1. Introduction
Pulpitis is a common oral disease, with dental caries and tooth trauma as the main causes [ 1 ]. Without medical intervention, dental pulp inflammation may further progress to irreversible pulpitis and necrosis, leading to intolerable pain and potential tooth loss, which will significantly affect the quality of life [ 2 ]. The inflammatory response in the dental pulp is typically triggered by the invasion of microorganisms, particularly Gram-negative bacteria, which release lipopolysaccharide (LPS) as a key pathogenic component [ 3 , 4 ]. LPS has been extensively studied for its ability to induce inflammation of various cell types in dental pulp, including human dental pulp stem cells (hDPSCs), making it a valuable tool for simulating the microenvironment of pulpitis in vitro and in vivo [ 5 , 6 , 7 ]. DPSCs, as progenitor cells within the dental pulp, possess remarkable multidirectional differentiation potential, playing a critical role in pulp tissue homeostasis [ 8 ]. In the initial stage of dental pulp inflammation, DPSCs migrate to inflammatory sites and differentiate into odontoblast-like cells, facilitating reparative dentin formation and blocking bacterial invasion [ 9 ]. However, further development of inflammation limits the function of DPSCs, suggesting that timely and effective control of inflammation is essential for the repair and regeneration of dental pulp [ 10 , 11 , 12 ]. Studies have shown that nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways are critical in the pathogenesis and development of DPSC inflammatory response [ 13 , 14 ]. Inhibition of the signaling activation can limit the synthesis and release of pro-inflammatory molecules, thus avoiding the aggravation of inflammation and protecting dental pulp from damage [ 15 , 16 , 17 ].
Accumulating studies demonstrate that epigenetic modification participates in multiple physiological processes [ 18 , 19 , 20 ]. The newly characterized mRNA modification, N4-acetylcytidine (ac 4 C), is crucial for modulating both mRNA stability and the efficiency of translation [ 21 ]. N-acetyltransferase 10 (NAT10), the sole identified ac 4 C acetyltransferase, is considered to be vital in various cellular biological processes, including periodontal stem cell osteogenic differentiation, gastric cancer cell metastasis, and multiple myeloma cell apoptosis [ 22 , 23 , 24 ]. Recently, emerging evidence suggests that NAT10-mediated ac 4 C modification is associated with inflammatory responses. Notably, the effects of NAT10 may vary in different cell types. In neutrophils, NAT10 has been reported as an important negative regulator of pyroptosis; downregulation of NAT10 exacerbates sepsis by increasing pyroptosis in neutrophils via the ULK1-STING-NLRP3 axis [ 25 ]. Inconsistent with this, a previous study found that overexpression of NAT10 aggravated LPS-induced inflammation through the NOX2-ROS-NF-κB axis in macrophages, and Remodelin, a specific inhibitor of NAT10, alleviated macrophage infiltration in mice with periodontitis [ 26 ]. While these results indicated that the regulatory role of NAT10 in inflammation may be cell-specific, whether and how NAT10 modulates inflammation in hDPSCs has not been elucidated.
In this study, the inflammatory model of hDPSCs in vitro and the pulpitis model of rats in vivo were constructed. By knocking down NAT10 and using its inhibitor Remodelin, this study investigated the regulatory effect and potential mechanism of ac 4 C modification mediated by NAT10 in the regulation of pulpitis.
2. Results
2.1. Expression of NAT10 Is Upregulated in Inflamed Dental Pulp and LPS-Treated hDPSCs
To determine the involvement of NAT10 in dental pulp inflammation, immunohistochemistry was conducted on healthy and inflamed human dental pulp ( Figure 1 A). The results showed that NAT10 expression was elevated in pulpitis tissues compared to healthy pulp tissues, which was in line with the expression of NAT10 mRNA ( Figure 1 B). Similar results were obtained when NAT10 expression was detected in rat dental pulp from the control group and pulpitis group by immunofluorescence ( Figure 1 C). hDPSCs were cultured from human dental pulp and identified through flow cytometric analysis of surface protein markers and an assessment of their multi-directional differentiation capacity ( Figure 1 D–F); the expression of NAT10 was then evaluated in LPS-treated hDPSCs in vitro. The data from qRT-PCR ( Figure 1 G) and Western blot ( Figure 1 H) revealed that LPS treatment caused a time-dependent increase in NAT10 expression, implying a putative role of NAT10 in modulating inflammation in hDPSCs.
2.2. NAT10 Knockdown Attenuates LPS-Induced Inflammation in hDPSCs
To explore the potential role of NAT10 in the inflammatory regulation of hDPSCs, siRNA transfected with plasmid was used to inhibit the expression of NAT10. NAT10 mRNA and protein expression significantly decreased in the siNAT10 groups ( Figure 2 A,B). Meanwhile, the ac 4 C level of mRNA was also reduced ( Figure 2 C), validating the effectiveness of NAT10 knockdown. NAT10-silenced hDPSCs were then stimulated with LPS to induce inflammation. The siNAT10 groups exhibited decreased mRNA and protein levels of the inflammatory cytokines, IL-6 and IL-8, compared to the negative control (NC) group ( Figure 2 D,E). This study then explored whether the chemotactic migration in macrophages can be inhibited by knocking down NAT10 in hDPSCs. As expected, the transwell assay demonstrated that NAT10-deficient hDPSCs in the lower chamber of transwells suppressed the chemotactic migration of macrophages from the upper to the lower chambers ( Figure 2 F,G). In addition, immunofluorescence and flow cytometry displayed decreased ROS production in the siNAT10 groups ( Figure 2 H–J), which also reflected suppressed inflammation.
