Skip to main content
Download PDF

Expression patterns of mechanosensitive ion channel PIEZOs in irreversible pulpitis

BMC Oral Health volume 24, Article number: 465 (2024) Cite this article

Abstract

Background

Mechanosensitive ion channel PIEZOs have been widely reported to involve inflammation and pain. This study aimed to clarify expression patterns of PIEZOs and their potential relations to irreversible pulpitis.

Materials and methods

Normal pulp tissues (n = 29) from patients with impacted third molars and inflamed pulp tissues (n = 23) from patients with irreversible pulpitis were collected. Pain levels were assessed using a numerical rating scale. PIEZO expressions were measured using real-time PCR and then confirmed using GEO datasets GSE77459, immunoblot, and immunohistochemistry staining. Correlations of PIEZO mRNA expression with inflammatory markers, pain markers, or clinical pain levels were evaluated using Spearman’s correlation analysis. Univariate analysis was conducted to analyze PIEZO expressions based on pain description and clinical examinations of cold test, percussion, palpation, and bite test.

Results

Compared with normal pulp tissues, mRNA expression levels of PIEZO1 were significantly increased in inflamed pulp tissues, while PIEZO2 was significantly decreased, which was further confirmed in GSE77459 and on a protein and histological level. The positive correlation of the mRNA expression levels between PIEZO1 and inflammatory markers, as well as between PIEZO2 and pain markers, was verified. PIEZO2 expression was also positively correlated with pain levels. Besides, irreversible pulpitis patients who reported continuous pain and who detected a positive response to cold stimulus exhibited a higher expression level of PIEZO2 in the inflamed pulp tissues. By contrast, patients reporting pain duration of more than one week showed a higher expression level of PIEZO1.

Conclusions

This study demonstrated the upregulation of PIEZO1 and the downregulation of PIEZO2 in irreversible pulpitis and revealed the potential relation of PIEZO1 and PIEZO2 to inflammation and pain. These findings suggested that PIEZOs might play critical roles in the progression of irreversible pulpitis and paved the way for further investigations aimed at novel therapies of irreversible pulpitis by targeting PIEZOs.

Peer Review reports

Background

Irreversible pulpitis, referring to the situation when the inflammation in pulp is not able to resolve, is a major dental disease affecting many adults. The inflammation in pulp results in pain, which is the most frequent chief complaint of irreversible pulpitis [1]. For patients, understandably, management of pain is given priority over all other considerations [2]. However, the unpredictable results of medications for inflammation and pain directly or indirectly limited the further development of treatment for irreversible pulpitis [3], implying an urgent need for a better understanding of irreversible pulpitis.

Pulpal inflammation could alter the dentinal sensitivity of nerve fibers and solicit varying degrees of pain. Inflammatory cytokines induced by invading pathogens could decrease the threshold of the nociceptors and cause pain through exciting or upregulating various ion channels mediating pain [4]. However, antibiotic drugs, non-steroidal drugs, and steroidal drugs aiming at arresting inflammation could erratically control pain [5,6,7]. Besides, local anaesthetics that block ion channels mediating pain may not achieve completely profound anaesthesia in patients with symptomatic pulpitis [8]. A better understanding of the pathological mechanism behind the disease is crucial for the improvement of treatment [9, 10]. Hence, exploring mechanisms underlying inflammatory pulpal pain may pave the way for novel therapies for irreversible pulpitis.

The mechanosensitive ion channel PIEZOs, PIEZO1 and PIEZO2, could convert mechanical signals to electrical or chemical signals by evoking cellular Ca2+ influx, and have been reported to participate in inflammatory processes and pain transmission [11]. For PIEZO1, more studies concentrated on its role in mechanical force-related inflammation. Reportedly, PIEZO1 could be upregulated by interleukin (IL) -1α, sense stiffness in macrophages and modulate their polarization, and mediate calcium-dependent injury and deformation microtrauma [12,13,14]. With regard to PIEZO2, accumulating evidence has shown its contribution to inflammatory pain, such as tactile allodynia and visceral hypersensitivity under inflammatory conditions [15,16,17]. Despite these important functions of PIEZOs in inflammatory diseases and pain, their potential role in irreversible pulpitis remains obscure.

Notably, emerging evidence has shown that PIEZOs might participated in the transduction of mechanical and sensory signals in pulp. PIEZO1 has been demonstrated to promote intercellular odontoblast-odontoblast and odontoblast-neuron communication and inhibit mechanically stimulated mineralization of odontoblast [18, 19]. PIEZO2 was found to be positive in dental primary afferent neurons with myelinated A-fibers and identified as a possible transducer for pulpal mechanical stimulation-activated inward currents [20]. Considering the contribution of PIEZOs in inflammatory diseases, it is implicated that PIEZOs may be involved in inflammatory pain in irreversible pulpitis.

