (GBP5) is an interferon (IFN)-inducible GTPase that plays a crucial role in the cell-autonomous immune response against microbial infections. In this study, we investigated the immunoregulatory role of GBP5 in the pathogenesis of dental pulpitis. METHODOLOGY: Gene-set enrichment analysis (GSEA) was utilized to evaluate the IFN-γ signalling pathway, and the differential expression of GBP mRNA in normal versus inflamed dental pulp tissues was screened, based on Gene Expression Omnibus (GEO) datasets associated with pulpitis. Both normal pulp tissues and inflamed pulp tissues were used for experiments. The expression of IFNs and GBPs was determined by qRT-PCR. Immunoblotting and double immunofluorescence were performed to examine the cellular localization of GBP5 in dental pulp tissues. For the functional studies, IFN-γ priming or lentivirus vector-delivered shRNA was used to, respectively, overexpress or knock down endogenous GBP5 expression in human dental pulp stem cells (HDPSCs). Subsequently, LPS was used to stimulate HDPSCs (overexpressing or with knocked-down GBP5) to establish an in vitro model of inflammation. qRT-PCR and ELISA were employed to examine the expression of proinflammatory cytokines (IL-6, IL-8 and IL-1ß) and cyclooxygenase 2 (COX2). Every experiment has three times of biological replicates and three technical replicates, respectively. Statistical analysis was performed using the Student's t-test and one-way ANOVA, and a p-value of <.05 was considered statistically significant. RESULTS: GSEA analysis based on the GEO dataset revealed a significant activation of the IFN-γ signalling pathway in the human pulpitis group. Among the human GBPs evaluated, GBP5 was selectively upregulated in inflamed dental pulp tissues and predominantly expressed in dental pulp cells. In vitro experiments demonstrated that IFN-γ robustly induced the expression of GBP5 in HDPSCs. Knockdown of GBP5 expression in HDPSCs significantly amplified the LPS-induced upregulation of inflammatory mediators (IL-6, IL-8, IL-1ß and COX2) both with and without IFN-γ priming. CONCLUSION: Our findings demonstrated that GBP5 partook in the pathogenesis of dental pulpitis. The involvement of GBP5 in pulpitis appeared to coordinate the regulation of inflammatory cytokines. Knockdown of GBP5 contributed to the exacerbation of LPS-mediated inflammation.

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research.

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms underlying pulpitis and its efficient treatment is an ongoing focus of endodontic research. Therefore, understanding the inflammatory process within the dental pulp is vital for improving pulp preservation. Compared to other in vitro experiments, a murine pulpitis model offers a more authentic and genetically diverse context to observe the pathological progression of pulpitis. However, using mice, despite their cost-effectiveness and accessibility, poses difficulties due to their small size, poor coordination, and low tolerance, complicating intraoral and dental procedures. This protocol introduces a novel design and application of a mouth-gag to expose mouse pulp, facilitating more efficient intraoral procedures. The mouth-gag, comprised of a dental arch readily available to most dentists and can significantly expedite surgical preparation, even for first-time procedures. Micro-CT, hematoxylin-eosin (HE) staining, and immunofluorescence staining were used to identify changes in morphology and cell expression. The aim of this article is to help researchers establish a more reproducible and less demanding procedure for creating a pulp inflammation model using this novel mouth-gag.

Mitochondrial Dysfunction in the Pathogenesis and Treatment of Oral Inflammatory Diseases.

Oral inflammatory diseases (OIDs) include many common diseases such as periodontitis and pulpitis. The causes of OIDs consist microorganism, trauma, occlusal factors, autoimmune dis-eases and radiation therapy. When treated unproperly, such diseases not only affect oral health but also pose threat to people's overall health condition. Therefore, identifying OIDs at an early stage and exploring new therapeutic strategies are important tasks for oral-related research. Mitochondria are crucial organelles for many cellular activities and disruptions of mitochondrial function not only affect cellular metabolism but also indirectly influence people's health and life span. Mitochondrial dysfunction has been implicated in many common polygenic diseases, including cardiovascular and neurodegenerative diseases. Recently, increasing evidence suggests that mitochondrial dysfunction plays a critical role in the development and progression of OIDs and its associated systemic diseases. In this review, we elucidated the critical insights into mitochondrial dysfunction and its involvement in the inflammatory responses in OIDs. We also summarized recent research progresses on the treatment of OIDs targeting mitochondrial dysfunction and discussed the underlying mechanisms.

