KEY POINTS

Question: What is the efficacy and underlying mechanism of Moutan Cortex and its active ingredients on chronic orofacial pain therapy? Findings: As 1 of the active components of Moutan Cortex, quercetin effectively alleviated chronic orofacial pain through blocking voltage-gated sodium channel (Nav) channel in trigeminal ganglion sensory neurons, while downregulating the expression level of proinflammatory cytokines-FOS and TNF-α. Meaning: Identifying Nav as the molecular target of quercetin clarifies the analgesic mechanism of Moutan Cortex, and provides ideas for the development of novel selective and efficient chronic pain relievers.

Chronic orofacial pain (COP) is a long-lasting and refractory pain syndrome affecting 7% to 11% of people worldwide.1 As a vital region for life-sustaining and social behavior, persistent pain condition of the orofacial area markedly impacts patients’ life quality.2 However, COP treatment is challenging due to its broad pathogenesis and complicated orofacial anatomy.3 At present, drugs for COP have poor efficacy and serious adverse reactions.4 Thus, development is urgently needed of novel analgesics that attenuate the progression and occurrence of COP with minimal side effects and off-target properties.

Although the pathophysiological basis of COP may be varied, trigeminal sensitization is generally regarded as a common mechanism behind this abnormal condition.5 Originally from the trigeminal ganglion (TG), sensitization of the trigeminal nociceptive primary afferents is responsible for orofacial allodynia or pain chronicity.6 Several studies clarified that chronic orofacial inflammation is 1 of the etiologies of COP as abnormal immune response and proinflammatory cytokines are blamed for trigeminal sensitization.7Porphyromonas gingivalis–derived lipopolysaccharide (LPS)–induced inflammatory pain models presented facial hyperalgesia and TG proinflammatory response,8 constituting a possible approach to understand pain mechanisms associated with orofacial disorders.

The various medicine herb-derived ingredients provide ways for novel analgesic development.9,10 Moutan Cortex (MC), root of Paeonia suffruticosa, is a traditional Chinese medicine (TCM) herb widely used for inflammatory diseases. Recent studies have declared MC an effective drug for orofacial inflammation.11 Intriguingly, MC and its active ingredients, such as quercetin, have also been proven as analgesics in inflammatory and neuropathic pain therapy.12 Several studies attribute its analgesic effect to rebalancing pro- and anti-inflammatory responses,13,14 while others connect such mechanisms to a nociceptive ion channel’s inhibition.15 However, whether and how MC or quercetin can relieve orofacial inflammatory-induced trigeminal sensitization is still not clearly understood.

Herein, we identified the main active ingredients of MC and predicted their potential targets for the treatment of COP through network pharmacology, a computational and simulated approach for evaluating the numerous component-target interactions and networks that accelerate the development of novel drugs.16 To validate the predicted compound-target network, we performed real-time quantitative polymerase chain reaction (RT-qPCR) and molecular docking analysis. A pain behavioral test was conducted to evaluate quercetin’s analgesic effect. Whole-cell voltage clamp was performed to clarify the inhibition property of quercetin on ion channels. All these findings provided evidence and theoretical basis for supporting our study hypothesis that quercetin, as a main active ingredient of MC, alleviates COP via block of the voltage-gated sodium channel (Nav).

METHODS Animals

This article adheres to the applicable Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The animal study protocol was approved by the Review Board for Animal Research of Southern University of Science and Technology. All experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals and the ethical guidelines of the International Association for the Study of Pain. Pathogen-free male Sprague-Dawley (SD) rats (age 6–8 weeks on arrival; Beijing Viton Lever Co Ltd) were housed in a light- (12 hours light/dark cycle) and temperature- (23 ± 3°C) controlled room. During the experiments, animals were assigned to groups using a random number generator (GraphPad Prism 9). All behavioral experiments were performed by experienced experimenters blinded to the treatment methods and groups.

Reagents

Reagents used in this study are listed under the form of “Reagent name (Company name, Item number)” as follows: Quercetin (MCE, HY-18085), LPS (InvivoGen, tlrl-pglps), IA collagenase (Worthington, LS004194), neutral protease (Worthington, LS02104), poly-d-lysine hydrobromide (PDL) (Sigma, P7886), fetal bovine serum (FBS) (Gibco, 2176404), Dulbecco’s modified Eagle medium (DMEM) (Gibco, 2318815), penicillin/streptomycin (Gibco, 2321127), iCell Primary Mesenchymal Stem Cells Serum-Free Media (iCell, PriMed-iCELL-012-SF), PrimeScipt RT Master Mix (Takara, RR047B), and TB Green (Takara, RR820B). Other reagents were obtained from Sigma-Aldrich Chemicals.

