Depending on the American Association of Endodontists (AAE) criteria in 2013, pulpitis is classified as a reversible or irreversible state based on clinical objective or subjective findings, accompanied by intensive pain and an increased risk of systematic diseases (1–3). If treated improperly, pulp necrosis and severe maxillofacial infections may develop (4). Clinically, pulpotomy, root canal therapy, and antibiotics are traditional processes (5, 6). However, our comprehension of the pathogenetic mechanisms is limited, leading to the lack of pertinent medicine for irreversible pulpitis (IP). Histologically, bacterial invasion and proliferation, tissue inflammation, and micro abscesses are major characteristics of IP (7). Several biological factors, including Toll-like receptors, P substance, IL-8, and TGF-β1, are closely associated with the disease (8, 9). As indicated in functional analyses, the following pathways are related to IP: leukocyte migration and adhesion, inflammation mediation, and osteoclast polarization (10). To date, major attention has been given to typical inflammatory reactions, but the latest mechanisms lack further investigation.

Cell death plays a crucial role in multicellular organism growth and homeostasis maintenance (11). Apoptosis was first discovered as a form of programmed cell death associated with inflammation pathways such as TNF and TRAIL receptors, together with downstream ROS/RNS signaling (12, 13). In particular, ferroptosis is newly defined as a specific mode of cell death (14). This process, related to integrated oxidation and antioxidation homeostasis, is iron-dependent and nonapoptotic. Furthermore, ferroptosis is accompanied by morphological manifestations such as mitochondrial contraction, increased membrane density, and reduced or absent mitochondrial cristae (15). From the initially defined ferroptosis-related protein erastin to the key repressed enzyme GPX4, accumulating evidence has focused on the ferroptosis mechanism (16, 17). Interestingly, excessive or deficient ferroptosis is associated with physiological and physio pathological mechanisms, coupled with immune responses and inflammation programs. Ferroptosis influences the amount and function of immune cells, triggering serial inflammation. It has been shown that a high-iron diet or conditional depletion of Gpx4 in the pancreas aggravates cerulein-induced pancreatitis in mice. Polyunsaturated fatty acid diet causes Crohn’s disease by promoting ferroptosis. A thorough understanding of ferroptosis may offer insight into the intricate immunomodulatory effects involved in the course of infection (18). To date, ferroptosis has not been reported in the study of IP. Thus, the current study aimed to discover the pathologic and functional mechanisms underlying IP and ferroptosis.

LncRNA, a kind of noncoding RNA (ncRNA) more than 200 bp in length, is involved in gene editing, transcription, and epigenetic mechanisms (19). Epigenetic methods were considered to further investigate the biochemical mechanism. The ceRNA theory, which links ncRNA with protein-coding RNA, reveals the competitive relationship between lncRNA and mRNA through their common binding miRNAs, which are involved in numerous biological processes (20). However, few reports have applied ceRNA theory to the study of the IP. Xi et al. screened several lncRNAs in the context of revealing the interaction mechanism between inflammation and IP (21). Meanwhile, Lei et al. constructed a ceRNA network for marker genes of IP but barely discussed the specific mechanisms or genetic pathways (10).

In summary, we aimed to elucidate the correlation between ferroptosis and IP, deepening the application of the ceRNA theory in this field. After identifying differentially expressed mRNAs and lncRNAs (DEmRNAs, DElncRNAs) from the GSE92681 set, we screened these data with ferroptosis marker genes. FR-DElncRNAs were identified by coexpression analysis of DE-FRGs and DElncRNAs. Then, miRNA predictions related to DE-FRGs or FR-DElncRNAs were performed. Two ceRNA networks were built based on DE-FRG/miRNA/FR-DElncRNA. Finally, 6 hub lncRNAs (LUCAT1 and AC106897.1 ↑; LINC00943, AL583810.1, AC068888.1, and AC125257.1↓) were selected for further verification. In addition, immune infiltration analysis revealed the relationship between hub lncRNAs and immune cells.

