A biomarker for evaluating the efficacy of hormone therapy for allergic rhinitis and its application

Abstract

This invention relates to the field of biomedical technology and proposes a biomarker for evaluating the efficacy of hormone therapy for allergic rhinitis and its application. The biomarker includes one or a combination of TFF3 and WFDC2. Both TFF3 and WFDC2 can differentiate between hormone-sensitive and hormone-insensitive rhinitis. Compared to a single biomarker, the combined use of the two significantly improves the differentiation efficacy and provides better differentiation between the two types of patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a biomarker for evaluating the efficacy of hormone therapy for allergic rhinitis and its application. Background Technology

[0002] With global environmental changes, lifestyle shifts, and an increase in the types of allergens exposed, the incidence of allergic rhinitis (AR) is on the rise globally. The disease is characterized by nasal congestion, itchy nose, runny nose, and sneezing, which have a comprehensive impact on patients' quality of life, mental health, and social activities. Long-term illness can also lead to complications such as asthma, sinusitis, and nasal polyps, placing a heavy burden on patients' families and the social healthcare system.

[0003] Currently, clinical treatment options for allergic rhinitis (AR) are diverse, including drug therapy, nasal irrigation, and allergen avoidance. Among these, intranasal corticosteroids (INCS) are a core first-line treatment. Their powerful anti-inflammatory effects effectively suppress nasal mucosal inflammation, reduce inflammatory cell infiltration and inflammatory mediator release, alleviate symptoms in most AR patients, and control disease progression. They are particularly suitable for treating moderate to severe persistent AR. Compared to oral corticosteroids, INCS have the advantages of fewer systemic adverse reactions and higher safety. Second-generation INCS are even more lipophilic and have stronger receptor binding affinity, resulting in more significant efficacy. However, clinical application has revealed that some patients do not respond well to INCS treatment. Approximately 15%-25% of patients do not experience effective symptom control after using standard doses of INCS and still experience recurrent relapses. The diagnosis and treatment of these hormone-insensitive allergic rhinitis patients remains a clinical challenge. The clinical manifestations of this type of patient are not particularly specific and are difficult to distinguish from hormone-sensitive patients through routine physical examination or allergen testing. Clinical diagnosis relies on long-term empirical treatment and observation, usually requiring continuous medication for more than two weeks to determine the patient's sensitivity to INCS. This not only increases the patient's medication burden but may also lead to a worsening of the condition due to delayed treatment. In addition, some patients may suddenly or gradually lose sensitivity to INCS during treatment, further increasing the difficulty of diagnosis and treatment. Currently, there are no specific biomarkers for rapid identification of hormone-insensitive AR patients, and there is a lack of convenient and accurate detection methods. The diagnostic process relies too much on subjective assessment and lacks objective indicators. This shortcoming seriously hinders the advancement of precision treatment for AR and limits further improvement in clinical treatment effects. In the past, through proteomics analysis of nasal secretions from AR patients who responded to and did not respond to nasal spray hormones, we found significant differences in viral infection, biosynthesis, metabolism, and innate immune pathways between the two groups of patients. Among them, AR patients who responded to nasal corticosteroids exhibited an abnormal mixed inflammatory pattern. In addition to type 2 inflammation, they also showed higher levels of inflammatory factors related to neutrophils (S100A8, S100A9), innate immunity (TLR2), and macrophages (MIF), as well as abnormal energy metabolism patterns (such as higher expression levels of PKM, MGLL, RDH10, and NQO2). Furthermore, they showed high expression of cellular stress / antioxidant pathways (such as SOD1, SOD2, GPX3, FTH1, HSPA8, HSP90AB4P, HSPB6, and HSBP1) and antiviral / interferon pathways (such as OAS2, IRF9, and RIG-I). Therefore, finding efficient identification methods and diagnostic indicators has become a key research focus and urgent need in the current field of AR diagnosis and treatment. Summary of the Invention

[0004] In view of this, the present invention proposes a biomarker for evaluating the efficacy of hormone therapy for allergic rhinitis and its application. The biomarkers TFF3 and WFDC2 can distinguish between hormone-sensitive and hormone-insensitive rhinitis and are significantly correlated with the hormone therapy response of the two types of AR patients.