MC and COP-Associated Target Screening and Network Construction

We used the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) to identify active ingredients of MC.17 Thresholds of both high oral bioavailability (OB) >30% and drug-like properties (DL) >0.18 were set to screen high-activity compounds.18 Then, compound-associated targets were collected in the TCMSP database. After removing duplicates and converting to unified gene names through the UniProt database,19 the list of compounds-targets was saved for further analysis. To identify potential target genes of COP, we used the GeneCards20 databases by searching the keywords “chronic orofacial pain” under the species of “Homo sapiens.” The obtained COP-related gene lists were collected, duplicates were removed, and the list was saved for further analysis. Next, we combined the compound-target list with the disease-target list to generate a Venn diagram. Overlapping targets among these 2 lists were identified, and the compound-target network of MC for COP therapy was constructed and presented through Cytoscape (version 3.9.1).21

Protein-Protein Interaction and Compound-Target Network Construction

Protein-protein interaction (PPI) network construction was analyzed on the Search Tool for the Retrieval of Interacting Genes (STRING, version 11.0).22 After inputting the overlapping targets in the STRING database, a threshold score value of 0.9 was set to provide a high level of confidence for protein interactions. A map of the PPI network was constructed, while nonconnected proteins were eliminated. The PPI data were then put into Cytoscape for visualization. A novel Cytoscape plugin CytoNCA 2.1.6 topological analysis method was used for ranking essential proteins in the PPI network by calculating degree, betweenness, and closeness centrality.23

Functional Enrichment Analysis

To further explore the function of the overlapping targets, we used the online tool Webgestalt (www.webgestalt.org) to analyze the gene ontology (GO) term (including biological process [BP], cellular component [CC], and molecular function [MF]) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), and the results were visualized through dplyr24 and ggplot2 packages in R software (version 4.1.3).

Molecular Docking Analysis

The protein’s crystal structure was obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) database25 and preprocessed using the Protein Preparation Wizard module of Schrödinger suite 2020-3. Meanwhile, the LigPrep module of Schrödinger was used to process the 2-dimensional (2D) structures of compounds and generate all 3-dimensional chiral conformations. After this, the Receptor Grid Generation module of Schrödinger was used to obtain the protein’s active site, and the compound was docked with the active site at the Glide extra precision (XP) level. Finally, the binding of compounds with low-docking scores to the protein were analyzed by the molecular mechanic energies with generalized born and surface area (MM-GBSA). Binding energy less than –7.0 kcal/mol indicated strong binding activity, binding energy between –5.0 and –7.0 kcal/mol indicated good binding activity, and less than –4.25 kcal/mol indicated low binding ability for the target.16

Oral Mucosa LPS Injection

Orofacial inflammation was induced by unilateral oral mucosa LPS injection according to previously described with a minor modification.5,26 Briefly, animals were anesthetized with isoflurane and hold the mouth open with a mouth opener. LPS 25 μg in a volume of 50 μL normal saline was injected into the right-side intraoral maxillary submucosa of the mouth through a 30-G needle. After the injection, animals were placed in an observation box until complete recovery.

Intraganglionar Injection

Intraganglionar TG injection was performed according to Hummig et al26 with minor adaptations. Seven days after oral mucosa LPS injection, animals with significant orofacial mechanical hyperalgesia were anesthetized with isoflurane. A 27-G needle connecting to a Hamilton syringe (10 μL) was carefully injected into the right-side infraorbital foramen with a 10° relative to the median of the head until reaching the TG. The drug (quercetin 10 mM in 10 μL 10% dimethyl sulfoxide (DMSO) or vehicle) was then slowly delivered. The needle was carefully removed 5 minutes after the injection, and animals were transferred to an observation box for recovery.

Orofacial Mechanical Allodynia Assessment

Orofacial mechanical allodynia assessment was evaluated through Von Frey filaments as previously reported.5 Briefly, rats were gently restrained by a soft cotton glove. After habituation, a set of 8 calibrated nylon monofilaments (Danmic, Aesthesio) was used with increasing order of strengths (0.6–15 g), pressing on the animals’ right face near the whisker pad. Each filament was conducted 5 times perpendicularly with a 3-second interval between each application. Forepaw flitting, stroking, or head shaking were regarded as positive responses. The mechanical allodynia threshold was defined as the lowest force evoking at least 2 positive responses out of 5 stimuli.

