Saliva biomarker and application thereof in rheumatoid arthritis disease activity assessment

Abstract

The invention discloses a saliva biomarker and application thereof in rheumatoid arthritis disease activity assessment, and relates to the technical field of biomarkers. The saliva biomarker is one of the secretion amount of saliva extracellular vesicles and the expression level of porphyromonas gingivalis peptidyl arginine deiminase in the saliva extracellular vesicles. Peptidyl arginine deiminase exists in extracellular vesicles secreted by porphyromonas gingivalis in saliva, and in a patient with rheumatoid arthritis, the secretion amount of the extracellular vesicles in the saliva is remarkably increased and is in positive correlation with disease activity; the expression level of porphyromonas gingivalis peptidyl arginine deiminase in saliva extracellular vesicles is remarkably improved and is in positive correlation with disease activity. According to the invention, real noninvasive and high-compliance detection is realized based on a saliva sample, and large-scale screening and long-term management can be realized.

Description

Technical Field

[0001] This invention relates to a saliva biomarker and its application in assessing the activity of rheumatoid arthritis, belonging to the field of biomarker technology. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis, joint destruction, and systemic complications. Early diagnosis and accurate assessment of disease activity are crucial for timely intervention, improved prognosis, and reduced disability rates. Current clinical diagnostic methods for RA primarily rely on a combination of clinical symptom assessment, imaging studies, and serological markers. However, this system has significant limitations and struggles to meet the demands for early diagnosis and precise monitoring.

[0003] Early-stage rheumatoid arthritis (RA) often presents with nonspecific symptoms (such as joint pain, swelling, and morning stiffness), easily confused with other joint diseases like osteoarthritis and psoriatic arthritis, leading to delayed diagnosis. Plain X-ray films are not sensitive enough for early erosive joint lesions, typically only showing clear changes when the disease progresses to the bone destruction stage, failing to meet the needs of early diagnosis. While ultrasound (US) and magnetic resonance imaging (MRI) can detect early synovitis, bone edema, and bone erosion, these techniques are highly dependent on equipment performance and operator experience, and are expensive and have limited availability, making them unsuitable as routine methods for large-scale screening or long-term frequent monitoring.

[0004] Currently, the most widely used serological markers for rheumatoid arthritis (RA) in clinical practice are rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA), such as anti-cyclic citrullinated peptide (CCP) antibody. RF has low specificity and can be seen in other autoimmune diseases (such as Sjögren's syndrome), chronic infections (such as hepatitis C), and even some healthy elderly people, leading to a high false-positive rate. While ACPA has high specificity for RA and is considered an important diagnostic marker and predictor of RA, approximately 20-40% of RA patients are ACPA negative, and these patients cannot obtain effective diagnostic evidence through ACPA testing.

[0005] Given that serological testing is invasive (venous blood collection), which may reduce patient compliance and is not conducive to large-scale population screening and long-term frequent monitoring, non-invasive biological sample testing (such as saliva) is receiving increasing attention. Saliva sampling is simple, safe, non-invasive, and highly accepted by patients, and has unique advantages in achieving early screening for RA, improving diagnostic accessibility, and long-term dynamic monitoring of disease activity.

[0006] In recent years, researchers have actively explored potential RA biomarkers in saliva. One study reported that IgA-type ACPA could be detected in the saliva of some RA patients, and its level was associated with higher disease activity, suggesting its potential as a disease monitoring biomarker. However, the positive detection rate of this biomarker in the saliva of RA patients was much lower than in serum (approximately 12% vs 45%), indicating insufficient sensitivity and limiting its clinical application value. Another study showed that the level of anti-cyclic citrullinated peptide antibody in the saliva of RA patients was significantly higher than that in healthy controls, but its positive rate was only 61.9%, also exhibiting insufficient sensitivity.