2.3. Inhibition of NAT10 Represses LPS-Activated NF-Kappa B and MAPK Signaling Pathways in hDPSCs
To elucidate the underlying molecular mechanisms of NAT10 in regulating hDPSC inflammation, RNA sequencing (RNA-seq) analysis was conducted to investigate the effects of NAT10 depletion ( Figure 3 A). KEGG pathway analysis indicated significant enrichment of differentially expressed genes in inflammatory response pathways, notably the NF-κB signaling pathway ( Figure 3 B). Western blot analysis confirmed that siNAT10 cells exhibited reduced NF-κB pathway activity, as evidenced by decreased Iκ B and p65 phosphorylation ( Figure 3 C). The MAPK signaling pathway was also evaluated, and the data showed that NAT10 depletion resulted in decreased phosphorylation of ERK and p38 ( Figure 3 D). These results indicated that LPS-induced activation of NF-κB and MAPK signaling in hDPSCs was suppressed by NAT10 knockdown.
2.4. PTX3 May Be an ac 4 C -Modified Target of NAT10 in hDPSC Inflammation
Sequencing analysis revealed reduced PTX3 expression, which was further confirmed by lower PTX3 mRNA and protein expression in the siNAT10 groups ( Figure 4 A,B). The ac 4 C enrichment in PTX3 was also decreased after NAT10 knockdown, as shown in acRIP-qRCR ( Figure 4 C). Following Actinomycin D treatment, PTX3 mRNA stability decreased upon NAT10 depletion, implying the role of acetylation in maintaining PTX3 mRNA stability ( Figure 4 D). To validate whether the inflammatory suppression induced by NAT10 knockdown was achieved by inhibiting PTX3, NAT10-knockdown cells were supplied with human recombinant PTX3 (rmPTX3) to promote its expression. As the results showed, the decreased expression levels of inflammatory cytokine were partially reversed by rmPTX3 ( Figure 4 E). Furthermore, rmPTX3 also partially restored the inhibition of macrophage chemotactic migration and ROS production induced by NAT10 knockdown ( Figure 4 F–H). In summary, these data indicated that NAT10 might regulate hDPSC inflammation by catalyzing ac 4 C modification of PTX3 mRNA.
2.5. Remodelin Alleviates hDPSC Inflammation Induced by LPS and Dental Pulp Inflammation of Rats with Pulpitis
Remodelin, a specific inhibitor of NAT10, was evaluated for its therapeutic potential in the inflammatory model of hDPSCs in vitro and the pulpitis model of rats in vivo. To maintain the viability of hDPSCs, Remodelin with a concentration of 10μM was chosen, as suggested by a CCK8 assay ( Figure 5 A). After treatment with Remodelin, the expression of ac 4 C modification was downregulated, indicating the inhibition of acetylation ( Figure 5 B). qRT-PCR and ELISA analyses revealed a significant decrease in IL-6 and IL-8 expression in the Remodelin-treated group compared to the control group, suggesting that LPS-induced hDPSC inflammation was attenuated by Remodelin ( Figure 5 C,D). The effect of Remodelin on rats with pulpitis in vivo was further investigated. Immunohistochemistry demonstrated that Remodelin treatment significantly suppressed the expression of pro-inflammatory cytokines TNF-α and IL-1β in the inflamed dental pulp tissue, relative to the LPS + DMSO group ( Figure 5 E). Meanwhile, H&E staining exhibited a marked reduction in inflammatory cell infiltration within the dental pulp of rats treated with Remodelin ( Figure 5 F). These results suggested a potential anti-inflammatory effect of Remodelin in the treatment of pulpitis.
3. Discussion
Although inflammatory response is a prerequisite for pulp healing and regeneration, severe or persistent inflammation can be detrimental [ 27 , 28 ]. Lipopolysaccharide (LPS), a well-established inflammatory stimulus, was chosen for this study due to its ability to mimic the pathological conditions of pulpitis by activating immune cells and dental pulp cells to produce a spectrum of inflammatory cytokines [ 29 ]. This makes LPS an ideal agent for investigating the molecular mechanisms underlying dental pulp inflammation. In this study, an inflammatory environment in vitro was induced by stimulating hDPSCs with LPS, in which increased NAT10 expression was observed.
Studies of ac 4 C modification initially focused on tRNA and 18S rRNA; however, it is now considered a widespread modification in the human transcriptome [ 30 ]. NAT10, the sole identified ac 4 C “writer” protein, plays a critical role in mRNA stabilization and translation by ensuring accurate decoding of specific nucleotide sequences [ 31 ]. Emerging evidence links NAT10-mediated ac 4 C modification to the pathogenesis of numerous disorders like gastric cancer, osteoporosis, and cardiac fibrosis [ 32 , 33 ]. In particular, the impact of NAT10 on inflammation, as revealed by recent studies, has broadened the understanding of its role in pathogenicity and underlying mechanisms of disease [ 34 , 35 , 36 ]. Nevertheless, the regulation of NAT10 on inflammation of different cell types is not yet conclusive, and its potential involvement in modulating hDPSC inflammation has not been reported.