Cell Culture

TG neurons were collected from 6- to 8-week-old male SD rats (150–200 g). Briefly, animals were anesthetized with isoflurane and decapitated. The acutely isolated TGs were digested in a mixture of enzyme (neutral protease + IA collagenase) at 37°C for 40 minutes, and centrifuged for 3 minutes (room temperature, 800 r/min). After the supernatant was discarded, TG neurons were resuspended in medium (90% DMEM, 10% FBS, 1% penicillin/streptomycin), inoculated on PDL (50 μg/mL)–pretreated glass crawl sheets, and cultured in the medium for further experiments. Four hours after the neurons were fully attached, LPS (100 ng/mL) with or without quercetin (20 μM) was administrated 72 hours for RT-qPCR, or overnight for patch clamp recording.

The primary cultured human periodontal ligament stem cells (hPDLSCs) were purchased from iCell Bioscience Inc (HUM-iCell-m002) and inoculated on 6-well plates in 1 ×106 per well density. All cells were cultured by iCell Primary Mesenchymal Stem Cells Serum-Free Media in an incubator at 37°C with 5% CO2 for further experiments. Twelve hours after the incubation, LPS (100 ng/mL) with or without quercetin (20 μM) was administrated 72 hours for RT-qPCR.

Real-Time Quantitative Polymerase Chain Reaction

After the cells were pretreated with LPS at a dose of 100 ng/mL or control for 72 hours, we used the RNAsimple Total RNA kit (Tiangen, DP419) to isolate the total RNA. After RNA extraction, we used PrimeScipt RT Master Mix to synthesize complementary DNA (cDNA) under 10 μL of qPCR reactions system: 1 μL cDNA (10 ng/μL), 0.2 μL primers (10 nM in final), 5 μL TB Green, and 3.6 μL diethyl pyrocarbonate (DEPC) water. Reactions were run in a LightCycler 480 qPCR instrument (Roche) using the standard conditions (95°C for 30 seconds), 40 cycles (95°C for 5 seconds, 60°C for 30 seconds, and 72°C for 30 seconds), and melting curve (95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds). Relative levels were quantified with the 2−ΔΔCT normalized to the control group. The primers used are listed in Supplemental Digital Content 1, Supplemental File 1, https://links.lww.com/AA/E558.

Whole-Cell Voltage Clamp Electrophysiology

Whole-cell voltage clamp recording was performed as previously described.27 Briefly, cell patch was conducted under a HEKA EPC 10-USB patch clamp amplifier (HEKA Instruments). Data were obtained by Patchmaster (HEKA) and analyzed with Fitmaster (HEKA). All experiments were performed at room temperature. The compositions of the extracellular and intracellular recording solutions are listed in Supplemental Digital Content 1, Supplemental File 1, https://links.lww.com/AA/E558. Current-voltage (I-V), activation, and inactivation recording protocols were conducted and are listed in Supplemental File 1, https://links.lww.com/AA/E558.

Data Analysis

Our analysis aimed at evaluating significant inhibition properties of quercetin on orofacial pain (primary outcome), proinflammatory factors (secondary outcome), and NaV channel (secondary outcome). To achieve this, we first conducted a normality test (Shapiro-Wilk test) on our original data of experiments for the selection of parametric/ nonparametric methods. Then, we analyzed orofacial mechanical hyperalgesia assessment data using the nonparametric Mann-Whitney test. To further investigate the mechanism of quercetin on COP alleviation, we performed a 1-way analysis of variance (ANOVA) test with Tukey’s multiple comparison on the mRNA expression level of hub genes in the RT-qPCR experiment. Then, we conducted a Kruskal-Wallis test with Dunn’s multiple comparison on peak current density of whole-cell clamp electrophysiology. A value of P < .05 was considered statistically significant in multiple comparisons.

Figure 1.:

Construction of the target genes set and compound-target network between MC and COP. A, Study flow diagram. B, Related genes from MC (blue) and COP (green) are intersected, and a total of 46 correlated genes is identified. C, The compound-target network consisting of 50 nodes and 62 edges, including 4 active components (hexagon) and 46 targets (circle). Arrows indicated interactions between compounds and targets. COP indicates chronic orofacial pain; MC, Moutan Cortex; MOL007374, 5-[[5-(4-methoxyphenyl)-2-furyl]methylene]barbituric acid.