[0007] Previous studies have shown that the oral pathogen *Porphyromonas gingivalis* (… Porphyromonasgingivalis, Pg Infection is potentially associated with the onset and progression of rheumatoid arthritis (RA). Given that saliva directly reflects the oral microenvironment, saliva contains... Pg Related indicators are considered highly promising biomarkers for rheumatoid arthritis (RA). Some studies have detected IgA antibodies in saliva. Pg The levels of the virulence factor RgpB antibody were found to be statistically significantly positively correlated with RA disease activity (e.g., Disease Activity Score 28, DAS28). However, the strength of this correlation was weak (Spearman correlation coefficient was only 0.2 to 0.3), suggesting that its predictive or reflective efficacy of disease activity is limited and its clinical applicability is not high. Currently, no salivary biomarkers have been successfully translated and applied to routine clinical diagnosis or disease activity monitoring of RA.

[0008] Therefore, developing novel salivary biomarkers with high sensitivity and specificity that can effectively reflect the activity of RA disease, in order to make up for the shortcomings of the existing diagnostic system and meet the urgent needs of early clinical diagnosis and accurate monitoring, remains a key issue that needs to be addressed. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a saliva biomarker and its application in assessing the activity of rheumatoid arthritis. Based on saliva samples, it achieves a truly non-invasive and highly compliant assessment of the activity of rheumatoid arthritis, enabling large-scale screening and long-term management.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: On one hand, the present invention provides a salivary biomarker, wherein the salivary biomarker is one of the secretion amount of salivary extracellular vesicles and the expression level of Porphyromonas gingivalis peptidylarginine deiminase in salivary extracellular vesicles.

[0011] Furthermore, the peptidylarginine deiminoase is present in extracellular vesicles secreted by *Porphyromonas salivaria*.

[0012] Furthermore, the method for detecting the secretion amount of salivary extracellular vesicles includes: Salivary extracellular vesicles were extracted from saliva. The protein concentration of extracellular vesicles in salivary cells was detected using the biuret protein quantification method. The protein concentration of salivary extracellular vesicles is used as the secretion amount of salivary extracellular vesicles.

[0013] Furthermore, the method for detecting the expression level of *Porphyromonas gingivalis* peptidylarginine deiminoase in salivary extracellular vesicles includes: Salivary extracellular vesicles were extracted from saliva. Preparation of biotin-labeled Porphyromonas gingivalis peptidylarginine deiminase antibody; The expression level of peptidylarginine deiminase in *Porphyromonas gingivalis* in salivary extracellular vesicles was detected using a double-antibody sandwich assay, specifically including: The antibody against Porphyromonas gingivalis peptidylarginine deiminase was coated onto an ELISA plate and left to stand overnight. Blocked with phosphate buffer containing bovine serum albumin, washed, and then added with salivary extracellular vesicles, and incubated with shaking at room temperature; After cleaning, add biotin-labeled Porphyromonas gingivalis peptidylarginine deiminase antibody and incubate with shaking at room temperature; After washing, add horseradish peroxidase-labeled streptavidin and incubate with shaking at room temperature. After cleaning, tetramethylbenzidine was added, and the OD value at a wavelength of 450 nm was measured after color development. The OD value at a wavelength of 450 nm was used as the expression level of peptidylarginine deiminoase in extracellular vesicles of Porphyromonas gingivalis in salivary cells.

[0014] Furthermore, the saliva was collected within one hour of the subject waking up in the morning, without brushing teeth, rinsing mouth, eating, or drinking water.

[0015] On the other hand, the present invention also provides an application of the above-mentioned salivary biomarkers in assessing the disease activity of rheumatoid arthritis.

[0016] Furthermore, the amount of secretion from the salivary extracellular vesicles is positively correlated with the disease activity of rheumatoid arthritis.

[0017] Furthermore, the expression level of Peptidylarginine deiminoase in the extracellular vesicles of salivary cells is positively correlated with the disease activity of rheumatoid arthritis.

