MT2A-Mediated Regulation of Cuproptosis in Human Dental Pulp Cells Modulates Macrophage M1 Polarization in Pulpitis

Abstract

Background

The progression of pulpitis is strongly affected by the fate of dental pulp cells, with their modes of cell death determining whether inflammation resolves or leads to irreversible necrosis. Cuproptosis, a copper-dependent form of regulated cell death, has been implicated in inflammatory and metabolic disorders. Given the high metabolic activity of pulp cells and dynamic copper fluctuations during inflammation, cuproptosis may play a role in pulpitis, though this remains unexplored.

Methods

Copper deposition was examined in inflamed dental pulp using copper staining, and cuproptosis activation was assessed by lipoylated Dihydrolipoamide S-acetyltransferase (lip-DLAT) expression through Western blot (WB) and immunofluorescence (IF). Key cuproptosis-related genes, including Metallothionein 2A (MT2A), were identified from Gene Expression Omnibus (GEO) datasets and validated. A mouse pulpitis model with MTII (MT2A ortholog in mouse) knockdown was used to evaluate pulp tissue changes via Hematoxylin and Eosin staining and IF staining, and immune infiltration analyses were performed to identify MT2A-related immune cells. Finally, an inflammatory human dental pulp cells (hDPCs) model was used to assess its effects on macrophages by flow cytometry.

Results

Inflamed human pulp tissues exhibited marked copper accumulation and significantly elevated lip-DLAT expression, indicating activation of cuproptosis. Bioinformatic analysis of GEO datasets identified 6 hub cuproptosis-related genes, which were validated by reverse transcription quantitative polymerase chain reaction. In hDPCs, the cuproptosis inhibitor tetrathiomolybdate reduced copper overload, cell death, and lip-DLAT expression. Conversely, MT2A knockdown enhanced cuproptosis, as evidenced by increased copper accumulation, lip-DLAT levels, and IL-6 expression. Moreover, MT2A knockdown in the inflammatory hDPCs model promoted M1 macrophage polarization.

Conclusion

Cuproptosis is activated in inflamed dental pulp and contributes to the pathogenesis of pulpitis. MT2A functions as a protective regulator, mitigating cuproptosis and modulating macrophage-mediated inflammatory responses. Targeting cuproptosis and MT2A may represent a novel therapeutic strategy for preserving pulp vitality.

Introduction

Pulpitis is a highly prevalent oral disease caused by microbial infection of dental plaque. Its pathological hallmark is irreversible inflammatory damage to the pulp tissue,^1,2 often resulting in destruction or even loss of tooth structure, which severely impairs oral function and quality of life.^3,4 Currently, clinical management primarily relies on root canal therapy to control infection; however, this approach may lead to long-term complications, such as increased tooth fragility and discoloration.5, 6, 7 Therefore, elucidating the molecular mechanisms underlying pulpitis and identifying novel therapeutic targets are of significant clinical importance.

Copper is an essential trace element involved in a wide range of biological processes, including mitochondrial respiration, antioxidant defense, enzymatic reactions, and cellular metabolism.^8,9 Beyond its physiological roles, copper also participates in immune regulation and inflammatory responses.¹⁰ At normal levels, copper supports host defense by promoting the activity of copper-dependent enzymes, such as superoxide dismutases, which mitigate oxidative stress and protect tissues from damage.¹¹ Furthermore, copper modulates immune cell activation, proliferation, and tissue repair through multiple signaling pathways.12, 13, 14 Conversely, dysregulation of copper homeostasis can have deleterious effects.¹⁵ Excess intracellular copper disrupts mitochondrial function and induces a specific form of regulated cell death termed cuproptosis.16, 17, 18 Mechanistically, cuproptosis arises from the binding of copper to lipoylated components of the tricarboxylic acid cycle—particularly dihydrolipoamide S-acetyltransferase (DLAT)—leading to protein aggregation, loss of iron-sulfur cluster proteins, proteotoxic stress, and ultimately cell death.¹⁹ In inflamed tissues, copper-induced cell death may exacerbate tissue injury, amplify inflammatory signaling, and alter the local immune microenvironment.20, 21, 22 This dual role of copper, as both an indispensable cofactor for normal immune and metabolic functions and as an inducer of cuproptosis under pathological conditions, highlights the importance of tightly controlling copper-induced cell death to maintain tissue and immune homeostasis.

Emerging evidence further suggests that copper-induced cell death not only affects the survival of resident tissue cells but also impacts the composition and functional state of local immune populations.^23,24 Cells undergoing copper-dependent death release intracellular signals that can influence immune cell recruitment and activation.^25,26 Among these, macrophages are particularly sensitive to immunomodulatory signals due to their central role in tissue homeostasis and inflammatory responses,^27,28 indicating a potential link between copper-induced cell death and immune modulation in inflamed tissues.

Although the role of cuproptosis in various inflammatory diseases has been increasingly recognized, its functional mechanisms in pulpitis remain largely unexplored. Investigating how cuproptosis shapes the immune microenvironment of human dental pulp cells (hDPCs) may reveal critical insights into the cellular and molecular mechanisms driving pulp inflammation, and could guide the development of novel strategies to preserve pulp tissue integrity and modulate immune responses. In this study, we investigated cuproptosis-related mechanisms in hDPCs under inflamed conditions using a combination of Gene Expression Omnibus (GEO) data mining, machine learning approaches, and experimental validation (Figure 1). This work aimed to explore key regulators associated with cuproptosis in hDPCs, providing a framework for understanding potential approaches to modulate the immune microenvironment in pulpitis and inform future therapeutic interventions.

Fig. 1.

