Annali di Stomatologia | 2026; 17(2): 417-424

ISSN 1971-1441 | DOI: 10.59987/ads/2026.2.417-424

Articles

Salivary glutamate and substance P response to ear acupuncture in healthy and temporomandibular disorders myalgia subjects: a pilot study

Emanuela Serritella^1–3, Hajer Jasim^1–4, Bijar Ghafouri², Carlo Di Paolo³, Malin Ernberg¹

¹Department of Dental Medicine, Karolinska Institutet, and the Scandinavian Center for Orofacial Neuroscience (SCON), Huddinge, Sweden

²Pain and Rehabilitation Center and Department of Health, Medicine and Caring Sciences, Linköping University, Linköping, Sweden

³Department of Oral and Maxillofacial Sciences, Sapienza University, Rome, Italy

⁴Department of Orofacial Pain and Jaw Function, Eastman Institute, Folktandvården AB, Stockholm, Sweden

^*Corresponding author: Emanuela Serritella - emanuela.serritella@uniroma1.it

Article History

Received: May 7, 2026

Accepted: June 15, 2026

Published: June 30, 2026

Abstract

Aim

Auricular acupuncture (AA) treats pain-related temporomandibular disorders (TMD). Studies have suggested gene and protein expression variations after acupuncture; however, the salivary protein response to AA in subjects with and without TMD has not been investigated. This study aimed to analyze salivary glutamate and substance P (SP) responses to AA in healthy and TMD myalgia subjects and to explore the effectiveness of AA on this type of pain.

Methods

This study included 30 healthy participants and 10 patients with myalgia caused by TMD. Healthy participants were randomly assigned to one of the following three groups (10 subjects per group): real AA, sham AA, or no treatment. Patients with TMD underwent actual AA treatment. Stimulated whole saliva was collected to analyze glutamate and SP levels before (T0) and 1 week after (T1) the intervention. Current, average, and worst pain intensities were assessed using 0–10 numeric rating scale (NRS).

Results

Two-way repeated-measures ANOVA did not reveal significant differences between groups or time points for glutamate or SP levels (p > 0.05). There were no differences in background variables between the groups (p > 0.05), but the characteristic pain intensity differed significantly between the patients and healthy subjects (p < 0.001). The pain intensity of the NRS in the TMD group decreased significantly after the AA administration (p < 0.01).

Conclusions

There was no difference in salivary glutamate and SP across levels after the one-week intervention in any group, despite a decrease in pain intensity in TMD patients.

Keywords: Temporomandibular disorders, myalgia, TMJ, pain, acupuncture, auriculotherapy, saliva, biomarker, glutamate, substance P.

Introduction

Temporomandibular disorders (TMD) are widespread worldwide and are characterized by complex signs and symptoms, representing the main form of musculoskeletal pain in the head and face region [1–2]. Concerning pain therapy related to TMD, different treatment modalities have obtained various degrees of acceptance in the literature, e.g., including occlusal appliances, drugs, physical therapy, and, last but not least, acupuncture [3–5].

Auricular Acupuncture (AA) represents an acupuncture technique that normalizes the body’s pain and dysfunction by stimulating points on the ear [6]. Several studies show that the stimulation of the acupoints determines a variation in gene and protein expression in treating neuropathic pain and various nerve pathologies [14–15]. Multiplex panels have been developed recently to allow simultaneous analysis of several markers, allowing a better understanding of the pathophysiological mechanisms underlying different pathologies. Studies concerning the proteomic analysis of saliva of subjects suffering from TMD myalgia and myofascial pain have shown an overexpression of various proteins involved in metabolic processes, in the immune response, and in the stress response, some of which (PGK1, GAPDH, sAA, CRISP3, FABP) have been proposed as candidates to assess pain in these patients objectively [10–19]. However, these studies highlight the lack of a significant correlation between the expression of these proteins and the clinical characteristics of myalgia, and there are no studies in the literature that evaluate the release of salivary markers in response to AA in subjects suffering from TMD-related pain.

Based on this evidence, we decided to undertake this pilot study as a collaboration between the Karolinska Institute in Huddinge, Sweden, and Sapienza University in Rome, Italy, with the following main purposes: 1) to analyze the response of salivary glutamate and substance P (SP) levels to AA stimulation in a group of healthy subjects and compare it with sham AA and no treatment, and 2) to explore whether AA can be an effective acupuncture method in patients with TMD myalgia through a better understanding of its neuromodulatory effects.