[0018] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: The salivary biomarkers provided by this invention rely on saliva samples to achieve truly non-invasive and highly compliant assessment of rheumatoid arthritis (RA) disease activity, enabling large-scale screening and long-term management. The secretion of extracellular vesicles in salivary tissue is significantly increased in RA patients, with ROC curve analysis showing an AUC of 0.76, sensitivity of 53.57%, and specificity of 86.67%. The expression level of *Porphyromonas gingivalis* peptidylarginine deiminoase in extracellular vesicles of salivary tissue is also significantly increased, with ROC curve analysis showing an AUC of 0.73, sensitivity of 57.69%, and specificity of [missing data]. The positive rate was 86.67%, demonstrating good diagnostic value in RA. Furthermore, the positive rate of Porphyromonas gingivalis peptidylarginine deiminoase in salivary extracellular vesicles was high at 93.3%, significantly higher than that of existing salivary biomarkers. Together, these findings address key bottlenecks in existing RA disease activity assessment systems (especially non-invasive salivary biomarkers), such as low positive rates, weak correlation with activity, and a lack of clinical translation. This provides a reliable, convenient, and promising new non-invasive detection tool for objective assessment of RA disease activity and prognostic management. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the morphological observation and particle size analysis of extracellular vesicles (EVs) in the salivary cells of patients with rheumatoid arthritis (RA) in Example 1 of the present invention. In this diagram, A is a morphological diagram of salivary EVs and B is a particle size distribution diagram of salivary EVs. Figure 2 This is a schematic diagram of the analysis of the secretion of salivary EVs in a disease group (RA group) and a healthy group (HC group) in some embodiments of the present invention. In this diagram, A is a schematic diagram comparing the protein content of salivary EVs in the RA group and the HC group, B is a schematic diagram of ROC curve analysis of the protein content of salivary EVs used to distinguish between RA patients and healthy individuals, and C is a schematic diagram of the correlation analysis between the protein content of salivary EVs in RA patients and the disease activity score 28 (DAS28). Figure 3 The sample prepared according to Example 2 of the present invention Pg EVs, prepared in Comparative Example 1 Pg The whole bacterial lysate and the Escherichia coli prepared in Comparative Example 2 (Escherichia coli, Ec) Peptidylarginine deiminoase in whole bacterial lysate ( Porphyromonas gingivalisSchematic diagram of peptidylarginine deiminase (PPAD) expression levels; Figure 4 This is a schematic diagram comparing the expression levels of PPAD in salivary EVs of a disease group (RA group) and a healthy group (HC group) in some embodiments of the present invention; Figure 5 This is a schematic diagram of ROC curve analysis of PPAD expression levels in salivary EVs used to distinguish between RA patients and healthy individuals in some embodiments of the present invention; Figure 6 This is a schematic diagram illustrating the correlation between PPAD expression levels in salivary EVs of RA patients and disease activity score 28 (DAS28), anti-cyclic citrullinated peptide antibody (Anti-CCP), and rheumatoid factor (RF) levels in some embodiments of the present invention. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention.

[0021] Example 1

[0022] This invention provides a salivary biomarker, which is the amount of extracellular vesicles (EVs) secreted in saliva. The following analysis examines the effectiveness of applying the amount of EVs secreted in saliva to assess the disease activity of rheumatoid arthritis (RA): Subject grouping and inclusion criteria: This embodiment sets up a disease group and a healthy group. The subjects of the disease group are RA patients who visited the Department of Rheumatology and Immunology of the Second Affiliated Hospital of Soochow University from November 1, 2024 to April 1, 2025. The subjects of the healthy group are healthy people who underwent physical examinations during the same period.

[0023] Inclusion criteria for the disease group (RA group): adherence to the 2010 revised diagnostic criteria for RA of the American College of Rheumatology, no past medical history, active disease, no concurrent autoimmune diseases, and no use of antibiotics within the past week.

[0024] Inclusion criteria for the healthy group (HC group): normal blood cell count, normal liver and kidney function, no history of autoimmune diseases, and negative for rheumatoid factor, antinuclear antibody and anti-cyclic citrullinated peptide antibody.

[0025] In this embodiment, there were 30 cases in the RA group, all of whom met the criteria for moderate to high disease activity and had systemic inflammatory response. There were 26 cases in the HC group.