Graphical abstract, Human dental pulp tissues and GEO datasets (GSE92681, GSE77459) were analyzed to identify hub cuproptosis-related genes. Subsequent functional validation in hDPCs and mouse pulpitis models demonstrated that MT2A/MTII regulates cuproptosis, inflammation, and macrophage polarization, highlighting its potential as a therapeutic target.

Materials and methods

Clinical sample collection

A total of 10 dental pulp samples were collected from 10 individuals aged 18–60 years, including 5 healthy samples and 5 samples with irreversible pulpitis. All samples were obtained from maxillary third molars. Healthy pulp was collected from intact, caries-free teeth without prior restorations, whereas pulpitis samples were obtained from teeth diagnosed with irreversible pulpitis according to the criteria of the American Association of Endodontists.²⁹ Subjects with periapical or active periodontitis (American Academy of Periodontology criteria³⁰) or other systemic or immune-related conditions were excluded from both groups. Only one tooth was collected per participant, and the entire pulp tissue from each tooth was considered an independent sample. Each pulp sample was subsequently divided into 3 fragments for histological staining, RNA extraction, and protein extraction. Participants in both groups were adults in generally good health, with no history of serious systemic diseases affecting the heart, brain, liver, kidney, or endocrine system, and none had immune dysfunction or were taking long-term immunomodulatory medications. Apart from disease status, all collection and handling procedures were identical between the healthy and pulpitis groups.

Hematoxylin and eosin (HE) staining

Healthy permanent teeth and molar teeth with pulpitis were extracted after obtaining consent and immediately fixed in 4% polyformaldehyde (Biosharp) solution for 24 h. Pulp tissues were removed from teeth, dehydrated with the concentration gradient ethanol, and made transparent with xylene. The paraffin wax was completely immersed in the tissues mass before embedding. The samples were routinely fixed in an embedded box, sliced, baked, dewaxed, hydrated, stained with HE (Biosharp), dehydrated, transparent, sealed and examined under microscope (Leica, Germany).

Copper ion staining of sample tissues

Samples were dehydrated, embedded, fixed, sliced, and dewaxed according to routine procedures. Copper ion staining was performed using a copper salt dyeing kit with the red amino acid method (Solarbio, Beijing, China). After staining, the samples were made transparent, sealed, and examined under a microscope (Leica, Germany).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from both pulpitis and healthy tissues using a tissues total RNA extraction kit (EZBioscience, USA). The RNA of hDPCs was extracted using cells RNA extraction kit (EZBioscience). The extracted RNA was reverse-transcribed into complementary DNA (cDNA) using the color reverse transcription kit (EZBioscience). RT-qPCR was performed using qantstudiotm Real-Time PCR softwareto (Thermo Fisher Scientific) to verify the expression of cuproptosis-related genes (CRGs). Primer sequences were designed using the primer tool available on the National Center for Biotechnology Information (NCBI) website and are provided in Supplement S1.

Western blot (WB)

Cells or tissues samples were collected and lysed using pre-cooled RIPA lysis buffer (Acmec). The protein concentration was quantified using bicinchoninic acid assay (Acmec). A standard curve was generated with bovine serum albumin (Solarbio) as the standard control and solvent as the blank control, allowing the estimation of protein content in the extracted samples. The extracted proteins were denatured, separated on a 10% SDS-PAGE gel, and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 5% skim milk at 37 °C for 1 h with gentle shaking, followed by elution. The membrane was then incubated with the primary antibody overnight at 4 °C. After washing with TBST 3 times for 15 minutes each, the membrane was incubated with the secondary antibody at room temperature for 1 h. WB analysis was performed using the chemiluminescence method. The proteins were quantified using ImageJ software (version 1.53t)

Immunohistochemistry (IHC) staining

After dewaxing, the tissues sections were followed by antigen retrieval, treatment with hydrogen peroxide (H₂O₂) to block endogenous peroxidase activity, and non-specific antigen blocking. The sections were incubated with lip-DLAT (Abcam, UK) antibodies overnight at 4°C. After washing the sections with phosphate-buffered saline (PBS) to remove any primary antibody, the sections were then incubated with a secondary antibody (Proteintech, Wuhan, China) at room temperature for 1 h. Signals were detected using an enhanced 3,3′-Diaminobenzidine staining kit (Servicebio, Wuhan, China).

Immunofluorescence (IF) staining

The first step was same as IHC from antigen retrieval to secondary antibody incubation, and then repeated the step after washing the section with PBS (change the second primary antibody with S100A4 (Abcam) antibodies). After adding DAPI staining solution (Abcam) under dark conditions, the sections were mounted using an anti-fade mounting medium (Abcam) antibodies. The antigen retrieval of animals’ tissue sections were as same as IHC, subsequent processes were conducted according to the manufacture’s protocol followed by multicolor immunofluorescence staining using the Treble-Fluorescence Immunohistochemistry Mouse/Rabbit Kit (Immunoway), The primary antibodies used were as follows: IL-6 (Abcam), Lipoic Acid (Abcam), MTII (Abcam), F4/80 (Affinity Biosciences), INOS (Affinity Biosciences). Finally, we used fluorescence microscope (Nikon) to observe the sections.

Isolation and culture of hDPCs

Extracted teeth were repeatedly washed with sterile PBS. Under aseptic conditions, dental pulp tissues were carefully isolated, and the apical portion was removed. The remaining pulp tissue was minced into approximately 1.0 mm³ fragments for primary explant culture. The tissue fragments were placed onto the bottom of a T25 culture flask and allowed to adhere. Subsequently, 500 μL of α-MEM solution (Gibco, USA) supplemented with 10% fetal bovine serum (Pricella), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) was gently added to cover the tissue fragments. After initial attachment, an additional 2 mL of complete culture medium was slowly added. Cells were incubated at 37°C in a humidified atmosphere with 5% CO₂, and the culture medium was replaced every 3 days. When cells reached 80-90% confluence, primary cells were digested using 0.25% trypsin (Gibco) and passaged at a 1:3 ratio. Cells at passages 4-6 were used for subsequent experiments.

