Evaluation of salivary nitric oxide levels and anxiety in multiple sclerosis patients, with and without Xerostomia: correlation with clinical variables.

Introduction

Multiple sclerosis (MS) is a prevalent chronic inflammatory disease of the central nervous system (CNS) that affects over 2 million individuals worldwide and is the leading cause of neurological impairment in young adults [ 1 – 4 ]. MS profoundly impacts individuals in various aspects, including personal, family, social, and professional life [ 5 ]. MS has a notable effect on oral health, particularly through the development of xerostomia [ 6 , 7 ]. Xerostomia significantly reduces the protective effects of saliva, increasing the risk of dental caries and periodontal disease [ 8 ]. A study utilizing ultrasonography on salivary glands revealed that the parotid and submandibular gland tissues in MS patients exhibited reduced homogeneity compared to the control group. Additionally, the salivary flow rate in MS patients was significantly decreased, leading to an increased risk of dental caries and oral hygiene issues [ 9 ]. Xerostomia is the subjective feeling of dry mouth, which may occur without reduced salivary flow, while hyposalivation is an objective decrease in saliva production that can alter the oral environment and increase the risk of dental and periodontal diseases [ 10 ]. Additionally, patients with MS face challenges in maintaining proper oral hygiene. Factors such as muscle weakness, fatigue, pain, and lack of coordination hinder their ability to effectively clean their teeth and gums, further exacerbating the effects of xerostomia [ 11 ]. As a result, the combination of xerostomia and difficulties in maintaining oral hygiene leads to a heightened risk of dental decay and gum disease in individuals with MS.

Xerostomia, common in MS, is also a key symptom of primary Sjögren’s syndrome. Some MS patients meet Sjögren’s syndrome criteria, suggesting shared immune mechanisms and pathological overlap [ 12 , 13 ]. Medications used to manage its symptoms can contribute to the development of xerostomia [ 14 – 16 ]. Xerostomia in MS patients is primarily caused by the side effects of medications such as anticholinergics, muscle relaxants, antidepressants, and immunomodulators, which are prescribed to manage symptoms and slow disease progression. These drugs reduce saliva production, making them the main cause of xerostomia in MS patients [ 16 ]. In patients with MS, changes in the composition of saliva may result from immune and inflammatory disorders observed in these individuals [ 17 ]. For example, alterations in the protein and electrolyte contents of saliva can reflect inflammatory and metabolic activities in the body [ 18 , 19 ]. These changes not only affect oral and dental health but also may contribute to the exacerbation of other symptoms of the disease. An increase in nitric oxide (NO) levels may indicate metabolic disorders and treatment responses in various tissues of the body. Consequently, these changes could be used to support therapeutic improvements and interventions [ 20 ].

The elevated NO levels observed in MS lesions are attributed to the release of NO by macrophages and astrocytes, which are specialized immune cells within the CNS [ 21 ]. Previous research has reported elevated NO levels in the cerebrospinal fluid, blood, and urine of individuals with MS [ 22 – 25 ]. NO is particularly important in MS because of its association with disruptions in metabolic energy and mitochondrial dysfunction in oligodendrocytes, which are key contributors to disease progression [ 26 – 28 ]. Its multifaceted role in MS warrants further investigation, as it presents promising opportunities for the development of novel diagnostic, preventive, and therapeutic strategies for demyelinating disorders [ 29 , 30 ]. Moreover, NO plays a vital role in salivary gland function and secretion, serving as an essential signaling molecule in the maintenance of oral health [ 31 , 32 ].

NO helps regulate salivary gland function by controlling salivary secretion and blood flow [ 31 , 33 ]. NO improves the activity of cells in the salivary glands, which are responsible for releasing fluids and electrolytes [ 34 ]. It also protects the salivary glands from damage caused by oxidative stress and inflammation. Studies show that NO is necessary for the proper response of salivary glands in both normal and disease conditions [ 34 ]. If NO signaling is disrupted, it can reduce saliva production and lead to problems in the glands’ function [ 35 ].