Materials and Methods

Healthy participants and TMD patients, both male and female, were recruited at the Specialist Clinic for Orofacial Pain and Jaw Function at the University Dental Clinic, Karolinska Institutet, Huddinge. The study protocol followed good clinical practice and guidelines according to the Declaration of Helsinki, and the STRICTA (Standards for Reporting Interventions in Clinical Trials of Acupuncture) criteria [20–21].

The inclusion criteria for healthy pain-free participants were age > 18 years and no known significant health problems; for patients aged > 18 years, a diagnosis of TMD myalgia according to the Diagnostic Criteria for TMD (DC/TMD) [1] and a minimum characteristic pain intensity (CPI) of 40/100. Exclusion criteria for both groups were: 1) current acute or chronic pain (except TMD pain for patients), 2) systemic inflammatory disease (e.g., rheumatoid arthritis), 3) severe psychiatric disease (e.g., schizophrenia, bipolar disorder), 4) pregnancy; and 5) current analgesic medication or other drugs that can affect the pain experience (e.g., anxiolytic drugs).

General procedure

The study was conducted between March 1 and August 31, 2021, and consisted of two parts. The first part was a randomized, participant-blinded, case-control study encompassing 30 healthy, pain-free participants, and the second part was an exploratory, non-blinded observational study encompassing patients with TMD myalgia. All experiments were performed in the morning with the patient seated upright on a conventional dental chair. The participants completed validated questionnaires [1], a clinical examination according to the DC/TMD was conducted, and saliva was collected to evaluate salivary marker changes before (T0) and one week after treatment (T1).

Part I

After a clinical examination confirmed that the participants had no TMD-related pain, they were randomly allocated into three groups using a random generator (www.randomization.com):

Study Group (SG): AA (N=10)
Placebo Group (ShG): sham AA (N=10)
Control Group (CG): No treatment (N=10)

Part II

This study included patients with TMD myalgia (N=10) who were treated with AA in the same manner as the SG group.

Auricular acupuncture and sham treatment

The treatment protocol was elaborated and defined according to Traditional Chinese Medicine (TCM) principles of treatment methods and point selection and according to the basis of auricular anatomy, physiology, and innervation [6,11–12,22]. The AA treatment was performed by a single experienced licensed acupuncturist (E.S.). The points used are shown in Figure 1A. After disinfection of the ear surface, the reactive point was identified and a thumb-tack needle was applied immediately afterward and fixed to the skin with a bandage. (Figure 1B-C) This procedure was repeated for all the points to be treated. The thumbtack needle remained in place for seven days after the application and was then removed by the same acupuncturist; the points where the needles were placed were pressed repeatedly every four hours by the participant for seven days. This technique was taught to all the participants, and it was confirmed that the patients correctly performed it at T1. The ShG group underwent the same procedure using the same auricular points, but the bandage did not have a thumbtack needle; a toothpick was used to create the sensation of needle perforation [23]. The CG group received no treatment and was included to observe the normal fluctuation of salivary markers.

**Figure 1:** (A) Map of auricular points used: (1) Shenmen; (2) Liver; (3) Spleen; (4) Gallbladder; (5) Occiput; (6) Temple. Example of (B) sensitive points research and of (C) thumbtack needle application.

Saliva sampling and Biochemical analysis

This procedure was conducted twice for each subject who participated, before (T0) and one week after (T1) the application of AA treatment. At both time points, 5 ml of the salivary fluid was collected. Stimulated whole saliva was collected in the morning using a standardized protocol, and the samples were thawed, blinded, and analyzed randomly for glutamate and SP, as described previously [17–19]. Analyses were performed at the Painomics Laboratory in Linköping, Sweden.

Statistical Analyses

The normality of the data distribution was checked using the Shapiro-Wilk test. In Part I, comparisons were made between the groups (SG, ShG, and CG), and in Part II, patient data were compared with the SG data from Part I. A chi-square test was used to analyze differences in frequencies in the background data and sex of the study participants. One-way ANOVA was used to compare age and BMI between groups in Part I, whereas Student’s t-test was used for these data in Part II. The Kruskal-Wallis test (Part I) or Mann-Whitney U-test (Part II) were used to compare axis II data among the groups. The Wilcoxon test was used in Part II to assess whether there were significant differences in the patient’s pain levels before and after treatment. A two-way repeated-measures ANOVA was used with group (SG, ShG, and CG in Part I; patients and SG in Part II) and time (T0, T1) as factors to analyze the differences in salivary markers. Data was analyzed using the Statistical Analysis System (SAS) software (SAS Institute Inc., Cary, NC, USA). For all tests, the significance level was set at p < 0.05.

Results

The background data of the participants and questionnaire results are presented in Table 1.

Table 2 lists the concentrations of Glutamate and SP. Glutamate was analyzed in all samples, whereas SP was analyzed in 71 of the 80 samples.

