Diabetes neurogenic bladder biomarker and screening method and application thereof

Abstract

The invention relates to a biomarker used as a diabetic neurogenic bladder (DNB) and a screening method and application thereof. Specifically, the invention provides a biomarker for the neurogenic bladder of diabetes, and the biomarker comprises one or more of ICAM2, VEGFA, JAML, PTPRJ, PTPRC, CD40, IL2RB and NCR1. By utilizing the biomarker provided by the invention, non-invasive and efficient identification of DNB can be realized, and a new molecular target is provided for complication prevention and control of diabetic patients.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to a biomarker of diabetic neurogenic bladder, a screening method and use thereof. Background Art

[0002] Diabetic neurogenic bladder (DNB) is a common and serious chronic complication of diabetic autonomic neuropathy. Its typical manifestations include damage to the sensory nerves of the bladder wall and detrusor muscle dysfunction, leading to symptoms such as urinary retention, incontinence, and dysuria. Epidemiological studies have shown that approximately 40%-80% of diabetic patients experience varying degrees of urinary dysfunction. Even with good blood sugar control, the incidence of DNB can be as high as 25%, and it increases annually with the duration of diabetes.

[0003] Because DNB has an insidious onset and its early symptoms are not obvious, it is often ignored by many patients, missing the best time for intervention. As the disease progresses, it will not only significantly reduce the patient's quality of life, but may also cause serious complications such as urinary tract infection, bladder decompensation, renal damage, and even uremia. However, the current diagnosis of DNB mainly relies on medical history analysis, neurological examination and bladder function assessment (such as residual urine volume detection). These methods are mostly invasive or complicated to operate, and their sensitivity and specificity are limited, which makes it difficult to meet the actual clinical needs for early, rapid, and non-invasive diagnosis.

[0004] The development of liquid biopsy technology has provided a new opportunity for the early diagnosis of DNB. Liquid biopsy collects samples of body fluids such as blood and urine and detects biomarkers in them. It has been widely used in the early screening and dynamic monitoring of cancer, neurological diseases, etc. Among them, exosomes, as "information carriers" of the state of the source cells, have significant advantages in stability, representativeness, and ease of acquisition. Exosomes are widely present in body fluids such as blood and urine, with a diameter of about 30-150nm. They are rich in functional molecules such as proteins and RNA and can reflect the dynamic changes in the occurrence and development of the disease. However, there are currently no systematic reports on DNB-specific body fluid exosome biomarkers.

[0005] Therefore, there is an urgent need in this field to develop biomarkers that can be effectively used for diabetic neurogenic bladder, so as to achieve better disease management in diabetic patients. Summary of the Invention

[0006] Given the current lack of efficient and non-invasive early molecular diagnostic methods for diabetic neurogenic bladder (DNB), the present invention utilizes a novel high-throughput method for single exosome analysis, namely proximity barcoding assay (PBA), to systematically analyze more than 200 proteins on the surface of single exosomes in urine, identifying several extracellular vesicle characteristic proteins and their combinations that are closely related to DNB, thereby providing clear biomarkers for non-invasive screening, disease monitoring and clinical intervention of DNB, and bringing a new technical path for the precise management of diabetic complications.

[0007] In a first aspect, the present invention provides a biomarker for diabetic neurogenic bladder, wherein the biomarker comprises one or more of ICAM2, VEGFA, JAML, PTPRJ, PTPRC, CD40, IL2RB and NCR1.

[0008] Optionally, the biomarker is selected from one or more of PTPRJ, ICAM2, PTPRC and CD40.

[0009] Optionally, the biomarkers are derived from urinary exosomes.

[0010] In a second aspect, the present invention provides a kit for detecting the biomarker of the first aspect, the kit comprising a DNA-coupled antibody against the biomarker and a sequencing or probe system for signal amplification and analysis.

[0011] In a third aspect, the present invention provides a method for screening biomarkers for diabetic neurogenic bladder, comprising: a) collecting a sample; b) extracting exosomes from the sample obtained in step a); c) detecting the target protein in the exosomes obtained in step b) using PBA technology; d) selecting proteins with significantly different expression levels as candidate markers based on the target protein expression levels in step c); e) selecting biomarkers for diabetic neurogenic bladder from the candidate markers of step d) based on the AUC values ​​calculated from the ROC curve.

[0012] Optionally, the sample is urine.

[0013] Optionally, the AUC value of step e) is greater than 0.75.

[0014] In a fourth aspect, the present invention provides use of the biomarker described in the first aspect or the kit described in the second aspect in preparing a product for early screening, diagnosis, efficacy evaluation, and prognosis monitoring of diabetic neurogenic bladder.

[0015] In a fifth aspect, the present invention provides a use of the biomarker described in the first aspect in the preparation of a medicament for the treatment of diabetic neurogenic bladder, wherein the medicament achieves therapeutic effect by regulating or intervening in the release or function of exosomes containing the biomarker.