Construction of cuproptosis model of hDPCs

P4-P6 hDPCs were seeded into 6-well plates according to 5 × 10⁴ cells/well. For the cuproptosis experimental group, 40 nM elesclomol (Ele; Medchemexpress, New Jersey, USA) and 40 nM CuCl₂ (Medchemexpress, New Jersey, USA) were used, while the inhibition group received 40 nM Ele, 40 nM CuCl₂ and 20 μM tetrathiomolybdate (TTM; Sigma-Aldrich, USA), with DMSO (Sigma-Aldrich, USA) as the solvent control. Cell samples were collected after 4 h of incubation.

A cell counting kit 8 (CCK-8) detection of the effect of cuproptosis inducer on cell viability

The hDPCs were seeded into 96-well plate (servicebio) with density of 2.5 × 10⁵ cells /mL each plate. After the cells adhered to the plate surface, 40 nM Ele and 40 nM CuCl₂ were added to the experimental group. In the inhibition group, 40 nM Ele, 40 nM CuCl₂ and 20 μM TTM were added, while DMSO was used for the control group. After 4 h of incubation, cells were collected, 20 μL CCK-8 (Dojindo, Japan) reagent and 100 μL α-MEM culture was added. The cells were incubated at 37 °C in the dark for 2 h, and the absorbance (OD value = 450 nm) was measured using a standard instrument.

Copper ion staining of hDPCs

The hDPCs slides were fixed in 4% paraformaldehyde at room temperature for 2 h. Copper ion staining was performed using a copper salt dyeing kit with the red amino acid method (Solarbio, Beijing, China). After staining, the samples were made transparent, sealed, and examined under a microscope (Leica, Germany).

Functional study of siRNA knockdown of MT2A in hDPCs

The small interfering RNA (siRNA) targeting the Metallothionein 2A (MT2A) gene (si-MT2A) obtained from Tsingke Biotechnology (Beijing, China), the sequence of si-MT2A was provided in Supplement S1. Cells were planted in 6-well plates with density of 1.5 × 10⁵/well and in 96-well plates with density of 5 × 10³/well. The cells were transfected when the density is between 70-80%. The cells were transfected with plasmid (lipo-3000, Thermo Fisher Scientific), and si-MT2A for 8 h. Serum-reduced medium (opti-MEM, Thermo Fisher Scientific), was added. Removing the supernatant after 72 h, 40 nM Ele and 40 nM CuCl₂ were added to stimulate, and cell samples were collected after 4 h of incubation.

Bulk sequencing data and microarray data processing

Two microarray datasets and corresponding clinical data of pulpitis patients were obtained from the GEO database. The dataset GSE92681 contained 7 human pulpitis and 5 healthy pulp tissues, and GSE77459 contained human 6 pulpitis and 6 healthy pulp tissues. A total of 52 CRGs were retrieved from the from previous studies^31,32 and combined with the Molecular Signature Database (MsigDB) v7.0 database. R packages "limma" and "sva" were used to eliminate batch effects. The data sets GSE92681 and GSE77459 were combined using the principal component analysis (PCA) to visualize before and after the batch. The R preprocessCore package was used to homogenize the data set. Differential analysis was performed using limma package in R language, and differentially expressed genes (DEGs) were screened (P-value < .05, |logFC| > 0). Volcano maps and heat maps showed DEGs between pulpitis and healthy pulp tissues.

Selection of CRGs and validation of hub-CRGs

CRGs and DEGs were intersected using Venn diagrams, identifying 12 differentially expressed cuproptosis-related genes (DE-CRGs). The results were visualized through volcano plots and heatmaps. Based on the expression values of DE-CRGs, least absolute shrinkage and selection operator (LASSO) regression was used to screen and identify 8 genes using “glmnet” R package. Additionally, 10 genes were selected according to their importance using the random Forest method with “randomForest” R package. 12 genes were identified using the support vector machine-recursive feature elimination (SVM-RFE) method with “kernlab” R package. The key genes obtained by the 3 machine learning methods were intersected, we validated 6 hub-CRGs. Finally, receiver operating characteristic (ROC) curves of the 6 hub-CRGs were generated using the “pROC” R package.

Gene ontology (GO) analysis and kyoto encyclopedia of genes and genomes (KEGG)

To investigate the potential biological functions of hub-CRGs, GO enrichment analysis and KEGG pathway enrichment analysis were performed by the R package to assess the function of the DE-CRGs.

Immune infiltration analysis

Gene set variation analysis was performed using the “ssGSEA” R package to analyze to observe the expression of immune cells in the healthy and pulpitis tissues. In additon, the correlation between immune cells and hub-CRGs were conducted using Spearman's correlation analyses, the results were visualized using bubble charts.

Establishment of pulpitis model in vivo

Animal experiments were approved by the Institutional Animal Care and Use Committee of the Nanfang Hospital in China (approved number: IACUC-LAC-20231030-003), and all experimental procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. In this study, male C57BL6/J mice (6-8 weeks old) were randomly divided into 3 groups (Healthy group, Pulpitis group, Pulpitis + TTM group), with 5 mice per group. Healthy group didn’t receive any treatment. The mouse pulpitis model was established by mechanical pulp exposure in mice.³³ Briefly, experimental pulpitis induction was accomplished by first intraperitoneal injection with 1.5% tribromoethanol (Avertin, 15 mL/kg) .The maxillary first molars were exposed using a 1/4 round bur (TC-11EF, MANI, Japan). The exposed pulp was then left open and exposed to the oral environment for 24 h. Pulpitis group was gavage with saline while the Pulpitis + TTM group was gavage with TTM (0.5 mg/kg) one day before and on the day of pulp exposure. Teeth were extracted from each group for further HE and IF staining.

