Children allergic asthma marker and application thereof

Abstract

The invention discloses a child allergic asthma marker and application thereof, and belongs to the technical field of biological medicine. Through detection of clinical samples, compared with healthy control children, the blood plasma BCAA level of children with allergic asthma is remarkably increased, and the kit has good diagnostic value. After medication treatment, the blood plasma BCAA level of children with allergic asthma is obviously reduced, and the composition has a good treatment and prognosis value. Therefore, the plasma BCAA can be used as a marker for diagnosis and prognosis of the children allergic asthma, and a foundation is laid for guiding clinical medication.

Description

Technical Field

[0001] The present invention relates to markers for childhood allergic asthma and their applications, belonging to the field of biomedical technology. Background Art

[0002] Allergic asthma is one of the diseases with the fastest growing incidence globally. Asthma is difficult to cure and prone to recurrence, seriously affecting the health and quality of life of patients and bringing a huge economic burden to society. If allergic asthma occurs in childhood, it still has a greater impact on adulthood. Glucocorticoids are currently commonly used anti-inflammatory drugs in clinical practice, however, this treatment mainly targets type 2 inflammatory responses. Many asthma patients still develop drug resistance and progress to severe asthma despite receiving regular inhaled treatment clinically; in addition, long-term use of hormones is prone to cause adverse reactions, especially affecting the growth and development of children. Therefore, timely judgment of treatment effects and standardized medication can effectively reduce the side effects of long-term hormone use on children's growth and development.

[0003] Research shows that the levels of various substances in the sera of patients with allergic asthma are abnormal. Typically, the expression of allergen-specific IgE increases. In addition, the increase in peripheral blood eosinophils can be seen in allergic or non-allergic asthma, and may be related to airway obstruction and can predict disease exacerbation. Some studies have shown that the peripheral eosinophil levels in patients with allergic asthma are higher than those in patients with non-allergic asthma. It has been found that Th2 cells in patients with allergic asthma undergo polarization reactions, with elevated plasma levels of IL-5 and IL-13, and decreased levels of INF-γ and IL-12 / 3. Reports show that various cytokines such as peripheral blood IL-21, L-10, IL-4, IL-5, and IL-13 are involved in the regulation of childhood allergic asthma. Clinically, the treatment of allergic asthma mainly targets inflammatory storms including cytokines, but when the levels of inflammatory factors decrease, asthma may only be temporarily relieved. Therefore, if children seek medical treatment during non-acute asthma periods, their conditions are easily overlooked, leading to the progression and aggravation of the disease and missing the best treatment opportunity. Searching for plasma diagnostic markers for allergic asthma that are not related to inflammation is expected to assist in the clinical diagnosis of childhood allergic asthma more efficiently, sensitively, and stably.

[0004] Clinically, most children with asthma also have allergic rhinitis, allergic conjunctivitis, and allergic constitutions, so they are collectively referred to as childhood allergic rhinitis-asthma syndrome. Airway inflammation triggered by repeated contamination with respiratory viruses is one of the important causes of recurrent asthma in patients. Timely judgment of treatment effects and standardized medication can effectively reduce the side effects of long-term hormone use on children's growth and development. Currently, there is no clear standard for the evaluation of the treatment prognosis of childhood allergic asthma. Summary of the Invention

[0005] In view of the deficiencies of the above-mentioned prior art, the present invention provides a biomarker for childhood allergic asthma and its applications, aiming to find a plasma biomarker with relatively high sensitivity as a biomarker for the diagnosis and prognosis of childhood allergic asthma, and laying a foundation for guiding clinical medication.

[0006] The first technical solution provided by the present invention is the application of a reagent for detecting the level of plasma branched-chain amino acids in the preparation of a product for detecting, evaluating or diagnosing childhood allergic asthma, and the branched-chain amino acids include leucine, isoleucine and / or valine.

[0007] In some embodiments, the product includes a biochip or a kit.

[0008] The second technical solution provided by the present invention is a biomarker for childhood allergic asthma, and the biomarker is plasma branched-chain amino acids, and the branched-chain amino acids include leucine, isoleucine and / or valine.

[0009] In some embodiments, the biomarker is derived from a blood sample.

[0010] The third technical solution provided by the present invention is a kit for detecting childhood allergic asthma, and the kit contains a reagent for detecting the biomarker described in the second technical solution.

[0011] The fourth technical solution provided by the present invention is the application of the biomarker described in the second technical solution in constructing a prediction model for childhood allergic asthma.

