Trigeminal nerve root compression induced neuroinflammatory response promotes mechanical allodynia through the CGRP/SP-Piezo2 axis via Ca²⁺ signaling

Abstract

Trigeminal neuralgia (TN) is one of the most severe types of neuropathic pain, but its pathological mechanisms remain unknown. In this study, we identified a unique neuroinflammatory response induced by chronic compression of trigeminal root entry zone (TREZ) in a TN rat model, establishing a connection between ATP-driven intracellular pathways and Piezo2-mediated mechanotransduction. Piezo2, the pain-related neuropeptide calcitonin gene-related peptide (CGRP) receptor complex CRLR-RAMP1 and the neuropeptide substance-P (SP) receptor NK1R are co-expressed on rat Merkel cells. Protein kinase C (PKC) plays a crucial role in upregulating Piezo2 and CGRP/SP expression in both the trigeminal ganglion (TG) and whisker pad, thereby facilitating orofacial mechanical allodynia in TN rats. Furthermore, the inhibition of cAMP signaling in the whisker pads effectively alleviated mechanical allodynia, while Piezo2 knockdown in both the TG and whisker pads significantly reversed db cAMP-induced allodynia. In vitro studies demonstrated that extracellular ATP not only enhances CGRP and SP expression but also induces Piezo2 expression through Ca²⁺-dependent activation of ERK1/2 and p38 MAPK cascades, mediated by specific transcription factors. These findings reveal that peripheral sensitization in TN is mediated through a Ca²⁺-CGRP/SP-Piezo2 positive feedback loop, dependent on the neuroinflammatory response along the TG neuron–Merkel cell axis as a prerequisite condition. This discovery provides a novel insight into the pathogenesis of TN.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s11658-025-00831-6.

Introduction

Trigeminal neuralgia (TN) is a type of severe neuropathic pain that is characterized by paroxysmal pain in the orofacial area and is triggered by light touches or other forms of mechanical stimulation at a specific trigger zone on the face [1, 2]. Although microvascular compression of the trigeminal root entry zone (TREZ) is a common etiology among most primary TN patients, the pathogenesis of TN remains unknown [3]. Currently, abnormal sensitivity to mechanical stimuli is considered a key characteristic of TN. The two main treatment methods for TN are surgery and a group of sodium channel inhibitors, such as carbamazepine [4, 5]. However, neither of these methods can be considered fully satisfactory. Emerging evidence indicates that neuroinflammation, characterized by glial activation and the release of neuropeptides and cytokines in the trigeminal ganglion (TG), contributes to the persistence of TN. However, its interaction with mechanical allodynia remains to be elucidated [6, 7].

Recent studies on mechanotransduction have identified Piezo2, a mechanosensitive ion channel that plays a crucial role in the sensation of touch and proprioception [8], as a treatment candidate for attenuating mechanical allodynia after nerve injury. Animals harboring Piezo2 mutation or Piezo2 knockout models exhibit abnormal touch perception and reduced mechanical allodynia following nerve injury [9, 10]. However, the intracellular molecular mechanisms of homeostatic changes in neuronal mechanosensitivity after nerve injury remain poorly understood, but they potentially involve intracellular calcium (Ca²⁺) signaling, which regulates Piezo2 transcription or cellular trafficking. Additionally, activation of Piezo2 also induces depolarization and Ca²⁺ influx, which may promote Ca²⁺-dependent signaling in addition to action potential (AP) transduction [8]. Thus, dysfunction of Ca²⁺-dependent pathways may underline the hyperexcitability of Piezo2 associated with mechanical allodynia.

Notably, Ca²⁺ signaling serves as a crucial regulator of neuroinflammatory processes [11, 12]. Previous studies from our lab have demonstrated that chronic compression of the trigeminal nerve root (CCT) in rat models induces the upregulation of Iba1, GFAP, and a series of cytokines [13, 14]. Specifically, neuronal Ca²⁺-dependent signaling plays a significant role in neurogenic inflammation, mediating the synthesis and release of neuropeptides and neurotrophic factors, including calcitonin gene-related peptide (CGRP) and substance-P (SP) [7, 15]. This dual regulatory function establishes Ca²⁺ as a potential molecular bridge between Piezo2-mediated mechanotransduction and neuroinflammation in TN. Furthermore, cAMP signaling has been demonstrated to induce hyperalgesia in primary afferent nociceptive neurons during inflammatory conditions [16–19]. Nevertheless, the details of cAMP- and Ca²⁺-dependent signaling pathways involved in the regulation of Piezo2-mediated mechanical allodynia remain unclear. Damaged axons and myelin sheaths release adenosine 5'-triphosphate (ATP) into the microenvironment during nerve injury [20, 21]. In the extracellular space, ATP acts as a transmitter coupled with purinergic P2 receptors (P2Rs), which play important roles in neuropathic pain [22]. P2Xs belong to the family of nonselective cationic permeable ligand-gated ion channels, and their permeability to Ca²⁺ is greater than that to Na⁺ and K⁺ ions [23]. The ionotropic P2X3, P2X4, and P2X7 subtypes, as well as the P2Y6 receptor, which is a G-coupled protein receptor, play critical roles in the sensitization of primary sensory afferents [24]. Moreover, several subtypes of metabotropic P2Y receptors reportedly mediate the Ca²⁺-PLC‒PKC axis and cAMP signaling pathway, which ultimately regulate gene expression and facilitate the release of neurotransmitters in nociceptive afferents [25].The downstream events of PKC signaling are closely related to the expression and release of CGRP and SP [26–28]. CGRP and SP play key roles in central sensitization to pain in the dorsal horn of the spinal cord and caudal spinal trigeminal nucleus caudalis (Sp5C) [29]. These proteins are also crucial in the neurogenic inflammation caused by skin injury [30]. However, the precise mechanism by which ATP mediates neuroinflammation through the release of CGRP and SP in innervated regions remains elusive, potentially representing a novel therapeutic target for TN management.

Previously, we revealed that extracellular ATP could increase mechanosensitivity and Piezo2 expression; it also induces dramatic Ca²⁺ influx into TG neurons, and this is thought to be a potential mechanism that regulates Piezo2 expression [31]. In this study, we demonstrated that the PKC signaling pathway plays a critical role in Piezo2-dependent mechanical allodynia and the regulation of CGRP/SP expression following trigeminal nerve root injury. We further confirmed a potential mechanism by which CGRP/SP reinforces the cAMP pathway in Merkel cells located in the whisker pads of TN rats. Moreover, we verified that extracellular ATP initiates the Ca²⁺ signaling pathway and upregulates Piezo2 and CGRP/SP expression, as well as Piezo2-dependent mechanical sensitivity in primary TG neurons. By targeting the dysregulated TG neuron–Merkel cell axis, we aim to elucidate a unified mechanism that integrates mechanotransduction with neuroinflammation, thereby identifying a potential Ca²⁺-PKC mediated therapeutic target for TN treatment.

Methods

Animals

Adult male Sprague–Dawley rats (160 ± 20 g; RGD Cat# 13525002) were obtained from the Experimental Animal Center of Fujian Medical University. The animals were housed under a 12:12 h light:dark cycle at constant room temperature (22 ± 2 °C), with food and water provided ad libitum. All animal experimental procedures were approved by the Animal Care and Use Committee of Fujian Medical University (Approval number: IACUC FJMU 2022-Y-0543) on June 6th, 2022. All the experiments followed the guidelines of the International Association for the Study of Pain (IASP). Given that experimental animals exhibit significant sex-based differences in drug sensitivity and stimulus responses, we exclusively used male rats in this study to eliminate potential variability associated with the female estrous cycle.

Establishment of TN animal model and shRNA injection

The rats were randomly divided into five groups (table of random digits), with 46 rats included in the final analysis: the TN group (n = 12), sham group (n = 12), TN + LV-Piezo2 shRNA-skin group (n = 5), TN + LV-scramble shRNA-skin group (n = 5), sham + LV-Piezo2 shRNA-TG-skin group (n = 6), and TN + Go6983-TG group (n = 6). The researchers were blinded to the treatment during the behavioral tests and biochemical assessments.

A modified animal model of TN induced by chronic compression of the trigeminal nerve root (CCT) was established following our previously described methods [32]. The rats of TN group were anesthetized with sodium pentobarbital (40 mg/kg, _i.p.), and an anterior–posterior curved skin incision was made above the right eye to expose the right infraorbital nerve traveling through the infraorbital groove. A homemade, curved, hollow, metal conduit was slowly pushed into the intracalvarium from the inferior orbital fissure to reach the right TREZ, and a 0.4 mm diameter nylon wire was inserted along the metal conduit to compress the TREZ. In the sham group, the right infraorbital nerve was exposed, but the trigeminal nerve was not subjected to nylon wire compression.

shRNA injection and compounds administration

shRNAs targeting murine Piezo2 (GenBank accession: XM_225880.10; 5′-CAATCTTCACTG CTGGGCACCTGAT-3′; HANBIO # HBLV-r-Piezo2 shRNA2-ZsGreen-PURO, Shanghai, China) and a scrambled shRNA (5′-TTCTCCGAACGTGTCACGT-3′; HANBIO # HBLV-ZsGreen-PURO) were designed and ligated into the lentiviral vector pHBLV driven by the U6 promoter (HANBIO, # hU6-MCS-EF1-ZsGreen-T2A-puromycin). During CCT surgery, lentiviral vectors (2 × 10⁵ TU, 5 µL) were administered into either the TG or orofacial whisker pad via the infraorbital foramen using a precision microinjector (Gaoge, Shanghai, China). The same procedure was used for Go 6983 (100 nM, MCE, Cat# HY-13689) injection.

