Salivary thioredoxin-1 as a non-invasive redox biomarker in oral diseases

Abstract

Thioredoxin-1 (Trx-1) is a key redox-regulating protein involved in antioxidant defense, inflammation, immune responses, and cell survival. Dysregulation of redox balance is increasingly being recognized as a central mechanism in the pathogenesis of a wide spectrum of oral diseases, ranging from inflammatory conditions to oral potentially malignant disorders and oral squamous cell carcinoma. Saliva, owing to its non-invasive, accessible, and disease-reflective nature, has emerged as a promising diagnostic medium for redox and inflammatory biomarkers. Limited but emerging evidence suggests that salivary Trx-1 levels may be altered in oral diseases associated with oxidative stress and chronic inflammation, reflecting underlying molecular and cellular changes within the oral microenvironment. From a clinical perspective, assessment of salivary Trx-1 may assist in early disease detection, chairside risk stratification, and longitudinal monitoring of disease activity and treatment response. Elevated salivary Trx-1 expression has been associated with disease presence, severity, and malignant transformation risk, highlighting its potential diagnostic and prognostic utility. Despite these advantages, variability in detection methods, population heterogeneity, and limited longitudinal data remain challenges to clinical translation. Overall, salivary Trx-1 represents a promising non-invasive redox biomarker with significant potential for improving early detection, risk assessment, and personalized management of oral diseases.

Key Words: Oral disease; Oxidative stress; Redox marker; Salivary biomarker; Thioredoxin-1

Core Tip: Redox imbalance is an important mechanism in the etiopathogenesis of various diseases, including periodontal diseases, oral potentially malignant disorders, and squamous cell carcinoma of the oral cavity. Thioredoxin-1 (Trx-1), which acts as a redox regulator, plays a crucial role in the body’s antioxidative protection, inflammation, apoptosis processes and tumor growth. The detection of Trx-1 in saliva may serve as a promising non-invasive method for early diagnostics and prognosis in the diseases of the oral cavity.

INTRODUCTION

Oxidative stress refers to an imbalance between the generation of reactive oxygen species (ROS) and the ability of biological systems to detoxify these reactive intermediates or repair the resulting cellular damage[1,2]. The alteration of this balance has been significantly linked with the development of several diseases associated with the mouth, from inflammatory states such as periodontal diseases to cancerous conditions such as oral squamous cell carcinoma (OSCC)[3-5]. The oral cavity is a dynamic environment that is known to house a diverse microbial population along with normal and inflammatory mediators. Inflammation caused by microbial biofilms that generate excessive ROS causes periodontal destruction in terms of destruction of connective tissue and bone resorption[6,7]. The same applies to oral potentially malignant disorder (OPMD), such as leukoplakia and oral submucous fibrosis, whereby changes occur in the redox state leading to epithelial dysplasia and progression to cancer. In OSCC, ROS not only increase genomic instability but also control crucial signal transduction pathways that play a role in carcinogenesis, angiogenesis, and chemoresistance. Redox-regulated molecules have been identified as important markers in predicting early diagnosis and prognosis of OSCC[8,9].

Over the past few decades, there has been a growing tendency to use saliva as a medium for diagnosis in oral as well as systemic diseases[10]. There are a number of benefits of using saliva such as easy availability, noninvasive sampling, low cost, and repetitive sampling. Moreover, saliva consists of a complex combination of biomolecules, both locally and systemically produced, including different types of proteins, enzymes, DNA and RNA molecules, and oxidative stress products[11]. The area of salivary diagnostic science has helped in the discovery of reliable biomarkers that can help detect early diseases and provide personalized treatments. One of the important biological processes that have been considered in this area is the redox regulating system[12]. The thioredoxin system is extremely important in order to maintain the physiological balance. Thioredoxin as a major member of the thioredoxin system plays a vital role in maintaining normal physiological function and oxidative responses to stress and is therefore a very promising tool in salivary biomarkers studies[13]. Protein thioredoxin-1 (Trx-1) is a small, ubiquitous molecule. In humans, Trx-1 acts as an antioxidant and reduces disulfide bonds by oxidizing dithiols and reducing disulfides[14,15]. Besides the main function, protein Trx-1 regulates intracellular signaling pathways, transcription factors activity, inhibits apoptosis and stimulates cell proliferation[16]. There are major changes in Trx-1 expression in various diseases. Increasing numbers of studies show that Trx-1 is also found in the extracellular fluids, including saliva, which may be used as a surrogate marker of local and systemic oxidative processes[17,18]. Thus, salivary Trx-1 can be considered as a biomarker of oxidative stress in oral pathologies. However, there is no consistency in the literature due to the differences in methodology, analysis and clinical applications. This review aims to be among the first focused investigations of the role of salivary Trx-1 as a non-invasive redox biomarker in oral diseases. The review will discuss biological significance of salivary Trx-1, its role in oral disorders and its clinical applications.