Figure 2.: Quercetin alleviates LPS-induced orofacial mechanical allodynia. A, Chemical structure of quercetin (National Center for Biotechnology Information [2023]. PubChem Compound Summary for CID 5280343, Quercetin. Retrieved April 17, 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Quercetin). B, Flow chart of LPS-induced rat model of chronic orofacial pain construction and orofacial mechanical nociceptive assessment. Rats were treated intraoral with LPS 25 μg/50 μL to construct chronic orofacial pain (blue stick). Seven days after oral mucosa LPS injection, animals with significant orofacial mechanical hyperalgesia were selected for intraganglionar injection with quercetin (10 mM/10 μL) or vehicle (yellow stick). C, Mechanical allodynia threshold of adult male rats (n = 6) was measured repeatedly according to the flow chart (red stick). Data are expressed as median and interquartile range. Asterisks indicate statistical significance compared with vehicle treatment (**PFigure 3.:

Functional enrichment analysis of potential target genes of Moutan Cortex and quercetin in COP therapy. A, Gene ontology enrichment analysis for the compound-target network in biological process (BP, blue point), cellular component (CC, red point), and molecular function (MF, green point). B, KEGG pathways analysis for the compound-target network. COP indicates chronic orofacial pain; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 4.:

PPIs network construction and hub target genes screening. A, PPI network of the 46 selected targets. B, Topological analysis of the selected targets. Top 10 ranking simultaneously in BC, CC, and DC were utilized as screening criteria. Five hub targets, FOS, TNF-α, IL-10, STAT1, and TP53, were identified. BC indicates betweenness centrality; CC, closeness centrality; DC, degree centrality; FOS, fos proto-oncogene, AP-1 transcription factor subunit; PPI, protein-protein interaction; IL-10, interleukin 10; STAT1, signal transducer and activator of transcription 1; TNF-α, tumor necrosis factor alpha; TP53, tumor protein 53.

Figure 5.:

Transcriptional level of hub targets in both hPDLSCs and rat TGs treated with LPS or quercetin. Relative transcriptional level of FOS (A), TNF-α (B), IL-10 (C), STAT1 (D), and TP53 (E) in hPDLSCs 72 h after treatment with vehicle (0.1% DMSO), LPS (100 ng/mL), or LPS + quercetin (20 μM). Relative transcriptional level of FOS (F), TNF-α (G), IL-10 (H), STAT1 (I), and TP53 (J) in rat TG primary cultural neurons 72 h after treatment with vehicle (0.1% DMSO), LPS (100 ng/mL), or LPS + quercetin (20 μM). Data are presented as mean ± SEM. Asterisks indicate statistical significance compared with different treatments (*P < .05, **P < .01, ***P < .001, and ****P < .0001; 1-way ANOVA with Tukey’s multiple comparison, n ≥ 4 per condition). ANOVA indicates analysis of variance; DMSO, dimethyl sulfoxide; FOS, fos proto-oncogene, AP-1 transcription factor subunit; hPDLSCs, human periodontal ligament stem cells; IL-10, interleukin 10; LPS, lipopolysaccharide; Q, quercetin; SEM, standard error of the mean; STAT1, signal transducer and activator of transcription 1; TG, trigeminal ganglion; TNF-α, tumor necrosis factor alpha; TP53, tumor protein 53.

We estimated sample sizes using the PS Power and Sample Size Calculation software. For behavioral test, we could obtain a power greater than 0.80 by using 6 samples for each group, assumed that α=0.05 and the ratio of control sample to test sample=1. This was based on preliminary experiments that the difference of mechanical allodynia threshold between control and Quercetin group was 6, and SD = 3.4. Similarly, for RT-qPCR test, the estimated sample sizes were 3 samples for each group, based on preliminary experiments that the difference of fos proto-oncogene, AP-1 transcription factor subunit (FOS) expression between control and LPS group was > 0.3, and SD = 0.1. Besides, the estimated sample sizes of whole-cell voltage clamp electrophysiology were 7 samples for each group, based on preliminary experiments that the difference of peak current density between LPS and Quercetin group was 419.8, and SD = 280.0.

