Application of biomarker detection reagent in preparation of xerophthalmia diagnosis and typing detection reagent

Abstract

According to the application of a reagent for detecting the biomarker in preparation of a xerophthalmia diagnosis and typing detection reagent, the galectin-9 plays an important role in the occurrence and development process of the xerophthalmia, the expression level of the galectin-9 has a certain correlation with the severity of the xerophthalmia, and the galectin-9 is expected to be used as the biomarker for diagnosis and treatment of the xerophthalmia and can be used for preparing the xerophthalmia diagnosis and typing detection reagent. By detecting the expression condition of Galectin-9 in tears, early and accurate diagnosis of the dry eye and quantitative evaluation of the disease degree are achieved, in addition, Galectin-9 expressed on T cells plays an important role in immune inflammation of the dry eye, ocular surface damage of the dry eye can be relieved by locally intervening Galectin-9 through conjunctiva, and Galectin-9 serves as a novel potential target and has a good application prospect. A new way is opened up for diagnosis and treatment of xerophthalmia, a therapeutic drug or an intervention means developed for Galectin-9 can regulate immune response, inhibit inflammatory response, improve tear secretion and the like from the etiological level, and an existing xerophthalmia treatment mode is expected to be changed, so that the treatment effect is improved, the prognosis of a patient is improved, and the clinical application prospect is broad. The application prospect in the field of diagnosis and treatment of the xerophthalmia is wide, and a certain significance is achieved for improving the overall prevention and treatment level of the xerophthalmia.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a reagent for detecting biomarkers in the preparation of reagents for the diagnosis and classification of dry eye syndrome. Background Technology

[0002] Dry eye disease (DED) is a chronic ocular surface disease caused by multiple factors, characterized by abnormalities in the quality and quantity of tears and dynamic imbalances. This leads to dryness and damage to the ocular surface, resulting in various uncomfortable symptoms such as dryness, burning, and a foreign body sensation. In severe cases, it can cause decreased vision and even corneal ulcers and perforation, significantly impacting patients' quality of life and visual function. With the increasing use of electronic screens in modern society, poor indoor air quality, and the accelerating aging of the population, the incidence of dry eye is rising year by year, becoming a common ocular surface disease worldwide.

[0003] Currently, clinically, dry eye syndrome (DED) is mainly classified into two types based on tear film composition: aqueous hypothermia (insufficient tear secretion) and evaporative dry eye (excessive tear evaporation). Evaporative dry eye caused by prolonged use of video display terminals (VDTs) accounts for over 60% of cases. Given the differences in treatment strategies for different types of DED, accurate classification using non-invasive methods is crucial for optimizing clinical diagnosis and treatment.

[0004] Existing diagnostic methods (such as the OSDI questionnaire, Schirmer test, tear film breakup time, and corneal fluorescein staining) rely on subjective assessment, which has limitations such as low accuracy, poor reproducibility, and large data variability, making it difficult to meet the needs of early, accurate diagnosis and dynamic assessment. In contrast, tear protein testing has significant advantages: it is non-invasive and convenient, requiring only a small amount of tear fluid for analysis, causing less patient discomfort, resulting in high compliance, facilitating widespread clinical application and promotion, and is also suitable for large-scale screening and long-term monitoring.

[0005] Artificial tears are currently a standard and fundamental treatment for dry eye, especially when the cause is unknown, making them the first-line treatment. However, the pathogenesis of dry eye is complex and diverse, involving multiple factors such as neurological abnormalities, immune dysregulation, tear hyperosmolarity, and inflammatory responses. Relying solely on artificial tears can only temporarily relieve symptoms and cannot address the underlying cause, making it difficult to achieve ideal treatment results and disease control. Long-term treatment outcomes for moderate to severe dry eye patients are also less than ideal. If we can gain a deeper understanding of the pathophysiological mechanisms of dry eye in each patient based on tear protein testing results, we can develop more targeted and personalized treatment plans. Furthermore, regular tear protein testing during treatment allows for dynamic monitoring of treatment effects, providing timely and objective evidence for clinical treatment and improving the success rate and efficacy of treatment. Summary of the Invention

[0006] To address the technical deficiencies of existing technologies, this invention provides an application of a reagent for detecting biomarkers in body fluids in the preparation of reagents for the diagnosis and classification of dry eye syndrome.