[0026] Saliva collection and processing Participants were required to naturally secrete at least 3 mL of saliva into a collector within 20 minutes of waking up in the morning, without brushing their teeth, rinsing their mouths, eating, or drinking. The saliva was then placed in an ice pack and transported to the laboratory within 45 minutes. A protease inhibitor (APExBIO, USA) was added, the mixture was thoroughly mixed, and the saliva was stored at -80°C.

[0027] Extraction of salivary EVs (1) Centrifuge 400 μL of saliva at 3000 rpm for 15 min at 4℃; (2) Mix the supernatant with ExoQuick™ extraction reagent (SBI, USA) at a ratio of 4:1 and incubate overnight at 4°C; (3) Centrifuge at 1500 rcf for 30 min at 4℃, and discard the supernatant; (4) Centrifuge again at 1500 rcf for 5 min at 4℃, and discard the supernatant; (5) Resuspend the precipitate in 40 μL PBS buffer and store at -80℃.

[0028] Protein quantification of salivary EVs To avoid the impact of differences in salivary EV loading volume on subsequent Porphyromonas gingivalis peptidylarginine deiminase (PPE) assays... Porphyromonas gingivalis The results of quantitative detection of peptidylarginine deiminase (PPAD) were affected, so the concentration of EVs protein in saliva samples was detected using the biuretine acid assay (BCA).

[0029] Protein concentration was determined using the BCA Protein Quantitative Kit (BOSTER, China). The procedure is as follows: (1) Take an appropriate amount of EVs and mix them with RIPA lysis buffer (KeyGEN BioTECH, China); (2) Shake for 1 minute, then ice bath for 10 minutes, repeat twice; (3) Shake for 1 min, centrifuge at 14000 rcf for 10 min at 4℃, and collect the supernatant; (4) Mix reagents A and B at a ratio of 50:1 to prepare the working solution; (5) Serially dilute the standard (20 μL / well) in a 96-well plate with PBS; (6) Add 20 μL of the serially diluted standard and the supernatant after centrifugation to the detection wells respectively; (7) Add 200 μL of working solution to each detection well and incubate at 37°C for 30 min; (8) The OD value at a wavelength of 570 nm was measured using an ELISA reader (Tecan, Switzerland); (9) Plot the standard curve and calculate the protein content.

[0030] Morphological observation and particle size analysis of salivary EVs Morphological observation was performed using a transmission electron microscope (Philips, Netherlands), and particle size analysis was conducted using a nanoparticle tracking analyzer (Particle Metrix, Germany). Figure 1 As shown, salivary EVs from RA patients exhibit a typical biconcave disc-like structure under electron microscopy. Figure 1 (Medium A), with a particle size in the range of 50~150 nm, conforms to the characteristics of small EVs ( Figure 1 (B)

[0031] Relationship between salivary EV secretion and RA disease activity The results of salivary EV secretion in 56 participants are shown in Table 1: Table 1: Results of EV secretion in the saliva of 56 participants .

[0032] There were no statistically significant differences in age and gender distribution among the 56 participants (P>0.05).

[0033] Combination Figure 2 The amount of salivary EVs secreted by RA patients was significantly higher than that in the HC group (P < 0.01). Figure 2 The results (A) suggest that the production of salivary EVs may be related to the pathological state of RA. Receiver operating characteristic (ROC) curves were plotted using Prism software version 9.0.0, and the area under the curve (AUC) was used to assess the diagnostic value of salivary EV secretion. The results showed that the AUC for salivary EV protein content in RA patients was 0.76, with a cutoff value indicating a sensitivity of 53.57% and a specificity of 86.67%. Figure 2 (Medium B) indicates that the amount of salivary EVs secreted has good diagnostic value in RA. Furthermore, the amount of salivary EVs secreted by RA patients was significantly positively correlated with the disease activity score DAS28 (R=0.5418, P=0.0020). Figure 2 (C), which effectively demonstrates that the amount of extracellular vesicles secreted in saliva can serve as a potential biomarker for the clinical diagnosis and disease assessment of RA.