Stress, particularly psychological stress, is recognized as an exacerbating factor in MS and can influence immune responses [ 36 ]. NO, a key signaling molecule in inflammation and oxidative stress, plays a role in the pathogenesis of MS, with elevated levels associated with disease activity [ 37 ]. Since salivary NO serves as a non-invasive biomarker for assessing inflammation and oxidative stress, its investigation in MS patients can provide valuable insights [ 38 ]. Xerostomia is a common comorbidity in MS patients, often linked to autonomic dysfunction or medication side effects [ 9 , 39 , 40 ], and can alter saliva composition, including NO levels. Research indicates that individuals with xerostomia, due to lower NO levels, are more susceptible to dental caries and gum disease [ 41 , 42 ]. Additionally, reduced salivary NO concentrations are associated with xerostomia symptoms and may serve as a predictor for this condition [ 43 , 44 ]. Changes in the oral microbiome due to xerostomia can also impair NO synthesis, as beneficial oral bacteria responsible for converting nitrate to NO are affected [ 45 , 46 ]. This dysbiosis (microbial imbalance) not only increases the risk of oral diseases but may also have systemic health implications [ 47 ].

On the other hand, psychological stress can influence salivary NO levels. Studies have shown that acute stress can temporarily increase salivary NO production, part of the body’s response to stressors [ 48 ]. This increase may be accompanied by hormonal changes, such as elevated cortisol levels, highlighting the link between stress and inflammation in MS patients. Therefore, investigating the relationship between stress and salivary NO levels in MS patients with and without xerostomia can enhance our understanding of the interplay between psychological stress, inflammation, and salivary changes.

This study aims to explore xerostomia as a clinical mediator in the relationship between stress and NO levels and evaluate salivary NO as a potential biomarker for monitoring inflammation and stress in MS patients. Clinically, these findings could contribute to identifying new biomarkers for disease progression, developing targeted stress management strategies, and improving therapeutic approaches for xerostomia. A deeper understanding of these relationships may lead to more effective disease management and improved quality of life for MS patients.

This observational cross-sectional study aimed to investigate the role of NO in the pathophysiology of MS and its significant implications for oral health, mainly focusing on the potential association between salivary NO levels and xerostomia in MS patients. The primary objective of this study was to compare salivary NO concentrations between MS patients with xerostomia and those without xerostomia. This study explores xerostomia as a clinical mediator in the relationship between stress and NO levels. It evaluates salivary NO as a potential biomarker for monitoring inflammation and stress in MS patients. Additionally, the study assessed the impact of medications commonly used by these patients on salivary NO levels and the prevalence of xerostomia. Clinically, these findings could contribute to identifying new biomarkers for disease progression, developing targeted stress management strategies, and improving therapeutic approaches for xerostomia. A deeper understanding of these relationships may lead to more effective disease management and improved quality of life for MS patients.

Methods

Ethics statement

This study was approved by the Ethics Committee of Tehran University of Medical Sciences (Ethical Code: IR.TUMS.DENTISTRY.REC.1400.185). All participants provided written informed consent after being fully briefed on the study objectives. All procedures were conducted in accordance with the relevant guidelines and regulations and all methods were performed in accordance with the Declaration of Helsinki [ 49 ].

Samples

This cross-sectional study, conducted in 2022 at the Neurology Research Center of Imam Khomeini Hospital (Tehran, Iran), included two groups of MS patients diagnosed by neurologists on the basis of MRI findings and the 2017 McDonald criteria [ 50 ]: one consisting of MS patients with xerostomia and the other comprising MS patients without xerostomia.The assessment of xerostomia in both patient groups was conducted using Thomson xerostomia inventory [ 51 ]. In this study, 11-item Xerostomia Inventory (XI) was used to assess xerostomia. This version of the questionnaire is a more comprehensive tool designed to assess the severity of xerostomia in detail [ 51 ] (Supplementary File 1 ). To assess the severity of xerostomia, the scores for the questions are collected. The total score is then calculated, and based on that, xerostomia severity can be divided into three categories; Low score (less than 10): mild xerostomia, moderate score (10–13): moderate xerostomia, and high score (14 or above): severe xerostomia. A cutoff score of 14 was used to determine the presence of xerostomia, which indicates the existence of xerostomia in patients. Additionally, for severe xerostomia, a cutoff score of 20 was used.

