A biomarker for reflecting the effect of hormone therapy for allergic rhinitis and application thereof

Abstract

This invention relates to the field of biomedical technology and proposes a biomarker for reflecting the efficacy of hormone therapy for allergic rhinitis and its application. The biomarker includes one or more combinations of IFNGR1, Furin, and CSF1R. The biomarkers IFNGR1, Furin, and CSF1R of this invention can all differentiate between hormone-sensitive and hormone-insensitive rhinitis. Their expression levels in the nasal secretions of patients with hormone-insensitive allergic rhinitis are significantly lower than those of patients with hormone-sensitive allergic rhinitis, thus reflecting the efficacy of hormone therapy for allergic rhinitis. Furthermore, compared with a single biomarker, the combination of the three biomarkers has better discriminative ability and better efficacy, providing important evidence for subsequent treatment of allergic rhinitis.

Description

Technical Field

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

[0002] Allergic rhinitis (AR) is a prevalent respiratory allergic disease worldwide, with an even higher incidence in children, and the incidence is showing an increasing trend year by year. Typical symptoms include nasal congestion, itchy nose, runny nose, and sneezing. It not only severely affects patients' sleep quality, learning and work efficiency, but may also trigger complications such as asthma, sinusitis, and conjunctivitis, placing a heavy burden on patients' families and the social healthcare system.

[0003] Currently, intranasal corticosteroids (INCS) are the first-line treatment for allergic rhinitis (AR), effectively relieving inflammatory symptoms and controlling disease progression in most patients. However, in clinical practice, approximately 15%-25% of AR patients do not respond well to standard-dose intranasal corticosteroids, experiencing persistent or recurrent symptoms. These patients are defined as hormone-insensitive allergic rhinitis. These patients lack specific clinical manifestations, and clinicians often need to rely on empirical treatment, i.e., continuous use of corticosteroids for 2 weeks or even longer, to determine hormone sensitivity. Some patients may also gradually or suddenly lose sensitivity to INCS as the disease progresses. Currently, there is a lack of specific biomarkers or detection methods for rapidly and accurately identifying hormone-insensitive patients. Related diagnoses rely on subjective symptom assessment and treatment response follow-up, lacking objective indicators, which has become a key bottleneck restricting the precise diagnosis and treatment of AR. Summary of the Invention

[0004] In view of this, the present invention proposes a biomarker for reflecting the effect of hormone treatment on allergic rhinitis and its application. This biomarker can distinguish between hormone-sensitive and hormone-insensitive rhinitis and has a significant correlation with the hormone treatment response of the two types of AR patients.

[0005] The technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a biomarker for reflecting the effect of hormone treatment for allergic rhinitis, said biomarker including one or more combinations of IFNGR1, Furin and CSF1R.

[0006] Based on the above technical solutions, preferably, the biomarker is composed of IFNGR1, Furin and CSF1R.

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

[0008] Secondly, the present invention provides the application of the above-mentioned biomarkers in the preparation of products for determining the sensitivity of patients with allergic rhinitis to nasal spray hormone treatment.

[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 is used to differentiate between hormone-sensitive allergic rhinitis and hormone-insensitive allergic rhinitis.

[0011] Thirdly, the present invention provides a detection product for determining the sensitivity of patients with allergic rhinitis to nasal spray hormone treatment, the product comprising reagents for detecting the expression levels of the aforementioned biomarker proteins.

[0012] The biomarker for reflecting the efficacy of hormone therapy for allergic rhinitis and its application, as described in this invention, have the following advantages over the prior art: The biomarkers IFNGR1, Furin, and CSF1R of this invention can all differentiate between hormone-sensitive and hormone-insensitive rhinitis. Their expression levels in the nasal secretions of patients with hormone-insensitive allergic rhinitis are significantly lower than those of patients with hormone-sensitive allergic rhinitis. Compared to a single biomarker, the combination of these three biomarkers demonstrates better discriminative ability and superior efficacy. Attached Figure Description

[0013] 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.