[0034] Example 2

[0035] This invention provides a salivary biomarker, namely the expression level of PPAD in salivary EVs. PPAD in salivary EVs is primarily composed of Porphyromonas gingivalis in saliva (…). Pg) secretion. Therefore, the following analysis examines the effect of PPAD expression levels in salivary EVs on the detection of rheumatoid arthritis (RA) disease activity: The subject grouping and inclusion criteria in this embodiment are the same as in Example 1, and will not be repeated here.

[0036] Extracellular vesicles secreted by Porphyromonas gingivalis ( Pg Extraction of EVs (1) Pg The W83 strain was anaerobically cultured in BHI medium until the optical density (OD) reached 1-2. (2) Centrifuge at 8500rcf for 10 min at 4℃, collect the supernatant, and filter the supernatant through a 0.22μm filter (Merck, USA); (3) Add the filtered supernatant to a 100 KD ultrafiltration tube (Merck, USA), centrifuge at 1500 rcf for 25 min at 4 °C, collect the concentrate, and filter it again with a 0.22 μm filter; (4) Mix the concentrate with ExoQuick™ extraction reagent (SBI, USA) at a ratio of 3:1 and incubate overnight at 4°C; (5) Centrifuge at 1500 rcf for 30 min at 4℃, and discard the supernatant; (6) Resuspend the precipitate in 40 μL PBS buffer and store at -80℃.

[0037] Comparative Example 1: This comparative example provides a *Porphyromonas gingivalis* ( Pg The bacterial cells were collected using the following method: (1) Pg The W83 strain was anaerobically cultured in BHI medium until the optical density (OD) reached 1-2. (2) Centrifuge at 8500rcf for 10 min at 4℃, collect the bacterial cells and set aside.

[0038] Comparative Example 2: This comparative example provides an Escherichia coli (E. coli) Ec The bacterial cells were collected using the following method: (1) Ec The DH5a strain was cultured in LB medium until the OD value reached 1-2; (2) Centrifuge at 8500rcf for 10 min at 4℃, collect the bacterial cells and set aside.

[0039] To clarify PgTo determine whether the derived EVs express PPAD and to verify the specificity of the PPAD antibody, Western blot was used to detect the EVs prepared in Example 2. Pg EVs, prepared in Comparative Example 1 Pg The whole-cell lysate and the solution prepared by Comparative Example 2 Ec The expression of PPAD in whole-cell lysate was determined by the following steps: (1) Sample preparation Take an appropriate amount of the above sample and mix it with RIPA lysis buffer (KeyGEN BioTECH, China). Incubate on ice for 10 min, then shake for 1 min. Repeat twice. Centrifuge at 14000 rcf for 10 min at 4℃ and collect the supernatant. Add SDS-PAGE protein loading buffer (KeyGEN BioTECH, China), mix well, and incubate in a water bath at 100℃ for 10 min. Cool on ice.

[0040] (2) Gel preparation A 12.5% ​​separating gel was prepared using the Omni-Easy™ kit (Epizyme, China). 4.0 mL of the lower gel solution was mixed with an equal volume of lower gel buffer, 80 μL of coagulant was added, and the mixture was poured into a glass plate until the liquid level was 1.5 cm from the top edge of the short plate.

[0041] Prepare a 5% stacking gel. Take 1.0 mL of the top gel solution and mix it with an equal volume of colored top gel buffer. Add 20 μL of coagulant, mix gently, and slowly pour into a glass plate. Immediately insert the comb teeth. Let stand at room temperature for 30 minutes. After the gel has completely solidified, carefully remove the comb teeth.

[0042] (3) Sample loading and electrophoresis Dilute 10×Tris-glycine-SDS electrophoresis buffer (Epizyme, China) with ultrapure water to prepare 1× electrophoresis buffer; install the gel into the electrophoresis tank and add 1× electrophoresis buffer; load 25 μL of protein (prepared in step (1)) into each well, and add pre-stained protein marker as a reference; adjust the voltage to 80V, electrophore for 30 min, then adjust the voltage to 120V, and stop electrophoresis when the indicator migrates to the bottom of the gel.