At 21 days post-operation, db cAMP (1 mM; APExBIO, Cat# B9001, Houston, TX, USA) was administered into the orofacial whiskers in the sham group, sham + LV-Piezo2 shRNA-TG-skin group, and TN + LV-Piezo2 shRNA-skin group. In parallel, SQ22536 (1 mM; MCE Cat# HY-100396, Monmouth Junction, NJ, USA) was injected into TN model animals. Behavioral assessments were performed 24 h after compounds administration.

Measurement of orofacial mechanical allodynia

The orofacial mechanical allodynia behavior tests were performed 3 days before the CCT operation to obtain baseline values (BL) and were repeated on days 7, 14, and 21 postoperation. The animals were allowed to acclimate to the testing environment for 3 days before baseline testing was conducted, and then the animals were placed in homemade metal boxes and allowed to adapt for 30 min before each behavioral test was conducted. To measure the degree of mechanical allodynia, von Frey filaments (Aesthesio, San Jose, CA, USA) were used to evaluate the orofacial mechanical threshold. The testing procedure followed a previously described protocol with some modifications [33]. Briefly, a 0.07 g von Frey filament was used to evaluate orofacial mechanical allodynia in the whisker pad. The behavioral response of the rats was scored as follows [34]: those that did not stir or only looked around received a score of 0; those that responded gently or withdrew their face received a score of 1; those that quickly shielded their face or slightly lifted a paw received a score of 2; those that exhibited swift evasive behavior or lifted a paw and wiped their face received a score of 3; those that wiped their face and lifted a paw received a score of 4.

Tissue preparation and immunofluorescence staining

The TG and orofacial skin tissues on the right side of the rats were harvested after transcardial perfusion with phosphate-buffered 4% (w/v) paraformaldehyde through the left ventricle, and then these tissues were cryoprotected by incubation in a 30% (w/v) sucrose solution in 0.1 M phosphate buffer for 24 h at 4 °C. Serial longitudinal sections were cut at a thickness of 10 μm for TG and 40 μm for orofacial skin using a cryostat microtome (Leica CM1950, Heidelberger, Germany).

After being washed with 0.1 M phosphate-buffered saline (PBS), the sections from each group were blocked with QuickBlock™ Blocking Buffer (Beyotime, Cat# P0260, Shanghai, China) at room temperature for 1 h and then incubated overnight at 4 °C with the following primary antibodies: goat anti-CGRP (1:100; Abcam, Cat# ab36001, RRID:AB_725807, Cambridge, UK), rabbit anti-Substance P (1:1000; Immunostar, Cat# 20064, RRID:AB_10717640, Hudson, WI, USA), rabbit anti-Piezo2 (1:200; Novus, Cat# NBP1-78624, RRID: AB_11005294, Littleton, CO, USA), chicken anti-NEFH (1:5000; Abcam, Cat# ab4680, RRID:AB_30456), mouse anti-Cytokeratin 8 (1:100; Abcam, Cat# ab9023, RRID:AB_306948), rabbit anti-CRLR (1:100; Bioss, Cat# BS-1860R, RRID:AB_10855106, Beijing, China), rabbit anti-RAMP1 (1:100; Abcam, Cat# ab203282, RRID:AB_2924360), and rabbit anti-NK1R (1:200; Sigma, Cat# S8305, RRID:AB_261562, Saint Louis, MO, USA). After being washed with 0.01 M PBS, the sections were further incubated with the following secondary antibodies: goat anti-rabbit Alexa Fluor Plus 555 (1:1000; Invitrogen, Cat# A32732, RRID:AB_2633281, Carlsbad, CA, USA), goat anti-mouse Alexa Fluor Plus 647 (1:1000; Invitrogen, Cat# A32728, RRID:AB_2633277), or goat anti-chicken Alexa Fluor 647 (1:1000; Invitrogen, Cat# A-21449, RRID:AB_2535866) at room temperature for 1 h. DAPI (1:1000, Beyotime, Cat# C1002) was used to stain the nuclei. Fluorescence images were captured with a confocal microscope (Leica TCS SP8, Heidelberger, Germany). Using ImageJ software with background subtraction, we quantified the average fluorescence intensity from 3 to 5 regions of interest (ROIs) corresponding to Merkel cells or TG neurons per image.

RNA sequencing and bioinformatic analysis

The ipsilateral TG and a part of its projection to the TREZ were collected and promptly frozen in liquid nitrogen. These tissues were then subjected to RNA sequencing (RNA-seq) analysis conducted by the Beijing Genomics Institute (BGI). Differentially expressed genes (DEGs) were identified via the DESeq package, with a discriminatory approach to exclude irrelevant genes. DEGs with P < 0.05 were considered significantly different in expression.

DAVID (http://david.abcc.ncifcrf.gov/) was used to perform the gene functional annotation analysis. All the genes of Rattus norvegicus were used as background genes. The KEGG pathway categories were selected as background databases, and visualization was performed via the ggplot package in R software. To further explore the interactions between DEGs, a protein‒protein interaction (PPI) network was constructed via the Search Tool for Interacting Genes (STRING) (http://string-db.org/) and visualized via Cytoscape software.

Primary culture of TG neurons and compound treatment

Primary TG neurons were harvested from the TG of neonatal Sprague‒Dawley rats (1–3 d) as previously described [31]. The isolated neurons were seeded on 29 mm glass-bottomed confocal dishes for immunocytochemistry experiments. Half of the culture medium was replaced every 3 days beginning on day 1 in vitro (DIV 1). At DIV 7, TG neurons from the same preparation were divided into different groups by simple randomization (drew lots), and then different compounds were administered to the primary TG neuron cultures according to their group. In brief, ATP (2 mM; MCE, Cat# HY-B0345A), the calcium ionophore ionomycin (500 nM; MCE, Cat# HY-13434), db cAMP (1 mM; APExBIO, Cat# B9001) or Yoda 1 (10 μM; MCE, Cat# HY-18723) were added to primary TG neuron cultures and incubated for 1 h, after which the medium was replaced with fresh culture medium. For hydrolysates, ADP (500 μM, MCE, Cat# HY-W010918) and adenosine (500 μM, MCE, Cat# HY-B0228) were added to the neuron cultures and incubated for 3 d. To identify signal transduction mechanisms and transcription factors that regulate ATP-induced Piezo2 expression, inhibitors were added and coincubated with ATP for 1 h. After changing the medium, the neurons were treated with inhibitors for 72 h. The inhibitors that were used in this study includes T-5224 (50 μM, APExBIO, Cat# B4664), 666–15 (100 nM, MCE Cat# HY-101120), BI-6015 (1.25 μM, MCE, Cat# HY-108469), YC-1 (10 μM, APExBIO, Cat# B7641), cyclic pifithrin-α hydrobromide (20 μM, APExBIO, Cat# A4477), SGC-CBP30 (100 nM, MCE, Cat# HY-15826), Go 6983 (100 nM, MCE, Cat# HY-13689), H89 (1 μM, APExBIO, Cat# B2190), SP600125 (1 μM, MCE, Cat# HY-12041), SB203580 (10 μM, MCE, Cat# HY-10256A) and U0126 (10 μM, MCE, Cat# HY-12031). Furthermore, α-CGRP (100 nM, MCE, Cat# HY-P0203) and Substance P (1 μM, MCE, Cat# HY-P0201) were added to the neuronal cultures and manitained for 3 days to assess their effects on Piezo2 expression.

RNA extraction and quantitative RT‒PCR analysis

Primary TG neurons were treated with ATP (2 mM/mL) for 1 h followed by culture in fresh medium for an additional 1 h. Total RNA was subsequently extracted with TRIzol reagent (Invitrogen, Cat #155596026). First-strand cDNA was synthesized via the Prime Script™ RT Reagent Kit (Vazyme Biotech, Cat #R222-01, Nanjing, China). Real-time qPCR was performed with SYBR Green PCR Master Mix (Vazyme Biotech, Cat #Q311-02). The primers that were used for qPCR are shown in Supplemental Table 1. The 2^−∆∆Ct method was used to calculate the CGRP and SP expression levels normalized to the GAPDH expression level.