[0005] The technical solution of the present invention is implemented as follows: On the one hand, the present invention provides a biomarker for evaluating the effect of hormone therapy for allergic rhinitis, said biomarker including one or a combination of two of TFF3 and WFDC2.

[0006] On the other hand, the present invention also provides the use of the above-mentioned biomarkers in the preparation of products for assessing the sensitivity of patients with allergic rhinitis to nasal spray hormone therapy.

[0007] Based on the above technical solutions, preferably, the expression level of the biomarker TFF3 in the nasal secretions of patients with hormone-insensitive allergic rhinitis is higher than that in patients with hormone-sensitive allergic rhinitis.

[0008] Based on the above technical solutions, preferably, the expression level of the biomarker WFDC2 in the nasal secretions of patients with hormone-sensitive allergic rhinitis is higher than that in patients with hormone-insensitive allergic rhinitis.

[0009] Based on the above technical solutions, preferably, the nasal spray hormone is mometasone furoate nasal spray.

[0010] Based on the above technical solutions, preferably, the product includes at least one of reagents, kits, and chips for detecting TFF3 expression levels.

[0011] Based on the above technical solutions, preferably, the product includes at least one of reagents, kits, and chips for detecting WFDC2 expression levels.

[0012] Based on the above technical solutions, preferably, the product includes at least one of reagents, kits, and chips for detecting the expression levels of TFF3 and WFDC2.

[0013] The biomarker for evaluating the efficacy of hormone therapy for allergic rhinitis and its application, as described in this invention, have the following advantages over existing technologies: Biomarkers TFF3 and WFDC2 can differentiate between hormone-sensitive and hormone-insensitive rhinitis. Specifically, TFF3 expression levels were significantly higher in the RAR group (hormone-insensitive allergic rhinitis group) than in the CAR group (hormone-sensitive allergic rhinitis group); WFDC2 expression levels were significantly higher in the CAR group than in the RAR group. Both TFF3 and WFDC2 were significantly correlated with hormone treatment response in both types of AR patients. Compared to using a single biomarker, the combined use of both significantly improved the differential diagnostic efficacy and provided better differentiation between the two patient groups. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 Proteomic expression diagrams of TFF3 and WFDC2; Figure 2 To validate the expression maps of TFF3 and WFDC2 for Luminex; Figure 3 ROC curves for CAR and RAR predictions by TFF3 and WFDC2; Figure 4 The ROC curves for the joint predictions of CAR and RAR by TFF3 and WFDC2; Figure 5 Forest plot of regression coefficients for TFF3 and WFDC2; Figure 6 A graph showing the PCA scores between the two groups; Figure 7 Box plot for predicting probability distribution; Figure 8 The model calibration curve is shown.

[0016] Figures 1-3 In the diagram, Figure A shows the test results for the TFF3 protein, and Figure B shows the test results for the WFDC2 protein. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] This invention discloses biomarkers for evaluating the efficacy of hormone therapy for allergic rhinitis and their applications. These biomarkers, TFF3 and WFDC2, can differentiate between hormone-sensitive and hormone-insensitive rhinitis and are significantly correlated with hormone therapy response in both types of AR patients. Specifically, the expression level of TFF3 protein in the nasal secretions of patients with hormone-insensitive allergic rhinitis is higher than that of patients with hormone-sensitive allergic rhinitis. Similarly, the expression level of WFDC2 protein in the nasal secretions of patients with hormone-sensitive allergic rhinitis is higher than that of patients with hormone-insensitive allergic rhinitis.

[0019] TFF3, or Trefoil Factor 3, is a protein primarily secreted by goblet cells in the mucosa. Its core function is to protect and repair the mucosa. When the nasal mucosa is damaged or irritated, TFF3 is secreted in large quantities to help epithelial cells heal and maintain the integrity of the mucosal barrier.

[0020] WFDC2 protein, also known as WAP Four-Disulfide Core Domain 2 or human epididymal protein 4 (HE4), is an anti-protease protein whose main function is to inhibit the activity of inflammatory enzymes (such as neutrophil elastase) to prevent excessive damage to the body's own tissues by the inflammatory response and to play a protective role in the mucosa.