[0012] In some embodiments, the prediction model is an ROC curve drawn with leucine, isoleucine and valine as plasma biomarkers. With a leucine concentration of 619.23 μmol / L as the optimal cut-off value, it can significantly distinguish the asthma group from the healthy control group; with an isoleucine concentration of 300.49 μmol / L as the optimal cut-off value, it can significantly distinguish the asthma group from the healthy control group; with a valine concentration of 294.53 μmol / L as the optimal cut-off value, it can significantly distinguish the asthma group from the healthy control group; with a branched-chain amino acid concentration of 1221.30 μmol / L as the optimal cut-off value, it can significantly distinguish the asthma group from the healthy control group.

[0013] The fifth technical solution provided by the present invention is the application of the biomarker described in the second technical solution in the preparation of a product for evaluating the efficacy and / or prognosis of drugs for treating childhood allergic asthma on childhood allergic asthma.

[0014] In some embodiments, the product includes a kit and a reagent.

[0015] The technical effects of the present invention are as follows:

[0016] By detecting clinical samples, the present invention found that the plasma BCAA level in children with allergic asthma was significantly higher than that in healthy control children, showing good diagnostic value. After drug treatment, the plasma BCAA level in children with allergic asthma decreased significantly, indicating good prognostic value for treatment. Therefore, the present invention can use plasma BCAA as a biomarker for the diagnosis and prognosis of childhood allergic asthma, laying a foundation for guiding clinical medication. Description of the Drawings

[0017] Figure 1 Plasma branched-chain amino acid levels in children with allergic asthma; (A) Leucine - ELISA kit was used to detect the content of leucine in plasma specimens; (B) Isoleucine - ELISA kit was used to detect the content of isoleucine in plasma specimens; (C) Valine - ELISA kit was used to detect the content of valine in plasma specimens; (D) The sum of the contents of the 3 branched-chain amino acids was the statistical total of branched-chain amino acid content; the control group was plasma specimens from healthy control children, n = 10; the asthma group was plasma specimens from children with allergic asthma, n = 39; ** represents P < 0.01, *** represents P < 0.001.

[0018] Figure 2 ROC curves of leucine, isoleucine, valine, and branched-chain amino acids for predicting childhood allergic asthma; (A) ROC curve of leucine for predicting childhood allergic asthma: AUC 0.842, 95% CI 0.735 - 0.950, P < 0.001; (B) ROC curve of isoleucine for predicting childhood allergic asthma: AUC 0.723, 95% CI 0.581 - 0.865, P < 0.05; (C) ROC curve of valine for predicting childhood allergic asthma: AUC 0.908, 95% CI 0.826 - 0.989, P < 0.001; (D) ROC curve of branched-chain amino acids for predicting childhood allergic asthma: AUC 0.887, 95% CI 0.796 - 0.979, P < 0.001.

[0019] Figure 3Correlation analysis of serum IL-10 level and serum BCAA level in children with allergic asthma; (A) Correlation analysis of serum IL-10 level and serum leucine level in children with allergic asthma, r = -0.5759, P = 0.0196; (B) Correlation analysis of serum IL-10 level and serum isoleucine level in children with allergic asthma, r = -0.4337, P = 0.0933; (C) Correlation analysis of serum IL-10 level and serum valine level in children with allergic asthma, r = -0.4751, P = 0.0629; (D) Correlation analysis of serum IL-10 level and serum branched-chain amino acid level in children with allergic asthma, r = -0.4751, P = 0.0629. n = 16.

[0020] Figure 4 Serum branched-chain amino acid levels in children with allergic asthma before and after allergen immunotherapy (AIT); (A) Serum leucine levels in children with allergic asthma before and after allergen immunotherapy, P < 0.05; (B) Serum isoleucine levels in children with allergic asthma before and after allergen immunotherapy, P < 0.01; (C) Serum valine in children with allergic asthma before and after allergen immunotherapy, P < 0.01; (D) Serum branched-chain amino acid levels in children with allergic asthma before and after allergen immunotherapy, P < 0.01. The asthma group was the control group before treatment, and the treatment group was after receiving the allergen immunotherapy protocol.