Immunocytochemistry

TG neurons were cultured in glass-bottom cell culture dishes. After compounds treatment, each group of cultured neurons was fixed with 4% PFA at room temperature for 15 min. For the cells designated for Piezo2 antibody incubation, antigen retrieval was performed via citrate buffer. The remaining cells were permeabilized with 0.2% Triton X-100 at room temperature for 5 min. The fixed cells were blocked with 5% (w/v) bovine serum albumin (BSA) (Phygene, Cat# PH0420, Shanghai, China) at room temperature for 30 min and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-Piezo2 (1:250; Novus, Cat# NBP1-78624, RRID: AB_11005294, Littleton, CO, USA), chicken anti-NEFH (1:5000; Abcam, Cat# ab4680), mouse anti-NeuN (1:500; Abcam, Cat# ab104224, RRID:AB_10711040), rabbit anti-NeuN (1:500; Abcam, Cat# ab177487, RRID:AB_2532109), mouse anti-GFAP-cy3 (1:1000; Sigma, Cat# c9205, RRID:AB_476889), rabbit anti-β-III-tubulin (1:100; Servicebio, Cat# GB11139, RRID:AB_2895013), and mouse anti-NSE (1:200; Proteintech, Cat# 66150-1-Ig, RRID:AB_2883272) and mouse anti-c-Fos (1:200; Abcam, Cat# ab208942, RRID: AB_2747772). After being washed with 0.01 M PBS, the cells were further incubated with the following secondary antibodies: goat anti-rabbit Alexa Fluor Plus 555 (1:1000; Invitrogen, Cat# A32732), goat anti-chicken Alexa Fluor 647 (1:1000; Invitrogen, Cat# A-21449), donkey anti-rabbit Alexa Fluor Plus 488 (1:1000; Invitrogen, Cat# A-21206) or goat anti-mouse Alexa Fluor 555 (1:1000; Invitrogen, Cat# A28180) at room temperature for 1 h. DAPI was used to stain the nuclei. Images were captured with a Leica TCS SP8 confocal microscope. Each NEFH-positive neuron was circled as a region of interest (ROI). Then, ImageJ software was used to determine the ROI integration density, and the noncellular background was subtracted in immunocytochemical applications, yielding individual measurement data points per cell.

cAMP level measurement by ELISA

Cultured TG neurons were incubated with ATP (2 mM) for 1 h on DIV 7, and samples from the ATP group and control group were harvested on DIV 10. cAMP activation was measured via a cAMP assay kit (Abcam, Cat# ab138880) according to the manufacturer's instructions. Standard cAMP solutions of different concentrations were prepared and added to a 96-well plate that was provided with the kit to determine the standard curve. Two sets of multiple wells were established to eliminate abnormal readings. The fluorescence of the end product was measured at Ex/Em = 540/590 nm via a Tecan Spark 20 M microplate reader (Tecan Trading AG, Switzerland).

Western blot

TG neurons were cultured until DIV7. They were then coincubated with ATP and a MEK 1/2 inhibitor (U0126) or p38 MAPK inhibitor (SB203580) for 1 h, the medium was changed, and protein extraction was performed after 1 h of culture. For the ipsilateral TG and oral facial tissues from each animal, homogenization was performed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (Meilunbio, Cat# MA0151) containing a protease inhibitor cocktail (APExBIO, Cat# K1015). The protein samples were obtained after the lysate containing the tissue or cells was centrifuged at 12,000 × g at 4 °C for 20 min. The total protein concentration was measured with a BCA kit (Beyotime, Cat# P0010). Protein samples (10 μg) were separated on 10% SDS–PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Merck, Cat# 03010040001). The PVDF membranes were blocked with 5% (w/v) nonfat milk for 1 h at room temperature (24 ± 2 °C) followed by incubation overnight at 4 °C with the following primary antibodies: mouse anti-CGRP (1:500; Abcam, Cat# ab81887, RRID:AB_1658411), rabbit anti-Piezo2 (1:1000), rabbit anti-phospho-ERK1/2 (1:2000; CST, Cat# 4370S, RRID:AB_2315112, Danvers, MA, USA), rabbit anti-ERK1/2 (1:1000; CST, Cat# 4965), rabbit anti-phospho-p38 MAPK (1:1000; CST, Cat# 4511, RRID:AB_2139682), rabbit anti-p38 MAPK (1:1000; CST, Cat# 8690S, RRID:AB_10999090), rabbit anti-GAPDH (1:10000; Bioworld Cat# AP0066, RRID:AB_2797448), or mouse anti-β tubulin (1:10000; Bioworld, Cat# BS1482M, Minneapolis, MN, USA). Next, the PVDF membranes were incubated with a horseradish peroxidase–conjugated goat anti-rabbit IgG (H + L) (1:10000; Bioworld, BS13278, RRID: AB_2773728) or goat anti-mouse IgG (H + L) (1:10000; Bioworld, Cat# BS12478, RRID: AB_2773727) secondary antibody at room temperature for 2 h. The protein bands were visualized with Immobilon Western Chemiluminescent Reagent (Millipore, Cat# 1925902) and a chemiluminescence image analysis system (Tanon, 4600SF, Shanghai, China). ImageJ software was used to analyze and normalize the band intensities.

Shaking experiment of TG neurons

TG neurons were cultured in glass-bottomed confocal dishes (DIV 7) that were tightly sealed with sealing film, and each Petri dish was fixed in a closed container and incubated in a constant-temperature rail setting shaker at 37 °C for the shaking experiment (70 rpm/min). To compare the effects of short-term (1 h) and long-term (3 d) shaking stimuli on the expression of Piezo2, immunocytochemical fluorescence intensities were analyzed. In addition, coadministration of db cAMP with shaking stimulation (70 rpm/min, 1 h) was performed to evaluate the role of cAMP signaling in regulating Piezo2 expression in TG neurons under shaking conditions. The fluorescence intensity of Piezo2 was subsequently measured 72 h later.

Calcium ion functional imaging

TG neurons were cultured in glass-bottomed confocal dishes (DIV 7) and loaded with Fluo-3 AM (5 μM; Invitrogen, Cat# F1241) in culture medium (Neurobasal + 2% (v/v) B27) for 30 min in the dark at 37 °C. After the cells were treated with the inhibitor GsMTx4 (3 μM, MCE, Cat# HY-P1410) for 30 min, they were loaded with Fluo-3 AM. After loading, the cells were washed three times with Tyrode's solution and then incubated in PBS for 30 min to allow for the de-esterification of the cytosolic dye. The excitation and emission wavelengths of Fluo-3 AM are 488 nm and 525 nm, respectively. Fluorescence images were captured under an inverted Nikon C2 confocal microscope and processed with Nikon NIS Elements software. After 5 min of stable baseline recording, glass beads with diameters ranging from 50–100 μm (Polysciences; Cat#15,926) were used for mechanical stimulation. For the Ca²⁺-free Tyrode solution, Ca²⁺ was buffered with 0.5 mmol/L EGTA. [Ca²⁺]_i was calculated via the equation , where K_d is a dissociation constant (K_d = 1100 nM), F is the fluorescence intensity, and F_max is the maximum fluorescence intensity determined in situ in cells superfused with 10 μM 4-bromo A-23187 and 20 mM Ca²⁺. When the fluorescence intensity decreased from the highest point, Mn²⁺ (10 mM) was added to detect the background fluorescence value (F_bg) [35].

Transcription factor array

TG neurons were incubated with ATP (2 mM) for 1 h and then cultured with fresh medium for another hour. Nuclear extracts of TG neurons were prepared using a Nuclear Extraction Kit (Signosis, Cat# SK-0001, Santa Clara, CA, USA), and then the transcription factor (TF) Activation Profiling Plate Array II (Signosis, Cat# FA-1002) was used to analyze 96 TFs simultaneously according to the manufacturer’s protocol.

Signosis TF Activation Profiling Arrays are used to analyze the activities of multiple TFs simultaneously. Briefly, the nuclear extracts were incubated with biotin-labeled probes that were designed based on the consensus sequences of the TF binding sites. The TF‒probe complexes were purified, and then the bound probes were separated from the complexes. The detached probes were hybridized in 96-well plates in which each well was specifically coated with sequences that were complementary to the probes. The bound DNA probes were mixed with horseradish peroxidase (HRP)-streptavidin conjugates, and the luminescence was measured via a microplate luminometer. The luminescence of the control and ATP-treated TG neurons was then compared.

Bioinformatics analysis of TF binding sites

TFs whose binding motifs increased by twofold or more, as shown by a TF array kit, were verified via JASPAR web-based tools. JASPAR is an open-access database of TF binding profiles, and the most recent version (9th release, 2022) was selected. The promoter region of Piezo2 in Rattus norvegicus [NC_051353.1:c56846984-56844485] was selected. The TF-binding sites were scored by constructing position weight matrices (PWMs) or position-specific scoring matrices (PSSMs), which were based on position frequency matrices (PFMs). The four top-scored TFs were selected for verification with inhibitors.

Statistics

The data are presented as the mean ± standard error of the mean (SEM). All the statistical analyses were performed via GraphPad Prism 9 (GraphPad Software, Inc.). Student’s two-tailed t test was used for two-group comparisons, whereas one-way ANOVA or two-way repeated-measures ANOVA was used for multiple comparisons, followed by Bonferroni correction. P < 0.05 was considered to indicate statistical significance.

Results

CCT induced Piezo2 upregulation in Merkel cells colocalized with CGRP and SP receptors

Piezo2 expression in the whisker pads of rats in both the TN group and the sham group was first measured in our study. The immunofluorescence results revealed that Piezo2 was widely distributed on Merkel cells at whisker follicles in vivo. Moreover, CCT injury markedly induced Piezo2 expression in the whisker pads of the TN model rats (Fig. 1). In addition, the CGRP receptor complex CRLR with RAMP1 and the SP receptor NK1R also colocalized with Krt8 on Merkel cells (Fig. 2).

Fig. 1.

The co-localization and expression of Piezo2 on whisker follicle Merkel cells after CCT. a Double immunostaining of Piezo2 (red) and Krt8 (green) in the whisker follicles of sham and TN model animals. Plot analysis was performed on the white line segment in (a), and the numbers 1–2 with a dashed line indicate that the plot analysis is 1 to 2. Scale bar = 50 μm. b Plot analysis revealed good colocalization of Krt8 and Piezo2 in whisker hair follicles, which confirmed the expression of Piezo2 on Merkel cells. c The histogram shows the fluorescence intensity of Piezo2 increased after the CCT operation (mean ± SEM, n indicates the number of Merkel cells in the independent tissue section from at least three animals; ***P < 0.001; unpaired t test)

Fig. 2.