Furthermore, a thorough clinical examination was performed, which included a detailed inspection of the oral mucosa, lip dryness, and the presence of saliva. Patients with chronic periodontitis were excluded due to its association with elevated local nitric oxide (NO) production from periodontal inflammation, which could confound systemic NO levels relevant to MS [ 41 , 52 ]. Similarly, individuals with active oral mucosal lesions (ulcers, lichen planus, and candidiasis) or acute/chronic salivary gland infections (sialadenitis and parotitis) were excluded, as these conditions induce localized inflammation or infection that may artificially alter salivary NO concentrations [ 53 – 55 ]. Finally, participants who underwent dental procedures (extractions and scaling) within the past three months were excluded to avoid transient disruptions to oral microbiota and inflammation that might influence salivary biomarkers [ 56 ]. Patients with known underlying conditions associated with xerostomia were excluded, including Sjögren’s syndrome [ 57 ], rheumatoid arthritis [ 58 ], chronic skin disorders, including systemic lupus erythematosus [ 59 ], vesiculobullous disorders [ 60 ], and malignancies or patients undergone cancer treatments such as surgery, chemotherapy, or radiotherapy [ 61 ]. Additionally, individuals with acute or chronic infections of the salivary glands (viral sialadenitis and bacterial parotitis) were excluded to eliminate potential confounding effects on salivary function [ 62 ]. Pregnant women [ 63 ] and patients with diabetes [ 64 ] were excluded due to their potential influence on salivary secretion. Individuals who used tobacco [ 65 ] or alcohol [ 66 ] were also excluded, as these substances can impact salivary gland function and affect the study findings. To account for recent interventions that might temporarily alter salivary flow, patients who underwent dental procedures or oral surgeries within the past three months were also excluded. Furthermore, the time from the definitive diagnosis of MS to saliva sampling was recorded based on the patient’s medical records. The anxiety levels of the individuals were assessed using the Beck Anxiety Inventory (BAI) questionnaire [ 67 , 68 ], and participants in both groups were matched for age and sex (Fig.  1 ). The BAI was selected as the primary tool for assessing anxiety in this study. This choice was based on the BAI’s emphasis on somatic symptoms of anxiety and its validation in MS patients [ 69 , 70 ]. The BAI’s reliability and clinical efficiency, owing to its brief and practical nature, enabled a precise assessment of the relationship between anxiety and xerostomia in this study. Patients were matched either one-to-one based on exact values for sex or closely matched age ranges (within ± 4 years). For instance, for each male patient aged 50 in the MS patients without xerostomia, a male patient aged 52 from the MS patients with xerostomia was selected. Additionally, the researchers responsible for the matching process were blinded to the outcome data to prevent any potential bias. This study has been structured in accordance with the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines (Supplementary File 2 : STROBE Checklist).

STROBE flow diagram illustrating the enrollment of MS patients with and without xerostomia

(STROBE: strengthening the reporting of observational studies in epidemiology)

All participants in the study were on medication, and the specific medications they were taking were documented. These medications were categorized into four groups: (1) Disease-modifying drugs (DMDs), including interferon beta-1a and interferon beta-1b; (2) Antidepressants; (3) The medications used for symptom management included anti-inflammatory drugs (corticosteroids); and (4) Drugs for managing relapses and remissions of MS (control of MS complications), such as beta-glutamate acetate and monoclonal antibodies (including natalizumab and daclizumab);

Saliva collection

Unstimulated whole saliva was collected after the participants spit into sterile containers. They were instructed to refrain from eating, drinking, brushing their teeth, and engaging in any activities that could stimulate salivation for one hour prior to sample collection to minimize potential confounding factors. Additionally, saliva sampling was conducted within a designated morning window (9:00 AM to 11:00 AM) to account for possible circadian rhythm effects on salivary composition. The participants were asked to collect and pool saliva in their mouths at one-minute intervals over a period of five minutes, with the goal of obtaining a minimum volume of 2 ml but smaller volumes were also accepted if participants could not meet the 2 ml goal. Saliva samples were carefully stored in a -80 °C refrigerator to prevent any changes in the composition of the saliva, ensuring the integrity of the samples for accurate analysis.