Establishment of pulpitis model of MTII knockdown in vivo

The siRNA targeting the MTII gene (si-MTII) and si-NC obtained from Tsingke Biotechnology (Beijing, China), the sequence of si-MTII was provided in supplement. In this study, the C57BL6/J mice (6–8 weeks old) were randomly divided into 3 groups (Healthy group, si-MTII + Pulpitis group, si-NC + Pulpitis group), invivo-siRNA-Mate purchased from GenePharma (Shanghai, China) was used for the in vivo siRNA transfection in accordance with the manufacturer’s instructions. The siRNA-Mate/siRNA complex (10 μg siRNA/10 μL total volume/ buccal and palatal sides of bilateral maxillary first molar) were administered via intralingual delivery of si-MTII and si-NC at Day 0 and 2, followed by standardized pulp exposure at Day 3, the animals were sampled at Day 4.

Transwell migration assay

To study the transmigration of THP-1 macrophages, 24-well Transwell inserts with pore sizes of 8 μm (Corning) were used. At first, THP-1 monocytes were resuspended at a density of 10⁶ cells/well in RPMI 1640 with 10 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) and loaded into the upper chamber of the transwell insert (24-well migration chambers, 8.0 μm pore membrane, Corning) and incubated for 24 h. After hDPCs were plated in the lower chamber, incubated for 24 h and treated as above cuproptosis model of hDPCs method, fresh DMEM was added to the hDPCs. And then the upper chamber loaded with THP-1 macrophages was transferred to allow migration. After 24 h, the media in the transwell inserts were carefully removed. The cells were fixed with 4% paraformaldehyde (Leagene Biotech, Beijing, China) and stained with 0.1% crystal violet (Solarbio). Cells that did not migrate across the Transwell membrane were gently removed with cotton swabs. Migrated cells were viewed and imaged with a microscope (IX63; Olympus) and measured using ImageJ software.

Flow cytometry

To study the polarization of THP-1 macrophages, we use DMSO, Ele-CuCl₂ and Ele-CuCl₂-TTM to treat the hDPCs as above cuproptosis model of hDPCs method. After incubation of 4 h, hDPCs were washed with PBS 3 times, the cells were supplied with fresh DMEM (500 μL/well). After 24 h, the culture supernatant was harvested and added into 24-well paltes (THP-1 macrophages, 10⁶ cells/well) with 100 μL per well. After receiving treatment, THP-1 macrophages were harvested through enzymatic digestion, washed with PBS for 3 times. The cells were resuspended with 300 μL PBS and incubated with antibodies against human CD163 (PTM Bio) and CD80 (Affinity Biosciences) for 1 h on ice. Cells were then washed with PBS for 3 times. And then, cells were stained with secondary antibodies (Abcam). After being washed with PBS, THP-1 macrophages were subjected to FCM analysis by Cytoflex N31376 (Beckman Coulter Life Sciences).

Statistical analysis

All data in the figures were expressed as the average ± SEM of independent experiments. Data were analyzed using Prism 9 (GraphPad software). Unpaired 2-tailed Student’s t test was used to compare the means between 2 independent groups, and one-way analysis of variance (ANOVA) was used to compare the means between 2 or more groups. All experiments were repeated 3 or more times. All results with a P < .05 were considered statistically significant. *, **, ***, and **** represent P < .05, P < .01, P < .001, and P < .0001, respectively.

Results

Cuproptosis is activated in human and mouse pulpitis

Histological examination of human pulp tissues confirmed immune cell aggregation in inflamed pulp compared with healthy control (HE staining, Figure 2A). Copper ion staining further revealed marked copper accumulation in pulpitis tissues (Figure 2B). WB and IHC analyses showed no significant change in total DLAT, but lip-DLAT, a key regulator of cuproptosis, was significantly elevated in inflamed pulp (Figure 2C–D). Immunofluorescence confirmed increased lipoylated proteins, colocalizing with the fibroblast marker S100A4, suggesting that cuproptosis mainly occurs in hDPCs (Figure 2E).

Fig. 2.

Cuproptosis is activated in inflamed human pulp tissues, A. HE staining of human healthy pulp tissues (n = 3) and pulpitis tissues (n = 3); B. Copper ion staining of human healthy pulp tissues (n = 3) and pulpitis tissues (n = 3) (Blue arrow represents copper ion enrichment); C. Immunohistochemical staining of lipoic acid protein in human healthy pulp tissues (n = 3) and pulpitis tissues (n = 3); D. WB detected the expression of lip-DLAT protein in human healthy pulp tissues (n = 3) and pulpitis tissues (n = 3); E. Immunofluorescence staining of lipoic acid and S100A4 protein in human healthy pulp tissues (n = 3) and pulpitis tissues (n = 3) (Yellow arrow represents hDPCs occurring cuproptosis). All error bars are mean ± standard error of the mean. P values were calculated by unpaired 2-tailed Student’s t test. *P < .05.