Colocalization of CGRP receptors and NK1R on whisker follicle Merkel cells. a Double immunostaining was performed to detect the expression of CRLR (red)/Krt8 (green) and RAMP1 (red)/Krt8 (green) in the whisker pads of naïve animals. Plot analysis was performed on the white line segment in (a), and the numbers 1–2 with a dashed line indicate that the plot analysis is 1 to 2. b Plot analysis indicated that CRLR and RAMP1 colocalized well with Krt8. c Double immunostaining of the SP receptors NK1R (red) and Krt8 (green) in the whisker pads of naïve animals. d NK1R colocalized well with Krt8 (green). Scale bar = 50 μm

CCT increased Piezo2, CGRP and SP in the TG and whisker pads by initiating PKC signaling and promoting Piezo2-dependent allodynia via cAMP signaling

The expression levels of CGRP and SP were measured in the whisker pads and TG of both groups (Fig. 3a–b). On the whisker follicle side, the fluorescence intensities of CGRP and SP were significantly greater in the TN group than in the sham group after CCT injury and colocalized well with Merkel cells (Fig. 3c). On the TG side, there were more CGRP- and SP-positive neurons in the TN group than in the sham group, as did the intensity of CGRP and SP fluorescence (Fig. 3b, d).

Fig. 3.

PKC signaling initiates Piezo2-dependent allodynia by activating the CGRP/SP intracellular pathway. a Representative images of triple immunostaining for CGRP (red), SP (green) and DAPI (blue) in the whisker pads of TN and sham animals after the CCT operation. b Representative images of triple immunostaining for CGRP (red), SP (green) and DAPI (blue) in the TG of TN and sham animals after the CCT operation. Scale bar = 50 μm. c–d The results of fluorescence intensity analysis showed that the expression of CGRP and SP both increased at 21 days after the CCT operation in the TG and whisker pads (mean ± SEM, n indicates the number of Merkel cells or TG neurons in the independent tissue section from at least three animals; **P < 0.01, ***P < 0.001; unpaired t test). e–h Western blot analysis of Piezo2 and CGRP in the sham group, TN group and TN + Go6983 group TG and whisker pad tissues at 21 days after CCT operation. The relative expression levels of Piezo2 and CGRP in the different groups were normalized to those of GAPDH. Injection of Go6983 into TG significantly attenuated the expression of CGRP and Piezo2 in both the TG and whisker pads at 21 days after CCT operation (mean ± SEM, n = 4; n indicates the number of independent animals; *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant difference; one-way ANOVA). i Measurement of allodynia grade in the three groups after the operation. The allodynia grade was significantly greater in the TN group than in the sham group at PODs 14 and 21, and Go6983 attenuated the effect of the CCT operation (TN or TN + Go6983 vs. Sham; mean ± SEM, n = 4–5; n indicates the number of independent animals; **P < 0.01, ns, no significant difference; two-way ANOVA). j The severity of allodynia was increased after CCT. Subcutaneous injection of LV-scramble shRNA into the whisker pad had no significant effect on allodynia severity after the CCT operation (TN vs. TN + LV-scramble shRNA; mean ± SEM, n = 5, n indicates the number of independent animals; **P < 0.01, ***P < 0.001, ns, no significant difference; two-way ANOVA). CCT-induced mechanical allodynia was markedly attenuated 14 d after CCT surgery when LV-Piezo2 shRNA was subcutaneously injected into whisker pads (TN + LV-scramble shRNA vs. TN + LV-Piezo2 shRNA-skin; mean ± SEM, n = 5, n indicates the number of independent animals; ns, no significant difference, **P < 0.01, **P < 0.001; two-way ANOVA). k Administration of SQ22536 into the whisker pads of TN model animals produced significant alleviation of mechanical allodynia at 24 h post-injection (mean ± SEM, n = 5, n indicates the number of independent animals; ***P < 0.001; unpaired t test). Intra-whisker pad injection of dbcAMP still significantly increased allodynia severity 24 h post-injection in Piezo2-knockdown animals (mean ± SEM, n = 5, n indicates the number of independent animals; ***P < 0.001; unpaired t test). l Coadministration of LV-Piezo2 shRNA into the TG and whisker pad had no significant effect on allodynia severity compared with that in the sham group (mean ± SEM, n = 6–9; n indicates the number of independent animals; ns, no significant difference; two-way ANOVA). m Intra-whisker pad injection of PBS (vehicle control) did not significantly affect mechanical allodynia severity when assessed 24 h post-injection (mean ± SEM, n = 4, n indicates the number of independent animals; ns, no significant difference; unpaired t test). Intra-whisker pad injection of db cAMP significantly induced mechanical allodynia at 24 h post-injection in sham-operated animals (mean ± SEM, n = 5, n indicates the number of independent animals; ***P < 0.001; unpaired t test). At 24 h after intra-whisker pad injection of db cAMP, sham-operated animals with concurrent LV-Piezo2 shRNA-mediated knockdown in both TG and whisker pad innervation zones exhibited no significant development of mechanical allodynia (mean ± SEM, n = 5; n indicates the number of independent animals; ns, no significant difference; unpaired t test)

The semiquantitative and immunostaining results revealed that intervention with the PKC signaling inhibitor Go6983 in the TG suppressed the expression of not only CGRP and SP (Fig. 3e, g and S1a, c, e, g) but also Piezo2 (Fig. 3f, h and S1b, d, f, h) in both the TG and whisker follicles. Behavior tests of orofacial mechanical allodynia also revealed that the PKC inhibitor significantly alleviated the allodynia grade after the CCT operation on PODs 14 and 21 (Fig. 3i).

To investigate whether the potential role of CGRP/SP in neuropathic pain was related to Piezo2, Piezo2 was then knocked down by injecting lentivirus (LV-shRNA Piezo2) into the whisker pad. The effects of knockdown by shRNA were validated in a previous publication in which the shRNA was injected into the TG [31]. Here, by injecting LV-Piezo2 shRNA or LV-scramble shRNA into the TG and whisker pad of rats, we revalidated the effectiveness of knockdown using western blotting or immunofluorescence staining, and the results revealed that LV-Piezo2 shRNA significantly inhibited the expression level of Piezo2 (Figure S2). Behavior tests revealed that knockdown of Piezo2 in whisker pads by LV-Piezo2 shRNA significantly ameliorated allodynia after the CCT operation on PODs 14 and 21 (Fig. 3j), and previous results indicated that both Piezo2 in Merkel cells and the TG were critical for the initiation of allodynia. We next analyzed the effect of cAMP on allodynia in TN rats. SQ22536, which is an inhibitor of adenylyl cyclase, was administered to the whisker pads of TN model animals, after which the von Frey test was conducted. The results showed that SQ22536 significantly reduced the severity of allodynia in TN model rats. However, an analog of cAMP, db cAMP, increased the severity of allodynia in the rats in the TN + LV-Piezo2 shRNA-skin group (Fig. 3k). Additionally, compared with sham treatment, Piezo2 knockdown in both the TG and whisker pads of sham rats resulted in no significant difference in the severity of allodynia (Fig. 3l). Moreover, db cAMP induced significant allodynia in the sham group, but it had no significant effect on mechanical allodynia in sham animals in which Piezo2 was knocked down in either the TG or whisker pads (Fig. 3m).

CGRP and SP are synthesized primarily by TG sensory neurons and are released into the innervated areas of the TG, where they may modulate Piezo2 on Merkel cells. Additionally, TG neurons themselves express Piezo2, which can be transported to the Aβ nerve endings that innervate Merkel cells [36]. Therefore, we believe that elucidating the intracellular mechanisms of TG neurons following CCT injury is key to understanding peripheral sensitization in TN.

Extracellular ATP is involved in calcium signaling, and MAPK signaling upregulates CGRP and SP in TG neurons

A defining characteristic of TN is the proximity of injury sites to TG. The CCT model effectively recapitulates this clinical feature. Consequently, injury-induced alterations in extracellular signaling molecules surrounding TG neurons may represent a key mechanism underlying TN-associated peripheral sensitization.

The bioinformatic methods for TN and sham rat TG-TREZ tissue RNA-seq results could predict the intracellular process after CCT injury, thereby facilitating the identification of potential extracellular signaling molecules involved. The DEGs identified via RNA-seq were significantly enriched in the MAPK signaling pathway, PKC pathway and Calcium signaling pathway (Fig. 4a). P2rx2, P2rx4 and P2rx6 are enriched in the calcium signaling pathway, which is consistent with the results of previous studies [31]. Additionally, the P2Y family (P2ry12, P2ry6 and P2ry2) was enriched mainly in efferocytosis and other inflammatory pathways.

Fig. 4.

CCT induced the expression of CGRP and SP in the TG of the TN model rats. a DEGs identified via RNA-seq were significantly enriched in nerve growth, metabolism, cytophagocytosis, pain sensitization and other related pathways, among which the MAPK pathway may be significantly related to TN (P = 2.85E-13, P < 0.0001). b Protein‒protein interaction network of the differentially expressed genes. c RT‒qPCR results showing that ATP significantly upregulated the mRNA expression levels of CGRP and SP in TG neurons. (mean ± SEM, n = 3; n indicates the number of independent cell culture preparations; **P < 0.01, ***P < 0.001; unpaired t test)

Protein interaction analysis revealed that three P2 receptors (P2ry1, P2rx4 and P2rx6) interact with Plcb2 and Ngf in the PKC/MAPK pathway. Specifically, the P2X family may participate in the PKC pathway through Plcb2 and mediate the MAPK pathway through Ngf, and receptor tyrosine kinases link Plcb2 and Ngf, thereby supporting P2X through PKC/MAPK-mediated pain sensitization (Fig. 4b).

