Application of urine exosome human hairy-like protein 1 (COTL1) and lymphocyte cytoplasm protein 1 (LCP1) in diagnosis and treatment of allergic rhinitis
By using COTL1 and LCP1 proteins in urinary exosomes as biomarkers, a non-invasive detection kit was developed, which solved the pain and risk problems in the diagnosis of allergic rhinitis and enabled non-invasive and sensitive disease monitoring and treatment effect evaluation.
Patent Information
- Application Number
- CN202410501526.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-25
- Publication Date
- 2025-10-28
AI Technical Summary
Current diagnostic methods for allergic rhinitis mainly rely on invasive testing, which carries the risks of pain and systemic allergic reactions. There is a lack of non-invasive, sensitive, and specific biomarkers for the diagnosis and evaluation of the disease state.
Using COTL1 and LCP1 proteins in urinary exosomes as biomarkers, we screened and verified their high expression in allergic rhinitis using data-independent mass spectrometry, and developed a non-invasive detection kit for diagnosis, differential diagnosis, disease assessment, treatment effect evaluation, and prognosis assessment.
This invention provides a non-invasive, suitable, sensitive, and specific biomarker detection method that can accurately diagnose and monitor the severity of allergic rhinitis and assess treatment effectiveness, thereby reducing patient suffering and risks.
Smart Images
Figure CN120847261A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of proteomics research, specifically to the application of human trichomeal protein 1 (COTL1) and lymphocyte cytoplasmic protein 1 (LCP1) from urinary exosomes in the preparation of reagents for the diagnosis, differential diagnosis, severity assessment, treatment efficacy evaluation, monitoring, prognostic assessment, and mechanism research of allergic rhinitis. Background Technology
[0002] Allergic rhinitis is caused by an abnormal immune response triggered by various external factors, mainly manifested as nasal congestion, sneezing, runny nose, nasal itching, and itchy eyes. Allergic rhinitis can be classified into seasonal allergic rhinitis and perennial allergic rhinitis based on the frequency and duration of symptoms. The incidence of allergic rhinitis is on the rise globally, which may be related to environmental pollution, changes in lifestyle, increased exposure to indoor and outdoor allergens, and insufficient public awareness of allergic diseases. Allergic rhinitis not only affects patients' quality of life but can also lead to a series of complications, such as asthma, sinusitis, and otitis media. Currently, the diagnosis of allergic rhinitis mainly relies on clinical history combined with laboratory tests such as skin prick tests and serum sIgE antibody detection. However, both of these methods are invasive, causing discomfort to patients and posing a risk of systemic allergic reactions. Therefore, exploring a non-invasive, appropriate, sensitive, and specific biomarker for the disease state is of great significance for the diagnosis of allergic rhinitis.
[0003] Urinary exosomes are released from cells of various organs in the body. Compared to urinary proteins, proteins in urinary exosomes have the advantages of stability, sensitivity, and long preservation time, making them promising biomarkers for diseases. Meanwhile, data-independent acquisition (DIA) as a mass spectrometry data acquisition method can acquire mass spectrometry data of all peptides in parallel, significantly improving the accuracy of protein identification and quantification, increasing detection coverage, and enhancing the analytical capabilities for complex samples. This allows it to capture subtle changes in disease states, making it a powerful tool for screening and validating disease biomarkers.
[0004] To explore non-invasive, appropriate, sensitive and specific biomarkers for the diagnosis and evaluation of allergic diseases, and to provide a basis and ideas for the selection of therapeutic targets and treatment regimens for type I hypersensitivity diseases, in this invention we focus on the proteomic changes in urinary exosomes in allergic rhinitis.
[0005] COTL1, coactosin-like protein 1, is an actin-binding protein, also known as a trichome-like protein, involved in actin binding and innate immune processes. COTL1 plays a crucial role in cell morphology; it participates in inflammatory responses and, as a companion to ALOX5 (5LO), influences its stability and activity in leukotriene synthesis. Studies have shown that COTL1 is associated with inflammatory diseases of the respiratory and digestive systems. Furthermore, COTL1 is associated with the development and progression of neurological diseases and cancer. Our results show that COTL1 expression was elevated in urinary exosomes from all three groups of patients with allergic rhinitis, without significant differences based on severity, suggesting its upregulation in allergic reactions and its potential as a biomarker for allergic diseases.