[0014] Figure 1 This is a diagram showing the expression of CSF1R, Furin, and IFNGR1 in proteomics. Figure 2 Expression maps of CSF1R, Furin, and IFNGR1 were validated for luminex. Figure 3 The ROC curves for CAR and RAR predictions by CSF1R, Furin, and IFNGR1 are shown. Figure 4The ROC curves for the combined predictions of CSF1R, Furin, and IFNGR1 for CAR and RAR are shown. Figure 5 A graph showing the PCA scores between the two groups; Figure 6 Box plot for predicting probability distribution; Figure 7 The model calibration curve is shown.

[0015] Figures 1-3 In the diagram, Figure A shows the test results for CSF1R, Figure B shows the test results for Furin, and Figure C shows the test results for IFNGR1. Detailed Implementation

[0016] 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.

[0017] Previous research has revealed potential differences in the inflammatory mechanisms and protein expression between hormone-insensitive and hormone-sensitive allergic rhinitis (AR). Based on this, our project team previously conducted proteomic analysis on nasal secretion samples from patients with both hormone-insensitive and hormone-sensitive AR, initially identifying IFNGR1, Furin, and CSF1R as potentially valuable in differentiating between hormone-sensitive and hormone-insensitive AR. Validation experiments showed that this combination of biomarkers was significantly correlated with hormone treatment response in both types of AR patients.

[0018] This invention identifies a combination of biomarkers that can reflect the sensitivity of patients with allergic rhinitis (AR) to nasal spray steroid treatment by screening and validating differentially expressed proteins in nasal secretions. The specific technical approach is as follows: 1. Screening and grouping of research subjects 1.1 Inclusion Criteria AR patients 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 were selected. The inclusion criteria are as follows: (1) Age 18-65 years old, gender not limited.

[0019] (2) Meets the diagnostic criteria of the Chinese Guidelines for the Diagnosis and Treatment of Allergic Rhinitis (2022 Revised Edition), is perennial, persistent, moderate to severe allergic rhinitis, has a history of ≥1 year, and has symptom attacks for ≥12 weeks per year and ≥4 days per week.

[0020] (3) At least two items in the Total Nasal Symptom Score (TNSS) are ≥2 points, and the total score is ≥6 points.

[0021] Note: The TNSS (Total Nasal Symptom Score) is a standardized tool that assesses the severity of nasal symptoms in patients with allergic rhinitis by evaluating the intensity of four nasal symptoms (nasal congestion, runny nose, nasal itching, and sneezing). Each symptom is scored from 0 to 3 (0: no symptoms; 1: mild (present but not bothersome); 2: moderate (obvious and affecting daily life); 3: severe (unbearable and significantly interferes with activities)). The daily total score ranges from 0 to 12, with higher scores indicating more severe symptoms.

[0022] (4) Skin prick test (SPT) and / or serum specific IgE antibody test, showing positive results for at least one perennial allergen (dust mites, cockroaches, animal dander, etc.).

[0023] 1.2 Exclusion Criteria (1) Individuals who have had upper or lower respiratory tract infections, fever, or other systemic symptoms within the past two weeks; (2) Patients with concurrent acute / chronic sinusitis, dry rhinitis, atrophic rhinitis, severe nasal septum deviation, or bronchial asthma; (3) Pregnant women, breastfeeding women, or those who plan to have children in the near future; (4) Patients with severe systemic diseases who are deemed unsuitable for participation in this study by a physician; (5) Patients who have participated in other drug clinical trials within the past 3 months.

[0024] All participants signed written informed consent forms and completed questionnaires on baseline clinical data and nasal symptoms.

[0025] 1.3 Nasal spray hormone therapy intervention Patients received a two-week standard treatment regimen using mometasone furoate nasal spray (trade name: Nasonex®, manufacturer: Merck Sharp & Dohme). The administration method was as follows: adults and adolescents aged 12 years and older, one spray (each spray containing 50 μg of mometasone furoate) was administered to each nostril once daily. The nasal cavity was cleaned before application, and the spray nozzle was directed towards the outer wall of the nasal cavity to avoid direct irritation of the nasal septum. Regular, uninterrupted use was maintained throughout the treatment.