The administration of ATP successfully upregulated the expression of CGRP and SP in primary TG neurons (Fig. 4c). ATP could also increase the expression level of Piezo2 and was revealed to be involved in the calcium signaling pathway. We investigated whether Ca²⁺-PKC-MAPK signaling is the key to the intracellular mechanism of ATP-induced Piezo2 and CGRP/SP upregulation in the TG. The involvement of PKC in the regulation of CGRP/SP has been revealed relatively clearly, and we focused on the intracellular mechanism regulating Piezo2.

Extracellular ATP induced Piezo2 expression and Piezo2-dependent Ca²⁺ influx in TG neurons

Given that several types of neurons and nonneuronal cells exist in the TG, we first identified whether the cell types were harvested from rat TG tissues via multi-immune staining. β-III-Tubulin, neurofilament heavy chain (NEFH), neuron-specific enolase (NSE) and neuronal nuclei (NeuN) antibodies were used to detect neurons, a glial fibrillary acidic protein (GFAP) antibody was used to detect satellite cells, and DAPI was used for all the cells in the culture. The results revealed that most of the cells in the TG cell culture were neurons, and very few cells were labeled with a GFAP antibody. We found that NEFH stains large neurons with typical pseudounipolar neuron morphology especially well. The NSE-positive, NeuN-positive and NEFH-positive signals were strongly colocalized on the soma of the neurons. Most of the neurons were smaller and morphologically similar to bipolar neurons, and NEFH fluorescence was evident only in the soma (Figure S3). To avoid species differences in subsequent experimental antibodies, NEFH was selected as the neuronal marker for subsequent experiments.

To assess the effect of extracellular ATP on Piezo2 expression, a series of immunostaining assays were performed on cultured TG neurons that were treated with ATP for different durations in vitro (Fig. 5a). ATP (2 mM) was administered at DIV 7 in the ATP treatment groups, and cell slides were prepared for immunostaining at DIV 8, DIV 9, DIV 10 and DIV 14. In addition, the target specificity of the Piezo2 antibodies was determined via negative control experiments (Figure S4). Piezo2 expression increased after ATP administration at DIV 8, 9 and 10 but decreased at DIV 14 (Fig. 5b). Compared with that of the control group, the fluorescence intensity of Piezo2 was significantly higher at DIV 9 and 10 (Fig. 5c). Therefore, DIV 10 (3 days after ATP treatment) was chosen as the default time point for the following in vitro experiments.

Fig. 5.

ATP increased the expression of Piezo2 in primary cultured TG neurons during the first 3 d and initiated Ca²⁺ influx. a Schematic of the timeline for the ATP administration experiments. ATP was added to the culture medium at DIV 7, and immunocytochemical analysis was performed at DIV 8, 9, 10 and 14. b–c Representative images showing triple immunostaining for Piezo2 (red)/NEFH (green)/DAPI (blue) in cultured TG neurons from the control group and ATP group at different time points. Analysis of Piezo2 fluorescence intensity at different time points. ATP induced a significant increase in Piezo2 expression compared with that in the control group at 1 d, 2 d, and 3 d (DIVs 8, 9, and 10) after ATP treatment for 1 h (mean ± SEM, n = 16–31, n indicates the number of independent cultured TG neurons; ***P < 0.001, ns, no significant difference; two-way ANOVA). d–e Triple immunostaining for Piezo2 (red)/NEFH (green)/DAPI (blue) in cultured TG neurons treated with a high concentration of ATP (2 mM) for 1 h or continuously treated with a low concentration of ATP (500 μM). The histogram shows the fluorescence intensity of Piezo2 in the three groups in d. Fluorescence intensity analysis revealed that both short-term treatment with ATP at 2 mM and long-term treatment with ATP at 500 μM had similar effects on the upregulation of Piezo2 expression. (mean ± SEM, n = 24–34; n indicates the number of independent cultured TG neurons; ***P < 0.001; ns, no significant difference; one-way ANOVA; scale bar = 10 μm). f Glass bead application induced a [Ca²⁺]_i increase that readily returned to a steady level close to the basal level in control and ATP group neurons. Fura-3AM fluorescence images show the change in Ca²⁺ concentration during ATP stimulation. (g-h) GsMTx4 efficiently inhibited the Δ[Ca²⁺] induced by ATP stimulation. A comparison of Δ[Ca²⁺] among the control group, ATP group and ATP with GsMTx4 preincubated group revealed that the ATP group presented significantly greater Ca²⁺ influx induced by glass beads and that GsMTx4 significantly inhibited Ca²⁺ influx. (mean ± SEM, n = 9–24, where n indicates the number of independent cultured TG neurons; **P < 0.01, ***P < 0.001, ns, no significant difference; one-way ANOVA; scale bar = 20 μm). i ELISA experiments revealed that ATP significantly increased cAMP levels in cultured TG neurons. (mean ± SEM, n = 3; n indicates the number of independent cell culture preparations; **P < 0.01; unpaired t test). j–k Triple immunostaining of Piezo2 (red)/NEFH (green)/DAPI (blue) in primary cultured TG neurons subjected to different treatments. The fluorescence intensity analysis revealed that ionomycin, but not cAMP, significantly increased the expression level of Piezo2. (mean ± SEM, n = 11–43; n indicates the number of independent cultured TG neurons; ***P < 0.001; ns, no significant difference; one-way ANOVA; scale bar = 10 μm). l–m Glass bead application induces a very large Piezo2-dependent [Ca²⁺]_i increase in the ionomycin/cAMP group, with a peak [Ca²⁺]_i near 4000 nM/2000 nM, after which it returns to an unstable level, which is greater than the basal level in ionomycin-treated neurons. Comparison of Δ[Ca²⁺] among the control, ionomycin, and ionomycin with GsMTx4 pretreated groups. The ionomycin group presented significantly greater Ca²⁺ influx induced by glass beads, and GsMTx4 inhibited Ca²⁺ influx (mean ± SEM, n = 9–26; n indicates the number of independently cultured TG neurons; **P < 0.01, ***P < 0.001; one-way ANOVA). (n–o) Fluorescence intensity analysis of the control, shaking and shaking + db cAMP groups revealed that shaking significantly upregulated Piezo2 expression in TG neurons in the presence or absence of db cAMP. There was no significant difference in Piezo2 expression between the shaking group and the db cAMP + shaking group (mean ± SEM, n = 20–37, where n indicates the number of independent cultured TG neurons; ***P < 0.001, ns, no significant difference; one-way ANOVA; scale bar = 10 μm)

Although short-term stimulation (1 h) with a relatively high concentration (2 mM) of ATP effectively upregulated Piezo2 expression, we were also interested in simulating another potential mode of ATP exposure following nerve injury, namely a more sustained, lower-level exposure. In this study, two different methods for ATP administration were compared: one was the administration of a relatively high concentration (2 mM) for a short time, and the other was the administration of a low concentration of 500 μM ATP every 12 h to simulate the continuous effects and subsequent hydrolysis of ATP that occur in vivo. The two distinct treatment methods were designed based on the possible local microenvironment in the acute versus chronic phase after nerve injury. It is well established that the concentration of ATP after tissue injury can reach millimolar levels [37]. We selected an ATP concentration of 2 mM to maintain consistency with previous experimental protocols, as also supported by prior publication [38]. In contrast, determining the ATP concentration during chronic nerve injury and regeneration was more challenging. Therefore, we developed a similar treatment protocol based on previous studies [39]. Our results revealed that there was no significant difference in the upregulation of Piezo2 expression between these two methods (Fig. 5d, e). Since ATP is hydrolyzed to ADP and adenosine in the extracellular space, the effects of ADP and adenosine in regulating Piezo2 expression should be considered. We next analyzed the effects of the individual use of 500 μM ATP, ADP or adenosine on the expression of Piezo2, and no significant differences were detected compared with those in the control group (Figure S5).

In our previous study, we investigated Ca²⁺ influx ([Ca²⁺]_i) after performing ATP with mechanical stimuli and reported that the promotion of mechanical sensitivity in TG neurons was Piezo2 dependent. The mechanical stimuli generated by a single layer of glass beads with a diameter of 100 μm would likely deliver mechanical pressure at approximately 10⁻⁵ atmospheres, initiating significantly greater mechanical stimulation.

In this study, Piezo2-dependent [Ca²⁺]_i was further characterized and quantified. ATP-treated TG neurons were treated with 2 mM ATP for 60 min and then replaced with normal culture medium for 3 days before measurement. The application of glass beads triggered a very large initial increase in [Ca²⁺]_i in TG neurons in both the control group and the ATP group, followed by restoration to a new equilibrium level, as described previously. The Δ[Ca²⁺] represents the difference between the peak level of [Ca²⁺]_i and rest level [Ca²⁺]_i after the glass beads were used. Significant differences in Δ[Ca²⁺] were detected between the control group and the ATP-stimulated group (Fig. 5f). Moreover, we also verified whether the mechanically initiated responses were Piezo2-dependent by incubation with the Piezo inhibitor GsMTx4 30 min before the experiment in ATP-treated cells and found that the Δ[Ca²⁺] was significantly lower than that in ATP-treated cells (Fig. 5g, h). Consequently, Δ[Ca²⁺] could be regarded as a parameter corresponding to the mechanosensitivity of TG neurons.