LITERATURE SEARCH

A literature search was performed employing electronic databases such as PubMed, Scopus, and Web of Science. The systematic search of these databases included studies from January 2000 to March 2026. Relevant studies that detailed upon Trx-1, including its significance as a salivary redox biomarker for oral diseases, were sought by utilizing the following keywords and Medical Subject Headings (MeSH) terms: “Thioredoxin-1”, “Trx-1”, “saliva”, “salivary biomarkers”, “oxidative stress”, “redox”, “oral diseases”, “periodontitis”, “oral potentially malignant disorders”, “oral submucous fibrosis”, “oral leukoplakia”, and “oral squamous cell carcinoma”. Relevant articles were also located through screening references in selected sources. Articles that discussed the biological functions of Trx-1, oxidative stress mechanisms, salivary analysis, and oral diseases were selected for this review. The inclusion criteria also expanded to include articles that evaluated the potential of salivary, serum, or cellular expression of thioredoxin and its biological effects within the oral cavity. Original research articles, clinical studies, experimental investigations, observational studies, and relevant review articles published in peer-reviewed English-language journals were considered. Articles written in any language other than English, conference abstracts, editorials, letters to editors, duplicate articles, and articles that did not have enough relevancy in terms of either Trx-1 or oral diseases were omitted from the review process. The titles and abstracts of articles that were identified during the database search were screened first for their relevancy to the review question; then, only those which were potentially eligible were read in full text. The final literature was synthesized narratively to summarize the biological functions of Trx-1, its role in oral disease pathogenesis, and its potential application as a salivary biomarker. In regard to this review paper, “saliva” refers to total mixed saliva (whole saliva). Total mixed saliva includes the fluid secretions from both major and minor salivary glands, together with gingival crevicular fluid, desquamated epithelial cells, bacteria, and other components of the mouth.

BIOLOGY OF TRX-1

Trx-1 is a highly conserved, small-sized protein, about 12 kDa, which plays an essential role in the maintenance of the cellular redox balance. It is classified among thioredoxin oxidoreductases based on the presence of a conserved motif, Cys-Gly-Pro-Cys, within the active site region, endowing it with the capability of catalyzing disulfide bonds' reduction[19,20]. This redox-active center allows Trx-1 to act as an antioxidant within cells, facilitating the reduction of oxidized cysteines within its target proteins, thus ensuring their proper structure and functionality. Trx-1 is primarily found within the cytoplasm under normal circumstances; however, it demonstrates intracellular movement and translocation into the nucleus in the presence of oxidative stress to regulate gene expression. Moreover, apart from its intracellular activity, Trx-1 is also found outside the cell, implicating its involvement in cell-cell interaction mechanisms and rendering it detectable within biological fluids like saliva[21,22].

Biological function of Trx-1 is directly related to the thioredoxin pathway which is one of the major pathways for the control of cellular redox balance and includes Trx-1, thioredoxin reductase (TrxR), and nicotinamide adenine dinucleotide phosphate (NADPH)[23]. The function of thioredoxin pathway is performed cyclically when Trx-1 reduces protein targets in the oxidized form through the action of its cysteine residues; Trx-1 itself becomes oxidized during the process. After that, the oxidized form of Trx-1 is converted to the reduced form of the protein with the help of TrxR and NADPH. The pathway plays an important role in the maintenance of reduced forms of proteins in the cell and protection from oxidative stress. Thioredoxin pathway cooperates with other systems of antioxidants, such as glutathione system, but differs from them in direct participation in the redox signaling and protein regulation[24]. Besides the antioxidant function, Trx-1 acts as a regulator of many cellular processes, such as synthesis, repair, and proliferation of DNA. Trx-1 provides reduction of ribonucleotide reductase that is responsible for synthesis of deoxyribonucleotides, thus, for DNA replication and repair, which function is especially necessary for proliferating cells under stress conditions[25,26].