[0016] In summary, the present invention includes at least one of the following beneficial technical effects: 1. By combining PBA technology with high-resolution, high-sensitivity proteomic analysis of exosomes in the urine of DNB patients, we systematically screened for DNB-specific exosome biomarkers and established a novel, efficient, and non-invasive method for early DNB screening. This method not only has significant scientific value but also has high clinical translational potential, filling the gap in DNB early screening methods and facilitating precise disease intervention and health management. 2. The urine exosome biomarkers provided by the present invention break through the specificity and sensitivity bottlenecks of traditional DNB detection, and provide new solutions for non-invasive screening, status monitoring and targeted treatment of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a PBA flow chart showing the process of high-throughput detection of single exosomes for biomarkers; Figure 2 shows the expression of multiple key proteins CD40 ( Figure 2A )、JAML( Figure 2B )、VEGFA( Figure 2C )、ICAM2( Figure 2D )、NCR1( Figure 2E )、PTPRJ( Figure 2F )、IL2RB( Figure 2G )、PTPRC( Figure 2H ) expression scatter plot; Figure 3 shows the candidate biomarkers ICAM2 ( Figure 3A )、IL2RB( Figure 3B )、PTPRC( Figure 3C )、VEGFA( Figure 3D )、JAML( Figure 3E )、NCR1( Figure 3F )、CD40( Figure 3G )、PTPRJ( Figure 3H )’s ROC curve. DETAILED DESCRIPTION

[0018] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0019] In the following examples, the experimental methods without specific conditions were carried out according to conventional procedures, and the materials and reagents used were all conventional commercial products.

[0020] The term "Proximity Barcoding Assay (PBA)" was developed by Shenzhen Mima Technology Co., Ltd. It uses antibody-DNA conjugates to label exosomal proteins or biomarkers and, combined with a high-throughput sequencing platform, enables parallel detection of multiple factors at the single exosome level. Compared to traditional protein analysis techniques such as mass spectrometry and flow cytometry, PBA offers the following advantages: single-exosome resolution; unlimited simultaneous detection of multiple factors; high sensitivity, theoretically achieving single-molecule protein detection; high throughput, enabling analysis of all single exosomes in a sample; parallel processing of samples using 96-well plates; direct capture of exosomes from samples without purification; and unrestricted exosome size.

[0021] The term "exosome" refers to tiny membrane vesicles approximately 30-150 nm in diameter, secreted by a variety of cells. These vesicles contain specific proteins (for example, the exosome membrane is rich in transmembrane proteins CD63, CD81, and CD9, which are involved in exosome transport), lipids, cytokines, or genetic material. Exosomes are secreted by a variety of cells under both normal and pathological conditions. They are widely present in body fluids such as blood, saliva, urine, cerebrospinal fluid, and breast milk. They are considered specialized secretory vesicles that participate in intercellular communication.

[0022] The term "biomarker" refers to a biochemical marker that can identify changes or potential changes in the structure or function of a system, organ, tissue, cell, or subcellular structure. Biomarkers have a wide range of applications. They can be used for early disease screening, diagnosis, efficacy assessment, prognosis monitoring, and as therapeutic targets for drug development.

[0023] Example 1 High-throughput single exosome protein detection in urine exosomes 1.1 Sample collection 200 urine samples were collected and divided into the following 4 groups, with 50 samples in each group: a. Control group (Healthy Control): healthy volunteers with no history of diabetes or urinary system diseases; bD group (type II diabetic patients without neurogenic bladder); c.DNB group (type II diabetic patients with neurogenic bladder); d. NB group (neurogenic bladder not caused by diabetes, caused by other factors).

[0024] All samples were collected from 8–50 mL of mid-morning urine, which was centrifuged at 2000 g for 15 minutes. The supernatant was transferred to a new tube and stored at −80°C for subsequent analysis.

[0025] 1.2 Extraction and characterization of urinary exosomes Urinary exosomes were extracted using differential centrifugation. Mid-morning urine samples were centrifuged at 300 g for 10 minutes, 2000 g for 10 minutes, and 10,000 g for 10 minutes to remove cells, debris, and large particles. The supernatant was then transferred to an ultracentrifuge tube and centrifuged at 100,000 g for 70 minutes (using a 32Ti rotor, Optima XPN ultracentrifuge, Beckman Coulter, Germany) to pellet the exosomes. The pellet was resuspended in PBS and centrifuged again under the same conditions for 70 minutes. The exosome pellet was finally resuspended in PBS and frozen at –80°C until further use.

[0026] The concentration and characterization of exosomes were confirmed using multiple methods. A nanoparticle tracking analyzer (NTA, NanoSight NS300, Malvern Panalytic, UK) was used to measure the size distribution and concentration of exosomes, revealing that the particle size was primarily concentrated in the 50–150 nm range. Western blot analysis of the exosomal marker proteins CD81 (sc-7637, Santa Cruz, USA) and TSG101 (sc-7964, Santa Cruz, USA) confirmed their typical exosomal characteristics. Transmission electron microscopy (TEM, JEOL JEM-1200EX, Japan) was used to observe the morphology of the purified exosomes, confirming their typical double-membrane vesicle structure.