To simulate Ca²⁺ influx induced by ATP, ionomycin was administered to primary TG neurons, and immunostaining assays were subsequently performed to determine the effect of ionomycin on the expression of Piezo2 at different time points (Figure S6a). Compared with the control group, the ionomycin-treated group presented a greater fluorescence intensity of Piezo2, which gradually increased over time (Figure S6b, c). To exclude the potential influence of Piezo1 activation in TG neurons, which might also induce Ca²⁺ influx, we added Yoda 1 to the culture medium and incubated the cells for 1 h to activate Piezo1. Immunostaining analysis demonstrated that the activation of Piezo1 had no effect on the expression of Piezo2 (Figure S6d, e).

Moreover, ATP was observed to induce the upregulation of intracellular cAMP (Fig. 5i). To evaluate the effect of cAMP on the expression of Piezo2, an analog of cAMP (db cAMP) was administered to TG neurons. Compared with those of the control group and the ionomycin-treated group at DIV 10 (3 days after componds treatment), the results revealed that db cAMP did not upregulate the expression of Piezo2, whereas ionomycin significantly increased the expression of Piezo2 (Fig. 5j, k).

We further applied Ca²⁺ imaging to control neurons, ionomycin-stimulated neurons and db cAMP-stimulated neurons at DIV 10. Compared with those in the control group, the Δ[Ca²⁺] of the glass beads in the ionomycin group were significantly greater, and GsMTx4 efficiently inhibited the influx of Ca²⁺, which was also consistent with our previous conclusions (Fig. 5l). Interestingly, Ca²⁺ imaging also revealed a significantly greater Δ[Ca²⁺] in db cAMP-treated neurons than in control neurons (Fig. 5m), indicating that cAMP might independently mediate the mechanosensitivity of TG neurons in response to extracellular ATP. Moreover, a mechanical stimulus itself might induce Ca²⁺ influx, which might in turn initiate Ca²⁺-dependent signaling, and an additional shaking stimulus test was performed to eliminate the influence of this factor in vitro. The immunostaining results revealed that shaking stimulation upregulated the expression of Piezo2 compared with that in the control group (Figure S6f-g). Nevertheless, there was no significant difference between the db cAMP + shaking group and the shaking group (Fig. 5n, o).

We also compared the rest [Ca²⁺]_i, peak [Ca²⁺]_i and Δ[Ca²⁺] across different groups of TG neurons subjected to different interventions (Figure S7). It was quite clear that ATP, ionomycin and db cAMP significantly induced greater Δ[Ca²⁺] in TG neurons than in control neurons, indicating greater mechanosensitivity. Moreover, the Piezo inhibitor GsMTx4 efficiently inhibited the influx of Ca²⁺, demonstrating that Piezo2 is essential for the mechanosensitivity of TG neurons.

Extracellular ATP upregulated Piezo2 by activating PKA/PKC-MAPK signaling

To investigate the extracellular ATP-mediated cellular signaling pathways that might be involved in regulating the expression of Piezo2, several kinase inhibitors were administered after ATP treatment. The immunostaining results revealed that Go 6983, H89, SB203580 and U0126 obviously reduced the fluorescence intensity of Piezo2 in TG neurons pretreated with ATP (Fig. 6a, b), indicating that PKC, PKA, p38-MAPK and MEK 1/2 might contribute to ATP-mediated Piezo2 expression. Further immunoblotting assays confirmed that ATP significantly induced the expression of p-ERK and p-p38 (Fig. 6c, d), which indicated that the ERK1/2 and p38 MAPK signaling cascades could be activated by extracellular ATP. In addition, we administered kinase inhibitors separately to cultured neurons without ATP pretreatment and found that none of these inhibitors had a significant effect on the expression of Piezo2 (Figure S8).

Fig. 6.

Extracellular ATP upregulated Piezo2 expression by activating PKC/PKA signaling and the ERK 1/2 and p38 signaling cascades. a Representative images of cultured TG neurons treated with different compounds. b The histogram shows the fluorescence intensity of Piezo2 in TG neurons treated with different inhibitors as indicated below. Inhibitors of PKC, PKA, p38-MAPK and MEK 1/2 significantly attenuated the ATP-induced increase in Piezo2 expression in TG neurons (2 mM ATP for 1 h and continued culture for 3 d). Compared with ATP, the JNK inhibitor SP600125 did not significantly affect the expression of Piezo2. c Activation of ERK 1/2 after ATP administration and the inhibitory effect of U0126 were confirmed by Western blot analysis. U0126 significantly reduced the level of p-ERK (p-ERK/ERK) after ATP treatment. d Activation of p38 MAPK after ATP administration and the inhibitory effect of SB203580 were confirmed by Western blot analysis. The histogram shows that SB203580 significantly reduced the level of phosphorylated p38 (p-p38/p38) after ATP treatment. (mean ± SEM, for ICC, n = 19–46, n indicates the number of independent cultured TG neurons; for WB, n indicates the number of independent cell culture preparations; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA; scale bar = 10 μm)

c-Fos is a well-known marker of activated neurons, so the expression of c-Fos was measured at different time points after treatment with 2 mM ATP to determine whether ATP could induce sustained activation of TG neurons. The results revealed transient upregulation of c-Fos expression in the nucleus at 0 h and 1 h but then dramatically decreased to a basal level that was similar to that of the control group. There was no significant difference in c-Fos expression at 3 h, 6 h, 12 h or 24 h compared with that in the control group (Figure S9a, b). In addition, changing the medium had no effect on the activation of c-Fos, suggesting that extracellular ATP independently induces TG neuron activation (Figure S9c). As a result, 1 h of treatment with 2 mM ATP followed by changing the culture medium was determined to be the proper compound delivery method for the subsequent TF array assays.

To screen the downstream TFs that might be involved in the regulation of Pizeo2 expression, primary TG neurons were treated with 2 mM ATP for 1 h at DIV 8, after which nuclear lysates were prepared and subjected to a 96-TF array assay. As a result, the top 20 upregulated TFs were screened (Fig. 7a), and 6 were predicted to be able to bind to the Piezo2 promoter according to JASPAR (http://jaspar.genereg.net/). To further validate whether HIF, AP1, p53, ATF2, HNF4 and CREB can regulate Piezo2 expression, the corresponding inhibitors were administered to TG neurons pretreated with ATP, and the expression of Piezo2 was measured by immunostaining. T-5224 (an inhibitor of AP1), 666-15 (an inhibitor of CREB), SGC-CBP30 (an inhibitor of ATF2) and BI-6015 (an inhibitor of HNF4) significantly decreased the fluorescence intensity of Piezo2 (Fig. 7b, c), indicating that AP1, CREB, ATF2 and HNF4 might be critical TFs that are downstream of extracellular ATP signaling and regulate Piezo2 transcription. The administration of TF inhibitors had no significant effect on the expression of Piezo2 in TG neurons without ATP pretreatment (Figure S10).

Fig. 7.

Identification of transcription factors (TFs) that bind to the Piezo2 promoter and regulate Piezo2 transcription a The activation of 96 TFs in response to ATP treatment was assessed; the TFs whose expression increased x-fold over that at baseline were ranked, and the top 20 are shown in the diagram. Note that for “JASPAR” below the bar diagram, +/− indicates whether the respective TF was found to have a predicted binding site in the proximal Piezo2 promoter via the JASPAR online TF-binding prediction tool. Note that “Inhibitor” indicates whether the respective TF can be inhibited with a well-established selective inhibitor. The six red arrows point toward the 6 TFs that meet all criteria, namely, HIF, AP1, p53, ATF2, HNF4 and CREB. b Representative images of cultured TG neurons treated with different kinds of TF inhibitors. c Analysis of Piezo2 fluorescence intensity in the different groups. The names of the TF inhibitors are noted below the histogram. AP1, ATF2, and CREB were confirmed to be involved, and the AP1 inhibitor T-5224, the ATF2 inhibitor CBP30, and the CREB inhibitor 666-15 significantly attenuated the expression of Piezo2 after ATP treatment; however, HIF and p53 were not confirmed to be relevant. d A schematic of the predicted binding sites of AP1 and CREB in the proximal Piezo2 promoter, as revealed by the bioinformatics platform JASPAR. (mean ± SEM, n = 25–45; n indicates the number of independent cultured TG neurons; ***P < 0.001; one-way ANOVA; scale bar = 10 μm)

In the above experiments, we employed multiple signaling pathways and transcription factor inhibitors to investigate the effects of ATP’s potential downstream signaling molecules on Piezo2 expression levels. These experimental interventions and corresponding signaling pathways are summarized in the schematic diagram (Figure S11).

Discussion

In recent decades, investigations on peripheral neuropathic pain have revealed several potential mechanisms involved. CGRP and SP, which are classical neurogenic inflammation-related factors, are released from peripheral nerve terminals following peripheral nerve injury [30, 40]. The function of Piezo2, which is a critical ion channel that is thought to be involved in neuropathic pain, has been widely investigated [41–44]. In this study, we observed the critical involvement of PKC signaling in the regulation of Piezo2- and CGRP/SP-related peripheral sensitization in a CCT animal model of TN. We then investigated the initiation of the intracellular mechanism by which Piezo2 is upregulated in TG neurons induced by extracellular ATP. In addition to its neuronal effects, extracellular ATP may also play a regulatory role in neuroimmune responses associated with TN.