A critical aspect of Trx-1 is its involvement in redox-sensitive signal transduction. It functions as a regulatory protein that regulates the function of multiple transcription factors, such as nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and p53, using a redox mechanism. Through maintaining these transcription factors in a reduced state and making them functionally active, Trx-1 controls the pattern of gene expression involved in inflammation, immune reactions, survival, and cell proliferation[27]. Trx-1 also has a key function in the regulation of apoptosis, mainly through its association with apoptosis signal-regulating kinase 1 (ASK-1). In its reduced state, Trx-1 interacts with ASK-1, blocking its kinase activity and thus preventing the induction of apoptosis. Oxidation of Trx-1 during oxidative stress induces its dissociation from ASK-1, which activates the apoptotic pathway downstream[28,29]. This mechanism is significant in chronic inflammatory conditions and cancers, where dysfunction in apoptotic processes is involved in their development. Another biological feature of Trx-1 involves the secretion of Trx-1 outside of cells and its cytokine-like activities. Although Trx-1 does not have a traditional signal sequence for secretion, it can still be released into extracellular compartments, and it has been found in many extracellular fluids, such as plasma, serum, and saliva[30]. Trx-1 in the extracellular milieu has immunomodulatory functions, including regulation of cytokine synthesis and antioxidative effects. The detection of Trx-1 in saliva is valuable in the field of oral medicine because it could indicate oxidative damage in the mouth and systemic redox homeostasis. This dual origin enhances its potential utility as a non-invasive biomarker.

SALIVA - A DIAGNOSTIC MEDIUM

Saliva is a clinically and biologically relevant fluid that may be used for the evaluation of redox-dependent biomarkers of oral pathology due to its direct exposure to the oral cavity environment and capability of reflecting current changes in local biochemical processes[31]. Saliva includes various proteins and enzymes that are produced by the salivary glands, gingival crevicular fluid, oral epithelial cells, and bacteria that inhabit the oral cavity[32]. Regarding oral biomarker studies, whole saliva is frequently used owing to its ability to mirror the contribution of both the saliva produced by salivary glands and that which is generated within the oral cavity itself. Consequently, the composition of saliva allows us to identify the current oxidative and inflammatory processes, which occur during such diseases as periodontitis, potentially malignant conditions, and squamous cell carcinoma[12,33]. Being a redox-dependent substance, Trx-1 is found in the saliva and it corresponds to the activity of the pathological process. Saliva provides numerous benefits for the analysis of redox biomarkers, such as Trx-1[34]. The non-invasive character of its collection, simplicity, and possibility for regular samples collection provide opportunities for screening procedures and regular testing of patients. However, various factors influencing the composition of saliva should be considered when collecting and analyzing the samples. Overall, the combination of non-invasiveness and biological significance of saliva makes the substance relevant to the study of Trx-1 in oral pathology.

MECHANISM OF ACTION OF TRX-1 IN ORAL DISEASE PATHOGENESIS

Trx-1 is a crucial mediator of oxidative stress-induced pathophysiological changes through a number of biological functions beyond those associated with antioxidative activity. Among other actions, Trx-1 regulates numerous signaling pathways that control cell proliferation and survival. Due to a significant role of oxidative stress as a common factor in pathogeneses of oral diseases, Trx-1 serves as a major mediator of biological processes induced by redox imbalance and underlying disease development. A main pathway through which Trx-1 induces pathological alterations is related to its capability to modulate redox-sensitive transcription factors. More specifically, Trx-1 helps maintain reduction state of certain transcription factors, such as NF-κB and AP-1, which facilitates the activation of DNA binding ability of the latter[35]. The activation of such transcription factors results in upregulation of inflammation and cytokine and cellular proliferation-related genes. Such processes become especially pronounced during chronic periodontitis where persistent inflammatory reactions take place. Moreover, activation of transcription factor is responsible for establishment of dysplastic environment within the affected tissues. The regulation of transcriptional activity through modulation by Trx-1 suggests that this enzyme represents a crucial link between oxidative stress and transcription.