In the present study, increased NAT10 expression was observed in pulp tissues from patients with pulpitis, which is consistent with the expression of NAT10 in LPS-stimulated hDPSCs. NAT10′s regulatory effect on hDPSC biological behavior was then investigated through NAT10 knockdown experiments, which revealed a significant downregulation of key inflammatory mediators, IL-6 and IL-8. Previous studies have demonstrated that dental pulp fibroblasts may influence the progression of pulpitis by regulating the polarization and localization of macrophages [ 37 ]. Specifically, it has been shown that the supernatant of LPS-stimulated dental pulp cells increases the number of recruited macrophages, suggesting that macrophages can migrate to the site of dental pulp inflammation [ 1 , 38 ]. The present data showed that knocking down NAT10 remarkably inhibited macrophage migration, which reflected the inhibitory effect of NAT10 knockdown on hDPSC inflammation. The observed reduction in ROS levels following NAT10 knockdown provides additional mechanistic insights, as ROS generation is not only a byproduct of mitochondrial oxidative metabolism but also a critical component of immune responses in inflammatory diseases [ 39 , 40 ]. The decreased ROS expression in our study suggests that NAT10 knockdown suppresses inflammation in LPS-stimulated hDPSCs through multiple pathways, including modulation of cytokine expression, macrophage migration, and oxidative stress responses.
To explore the potential mechanisms by which NAT10 regulates inflammation in hDPSCs, RNA sequencing was performed with and without NAT10 knockdown. KEGG analysis identified significant enrichment of differentially expressed genes in the NF-κB pathway, a classical inflammatory reaction pathway; the MAPK signaling pathway was also tested for its role in hDPSC inflammation modulation [ 41 , 42 ], despite no enrichment found in the sequencing. As verified by Western blot, knocking down NAT10 inhibited the phosphorylation of key components in NF-κB and MAPK signaling pathways.
Pentraxin 3 (PTX3), a soluble pattern recognition molecule, belongs to an evolutionarily conserved superfamily of soluble pattern recognition molecules in the innate immune system [ 43 ]. As a multifunctional protein, PTX3 has been shown to be actively involved in cardiovascular diseases, tumorigenesis, and inflammatory disorders [ 44 , 45 , 46 ]. Notably, according to Kim et al., the expression of PTX3 was elevated in TNF-α-treated dental pulp cells, and PTX3 knockdown led to a substantial downregulation of IL-6 and IL-8, indicating the regulatory role of PTX3 in pulpitis [ 47 ]. Other than that, Liu et al. characterized PTX3 as the direct target of NAT10 through ac 4 C-seq and RNA-seq in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs); the mutant of PTX3 engineered to replace the ac 4 C consensus sequence CXX with GXX proved that the modification of ac 4 C mediated by NAT10 directly regulated PTX3 expression [ 48 ]. Together with the role of PTX3 in regulating the inflammation of dental pulp cells, it can be reasonably inferred that PTX3 may be a target gene for NAT10 to regulate inflammation in hDPSCs. In this study, the inhibition of inflammation caused by NAT10 knockdown was accompanied by a decreased PTX3 expression level, and the provision of exogenous PTX3 partially reversed the effect of NAT10 knockdown on inflammation. It was further demonstrated that ac 4 C acetylation mediated by NAT10 regulated the expression of PTX3 by stabilizing its mRNA. While this study provides evidence for PTX3 as the target of NAT10 in hDPSC inflammation regulation, the involvement of other unidentified proteins acting as potential NAT10 targets in this process cannot be ruled out. Further studies aimed at identifying and characterizing these additional targets will be essential to fully elucidate the mechanistic complexities of NAT10-mediated inflammation and to refine therapeutic approaches for inflammatory conditions in dental pulp and beyond.
To further investigate the therapeutic potential of Remodelin, a specific NAT10 inhibitor, we applied it to a LPS-stimulated DPSC and pulpitis model of rats. Results suggested that Remodelin treatment downregulated inflammatory factor expression in LPS-treated hDPSCs. Furthermore, Remodelin significantly lowered TNF-α and IL-1β protein levels in the dental pulp of rats with pulpitis, concurrently reducing inflammatory cell infiltration within the dental pulp tissue. These results indicated that NAT10-mediated ac 4 C modification may represent a viable therapeutic target for pulpitis, and Remodelin exhibited the potential as a pulp capping agent. For in vivo experiments, rats were selected due to the similarity of their tooth anatomy and cellular processes to those of humans [ 49 ]. However, it is important to note that there are physiological and metabolic distinctions between rats and humans, which is a potential limitation of this study. These interspecies differences may influence the interpretation and translational relevance of the results.
In conclusion, this study showed that NAT10 expression was elevated in inflamed human dental pulp and LPS-stimulated hDPSCs. Both NAT10 knockdown and pharmacological inhibition with Remodelin effectively attenuated hDPSC inflammation. Mechanistically, NAT10 regulates cell inflammation by promoting the mRNA stability of N4-acetylated PTX3. In vivo experiments further validated these findings, as Remodelin treatment markedly alleviated the severity of dental pulp inflammation in rats with pulpitis. Collectively, these findings revealed the potential role of RNA epigenetic modification in regulating hDPSC inflammatory response, and Remodelin demonstrated promising characteristics as a pulp capping agent, warranting further investigation into its clinical application.