Pulpitis derives from bacterial-induced infection and necroptosis and is characterized by acute pain (22). Inflammatory processes and pathways are key to the mechanistic mechanisms of pulpitis, including specific PRR receptors, NF-kB signaling, and cytokine secretion (23, 24). Intriguingly, cell death is closely related to inflammation as well. Ferroptosis is a newly discovered method of cell death (25). Serving as a nonapoptotic and iron-dependent process, it is triggered by iron-induced lipid peroxidation and stimulates the downstream GPX4 pathway and AMPK signaling (26, 27). Emerging evidence indicates that ferroptosis participates in various diseases. For periodontitis, biochemical analyses indicated that ferroptosis marker genes are differentially expressed among experimental and control groups (28). However, the mutual mechanism between IP and ferroptosis remains unclear. Thus, the current study aims to fill this gap and provide aetiological hypotheses.

Noncoding genes are involved in various aspects of physiological and pathophysiological processes through gene regulation and epigenetic mechanisms (29). LncRNA, a subtype of ncRNA more than 200 bp in length, is a hotspot for epigenetic mechanism investigations (30, 31). Among multifarious regulatory methods, the ceRNA regulatory network has a great reputation. lncRNAs and mRNAs mutually regulate each other by competing for common miRNAs at the posttranscriptional level (32). Thus, this theory sheds light on the epigenetic mechanism in multiple vital processes. Recently, Lei et al. built a ceRNA network based on differentially expressed data of pulpitis, containing lncRNA PVT1, hsa-miR-455-5p, and 2 mRNAs (SOCS3 and PLXNC1) (10). The enriched GO terms inflammatory response and positive regulation of leukocyte chemotaxis are identified in this report, which is similar to the immune regulation term identified in our study. Moreover, an immune infiltration experiment was conducted in our study to further ensure the connection between selected hub lncRNAs and various immune cells, including activated B cells and T cells. Meanwhile, another study searched for pulpitis marker genes based on the ceRNA theory by combining differentially expressed lncRNA, mRNA, and miRNA data (21). To our surprise, hsa-miR-340-5p, hsa-miR-5590-3p, hsa-miR-424-5p, and hsa-miR-515-5p were identified in both their networks and our networks. Among them, miR-340-5p was found to target LIMS, a coding protein participating in integral protein signaling pathways; thus, this miRNA influences integral-linked odontogenic stimulation of human dental pulp stem cells (33, 34). MiRNAs may be involved in pulp tissue healing and reconstruction in pathological processes. Based on limited previous evidence, the ceRNA theory may provide us with potential mechanistic methods and clinical targets worth further exploration.

In the current study, we built ceRNA networks based on DE-FRGs, FR-DElncRNAs and the intersection of predicted miRNAs. Several hub lncRNAs, LUCAT1, AC106897.1 (↑) and LINC00943, AL583810.1, AC068888.1, AS125257.1 (↓), were identified for further discussion and experimental verification. Three of them were previously investigated, and LINC00943 and LUCAT1 were the most important. Taking LINC00943 as the first example, a ceRNA theory article for breast cancer found that LINC00943 is elevated in the high CD8 T-cell group compared with the low CD8 T-cell group, proving its close relationship with CD8 T cells (35). The expression of CD8 T cells was significantly increased in pulpitis tissue as a result of the immune infiltration experiment, indicating the possibility that LINC00943 may be involved in the pulpitis mechanism. Simultaneously, LUCAT1 is defined as an oncogenic molecule closely associated with ferroptosis (36). Based on bioinformatic methods, previous articles have discovered an accurate model for cell viability, hepatocellular carcinoma progression, and the microenvironment as a part of the ferroptosis mechanism (37, 38). However, in vivo or in vitro experiments to further verify the relationship between the two lncRNAs and these functions are limited.