Given that PIEZOs play a vital role in inflammation and pain, the current study assumed that PIEZO expressions were altered in irreversible pulpitis, and primarily compared expression patterns of PIEZO1 and PIEZO2 between irreversible pulpitis and normal pulp. To analyze their relations to inflammation and pain, the correlation of PIEZO expressions with the inflammatory mediators, IL-1β, tumor necrosis factor (TNF) -α, and IL-6 and pain markers, Neuropeptide Y (NPY) and tachykinin precursor 1 (TAC1) were assessed. This study also assessed the correlation between PIEZO expressions and clinical pain levels. Further, the expressional features of PIEZOs were analyzed based on pain description and clinical examinations of cold test, percussion, palpation, and bite test. Collectively, these results suggested the potential roles of PIEZO1 and PIEZO2 in inflammation and pain in irreversible pulpitis, which pave the way for further investigations aimed at clarifying the underlying mechanisms and achieving corresponding clinical outcomes for irreversible pulpitis.

Materials and methods

Sample collection

Twenty-nine normal pulp tissue samples were obtained from patients with impacted third molar teeth, and twenty-three inflamed pulp tissues were obtained from patients with irreversible pulpitis. The study was performed in accordance with the Declaration of Helsinki and received ethical approval from Ethics Committee of Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Informed consent was obtained for experimentation with human subjects.

The clinical diagnosis of normal pulp and irreversible pulpitis is made according to the identification and definition of pulpal health and disease states approved by American Association of Endodontics Consensus Conference [1]. For patients with irreversible pulpitis, only those who developed irreversible pulpitis secondary to caries were included. For each patient, a standardized survey on the demographic information, history of dental pain (including pain categories, pain duration, and whether the pain was radiating), and medical/dental history were verbally administered. Tooth number was recorded, and examination procedures consisted of intraoral examinations to observe sinus tract, soft tissue, caries, and periodontal status, sensitivity cold tests, periapical tests to observe percussion, palpation and biting, and radiographs. Demographic information was sought by interviewing. Demographic and clinical information of the subjects was described in Table 1.

Table 1 Demographic and clinical information of the subjects
Full size table

Patients with the following situation were included for the normal group: (i) aged between 18 and 60; (ii) with impacted teeth and presenting for tooth extraction. Patients with the following situation were excluded: (i) any disease or medication history affecting pulpal status; (ii) oral or systemic diseases that would cause chronic or acute orofacial pain; (iii) mental health disorders, such as anxiety and depression, which cause pain; (iv) undergoing medical/dental treatment to manage pain; (v) smoking or drinking history; (vi) pregnant, lactating, or menopausal status.

Patients with the following situation were included for the diseased group: (i) aged between 18 and 60; (ii) diagnosed with irreversible pulpitis and presenting for endodontic treatment or tooth extraction with no evidence of periapical lesions and no previous pulp therapy. Patients with the following situation were excluded: (i) medication history affecting pulpal pain status; (ii) oral or systemic diseases that would cause chronic or acute orofacial pain; (iii) mental health disorders, such as anxiety and depression, which cause pain; (iv) undergoing medical/dental treatment to manage pain; (v) smoking or drinking history; (vi) pregnant, lactating, or menopausal status.

For RNA or protein isolation, normal pulp tissue samples were collected during extractions of impacted third molar teeth, and inflamed pulp tissue samples were obtained during pulpectomy. Subsequently, the pulp tissues were rinsed with normal saline, precooled, and stored using liquid nitrogen for further analysis. For histologic analysis, teeth from patients with extraction indications were extracted, rinsed with normal saline, and fixed in 10% neutral buffered formalin.

This laboratory study has been reported according to The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines [21] (Fig. 1).

Fig. 1
figure 1

The STROBE flowchart

Full size image

Real-time Polymerase Chain Reaction (real-time PCR)

The mRNA expressions of PIEZO1, PIEZO2, IL-1β, TNF-α, IL-6, NPY, CALCA, CALCB, TAC1, and ACTB in the pulp tissues were measured using real-time PCR. Briefly, total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and qualified using NanoDrop Spectrophotometer (Thermo Fisher Scientific, USA). For each sample, 1-μg total RNA was added to a 20-μL reaction mixture using PrimeScript™ RT Master Mix kit (Takara Bio, Otsu, Shiga, Japan), and reverse-transcribed into cDNA using T100 Thermal Cycler (BIO-RAD, Hercules, CA, USA). Real-time PCR reaction mixture was prepared using FastStart Universal SYBR Green Master Mix reagent (Roche, Nutley, NJ, USA) and analyzed on a QuantStudio7 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative mRNA expression levels of PIEZO1, PIEZO2, IL-1β, TNF-α, IL-6, NPY, CALCA, CALCB, and TAC1 were normalized to the mRNA level of human ACTB using the 2-DeltaDeltaCt method. Sequences of the primers are provided in Supplementary Table S1.

Analysis of Gene Expression Omnibus (GEO) dataset GSE77459

GEO, a public genomics database of the National Center of Biotechnology Information, was thoroughly queried for datasets involving studies on inflamed pulp or pulpitis. The processed dataset of GSE77459 which measured the expression profile in human inflamed pulp and normal pulp tissues, was downloaded [22]. Data were analyzed in a log2 scale, and expression data on the probe level were transformed to gene level utilizing GPL17692.