RESULTS Main Active Ingredients of MC and Potential Targets Screening and Network Construction

A flow diagram of this study is presented in Figure 1A. Through the TCMSP database and the screening strategy, we recognized 11 compounds with high activity as main active ingredients of MC (Supplemental Digital Content 2, Supplemental File 2, https://links.lww.com/AA/E559) and 171 associated targets after removal of duplicates (Supplemental Digital Content 3, Supplemental File 3, https://links.lww.com/AA/E560). Next, to explore potential targets for COP treatment, 1164 COP-related genes were acquired through the GeneCards database (Supplemental Digital Content 4, Supplemental File 4, https://links.lww.com/AA/E561). Finally, related targets from MC and COP were intersected and a total of 46 correlated targets were identified for compound-target network construction (Figure 1B), which consisted of 50 nodes and 62 edges, including 4 active components and 46 targets (Figure 1C and Supplemental Digital Content 5, Supplemental File 5, https://links.lww.com/AA/E562). Since quercetin is 1 of the active components that correlated with most of the related targets (41 of 46), we took this small-molecule natural compound under further investigation.

Quercetin Alleviated Mechanical Allodynia in LPS-Induced Rat Model of COP

Quercetin (C15H10O7, Figure 2A) is a flavonoid that has been reported with anti-inflammatory and analgesic properties.28 To investigate quercetin’s potential antinociceptive activity on orofacial pain, we selected the LPS-induced COP model, a commonly used inflammatory pain model characterized by trigeminal sensitization of the orofacial area.26 Rats received the mechanical nociceptive threshold measurement 0, 1, 3, and 7 days after oral mucosa LPS injection, and those that presented sustained mechanical allodynia until the 7th day were intraganglionar treated with vehicle or quercetin (10 mM/10 μL) (Figure 2B). As a result, after half an hour’s treatment, quercetin significantly alleviated the orofacial mechanical allodynia of rats (mechanical allodynia threshold median [IQR] 0.5 hours after drug administration: vehicle 1.3 [0.6–2.0] g vs quercetin 7.0 [6.0–8.5] g, P = .002, Mann-Whitney test). Such an effect can last for 3 hours (Figure 2C). These data demonstrated that quercetin is an efficient COP reliever.

Functional Enrichment Analysis of Potential Target Genes of MC and Quercetin in COP Therapy

To uncover the underlying mechanism of quercetin, the main active ingredient of MC, in COP therapy, we performed the functional enrichment analysis for the compound-target network in BP, CC, MF, and KEGG pathways. GO enrichment analysis indicated that these targets mainly enriched in regulation of oxidative stress process, membrane functions, and intracellular signal transduction (Figure 3A). In addition, KEGG pathways analysis reviewed these targets involved in cancer-related signaling pathways, IL-17 signaling pathway, and tumor necrosis factor (TNF) signaling pathway (Figure 3B). Among these results, we found that immune response and membrane functions play an essential role in COP therapy.

PPIs Network Construction and Hub Targets Screening

To identify hub targets among the compound-target network, we imported all 46 targets into the STRING database to acquire the PPI network (Figure 4A). Topological analysis was performed to screen hub targets. As a result, 5 targets (FOS, tumor necrosis factor alpha [TNF-α], interleukin 10 [IL-10], STAT1, and tumor protein 53 [TP53]) simultaneously ranked at top 10 in betweenness, closeness, and degree centrality (Supplemental Digital Content 6, Supplemental File 6, https://links.lww.com/AA/E563). This process is shown in Figure 4B, and information from the 5 core targets is listed in Supplemental Digital Content 7, Supplemental Table 1, https://links.lww.com/AA/E564.

In Vitro Experiments Verified FOS and TNF-α Are Potential Targets of Quercetin in COP Therapy