[0021] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0022] This invention focuses on patients with allergic rhinitis (AR). By screening and validating differentially expressed proteins in nasal secretions, a combination of biomarkers reflecting patients' sensitivity to nasal spray hormone therapy was successfully constructed. The specific technical approach is as follows: 1. Screening and grouping of research subjects 1.1 Inclusion and Exclusion Criteria This study selected patients with acute rheumatoid arthritis (AR) who visited the Department of Otolaryngology-Head and Neck Surgery at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, between January 2024 and August 2025 as the study subjects. The specific inclusion and exclusion criteria are as follows: (1) Age range: 18-65 years old, gender not limited; (2) Meets the diagnostic criteria of the Chinese Guidelines for the Diagnosis and Treatment of Allergic Rhinitis (2022 Revised Edition), is diagnosed with perennial, persistent, moderate to severe allergic rhinitis, and meets the conditions of a history of ≥1 year, ≥12 weeks of symptom onset per year and ≥4 days of symptom onset per week; (3) At least two symptoms in the Total Nasal Symptom Score (TNSS) are ≥2 points, and the total score is ≥6 points; Note: The TNSS is a standardized scale for assessing the severity of nasal symptoms in patients with acute rhinitis (AR). It is achieved by quantifying four core symptoms: nasal congestion, runny nose, nasal itching, and sneezing. Each symptom is scored from 0 to 3 points (0 = no symptoms, 1 = mild symptoms, 2 = moderate symptoms affecting daily life, 3 = severe symptoms significantly interfering with daily activities). The total score ranges from 0 to 12 points, with higher scores indicating more severe symptoms.

[0023] (4) Skin prick test (SPT) and / or serum specific IgE antibody test results show a positive reaction to at least one perennial allergen (dust mites, cockroaches, animal dander, etc.).

[0024] Exclusion criteria: (1) Individuals who have experienced upper and lower respiratory tract infections, fever, or other systemic infection symptoms within the past two weeks; (2) Patients with concurrent acute / chronic sinusitis, dry rhinitis, atrophic rhinitis, severe nasal septum deviation, bronchial asthma, etc. (3) Pregnant women, breastfeeding women, or subjects who have plans to have children in the near future; (4) Patients with severe systemic diseases who are deemed unsuitable for participation in this study by a clinician; (5) Subjects who have participated in other drug clinical trials within the past 3 months.

[0025] All participants included in the study signed written informed consent forms and completed baseline clinical data collection and a nasal symptom questionnaire.

[0026] 1.2 Nasal spray hormone therapy intervention All AR patients meeting the inclusion criteria underwent a 2-week course of standard nasal corticosteroid treatment. The treatment drug used was mometasone furoate nasal spray (trade name: Nasonex®, manufacturer: Merck Sharp & Dohme). The specific dosage regimen was: one spray (each spray contains 50 μg of mometasone furoate) into each nostril once daily for adults and adolescents aged 12 years and older.

[0027] Before using the medication, the nasal cavity should be cleaned. When using the spray, the nozzle should be pointed towards the outer wall of the nasal cavity to avoid direct contact with the nasal septum and reduce mucosal irritation. During the treatment period, it is necessary to ensure regular medication without interruption or missed doses.

[0028] 1.3 Treatment effect grouping The decrease in TNSS score after treatment was used as the core efficacy evaluation indicator. Two weeks after treatment, the improvement of patients' symptoms was reassessed using the TNSS questionnaire, and patients were divided into two groups accordingly: Hormone-sensitive allergic rhinitis group (CAR group): TNSS score improvement rate ≥30%, 20 patients were included.

[0029] Hormone-insensitive allergic rhinitis group (RAR group): TNSS score improvement rate <30%, 20 patients were included.

[0030] Note: The formula for calculating the TNSS improvement rate is: (TNSS score before treatment - TNSS score after treatment) / TNSS score after treatment × 100%.

[0031] 2. Nasal secretion sample collection and processing 2.1 Sample Collection A PU sponge measuring 2.5cm×1cm×0.5cm was placed between the nasal septum and inferior turbinate in both nostrils of the subject. The subject was instructed to gently pinch the nostrils for 5 minutes to allow the PU sponge to fully absorb nasal secretions.