[0006] LCP1, L-Plastin, is a lymphocyte cytoplasmic protein 1 belonging to the actin-binding protein family and is expressed in most tissues of higher eukaryotes. Its associated pathways include cytokine signaling and NF-kappaB signaling in the immune system. LCP1 participates in calcium ion binding and actin binding processes, playing a role in T cell activation via TCR / CD3 and CD2 or CD28. Our results show that LCP1 expression in urinary exosomes of female patients with allergic rhinitis increases with disease severity, while expression is relatively stable in men, and the degree of LCP1 protein elevation is significantly higher in women than in men. LCP1 is upregulated in allergic reactions and has the potential to reflect the severity of allergic diseases in women.
[0007] This invention leverages the advantages of urine sample acquisition—non-invasiveness, large-scale repeatability, and convenient preservation—to detect multiple biomarkers of allergic rhinitis using urine samples. Research has revealed that urinary exosome proteins COTL1 and LCP1 are key proteins in the development and progression of allergic rhinitis. Compared to the general population, their expression was significantly increased in urinary exosome samples from patients with allergic rhinitis of varying degrees and in different genders. COTL1 and LCP1 are of significant importance for the diagnosis, differentiation, monitoring, and prognostic assessment of allergic rhinitis. Summary of the Invention
[0008] To address the aforementioned problems, the present invention aims to provide one or two urinary exosome protein markers, COTL1 and LCP1, for the application in the preparation of formulations for the diagnosis, differential diagnosis, severity assessment, treatment efficacy evaluation, monitoring, prognostic evaluation, and mechanism research of allergic rhinitis.
[0009] Preferably, the amino acid sequences of COTL1 in the urinary exosomes are as shown in SEQ ID NO. 1-2. NO.1 (COTL1): (1 matkidkeac raaynlvrdd gsaviwvtfk ydgstivpge qgaeyqhfiq qtddvrlfa 61 fvrfttgdam skrskfalit wigenvsglq raktgtdktl vkevvqnfak efvisdrkel 121 eedfikselk kagganydaq te NO.2 (LCP1) 1 margsvsdee mmelreafak vdtdgngyis fnelndlfka aclplpgyrv reitenlmat 61 gdldqdgris fdefikifhg lkstdvaktf rkainkkegi caiggtseqs svgtqhsyse 121 eekyafvnwi nkalendpdc rhvipmnpnt ndlfnavgdg ivlckminls vpdtiderti 181 241 elsrnealia llregesled lmklspeell lrwanyhlen agcnkignfs tdikdskayy 301 hlleqvapkg deegvpavvi dmsglrekdd iqraecmlqq aerlgcrqfv tatdvvrgnp 361 klnlafianl fnrypalhkp enqdidwgal egetreeertf rnwmnslgvn prvnhlysdl 421 sdalvifqly ekikvpvdwn rvnkppypkl ggnmkklenc nyavelgknq akfslvgigg 481 qdlnegnrtl tlaliwqlmr rytlnileei gggqkvnddi ivnwvnetlr eakksssiss 541 fkdpkistsl pvldlidaiq pgsinydllk tenlnddekl nnakyaisma rkigarvyal 601 pedlvevnpk mvmtvfaclm gkgmkrv).
[0010] Preferably, the preparation is a kit for detecting the expression levels of COTL1 and LCP1 in urinary exosomes of patients with allergic rhinitis.
[0011] Preferably, the kit includes an immunization method for antigen-antibody reaction and a kit thereof, such as one or more aptamer antibodies or antibody fragments capable of specifically binding to COTL1.
[0012] Preferably, the kit further includes any one or a combination of several of the following components: solid support, diluent, control, standard, quality control, detection antibody, second antibody, second antibody diluent, luminescent reagent, washing solution, colorimetric solution, and stop solution.
[0013] Preferably, the standards include COTL1 and LCP1 standards and humanized labeled antibody standards; more preferably, the quality control products include COTL1 and LCP1 quality control products and humanized labeled antibody quality control products; more preferably, the solid-phase carrier includes microparticles, microspheres, glass slides, test strips, plastic beads, liquid phase chips, microplates or affinity membranes, and other carriers with equivalent functions.
[0014] Preferably, the solid support is made of any one of polyvinyl chloride, polystyrene, polyacrylamide, cellulose, or a similar carrier.
[0015] The inventors first collected urine samples from healthy individuals and patients with mild, moderate, and severe allergic rhinitis, extracted urinary exosome specimens, and used data-independent scanning mode (DIA) mass spectrometry to analyze proteins in the urinary exosome specimens from normal individuals and patients with mild, moderate, and severe allergic rhinitis. Significantly differentially expressed proteins were screened and analyzed. Ultimately, the target proteins COTL1 and LCP1 were obtained.