To reveal the core process of the interaction between ferroptosis and pulpitis, DE-FRGs were identified through interaction analysis of DEGs and FRGs. Among them, PDK4 related to LINC00943 is worth further discussion. As discovered by Liu et al, PDK4-mediated pyruvate oxidation inhibition participates in ferroptosis repression in pancreatic ductal adenocarcinoma (PDAC) cells (39). PDK4 is involved in ferroptosis resistance by suppressing both pyruvate oxidation and the subsequent TCA cycle and lipid peroxidation, thus hindering the driving factors of ferroptosis (40). Additionally, PDK4 has a complex relationship with the energy sensor AMP-activated protein kinase (AMPK), which is involved in ferroptosis by its phosphorylated substrate (41). In addition, PDK4 was found to be related to chronic inflammation since LPS induces its expression via the Jun N-Terminal Kinase pathway (42). Since LPS has been widely proven to stimulate inflammation in human tissues, including dental pulp, we indicated that PDK4 may participate in the progression of pulpitis (43). Collectively, these results show that PDK4 may serve as a key resistance factor for ferroptosis via various pathways and may be involved in pulpitis progression. Interestingly, Liu et al. found that LINC00662 may modulate cell apoptosis by sponging miR-103a-3p and upregulating PDK4, indicating potential lncRNA-related regulation of PDK4 (44).

According to previous studies, LINC00943 and PDK4 may reveal the correlation between ferroptosis and pulpitis; however, their interaction has not yet been discussed. Twenty-three miRNAs were identified in our network, which may explain the interaction via ceRNA theory. Against the theoretical background, these miRNAs can bind to both LINC00943 and PDK4, resulting in a competitive relationship. Among the 23 miRNAs, hsa-miR-15b-5p and hsa-miR-29a-3p were determined to be significant for discussion. Initially, hsa-miR-15b-5p serves as a MAPK-related molecule, while the MAPK pathway induces ferroptosis by promoting free radical production after ICH and regulating TNF signaling (45, 46). Hu et al. indicated that hsa-miR-29a-3p correlates with ferroptosis via bioinformatic analyses (47). Simultaneously, an integrative analysis of H. pylori-infected peptic ulcer disease and periodontitis revealed its potential role in both diseases. By conducting RNA-seq dataset analysis of severe COVID-19 patients, Kim et al. indicated that hsa-miR-29a-3p may have a potential anti-inflammatory function (48). Because of the analogical relationship between pulpitis and periodontitis, hsa-miR-29a-3p may be at the core of the interaction between pulpitis and ferroptosis (49). Considering the functions of the ceRNA network, the cellular location of these genes needs to be in the cytoplasm. According to GeneCards, PDK4 is mainly localized in the mitochondrion, hsa-miR-29a-3p is mainly found in the nucleus and lysosomes, and LINC00943 is associated with the mitochondrion. Furthermore, three of them have been identified in different ceRNA axes according to other studies (50–52). Our qRT-PCR results also revealed that the expression levels of LINC00943, PDK4, and hsa-miR-29a-3p matched the corresponding changes based on the ceRNA theory.

Several limitations in the current study need to be mentioned. Initially, the lack of a differentially expressed miRNA database may result in some deviation. In vivo or in vitro experiments are needed to verify the specific functions and interactions of LINC00943, hsa-miR-29a-3p, and PDK4.

In summary, we screened six hub lncRNAs based on ceRNA networks to explore the link between ferroptosis and IP. Additionally, the ceRNA network of LINC00943/hsa-miR-29a-3p/PDK4 might play a key role in the interactive mechanism of ferroptosis and IP.

The original contributions presented in the study are included in the article/supplementary materials. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Ethics Statement. The research section about human pulp tissues was approved by the Ethics Committee of Southern Medical University (NFEC-2023-079). The patients/participants provided their written informed consent to participate in this study.

HW contributed to the conception and design of the research. QX contributed to the writing and drafting of the manuscript. HY and ZL contributed to drawing the figures and tables and analyzing the data. BZ contributed to performing the experimental work. FF and WQ contributed to the critical revision of the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.