IPO7 promotes lipopolysaccharide-induced inflammatory responses in human dental pulp cells via p38 MAPK and NF-κB signaling pathways.

Pulpitis is a chronic inflammatory process that greatly affects the physical, mental health and life quality of patients. Human dental pulp cells (hDPCs) are essential components of dental pulp tissue and play a significant role in pulpitis. Lipopolysaccharide (LPS) is an initiator of pulpitis and can induce the production of inflammatory cytokines in hDPCs by activating p38 MAPK and NF-κB signaling pathways. Importin7 (IPO7), a member of the importin-ß family, is widely expressed in many tissues. Previous studies have shown that IPO7 mediated nuclear translocation of p-p38 after stimulation, and IPO7 homologous protein IPO8 participated in human dental pulp inflammation. This research aims to investigate whether IPO7 is involved in pulpitis and explore its underlying mechanisms. In the current study, we found the expression of IPO7 was increased in pulpitis tissue. In vitro, hDPCs treated with LPS to mimic the inflammatory environment, the expression of IPO7 was increased. Knockdown of IPO7 significantly inhibited the production of inflammatory cytokines and suppressed the p38 MAPK and NF-κB signaling pathways. Activating the p38 MAPK and NF-κB signaling pathways by the p38 activator and p65 activator reversed the inflammatory responses. IPO7 interacted with p-p38 under LPS stimulation in hDPCs. In addition, the increased binding between IPO7 and p-p38 is associated with the decreased binding ability of IPO7 to Sirt2. In conclusion, we found that IPO7 was highly expressed in pulpitis and played a vital role in modulating human dental pulp inflammation.

Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions.

BACKGROUND: Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. OBJECTIVE: To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. METHODOLOGY: HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). RESULTS: After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. CONCLUSION: The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.

Dysregulation of MicroRNA-152-3p is Associated with the Pathogenesis of Pulpitis by Modulating SMAD5.

PURPOSE: To research the role of microRNA (miR)-152 in the pathogenesis of pulpitis using a cell model based on human dental pulp cells (HDPCs) treated with lipopolysaccharides (LPS). MATERIALS AND METHODS: The biological activity of HDPCs infected by LPS was measured using a cell counting kit (CCK-8), Transwell test, flow cytometry, and fluorescent quantitative PCR. The concentration of superoxide dismutase (SOD) and malondialdehyde (MDA) was evaluated using an assay kit, the levels of interleukin (IL)-1ß and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA), and the targeting relationship between SMAD5 and miR-152 was measured by the double-luciferase report test. The expression of cell cycle-related CyclinD1 and BAX was assessed by PCR. By plotting a receiver operating characteristic (ROC) curve, the diagnostic value of miR-152 was shown. RESULTS: The level of miR-152 in HDPCs induced by LPS decreased, while the level of SMAD5 increased. After overexpressing miR-152 in LPS-induced HDPCs, the viability was elevated, the apoptosis rate decreased, CyclinD1 was elevated, BAX diminished, the inflammatory cytokines (IL-6 and IL-1ß) were inhibited, the activity of SOD increased, and the MDA content decreased. miR-152 targeted regulation of SMAD5, and SMAD5 modulated the effects of miR-152 on cell viability, apoptosis, inflammation, and the oxidative response of HDPCs. Reduced miR-152 expression was verified in patients with pulpitis, which could be a biomarker for pulpitis. CONCLUSION: miR-152 was found to be a biomarker correlated with the pathogenesis of pulpitis and the biological behaviour of HDPCs.

Non-Psychoactive Cannabinoid Modulation of Nociception and Inflammation Associated with a Rat Model of Pulpitis.

Despite advancements in dental pain management, one of the most common reasons for emergency dental care is orofacial pain. Our study aimed to determine the effects of non-psychoactive Cannabis constituents in the treatment of dental pain and related inflammation. We tested the therapeutic potential of two non-psychoactive Cannabis constituents, cannabidiol (CBD) and ß-caryophyllene (ß-CP), in a rodent model of orofacial pain associated with pulp exposure. Sham or left mandibular molar pulp exposures were performed on Sprague Dawley rats treated with either vehicle, the phytocannabinoid CBD (5 mg/kg i.p.) or the sesquiterpene ß-CP (30 mg/kg i.p.) administered 1 h pre-exposure and on days 1, 3, 7, and 10 post-exposure. Orofacial mechanical allodynia was evaluated at baseline and post-pulp exposure. Trigeminal ganglia were harvested for histological evaluation at day 15. Pulp exposure was associated with significant orofacial sensitivity and neuroinflammation in the ipsilateral orofacial region and trigeminal ganglion. ß-CP but not CBD produced a significant reduction in orofacial sensitivity. ß-CP also significantly reduced the expression of the inflammatory markers AIF and CCL2, while CBD only decreased AIF expression. These data represent the first preclinical evidence that non-psychoactive cannabinoid-based pharmacotherapy may provide a therapeutic benefit for the treatment of orofacial pain associated with pulp exposure.

