RBP4, ferritin as biomarkers for evaluating the effect of hormone therapy for allergic rhinitis

Abstract

This invention relates to the field of biomedical technology and proposes RBP4 and Ferritin as biomarkers for evaluating the efficacy of hormone therapy for allergic rhinitis. The biomarkers include one or a combination of RBP4 and Ferritin. Studies have confirmed that both RBP4 and Ferritin are capable of distinguishing between hormone-sensitive and hormone-insensitive allergic rhinitis. RBP4 is expressed at higher levels in the hormone-sensitive allergic rhinitis group, while Ferritin is expressed at higher levels in the hormone-insensitive allergic rhinitis group. Compared to single biomarker detection, the combined use of RBP4 and Ferritin significantly improves the differential diagnostic efficacy, providing better differentiation between the two patient groups.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to RBP4 and Ferritin as biomarkers for evaluating the efficacy of hormone therapy for allergic rhinitis. Background Technology

[0002] Allergic rhinitis (AR), a prevalent respiratory allergic disease worldwide, has a continuously rising prevalence globally, particularly among children. The incidence rate is significantly higher in children than in adults and continues to increase annually, making it one of the major chronic diseases threatening children's respiratory health. Typical clinical manifestations include nasal congestion, nasal itching, paroxysmal sneezing, and clear runny nose. These symptoms not only directly affect patients' daytime activities, learning, and work efficiency, but nighttime nasal congestion also severely disrupts sleep quality, leading to problems such as daytime sleepiness and poor concentration. Long-term, recurrent AR can also induce various complications such as asthma, sinusitis, and allergic conjunctivitis, placing a heavy caregiving burden and financial strain on patients' families and significantly depleting social medical resources.

[0003] In the clinical treatment of allergic rhinitis (AR), nasal corticosteroids (INCS) have significant anti-inflammatory and anti-allergic effects, acting on multiple stages of the inflammatory cascade to effectively relieve nasal symptoms and control disease progression in most patients. INCS have the advantages of low systemic bioavailability and good safety profile, acting directly on the nasal mucosa lesions to exert a highly effective local anti-inflammatory effect. However, a significant treatment bottleneck exists in clinical practice: approximately 15%-25% of AR patients do not respond well to standard-dose INCS treatment, with persistent or recurrent symptoms. These patients are defined as steroid-insensitive allergic rhinitis. Because these patients lack specific clinical symptoms and signs compared to hormone-sensitive AR patients, they cannot be distinguished through routine examinations. Clinicians can only employ empirical treatment plans, such as having patients continuously use INCS for 2 weeks or even longer, and assessing hormone sensitivity based on symptom improvement. This approach not only delays precise treatment for some patients but may also increase the risk of local adverse reactions such as nasal dryness and nosebleeds due to long-term inappropriate use of hormones. Some patients may also gradually lose sensitivity to INCS as the disease progresses or with long-term medication. Currently, the clinical field still lacks specific biomarkers and convenient testing methods for rapidly and accurately identifying hormone-insensitive AR patients. Related diagnoses mainly rely on physicians' assessment of patients' subjective symptoms and long-term follow-up of treatment responses, lacking objective indicators. This situation severely restricts the development of precision diagnosis and treatment for AR and has become a key problem that urgently needs to be solved in clinical practice. 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. The combination of RBP4 protein and Ferritin protein can distinguish between hormone-sensitive and hormone-insensitive rhinitis, and is significantly correlated with the hormone therapy response of both types of AR patients.

[0005] The technical solution of the present invention is implemented as follows: On the one hand, the present invention provides the application of a biomarker in the preparation of products for evaluating the efficacy of hormone therapy for allergic rhinitis, said biomarker including one or a combination of RBP4 and Ferritin.

[0006] Based on the above technical solutions, preferably, the expression level of the biomarker RBP4 in the nasal secretions of patients with hormone-sensitive allergic rhinitis is higher than that in patients with hormone-insensitive allergic rhinitis.

[0007] Based on the above technical solutions, preferably, the expression level of the biomarker Ferritin 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 hormone is mometasone furoate nasal spray.

[0009] On the other hand, the present invention also provides a product for evaluating the efficacy of hormone therapy for allergic rhinitis, the product comprising at least one of a reagent, a kit, and a chip for detecting RBP4 expression levels.

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

[0011] Based on the above technical solutions, preferably, the product includes at least one of reagents, kits, and chips for detecting the expression levels of RBP4 and Ferritin.

[0012] Based on the above technical solutions, preferably, the sample to be tested is nasal secretions.