4. Materials and Methods
4.1. Collection of Clinical Samples
This research was conducted with ethical approval from the Ethics Committee of the Affiliate Stomatology Hospital of Sun Yat-sen University (KQEC-2025-035-01). All participants gave written informed consent. The dental pulp tissues in this study were extracted from premolars and third molars extracted for orthodontic or surgical purposes. Samples were categorized into healthy and pulpitis groups based on clinical presentation.
4.2. Culture of Primary Human Dental Pulp Stem Cells (hDPSCs)
Healthy human dental pulp tissues were obtained from people aged 18–25 years old. Cell suspensions were generated by digesting washed and minced tissues with collagenase I (Sigma-Aldrich, St. Louis, MO, USA). The suspension was subsequently filtered and centrifuged before being cultured in α-MEM (Gibco, New York, NY, USA) with 20% FBS (Cellmax, Lanzhou, China) and 1% penicillin-streptomycin (NCM, Suzhou, China) supplementation. Cells at passages 3–5 were used throughout the study.
4.3. Induction of Osteogenic and Adipogenic Differentiation of hDPSCs
The culture medium, comprising α-MEM with the addition of 50 μg/mL ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), was used to induce osteogenic differentiation of hDPSCs. hDPSCs were cultured for 7 or 21 days. Adipogenic differentiation of hDPSCs was induced by α-MEM supplemented with 500 μM IBMX, 10 μg/mL insulin, 100 μM indomethacin, and 1 μM dexamethasone. The culture of hDPSCs was sustained for 21 days.
4.4. Alizarin Red (ARS) and Oil Red O Staining
Following fixation in 4% paraformaldehyde, cells were subjected to specific staining protocols. Calcium deposits were visualized using 1% Alizarin Red staining (GL Biochem, Shanghai, China). Lipid droplet formation was tested through Oil Red O staining. All stained cells were subsequently photographed using an inverted phase-contrast microscope (IX83 + DP74, Olympus, Tokyo, Japan).
4.5. Flow Cytometric Analysis
For cell surface marker analysis, 5 × 10 5 hDPSCs were collected, suspended in 75 μL PBS, and then incubated in darkness with fluorochrome-conjugated antibodies against human CD34, CD45, CD29, CD44, and IgG. After incubation, hDPSCs were prepared for flow cytometric analysis by washing with 800 μL PBS via centrifugation (300× g , 5 min), followed by resuspension in 500 μL PBS. Cellular analysis was performed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
4.6. Immunohistochemistry
The healthy and inflamed human dental pulp was processed for immunohistochemical analysis. Following dehydration and paraffin embedding, 5 μm sections were prepared. These sections underwent deparaffinization, rehydration, and antigen retrieval in a citric acid buffer. The following steps were conducted using the Streptavidin-Peroxidase kit (ZSGB-Bio). Sections were incubated overnight in primary antibody solution against NAT10 (1:250; ABclonal, Wuhan, China) at 4 °C. DAB was used for staining, with an approximate incubation time of 20 s. Hematoxylin was applied as a counterstain for 10 s. Nuclear staining ranging from light yellow to brown was considered indicative of NAT10 positivity. The sections were scanned with a slice scanner (Leica Biosystems, Buffalo Grove, IL, USA).
4.7. Cell Proliferation Assay
hDPSCs cultured in 96-well plates were exposed to stimuli for designated periods. The viability was detected via a CCK-8 assay (GLPBIO, Montclair, CA, USA) by measuring absorbance at 450 nm.
4.8. Cell Transfection
A total of 50 nM NAT10 specific siRNAs or a nonspecific (Hippobio, Huzhou, China) and Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, USA) were transfected into hDPSCs following the protocol to establish knockdown cell lines. The knockdown efficiency was measured via qRT-PCR and Western blot. The siRNA sequences are detailed in Table 1 .
4.9. Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR)
RNA isolation was performed using RNAzol (MRC, Cincinnati, OH, USA). Subsequently, ABScript III RT Master Mix was employed to generate cDNA (ABclonal, Wuhan, China). A LightCycler 480 (Roche, Basel, Switzerland) was used to conduct qPCR. The primers used in this study are listed in Table 2 .
4.10. Western Blot
Cells were lysed for 30 min on ice using RIPA buffer (Beyotime, Haimen, China) containing protease and phosphatase inhibitors (Cwbiotech, Beijing, China). Protein concentrations were evaluated by a BCA kit (Cwbiotech, Beijing, China); 25 μg of protein per lane were separated by 10% SDS-PAGE. Polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), onto which proteins had been transferred, were blocked with 5% bovine serum albumin for 1 h. Next, primary antibodies against NAT10, PTX3 (1:1000; ABclonal, Wuhan, China), ERK, p-ERK, p38, p-p38, p65, p-p65, IκB, p-IκB, (1:800; Cell Signalling Technology, Boston, MA, USA), and β-actin (1: 50000; ABclonal, Wuhan, China), together with the membranes, were held at 4 °C for an overnight period. Proteins were observed by an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA) following secondary antibody incubation.
4.11. Enzyme-Linked Immunosorbent Assay (ELISA)
The concentration of IL-6 and IL-8 in hDPSC culture supernatant was analyzed via ELISA kits (ABclonal, Wuhan, China) as the recommended procedure.
4.12. Transwell Chemotactic Migration Assay
NAT10 siRNA or a negative control siRNA was used to transfect hDPSCs. Following a 12 h stimulation with LPS, the supernatant was collected. Then, seeded RAW 264.7 cells were placed into the upper compartment of transwell inserts (Corning, NY, USA), with the collected supernatant placed in the lower compartment. Migrated cells, collected from the membrane surface after a 24 h incubation, were fixed using 4% paraformaldehyde and then stained by crystal violet. The cells were subjected to quantification using phase-contrast microscopy (Axiovert 40; Zeiss, Jena, Germany).