[0021] Figure 5 Plasma branched-chain amino acid levels in a murine model of allergic asthma; (A) A murine model of allergic asthma was constructed, and an enzyme-linked immunosorbent assay kit was used to detect the content of murine plasma IgE. Control group n = 5; Asthma group n = 5; Dexamethasone treatment group n = 5. ** represents P < 0.01; (B) A murine model of allergic asthma was constructed, and an enzyme-linked immunosorbent assay kit was used to detect the contents of murine plasma leucine, isoleucine, and valine. Control group n = 5; Asthma group n = 5; Dexamethasone treatment group n = 5. ** represents P < 0.01. Detailed implementation manners

[0022] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0023] Example 1 Detection of plasma BCAA levels in children with allergic asthma

[0024] 1. Collection of research subject information

[0025] (1) Inclusion criteria:

[0026] ①For individuals aged ≥6 years and <18 years, the diagnostic criteria for asthma refer to the Global Initiative for Asthma (GINA) guidelines and the "Guidelines for the Prevention and Treatment of Bronchial Asthma" formulated by the Asthma Group of the Chinese Medical Association's Respiratory Disease Branch in 2020. These criteria include clinical symptoms such as intermittent shortness of breath, chest tightness, and cough, as well as objective evidence of varying degrees of expiratory airflow limitation. In addition, other conditions that may cause asthma-like symptoms are excluded;

[0027] ②Positive serum specific IgE for dust mite allergens;

[0028] ③The child or their legal guardian is able to understand the potential risks and limitations related to treatment and is able to cooperate with medical staff to complete the questionnaire;

[0029] (2) Exclusion criteria:

[0030] ①Patients with moderate to severe asthma and those in the acute exacerbation or acute attack phase of asthma;

[0031] ②Having a history of other chronic diseases or acute infections within 1 week;

[0032] ③Having immune deficiencies or other diseases not suitable for desensitization treatment.

[0033] (3) Collect plasma specimens from healthy children as the control group.

[0034] Table 1 Analysis of clinical characteristics of children with asthma and the control group

[0035]

[0036] 2. Detect the plasma BCAA levels in children with allergic asthma

[0037] The plasma levels of three BCAAs in the collected children without allergic asthma in the control group and children with allergic asthma were detected using ELISA kits for leucine, isoleucine, and valine respectively (Wuhan Aidy Anti Co., Ltd.: the catalog number of the leucine detection kit is AD9252; the catalog number of the isoleucine detection kit is AD9258; the catalog number of the valine detection kit is AD9261; the branched-chain amino acid value is the sum of leucine, isoleucine, and valine). The results showed that the plasma leucine in the children with allergic asthma increased to 1.37 times that of the control group, isoleucine increased to 1.15 times that of the control group, and valine increased to 1.46 times that of the control group ( Figure 1 A - C). At the same time, the cumulative content of the three BCAAs in the children with allergic asthma was also 1.33 times that of the healthy control group children ( Figure 1 D).

[0038] Example 2 Construction of a good prediction model for childhood allergic asthma

[0039] Based on the detection of the contents of 3 branched-chain amino acids in Example 1, the corresponding ROC curve was constructed using the ROC curve in the classification in SPSS software to detect whether the plasma BCAA level can be used as a good predictive marker for childhood allergic asthma. The results showed that the area under the curve (AUC) of Leucine for predicting childhood allergic asthma was 0.842 (95% CI 0.735 - 0.950, P < 0.001)( Figure 2 A); the area under the curve (AUC) of Isoleucine for predicting childhood allergic asthma was 0.723 (95% CI 0.581 - 0.865, P < 0.05)( Figure 2 B); the area under the curve (AUC) of Valine for predicting childhood allergic asthma was 0.908 (95% CI 0.826 - 0.989, P < 0.001)( Figure 2 C); the area under the curve (AUC) of BCAA for predicting childhood allergic asthma was 0.887 (95% CI 0.796 - 0.979, P < 0.001)( Figure 2 D). Among them, the area under the curve of Valine for predicting childhood allergic asthma was the largest and > 0.9, indicating a relatively high diagnostic accuracy, and its detection sensitivity was 79.5%. In summary, both the increase in the single plasma BCAA level and the total plasma BCAA content can indicate the occurrence of childhood allergic asthma, and Valine has the highest diagnostic accuracy under this sample cohort model.