Determination of salivary NO levels

To prepare the unstimulated saliva samples for analysis, they were first subjected to centrifugation at 2500 × g for 10 min (DM0424, DLAB, China). This process helps separate the cellular components and any particulate matter from the liquid portion of the saliva, resulting in a clearer supernatant for further examination. Following centrifugation, the samples were diluted in saline phosphate buffer at a ratio of 1:10, and 10 mL of the buffer was added to each sample. The diluted samples were then centrifuged again for 5 min to remove any remaining debris, ensuring that the supernatant was as clean as possible. Once the samples were prepared, the total concentration of salivary NO was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit based on the Griess reaction. This particular kit, supplied by ZellBio (Enzo NO parameter assay, Cat No. ZX-44107‐96, ZellBio GmbH, Germany), is designed for accurate quantification of NO levels in biological samples. The Griess reaction is a well-established method for detecting nitrite, a stable end product of NO metabolism. The unit of measurement for salivary NO levels was nanograms per milliliter (ng/mL).

Statistical analysis methods

The collected data were systematically entered into SPSS software, version 19, for analysis. Descriptive statistics, including means and standard deviations for continuous variables and frequencies for categorical variables, were used to summarize the data. The normality of quantitative variables was assessed using the Kolmogorov-Smirnov test. Specifically, the chi-square test was applied to compare proportions or distributions across categorical variables, whereas the Mann‒Whitney U test was utilized for comparing distributions between two independent samples that did not meet the assumption of normality. For the comparison of means between groups, the independent samples t-test was employed. The significance of the results was determined using a p value threshold of 0.05, with values above this threshold indicating nonsignificant differences between the groups being compared. Additionally, the correlation between each pair of variables was analyzed using the Spearman correlation coefficient. For multivariable analysis, multiple linear regression and logistic regression models were employed to identify factors associated with xerostomia. Variable selection for the regression models was not limited to those that were statistically significant in univariate analysis. Variables that were clinically relevant or theoretically justified based on previous studies were included in the models, regardless of their significance in univariate tests. Furthermore, a stepwise regression approach was also utilized to optimize the model by including significant predictors while considering potential interactions.

Sample size calculation

The sample size for this study was determined based on the results of a pilot study conducted on two groups of patients: those with MS and dry mouth, and those with MS without dry mouth. The pilot study included 10 participants in each group, and the data from this preliminary investigation were used to estimate the effect size and calculate the required sample size. Using standard statistical methods, with a significance level of 0.05 and a statistical power of 80%, it was determined that a minimum of 27 participants per group would be required to ensure adequate power for detecting meaningful differences between the groups. To strengthen the robustness of our findings, we ultimately included 30 participants in each group. Further details on the pilot study and the methodology used for sample size calculation are provided in Supplementary File 3 .

Results

Patient characteristics

Table  1 presents the demographics, clinical characteristics, and laboratory findings of MS patients both with and without xerostomia. Within the characteristics detailed in Table  1 , a significant difference was found in salivary NO levels between the two groups. Specifically, MS patients who experienced xerostomia had a mean salivary NO level that was significantly lower than that of those without xerostomia ( p  < 0.001). Among these patients, Celiac disease, Inflammatory Bowel Disease (IBD), and hypothyroidism were the most common coexisting conditions.

Table 1 Demographics, clinical characteristics, and laboratory findings of MS patients with and without Xerostomia Characteristics MS patients without xerostomia ( n  = 30) MS patients with xerostomia ( n  = 30)
p
*