In addition to transcription, Trx-1 has a significant effect on regulation of the processes of apoptosis through the mechanism involving ASK-1. At baseline, reduced Trx-1 interacts with ASK-1 and prevents its activation, which consequently suppresses the process of programmed cell death. In case of oxidative stress, Trx-1 becomes oxidized and separated from ASK-1, resulting in its activation and initiation of apoptotic pathways. Such a redox-dependent switch can be considered a molecular indicator of the level of cellular stress. In oral pathologies, dysregulation of this mechanism can have dual consequences: Excessive apoptosis may contribute to tissue destruction in inflammatory conditions, whereas inhibition of apoptosis may allow survival of genetically damaged cells, facilitating malignant transformation[36-38].

Trx-1 is highly related to proliferation and DNA synthesis, whereby Trx-1 provides reducing power to ribonucleotide reductase to synthesize deoxyribonucleotides used in DNA synthesis and DNA repair[39]. In case of chronic oxidative stress, which is seen in oral potentially malignant lesions and oral cancer, this process becomes essential for the survival and proliferation of mutated cells. Also, redox control of signal transduction by Trx-1 promotes cell cycle progression, thus causing uncontrolled cell proliferation. Furthermore, another important role of Trx-1 is the formation of tumor microenvironment in the case of malignancy. Trx-1 has been shown to contribute to angiogenesis by modulating the redox-sensitive signaling pathways and pro-angiogenic factors[40,41]. Trx-1 causes resistance to oxidative damage thus making the malignancies able to survive under stressful situations such as hypoxia and chemotherapeutic treatments. Besides its intracellular functions, the extracellular Trx-1 acts as a cytokine and takes part in intercellular communication in the mouth. There have been studies on the role of extracellular Trx-1 in the regulation of the immune and inflammatory reactions that cause oral diseases. The secretion of Trx-1 in saliva results from the biological functions described above, which include secretion from local cells and systemic circulation[42]. Thus, the extracellular existence of Trx-1 establishes the link between the biological functions and the potential diagnostic value of the biomarker. In summary, the biological functions of Trx-1 include oxidative stress regulation, transcription factor modification, apoptosis regulation, cell proliferation, and tumor biology (Table 1).

Table 1 Molecular actions of thioredoxin-1 and their relevance in oral diseases.

Molecular function of thioredoxin-1	Associated pathway or target	Biological effect	Relevance in oral diseases
Antioxidant activity	Reactive oxygen species scavenging pathways	Reduction of oxidative stress and maintenance of redox balance	Protection against periodontal tissue destruction and oxidative cellular injury
Redox regulation	Nuclear factor kappa B and activator protein-1 signaling pathways	Regulation of inflammatory cytokine production and inflammatory signaling	Contribution to chronic inflammation in periodontal diseases
Inhibition of apoptosis	Apoptosis signal-regulating kinase 1 pathway	Prevention of programmed cell death and promotion of cell survival	Survival of dysplastic and malignant cells in oral lesions
Support of DNA synthesis and repair	Ribonucleotide reductase pathway	Promotion of cellular proliferation and genomic maintenance	Facilitation of progression in oral potentially malignant disorders and oral squamous cell carcinoma
Promotion of angiogenesis	Redox-sensitive angiogenic signaling pathways	Formation of new blood vessels and enhancement of tumor microenvironment	Tumor progression and metastatic potential in oral squamous cell carcinoma
Extracellular signaling activity	Cytokine-like immunomodulatory pathways	Regulation of immune and inflammatory responses	Basis for its detectability and utility as a salivary biomarker

TRX-1 IN ORAL DISEASES

Periodontal diseases

Periodontal diseases represent a state of chronic inflammatory condition where microbial biofilms and an over-exuberant immune response from the host contribute towards disease progression in a manner where oxidative stress serves as an underlying mechanism. Inflammation involving persistent neutrophil and macrophage activity in periodontal tissues results in the overproduction of ROS that cause oxidative damage in lipids, proteins, and DNA[43,44]. Oxidative damage in turn results in tissue damage such as the breakdown of connective tissue and alveolar bone[45]. Redox imbalance in the local environment can lead to dysfunction of antioxidants and makes redox regulating proteins like Trx-1 important targets. Trx-1 is an important antioxidant both inside and outside cells that plays a critical role in regulating oxidative stress and inflammatory signaling in periodontal tissues. The elevated expression of Trx-1 has been found in inflammatory states as a defensive strategy to reduce the excessive production of ROS. Elevated expression of Trx-1 in periodontitis patients is evident from increased Trx-1 expression in the gingiva as well as the presence of Trx-1 in extracellular compartments like saliva and gingival crevicular fluid. Thus, salivary Trx-1 concentration can serve as an indicator of oxidative stress and inflammation in periodontal tissues[34].