Immunoblot

Pulp tissues were frozen in radioimmunoprecipitation lysis buffer containing 1% phenylmethylsulfonyl fluoride, ground in liquid nitrogen, and lysed on ice for 30 min. Total protein concentration was detected using BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Then sodium dodecyl sulfate loading buffer (Takara Bio, Otsu, Shiga, Japan) was added to protein samples, and the mixture was boiled at 100 ℃ for 5 min. Denatured protein samples were then loaded onto sodium dodecyl sulfate–polyacrylamide gels for separation and transferred onto polyvinylidene fluoride membranes. Membranes were blocked using bovine serum albumin blocking buffer for 1 h at room temperature, incubated in solutions containing primary antibodies against PIEZO1 (Novus Biologicals, MO, USA), PIEZO2 (Novus Biologicals), or GAPDH (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C, washed with phosphate-buffered saline with Tween detergent, and then incubated in solution containing horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology) for 1 h at room temperature. After washing, membranes were exposed using chemiluminescence reagents (Millipore, Billerica, MA, USA) on ChemiDoc™ Imaging System (BIO-RAD, CA, USA). Immunoreactive bands were quantified using ImageJ software (X64; version 2.1.4).

Histologic processing

Teeth were fixed adequately with 10% neutral buffered formalin. To reduce the time required for decalcification, the enamel parts of the fixed teeth were removed previously. Then the specimens were decalcified in 10% ethylene diamine tetraacetic acid, embedded in paraffin blocks, and cut into 2.5-μm sections. Subsequently, the sections were deparaffinized, hydrated and underwent Hematoxylin and eosin (H&E) staining or immunohistochemistry. For immunohistochemistry, briefly, the sections underwent antigen retrieval, blocking, antibody incubation, and visualization. Finally, the images were scanned and analyzed by a digital scanner (3DHISTECH, Pannoramic DESK, Budapest, Hungary).

Pain intensity assessment

All the patients were asked to report the intensity of ongoing pain of the teeth immediately before the clinical treatment to collect the pulp tissues. The current pain levels were quantified using the 0–10 numerical rating scale (NRS), which has been reported previously [23]. In an NRS “0” denoted “no pain”, and “10” denoted “worst possible pain”.

Statistical analysis

Data were analyzed using GraphPad Prism 9.0 and represented by mean ± standard error of mean (S.E.M). Significance of statistical differences between the normal pulp group and the inflamed pulp group was evaluated using the Mann–Whitney U test. The result was considered statistically significant when P-value was less than 0.05. The symbols of “*” indicated P < 0.05; “**” indicated P < 0.01; “***” P < 0.001 indicated P < 0.001. Correlations between mRNA expression levels were assessed using Spearman's correlation analysis. The absolute magnitude of Spearman's correlation coefficient R-value (R) was interpreted as follows: negligible (R = 0.00–0.10), weak (R = 0.10–0.39), moderate (R = 0.40–0.69), strong (R = 0.70–0.89), and very strong (R = 0.90–1.00) [24].

Results

Expression profile of PIEZO1 and PIEZO2 in normal and inflamed human pulp tissues

We first evaluated the mRNA expression levels of PIEZO1 and PIEZO2 in normal dental pulp tissues (n = 21) and inflamed pulp tissues (n = 15) using real-time PCR. The results showed that PIEZO1 mRNA expression in the inflamed pulp tissues was significantly increased compared to normal pulp tissues, while PIEZO2 mRNA expression was significantly decreased (Fig. 2A), which was in line with the analysis of the GEO dataset, GSE77459 (Fig. 2B, C). The upregulation of PIEZO1 and the downregulation of PIEZO2 in the inflamed pulp tissues were further validated using immunoblot (Fig. 2D, E). Besides, histological analysis was performed to examine the distribution of PIEZO expressions. H&E staining showed the infiltration of immune cells and more blood vessels in inflamed pulp tissues (Fig. 2F), which confirmed the inflammatory status in the pulp tissues. The results of immunohistochemistry staining indicated that the upregulation of PIEZO1 and the downregulation of PIEZO2 were mainly in the odontoblast layer (Fig. 2F). Overall, these data revealed that PIEZO1 expression increased while PIEZO2 expression decreased in irreversible pulpitis.

Fig. 2
figure 2

The expression patterns of Mechanosensitive ion channel PIEZOs in human irreversible pulpitis. A The mRNA expression levels of PIEZO1 and PIEZO2 in normal pulp tissues (n = 21) and inflamed pulp tissues (n = 15) were examined using real-time PCR. B, C The gene expression levels of PIEZO1 and PIEZO2 in normal pulp tissues (n = 6) and inflamed pulp tissues (n = 6) in the public database, GSE77459, were analyzed. D The protein expression levels of PIEZO1 and PIEZO2 in normal pulp tissues (n = 5) and inflamed pulp tissues (n = 5) were examined using immunoblot. E The immunoblot results of protein expression levels of PIEZO1 and PIEZO2 were quantified. F Representative images of H&E staining and immunohistochemistry of the pulp are presented. Inflammatory cells (green arrows) and more blood vessels (blue arrows) are shown in inflamed pulp using H&E staining. Highly expressed PIEZO1 (yellow arrows) and highly expressed PIEZO2 (red arrows) are shown in immunohistochemistry images. P, pulp; D, dentin. * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001