4.13. Measurement of Reactive Oxygen Species (ROS) Expression
Following LPS stimulation, hDPSCs were incubated with 20 μM DHE (Vigorous, Beijing, China) for 1 h. Flow cytometry (LSRFortessa, Becton Dickinson, Franklin Lakes, NJ, USA) was used to identify labeled cells, and fluorescence emission was analyzed by an inverted fluorescence microscope (Olympus IX83 + DP74, Tokyo, Japan) equipped with a TRITC filter set.
4.14. RNA Sequencing
Following total RNA extraction, mRNA was isolated and purified. The resulting RNA samples underwent fragmentation and were subsequently converted into double-stranded cDNA. Illumina sequencing was performed on the cDNA libraries (Metware Biotechnology Co., Ltd., Wuhan, China). Statistically significant differential expression was defined as a p -value < 0.05 and |FC| > 2. KEGG pathway enrichment analysis, performed with the BGI platform, was used to explore the functional implications of DEGs.
4.15. RNA ac 4 C Dot Blot Assay
Hybond-N+ membranes (Solarbio, Beijing, China) were used to capture mRNA, which had been heat-denatured at 95 °C for 3 min. Crosslinking was performed for 45 min using UV light. The membranes were blocked with 5% BSA for 1 h before overnight incubation with anti-ac 4 C antibody (1:1000; ABclonal, Wuhan, China) at 4 °C. Subsequently, membranes were incubated for 1 h with a 1:2000 dilution of secondary antibody (Cell Signaling Technology, Boston, MA, USA); then, the membranes were observed via the enhanced chemiluminescence kit (Millipore, Billerica, MA, USA). Total input RNA was quantified by staining membranes with 0.2% methylene blue.
4.16. RNA-Binding Protein Immunoprecipitation (RIP)
This experiment was conducted using the RIP Kit (Merck Millipore, MA, USA). Cell lysates, prepared from washed cells using RIP lysis buffer, were incubated overnight at 4 °C to bind either an anti-ac 4 C antibody (1:1000, ABclonal, Wuhan, China) or IgG antibody to magnetic beads. Following incubation, beads were washed extensively with PBS to remove non-specifically bound material. Following proteinase K digestion and RNAzol extraction, bound RNA was quantified by qRT-PCR.
4.17. Assessment of the mRNA Stability
To assess mRNA stability, global mRNA transcription was inhibited by treating stimulated hDPSCs with actinomycin D (Sigma, St. Louis, MO, USA). RNA was extracted at defined time intervals, and target gene mRNA stability was then determined by measuring relative mRNA levels.
4.18. Pulpitis Model in Vivo
The in vivo study was approved by the Animal Care and Use Committee of Sun Yat-sen University (SYSU-IACUC-2024-002551). Eight-week-old male SD rats, weighing 180–250 g, were purchased from the Laboratory Animal Center of Sun Yat-sen University. The rats were randomly divided into three groups (n = 6): the control group, the LPS + DMSO group, and the LPS + Remodelin group. An occlusal cavity was created on the maxillary first molar with a small dental circular drill and an endodontic hand file. The exposed cavity was stimulated with LPS for 15 min to simulate the inflammatory environment, as described in a previous study [ 50 ]. The untreated first molar was used as a healthy control. Rats were euthanized after 1 w, and their maxillaries were extracted for subsequent analysis.
4.19. H&E, Immunohistochemistry and Immunofluorescence Staining
The maxillaries were fixed in 4% paraformaldehyde at 4 °C for 24 h. Following decalcification in 10% EDTA for two months, samples were paraffin-embedded and sectioned at 4 μm. Hematoxylin and eosin (H&E) and immunohistochemistry staining were performed on deparaffinized and rehydrated sections, adhering to the manufacturer’s recommended procedure. The protocol of immunohistochemistry staining was the same as described above. For immunofluorescence, sections underwent antigen retrieval using a pepsin solution (Servicebio, Wuhan, China). Then, sections were washed with PBS containing 0.1% Triton X-100 (PBST) and blocked with QuickBlock™ (Beyotime, Haimen, China) for 15 min at room temperature. Subsequently, sections were incubated with primary antibodies overnight at 4 °C. After three washes with PBST, secondary antibodies were applied. Finally, cell nuclei were counterstained with DAPI (ABclonal, Wuhan, China). The samples were then analyzed using a fluorescence microscope (FV3000, Olympus, Tokyo, Japan).
4.20. Statistical Analysis
Statistical analyses were conducted using GraphPad Prism 9 (GraphPad, San Diego, CA, USA). Data are presented as the mean ± SD. Student’s t-tests or one-way ANOVA were utilized for comparisons. The criterion for statistical significance was set at p < 0.05.
Acknowledgments
This work was supported by the Guanghua School of Stomatology & Guangdong Provincial Key Laboratory of Stomatology.
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Author Contributions
Z.N., Q.X. and C.T. conceived and designed the experiments; Z.N., L.C. and I.-C.T. performed the in vitro experiments; Z.N., L.C. and I.-C.T. performed the animal experiments. Z.N., W.D. and D.L. analyzed the data; Q.X. and C.T. contributed the reagents; Z.N. and Q.X. wrote the paper. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Rats used for in vivo experiments were supplied by the Laboratory Animal Center of Sun Yat-sen University, and the animal study protocol was approved by the Animal Ethical and Welfare Committee (approval number: SYSU-IACUC-2023000134, date of approval 12 October 2024).