One main source of CGRP and SP released in the whisker pad is peptidergic neurons in the TG, which are initiated during nerve injury and regeneration, inducing the upregulation and release of CGRP and SP [45, 46]. However, normally, peptidergic neurons are nociceptive small neurons with C fibers or Aδ fibers terminated at free nerve endings, which should not dominate Merkel cells (innervated by Aβ fibers originating from nonnociceptive neurons normally large) [47, 48]. This result indicated that nonnociceptive neurons may be changed into those expressing CGRP and SP during pathophysiological changes after nerve injury. We did observe that CGRP and/or SP immune-positive cells were increased in the TN group, especially in large neurons (Figure S1e, f). In our previous study, we confirmed that Piezo2 was upregulated more significantly in CGRP-positive neurons than in CGRP-negative neurons after nerve injury [31]. These results indicate that nonnociceptive neurons may switch to synthesizing and releasing CGRP/SP to act on Merkel cells after nerve injury. In addition to the upregulation of Piezo2 in TG and whisker pad Merkel cells, the mechanisms underlying mechanical allodynia in TN animals can be divided into two closely related pathways. First, Piezo2 and CGRP/SP are upregulated in TG. Second, Piezo2 interacts with CGRP/SP intracellular signaling in Merkel cells.

The synthesis and release of CGRP and SP are modulated by intracellular Ca²⁺-PKC signaling [46, 49, 50]. The activation of many kinds of G protein (Gs)-coupled receptors and cytokine receptors can increase the concentration of Ca²⁺, which not only initiates PLC‒PKC signaling but also initiates the processing of vesicle fusion and neuropeptide release from terminals after nerve injury [51, 52]. PKC also initiates the downstream MAPK cascade, upregulating CGRP and SP by activating specific transcription factors [53].

The intracellular effects of CGRP converge on the cAMP pathway [54]. Activation of NK1R recruits and binds Gq/11 family G proteins in the cytoplasm, further initiating the PLC-IP3/DAG signaling cascade, and increasing the intracellular [Ca²⁺] [55]. This change may potentially contribute to the increased Piezo2 expression. By interfering with cAMP signaling and Piezo2 in the whisker pad, we revealed that mechanical allodynia requires both Piezo2 and cAMP to be functionally intact during TN (Fig. 3j–m). These findings indicate that the CGRP receptors may represent potential drug targets for alleviating TN and other types of neuropathic pain. To preliminarily investigate whether CGRP/SP could increase Piezo2 expression, we supplemented the experiments with sustained CGRP/SP treatment in cultured neurons in vitro. However, the results showed no direct effect on Piezo2 expression levels (Figure S12). A previous study reported that CGRP does not induce intracellular [Ca²⁺] increase in TG neurons [56], which aligns with our subsequent finding that cAMP signaling primarily affects mechanotranduction rather than Pizeo2 expression. The absence of SP’s effect on Piezo2 expression may be attributed to the relatively low NK1R expression in primary sensory neurons, as demonstrated by several single-cell studies [57]. Furthermore, these results exclude potential autocrine effects of CGRP and SP on Piezo2 expression following ATP and ionomaycin treatment in TG neurons. However, whether the increase in Piezo2 expression in Merkel cells is related to CGRP and SP needs to be further investigated in the future, and our prediction is very likely based on the following results, while not in a direct way.

To elucidate the mechanisms that initiate the increase in Piezo2 and CGRP/SP expression levels, in vitro studies were performed in primary cultured TG neurons. Axonal injury induces the release of ATP into the extracellular matrix, where ATP acts as a ligand, generating many intracellular processes in neurons [58, 59]. Compared with other TN animal models, the CCT model better reproduces the clinical etiology of primary TN patients, in which axon injury is near the TG in the TREZ [32, 33]. This leads to the direct action of ATP on TG neurons. Our previous study verified that extracellular ATP potentially induced Piezo2 expression via intracellular Ca²⁺, but the specific intracellular signaling mechanism involved remains unclear [31]. In this study, bioinformatic analysis of RNA-seq data revealed potential intracellular processing between purinergic receptors and PKC-MAPK pathway, forming a highly interconnected regulatory network. This network is implicated in the modulation of both neuronal sensitization and neuroimmune responses (Fig. 4a). The protein–protein interaction analysis identified Plcb2 and Ngf as hub genes that bridge PKC signaling with inflammatory mediators (Fig. 4b), providing further evidence for the activation of the neuroimmune response axis in TN. The real-time PCR results revealed that ATP increased the expression of CGRP and SP in cultured TG neurons (Fig. 4c). These results indicate that ATP likely regulates CGRP/SP and Piezo2 via similar intracellular signaling pathways in sensory neurons, potentially through PKC signaling.

Different subtypes of purinergic receptors reportedly mediate different intracellular effects [23, 60, 61]. The activation of P2Xs induces an influx of cations, especially Ca²⁺ cations, which act mainly as intracellular messengers to activate PKC signaling [24, 62]. However, the effects of activating P2Ys are much more complex. P2Y1, P2Y2, P2Y4, and P2Y6 are known Gq-coupled receptors, and the activation of these receptor subtypes typically increases the intracellular Ca²⁺ concentration and initiates Ca²⁺-dependent PKC signaling. Other P2Y receptor subtypes, including P2Y12, P2Y13, and P2Y14, are inhibitory adenylate cyclase G protein (Gi)-coupled receptors that mediate the inhibition of adenylyl cyclase (AC) and decrease intracellular cAMP levels [22, 23]. For adenosine, the metabolite ATP may bind to P1 receptors and upregulate intracellular cAMP signaling [63]. Our results demonstrated that Ca²⁺ signaling is sufficient to increase the expression level of Piezo2 but also somewhat differs from the effects of ATP. After treatment with ATP at DIV 7, the expression level of Piezo2 first increased but then decreased (Fig. 5a, b, c), but ionomycin had a sustained effect on Piezo2 upregulation (Figure S6a, b, c). The different effects of ATP and ionomycin might be attributed to the complex interactions between intracellular signaling pathways that are activated by different P2 receptors.

Interestingly, an in vitro study revealed that transient shaking promoted sustained Piezo2 expression in TG neurons, indicating that the Ca²⁺ influx induced by Piezo2 activation could initiate the expression of Piezo2, which provided a strong complement to a positive feedback process between the expression level of Piezo2 and the influx of Ca²⁺ [31] (Fig. 5n, o and S6 f, g). In brief, the activation of Piezo2 increased the [Ca²⁺]_i level, further enhancing the expression of Piezo2 itself, which supports our previous prediction in Merkel cells that CGRP/SP promotes the expression of Piezo2 by sensitizing its mechanotransduction.

The other member of the Piezo protein family is Piezo1, which mainly converts mechanical stimulation into elevated [Ca²⁺]_i levels and promotes Ca²⁺-dependent signaling, which causes a wide variety of intracellular effects in different cell types[64, 65]. Recent studies have shown that sensory neurons also express Piezo1, which transduces mechanical stimuli into itching sensations[66–68]. A previous study revealed positive feedback between Ca²⁺ and Piezo1[65]. However, Yoda1, which is an agonist of Piezo1, failed to upregulate the expression of Piezo2 in cultured TG neurons (Figure S6d, e), indicating that the activation of Piezo1 was not correlated with Piezo2 expression in TG neurons.

Another surprising finding was that there was no significant difference in the effect of high concentration of short duration ATP (2 mM) treatment versus long duration of relatively low concentration of ATP (500 μM) treatment on Piezo2 expression (Fig. 5d, e), which may be related to the fact that continuous exposure to ATP leads to remodeling of P2 receptor function and expression, such as desensitization and internalization [69]. In contrast, short treatments are limited by the timing of P2 receptor activation. This will be further elaborated in future studies on the different roles of different P2 receptors on Piezo2 expression or function.

Our previous study revealed that CCT increased the concentration of cAMP in the TG [31]. This study demonstrated that ATP increases the level of intracellular cAMP in cultured TG neurons (Fig. 5i). The administration of db cAMP for 1 h significantly increased the mechanical sensitivity of TG neurons (Fig. 5m) but had no prominent effect on regulating Piezo2 expression (Fig. 5j–k). However, db cAMP coadministration with shaking increased the expression of Piezo2 (Fig. 5n, o). Moreover, PKA inhibition reversed the upregulation of Piezo2 induced by ATP (Fig. 6a, b). Importantly, previous studies demonstrated that the Epac1‒Rap1 pathway, rather than the PKA signal, was activated by cAMP signaling, resulting in enhanced Piezo2-dependent current [17, 18, 70–72]. These findings suggest that ATP-activated PKA signaling may be coupled with Ca²⁺-PKC signaling, which contributes mainly to the upregulation of Piezo2 expression, whereas cAMP signaling affects mainly the mechanotransduction function of Piezo2 by activating Epac1-Rap1. In line with this functional divergence, the cAMP treatment results are consistent with the previous findings that CGRP treatment did not affect Piezo2 expression in cultured TG neurons in vitro (Fig. 5k, S12). PKA and PKC are both critical for the upregulation of Piezo2 expression, which may be attributed to the synergistic regulation of Piezo2 by multiple TFs [65, 73, 74]. In this study, several TFs, including AP1, CREB, ATF2 and HNF4, are likely involved in the ATP-mediated upregulation of Piezo2 expression (Fig. 7). Further investigation will employ direct experimental approaches, such as ChIP or CUT&Run to validate the binding of these TFs to specific recognition sequence in the Piezo2 promoter region. Among these factors, CREB can be directly enhanced by PKA [75, 76]. ATF2 and AP1 are mainly activated by MAPK signaling [77, 78]. Additionally, the p38-MAPK pathway and the ERK 1/2 signaling pathway were upregulated by extracellular ATP stimuli (Fig. 6c–d). Previous studies have shown that both ERK 1/2 and p38 MAPK can be activated by PKA and PKC [79, 80]. Taken together, these results help elucidate the relatively intact intracellular mechanism by which ATP regulates Piezo2 expression.