[0016] This invention demonstrates through research that, compared to healthy individuals, COTL1 and LCP1 are highly expressed in the urinary exosomes of patients with allergic rhinitis. These findings can be used for the application of formulations in disease diagnosis, differential diagnosis, disease severity assessment, treatment efficacy evaluation, monitoring, prognostic evaluation, and mechanism research.
[0017] This invention leverages the advantages of urine sample acquisition being non-invasive, allowing for large-scale repeated sampling, and being easy to preserve, to detect COTL1 and LCP1 in urinary exosomes using urine samples.
[0018] To make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description
[0019] Figure 1 This is a graph showing the COTL1 levels in urinary exosomes in patients with different degrees of allergic rhinitis and healthy controls. (A) Compared with the NC group, COTL1 expression was upregulated in the total AR group. (B) Compared with the NC group, COTL1 was upregulated in the AR1, AR2, and AR3 groups. (C) In males, compared with the NC group, COTL1 was upregulated in the AR1, AR2, and AR3 groups. (D) In females, compared with the NC group, COTL1 was upregulated in the AR1, AR2, and AR3 groups. NC: Healthy control group; AR: Allergic rhinitis summary group; AR1: Mild allergic rhinitis group; AR2: Moderate allergic rhinitis group; AR3: Severe allergic rhinitis group. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.
[0020] Figure 2 This is a graph showing the LCP1 content in urinary exosomes in patients with different degrees of allergic rhinitis and healthy controls. (A) Statistical analysis of LCP1 expression in the NC group and the total AR group. (B) Statistical analysis of LCP1 expression in the NC group, AR1 group, AR2 group, and AR3 group, regardless of gender. (C) In males, there was no significant difference in LCP1 expression in the AR1 group, AR2 group, and AR3 group compared with the NC group. (D) In females, compared with the NC group, LCP1 expression was upregulated with disease severity. NC: Healthy control group; AR: Allergic rhinitis summary group; AR1: Mild allergic rhinitis group; AR2: Moderate allergic rhinitis group; AR3: Severe allergic rhinitis group. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.
[0021] Figure 3 The expression levels of allergens (trypsin-like enzymes) in a zebrafish model by knocking down the cotl1 and lcp1 genes were compared. Trypsin-like enzyme levels before and after sensitization were compared in the cotl1(A) and lcp1(B) gene knockdown groups. Data are presented as mean ± standard deviation; ns: p > 0.05, *: p < 0.05, **: p < 0.01. Detailed Implementation
[0022] Example 1 Collection and processing of urinary exosome specimens A total of 36 patients with different levels of allergic rhinitis were included in the study: mild group (6 males and 6 females), moderate group (6 males and 6 females), and severe group (6 males and 6 females). All patients presented with typical paroxysmal cough, nasal itching, nasal congestion, and runny nose, lasting for more than three months; all were Han Chinese and permanent residents of the area for more than six months; all patients met the diagnostic criteria for allergic rhinitis (AR) approved by the editorial board of the Chinese Journal of Otorhinolaryngology Head and Neck Surgery. Patients were graded according to the results of the Artemisia argyi allergen test: mild group (AR1): Artemisia argyi allergen test +~++; moderate group (AR2): Artemisia argyi allergen test 3+~4+; severe group (AR3): Artemisia argyi allergen test 5+~6+. A healthy control group (NC) of 12 patients (6 males and 6 females), age-matched to the case groups, was also included. Exclusion criteria for all enrolled cases: chronic diseases such as asthma, chronic sinusitis, atopic dermatitis, chronic kidney disease, hypertension, diabetes, rheumatic diseases, and hepatitis; patients with a history of smoking and alcohol abuse; patients with white blood cells and red blood cells in routine urinalysis or a urine albumin-to-creatinine ratio >30 mg / g; and patients who received nasal or oral treatment for nasal or allergy-related diseases within two weeks prior to urine sample collection. All subjects were prohibited from any abnormal or strenuous physical activity the day before urine collection. Midstream urine samples were collected in the morning from all subjects in allergic rhinitis patients and healthy controls and placed in sterile polypropylene catheters for further processing to extract urinary exosomes.
[0023] Example 2 Differential peptide data analysis Quantitative proteomic analysis (DIA) was used to identify and analyze urinary exosome proteins in patients with mild, moderate, and severe allergic rhinitis and healthy controls. Separately, six samples from each group were pooled, and each group was analyzed three times. DIA identified a total of 48,057 peptides and 5,037 proteins. The screening criteria for differentially expressed proteins were: fold change (FC) > 1.5, and p < 0.05, indicating significant difference in expression. Analysis of the urinary exosome protein quantification results revealed a group of immune-related proteins that were upregulated or downregulated in different groups; COTL1 and LCP1 were identified as the target proteins.