Informed Consent Statement
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement
Data supporting the reported results are available from the corresponding authors to all interested researchers.
Conflicts of Interest
The authors declare no conflicts of interest.
References
1. Guo D. Dong W. Cong Y. Liu Y. Liang Y. Ye Z. Zhang J. Zhou Y. LIF Aggravates Pulpitis by Promoting Inflammatory Response in Macrophages Inflammation 2024 47 307 322 10.1007/s10753-023-01910-6 37782452
2. Chang M.C. Chang S.H. Tsai Y.L. Pan Y.H. Yeung S.Y. Chang H.H. Jeng J.H. Inducing phospholipase A2 and cyclooxygenase-2 expression and prostaglandins’ production of human dental pulp cells by activation of NOD receptor and its downstream signaling Int. J. Biol. Macromol. 2025 292 139193 10.1016/j.ijbiomac.2024.139193 39730047
3. Mo Z. Li Q. Cai L. Zhan M. Xu Q. The effect of DNA methylation on the miRNA expression pattern in lipopolysaccharide-induced inflammatory responses in human dental pulp cells Mol. Immunol. 2019 111 11 18 10.1016/j.molimm.2019.03.012 30952010
4. Sun G. Ren Q. Bai L. Zhang L. Phoenixin-20 suppresses lipopolysaccharide-induced inflammation in dental pulp cells Chem.-Biol. Interact. 2020 318 108971 10.1016/j.cbi.2020.108971 32017913
5. Zhang X. Tian X. Wang Y. Yan Y. Wang Y. Su M. Lv H. Li K. Hao X. Xing X. Application of lipopolysaccharide in establishing inflammatory models Int. J. Biol. Macromol. 2024 279 135371 10.1016/j.ijbiomac.2024.135371 39244120
6. Bindal P. Ramasamy T.S. Kasim N.H.A. Gnanasegaran N. Chai W.L. Immune responses of human dental pulp stem cells in lipopolysaccharide-induced microenvironment Cell Biol. Int. 2018 42 832 840 10.1002/cbin.10938 29363846
7. Nara K. Kawashima N. Noda S. Fujii M. Hashimoto K. Tazawa K. Okiji T. Anti-inflammatory roles of microRNA 21 in lipopolysaccharide-stimulated human dental pulp cells J. Cell. Physiol. 2019 234 21331 21341 10.1002/jcp.28737 31042008
8. Gronthos S. Mankani M. Brahim J. Robey P.G. Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo Proc. Natl. Acad. Sci. USA 2000 97 13625 13630 10.1073/pnas.240309797 11087820 PMC17626
9. Zhang L. Lin C. Chen Z. Yue L. Yu Q. Hou B. Ling J. Liang J. Wei X. Chen W. Expert consensus on pulpotomy in the management of mature permanent teeth with pulpitis Int. J. Oral. Sci. 2025 17 4 10.1038/s41368-024-00333-9 39762213 PMC11704326
10. Bucchi C. Bucchi A. Martínez-Rodríguez P. Biological Properties of Dental Pulp Stem Cells Isolated from Inflamed and Healthy Pulp and Cultured in an Inflammatory Microenvironment J. Endod. 2023 49 395 401.e6 10.1016/j.joen.2023.02.002 36828285
11. Ma Y. Liu X. Dai R. Li Q. Cao C.Y. LL-37 regulates odontogenic differentiation of dental pulp stem cells in an inflammatory microenvironment Stem. Cell Res. Ther. 2024 15 469 10.1186/s13287-024-04075-7 39696668 PMC11656940
12. Sonmez Kaplan S. Sazak Ovecoglu H. Genc D. Akkoc T. TNF-α, IL-1B and IL-6 affect the differentiation ability of dental pulp stem cells BMC Oral. Health 2023 23 555 10.1186/s12903-023-03288-1 37568110 PMC10422753
13. Zhang W. Xu T. Li X. Zhang Y. Zou X. Chen F. Yue L. Single-cell atlas of dental pulp stem cells exposed to the oral bacteria Porphyromonas gingivalis and Enterococcus faecalis Front. Cell Dev. Biol. 2023 11 1166934 10.3389/fcell.2023.1166934 37287452 PMC10242116
14. Cooper P.R. Takahashi Y. Graham L.W. Simon S. Imazato S. Smith A.J. Inflammation–regeneration interplay in the dentine–pulp complex J. Dent. 2010 38 687 697 10.1016/j.jdent.2010.05.016 20580768
15. Wang F. Han Y. Xi S. Lu Y. Catechins reduce inflammation in lipopolysaccharide-stimulated dental pulp cells by inhibiting activation of the NF-κB pathway Oral. Dis. 2020 26 815 821 10.1111/odi.13290 31999881
16. Xiao Z. Zhang Y. She Y. Yuan G. Yang G. IPO7 promotes lipopolysaccharide-induced inflammatory responses in human dental pulp cells via p38 MAPK and NF-κB signaling pathways Mol. Immunol. 2023 163 116 126 10.1016/j.molimm.2023.09.011 37769576