Gender
0.79  Female 21 (70.00%) 20 (66.67%)  Male 9 (30.00%) 10 (33.33%) Age , years 40.27 ± 9.32 34.63 ± 10.81 0.06 • Duration of MS disease (year) 3.72 ± 2.65 5.35 ± 6.07 0.18 • Other systemic diseases 7 (23.33%) 6 (20.00%) 0.76  Celiac disease 3 (10.00%) 2 (6.67%)  Inflammatory Bowel Disease (IBD) 2 (6.67%) 3 (10.00%)  Hypothyroidism 2 (6.67%) 1 (3.33%) • Medications  Disease-modifying drugs 11 (36.67%) 16 (53.33%) 0.20  Antidepressants use 11 (36.67%) 7 (23.33%) 0.27  Corticosteroid use 18 (60.00%) 18 (60.00%) 0.45  Control of MS complications 12 (40.00%) 8 (26.67%) 0.28
Xerostomia Severity
< 0.001  Low (less than 5) 19 (63.3%) 0 (0.0%)  Moderate (5–7) 8 (26.7%) 0 (0.0%)  High (8 or above) 3 (10.0%) 7 (23.3%)  Severe xerostomia (> 20) 0 (0%) 23 (76.7%)
XI scores
4.40 ± 2.01 16.97 ± 5.50
Stress level**
11.77 ± 8.30 17.23 ± 10.21 0.03 Salivary NO , ng/mL 227.47 ± 183.84 102.37 ± 70.87 < 0.001 For ordinal or discrete data, the number of data points is provided; For non-ordinal or continuous data, the mean of the data along with the standard deviation (mean ± standard deviation (SD)) or percent (%) is reported. * p: P value for comparing MS patients with xerostomia and without xerostomia. ** Stress level : An assessment of the participants’ anxiety levels was conducted using the Beck Anxiety Inventory (BAI) questionnaire [67, 68].

Demographics, clinical characteristics, and laboratory findings of MS patients with and without Xerostomia

For ordinal or discrete data, the number of data points is provided; For non-ordinal or continuous data, the mean of the data along with the standard deviation (mean ± standard deviation (SD)) or percent (%) is reported.

* p: P value for comparing MS patients with xerostomia and without xerostomia.

** Stress level : An assessment of the participants’ anxiety levels was conducted using the Beck Anxiety Inventory (BAI) questionnaire [67, 68].

However, there were no statistically significant differences noted between patients with xerostomia and those without xerostomia in terms of the duration of MS, the presence of other systemic diseases (including celiac disease, IBD, and hypothyroidism), or the medications they were taking. The stress level was significantly higher in MS patients with xerostomia (17.23 ± 10.21) than in those without xerostomia (11.77 ± 8.30) ( p  = 0.03) (Table  1 ).

Associations between Xerostomia and related characteristics

The correlation coefficient between salivary NO levels and the presence of xerostomia in MS patients was − 0.44, indicating a negative and significant correlation ( p  < 0.001). These findings suggest that lower levels of salivary NO are associated with an increased likelihood of the presence of xerostomia in MS patients. Additionally, the correlation between stress level and xerostomia manifestation was 0.28, indicating a positive relationship ( p  = 0.03). This implies that higher stress levels in MS patients may be linked to a higher incidence of xerostomia Manifestation. Furthermore, Fig.  2 illustrates the correlations and p values between xerostomia occurrence and other patient characteristics.

Heatmap showing the correlation coefficients and p values for xerostomia, salivary NO, and other features in MS patients

* Other systemic diseases is consist of celiac disease, IBD, and hypothyroidism

Associations between the evaluated variables and the risk of Xerostomia in MS patients

The multivariate binary logistic regression analysis identified salivary NO level (OR = 0.99, p  = 0.003), and age (OR = 1.10, p  = 0.016) as significant predictors of xerostomia in MS patients (Table  2 ). However, no significant association was found between xerostomia and the presence of other systemic diseases.

Table 2 Univariate binary logistic regression analysis of the association between evaluated variables and the risk of developing Xerostomia in MS patients Characteristics Odds ratio (OR) (95% CI for OR) p value Age 1.103 (1.025 to 1.206) 0.016 Gender 0.767 (0.172 to 3.391) 0.723 Other systemic diseases * 1.294 (0.221 to 9.037) 0.780 Duration of MS 0.891 (0.743 to 1.023) 0.131 Nitric oxide (NO), ng/mL 0.986 (0.975 to 0.994) 0.003 Stress level 1.081 (1.006 to 1.180) 0.050 * Other systemic diseases is consist of celiac disease, IBD, and hypothyroidism

Univariate binary logistic regression analysis of the association between evaluated variables and the risk of developing Xerostomia in MS patients

* Other systemic diseases is consist of celiac disease, IBD, and hypothyroidism

Figure 3 A and 3 B illustrate the salivary NO and stress levels in MS patients with and without xerostomia, respectively. By combining the effects of NO and stress levels using a logistic regression model, we can predict the incidence of xerostomia in MS patients with 80% accuracy, yielding an odds ratio of 16.43 (AUC = 0.88).