Differentially Expressed Genes in Dental Pulp Tissues of Individuals With Symptomatic Irreversible Pulpitis With and Without History of COVID-19.

INTRODUCTION: Increased levels of proinflammatory markers have been reported in tissues of individuals with Coronavirus Disease 2019 (COVID-19). We hypothesize that inflamed dental pulp tissues of individuals with previous history of COVID-19 may present a differential inflammatory gene expression profile in comparison with individuals who never had COVID-19. MATERIALS AND METHODS: Dental pulp tissues were collected from 27 individuals referred for endodontic treatment due to symptomatic irreversible pulpitis. Of these, 16 individuals had a history of COVID-19 (6 months to 1 year post infection) and 11 individuals had no previous history of COVID-19 (controls). Total RNA from pulp tissue samples was extracted and subjected to RNA sequencing for comparison of differentially expressed genes (DEGs) among groups. DEGs showing log2(fold change) > 1 or < -1, and P < .05 were considered significantly dysregulated. RESULTS: RNA sequencing identified 1461 genes as differentially expressed among the groups. Of these, 311 were protein coding genes, 252 (81%) that were upregulated and 59 (19%) that were downregulated in the COVID group compared with controls. The top upregulated genes in the COVID group were HSFX1 (4.12-fold change) and LINGO3 (2.06-fold change); significantly downregulated genes were LYZ (-1.52-fold change), CCL15 and IL8 (-1.45-fold change). CONCLUSIONS: Differential gene expression in dental pulp tissues of COVID and non-COVID groups suggests potential contribution of COVID-19 on dysregulating inflammatory gene expression in the inflamed dental pulp.

Overexpression of MicroRNA-155 aggravates pulpitis by targeting kinesin superfamily Proteins-5C based on illumina high-throughput sequencing.

AIM: To investigate the regulatory role of miR-155 and Kinesin Superfamily Proteins-5C (KIF-5C) in the progression of pulpitis based on bioinformatic analysis. METHODOLOGY: Normal pulp tissues and pulpitis pulp tissues were collected and subjected to high-throughput sequencing and the differentially expressed miRNAs were determined. An in vitro and in vivo pulpitis model was established. HE, IHC staining and histological evaluation were used to verify the inflammatory state of human and mouse pulp tissues. The mRNA expression of IL-1ß and TGF-ß1 were determined by RT-qPCR and protein expression of IL-1α, IL-4, IL-8, IL-13, IFN-γ, IL-6, IL-10 and MCP-1 were determined by protein chip. The target genes of miR-155 were predicted by miRanda database and verified by Dual-luciferase reporter assay, RT-qPCR and western blotting. MiR-155 lentivirus were used to upregulate or downregulate miR-155 and the siRNA of KIF-5C was used to downregulate KIF-5C. The expression of miR-155 or KIF-5C was determined by RT-qPCR. All statistics were analysed using GraphPad prism 8.2. RESULTS: The high-throughput sequencing results showed that 6 miRNAs (miR-155, miR-21, miR-142, miR-223, miR-486, miR-675) were significantly upregulated in diseased human pulp tissues, and miR-155 was significantly elevated among the six miRNAs. RT-qPCR results demonstrated that miR-155 expression was upregulated in human pulpitic tissue, mice pulpitic tissue and LPS-HDPCs. IL-1ß was increased while TGF-ß1 was decreased in lenti-miR-155 transfected LPS-HDPCs. Analysis of protein chip results indicated that lenti-miR-155 transfected LPS-HDPCs produced higher levels of IL-8, IL-6, MCP-1. The opposite results were obtained when miR-155 was inhibited. Through miRanda database screen and Dual-luciferase reporter assay, the target gene (KIF-5C) of miR-155 was identified. In lenti-miR-155 transfected LPS-HDPCs, the expression of KIF-5C was downregulated. However, when shRNA-miR-155 was transfected to LPS-HDPCs, the opposite result was obtained. Silent RNA was used to knock down KIF-5C, the results showed that when both KIF-5C and miR-155 were knocked down simultaneously, the downregulated expression of inflammatory factors observed in LPS-HDPCs following miR-155 knockdown was rescued. CONCLUSION: MiR-155 plays an important role in promoting pulpitis through targeting KIF-5C and may serve as a potential therapeutic target.