17. Cooper P.R. Chicca I.J. Holder M.J. Milward M.R. Inflammation and Regeneration in the Dentin-pulp Complex: Net Gain or Net Loss? J. Endod. 2017 43 S87 S94 10.1016/j.joen.2017.06.011 28844308
18. Zhu M. Ding Q. Lin Z. Chen X. Chen S. Zhu Y. New insights of epigenetics in vascular and cellular senescence J. Transl. Intern. Med. 2021 9 239 248 10.2478/jtim-2021-0049 PMC8802399 35136723
19. Feng J. Liu Y. Li K. Wu Y. Challenges and opportunities in targeting epigenetic mechanisms for pulmonary arterial hypertension treatment Int. J. Pharm. 2025 672 125332 10.1016/j.ijpharm.2025.125332 39929327
20. Li P. Lin Y. Ma H. Zhang J. Zhang Q. Yan R. Fan Y. Epigenetic regulation in female reproduction: The impact of m6A on maternal-fetal health Cell Death Discov. 2025 11 43 10.1038/s41420-025-02324-z 39904996 PMC11794895
21. Zhang Y. Lei Y. Dong Y. Chen S. Sun S. Zhou F. Zhao Z. Chen B. Wei L. Chen J. Emerging roles of RNA ac4C modification and NAT10 in mammalian development and human diseases Pharmacol. Ther. 2024 253 108576 10.1016/j.pharmthera.2023.108576 38065232
22. Cui Z. Xu Y. Wu P. Lu Y. Tao Y. Zhou C. Cui R. Li J. Han R. NAT10 promotes osteogenic differentiation of periodontal ligament stem cells by regulating VEGFA-mediated PI3K/AKT signaling pathway through ac4C modification Odontology 2023 111 870 882 10.1007/s10266-023-00793-1 36879181
23. Zhang Y. Jing Y. Wang Y. Tang J. Zhu X. Jin W.-L. Wang Y. Yuan W. Li X. Li X. NAT10 promotes gastric cancer metastasis via N4-acetylated COL5A1 Sig. Transduct. Target. Ther. 2021 6 173 10.1038/s41392-021-00489-4 PMC8093205 33941767
24. Wei R. Cui X. Min J. Lin Z. Zhou Y. Guo M. An X. Liu H. Janz S. Gu C. NAT10 promotes cell proliferation by acetylating CEP170 mRNA to enhance translation efficiency in multiple myeloma Acta. Pharm. Sin. B 2022 12 3313 3325 10.1016/j.apsb.2022.01.015 35967285 PMC9366180
25. Zhang H. Chen Z. Zhou J. Gu J. Wu H. Jiang Y. Gao S. Liao Y. Shen R. Miao C. NAT10 regulates neutrophil pyroptosis in sepsis via acetylating ULK1 RNA and activating STING pathway Commun. Biol. 2022 5 916 10.1038/s42003-022-04045-w 36068299 PMC9448771
26. Zhang Z. Zhang Y. Cai Y. Li D. He J. Feng Z. Xu Q. NAT10 regulates the LPS-induced inflammatory response via the NOX2-ROS-NF-κB pathway in macrophages Biochim. Biophys. Acta (BBA)—Mol. Cell Research 2023 1870 119521 10.1016/j.bbamcr.2023.119521 37307924
27. Galler K.M. Weber M. Korkmaz Y. Widbiller M. Feuerer M. Inflammatory Response Mechanisms of the Dentine–Pulp Complex and the Periapical Tissues IJMS 2021 22 1480 10.3390/ijms22031480 33540711 PMC7867227
28. Giraud T. Pulp capping materials modulate the balance between inflammation and regeneration Dent. Mater. 2019 35 24 35 10.1016/j.dental.2018.09.008 30269862
29. Chen J. Xu H. Xia K. Cheng S. Zhang Q. Resolvin E1 accelerates pulp repair by regulating inflammation and stimulating dentin regeneration in dental pulp stem cells Stem. Cell Res. Ther. 2021 12 75 10.1186/s13287-021-02141-y 33482900 PMC7821538
30. Arango D. Sturgill D. Alhusaini N. Dillman A.A. Sweet T.J. Hanson G. Hosogane M. Sinclair W.R. Nanan K.K. Mandler M.D. Acetylation of Cytidine in mRNA Promotes Translation Efficiency Cell 2018 175 1872 1886 10.1016/j.cell.2018.10.030 30449621 PMC6295233
31. Luo J. Cao J. Chen C. Xie H. Emerging role of RNA acetylation modification ac4C in diseases: Current advances and future challenges Biochem. Pharmacol. 2023 213 115628 10.1016/j.bcp.2023.115628 37247745
32. Yang W. Li H.Y. Wu Y.F. Mi R. Liu W. Shen X. Lu Y. Jiang Y. Ma M. Shen H. ac4C acetylation of RUNX2 catalyzed by NAT10 spurs osteogenesis of BMSCs and prevents ovariectomy-induced bone loss Mol. Ther.—Nucleic Acids 2021 26 135 147 10.1016/j.omtn.2021.06.022 34513300 PMC8413676
33. Li J. Yushanjiang F. Fang Z. Liu W. NAT10-mediated RNA ac4C acetylation contributes to the myocardial infarction-induced cardiac fibrosis J. Cell. Mol. Med. 2024 28 e70141 10.1111/jcmm.70141 39482983 PMC11528131