To validate these findings in vivo, a mouse pulpitis model was established by pulp exposure (Figure 3A). Inflammatory mediators IL-6 and IL-1β were significantly upregulated in pulpitis tissues, while treatment with the copper chelator tetrathiomolybdate (TTM) suppressed their expression (RT-qPCR, Figure 3B). Consistently, WB analysis revealed increased lip-DLAT and IL-6 protein levels in pulpitis tissues, both of which were reduced by TTM treatment (Figure 3C). Histological analysis showed dense inflammatory infiltration in pulpitis tissues, which was alleviated by TTM (HE staining, Figure 3D). Immunofluorescence further demonstrated increased lipoylated proteins and IL-6 expression in inflamed pulp, both of which were suppressed by TTM (Figure 3E).

Fig. 3.

Cuproptosis is activated in mouse pulpitis model, A. Schematic illustration of pulpitis induction in mice; B. RT-qPCR detected the expression of IL-6 and IL1-β mRNA (n = 5); C. WB detected the expression of lip-DLAT and IL-6 protein (n = 5); D. HE staining of mouse pulp tissue (n = 5); E. IF staining of lipoic acid and IL-6 protein (n=5). All error bars are mean ± standard error of the mean. P values were calculated by one-way ANOVA. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Together, these findings indicate that cuproptosis is activated in inflamed human and mouse pulp tissues and can be mitigated by copper chelation, supporting a pathogenic role of cuproptosis in pulpitis.

Screening and enrichment analysis of hub-CRGs

To investigate the expression of CRGs in pulpitis tissues, transcriptome sequencing data from human pulpitis and healthy pulp tissues (GSE92681 and GSE77459) were obtained from GEO database. To minimize experimental variation and remove batch effect, the R packages "limma" and "sva" were used. DEGs were then identified using the "limma" package. Volcanic plots (Figure 4A) and heat maps (Figure 4B) were generated to visualize the DEGs, revealing 1711 upregulated and 1995 downregulated genes in inflammatory pulp tissues compared to healthy pulp tissues. To screen DE-CRGs, we intersected DEGs with 52 CRGs using Venn analysis, identifying 12 DE-CRGs, which were displayed by Venn diagrams and heat maps (Figure 4C and D), including 6 upregulated and 6 downregulated genes.

Fig. 4.

Identification of hub-CRGs in human pulpitis and healthy pulp tissues, A. Volcano plot of DEGs; B. Heatmap of DEGs; C. Venn diagram showing DE-CRGs; D. Heatmap of DE-CRGs; E. Screen of hub-CRGs by LASSO regression; F. Screen of hub-CRGs by SVM-RFE method; G. Screen of hub-CRGs by random forest analysis; H. Venn diagram of hub-CRGs by the 3 algorithms; I. Correlation analysis of the 6 hub-CRGs; J. ROC curve of the 6 hub-CRGs.

To further identify hub-CRGs, 3 machine learning algorithms were used to screen hub-CRGs in pulpitis. First, eight genes, including Microtubule Associated Protein 1 Light Chain 3 Alpha (MAP1LC3A), Metallothionein 1H (MT1H) and MT2A, were screened by LASSO regression (Figure 4E). The SVM-RFE method selected the all of 12 genes (Figure 4F). Using random forest analysis (Figure 4G), the ten genes, such as Cytochrome P450 Family 1 Subfamily A Member 1 (CYP1A1), Lipoic Acid Synthetase (LIAS), and Dihydrolipoamide Branched Chain Transacylase E2 (DBT), were selected as being closely related to the pulpitis.

Finally, by combining results from the 3 algorithms, 6 hub-CRGs were identified (Figure 4H). Correlation analysis revealed that MT2A and MAP1LC3A exhibited the strongest negative correlation, while MT2A with MT1H had the strongest positive correlation (Figure 4I). Subsequently, Area under the curve (AUC) values were used to evaluate the diagnostic potential of the 6 hub-CRGs. Among them, DBT, LIAS, MT1H and MT2A exceed 0.9, suggesting that the 4 hub-CRGs may be significantly involved in pulpitis progression (Figure 4J).

RT-qPCR validation confirmed that CYP1A1, MT1H, and MT2A were upregulated, whereas MAP1LC3A, LIAS, and DBT were downregulated in inflamed pulp tissues compared with healthy control (Figure 5), consistent with bioinformatics predictions. Together, these results indicated that the 6 hub-CRGs are significantly associated with pulpitis.

Fig. 5.

Differential mRNA expression of 6 hub CRGs in normal and inflamed human dental pulp, RT-qPCR detected 6 hub-CRGs (DBT, MT1H, MT2A, LIAS, CYP1A1 and MAP1LC3A) mRNA expression in human healthy pulp tissues (n = 3) and pulpitis tissues (n = 3). All error bars are mean ± standard error of the mean. P values were calculated by unpaired 2-tailed Student’s t test. *P < .05, **P < .01, ***P < .001.

MT2A knockdown enhances cuproptosis and inflammation in hDPCs in vitro

Primary hDPCs were isolated using enzymatic digestion, and cells at passages 4-6 were used for subsequent experiments. An in vitro cuproptosis model was established by treating hDPCs with elesclomol (Ele) and CuCl₂. CCK-8 assay showed that Ele-CuCl₂ significantly reduced cell viability, whereas tetrathiomolybdate (TTM) treatment restored viability (Figure 6A). Consistently, WB and copper staining confirmed increased lip-DLAT expression and copper accumulation in the Ele-CuCl₂ group, both of which were attenuated by TTM (Figure 6B and C).

Fig. 6.