As 1 of the main pathogeneses of COP, LPS has been widely used to establish in vitro cell models of orofacial inflammatory disease.29,30 To verify these 5 genes are potential targets of MC in COP treatment, we performed an in vitro experiment to analyze the differential transcription of target mRNAs in LPS-preconditioned hPDLSCs and rats’ primary cultural TG neurons with or without treatment of the active component of MC, quercetin. As a result, LPS significantly increased the transcriptional level of FOS and TNF-α in both the hPDLSC cell line (Figure 5A, mean ± SEM, FOS: LPS [2.09 ± 0.15] vs control, P < .0001, vs LPS + quercetin [0.03 ± 0.01], P < .0001; TNF-α: LPS [2.67 ± 0.35] vs control, P = .0007, LPS + quercetin [0.23 ± 0.06], P < .0001, 1-way ANOVA test with Tukey’s multiple comparison) and rat TG neurons (Figure 5B, FOS: LPS [2.22 ± 0.33] vs control, P = .006, vs LPS + quercetin [1.33 ± 0.14], P = .034; TNF-α: LPS [8.93 ± 0.78] vs control, P < .0001, vs LPS + quercetin [3.77 ± 0.49], P < .0001, 1-way ANOVA test with Tukey’s multiple comparison), while administration of quercetin blunted such increments. These results not only verified FOS and TNF-α are potential targets of MC in trigeminal neuralgia therapy, but also indicated quercetin can improve the LPS-induced neuroinflammation in TG neurons. Thus, quercetin may play a pivot role in COP therapy by targeting FOS and TNF-α, and inhibiting the LPS-induced inflammatory response of hPDLSCs and TG neurons.

LPS-Induced Increment of Sodium Current in TG Sensory Neuron Is Blunted by Quercetin

To further explore whether quercetin acts through these targets directly to regulate signaling pathways involved in immune response, we performed molecular docking between these targets and quercetin. However, the binding affinity of quercetin was higher than −4.5 kcal/mol with both FOS and TNF-α, indicating poor binding interactions between quercetin and these targets (Supplemental Digital Content 8, Supplemental Figure 1, https://links.lww.com/AA/A1). Thus, quercetin showed unstable binding interactions with FOS and TNF-α, suggesting it may act on these 2 targets in an indirect way.

Figure 6.:

LPS-induced increment of Nav current in TG sensory neuron is blunted by quercetin. A, Representative traces of the Nav currents (−70 to 0 mV) from TG sensory neurons treated with 0.1% DMSO (control), LPS 100 ng/mL, or LPS with quercetin 20 µM. B, Current-voltage protocol for activation and inactivation. Currents were evoked by 200 ms pulse between −70 and +60 mV. Summary of the normalized (pA/pF) Nav current density versus voltage relationship (C) and peak Nav current density at −10 mV (D) from TG neurons treated as indicated. Boltzmann fits for normalized conductance, G/Gmax voltage relationships for voltage-dependent activation (E) and inactivation (F) sensory neurons. The half-maximal activation (V1/2) and slope values (k) for activation and inactivation are summarized in Supplemental Table 2. Data are expressed as mean ± SEM (C, E, F) or median and interquartile range (D). Asterisks indicate statistical significance compared with different treatments (**P < .01, Kruskal-Wallis test with Dunn’s multiple comparison). DMSO indicates dimethyl sulfoxide; LPS, lipopolysaccharide; Nav, voltage-gated sodium channel; pA, picoampere; pF, picofarad; Q, quercetin; SEM, standard error of the mean; TG, trigeminal ganglion.

As a critical component in generating action potentials and propagating nociceptive signaling, the voltage-gated sodium (Nav) channel plays an essential role in neuroinflammation progression. Hyperactivation of the Nav channel contributes to excessive release of major proinflammatory cytokines TNF-α and FOS.31,32 Thus, we hypothesized that quercetin might blunt the expression of FOS and TNF-α via inhibiting Nav activation. To consolidate our theory, we evaluated the property of Nav currents with or without quercetin treatment in LPS-pretreated TG nociceptive neurons. Representative traces of Nav currents from TG neurons treated with different conditions and related current-voltage protocols are shown in Figure 6A, B. Compared with the control group (0.1% DMSO), LPS increased Nav current density with a ~2.0-fold change in peak current density. Such an increment was blunted by quercetin administration (Figure 6C, D, median [IQR], control = −449.0 [−584.7 to −365.0] mV, LPS = −850.2 [−983.6 to −660.7] mV, LPS + quercetin = −589.6 [−711.0 to −147.8] mV, LPS vs control, P = .003, LPS vs LPS + quercetin, P = .006, Kruskal-Wallis test with Dunn’s multiple comparison). To rule out whether the channel gating property was modified by quercetin, a possible cause of the inhibitory effect on TG Nav currents, we recorded the activation and inactivation properties of TG Nav currents. As a result, quercetin significantly triggered a rightward shift of the activation curve of the Nav channel (Figure 6E, F, Supplemental Table 2, https://links.lww.com/AA/E566). Altogether, these findings suggested that quercetin, 1 of the main active ingredients of MC, plays a pivot role in COP therapy through blunting the increment of Nav current density in TG sensory neurons and inhibiting the LPS-induced inflammatory response of hPDLSCs and TG neurons.