A ) Comparison of salivary NO levels between MS patients with and without xerostomia

B ) Comparison of stress levels between MS patients with and without xerostomia

Discussion

This study aimed to investigate and compare salivary NO levels between MS patients with and without xerostomia. Upon analyzing the data, no significant differences were found in age, sex, MS disease duration, or the presence of other systemic diseases (including celiac disease, IBD, and hypothyroidism) between the two groups. Furthermore, the use of antidepressants, DMDs for MS control, corticosteroids, or other control medications did not differ significantly between MS patients with xerostomia and those without xerostomia. However, a notable finding was the significant difference in salivary NO levels ( p  < 0.001), with MS patients suffering from xerostomia exhibiting considerably lower salivary NO levels than those without xerostomia.

In MS patients, elevated levels of NO within inflammatory lesions exacerbate the disease process [ 71 , 72 ]. As a reactive free radical, NO can compromise the integrity of the blood‒brain barrier, increasing its permeability [ 73 , 74 ]. This weakened barrier allows for the leakage of antibodies into the CNS and impairs the regulation of T-cell activation, both of which intensify the inflammatory cascade characteristic of MS [ 75 ]. These increased NO levels play a crucial role in promoting inflammation within MS lesions, confirming its involvement in disease pathogenesis [ 48 , 76 ]. Covello et al. reported that xerostomia is a prevalent symptom among patients with MS, with a prevalence rate of 62% [ 6 ]. Xerostomia, a common manifestation in MS patients, can significantly impact their quality of life and increase the risk of dental issues, such as tooth decay and other oral complications [ 11 , 77 ]. Additionally, Cockburn et al. reported that xerostomia is the most frequently reported oral side effect associated with MS therapies, primarily caused by the use of anticholinergic medications. These drugs can lead to reduced saliva production, exacerbating the symptoms of xerostomia. Furthermore, the use of immunosuppressive agents can contribute to an increased risk of infections and other complications in the oral cavity [ 16 ].

NO serves as a crucial mediator in both physiological and pathological processes within the human body [ 78 ]. Its physiological effects primarily involve vascular processes, including vasodilation, vasoconstriction, and protection of blood vessels [ 79 , 80 ]. Additionally, NO plays a significant role in the immune response by activating immune cells and demonstrating antimicrobial properties [ 81 , 82 ]. Moreover, NO functions as a messenger or modulator in various biological processes, such as inflammatory responses, pain signaling, and neurotransmitter activity [ 83 , 84 ]. Its involvement extends to tumor proliferation, tissue damage, and bone loss, underscoring its diverse roles in health and disease [ 85 ]. The physiological effects of NO can be categorized into two main groups: those resulting from interactions with the enzyme guanylate cyclase [ 86 ] and those resulting from cGMP-independent effects. The latter includes regulating vascular tone and serving as a neural modulator in the CNS [ 87 , 88 ]. Owing to its vasodilatory effects, NO is regarded as the primary regulator of blood pressure, a function largely mediated by endothelial cells [ 89 ]. Given its role in vasodilation, it is plausible that elevated levels of NO could increase salivary secretion by increasing blood flow to the salivary glands [ 35 , 90 ]. This relationship highlights the importance of NO in maintaining oral health and its potential implications for conditions characterized by xerostomia.