TTM inhibits cuproptosis of hDPCs in vitro, Ele combined with CuCl₂ with or without TTM was used to treat hDPCs for 4 h (n = 3). A. CCK-8 detected the cell viability (n = 3); B. WB detected the expression of lip-DLAT protein (n = 3); C. Copper ion staining method detected the copper ion enrichment (n = 3). (Red arrow represents dead cells and blue arrow represents copper ion enrichment); D. RT-qPCR detected the expression of MAP1LC3A, CYP1A1, DBT, LIAS, MT1H and MT2A mRNA (n = 3). Data are from 3 independent experiments. All error bars are mean ± standard error of the mean. P values were calculated by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

RT-qPCR analysis demonstrated that CYP1A1 and MT2A were upregulated, while MAP1LC3A was downregulated in the Ele-CuCl₂ group compared to control; these alterations were reversed by TTM (Figure 6D). Among the 3 hub-CRGs, MT2A exhibited the highest diagnostic value (AUC) (Figure 4J), and was therefore selected for functional validation.

MT2A knockdown was achieved in hDPCs using siRNA, with an efficiency of >60% confirmed by RT-qPCR. Following Ele-CuCl₂ induction, si-MT2A cells displayed markedly reduced viability compared to si-NC cells (Figure 7A). RT-qPCR further revealed decreased MT2A expression and significantly elevated IL-6 levels (Figure 7B). At the protein level, lip-DLAT was strongly upregulated in si-MT2A cells (Figure 7C). Copper staining and cell death assays confirmed enhanced copper accumulation and increased cell mortality in the MT2A-silenced group (Figure 7D). Together, these findings indicate that MT2A mitigates cuproptosis and inflammatory responses in hDPCs, highlighting its protective role in pulpitis.

Fig. 7.

Knockdown of MT2A enhances cuproptosis and inflammatory response of hDPCs in vitro, After transfected with 100nM si-MT2A or si-NC, Ele combined with CuCl₂ was used to induce cuproptosis of hDPCs for 4 h. A. CCK-8 detected the cell viability; B. RT-qPCR detected the expression of MT2A and IL-6 mRNA; C. WB detected the expression of lip-DLAT protein; D. Copper ion staining method detected the copper ion enrichment (Red arrow represents dead cells and blue arrow represents copper ion enrichment). Data are from 3 independent experiments. All error bars are mean ± standard error of the mean. P values were calculated by unpaired 2-tailed Student’s t test. *P < .05, ***P < .001.

MTII knockdown exacerbates cuproptosis and pulp inflammation in vivo

Having demonstrated that the knockdown of MT2A could aggravate cuproptosis and inflammatory response in vitro, the function of MT2A was evaluated in vivo. Notably, MT2A refers to the human gene, while its murine homolog is denoted as MTII. We further established a mouse pulpitis model with MTII knockdown to investigate its role in vivo (Figure 8A). The HE staining shows intact odontoblast layers adjacent to dentin and normal vasculature in control. In contrast, the si-MTII group exhibits severe pulpitis, characterized by dense polymorphonuclear leukocyte infiltration predominantly in the coronal pulp region, accompanied by disruption of the odontoblast layer. The si-NC group shows moderate inflammatory infiltration (Figure 8B). The fluorescence staining reveals that the si-MTII group shows a significant reduction in MTII expression, accompanied by increased lipoic acid intensity and IL-6 expression. In contrast, the si-NC group demonstrates attenuated cuproptosis and reduced inflammatory response compared to si-MTII group (Figure 8C).

Fig. 8.

Knockdown of MTII enhances cuproptosis and inflammatory response of pulpitis, A. Establishment of pulpitis model of MTII knockdown in vivo; B. HE staining detected the expression of Healthy (n = 5), si-NC + Pulpitis (n = 5) and si-MTII + Pulpitis (n = 5) in mouse pulp tissue; C. IF staining of Healthy (n = 5), si-NC + Pulpitis (n = 5) and si-MTII + Pulpitis (n = 5).

MT2A attenuates cuproptosis-induced macrophage recruitment and M1 polarization in vitro

Bioinformatic analysis of GEO datasets (GSE92681 and GSE77459) revealed a strong positive correlation between MT2A expression and immune cell infiltration in human pulp tissues, with macrophages showing the strongest association (Figure 9A). Immunofluorescence staining of CD68 further confirmed abundant macrophage infiltration in inflamed pulp tissues (Figure 9B).

Fig. 9.

MT2A knockdown in hDPCs cuproptosis model enhances macrophage migration and M1 polarization, A. Pearson correlation coefficients among immune cells and the MT2A in human pulp tissues from GEO database (GSE92681 and GSE77459); B.Immunofluorescence staining of CD68 protein in human healthy pulp tissues (n = 3) and pulpitis tissues (n = 3); C. Transwell assays detected the migration of macrophages (n = 3); D. Flow cytometry detected the polarization of macrophages by the expression of CD80 and CD163 (n = 3).

Functionally, Transwell assays demonstrated that Ele-Cu-treated hDPCs significantly promoted macrophage chemotaxis, whereas TTM treatment markedly attenuated this effect (Figure 9C). Flow cytometry showed that macrophages recruited by Ele-Cu-stimulated hDPCs exhibited enhanced M1 polarization, while TTM alleviated this response (Figure 9D). To directly assess the role of MT2A, hDPCs with MT2A knockdown were cocultured with macrophages. Compared with si-NC, si-MT2A significantly increased macrophage migration and M1 polarization, whereas control hDPCs displayed minimal effects (Figure 10A, B).

Fig. 10.

MTII knockdown enhances macrophage infiltration and M1 polarization in the mouse pulpitis model, A. Transwell assays detected the migration of macrophages (n = 3); B. Flow cytometry detected the polarization of macrophages by the expression of CD80 and CD163 (n = 3); C. IF staining of MTII, F4/80 and iNOS protein (n = 5).