[0013] The RBP4 and Ferritin of this invention, as biomarkers for evaluating the efficacy of hormone therapy for allergic rhinitis, have the following advantages over existing technologies: Studies have confirmed that both RBP4 and Ferritin can distinguish between hormone-sensitive and hormone-insensitive allergic rhinitis. RBP4 expression is higher in the hormone-sensitive allergic rhinitis group, while Ferritin expression is higher in the hormone-insensitive allergic rhinitis group. Compared to single biomarker testing, the combined use of RBP4 and Ferritin significantly improves the differential diagnostic efficacy, providing better differentiation between the two types of patients. 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 diagram of RBP4 and Ferritin; Figure 2 To validate the expression maps of RBP4 and Ferritin for Luminex; Figure 3 ROC curves for CAR and RAR predictions using RBP4 and Ferritin; Figure 4 The ROC curves for CAR and RAR predictions by the combined use of RBP4 and Ferritin. Figure 5 Forest plot of RBP4 and Ferritin regression coefficients; 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 RBP4 protein, and Figure B shows the test results for Ferritin 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] 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.

[0019] This invention discloses the application of RBP4 and Ferritin proteins as biomarkers in the preparation of products for evaluating the efficacy of hormone therapy for allergic rhinitis. Specifically, the expression level of RBP4 protein in the nasal secretions of patients with hormone-sensitive allergic rhinitis is higher than that of patients with hormone-insensitive allergic rhinitis. The expression level of Ferritin protein in the nasal secretions of patients with hormone-insensitive allergic rhinitis is also higher than that of patients with hormone-sensitive allergic rhinitis.

[0020] RBP4, or retinol-binding protein 4, is mainly secreted by the liver and adipose tissue. Its core function is to transport vitamin A (retinol) in the blood to various tissues throughout the body, which is crucial for maintaining vision, immune function, and embryonic development. Scientific research has found that RBP4 has pro-inflammatory effects and can induce inflammatory responses and oxidative stress.

[0021] Ferritin protein is the body's main form of iron storage, and its level reflects the body's iron reserves. It is also an acute-phase reactant protein; during allergic rhinitis attacks, the inflammatory state of the nasal mucosa may lead to a certain degree of increase in ferritin levels in nasal secretions. Therefore, changes in ferritin levels can indirectly reflect inflammatory activity in the body.

[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) Those who have developed 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 RBP4 and Ferritin were finally identified as potential biomarkers.

[0036] Figure 1 The expression results of RBP4 and Ferritin in the two groups of subjects were presented by proteomics analysis. The results showed that RBP4 was expressed at a higher level in the CAR group and Ferritin was expressed at a higher level in the RAR group, with significant differences in protein expression between the two groups (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 with shaking 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 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 dual lasers. The data analysis was performed using Milliplex Analyst Version 5.1 software to achieve accurate quantitative detection of RBP4 and Ferritin proteins.

[0041] Figure 2 The expression results of RBP4 and Ferritin detected by the Luminex liquid-phase suspension chip are presented. Validation data show that the expression trends of RBP4 and Ferritin in the two groups of subjects are consistent with the previous proteomics detection results, and there are significant differences between the groups (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 RBP4 and Ferritin 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))=0.289528+0.000322×Ferritin−0.001604×RBP4.

[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 0.51 using the maximum Youden index method. At this threshold, the model sensitivity was 75.86%, the specificity was 82.76%, and the Youden index was 0.586. It can balance the missed diagnosis rate and the false diagnosis 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 RBP4 and Ferritin's predictions of CAR and RAR are shown. Figure 3 As shown, the areas under the ROC curves of RBP4 and Ferritin are both greater than 0.6, indicating good discriminative ability.

[0048] Figure 4 The ROC curves for the combined predictions of CAR and RAR using RBP4 and Ferritin are shown. Figure 4As shown, the combined curve of RBP4 and Ferritin has an area under the curve of 0.82, indicating better discriminative ability.

[0049] Figure 5 This is a forest plot of the RBP4 and Ferritin regression coefficients. In the plot, the confidence intervals of the two indicators do not cross the dashed line, indicating that the coefficients are significant; in addition, the position of the coefficient points visually reflects the direction of the effects of the two.

[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 RBP4 and Ferritin 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. The application of a biomarker in the preparation of products for evaluating the efficacy of hormone therapy for allergic rhinitis, characterized in that, The markers include one or a combination of RBP4 and Ferritin.

2. The application as described in claim 1, characterized in that: The expression level of the biomarker RBP4 in the nasal secretions of patients with hormone-sensitive allergic rhinitis was higher than that in patients with hormone-insensitive allergic rhinitis.

3. The application as described in claim 1, characterized in that: The expression level of the biomarker Ferritin 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 1, characterized in that: The hormone in question is mometasone furoate nasal spray.

5. A product for evaluating the efficacy of hormone therapy for allergic rhinitis, characterized in that, The product includes at least one of reagents, kits, and chips for detecting RBP4 expression levels.

6. The product as described in claim 5, characterized in that, The product includes at least one of reagents, kits, and chips for detecting Ferritin expression levels.

7. The product as described in claim 5, characterized in that, The product includes at least one of reagents, kits, and chips for detecting the expression levels of RBP4 and Ferritin.

8. The product as described in claim 5, characterized in that, The sample to be tested was nasal secretions.

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