Full size image

PIEZO1 expression was positively correlated with IL-1β, TNF-α, and IL-6

IL-1β, TNF-α, and IL-6 are inflammatory mediators that have been widely reported to involve the inflammatory process and potentiate pain in pulpitis [25]. Expectedly, our results showed the significant upregulation of IL1B, TNFA and IL6 in inflamed pulp tissues (Fig. 3A-C). Since we have demonstrated the alteration of PIEZO expressions in irreversible pulpitis, we then utilized Spearman’s correlation analysis and found that PIEZO1 mRNA expression was significantly and positively correlated with IL1B (R = 0.5607, P = 0.0322, moderate), TNFA (R = 0.7679, P = 0.0013, strong), and IL6 (R = 0.6250, P = 0.0148, moderate), but PIEZO2 was not significantly correlated with IL1B, TNFA, or IL6 (Fig. 3D-F). Therefore, our data revealed that PIEZO1 might be associated with inflammatory markers, implying that PIEZO1 presumably involved inflammatory processes in irreversible pulpitis.

Fig. 3
figure 3

The mRNA expression levels of inflammatory markers IL1B, TNFA, and IL6 and the correlations between PIEZOs and these inflammatory markers. A-C The mRNA expression levels of IL1B, TNFA, and IL6 in normal pulp tissues (n = 21) and inflamed pulp tissues (n = 15) were presented, respectively. D The correlation of the mRNA expression levels between PIEZO1 and IL1B, as well as PIEZO2 and IL1B, was assessed using Spearman’s correlation analysis. E The Correlation of the mRNA expression levels between PIEZO1 and TNFA, as well as PIEZO2 and TNFA, was assessed using Spearman’s correlation analysis. F The Correlation of the mRNA expression levels between PIEZO1 and IL6, as well as PIEZO2 and IL6, was assessed using Spearman’s correlation analysis. * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001

Full size image

PIEZO2 expression was positively correlated with NPY and substance P

NPY, calcitonin related polypeptide alpha (CALCA), calcitonin related polypeptide beta (CALCB), and TAC1 are genes that encode neuropeptides NPY, calcitonin gene-related peptide, and substance P, which are critical mediators of pain [25]. We first confirmed the upregulation of NPY, CALCA, CALCB, and TAC1 in inflamed pulp tissues (Fig. 4A-D). Further, by using Spearman’s correlation analysis, we demonstrated the significant and positive correlation of PIEZO2 expression with NPY (R = 0.7571, P = 0.0016, strong) (Fig. 4E) and TAC1 (R = 0.5321, P = 0.0438, moderate) (Fig. 4H). However, no significant correlation was observed between PIEZO1 and NPY, CALCA, CALCB, or TAC1 (Fig. 4E-H). Therefore, PIEZO2 might be related to pain mediator NPY and substance P, indicating the potential involvement of PIEZO2 in pain progression in irreversible pulpitis.

Fig. 4
figure 4

The mRNA expression levels of pain markers NPY, CALCA, CALCB, and TAC1 and the correlations between PIEZOs and these pain markers. A-D The mRNA expression levels of NPY, CALCA, CALCB, and TAC1 in normal pulp tissues (n = 21) and inflamed pulp tissues (n = 15) were presented, respectively. E The correlation of the mRNA expression levels between PIEZO1 and NPY, as well as PIEZO2 and NPY, was assessed using Spearman’s correlation analysis. F The correlation of the mRNA expression levels between PIEZO1 and CALCA, as well as PIEZO2 and CALCA, was assessed using Spearman’s correlation analysis. G The correlation of the mRNA expression levels between PIEZO1 and CALCB, as well as PIEZO2 and CALCB, was assessed using Spearman’s correlation analysis. H The correlation of the mRNA expression levels between PIEZO1 and TAC1, as well as PIEZO2 and TAC1, was assessed using Spearman’s correlation analysis. * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001

Full size image

PIEZO2 expression was positively correlated with clinical pain levels

In this study, the correlation between PIEZO expressions and pain levels was also performed using Spearman’s correlation analysis. As shown in Fig. 5, there was a significant and positive correlation between PIEZO2 expression and pain levels of patients with irreversible pulpitis, but no significant correlation was found between PIEZO1 and pain levels. In addition, univariate analysis was conducted to analyze PIEZO expressions based on the patient-reported pain description and responses to clinical examinations of cold test, percussion, palpation, and bite test (Table 2). Strikingly, PIEZO2 expression was elevated in the pulp tissues from patients who reported continuous pain and who presented a positive cold response, while PIEZO1 exhibited a significantly higher expression level in pulp tissues from patients who reported pain duration of more than one week. Collectively, these results indicated that PIEZOs might play a role in pain progression in irreversible pulpitis.