Together, these results demonstrate that cuproptosis in hDPCs promotes macrophage recruitment and M1 polarization, thereby amplifying inflammatory responses, whereas MT2A suppresses this process by limiting cuproptosis-induced pro-inflammatory signaling.

MTII knockdown enhances macrophage infiltration and M1 polarization in vivo

In the pulpitis mouse model with MTII knockdown, triple immunofluorescence staining for MTII, the pan-macrophage marker F4/80, and the M1 polarization marker iNOS revealed that the si-MTII group exhibited markedly increased macrophage infiltration and the highest proportion of M1-polarized cells. In contrast, the si-NC group showed reduced macrophage recruitment and lower M1 polarization (Figure 10C).

These findings provide in vivo evidence that MTII protects pulp tissues from excessive cuproptosis, thereby indirectly limiting macrophage infiltration and pro-inflammatory M1 polarization during pulpitis.

Discussion

This study demonstrates significant copper accumulation and upregulation of lip-DLAT in both human and mouse pulpitis tissues, predominantly enriched in hDPCs, indicating activation of cuproptosis in pulpitis. Dysregulated expression of the key cuproptosis-related gene MT2A in inflamed pulp was further confirmed through bioinformatic analyses and clinical sample validation. In vitro experiments showed that the cuproptosis inhibitor TTM effectively alleviated copper-induced hDPCs death and inflammatory responses. Collectively, these findings suggest that cuproptosis in hDPCs contributes to the pathogenesis of pulpitis, and that targeting the cuproptosis pathway may provide a novel therapeutic strategy for this disease.

In recent years, accumulating evidence has indicated that cuproptosis plays a critical role in the progression of various diseases, particularly exhibiting unique diagnostic and therapeutic potential in inflammatory disorders. For instance, cuproptosis can participate in the inflammatory cascade of diseases such as periodontitis and osteoarthritis by inducing the release of numerous immunoregulatory factors, and may further drive inflammation through the combined effects of immune activation and metabolic dysregulation. Although copper overload and cuproptosis have been implicated in multiple inflammatory and metabolic diseases, their specific mechanisms in pulpitis remain unclear. In this study, we systematically investigated cuproptosis in hDPCs by integrating clinical samples, RNA-seq data, and functional experiments. Our study demonstrates that copper-dependent cell death contributes to the pathogenesis of pulpitis, consistent with previous reports.³⁴ These findings establish hDPC cuproptosis as a critical mediator of tissue injury in inflamed pulp.³⁴ Nevertheless, the precise mechanisms by which copper-induced cell death operates at different stages of pulpitis remain largely unclear. Notably, evidence from other disease contexts suggests a dual role of copper in pathological processes: for instance, elevated extracellular copper can promote tumor growth and invasiveness through the MYC/CTR1 axis in liver cancer.³⁵ whereas copper complexes such as DSF/Cu can activate antitumor immunity by enhancing dendritic cell maturation and improving the efficacy of CD47 blockade therapy.³⁶ Analogously, it is plausible that copper-dependent cell death may exert context-dependent effects in pulpitis, potentially varying between early and late stages of inflammation. These considerations highlight the need for further investigation into the stage-specific roles of cuproptosis in pulpitis.

Through bioinformatic analysis, MT2A was identified as the gene most significantly associated with cuproptosis in hDPCs. In this study, we confirmed that MT2A expression was markedly upregulated in both human pulpitis tissues and cuproptosis models. Knockdown of MT2A resulted in increased copper accumulation, elevated levels of cuproptosis, and enhanced secretion of inflammatory factors in hDPCs, suggesting that MT2A may exert a protective effect by negatively regulating cuproptosis. As a member of the metallothionein family, MT2A possesses detoxifying, antioxidant, and anti-inflammatory functions. It regulates intracellular copper homeostasis, interacts with the reactive oxygen species (ROS) antioxidant system, suppresses inflammatory signaling pathways, and reduces the release of inflammatory mediators.³⁷ Its expression is regulated by metal ion concentrations and various stress signals. Under stimuli such as infection and inflammation, MT2A enhances antioxidant and anti-inflammatory responses by scavenging ROS.³⁸ Previous studies have also shown that high MT2A expression in mouse hepatocytes mitigates drug-induced hepatotoxicity by sequestering ROS.³⁹ Moreover, MT2A can inhibit the overexpression of LPS-related factor-1 in endothelial cells, thereby negatively regulating LPS-induced inflammatory responses.⁴⁰ Our findings are consistent with these reports, further supporting a protective role of MT2A in cuproptosis during pulpitis.