Salivary NO holds promise as a biomarker for MS, reflecting the disease’s oxidative stress and immune response [ 37 , 75 ]. Research has highlighted the involvement of nitric oxide synthase (NOS), the enzyme responsible for NO production, in both the early and chronic phases of MS, further supporting its role in disease progression [ 21 , 91 ]. Moreover, elevated salivary NO levels correlate with the characteristic inflammatory processes of the disease [ 92 , 93 ]. Higher NO concentrations in inflammatory MS lesions underscore its importance as a biomarker for oxidative stress and the immune response, providing a non-invasive method for assessing disease activity [ 94 ]. Given insights into the underlying pathophysiology of MS, salivary NO levels may emerge as a valuable tool for monitoring disease activity and progression. Cockburn et al. noted that the medications used to manage MS often cause oral complications, with dry mouth being the most prevalent side effect [ 16 ]. Sexton et al. reported that the most common oral manifestations in MS patients were dry mouth, tooth sensitivity, changes in taste, and oral pain [ 95 ].

Tewari et al. described the role of oxidative stress in the pathogenesis of cognitive disorders, neurological damage, and pathological conditions [ 88 ]. These findings indicated that NO levels, which increase in response to increased nitrosative/oxidative stress, were elevated in affected individuals. In the present study, salivary NO levels nearly doubled in patients taking both DMDs and MS control medications compared with those not taking these medications. This aligns with the findings of Abadi et al., who noted that NO, as both a free radical and a biological messenger, plays a significant role in salivary secretion in diabetic patients [ 96 ]. The findings of this study indicate a clear association between reduced salivary NO levels and xerostomia in MS patients. These findings suggest that NO may play a role in the pathophysiology of xerostomia in individuals with MS. Future research should aim to further investigate the relationship between salivary NO and the mechanisms underlying xerostomia. Additionally, broader studies that examine other contributing factors, such as alterations in muscarinic receptor function, could provide a more comprehensive understanding of the pathways involved in the development of xerostomia in MS and other conditions. Expanding the scope of research will help clarify the interactions between salivary NO levels and various physiological processes that lead to xerostomia.

The significantly higher stress levels observed in MS patients with xerostomia highlight a clinically relevant interplay between psychological distress and salivary dysfunction. Stress may exacerbate xerostomia via sympathetic dominance, hypothalamo-pituitary-adrenal (HPA) axis dysregulation, or oxidative stress-mediated NO depletion, while xerostomia-related discomfort may further amplify anxiety. These findings advocate for holistic management strategies that address both neurological and psychosocial dimensions of MS. Elevated stress levels may exacerbate autonomic dysfunction in MS, impairing salivary gland function via sympathetic overactivity and reduced parasympathetic signaling [ 36 , 97 ]. Stress-induced activation of inflammatory pathways likely amplifies inducible nitric oxide synthase (iNOS) activity, driving excessive NO production and oxidative damage in salivary tissues [ 32 , 98 ]. However, compensatory antioxidant mechanisms may mask stress-related NO fluctuations in some populations, underscoring the complexity of NO regulation.

Several medications commonly used in the treatment of MS and its associated conditions, such as depression, can contribute to xerostomia or hyposalivation [ 99 ]. Antidepressants, including tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs), are well-known to reduce salivary gland activity due to their anticholinergic effects, making them a significant cause of dry mouth [ 100 , 101 ]. Muscle relaxants like baclofen, often prescribed for spasticity in MS patients, can also lead to hyposalivation [ 102 ]. Additionally, anticholinergic drugs used for managing bladder dysfunction in MS inhibit parasympathetic stimulation, further decreasing salivary secretion [ 103 , 104 ]. Corticosteroids, though essential for managing acute MS relapses, and immunosuppressive agents can alter the oral environment and potentially exacerbate xerostomia [ 105 ]. Moreover, the combined use of multiple medications in MS patients can have a cumulative effect, increasing the risk of xerostomia or hyposalivation, particularly in individuals with predisposing factors [ 99 ]. These factors highlight the complex, multifactorial etiology of xerostomia and hyposalivation in MS patients and underscore the importance of considering medication side effects when evaluating these conditions.