[0026] 1.4 Treatment effect grouping The degree of decrease in TNSS score after treatment was used as an indicator of efficacy. Symptom improvement was assessed using the TNSS questionnaire two weeks after treatment. Hormone-sensitive allergic rhinitis group (CAR group): TNSS improvement rate ≥30%, 20 cases included.

[0027] Hormone-insensitive allergic rhinitis group (RAR group): TNSS improvement rate <30%, 20 cases included.

[0028] Note: TNSS improvement rate = (TNSS before treatment - TNSS after treatment) / TNSS after treatment × 100%.

[0029] 2. Nasal secretion sample collection and processing A 2.5cm × 1cm × 0.5cm PU sponge was placed between the nasal septum and inferior turbinate in both nasal cavities of the subject. The subject was instructed to pinch the nostrils for 5 minutes to allow the sponge to fully absorb nasal secretions. After removing the sponge, it was quickly placed into a filter tube, sealed with sealing film, and stored at 4°C. Within 4 hours, the sample was transferred to the laboratory on ice and centrifuged at 16000g for 20 minutes at 4°C to separate nasal secretions (NS). The volume of NS was recorded, aliquoted, and stored at -70°C for subsequent testing.

[0030] 3. Proteomics detection and differential biomarker screening 3.1 Sample Pretreatment Nasal secretion samples stored at -70℃ were reconstituted at room temperature for 2 hours and vortexed. Proteins were extracted by adding SDT lysis buffer (4% SDS, 100mM Tris-HCl, pH 7.6) and quantified using the BCA method. 15μg of protein was mixed with 5× loading buffer, boiled in water for 5 minutes, and then subjected to SDS-PAGE electrophoresis (4%-20% pre-prepared gradient gel, constant voltage 180V, 45 minutes) and stained with Coomassie Brilliant Blue R-250.

[0031] 3.2 Enzymatic hydrolysis and desalting A suitable amount of protein from each sample was mixed to form a pool sample for QC. All samples (including the pool sample) were digested with trypsin using the filter-assisted proteome preparation (FASP) method. The digested peptides were desalted using a C18 cartridge, lyophilized, and then reconstituted with 40 μL of 0.1% formic acid solution. The OD was then analyzed. 280 Determine peptide concentration.

[0032] 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 and DIA mode detection were performed using an Astral high-resolution mass spectrometer combined with a nanoliter flow rate VanquishNeo system (Thermo Fisher Scientific). The mass spectrometry data were processed using DIA-NN software to analyze the differences in protein expression profiles between the RAR group and the CAR group. IFNGR1, Furin, and CSF1R were screened as proteins with significant differential expression between the two groups (P_value < 0.05; Fold_Change < 0.667) as potential biomarkers.

[0033] Figure 1 This is a graph showing the expression of CSF1R, Furin, and IFNGR1 in proteomics. The results showed that CSF1R, Furin, and IFNGR1 were expressed at higher levels in the CAR group, and there were significant differences between the groups (P < 0.05).

[0034] 4. Validation and correlation analysis of biomarker combinations 4.1 Sample Expansion Twenty-nine patients from each of the CAR and RAR groups were re-collected, and nasal secretion samples were collected and processed according to the above method.

[0035] 4.2 Luminex liquid suspension chip detection (1) Reagent pretreatment: Before the experiment, the reagents were equilibrated to room temperature for 30 minutes; 7 concentration gradient standards and blank control were prepared; the microspheres (Beads), detection antibody and washing solution were diluted and 1×PE-streptavidin working solution was prepared; the sample supernatant was diluted to an appropriate multiple and 50 μL was loaded.

[0036] (2) Testing process ①Capture Incubation: Add microspheres, standards, quality control materials, and samples to each well, and incubate at room temperature (800 rpm) for 0.5-1 h or at 4°C overnight; ② After washing, add the detection antibody and incubate at room temperature (800 rpm) for 0.5-1 h; ③ After cleaning, add PE-streptavidin and incubate at room temperature (800 rpm) for 10-30 minutes; ④ After cleaning, add rinsing solution / sheath solution and incubate at room temperature (800 rpm) for 0.5-2 min.