Intercellular mitochondrial transfer alleviates pyroptosis in dental pulp damage.

Mitochondrial transfer is emerging as a promising therapeutic strategy for tissue repair, but whether it protects against pulpitis remains unclear. Here, we show that hyperactivated nucleotide-binding domain and leucine-rich repeat protein3 (NLRP3) inflammasomes with pyroptotic cell death was present in pulpitis tissues, especially in the odontoblast layer, and mitochondrial oxidative stress (OS) was involved in driving this NLRP3 inflammasome-induced pathology. Using bone marrow mesenchymal stem cells (BMSCs) as mitochondrial donor cells, we demonstrated that BMSCs could donate their mitochondria to odontoblasts via tunnelling nanotubes (TNTs) and, thus, reduce mitochondrial OS and the consequent NLRP3 inflammasome-induced pyroptosis in odontoblasts. These protective effects of BMSCs were mostly blocked by inhibitors of the mitochondrial function or TNT formation. In terms of the mechanism of action, TNF-α secreted from pyroptotic odontoblasts activates NF-κB signalling in BMSCs via the paracrine pathway, thereby promoting the TNT formation in BMSCs and enhancing mitochondrial transfer efficiency. Inhibitions of NF-κB signalling and TNF-α secretion in BMSCs suppressed their mitochondrial donation capacity and TNT formation. Collectively, these findings demonstrated that TNT-mediated mitochondrial transfer is a potential protective mechanism of BMSCs under stress conditions, suggesting a new therapeutic strategy of mitochondrial transfer for dental pulp repair.

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.

Immunohistochemical Analysis of S100 Proteins in Normal and Irreversibly Inflamed Human Dental Pulps.

INTRODUCTION: S100 proteins convey important roles in innate immune responses to infection and regenerative processes. However, their role in inflammatory or regenerative processes of the human dental pulp is poorly elucidated. The aim of the present study was to detect, localize, and compare the occurrence of 8 S100 proteins in normal, symptomatic, and asymptomatic irreversibly inflamed dental pulp specimens. METHODS: Human dental pulp specimens from 45 individuals were clinically assigned to 3 groups of pulpal diagnosis: normal pulp (NP, n = 17), asymptomatic irreversible pulpitis (AIP, n = 13), and symptomatic irreversible pulpitis (SIP, n = 15). The specimens were prepared and immunohistochemically stained for proteins S100A1, -A2, -A3, -A4, -A6, -A7, -A8, and -A9. Staining was classified using semiquantitative analysis and a 4-degree staining score (ie, no, decent, medium, and intense staining) at 4 different anatomic or functional regions (ie, the odontoblast layer [OL], pulpal stroma [PS], border area of calcifications [BAC], and vessel walls). The distribution of staining degrees between the 3 diagnostic groups was calculated using the Fisher exact text (P ≤ .05) at the 4 regions. RESULTS: Significant differences in staining were observed mainly in the OL and PS and at the BAC. The most significant differences were detected in the PS and when comparing NP with 1 of the 2 irreversibly inflamed pulpal tissues (AIP or SIP). The inflamed tissues were then invariably stained more intensely than their normal counterparts at this location (S100A1, -A2, -A3, -A4, -A8, and -A9). In the OL, NP tissue was significantly stronger stained for S100A1, -A6, -A8, and -A9 compared with SIP and for S100A9 when compared with AIP. Differences between AIP and SIP in direct comparison were rare and were found only for 1 protein (S100A2) at the BAC. Also, at the vessel walls, only 1 statistical difference in staining was observed (SIP was stronger stained than NP for protein S100A3). CONCLUSIONS: The occurrence of proteins S100A1, -A2, -A3, -A4, -A6, -A8, and -A9 is significantly altered in irreversibly inflamed compared with normal dental pulp tissue at different anatomic localizations. Some members of S100 proteins obviously participate in focal calcification processes and pulp stone formation of the dental pulp.