[0032] 2.2 Sample Preprocessing Immediately after removing the sponge, place it in a filter tube, seal it with sealing film, and temporarily store it at 4°C. Within 4 hours, transport the sample to the laboratory on ice and centrifuge at 16000g for 20 minutes at 4°C to obtain the nasal secretion (NS) supernatant. Record the NS volume, aliquot the sample, and store it at -70°C for long-term storage, ready for subsequent testing.

[0033] 3. Proteomics detection and differential biomarker screening 3.1 Sample Pretreatment Nasal secretion samples stored at -70℃ were removed and reconstituted at room temperature for 2 hours, followed by vortexing to mix thoroughly. Total protein was extracted by adding SDT lysis buffer (containing 4% SDS, 100mM Tris-HCl, pH 7.6) and quantified using the BCA method. 15μg of protein sample was accurately pipetted, mixed with 5× loading buffer in the specified ratio, heated in a boiling water bath for 5 minutes, and then subjected to SDS-PAGE electrophoresis (using a 4%-20% pre-prepared gradient gel, constant voltage 180V, electrophoresis for 45 minutes). After electrophoresis, Coomassie Brilliant Blue R-250 was used for staining and development.

[0034] 3.2 Enzymatic hydrolysis and desalting A pool sample was prepared by mixing appropriate amounts of protein from all samples and used as a quality control (QC) sample. All subject samples (including the pool sample) were digested with trypsin using the filter-assisted proteome (FASP) method. The digested peptides were desalted and purified using a C18 Cartridge column, lyophilized, and then reconstituted with 40 μL of 0.1% formic acid solution. Odulosic acid (OD) was measured. 280 The value determines the peptide concentration.

[0035] 3.3 DIA Mass Spectrometry Detection and Analysis An appropriate amount of iRT standard peptide was added to the enzymatically digested peptides of each sample. Liquid chromatography separation was performed using an Astral high-resolution mass spectrometer with a nanoliter flow rate Vanquish Neo system (manufactured by Thermo Fisher Scientific), and mass spectrometry detection was performed in DIA mode. The raw mass spectrometry data were processed using DIA-NN software, and the differences in protein expression profiles between the RAR group and the CAR group were compared and analyzed. Significantly differentially expressed proteins that met the criteria of P_value < 0.05 and Fold_Change < 0.667 were screened out, and TFF3 and WFDC2 were finally identified as potential biomarkers.

[0036] Figure 1 The expression results of TFF3 and WFDC2 in the two groups of subjects were presented by proteomics analysis. The results showed that the expression level of TFF3 in the RAR group was significantly higher than that in the CAR group; the expression level of WFDC2 in the CAR group was significantly higher than that in the RAR group; the differences between the groups were statistically significant (P<0.05).

[0037] 4. Validation and correlation analysis of biomarker combinations 4.1 Sample Expansion Twenty-nine new patients were recruited for both the CAR and RAR groups. Nasal secretion samples from both groups were collected again, strictly following the above sample collection and processing methods.

[0038] 4.2 Luminex liquid suspension chip detection 4.2.1 Reagent Pretreatment Before the experiment, all reagents were brought to room temperature and equilibrated for 30 minutes; standards and blank controls with 7 concentration gradients were prepared; microspheres (Beads), detection antibodies, and washing solutions were prepared separately to prepare 1×PE-streptavidin working solution; the sample supernatant was diluted to an appropriate factor and 50 μL was pipetted into the reaction wells.

[0039] 4.2.2 Testing Procedure Capture incubation: Add microspheres, standards, quality control and sample to each well in sequence, and incubate at 800 rpm for 0.5-1 hour at room temperature, or overnight at 4°C; Washing process: After incubation, perform a washing operation, then add the detection antibody and incubate at room temperature with shaking at 800 rpm for 0.5-1 hour; After the second cleaning, add PE-streptavidin and incubate at room temperature with shaking at 800 rpm for 10-30 minutes; After final cleaning, add rinsing solution / sheath solution and incubate at room temperature with shaking at 800 rpm for 0.5-2 minutes.