In addition to its function as an antioxidant, Trx-1 has been implicated in important signaling pathways related to periodontal inflammation through the regulation of redox-sensitive transcription factors, such as NF-κB and AP-1. This implies that Trx-1 could be involved in the regulation of the expression of pro-inflammatory cytokines and matrix metalloproteinases, as well as other mediators of tissue damage. As such, Trx-1 is positioned not only as a simple biomarker but as an important factor in the development of periodontal diseases. Additionally, involvement in apoptosis pathways may affect cell viability and influence the course of periodontal disease processes. Specifically, Trx-1 has been shown to play a pivotal role in alveolar bone homeostasis. Research by Wu et al[34] demonstrated that Trx-1 promotes the osteogenic differentiation of periodontal ligament stem cells by activating the Wnt/β-catenin signaling pathway. Furthermore, their therapeutic model showed that the injection of recombinant human Trx-1 significantly improved alveolar bone restoration in periodontitis subjects with comorbid diabetes, highlighting its potential not just as a marker, but as a therapeutic mediator in complex systemic-oral interactions.

Clinically, the detection of Trx-1 levels in saliva provides an important non-invasive means of evaluation for disease activity. Research into salivary oxidative stress biomarkers has found their correlation with disease severity. Recent findings indicate that Trx-1 can act as a promising candidate for diagnosing redox status in periodontal diseases. Despite the findings from early research linking increased levels to an adaptive mechanism, more recent high-level prospective studies have further defined this approach. In one such study using a sample size of 144 patients, Lorente et al[46] found an inverse correlation between low salivary Trx-1 levels and not only the incidence (P = 0.04) but also the severity (rho = -0.47) of periodontitis. The receiver operating characteristic curve generated by their study came up with an area under the curve (AUC) of 75%, thereby indicating a reduction in the antioxidant level as a better indicator of the condition than an increase. This stark contrast demonstrates that there is a two-phased biological response as the disease progresses. The reason why the production of Trx-1 is increased in acute phase of periodontal inflammation is because of the compensatory defense response produced by the cell in order to neutralize the increasing amount of ROS in the body. However, when the disease progresses to a chronic and highly destructive stage, where there is attachment loss and the continued presence of neutrophils, the antioxidants become exhausted due to the constant oxidative stress and tissue destruction. This makes it impossible for the cells to secrete Trx-1 extracellularly. Hence, while salivary Trx-1 is a feasible tool to assess redox status in periodontal diseases, it should be interpreted based on the phase of the disease.

Oral premalignant disorders

The OPMDs such as leukoplakia and oral submucous fibrosis are associated with epithelial changes and are known to pose a risk for subsequent malignant transformation[47]. Many studies have reported oxidative stress as a main contributing factor for development and progression of these lesions. The continuous exposure to various etiological agents including tobacco, areca nuts, and alcohol leads to constant production of free radicals and ROS[48]. Evidence of this oxidative shift is clearly detectable in the oral environment from previous studies. For instance, Vlková et al[49] demonstrated that patients with OPMDs exhibit significantly higher salivary markers of lipoperoxidation and carbonyl stress, alongside a decreased total antioxidant capacity and lower expression of superoxide dismutase compared to healthy controls. In this way, oxidative stress causes oxidative damage to DNA and cell membranes, ultimately disturbing cellular functions and inducing dysplastic changes. Although oxidative stress has been clearly established as an important contributing factor in the development of OPMDs, there is currently no existing literature on the correlation between salivary Trx-1 and OPMDs. In addition, this issue needs to be investigated since the biological importance of the thioredoxin system in terms of oxidative stress is quite high. As it is well-known, Trx-1 has been shown to increase in response to ROS as a cellular defense mechanism, which suggests its increase in the early stage of oxidative stress during OPMDs. Based on the above rationale, Trx-1 seems to be promising in determining the presence of oxidative stress before malignant transformation. Mechanistically, Trx-1 is involved in the regulation of redox-sensitive transcription factors and signaling pathways that play an important role in regulating cell proliferation, differentiation, and apoptosis. As a result of maintenance of redox-sensitive transcription factors, such as NF-κB and AP-1, in a reduced state, Trx-1 could affect the patterns of gene expression that lead to enhanced viability and dysplasia in epithelial cells. Moreover, by interfering with apoptosis-regulatory pathways, including inhibition of ASK-1, Trx-1 would allow damaged cells to avoid apoptosis and increase mutations leading to malignancy. Both pathways are quite relevant to OPMDs biology and could contribute to the mechanism of their development.