Table 1. Overview of the participants’ general characteristics of Parts I-II and questionnaires’ scores.

Variable	Healthy (Part I)			p-value 1	TMD (Part II)	p-value 2
Variable	SG	ShG	CG	p-value 1		p-value 2
Gender (n of F)	7	5	7	0.563	6	0.639
Age (yrs)	35.7 (± 7.9)	30.80 (± 7.3)	28.1 (± 5.8)	0.068	35.2 (± 7.4)	0.443
BMI (kg/m2)	24.5 (± 4.5)	25.1 (± 5.6)	22.6 (± 4.8)	0.382	24.6 (± 4.0)	0.125
CPI (0–100)	0	0	0	NA	64.3 (19.9)	<0.001
OBC-6 (0–24)	5.5 (3.5)	3.0 (4.5)	5.5 (8.8)	0.306	6.5 (5.3)	0.568
JFLS-8 (0–10)	0.1 (1.0)	0.0 (0.8)	0.1 (0.3)	0.453	0.4 (5.5)	0.129
PHQ-4 (0–12)	0.5 (4.5)	1.0 (2.3)	1.5 (4.3)	0.539	2.0 (3.3)	0.460
PSS-4 (0–16)	2.0 (4.8)	2.0 (5.3)	4.0 (5.8)	0.550	3.5 (5.5)	0.620
OHIP-5 (0–20)	0.0 (1.5)	0.0 (2.3)	1.0 (2.3)	0.686	3.5 (2.8)	0.032
GCPS (n)
Grade 0	10	10	10		0
Grade I	NA	NA	NA		5
Grade II	NA	NA	NA		2
Grade III	NA	NA	NA		1
Grade IV	NA	NA	NA		2

SG, study group (acupuncture; N=10); ShG = Sham group (sham acupuncture; N=10); CG = Control group (no acupuncture; N=10); TMD= Myalgia patients (acupuncture; N=10). BMI, body mass index; CPI = Characteristic Pain intensity; OBC, oral behavior checklist; JFLS, jaw functional limitation scale; PHQ, Patient History Questionnaire; PSS, Perceived Stress Scale; OHIP, Oral Health Impact Profile; GCPS, Graded Chronic Pain Scale.

Data are presented as number (sex, GCPS), mean (± SD), or median (IQR). P-value 1 shows comparisons between groups in Part I, and P-value 2 shows comparisons between the TMD and SG groups in Part II. Statistical comparisons (p < 0.05) were performed with a chi-square test for sex, a one-way ANOVA or Kruskal-Wallis test for all other comparisons in Part I, and a Student’s t-test or Mann-Whitney U-test for other comparisons in Part 2.

Table 2. Concentration of Glutamate and SP before (T0) and after (T1) treatment with acupuncture (SG; N=10), sham acupuncture (N=10), and no treatment (N=10) in healthy individuals (Part I) and in temporomandibular disorders (TMD) myalgia patients (N=10) after acupuncture treatment (Part II).

	Glutamate (nM)			Substance P (pg/ml)
Part I		T0	T1	T0	T1
	SG	19.2 (22.1)	13.2 (10.0)	33.3 (24.2)	28.3 (9.2)”
	ShG	19.7 (15.1)	20.8 (16.0)	29.5 (29.0)”	35.8 (25.5)
	CG	17.1 (12.6)	16.2 (8.4)	32.5 (23.3) #	46.3 (40.2) #
Part II
	TMD	16.3 (7.0)	19.7 (12.5)	38.9 (18.4)	42.6 (28.4)

SG, study group; ShG = Sham group; CG = Control group. ” n=9; # n=8.

Data are presented as mean (SD). There were no significant treatment effects between groups or with time for Part I or Part II (two-way repeated-measures ANOVA, p > 0.05).

In Part I, two-way ANOVA for glutamate showed no significant effects for either group (F = 0.27, p = 0.766) or time (F = 0.89, p = 0.367). Similarly, there were no significant effects of group (F = 0.01, p = 0.999) or time (F = 0.39, p = 0.424) on the SP.

In Part II, the comparison before and after AA between patients with TMD and controls (SG), two-way ANOVA showed no effect of group or time for either glutamate (group: F = 0.16, p = 0.691; time: F = 0.09, p = 0.766) or SP (group: F = 1.36, p = 0.253; time: F = 0.17, p = 0.683).

In Part II, the patient’s pain perception significantly decreased from baseline (T0) to the end of therapy (T1) (p = 0.009). Figure 2A-B shows the patients’ pain perception at T0 and T1 and the pain trend during the seven days of therapy.