34. Xing P. Zhou M. Sun J. Wang D. Huang W. An P. NAT10-mediated ac4C acetylation of TFRC promotes sepsis-induced pulmonary injury through regulating ferroptosis Mol. Med. 2024 30 140 10.1186/s10020-024-00912-w 39251905 PMC11382515
35. Wang C. Liu Y. Zhang Y. Wang D. Xu L. Li Z. Bai X. Wang Y. Targeting NAT10 protects against sepsis-induced skeletal muscle atrophy by inhibiting ROS/NLRP3 Life Sci. 2023 330 121948 10.1016/j.lfs.2023.121948 37467885
36. Li W.-P. Mao X.-T. Xie J.-H. Li J.-Y. Liu B.-Q. Wu L.-X. Yang B. Li Y.-Y. Jin J. N-acetyltransferase 10 is implicated in the pathogenesis of cycling T cell-mediated autoimmune and inflammatory disorders in mice Nat. Commun. 2024 15 9388 10.1038/s41467-024-53350-x 39477944 PMC11525920
37. Zhao Y. Wang C.-L. Li R.-M. Hui T.-Q. Su Y.-Y. Yuan Q. Zhou X.-D. Ye L. Wnt5a Promotes Inflammatory Responses via Nuclear Factor κB (NF-κB) and Mitogen-activated Protein Kinase (MAPK) Pathways in Human Dental Pulp Cells J Biol. Chem. 2014 289 21028 21039 10.1074/jbc.M113.546523 PMC4110308 24891513
38. Le Fournis C. Jeanneau C. Giraud T. El Karim I. Lundy F.T. About I. Fibroblasts Control Macrophage Differentiation during Pulp Inflammation J. Endod. 2021 47 1427 1434 10.1016/j.joen.2021.06.015 34181951
39. Chen A. Huang H. Fang S. Hang Q. ROS: A “booster” for chronic inflammation and tumor metastasis Biochim. Biophys. Acta (BBA)—Rev. Cancer 2024 1879 189175 10.1016/j.bbcan.2024.189175 39218404
40. Zhao W. Zhang Y. Chen J. Hu D. Revolutionizing oral care: Reactive oxygen species (ROS)-Regulating biomaterials for combating infection and inflammation Redox Biol. 2025 79 103451 10.1016/j.redox.2024.103451 39631247 PMC11664010
41. Botero T.M. Son J.S. Vodopyanov D. Hasegawa M. Shelburne C.E. Nör J.E. MAPK Signaling Is Required for LPS-induced VEGF in Pulp Stem Cells J. Dent. Res. 2010 89 264 269 10.1177/0022034509357556 20110511
42. Liu M. Chen L. Wu J. Lin Z. Huang S. Long noncoding RNA MEG3 expressed in human dental pulp regulates LPS-Induced inflammation and odontogenic differentiation in pulpitis Exp. Cell Res. 2021 400 112495 10.1016/j.yexcr.2021.112495 33524362
43. Ye X. Wang Z. Lei W. Shen M. Tang J. Xu X. Yang Y. Zhang H. Pentraxin 3: A promising therapeutic target for cardiovascular diseases Ageing Res. Rev. 2024 93 102163 10.1016/j.arr.2023.102163 38092307
44. Massimino A.M. Colella F.E. Bottazzi B. Inforzato A. Structural insights into the biological functions of the long pentraxin PTX3 Front. Immunol. 2023 14 1274634 10.3389/fimmu.2023.1274634 37885881 PMC10598717
45. Bozza S. Bistoni F. Gaziano R. Pitzurra L. Zelante T. Bonifazi P. Perruccio K. Bellocchio S. Neri M. Iorio A.M. Pentraxin 3 protects from MCMV infection and reactivation through TLR sensing pathways leading to IRF3 activation Blood 2006 108 3387 3396 10.1182/blood-2006-03-009266 16840729
46. Deban L. Russo R.C. Sironi M. Moalli F. Scanziani M. Zambelli V. Cuccovillo I. Bastone A. Gobbi M. Valentino S. Regulation of leukocyte recruitment by the long pentraxin PTX3 Nat. Immunol. 2010 11 328 334 10.1038/ni.1854 20208538
47. Kim Y. Park J.-S. Park H.-J. Kim M.-K. Kim Y.-I. Bae S.-K. Kim H.J. Jeong C.-H. Bae M.-K. Pentraxin 3 Modulates the Inflammatory Response in Human Dental Pulp Cells J. Endod. 2018 44 1826 1831 10.1016/j.joen.2018.08.003 30477668
48. Liu D. Kuang Y. Chen S. Li R. Su F. Zhang S. Qiu Q. Lin S. Shen C. Liu Y. NAT10 promotes synovial aggression by increasing the stability and translation of N4-acetylated PTX3 mRNA in rheumatoid arthritis Ann. Rheum. Dis. 2024 83 1118 1131 10.1136/ard-2023-225343 38724075
49. Huang H. Okamoto M. Watanabe M. Matsumoto S. Moriyama K. Komichi S. Ali M. Matayoshi S. Nomura R. Nakano K. Development of Rat Caries-Induced Pulpitis Model for Vital Pulp Therapy J. Dent. Res. 2023 102 574 582 10.1177/00220345221150383 36913545 PMC10152557
50. Zhai Y. Yuan X. Zhao Y. Ge L. Wang Y. Potential Application of Human β-Defensin 4 in Dental Pulp Repair Front. Physiol. 2020 11 1077 10.3389/fphys.2020.01077 32973567 PMC7472722