Furthermore, while our study found no significant differences in medication use or co-existing conditions (Celiac disease, IBD) between groups, the interplay between systemic inflammation, pharmacotherapy, and salivary NO warrants deeper exploration. For example, anticholinergic drugs, even at comparable usage rates between groups, may exert cumulative effects on salivary gland function, indirectly reducing NO bioavailability. Similarly, systemic inflammation from conditions like IBD could synergize with MS-related neuroinflammation, creating a milieu that exacerbates salivary gland dysfunction. Future studies with larger cohorts should stratify patients by medication profiles and comorbidities to elucidate these interactions. While our findings align with studies linking stress to salivary dysfunction in chronic inflammatory conditions [ 106 ], contradictory evidence highlights the complexity of this relationship. Researchers reported weak associations between stress and objective hyposalivation, suggesting that subjective xerostomia may reflect broader psychosocial distress rather than direct physiological effects [ 107 ]. Additionally, others emphasized medication-driven salivary hypofunction in MS, which may overshadow stress-related contributions [ 108 ]. However, the synergy between neuroinflammation, oxidative stress, and HPA axis dysregulation in MS likely creates a unique milieu where psychological stress exacerbates NO depletion and glandular impairment. Future research should disentangle these interactions using longitudinal designs and multimodal stress assessments.

The limitations of this study include the relatively small sample size, which may restrict the generalizability of the findings. Additionally, reliance on subjective assessments of xerostomia, even when using validated tools, introduces an element of subjectivity into the results. The exclusion of patients with certain comorbidities, such as diabetes or Sjögren’s syndrome, may also limit the applicability of the findings to all MS patients. Moreover, unmeasured confounding factors, such as dietary habits, might have influenced the outcomes. Finally, despite standardizing the timing of saliva collection to reduce variability, circadian fluctuations in salivary nitric oxide levels may still have impacted the results. These limitations highlight the need for further research to provide a more comprehensive understanding of the relationships examined in this study.

Conclusion

This study examined the relationship between salivary NO levels and xerostomia in patients with MS. Although no significant differences were found regarding disease duration, medication usage, or most co-existing conditions, salivary NO levels were notably lower in MS patients with xerostomia compared to those without xerostomia. These findings suggest the potential involvement of salivary NO in the development of xerostomia among MS patients. A negative correlation was observed between salivary NO levels and xerostomia, indicating that reduced NO levels may be associated with an increased risk of xerostomia. Additionally, stress levels were significantly positively correlated with xerostomia, suggesting a potential link between stress and a heightened risk of xerostomia in MS patients. Further research is essential to explore the underlying mechanisms connecting salivary NO and xerostomia. Additionally, studies aimed at developing strategies to modulate salivary NO levels could provide valuable insights for managing xerostomia in MS patients.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

Acknowledgements

Not applicable.

Abbreviations

Beck Anxiety Inventory

Confidence interval

Central nervous system

Disease-modifying drugs

Enzyme-linked immunosorbent assay

Hypothalamo-pituitary-adrenal

Inflammatory Bowel Disease

Multiple Sclerosis

Nanograms per milliliter

Nitric oxide

Nitric oxide synthase

Odds ratio

Selective serotonin reuptake inhibitors

Standard deviation

Strengthening the Reporting of Observational Studies in Epidemiology

Tricyclic antidepressants

Xerostomia inventory

Author contributions

MK, TKH, AKH, and SM conceived the study idea and led the data collection. MK, AKH, and SM created the study protocol and wrote the original draft. MK and AKH contributed to the data analysis/interpretation and manuscript preparation. MK, TKH, SK and AKH led the writing, review, and editing. MK, AKH, and AT interpreted the results. All the authors read and approved the final manuscript.

Funding

Not applicable.

Data availability

The data used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the Tehran University of Medical Sciences Ethical Committee (Ethical code: IR.TUMS.DENTISTRY.REC.1400.185). After the study objectives were described, all the participants signed informed consent before participating in this study. All methods were performed in accordance with the relevant guidelines and regulations and all methods were performed in accordance with the Declaration of Helsinki [ 49 ].

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Online version

https://ethics.research.ac.ir/ProposalCertificateEn.php? id=240623&Print=true&NoPrintHeader=true&NoPrintFooter=true&NoPrintPageBorder=true&LetterPrint=true.

Clinical trial number

Not applicable.

Footnotes

Publisher’s note

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Aynaz Khabazian, Maryam Koopaie and Tanaz Khabazian contributed equally to this work.

References

Associated Data

Supplementary Materials

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

Data Availability Statement

The data used and/or analyzed during the current study are available from the corresponding author upon reasonable request.