[0040] Example 3 Correlation between the contents of 3 plasma BCAA and plasma IL-10 concentration in children with allergic asthma

[0041] The levels of various inflammatory factors in the plasma of patients with allergic asthma will change significantly, among which the level of IL-10 will decrease significantly. We used the Luminex liquid suspension chip method (customized multi-factor chip) to detect the levels of various serum cytokines, including the content of IL-10 in the plasma, and performed a correlation analysis with the plasma BCAA content. The specific operations are as follows:

[0042] (1) Equilibrate the kit to room temperature: Half an hour before the experiment, take out the kit (Luminex Assays) from the 4°C refrigerator, check the kit and equilibrate it to room temperature;

[0043] (2) Sample preparation: After confirming that the kit is correct, take out the thawed sample to be tested from the 4°C refrigerator, mix it well with a shaker, and centrifuge it at 4°C and 450 g / min for 10 min;

[0044] (3) Prepare the microspheres: After ultrasonic oscillation and washing of the microspheres for 1 min, take out 500 ul and add it to the mixing bottle, and make up the volume to 5 ml with the microsphere diluent;

[0045] (4) Prepare standards and quality control products: Add 100 μL of diluent to the standards, invert and mix well, shake for 10 s, let stand for 10 min to fully dissolve the standards, and perform gradient dilution according to the instructions.

[0046] (5) Add magnetic beads: Add 50 μL of microsphere mixture to each well plate, and filter to remove the liquid in the wells.

[0047] (6) Sample addition: Add 50 μL of the diluted sample or standard to the corresponding well plate, and incubate with shaking in the dark at room temperature for 120 min.

[0048] (7) Wash the plate: Filter the liquid in the well plate and wash 3 times with 100 μL of washing solution per well.

[0049] (8) Add detection antibody: Add 50 μL of detection antibody to each well of the well plate, and incubate with shaking in the dark at room temperature for 60 min.

[0050] (9) Add Streptavidin-PE: Add 50 μL of Streptavidin-PE to each well of the well plate, and incubate with shaking in the dark at room temperature for 30 min.

[0051] (10) Wash the plate: Filter the liquid in the well plate and wash 3 times with 200 μL of washing solution per well.

[0052] (11) Add sheath fluid: Add 100 μL of sheath fluid to each well, shake for 2 min in the dark at room temperature, mix well and aspirate into the flow tube.

[0053] (12) Detection on the machine: Use the MAGPIX liquid-phase multi-factor analyzer to perform detection and analysis on the machine, export the data and perform analysis.

[0054] The results showed that there was a negative correlation between the plasma Leucine level and the IL-10 level in children with allergic asthma, r = -0.5759, P = 0.0196 ( Figure 3 A); there was a negative correlation between the plasma Isoleucine level and the IL-10 level in children with allergic asthma, r = -0.4337, P = 0.0933 ( Figure 3 B); there was a negative correlation between the plasma Valine level and the IL-10 level in children with allergic asthma, r = -0.4751, P = 0.0629 ( Figure 3 C); there was a negative correlation between the plasma BCAA level and the IL-10 level in children with allergic asthma, r = -0.5787, P = 0.0188 ( Figure 3 D). In summary, there was a significant correlation between the plasma Leucine level and the BCAA level and the IL-10 content in children with allergic asthma.

[0055] Plasma BCAA level in children with allergic asthma after treatment in Example 4

[0056] Children who started HDM-SLIT (house dust mite protein (HDM)-sublingual immunotherapy (SLIT), one of the two current mainstream AITs, with the drug brand Changdi used clinically and administered according to the drug instructions, see Table 2) in the Department of Pediatrics of Affiliated Hospital of Jiangnan University were selected, and children who used symptomatic drugs for allergic asthma (without a history of allergen immunotherapy) were selected as the control group. The index dates were respectively the dates of the first issuance of SLIT or allergic asthma drugs, and clinical data were continuously collected through follow-up (Table 3). A total of 40 children were obtained (20 in the treatment group and 20 in the control group). All inclusion and exclusion criteria are detailed below.

[0057] The inclusion period was extended from December 1, 2022 to March 31, 2023. The retrospective period included the three years before the index date. The monitoring of all patients started from the indication date until loss to follow-up (defined as not receiving reimbursed treatment for 3 consecutive months) or the end of the study (April 30, 2024), whichever occurred first.

[0058] This example study was approved by the Academic Ethics Committee of Affiliated Hospital of Jiangnan University (approval number: LS2022112), and informed consent forms were signed by the guardians of all children.