[0040] 4.2.3 On-machine testing The processed reaction plates were placed in the Luminex X-200 detection system, and the microsphere coding signal and fluorescence intensity signal were acquired by red and green dual lasers. The data analysis was performed using Milliplex Analyst Version 5.1 software to achieve accurate quantitative detection of TFF3 and WFDC2 proteins.

[0041] Figure 2 The expression results of TFF3 and WFDC2 detected by the Luminex liquid phase suspension chip are presented. In the Luminex validation results, the expression trends of TFF3 and WFDC2 are completely consistent with the proteomics results; the expression differences between the two groups are statistically significant (P < 0.05).

[0042] 4.3 Statistical Analysis SPSS and R software were used to perform statistical analysis on the detection data to verify the correlation between the expression level of the TFF3 and WFDC2 biomarker combination and the hormone treatment response of AR patients, and to confirm that the combination can effectively distinguish between hormone-sensitive and hormone-insensitive allergic rhinitis.

[0043] Calculate the Logistic Regression Model: logit(P(RAR))=7.7741534−0.0015710×WFDC2+0.0017170×TFF3.

[0044] The higher the score, the more likely it is to be RAR.

[0045] The results are shown in Table 1. The optimal decision threshold for the joint prediction model was determined to be 0.53 using the maximum Youden index method. At this threshold, the model's sensitivity and specificity both reached 82.76%, and the Youden index was 0.655. This approach can balance the false negative rate and the false positive rate, accurately distinguish between Order=0 and Order=1 samples, and the decision criteria are objective and repeatable.

[0046] Table 1 Regression Model Coefficient Data

[0047] Figure 3 The ROC curves for CAR and RAR predictions by TFF3 and WFDC2 are shown. Figure 3 As shown, the areas under the ROC curves of both TFF3 and WFDC2 are greater than 0.6, indicating good discriminative ability.

[0048] Figure 4 The ROC curves for the joint predictions of CAR and RAR by TFF3 and WFDC2 are shown. Figure 4 As shown, the combined curve of RBP4 and Ferritin has an area under the curve of 0.889, indicating better discriminative ability.

[0049] Figure 5 The graph shows the forest plots of the regression coefficients for TFF3 and WFDC2. In the plot, the confidence intervals for both indicators do not cross the dashed line, indicating that the coefficients are significant. Furthermore, the positions of the coefficient points visually represent the direction of their effects.

[0050] Figure 6 This is a graph showing the PCA scores between the two groups. Figure 6 As shown, the samples in Order=0 (CAR group) and Order=1 (RAR group) are clearly distinguishable in the PCA space, indicating that the combination of TFF3+WFDC2 can effectively cluster samples from different groups. Figure 7 This is a box plot for predicting the probability distribution. Figure 7 As shown, the median predicted probability of the Order=1 group (RAR group) is much higher than that of the Order=0 group, indicating that the model has a significant ability to distinguish the predicted probabilities of the two groups.

[0051] Figure 8 The model calibration curve is shown. Figure 8 As shown, the curve deviates little from the diagonal, indicating that the model's predicted probability is reliable.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A biomarker for evaluating the efficacy of hormone therapy for allergic rhinitis, characterized in that: The biomarkers include one or a combination of two of TFF3 and WFDC2.

2. The use of the biomarker as described in claim 1 in the preparation of a product for assessing the sensitivity of patients with allergic rhinitis to nasal spray hormone therapy.

3. The application as described in claim 2, characterized in that: The expression level of the biomarker TFF3 in the nasal secretions of patients with hormone-insensitive allergic rhinitis was higher than that in patients with hormone-sensitive allergic rhinitis.

4. The application as described in claim 2, characterized in that: The expression level of the biomarker WFDC2 in the nasal secretions of patients with hormone-sensitive allergic rhinitis was higher than that in patients with hormone-insensitive allergic rhinitis.

5. The application as described in claim 2, characterized in that: The nasal spray hormone is mometasone furoate nasal spray.

6. The application as described in claim 2, characterized in that: The product includes at least one of reagents, kits, and chips for detecting TFF3 expression levels.

7. The application as described in claim 2, characterized in that: The product includes at least one of reagents, kits, and chips for detecting WFDC2 expression levels.

8. The application as described in claim 2, characterized in that: The product includes at least one of reagents, kits, and chips for detecting the expression levels of TFF3 and WFDC2.

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