DISCUSSION

In this study, we discovered 2 salient findings: (i) the small-molecule natural flavonoid quercetin is 1 of the active components of MC that exerts prominent effects in LPS-induced COP therapy, and (ii) quercetin targets Nav channel to achieve this effect on anti-inflammation and chronic pain alleviation. We summarized the above conclusions by noting (i) the network pharmacology analysis reviewing quercetin’s high activity and broad interactions with the component-target network, and (ii) the prominent blocking effect of quercetin on the Nav channel in TG sensory neurons and the significant inhibition property on proinflammatory cytokines.

TCMs are major components of complementary and alternative medicine that play essential roles in various diseases. Although plenty of TCM decoctions and herbs have shown promising therapeutic effects, their complex compositions and undefined mechanisms limit their clinical application. MC has been broadly used as a traditional herbal medication in East Asia for thousands of years. Although a wide variety of pharmacological effects of MC have been reported, it is hard to screen out the practical active components with potential treatment for COP through in vivo and in vitro experiments. To address this, taking advantage of various computational and simulated approaches, network pharmacology is ideal for evaluating the numerous component-target interactions, biological pathways, and networks that accelerate the development of safe and effective TCM drugs for various diseases. Herein, we identified 4 primary active components of MC, quercetin, kaempferol, (+)-catechin, and 5-[[5-(4-methoxyphenyl)-2-furyl]methylene] barbituric acid, that presented therapeutic potential in the treatment of COP. Among those components, quercetin, kaempferol, and (+)-catechin correlated with most of the related targets and are all flavonoids, which have been verified in previous studies to possess potent anti-inflammatory and analgesic effects through decreasing inflammatory cytokines and blocking of the TRPV1 channel.15,33,34 This suggests that flavonoids may exert crucial roles in the treatment of COP.

As an active flavonoid constituent isolated from MC, quercetin has been reported to involve a variety of BPs including inflammation, apoptosis, and cancer therapy.35,36 Moreover, recent studies also demonstrate that quercetin is a potential inflammatory pain killer. Hannan et al reported that quercetin ameliorates adjuvant-induced arthritis through regulating antioxidant enzymes and inhibiting proinflammatory cytokines.14 Ji et al demonstrated that quercetin exerts anti-inflammatory effects and alleviates neuropathic pain by inhibiting toll-like receptor signaling pathway.37 Meanwhile, in our in vivo and in vitro experiments, quercetin not only was identified as 1 of the MC active components that correlated with most of the related targets, but also presented prominent effects on anti-inflammation and pain alleviation. Therefore, we hypothesize that quercetin may relieve COP by targeting proinflammatory cytokines.

As neuronal activation and proinflammatory markers,38 FOS and TNF-α are 2 of the 5 hub targets that are highly expressed in both LPS-pretreated hPDLSCs and rat TG neurons, and can be reduced by quercetin administration. We performed molecular docking between these targets and quercetin to evaluate whether quercetin exerts its analgesic effect by directly targeting on FOS and TNF-α. Nevertheless, quercetin has shown unstable binding interactions with both FOS and TNF-α, suggesting it may act on the regulation of COP inflammation in an indirect way.

GO and KEGG pathway analysis identified COP pathways related to immune regulation, intracellular signaling transduction, and membrane functions. As a transmembrane protein, the Nav channel plays fundamental roles in initiating and propagating action potentials in peripheral electrically excitable neurons, and involves abnormal pain signaling transmission.39 The function of Nav is regulated by membrane endocytosis,40 while hyperactivation of the Nav channel contributes to excessive release of major proinflammatory cytokines such as TNF-α.31 Thus, based on these findings, we investigated quercetin’s effect on the Nav channel in rat TG neurons to uncover the connection between quercetin and these proinflammatory cytokines. As expected, quercetin significantly blunted the LPS-induced increment of Nav currents. Such ion channel inhibiting property of quercetin has also been reported in a study showing that local administration of quercetin inhibited the Nav channel on nociceptive primary sensory neurons in the TG of naïve rats.12 These findings may uncover a possible mechanism through which quercetin may alleviate COP via inhibiting the hyperactivation of Nav and inflammatory response.