OSCC

OSCC is considered the most prevalent malignancy occurring in the mouth and involves a multitude of mechanisms of interaction between genomic aberrations, environmental exposures, and microenvironmental modifications within the tumor[8]. Oxidative stress acts as a key factor in OSCC development due to its ability to promote genomic instability, DNA mutations, and the activation of oncogenic signaling pathways[50]. Continuous redox imbalance leads to initiation and further development of OSCC along with angiogenesis and therapeutic resistance. Trx-1 is overexpressed in a wide variety of malignancies, including OSCC, and participates in cancer cell growth and survival. Increased concentration of Trx-1 results in increased resistance to oxidative stress of cancer cells, which allows their viability in an unfavorable microenvironment[51]. Furthermore, Trx-1 stimulates transcription factors, like NF-kB and AP-1, that act on genes associated with cellular proliferation, inflammation, and angiogenesis. Amirchaghmaghi et al[52] conducted a comparative analysis of salivary Trx-1 levels in patients with OSCC and healthy controls. Their findings indicated that salivary Trx-1 levels in the healthy cohort were actually higher than those observed in the OSCC group. This unexpected trend in saliva suggests a potential antioxidant exhaustion or a high intracellular utilization rate by the tumor itself, which may limit the release of the protein into the salivary environment. Notably, within the OSCC group, the highest concentrations of salivary Trx-1 occurred in patients over 65 years of age, suggesting that age-related redox shifts may influence biomarker detection[52]. The salivary findings observed in oral diseases appear to differ from the well-established intracellular upregulation of Trx-1 under oxidative stress conditions. While Trx-1 expression is generally increased within cells as an adaptive antioxidant response, certain salivary studies in periodontitis and OSCC have demonstrated comparatively lower salivary levels. This apparent discrepancy may reflect compartment-specific variations, altered extracellular secretion, increased intracellular utilization, or antioxidant depletion during advanced disease stages. On the mechanistic level, this inconsistency is related to an intracellular “tumor trap” effect. Fast-growing tumor cells suffer from high metabolic stress and require huge amounts of reduced Trx-1 inside the cell in order to provide ribonucleotide reductase with the needed amount of Trx-1 for fast DNA replication and to block apoptosis via ASK-1 pathway. The strong demand of tumor cells on Trx-1 forces the tumor to trap and use the local Trx-1 pool, which is confirmed by increased expression of thioredoxin reductase 1 (TrxR1) in invasive OSCC. The trapping of Trx-1 makes this protein unavailable for the release in the extracellular matrix and leads to decreased salivary levels of this protein during progression of malignancy.

The anti-apoptotic activity of Trx-1 is especially important in OSCC since it functions by suppressing ASK-1 and preventing apoptosis in cells. Thus, Trx-1 facilitates the growth of tumor by inhibiting cell death and, in addition, due to its involvement in DNA replication and repair, contributes to the aggressiveness of cancerous cells and their resistance to traditional treatments, namely chemo- and radiotherapy. In addition, Trx-1 was found to enhance angiogenesis and metastasis of OSCC. The analysis of salivary Trx-1 as a tool for OSCC diagnostics and monitoring could be considered one of the most promising directions in the investigation of the molecule. The redox environment of OSCC primary tumors was defined by Iwasawa et al[53], revealing a marked increase in TrxR1, which is crucial for the reduction and activation of oxidized Trx-1. Using immunohistochemistry on 50 patients, Iwasawa et al[53] showed that increased TrxR1 expression also had a strong correlation with regional lymph node metastases (P < 0.05) and clinical stage advancement (P < 0.01). The study also showed that this biomarker can be responsible for cancer progression in the oral cavity and perhaps can be used for molecular therapy in oral cancer. In addition to the known fact that elevated concentrations of redox-related biomarkers are characteristic of patients with oral cancer, the importance of Trx-1 can be attributed to its crucial involvement in the redox regulation in the body. Nevertheless, in spite of its biological significance, further research is needed before the diagnostic relevance of salivary Trx-1 in OSCC can be evaluated (Figure 1).