Pulpitis development is characterized by dynamic remodeling of the immune microenvironment,⁴¹ in which macrophages act as key immune regulators. Their recruitment and polarization play central roles in inflammation initiation, amplification, and tissue repair. Bioinformatic analysis revealed a significant positive correlation between MT2A expression and macrophage infiltration, suggesting that MT2A may participate in regulating macrophage function. In vitro experiments further demonstrated that cuproptosis-induced hDPCs actively recruit macrophages and promote their M1 polarization, and this effect was significantly enhanced upon MT2A knockdown, indicating that loss of MT2A can exacerbate macrophage-mediated inflammatory responses by amplifying cuproptosis. Notably, in a pulpitis model with MTII (the murine homolog of MT2A) knockdown, increased macrophage infiltration and enhanced M1 polarization were also observed, providing in vivo evidence that MT2A/MTII regulates macrophage behavior. These findings challenge the conventional view of copper as merely cytotoxic, suggesting that the cuproptosis-MT2A axis may play a crucial role in shaping the pulp immune microenvironment by modulating macrophage recruitment and polarization. We propose that MT2A may indirectly restrain excessive macrophage activation and M1 polarization by inhibiting cuproptosis in hDPCs, thereby slowing the progression of pulpitis. Interestingly, the immunomodulatory effects mediated by MT2A in our study show conceptual similarity to those of mineral trioxide aggregate (MTA), a bioactive material widely used in clinical pulp therapy. Previous studies have demonstrated that MTA can regulate macrophage behavior within the pulp microenvironment. Yeh et al. found that MTA can promote the polarization of macrophages to the M2 type and upregulate anti-inflammatory factors such as interleukin.⁴² Meanwhile, another study has demonstrated that bioceramic cements, including MTA, significantly increase ROS production by macrophages under inflammatory conditions, thereby enhancing pathogen clearance.⁴³ Although there is currently no direct evidence that MTA regulates intracellular copper homeostasis or MT2A expression, both MT2A-mediated suppression of cuproptosis and MTA-mediated bioactivity appear to converge functionally on the regulation of macrophages. Specifically, our findings suggest that MT2A alleviates pulp inflammation by limiting excessive macrophage recruitment and M1 polarization, a mechanism that parallels the immunoregulatory effects observed with MTA in pulp healing. These observations suggest a potential association between copper homeostasis-related pathways and the immunomodulatory effects of bioactive pulp-capping materials, which warrants further investigation.

Although this study systematically identified hub-CRGs in pulpitis by integrating multi-omics data with machine learning, and revealed the role of MT2A in regulating cuproptosis in hDPCs and macrophage-mediated immune responses, several limitations remain. First, the transcriptomic data derived from public databases were generated using gene probe-based sequencing technologies, which may have limited specificity and sensitivity, posing a potential risk of false-positive results. Second, although the machine learning algorithms employed enabled the identification of high-confidence hub-CRGs, no independent external validation cohort was included, and thus the robustness of the findings requires further confirmation. Furthermore, while cuproptosis has been reported to play roles in inflammatory diseases such as periodontitis and rheumatoid arthritis^31,44,45 this study has not yet fully elucidated the direct regulatory relationships between immune cells and hub-CRGs in in vivo or in vitro systems, and the underlying molecular mechanisms remain to be explored. In the pathological environment of pulpitis, MT2A can inhibit the copper-induced cell death process in hDPCs and protect the survival and function of dental pulp cells. It is worth noting that MT2A also exhibits similar metal regulation and protective effects in other disease models. In the rat Parkinson's disease model, Artesunate increases the expression of MT2A in astrocytes, reduces intracellular Cu²⁺ levels, and alleviates copper-induced damage to dopaminergic neurons, providing potential evidence for its therapeutic application in neurological diseases.⁴⁶ Based on the above mechanism, MT2A also has clinical translational potential in the treatment of pulpitis. TTM is a copper ion chelator that inhibits copper death by binding copper ions. According to its mode of action, it can be divided into non-absorbent and absorbent types. Currently, clinical research on non-absorbent TTM has made progress. The study by Ala et al. evaluated 8 Wilson disease patients who completed copper balance tests, and the results showed that TTM treatment could significantly lower the copper balance level of the patients, supporting its potential as a new treatment option for Wilson disease.⁴⁷ Absorbent TTM mainly functions by entering the circulatory system and reducing the level of "free copper" with biological activity in the serum. Based on this mechanism, this study selected absorbent TTM as a copper death inhibitor to systematically explore the impact and mechanism of inhibiting copper death on the pathological development process of pulpitis. This also supports the translational potential of TTM in pulpitis, particularly when combined with localized delivery strategies to achieve site-specific copper modulation. To further clarify the functional significance of cuproptosis in the development of pulpitis, future studies should experimentally validate the regulatory mechanisms of hub-CRGs and expand clinical sample analyses to assess their translational potential.

In summary, our findings demonstrate that cuproptosis plays a critical role in the progression of pulpitis, with 6 hub-CRGs acting as key mediators under copper-induced stress. MT2A was identified as an important endogenous inhibitor in hDPCs, capable of modulating immune cell infiltration and inflammatory responses by alleviating cuproptosis. These results not only deepen our understanding of the pathogenesis of pulpitis but also highlight the MT2A-mediated cuproptosis pathway as a potential therapeutic target.

Conclusion

This study demonstrates that cuproptosis is activated in human and mouse pulpitis, predominantly affecting dental pulp cells. The copper-dependent lipoylation of DLAT drives cell death and amplifies local inflammation. MT2A functions as a critical protective regulator of cuproptosis, mitigating cell death in hDPCs and indirectly limiting macrophage recruitment and M1 polarization. These findings highlight MT2A-mediated regulation of cuproptosis as a potential therapeutic target for preserving pulp vitality and controlling inflammatory progression in pulpitis.

Ethics approval

This research has been approved by the Ethics Committee of Shenzhen Stomatology Hospital (Pingshan) of Southern Medical University, and informed consent was obtained from all participating patients (Ethics approval number: 202415A). The processing of clinical samples had been in strict compliance with the ethical standards of the Declaration of Helsinki. All written informed consent was obtained from all participants.

CRediT authorship contribution statement

Yiming Zhong: Conceptualization, Data curation, Formal analysis, Writing – original draft. Xiaofei Qu: Investigation, Methodology, Visualization, Writing – original draft. Kailun Wu: Data curation, Formal analysis. Longrui Dang: Data curation, Formal analysis. Sitong Liu: Conceptualization, Data curation, Visualization. Xiaolan Guo: Data curation, Visualization. Zhao Chen: Resources, Writing – review & editing. Buling Wu: Conceptualization, Resources, Supervision, Writing – review & editing.

Conflict of interest

None disclosed.