[0027] 1.3 Proximity Block Analysis (PBA) The exosomes (EVs) samples extracted from the subjects' urine samples were analyzed using proximity coding analysis (PBA). The specific steps refer to the PBA technology patent CN105745334B; the PBA reaction product was sequenced using Illumina Nextseq to sequence the library. After DNA sequencing, the sample data was obtained in the form of a bcl file. Data analysis was performed according to the standard operating procedure provided by Mima Technology, namely PBA proteomics analysis software V1.0 (software copyright registration number 2020SR0822591). For specific procedures, see Figure 1 .

[0028] Example 2 Biomarker Screening Methods and Results Potential exosomal biomarkers of DNB were systematically screened and verified from multiple dimensions including expression differences, statistical tests, and ROC performance evaluation.

[0029] As can be seen from Figure 2, compared with the control group, the DNB group showed significant downregulation in proteins CD40 and PTPRJ, while significant upregulation in proteins ICAM2, IL2RB, JAML, PTPRC, NCR1, and VEGFA (p<0.05), indicating that these proteins may serve as potential clinical screening and diagnostic biomarkers for DNB.

[0030] Tables 1 and 2 show the statistical analysis results of differentially expressed proteins in DNB vs. D and DNB vs. Control, respectively. In Table 1, significantly differentially expressed proteins in DNB vs. D include PTPRJ, CD40, ICAM2, PTPRC, and JAML; in Table 2, significantly upregulated proteins in DNB vs. Control include IL2RB, AQP2, CD36, ITGB1, ENG, JAML, VEGFA, and NCR1.

[0031] Table 1 Statistics of differentially expressed proteins between DNB and D Table 2 Statistics of differentially expressed proteins between DNB and Control Figure 3 shows receiver operating characteristic (ROC) curves. The pROC package (v.1.18.0) was used to construct ROC curves and calculate the area under the curve (AUC), reflecting the diagnostic efficacy of each candidate biomarker for DNB identification. As shown in Figure 3, some proteins, such as PTPRJ (AUC = 0.853), ICAM2 (AUC = 0.817), PTPRC (AUC = 0.7896), and CD40 (AUC = 0.8333), demonstrated good discriminatory ability.

[0032] Tables 3 and 4 show the ROC analysis results for combined biomarkers for DNB vs. D and DNB vs. Control, respectively. In Table 3, combinations such as CADM3 & CUBN (AUC = 0.905) and DPP4 & HLA-DRA (AUC = 0.855) performed best in the DNB vs. D setting; in Table 4, combinations such as AQP2 & SLC12A1 (AUC = 0.889) and ITGB2 & SIGLEC5 (AUC = 0.878) showed high predictive power in the DNB vs. Control setting. Table 5 further summarizes the discriminatory power of combined biomarkers across different groups, with some combinations, such as IL2RB & VEGFA, showing some discriminatory power across multiple groups.

[0033] Table 3 ROC analysis of combined biomarkers of DNB vs. D Table 4 ROC analysis of combined protein markers of DNB vs Control Table 5 ROC analysis of combined biomarkers The above-described embodiments are merely preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A biomarker for diabetic neurogenic bladder, characterized by: The biomarkers include one or more of ICAM2, VEGFA, JAML, PTPRJ, PTPRC, CD40, IL2RB and NCR1.

2. The biomarker according to claim 1, characterized in that The biomarker is selected from one or more of PTPRJ, ICAM2, PTPRC and CD40.

3. The biomarker according to claim 1, characterized in that The biomarkers are derived from urinary exosomes.

4. A kit for detecting the biomarker according to any one of claims 1 to 3, characterized in that: The kit includes DNA-coupled antibodies against the biomarkers and a sequencing or probe system for signal amplification and analysis.

5. A method for screening the biomarker according to any one of claims 1 to 3, characterized in that: include: a) Collect samples; b) extracting exosomes from the sample obtained in step a); c) detecting the target protein in the exosomes obtained in step b) using PBA technology; d) selecting proteins with significantly different expression levels as candidate markers based on the target protein expression levels in step c); e) selecting biomarkers for diabetic neurogenic bladder from the candidate markers of step d) based on the AUC values ​​calculated from the ROC curve.

6. The method according to claim 5, characterized in that The sample is urine.

7. The method according to claim 5, characterized in that The AUC value of step e) is greater than 0.

75.

8. Use of the biomarker according to any one of claims 1 to 3 or the kit according to claim 4 in the preparation of a product for early screening, diagnosis, efficacy evaluation, and prognosis monitoring of diabetic neurogenic bladder.

9. Use of the biomarker according to any one of claims 1 to 3 in the preparation of a medicament for treating diabetic neurogenic bladder, characterized in that: The drug achieves therapeutic effect by regulating or intervening in the release or function of exosomes containing the biomarker.

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