**Figure 2:** A) Pain values of myalgia patients before (T0) and after (T1) AA treatment (NRS); B) pain trend during 7 days of AA therapy in myalgia patients (NRS).

Discussion

The present study is the first to evaluate the release of glutamate and SP in saliva in response to AA in healthy subjects and patients with TMD myalgia.

It has been shown that both glutamate and SP appear to increase with noxious stimulations and after tissue and/or nerve injuries and a previous study showed that patients with TMD myalgia have a higher salivary concentration of glutamate than healthy subjects [19] Salivary levels of SP, however, were not different from healthy controls [24]. However, the results obtained in this study do not appear to be consistent with these findings since glutamate and SP concentrations at baseline did not differ between patients and healthy subjects. One possible explanation may be the great variability in the expression of these proteins and the small sample size of the present study, which makes a systematic comparison with the reference study needs to be revised.

Very few studies have investigated the fluctuation of glutamate and SP in relation to acupuncture treatments, and none of them used salivary fluid as a detection tool [25–27]. Concerning SP, both studies of Karatay et al. and Mohammed et al. reported reduced serum levels of SP after acupuncture treatment, in groups of patients with fibromyalgia and with knee osteoarthritis, respectively [26–27]. Thus, these results do not agree with the present study’s results.

A single MRI study investigated the relationship between changes in glutamate levels within the insula and in multiple pain domains in patients with fibromyalgia in response to acupuncture and reported an increase in glutamate levels within the insula after treatment, which was related to a decrease in pain domains [25]. The results align with those of the present study regarding clinical variables since patients with TMD myalgia treated with AA reported a significant improvement in pain perception after treatment. However, glutamate levels did not coincide.

The different results between studies may be due to the short duration of AA (1 week), whereas in clinical practice, acupuncture treatment lasts 3–4 weeks on average. However, given the characteristic efficacy of the ear microsystem in treating pain in a very short time [23–24], as shown in this study in patients with TMD myalgia, we hypothesized that any molecular changes would be measurable after this short time. Since this was not the case, the reduction in pain levels may have other explanations; for example, the altered release of other pain markers, such as serotonin, NGF, CGRP, NPY, and VIP, which were not analyzed in this study, or that AA does not influence the salivary levels of algesic biomarkers. Of course, one cannot rule out the placebo effect on pain because the patients were aware of the treatment they received. Furthermore, as a pilot test, the sample size per group was very small (10 subjects per group). Given the high individual variability in the composition of the salivary proteome, a type II error cannot be ruled out. Another factor to consider is the clinical characteristics of the patients with TMD myalgia. They presented low pain interference values (GCPS), and the comparison with controls through the questionnaires was insignificant for all aspects investigated except for OHIP-5. This could also be a limitation because patients cannot be differentiated from healthy subjects due to the results’ generalizability.

However, given the different trends of salivary values of glutamate and SP depending on the intervention (AA, sham AA, or no treatment) and the contrast of this trend between patients with TMD and healthy controls who underwent the same treatment, further studies should be carried out involving the application of AA for longer times to verify the obtained results and explore the possible molecular mechanisms underlying acupuncture treatments.

Conclusions

This study showed no difference in the levels of salivary glutamate and SP after one week of AA stimulation in healthy subjects and patients with TMD myalgia. In addition, AA was effective in decreasing TMD myalgia-related pain; however, no relationship was detected between pain reduction, salivary glutamate, and SP expression. The results suggested different glutamate and SP salivary fluctuation trends according to different interventions (real AA, sham AA, and no treatment). Considering the significant variability in salivary protein expression and AA’s tremendous timing and application methods, the need for further investigations with a larger sample size and longer treatment time is highlighted.

Author’s contribution

E.S.: Conceptualization, Methodology, Investigation, Data Curation, Writing – Review & Editing. H.J.: Investigation, Methodology, Data curation; Editing. B.G.: Data Curation & Formal Analysis, Editing. C.DP.: Conceptualization, Methodology, Review & Editing. M.E.: Conceptualization, Methodology, Writing – Review & Editing and Supervision.

Funding

This study was supported by PhD international mobility projects grant of Sapienza University of Rome (2020) and by the “Academy Research Call 2021” of Sapienza University of Rome (00616_22).

Conflicts of interest

The authors declare that there are no conflicts of interest.

Ethical Approval

All participants received careful information regarding the aims and procedures of the study and signed an informed consent form before participation. The Swedish Ethical Review Authority (2021-00427, 2021-03-24).

Data availability Statement

The data that supports the findings of this study are available upon reasonable request from the corresponding author. The data is not publicly available due to privacy and ethical restrictions.

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