Fig. 5
figure 5

The correlation between PIEZO1 mRNA expression level and pain levels, as well as PIEZO2 and pain levels, was assessed using Spearman’s correlation analysis. * represents P < 0.05

Full size image
Table 2 Univariate analysis of PIEZO expressions
Full size table

Discussion

In this study, the upregulation of PIEZO1 and the downregulation of PIEZO2 were first confirmed in inflamed pulp tissues from patients with irreversible pulpitis. Consistently, chondrocytes in osteoarthritis cartilage displayed an increase in PIEZO1 expression upon IL-1α stimulation [12]. Besides, in cells exerted by mechanical stimulation, such as fluid sheer stress-treated osteocytes [26] and cyclic stretch-treated pulmonary microvascular endothelial cells [27], the increased expression of PIEZO1 was also observed. Hence, it is possible that the upregulation of PIEZO1 was a result of mechanical or inflammatory stimuli. Conversely, the reduced expression of PIEZO2 was demonstrated in irreversible pulpitis. Previous study has reported similar results that PIEZO2 was downregulated in pulmonary microvascular endothelial cells in response to abnormal shear stress, hypoxia, and TGF-β stimulation [28]. Considering that PIEZO2 has been widely reported to contribute to inflammation-induced sensitized mechanical pain [16], we speculated that the downregulation of PIEZO2 in inflamed pulp was a protective compensatory mechanism of the body to reduce harmful responses and relief pain, but it requires further validation.

We further demonstrated the positive correlation of the expression levels between PIEZO1 and inflammatory mediators, IL-1β, TNF-α, and IL-6. Reportedly, PIEZO1 could facilitate inflammation by transducing mechanical signals into inflammatory signals. For example, cyclical hydrostatic pressure could activate PIEZO1 in macrophages, thereby activating bacterial toxins-primed NOD-like receptor protein 3 inflammasomes and aggravating inflammation [29]. Besides, mechanical forces generated by cell adhesion molecule aggregation and blood flow could also activate PIEZO1 and induce downstream signalling events in endothelial cells, resulting in the opening of the endothelial barrier and leukocyte extravasation [30]. In infectious pulp tissues, cells could also experience mechanical force changes exerted by hydrostatic pressure changes or extravasation of leukocytes [23]. Therefore, PIEZO1 might also mediate the conversion of mechanical signals to inflammatory signals in irreversible pulpitis, but it requires further confirmation.

Herein, we also found that the expression level of PIEZO2 was positively correlated with pain markers, NPY and TAC1. PIEZO2 was demonstrated to be localized to low-threshold mechanoreceptors (LTMs) in sensory dorsal root ganglion neurons [31], which have been proven to produce substance P [32]. Besides, PIEZO2 was also localized to dental primary afferent neurons in trigeminal ganglions [20], which exhibited higher expression of LTM marker NPY [4]. Hence, future studies are required to study whether PIEZO2 acts on the function of LTMs to sense weak stimuli in inflamed pulp and whether it participates in the modulation of nociceptive neuropeptides.

Expectedly, the expression level of PIEZO2 was positively correlated with clinical pain levels, and PIEZO2 showed a higher expression level in patients with continuous pain and with positive responses to cold stimulation. This might be attributable to the potential association of PIEZO2 with nociceptive transmitters. Besides, PIEZO2 has been identified as a potential transducer to contribute to PIEZO2-like inward currents in dental primary afferent neurons [20] and mechanically activated currents of different kinetics in corneal trigeminal neurons [33]. Thus, it is also possible that PIEZO2 excitation could directly lead to subsequent inward currents to cause pulpal pain in pulpitis. By contrast, PIEZO1 exhibited elevated expression in irreversible pulpitis patients with pain duration of more than one week. As pain may come from inflammation, longer pain duration might reflect a longer inflammatory state of the pulp tissues. Combined with the correlation analysis that PIEZO1 expression was positively correlated with inflammatory mediators, IL-1β, TNF-α, and IL-6, it is speculated that PIEZO1 might play a vital role in the development of pulpal inflammation.

We also noticed that although PIEZO2 was found associated with pain in irreversible pulpitis, pain does not normally occur in normal pulp with higher expression of PIEZO2. This might be attributable to the functional activation of PIEZO2 under pathological conditions, which has indeed been observed previously. In dehydration, PIEZO2 was downregulated in kidney while PIEZO2 was significantly activated in juxtaglomerular renin-producing cells [34]. Besides, PIEZO2 has been demonstrated to be excited in nociceptive neurons under inflammatory conditions, although it was also highly expressed under normal conditions [35]. Evidence has shown that PIEZO2 mechanosensitivity could be potentiated by the cAMP pathway in inflammation [36]. Intriguingly, the quiescence of PIEZO2 was also observed in dental primary afferent upon mechanical stimulation under non-inflammatory conditions, by which approximately 43% of the PIEZO2-positive did not exhibit PIEZO2-like currents [20]. Hence, PIEZO2 might be functionally activated and mediate pain in irreversible pulpitis, but it requires further confirmation.