Several limitations exist in this study. First, Ca2+ and K+ channels are 2 other voltage-gated channels that play crucial roles in pain signaling progression, while the effect of quercetin on these channels remains unknown. Second, although we summarized quercetin’s inhibiting property on the Nav channel through patch recording, how quercetin directly or indirectly acts on Nav and its subtype channels has remained unclear. Third and last, although quercetin shows a prominent analgesic ability on rats, whether it has equivalent efficacy on humans should be further consolidated through clinical trials.

To sum up, we uncovered evidence supporting the active ingredient of MC-quercetin’s analgesic property on treatment of inflammatory orofacial pain disease. Identifying Nav as the molecular target of quercetin clarifies the analgesic mechanism of MC, and provides ideas for the development of novel selective and efficient chronic pain relievers.

DISCLOSURES

Name: Zhanli Liu, MD.

Contribution: This author helped in study design, conducting experiments, and data analysis.

Name: Zhiming Shan, MD.

Contribution: This author helped in study design, conducting experiments, data acquisition, data analysis, and article preparation.

Name: Haoyi Yang, MPhil.

Contribution: This author helped in conducting experiments, data acquisition, and data analysis.

Name: Yanmei Xing, MPhil.

Contribution: This author helped in conducting experiments and data analysis.

Name: Weijie Guo, MPhil.

Contribution: This author helped in conducting experiments and data analysis.

Name: Jing Cheng, MD.

Contribution: This author helped in data analysis.

Name: Yuanxu Jiang, MD, PhD.

Contribution: This author helped in data analysis.

Name: Song Cai, PhD.

Contribution: This author helped in study design and article preparation.

Name: Chaoran Wu, MD, PhD.

Contribution: This author helped in study design and data interpretation.

Name: Jessica Aijia Liu, PhD.

Contribution: This author helped with data interpretation and article revision.

Name: Chi Wai Cheung, MD, PhD.

Contribution: This author helped in study design and article revision.

Name: Yunping Pan, MD, PhD.

Contribution: This author helped in study design, data analysis, article revision, and study supervision.

This manuscript was handled by: Jianren Mao, MD, PhD.

ACKNOWLEDGMENTS

The authors are grateful to Zhongjun Zhang for excellent technical assistance, Xiongxiong Zhong for cell culture, and Xiying Chen and Qiuyin Xu for animal care. The authors also thank the Center of Pharmaceutical Technology of Tsinghua University for the support of Schrödinger software and molecular docking. All schematic plots in figures were created with BioRender.com.

REFERENCES 1. Zheng W, Huang X, Wang J, et al. The chronification mechanism of orofacial inflammatory pain: facilitation by GPER1 and microglia in the rostral ventral medulla. Front Mol Neurosci. 2022;15:1078309. 2. Iwata K, Sessle BJ. The evolution of neuroscience as a research field relevant to dentistry. J Dent Res. 2019;98:1407–1417. 3. Handa S, Keith DA, Abou-Ezzi J, Rosen A. Neuropathic orofacial pain: characterization of different patient groups using the ICOP first edition, in a tertiary level orofacial pain clinic. Oral Surg Oral Med Oral Pathol Oral Radiol. 2021;132:653–661. 4. Crescente G, Minervini G, Spagnuolo C, Moccia S. Cannabis bioactive compound-based formulations: new perspectives for the management of orofacial pain. Molecules. 2022;28:106. 5. Aczel T, Kun J, Szoke E, et al. Transcriptional alterations in the trigeminal ganglia, nucleus and peripheral blood mononuclear cells in a rat orofacial pain model. Front Mol Neurosci. 2018;11:219. 6. Shinoda M, Kubo A, Hayashi Y, Iwata K. Peripheral and central mechanisms of persistent orofacial pain. Front Neurosci. 2019 ;13 :1227. 7. Aczel T, Kecskes A, Kun J, et al. Hemokinin-1 gene expression is upregulated in trigeminal ganglia in an inflammatory orofacial pain model: potential role in peripheral sensitization. Int J Mol Sci . 2020;21:2938. 8. Uemura Y, Hiroshima Y, Tada A, et al. Porphyromonas gingivalis outer membrane vesicles stimulate gingival epithelial cells to induce pro-inflammatory cytokines via the MAPK and STING pathways. Biomedicines. 2022;10:2643. 9. Khan FA, Ali G, Rahman K, et al. Efficacy of 2-hydroxyflavanone in rodent models of pain and inflammation: involvement of opioidergic and GABAergic anti-nociceptive mechanisms. Molecules.