Figure 1 Schematic representation of the biological basis and potential clinical utility of salivary thioredoxin-1 in oral diseases. ROS: Reactive oxygen species.

CURRENT EVIDENCE AND RESEARCH GAP

The current literature on the application of Trx-1 as a biomarker for oral diseases appears sparse and incomplete. While various studies have focused on oxidative stress markers as potential biomarkers in different oral diseases, only a few studies specifically evaluate salivary Trx-1. This gap is especially evident in OPMDs, where no dedicated studies have evaluated its salivary levels despite strong biological significance. This trend applies even in the case of other diseases, such as periodontal disease and OSCC, wherein studies exist only through indirect methods like serum or tissue analysis.

The limitation associated with current research on this biomarker is due to small sample sizes and cross-sectional study design, limiting the extent to which causality can be established and temporal changes in biomarker levels identified. Variation in patient groups across different studies, along with the lack of clear definition of diseases and disease stages, makes it difficult to compare the results of each individual study. The use of different methods for biomarker estimation, ranging from enzyme-linked immunosorbent assay to varying sample collection and storage techniques, further exacerbates inconsistencies in results. Another critical issue is the lack of standardization in salivary diagnostics. Factors such as salivary flow rate, circadian variation, oral hygiene status, and systemic health conditions can significantly influence biomarker concentrations, yet these variables are not consistently controlled across studies[54]. The absence of established reference ranges or cutoff values for salivary Trx-1 further limits its clinical interpretation and applicability. In addition, given that Trx-1 is highly sensitive to oxidative changes, pre-analytical variables assume particular importance. Improper sample handling may lead to artificial oxidation or degradation, thereby altering measured levels. This necessitates strict standardization of salivary collection and processing protocols, including the use of protease inhibitors, rapid cooling or immediate storage at low temperatures, and minimal delay between collection and analysis[55,56]. Ensuring such methodological consistency is essential for obtaining reliable and reproducible measurements of salivary Trx-1 and for facilitating its translation into clinical practice. The available literature for the use of salivary Trx-1 reports encouraging findings, however, most studies are cross-sectional and observational. This largely restricts the capacity to adapt a causal relationship between altered levels of Trx-1 and oral disease as such. Additionally, variations in terms of disease categorization, patient samples, and laboratory methods add to the complexities when comparing results from various studies. Consequently, the currently available information must be interpreted cautiously, and the use of salivary Trx-1 as a potential diagnostic and prognostic biomarker should be seen as preliminary until validated through large-scale multicenter prospective studies. To provide a summary of the limitations, along with biological discrepancies involved, a comprehensive review of the relevant studies is provided in Table 2.

Table 2 Review of studies evaluating the role of theoredoxin-1 in oral diseases.