[0059] Table 2 Instructions for drug use

[0060]

[0061] Table 3 Analysis of clinical characteristics of asthmatic children before and after treatment

[0062]

[0063]

[0064] Regarding the test results of Examples 1 to 3, this example detected the changes in the plasma BCAA levels of children with allergic asthma before and after allergen immunotherapy (AIT). It was found that compared with before treatment, the plasma levels of Leucine (P < 0.05), Isoleucine (P < 0.01), Valine (P < 0.01), and BCAA (P < 0.01) in children with allergic asthma were significantly down-regulated after AIT treatment ( Figure 4 A-D). In summary, the above results suggest that the plasma BCAA level may be an important indicator for the treatment and prognosis monitoring of AIT in children with allergic asthma.

[0065] Application of plasma BCAA level as a prognostic biomarker for allergic asthma

[0066] Based on the detection of clinical specimens, a mouse model of allergic asthma was constructed in this study to verify the above results in the animal model. The specific steps are as follows:

[0067] Animal groups: control group, asthma group, dexamethasone treatment group (n = 5 in each group).

[0068] The HDM nasal drip method was used to construct the asthma mouse model. The specific steps are as follows.

[0069] (1) Grouping: After one week of adaptive feeding, we grouped the C57BL / 6 mice as follows: normal control group (CON group), asthma model group (HDM group), dexamethasone treatment group (Dex group);

[0070] (2) Sensitization: On the 8th - 10th day of the experiment, the mice were sensitized. The mice in the HDM group and Dex group were sensitized by nasal drip (0.1 mg / mL, 50 μL), and the mice in the CON group were given an equal volume of PBS by nasal drip;

[0071] (3) Challenge: From the 8th to the 10th day after the start of the experiment, the mice in the HDM group and Dex group were treated with nasal drip sensitization (1 mg / mL, 50 μL), and the mice in the CON group were given an equal volume of PBS by nasal drip;

[0072] (4) Treatment: The Dex group was intraperitoneally injected with Dex (1 mg / kg, dissolved in 500 μL PBS) on the 24th day of the experiment. For the other groups, an equal volume of PBS was given as a control; on the 26th day, the mice were euthanized, samples were collected, and various indicators were detected.

[0073] Detect the content of IgE in the plasma of mice in different groups in the constructed mouse model to verify the successful construction of the model ( Figure 5 A). The results showed that the plasma levels of Leucine, Isoleucine, Valine, and BCAA in the allergic asthma group were significantly higher than those in the healthy control group, while the plasma levels of the 3 BCAAs in the allergic asthma mice after dexamethasone treatment decreased significantly and were not different from the plasma levels of the 3 BCAAs in the healthy group ( Figure 5 B). Therefore, the plasma BCAA level was significantly upregulated in the allergic mouse model and significantly decreased after dexamethasone treatment, which is a potential therapeutic prognostic marker.

[0074] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Use of a reagent for detecting plasma branched-chain amino acid levels in the preparation of a product for detecting, evaluating or diagnosing allergic asthma in children, characterized in that: The branched-chain amino acids include leucine, isoleucine and / or valine.

2. The use according to claim 1, characterized in that: The product includes a biochip or a kit.

3. A biomarker for allergic asthma in children, characterized in that: The biomarker is plasma branched-chain amino acids, which include leucine, isoleucine and / or valine.

4. The marker according to claim 3, characterized in that The biomarkers are derived from blood samples.

5. A kit for detecting allergic asthma in children, characterized in that: The kit contains reagents for detecting the biomarkers according to claim 3 or 4.

6. Use of the biomarker described in claim 3 or 4 in constructing a prediction model for childhood allergic asthma.

7. The use according to claim 6, characterized in that: The prediction model is a ROC curve drawn with leucine, isoleucine and / or valine as plasma markers, with the leucine concentration of 619.23 μmol / L being the optimal cutoff value, which can clearly distinguish the asthma group from the healthy control group; the isoleucine concentration of 300.49 μmol / L being the optimal cutoff value, which can clearly distinguish the asthma group from the healthy control group; the valine concentration of 294.53 μmol / L being the optimal cutoff value, which can clearly distinguish the asthma group from the healthy control group; and the branched-chain amino acid concentration of 1221.30 μmol / L being the optimal cutoff value, which can clearly distinguish the asthma group from the healthy control group.

8. Use of the biomarker according to claim 3 or 4 in the preparation of a product for evaluating the efficacy and / or prognosis of a drug for treating allergic asthma in children.

9. The use according to claim 8, characterized in that: The products include kits and reagents.

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