As the potential role of PIEZOs and their associations with inflammation and pain were indicated in this study, PIEZOs might serve as potential targets for irreversible pulpitis. Notably, the therapeutical effect of the non-specific inhibitor of PIEZO1, Gsmtx4, has been reported to ameliorate osteoarthritis in the animal model [37]. Gsmtx4 is also an inhibitor for PIEZO2 and could reduce adenosine 5'-triphosphate-induced Ca2+ influx in neuron tissues in-vitro, which indicated its therapeutic potential in mechanical allodynia [38]. Hence, the pharmacological inhibitors targeting PIEZOs may hold promise for novel therapies for irreversible pulpitis. However, small molecule inhibitors that selectively inhibit PIEZO1 or PIEZO2 are still in their infancy, and more in-depth studies and a clear theoretical basis to evaluate their safety are needed before the clinical trials.

Conclusions

This study depicted the expression profile of mechanosensitive ion channel PIEZOs in pulp tissues from patients with irreversible pulpitis. The results identified the upregulation of PIEZO1 and the downregulation of PIEZO2. Furthermore, we verified the positive correlations of the mRNA expression levels between PIEZO1 and inflammatory mediators, IL-1β, TNF-α, and IL-6, as well as between PIEZO2 and pain markers, NPY and TAC1, in inflamed pulp tissues. We also demonstrated that PIEZO2 expression was positively correlated with pain levels. Besides, a higher expression level of PIEZO2 was found in the inflamed pulp tissues from the patients who described continuous pain and who responded to cold stimulus, while a higher expression level of PIEZO1 was found in patients with pain duration of more than one week. In summary, our findings provided new insights into the understanding of irreversible pulpitis pathogenesis, and future studies on PIEZOs and irreversible pulpitis might be helpful in leading to novel therapeutic approaches to relieve inflammation and pain in irreversible pulpitis.

Availability of data and materials

The data and materials that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

IL:

Interleukin

TNF:

Tumor necrosis factor

NPY:

Neuropeptide Y

TAC1:

Tachykinin precursor 1

STROBE:

The Strengthening the Reporting of Observational Studies in Epidemiology

PCR:

Polymerase Chain Reaction

GEO:

Gene Expression Omnibus

H&E:

Hematoxylin and eosin

S.E.M:

Standard error of mean

CALCA:

Calcitonin related polypeptide alpha

CALCB:

Calcitonin related polypeptide beta

TAC1:

Tachykinin precursor 1

LTM:

Low-threshold mechanoreceptor

References

  1. Levin LG, Law AS, Holland GR, Abbott PV, Roda RS. Identify and define all diagnostic terms for pulpal health and disease states. J Endod. 2009;35(12):1645–57.

    Article  PubMed  Google Scholar 

  2. Nosrat A, Dianat O, Verma P, Nixdorf DR, Law AS. Postoperative pain: an analysis on evolution of research in half-century. J Endod. 2021;47(3):358–65.

    Article  PubMed  Google Scholar 

  3. Abbott PV. Present status and future directions: managing endodontic emergencies. Int Endod J. 2022;55(Suppl 3):778–803.

    Article  PubMed  Google Scholar 

  4. Aminoshariae A, Kulild JC. Current concepts of dentinal hypersensitivity. J Endod. 2021;47(11):1696–702.

    Article  PubMed  Google Scholar 

  5. Agnihotry A, Thompson W, Fedorowicz Z, van Zuuren EJ, Sprakel J. Antibiotic use for irreversible pulpitis. Cochrane Database Syst Rev. 2019;5(5):Cd004969.

    PubMed  Google Scholar 

  6. Ribeiro-Santos FR, Arnez MFM, de Carvalho MS, da Silva RAB, Politi MPL, de Queiroz AM, et al. Effect of non-steroidal anti-inflammatory drugs on pulpal and periapical inflammation induced by lipopolysaccharide. Clin Oral Investig. 2021;25(11):6201–9.

    Article  PubMed  Google Scholar 

  7. Nogueira BML, Silva LG, Mesquita CRM, Menezes SAF, Menezes TOA, Faria AGM, et al. Is the use of dexamethasone effective in controlling pain associated with symptomatic irreversible pulpitis? A systematic review. J Endod. 2018;44(5):703–10.

    Article  PubMed  Google Scholar 

  8. Parirokh M, Abbott PV. Present status and future directions-mechanisms and management of local anaesthetic failures. Int Endod J. 2022;55(Suppl 4):951–94.

    Article  PubMed  Google Scholar 

  9. Afrah AA, Hamid HE, Tahrir NA, Khalil AM. Tumors of craniofacial region in Iraq (clinicopathological study). J Res Med Dent Sci. 2021;9(1):66–71.

    Google Scholar 

  10. Aldelaimi TN, Khalil AA. Clinical application of diode laser (980 nm) in maxillofacial surgical procedures. J Craniofac Surg. 2015;26(4):1220–3.