[0037] On-machine detection: The sample plate was placed in the Luminex X-200 detection system, and the microsphere coding signal and fluorescence intensity signal were acquired by red and green lasers; the data were analyzed using Milliplex Analyst Version 5.1 software to complete the quantitative detection of IFNGR1, Furin and CSF1R.

[0038] Figure 2 The expression profiles of CSF1R, Furin, and IFNGR1 were validated using Luminex. Luminex results showed that the expression trends of CSF1R, Furin, and IFNGR1 between the two groups were consistent with proteomics, and there were significant differences between the groups (P < 0.01).

[0039] 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 levels of the combined biomarkers IFNGR1, Furin, and CSF1R and the hormone treatment response in AR patients, confirming that this combination can effectively differentiate between hormone-sensitive and hormone-insensitive allergic rhinitis.

[0040] Calculate the Logistic Regression Model: logit(P(RAR)) = 9.427 - 0.07728 × IFNGR1 - 0.000229 × Furin - 0.000018 × CSF1R, the results are shown in Table 1. The optimal decision threshold for the joint prediction model was determined to be 0.58 using the Youden index maximization method. At this threshold, the model's sensitivity and specificity were both 89.66%, and the Youden index was 0.793, which balanced the false negative and false positive rates, accurately distinguishing between Order=0 and Order=1 samples. The decision criteria were objective and repeatable.

[0041] Table 1 Regression Model Coefficient Data

[0042] Figure 3 The ROC curves for CAR and RAR predictions by CSF1R, Furin, and IFNGR1 are shown. Figure 3 As shown, the areas under the ROC curves of CSF1R, Furin, and IFNGR1 are all greater than 0.7, indicating good discriminative ability.

[0043] Figure 4 The ROC curves for the combined predictions of CSF1R, Furin, and IFNGR1 for CAR and RAR are shown. Figure 4 As shown, the area under the combined curve of CSF1R, Furin, and IFNGR1 is 0.961, indicating better discriminative ability.

[0044] Figure 5 This is a graph showing the PCA scores between the two groups. Figure 5 As shown, the samples of Order=0 (CAR group) and Order=1 (RAR group) are clearly distinguishable in the PCA space, indicating that the combination of IFNGR1, Furin, and CSF1R can effectively cluster samples of different groups.

[0045] Figure 6 This is a box plot for predicting the probability distribution. Figure 6 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.

[0046] Figure 7 The model calibration curve is shown. Figure 7As shown, the curve deviates little from the diagonal, indicating that the model's predicted probability is reliable.

[0047] 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 reflecting the efficacy of hormone therapy for allergic rhinitis, characterized in that: The biomarkers include one or more combinations of IFNGR1, Furin, and CSF1R.

2. A biomarker for reflecting the efficacy of hormone therapy for allergic rhinitis as described in claim 1, characterized in that: The biomarkers consist of IFNGR1, Furin, and CSF1R.

3. A biomarker for reflecting the efficacy of hormone therapy for allergic rhinitis as described in claim 1, characterized in that: The expression levels of the biomarkers in the nasal secretions of patients with hormone-insensitive allergic rhinitis were significantly lower than those in patients with hormone-sensitive allergic rhinitis.

4. The use of the biomarker according to any one of claims 1 to 3 in the preparation of a product for determining the sensitivity of patients with allergic rhinitis to nasal spray hormone therapy.

5. The application according to claim 4, characterized in that, The nasal spray hormone is mometasone furoate nasal spray.

6. The application according to claim 4, characterized in that, The product is intended to differentiate between hormone-sensitive and hormone-insensitive allergic rhinitis.

7. A testing product for determining the sensitivity of allergic rhinitis patients to nasal spray hormone therapy, characterized in that, The product contains reagents for detecting the expression level of the biomarker protein according to any one of claims 1 to 3.

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