[0043] (4) Transfer membrane Prepare the transfer working solution by mixing 10× ice-free rapid transfer buffer (Epizyme, China), ultrapure water, and ethanol in a ratio of 1:8:1; immerse the PVDF membrane (0.2μm, Millipore Sigma, China) in ethanol for 30s; assemble the transfer clamp, placing the sponge pad, gel, PVDF membrane, and sponge pad in sequence, ensuring no air bubbles between each layer; add the transfer working solution and transfer at 400mA for 57min.

[0044] (5) Closed Dilute 10×TBS / Tween buffer (Epizyme, China) with ultrapure water to prepare 1×TBST buffer, add 5% skim milk, and block the PVDF membrane with 5% skim milk-TBST buffer at room temperature for 1 hour.

[0045] (6) Primary antibody incubation Dilute the PPAD primary antibody (Cayman, USA) 1:2000 with 1×TBST buffer; blot dry the PVDF membrane, add an appropriate amount of primary antibody to cover the entire membrane, and incubate overnight at 4°C; place the membrane in 1×TBST buffer, place it on a shaker, and wash it 3 times at room temperature for 10 min each time.

[0046] (7) Secondary antibody incubation Dilute the HRP-labeled secondary antibody (KeyGEN BioTECH, China) 1:5000 with 1×TBST buffer; blot dry the PVDF membrane, add an appropriate amount of secondary antibody to cover the entire membrane, and incubate at room temperature for 1 hour; place the membrane in 1×TBST buffer, place it on a shaker, and wash it 3 times at room temperature for 10 minutes each time.

[0047] (8) Image acquisition Mix ECL working solutions A and B (Vazyme, China) at a 1:1 ratio, evenly cover the PVDF film, and incubate in the dark for 1 min; acquire images using an e-BLOT imaging system (E-Blot, China).

[0048] The results are as follows Figure 3 As shown, under the condition of equal protein, Pg and Pg EVs all express PPAD, and Pg EVs showed higher PPAD expression levels, while Ec No PPAD expression was detected, which effectively proves that the PPAD antibody used in this invention is reliable and that PPAD is present in... Pg In secreted EVs.

[0049] Next, the expression levels of PPAD in salivary EVs from the RA and HC groups were detected, as follows: First, biotinylate the PPAD antibody, following the instructions of the biotinylation kit (Abcam, UK): (1) Add 1 μL of modifier reagent to every 10 μL of PPAD antibody (1 mg / mL) (Cayman, USA) and mix gently.

[0050] (2) Add the antibody sample containing the modifier directly into the dry biotinylated material and gently blow it 1-2 times.

[0051] (3) Let stand at room temperature away from light for 15 minutes.

[0052] (4) After incubation, add 1 μL of quencher reagent to every 10 μL of antibody and mix gently.

[0053] (5) Biotinylated antibodies can be used after 5 minutes and can usually be stored at 4°C for up to 18 months.

[0054] Then, the expression of PPAD in salivary EVs was detected using a double-antibody sandwich method. The specific steps are as follows: (1) Coat 5 μg / mL PPAD antibody (Cayman, USA) onto a 96-well microplate, 50 μL per well, and incubate overnight; (2) Block the ELISA plate coated with PPAD antibody in step (1) with PBS buffer containing 5% bovine serum albumin for 2 hours. After washing 4 times with washing buffer, add 400 μg of saliva EVs and incubate at room temperature with shaking for 90 min. (3) After washing 4 times with the washing solution, add 100 mL of biotin-labeled PPAD antibody (500 ng / mL) and incubate at room temperature with shaking for 60 min; (4) After washing 4 times with the washing solution, add 100 mL of horseradish peroxidase (HRP) labeled streptavidin (Proteintech, USA) and incubate at room temperature with shaking for 30 min. (5) After washing 4 times with the cleaning solution, add 100 mL of tetramethylbenzidine (TMB) substrate (Proteintech, USA) for color development for 15 min. After terminating the reaction, immediately measure the OD value at a wavelength of 450 nm.

[0055] The results of PPAD expression level detection in the RA group and HC group are shown in Table 2.