    Article  PubMed  Google Scholar 

  11. Kefauver JM, Ward AB, Patapoutian A. Discoveries in structure and physiology of mechanically activated ion channels. Nature. 2020;587(7835):567–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lee W, Nims RJ, Savadipour A, Zhang Q, Leddy HA, Liu F, et al. Inflammatory signaling sensitizes Piezo1 mechanotransduction in articular chondrocytes as a pathogenic feed-forward mechanism in osteoarthritis. Proc Natl Acad Sci U S A. 2021;118(13):e2001611118.

  13. Atcha H, Jairaman A, Holt JR, Meli VS, Nagalla RR, Veerasubramanian PK, et al. Mechanically activated ion channel Piezo1 modulates macrophage polarization and stiffness sensing. Nat Commun. 2021;12(1):3256.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Swain SM, Romac JM, Shahid RA, Pandol SJ, Liedtke W, Vigna SR, et al. TRPV4 channel opening mediates pressure-induced pancreatitis initiated by Piezo1 activation. J Clin Invest. 2020;130(5):2527–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Szczot M, Liljencrantz J, Ghitani N, Barik A, Lam R, Thompson JH, et al. PIEZO2 mediates injury-induced tactile pain in mice and humans. Sci Transl Med. 2018;10(462):eaat9892.

  16. Murthy SE, Loud MC, Daou I, Marshall KL, Schwaller F, Kühnemund J, et al. The mechanosensitive ion channel Piezo2 mediates sensitivity to mechanical pain in mice. Sci Transl Med. 2018;10(462):eaat9897.

  17. Xie Z, Feng J, Hibberd TJ, Chen BN, Zhao Y, Zang K, et al. Piezo2 channels expressed by colon-innervating TRPV1-lineage neurons mediate visceral mechanical hypersensitivity. Neuron. 2023;111(4):526-38.e4.

    Article  CAS  PubMed  Google Scholar 

  18. Matsunaga M, Kimura M, Ouchi T, Nakamura T, Ohyama S, Ando M, et al. Mechanical stimulation-induced calcium signaling by Piezo1 channel activation in human odontoblast reduces dentin mineralization. Front Physiol. 2021;12:704518.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Ohyama S, Ouchi T, Kimura M, Kurashima R, Yasumatsu K, Nishida D, et al. Piezo1-pannexin-1-P2X(3) axis in odontoblasts and neurons mediates sensory transduction in dentinal sensitivity. Front Physiol. 2022;13:891759.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Won J, Vang H, Lee PR, Kim YH, Kim HW, Kang Y, et al. Piezo2 expression in mechanosensitive dental primary afferent neurons. J Dent Res. 2017;96(8):931–7.

    Article  CAS  PubMed  Google Scholar 

  21. Ghaferi AA, Schwartz TA, Pawlik TM. STROBE reporting guidelines for observational studies. JAMA Surg. 2021;156(6):577–8.

    Article  PubMed  Google Scholar 

  22. Galicia JC, Henson BR, Parker JS, Khan AA. Gene expression profile of pulpitis. Genes Immun. 2016;17(4):239–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Erdogan O, Malek M, Gibbs JL. Associations between pain severity, clinical findings, and endodontic disease: a cross-sectional study. J Endod. 2021;47(9):1376–82.

    Article  PubMed  Google Scholar 

  24. Schober P, Boer C, Schwarte LA. Correlation coefficients: appropriate use and interpretation. Anesth Analg. 2018;126(5):1763–8.

    Article  PubMed  Google Scholar 

  25. Galler KM, Weber M, Korkmaz Y, Widbiller M, Feuerer M. Inflammatory response mechanisms of the dentine-pulp complex and the periapical tissues. Int J Mol Sci. 2021;22(3):1480.

  26. Li X, Han L, Nookaew I, Mannen E, Silva MJ, Almeida M, et al. Stimulation of Piezo1 by mechanical signals promotes bone anabolism. eLife. 2019;8:e49631.

  27. Zhang Y, Jiang L, Huang T, Lu D, Song Y, Wang L, et al. Mechanosensitive cation channel Piezo1 contributes to ventilator-induced lung injury by activating RhoA/ROCK1 in rats. Respir Res. 2021;22(1):250.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Tian S, Cai Z, Sen P, van Uden D, van de Kamp E, Thuillet R, et al. Loss of lung microvascular endothelial Piezo2 expression impairs NO synthesis, induces EndMT, and is associated with pulmonary hypertension. Am J Physiol Heart Circ Physiol. 2022;323(5):H958–74.