[0024] Example 3 Comparison of COTL1 and LCP1 levels in urinary exosomes COTL1 protein expression was significantly different between the NC group and the total AR group (p < 0.0001). Regardless of sex, COTL1 was upregulated in all AR1, AR2, and AR3 groups compared to the NC group. COTL1 protein was upregulated in all AR groups relative to the NC group in both sexes, but there were no significant differences within each AR group. Quantitative results showed that COTL1 expression was elevated in urinary exosomes of all three groups of allergic rhinitis patients (see [link to data]). Figure 1 Regardless of gender, LCP1 expression was upregulated in the AR3 group compared to the NC group. In males, LCP1 protein expression did not differ significantly across AR groups, but in females, LCP1 protein expression was upregulated with disease severity. Quantitative results for each group showed that LCP1 expression in urinary exosomes of female patients with allergic rhinitis increased with disease severity, while expression was relatively stable in males, and the degree of increase in LCP1 protein was significantly higher in females than in males. (See [link to relevant documentation]). Figure 2 .
[0025] Example 4 Construct a zebrafish model to verify the mechanisms of action of cotl1 and lcp1. Regarding sensitization indicators, trypsin is a neutral protease pre-synthesized within mast cells and is an important clinical indicator for testing whether a person is allergic. When zebrafish experience an allergic reaction, mast cells are also induced to secrete trypsin. To investigate whether cotl1 and lcp1 are key genes involved in allergic reactions, this invention used a CRISPR / Cas9 system to inject 4gRNA into wild-type zebrafish embryos, constructing F0 generations with knockdown of cotl1 and lcp1, respectively. Subsequently, the embryonic trypsin levels of the cotl1 or lcp1 knockdown zebrafish were measured. The increase in trypsin levels in the F0 chimeras of cotl1 and lcp1 after sensitization was significantly lower than that in the wild type, indicating that cotl1 and lcp1 are key factors in allergic reactions.
[0026] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.
Claims
1. The application of urinary exosome proteins in the preparation of formulations for the diagnosis, differential diagnosis, severity assessment, treatment efficacy evaluation, monitoring, prognostic evaluation, and mechanism research of allergic rhinitis, characterized in that... The biomarkers include one or two of the following proteins: human trichomorph protein 1 (COTL1) and lymphocyte cytoplasmic protein 1 (LCP1).
2. The application according to claim 1, characterized in that, The biomarkers may include one or two of them; preferably, the biomarkers include at least COTL1, and more preferably, the biomarkers include COTL1 and LCP1.
3. The application according to claim 1, characterized in that, The amino acid sequences of COTL1 and LCP1 in the urine are shown in SEQ ID NO:1~2.
4. The application according to claim 1, characterized in that, The formulation is used to detect the expression levels of one or both of COTL1 and LCP1.
5. The application according to claim 4, characterized in that, in, The formulation is used for the diagnosis, differential diagnosis, severity assessment, treatment efficacy evaluation, monitoring, prognostic evaluation, and mechanism research of allergic rhinitis. And / or, the formulation includes an antigen-antibody detection immunoassay reagent; preferably, it includes a urine antigen-antibody detection immunoassay reagent.
6. The application according to claim 5, characterized in that, The reagent kits prepared by the reagents include an immunoassay method for antigen-antibody reactions and a kit thereof, which can specifically bind one or more of COTL1 and LCP1 aptamer antibodies or antibody fragments.
7. The application according to claim 5, characterized in that, The kit also includes any one or a combination of several of the following components: solid support, diluent, control, standard, quality control, detection antibody, secondary antibody, secondary antibody diluent, luminescent reagent, washing solution, colorimetric solution, and stop solution.
8. The application according to claim 7, characterized in that, The standards include COTL1 and LCP1 standards, and humanized labeled antibody standards; the quality control materials include COTL1 and LCP1 quality control materials, and humanized labeled antibody quality control materials; the solid-phase carriers include microparticles, microspheres, glass slides, test strips, plastic beads, liquid phase chips, microplates or affinity membranes, and other carriers with equivalent functions.
9. The application according to claim 7, characterized in that, The solid support is made of any one of polyvinyl chloride, polystyrene, polyacrylamide, cellulose, or a similar carrier.