[0056] Table 2: Results of PPAD expression levels in the disease group and the healthy group .

[0057] Combination Figure 4 From Table 2 and Figure 4 It can be seen that the positive rate of PPAD detection in the saliva of RA patients (P<0.05) and the expression level were significantly higher than those of healthy individuals (P<0.05).

[0058] The ability of salivary EVs PPAD expression levels to distinguish between RA patients and healthy individuals was analyzed using ROC curves. The results are as follows: Figure 5 As shown, the AUC of PPAD expression level in salivary EVs of RA patients was 0.73, and the cutoff value showed a sensitivity of 57.69% and a specificity of 86.67%.

[0059] The correlation between salivary EVs PPAD expression levels and disease activity score 28 (DAS28), anti-cyclic citrullinated peptide antibody (Anti-CCP), and rheumatoid factor (RF) levels in RA patients is as follows: Figure 6 As shown, the expression level of salivary EVs PPAD in RA patients was significantly positively correlated with disease activity score 28 (R=0.6679, P<0.001) and anti-cyclic citrullinated peptide antibody level (R=0.4745, P<0.05), but not significantly correlated with rheumatoid factor level (R=0.0570, P>0.05). These results indicate that the expression level of salivary EVs PPAD has good diagnostic value in RA and is a potential biomarker for the clinical diagnosis and treatment of RA.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A saliva biomarker, characterized in that, The salivary biomarker is one of the following: the amount of secretion from salivary extracellular vesicles and the expression level of Porphyromonas gingivalis peptidylarginine deiminase in salivary extracellular vesicles.

2. The saliva biomarker according to claim 1, characterized in that, The peptidylarginine deiminoase is present in extracellular vesicles secreted by *Porphyromonas salivaria*.

3. The salivary biomarker according to claim 1, characterized in that, The method for detecting the secretion amount of salivary extracellular vesicles includes: Salivary extracellular vesicles were extracted from saliva. The protein concentration of extracellular vesicles in salivary cells was detected using the biuret protein quantification method. The protein concentration of salivary extracellular vesicles is used as the secretion amount of salivary extracellular vesicles.

4. The salivary biomarker according to claim 1, characterized in that, The method for detecting the expression level of Porphyromonas gingivalis peptidylarginine deiminase in salivary extracellular vesicles includes: Salivary extracellular vesicles were extracted from saliva. Preparation of biotin-labeled Porphyromonas gingivalis peptidylarginine deiminase antibody; The expression level of peptidylarginine deiminase in *Porphyromonas gingivalis* in salivary extracellular vesicles was detected using a double-antibody sandwich assay, specifically including: The antibody against Porphyromonas gingivalis peptidylarginine deiminase was coated onto an ELISA plate and left to stand overnight. Blocked with phosphate buffer containing bovine serum albumin, washed, and then added with salivary extracellular vesicles, and incubated with shaking at room temperature; After cleaning, add biotin-labeled Porphyromonas gingivalis peptidylarginine deiminase antibody and incubate with shaking at room temperature; After washing, add horseradish peroxidase-labeled streptavidin and incubate with shaking at room temperature. After cleaning, tetramethylbenzidine was added, and the OD value at a wavelength of 450 nm was measured after color development. The OD value at a wavelength of 450 nm was used as the expression level of peptidylarginine deiminoase in extracellular vesicles of Porphyromonas gingivalis in salivary cells.

5. The salivary biomarker according to claim 3 or 4, characterized in that, The saliva was collected within one hour of the subject waking up in the morning, before brushing teeth, rinsing mouth, eating, or drinking water.

6. The application of a salivary biomarker according to any one of claims 1 to 5 in assessing the disease activity of rheumatoid arthritis.

7. The application according to claim 6, characterized in that, The amount of extracellular vesicles secreted by salivary cells is positively correlated with the disease activity of rheumatoid arthritis.

8. The application according to claim 6, characterized in that, The expression level of Peptidylarginine deiminoase in extracellular vesicles of salivary cells was positively correlated with the disease activity of rheumatoid arthritis.

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