    Article  CAS  PubMed  Google Scholar 

  29. Solis AG, Bielecki P, Steach HR, Sharma L, Harman CCD, Yun S, et al. Mechanosensation of cyclical force by PIEZO1 is essential for innate immunity. Nature. 2019;573(7772):69–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wang S, Wang B, Shi Y, Möller T, Stegmeyer RI, Strilic B, et al. Mechanosensation by endothelial PIEZO1 is required for leukocyte diapedesis. Blood. 2022;140(3):171–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ranade SS, Woo SH, Dubin AE, Moshourab RA, Wetzel C, Petrus M, et al. Piezo2 is the major transducer of mechanical forces for touch sensation in mice. Nature. 2014;516(7529):121–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Nikolenko VN, Shelomentseva EM, Tsvetkova MM, Abdeeva EI, Giller DB, Babayeva JV, et al. Nociceptors: their role in body’s defenses, tissue specific variations and anatomical update. J Pain Res. 2022;15:867–77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Fernández-Trillo J, Florez-Paz D, Íñigo-Portugués A, González-González O, Del Campo AG, González A, et al. Piezo2 mediates low-threshold mechanically evoked pain in the cornea. J Neurosci. 2020;40(47):8976–93.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Mochida Y, Ochiai K, Nagase T, Nonomura K, Akimoto Y, Fukuhara H, et al. Piezo2 expression and its alteration by mechanical forces in mouse mesangial cells and renin-producing cells. Sci Rep. 2022;12(1):4197.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Prato V, Taberner FJ, Hockley JRF, Callejo G, Arcourt A, Tazir B, et al. Functional and molecular characterization of mechanoinsensitive “silent” nociceptors. Cell Rep. 2017;21(11):3102–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Szczot M, Nickolls AR, Lam RM, Chesler AT. The form and function of PIEZO2. Annu Rev Biochem. 2021;90:507–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ren X, Zhuang H, Li B, Jiang F, Zhang Y, Zhou P. Gsmtx4 alleviated osteoarthritis through Piezo1/calcineurin/NFAT1 signaling Axis under excessive mechanical strain. Int J Mol Sci. 2023;24(4):4022.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Luo Z, Liao X, Luo L, Fan Q, Zhang X, Guo Y, et al. Extracellular ATP and cAMP signaling promote Piezo2-dependent mechanical allodynia after trigeminal nerve compression injury. J Neurochem. 2022;160(3):376–91.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

The work was supported by National Natural Science Foundation of China [grant number 52171075]; Science and Technology Commission of Shanghai Municipality [grant number 21DZ2294700, 21ZR1439400, 201409006300, 20YF1424800]; Clinical Research Plane of Shanghai Hospital Development Center [grant number 2020CR5015]; Opening Project of Shanghai Key Laboratory of Orthopaedic Implant [grant number KFKT2021001]; Incubating Program for National Program of Renji Hospital, School of Medicine, Shanghai Jiao Tong University [grant number RJTJ23-PY-052].

Author information

Author notes

  1. Wenying Yang and Lu Lin contributed equally to this work.

Authors and Affiliations

  1. Department of Stomatology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, 160 Pujian Road, Shanghai, 200127, China

    Wenying Yang, Lu Lin, Shucheng Hu, Bin Jiang, Ruhan Yang, Weijun Yu, Jiaqi Tang, Dan Zhao, Yuting Gu, Min Jin & Eryi Lu

  2. Department of Ophthalmology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, 160 Pujian Road, Shanghai, 200127, China

    Jin Li

Authors
  1. Wenying Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lu Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shucheng Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bin Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ruhan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Weijun Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jiaqi Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dan Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yuting Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Min Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Eryi Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Wenying Yang and Lu Lin contributed equally to this study. Conceptualization: Wenying Yang, Lu Lin, Yuting Gu, Min Jin, Jin Li, and Eryi Lu; Data curation: Wenying Yang, Lu Lin, and Dan Zhao; Methodology: Wenying Yang, and Lu Lin; Validation: Wenying Yang, and Lu Lin; Visualization: Wenying Yang, and Lu Lin; Writing—original draft: Wenying Yang, and Lu Lin; Formal analysis: Shucheng Hu, Bin Jiang, Ruhan Yang, and Weijun Yu; Investigation: Wenying Yang, Lu Lin, Shucheng Hu, Bin Jiang, Ruhan Yang, and Weijun Yu; Resources: Shucheng Hu, Bin Jiang, Ruhan Yang, Weijun Yu, and Jiaqi Tang; Software: Dan Zhao; Funding acquisition: Lu Lin and Eryi Lu; Project administration: Yuting Gu, Min Jin, Jin Li, and Eryi Lu; Supervision: Yuting Gu, Min Jin, Jin Li, and Eryi Lu; Writing—review & editing: Wenying Yang, Lu Lin, Shucheng Hu, Bin Jiang, Ruhan Yang, Weijun Yu, Jiaqi Tang, Dan Zhao, Yuting Gu, Min Jin, Jin Li, and Eryi Lu. All authors read and approved the final version of the manuscript and agreed to be accountable for all aspects of the work.

Corresponding authors

Correspondence to Yuting Gu, Min Jin, Jin Li or Eryi Lu.

Ethics declarations

Ethics approval and consent to participate

The experiment was approved by Ethics Committee of Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (LY2023-197-B). The study was performed according to Helsinki Declaration. Informed consent was obtained from all individual participants included in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, W., Lin, L., Hu, S. et al. Expression patterns of mechanosensitive ion channel PIEZOs in irreversible pulpitis. BMC Oral Health 24, 465 (2024). https://doi.org/10.1186/s12903-024-04209-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12903-024-04209-6

Keywords

BMC Oral Health

ISSN: 1472-6831

Contact us