Here, we demonstrated in vitro that both PKC and PKA are involved in ATP-mediated regulation of Piezo2. However, we selectively validated PKC in vivo based on our RNA-Seq and protein interaction network analyses, which identified the PKC pathway as a central hub in the CCT-induced pathological network (Fig. 4b). This selection is further supported by extensive literature establishing PKC as a critical regulator in various neuropathic pain and neuroinflammation models [81, 82].

AP1, CREB and ATF2, along with their upstream ERK1/2 and p38 MAPK, are critically involved in regulating the expression of CGRP and SP [83, 84]. The promoter regions of both the substance P (Tac1) and CGRP (Calca) genes contain consensus binding sites for multiple activity-dependent transcription factors, including CREB, AP-1 and ATF2 [85]. Owing to the wide involvement of CGRP and SP in neuropathic pain[86, 87], different types of nerve injury typically manifest as mechanical allodynia because of their potential interaction with Piezo2. In addition to their role in nociceptive sensory coding, these TFs have been shown to function as intranuclear signaling molecules in neuroinflammation, reflecting the extensive neuroimmune crosstalk between neurons and glial cells during nerve injury. Specifically, AP1 drives interleukin-6 (IL-6) production in astrocytes [88], while CREB activation in microglia enhances the release of tumor necrosis factor-alpha (TNF-α) and IL-6 [89]. Collectively, our findings demonstrate that PKC-MAPK signaling promotes a board neuroinflammatory response in TN, encompassing CGRP/SP mediated neurogenic inflammation.

In summary, our study elucidates a potential mechanism underlying peripheral sensitization in a TN rat model induced by TREZ compression. In this model, ATP acts as the primary initiator, triggering three interconnected processes: (1) Piezo2 upregulation, (2) CGRP/SP mediated neuroinflammation, and (3) Piezo2-dependent mechanical allodynia. These processes promote two closely linked positive feedback loops. The first loop involves reciprocal regulation between Ca²⁺ influx and Piezo2 expression upregulation in both Merkel cells and TG neurons. The second loop encompasses a neuroinflammatory cascade extending from the TG to the whisker pad, characterized by Ca²⁺-PKC signaling activation in the TG that elevates CGRP/SP levels in the whisker pad and initiates cAMP signaling in Merkel cells, thereby amplifying Piezo2-mediated mechanotransduction. These effects lead to enhanced Piezo2-mediated ion influx (particularly Ca²⁺) in TG neurons, driving further upregulation of CGRP/SP in both the TG and whisker pad. This process establishes a self-perpetuating cycle of neuronal hypersensitivity and neurogenic inflammation, ultimately resulting in the mechanical allodynia characteristic of TN. Our demonstration of Ca²⁺-PKC as a central hub integrating these pathological pathways underscores its potential as a promising therapeutic target for TN management (Fig. 8).

Fig. 8.

Positive feedback-mediated TN pathogenesis involves TG neurons and Merkel cell intracellular signaling, which increases the expression and function of Piezo2. Extracellular ATP activates Ca²⁺-dependent PKC and PKA signaling, driving downstream p38 and ERK1/2 MAPK cascades that upregulate the transcription factors AP1, ATF2, CREB and HNF4, increasing the expression of Piezo2. The ATP metabolism adenosine activated cAMP-Epac1-Rap1 to increase the function of Piezo2. MAPK signaling also increases the levels of the pain-related neural transmitters SP and CGRP. Once released, CGRP/SP acts on Merkel cells in the trigeminal nerve-innervated area of whisker pads to activate cAMP signaling and intracellular Ca²⁺. cAMP signaling promotes the sensitization of Piezo2. Upregulated Piezo2 increases the intracellular Ca²⁺ concentration, which initiates a positive feedback mechanism, leading to further upregulation of Piezo2 and CGRP/SP in TG neurons and whisker pads

Nevertheless, P2Xs or P2Ys might not be ideal targets. First, our bioinformatics prediction revealed that the downstream signaling cascades of different P2 receptors could vary greatly, while the effect of Ca²⁺-PKC is the result of a combination of multiple P2 receptors. Second, several types of P2Xs or P2Ys have similar intracellular effects, but the exact differences have not yet been revealed [22–24, 62].

There are also several limitations in this study. We have not demonstrated the intracellular mechanism induced by CGRP/SP, which increases Piezo2 expression in Merkel cells. The distinct contributions of Piezo2 expression levels and neuronal excitability to mechanotransduction could not be differentiated between in vitro and in vivo conditions. Different types of neurons were not separated in the series of experiments. ATP and its metabolic products target a wide range of receptors, and it is difficult to differentiate the precise roles of these diverse receptors. The specific receptors and signaling molecules that play key roles in vivo in different phases of TN, as well as the effects and mechanisms of P2 receptors in the treatment of TN, need further investigation in our future work. Additionally, our study did not investigate the potential interactions between CGRP/SP signaling in Merkel cells and local immune cells (e.g., macrophages and mast cells) in modulating Piezo2-mediated mechanotransduction. Future studies should examine potential correlations between specific cytokine profiles (particularly IL-1β and TNF-α) in the TG-whisker pad axis and Piezo2 expression or function. Furthermore, given the well-documented female predominance in clinical TN presentations, we will systematically investigate sex differences in Piezo2-depedent mechanical allodynia using our TN animal model. This integrated approach will help elucidate both the neuroimmune mechanisms and sex-dependent factors underlying TN pathophysiology.

Abbreviations

ADP

Adenosine diphosphate

ATP

Adenosine 5′-triphosphate

BL

Baseline values

BSA

Bovine serum albumin

cAMP

Cyclic adenosine monophosphate

CCT

Chronic compression of the trigeminal nerve root

c-Fos

Fos proto-oncogene, AP-1 transcription factor subunit

CGRP

Calcitonin gene-related peptide

CREB

CAMP response element binding protein

CRLR

Calcitonin-receptor-like receptor

DAPI

4′,6-Diamidino-2-phenylindole

DEGs

Differentially expressed genes

DIV

Days in vivo

DRG

Dorsal root ganglion

Epac1

Exchange protein directly activated by cAMP 1

ERK

Extracellular regulated protein kinases

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

GFAP

Glial fibrillary acidic protein

GPCRs

G protein-coupled receptors

HRP

Horseradish peroxidase

IASP

International Association for the Study of Pain

KEGG

Kyoto Encyclopedia of Genes and Genomes

Krt8

Cytokeratin 8

MAPK

Mitogen-activated protein kinase

NEFH

Neurofilament, heavy polypeptide

NeuN

Neuronal nuclei

NK1R

Neurokinin-1 receptor\

NSE

Neuron-specific enolase

P2Rs

Purinergic 2 receptors

PBS

Phosphate-buffered saline

PKC

Protein kinase C

RAMP1

Receptor-activity-modifying protein 1

Rap1

Rhoptry-associated protein1

ROI

Region of interest

SD

Sprague–Dawley

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEM

Standard error of mean

shRNA

Short hairpin ribonucleic acid

SP

Substance P

Sp5C

Caudal spinal trigeminal nucleus caudalis

TF

Transcription factor

TG

Trigeminal ganglion

TN

Trigeminal neuralgia

TREZ

Trigeminal root entry zone

Author contributions

X.Y.L. and Z.K.L. performed most of the in vivo and in vitro experiments. F.H. was responsible for the molecular biology experiments and most of the data analysis. X.Y.L. and Z.K.L. wrote the first draft of the manuscript. Y.Q.W. was mainly involved in Ca²⁺ imaging experiments. Z.Y.Z. and W.H.L. helped to establish the TN animal model and confocal imaging. X.M.W. and Y.T.O. assisted with morphological experiments and behavior tests. F.W. provided the experimental scheme of cell intervention, data analysis, and guidance on draft. D.S.L. designed the experiment, analyzed the data and revised the manuscript. All the authors read and approved the final version of the manuscript.

Funding

This work was funded by the National Natural Science Foundation of China (Grant number: 82171213) and Joint Funds for the innovation of science and Technology, Fujian province (Grant number: 2023Y9005).

Data availability

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

Declarations

Ethics approval and consent to participate

The animal experiments included in this study have obtained the ethical approval of the Animal Care and Use Committee of Fujian Medical University with the approval number IACUC FJMU 2022-Y-0543. All animal-related experimental protocols comply with animal welfare and ethical requirements and follow ethical guidelines. The Animal Care and Use Committee of Fujian Medical University follows the rules of the Basel Declaration.

Consent for publication

The manuscript was critically reviewed and endorsed for publication by all contributing authors.

Competing interests

The authors declare that there are no competing interests.