Ref.	Study design & cohort/model	Biomarker & analyzed biofluid matrix	Key clinical findings/diagnostic & therapeutic metrics	Methodological weaknesses & risks of bias	Biological resolution of discrepancies (high vs low levels)
Lorente et al[46], 2026	Prospective observational clinical study (n = 144)	Salivary Trx-1 measured in unstimulated whole cell-free supernatant	Salivary Trx-1 levels were significantly lower in patients with periodontitis than in healthy controls (P < 0.001). Trx-1 showed a moderate negative correlation with tissue destruction severity (ρ = –0.47, P < 0.001). A diagnostic cut-off of ≤ 41.55 ng/mL achieved an AUC of 75%, with 83% sensitivity and 63% specificity	Participants were not adequately controlled or stratified for chronic systemic inflammatory comorbidities, introducing potential selection bias. In addition, no a priori sample size or statistical power calculation was reported	Chronic periodontal inflammation produces sustained oxidative stress and neutrophil activation, progressively exhausting the local antioxidant reserve. Consequently, extracellular salivary Trx-1 decreases because antioxidant consumption exceeds its production and secretion
Amirchaghmaghi et al[52], 2020	Comparative cross-sectional clinical study (n = 88)	Salivary Trx-1 measured in unstimulated whole cell-free supernatant	Salivary Trx-1 levels followed the trend healthy > OSCC > OLP, although the overall difference was not statistically significant (P = 0.135). Keratotic OLP demonstrated significantly higher Trx-1 levels (P = 0.003), while patients with OSCC aged > 65 years showed markedly elevated concentrations (P = 0.001)	A considerable age imbalance between the study groups introduced potential age-related redox confounding. Disease subgroup analyses were also limited by relatively small sample sizes	Trx-1 demonstrates a biphasic biological response. During early inflammatory and premalignant stages, Trx-1 is upregulated to protect against oxidative DNA damage. As malignancy progresses, tumor cells increasingly retain intracellular Trx-1 to support proliferation and survival, reducing its extracellular release into saliva
Iwasawa et al[53], 2011	Retrospective molecular validation and immunohistochemical tissue study (n = 50).	TrxR1 gene and protein expression evaluated in primary OSCC tissue and matched healthy surgical margins	TXNRD1 expression was increased by approximately 5.10-fold in OSCC tissues compared with healthy controls (P < 0.01). High cytoplasmic TrxR1 protein expression was significantly associated with lymph node metastasis (P = 0.027) and advanced clinical stage III/IV disease (P = 0.007)	The investigation evaluated only intracellular tissue expression and therefore could not determine trans-epithelial transfer into saliva. Semi-quantitative immunohistochemical scoring also introduced potential observer bias	The findings support intracellular sequestration of the thioredoxin system within malignant cells. Increased TrxR1 activity enables tumor survival under oxidative stress while limiting extracellular diffusion of Trx-1 into saliva, thereby explaining reduced salivary concentrations despite high tissue expression
Wu et al[34], 2023	Experimental pre-clinical in vivo diabetic mouse model combined with in vitro periodontal ligament stem cell experiments	Trx-1 expression evaluated in periodontal tissues and cultured periodontal ligament stem cells	Knockdown of Trx-1 inhibited osteogenic differentiation through disruption of the Wnt/β-catenin signaling pathway. Administration of recombinant human Trx-1 reduced reactive oxygen species, restored Wnt/β-catenin signaling, and significantly rescued alveolar bone loss in diabetic mice	Animal models cannot fully replicate the complexity of the human oral environment, including salivary matrix effects, systemic influences, lifestyle factors, and enzymatic degradation	These experimental findings demonstrate that excessive oxidative stress directly suppresses endogenous Trx-1 expression. Restoration of Trx-1 activity reverses oxidative injury, supports osteogenesis, and validates antioxidant depletion as a major mechanism underlying severe periodontal disease

Trx-1: Thioredoxin-1; TrxR1: Thioredoxin reductase-1; OSCC: Oral squamous cell carcinoma; OLP: Oral lichen planus; TXNRD1: Thioredoxin reductase 1 gene; Wnt: Wingless/integrated signaling pathway; β-catenin: Beta-catenin; AUC: Area under the curve.

Research for the future needs to focus on creating standardized methods for collecting saliva samples in order to control biological and procedural variation. Documentation of various factors such as the timing of the sample collection, whether or not the individual has fasted before sampling, the type of food consumed, oral hygiene, use of any medication, exposure to any substance, and the state of the individual’s health. The use of standardized sampling times for salivary samples is necessary to increase reproducibility and make inter-study comparisons possible because of the effects of circadian rhythm on salivary contents. Furthermore, the establishment of validated reference ranges and clinically meaningful cutoff values for salivary Trx-1 remains an essential prerequisite for its future clinical application.

CONCLUSION

The present study is one of the initial efforts in summarizing the possible role of salivary Trx-1 as a non-invasive redox biomarker in various oral conditions. The physiological functions of Trx-1 in maintaining the redox status, as well as promoting inflammation and cell survival, support the scientific basis of this biomolecule's potential use in periodontal diseases, OPMDs, and OSCC. The presence of Trx-1 in the saliva and its dual role as an antioxidant and signal transduction agent may possibly make it a suitable candidate for representing redox perturbation in the oral environment. Nevertheless, existing studies on this topic remain scarce, especially considering no available salivary research in some oral diseases like oral submucous fibrosis. Research should aim at conducting highly designed and long-term large sample sizes studies to verify the diagnostic and prognostic importance of salivary Trx-1, as well as to develop standardized procedures for collecting samples and analyzing the results. Future biomarker panels integrating salivary Trx-1 with established molecular, inflammatory, and immunological markers may provide better diagnostic and prognostic performance than individual biomarkers alone. In addition, advancements in point-of-care devices as well as biosensors could make it easy to translate the biomarker into practical clinical use through chair-side testing. Combining salivary markers with artificial Intelligence based analysis models might provide personalized diagnosis and risk prediction in oral health care.