[0007] The technical solution adopted in this invention is: the application of reagents for detecting biomarkers in body fluids in the preparation of reagents for the diagnosis and classification of dry eye syndrome, wherein the biomarker is galectin-9 in body fluids (preferably tears).

[0008] The primary objective is to provide a biomarker or an in vitro diagnostic reagent for detecting a biomarker for the diagnosis and classification of dry eye syndrome, wherein the biomarker is galectin-9.

[0009] Furthermore, the biomarker Galectin-9 is the Galectin-9 protein (LGALSL9 or Lgalsl9) or the Galectin-9 gene ( LGALSL9 or Lgalsl9 ).

[0010] Furthermore, the reagents for detecting biomarkers are reagents used to detect the mRNA expression level and / or the protein expression level of Galectin-9.

[0011] Furthermore, the biomarker detection is used to quantitatively assess the severity of dry eye to guide clinical treatment plans, such as for mild dry eye requiring only tear replacement and moderate to severe dry eye requiring adjunctive anti-inflammatory treatment.

[0012] Furthermore, the biomarker detection is used to differentiate between different subtypes of dry eye, such as video-terminal-related evaporative dry eye and non-evaporative dry eye.

[0013] The second objective is to provide an ophthalmic reagent that intervenes in the preparation and application of products for treating dry eye, wherein the biomarker is galectin-9. Furthermore, the ophthalmic reagent is a reagent used to block or antagonize the mRNA expression level and / or the protein expression level of Galectin-9.

[0014] Application of Galectin-9 protein expression inhibitors in the preparation of drugs for treating dry eye syndrome.

[0015] The beneficial effects of this invention are as follows: This invention provides an application of a reagent for detecting biomarkers in the preparation of diagnostic and subtyping reagents for dry eye syndrome. By detecting the expression of Galectin-9 in body fluids (preferably tears), early and accurate diagnosis of dry eye and quantitative assessment of the severity of the condition can be achieved. Furthermore, research reveals that Galectin-9 expressed on the surface of T cells plays a crucial role in the immune-inflammatory process of dry eye syndrome. Local blocking of Galectin-9 through the conjunctiva can effectively alleviate ocular surface damage caused by dry eye syndrome. Therefore, targeted therapies or interventions developed based on Galectin-9 can regulate immune responses, inhibit inflammation, and improve tear secretion at the etiological level. Such strategies are expected to change the existing treatment model for dry eye syndrome, thereby significantly improving treatment efficacy, patient prognosis, and reducing the adverse effects of dry eye syndrome on patients' quality of life and visual function. In summary, Galectin-9 has broad application prospects in the field of dry eye syndrome diagnosis and treatment, and is of great significance for improving the overall prevention and treatment level of dry eye syndrome. Attached Figure Description

[0016] Figure 1 This study presents proteomics data on tears from patients with dry eye. (A. Number of patients and specific experimental procedures; B. Differences in the expression levels of Galectin-9 protein in the tears of normal individuals, patients with mild dry eye, and patients with moderate to severe dry eye; C. Correlation between Galectin-9 protein expression levels and clinical symptoms and signs).

[0017] Figure 2 ROC curves for Galectin-9 protein content used in dry eye diagnosis; (D. ROC curve for tear Galectin-9 expression level to differentiate between healthy individuals and dry eye patients, AUC=0.823; E. ROC curve for tear Galectin-9 expression level to differentiate between healthy individuals, mild and moderate to severe dry eye, AUC=0.859).

[0018] Figure 3 Transcriptomic data were collected from VDT dry eye patients. (A. Thirty healthy volunteers underwent washing for 3 hours daily on a video terminal, followed by more than 12 hours of daily use of the video terminal device. Their ocular condition was assessed at 0, 1 week, and 3 weeks, and tear fluid was collected for transcriptomic analysis. B. Eight Galcetin protein family mRNAs were detected in the tear fluid of the above population, among which...) LGALSL9 The expression level increases with the extension of VDT time.

[0019] Figure 4 ROC curve for Galectin-9 mRNA content used in dry eye diagnosis; (A. tear film) LGALSL9The ROC curve AUC of expression levels at week 0 and week 3 of VDT was 0.70; B. The ROC curve AUC of Galectin-9 expression levels in tears, used to differentiate between healthy individuals and those with mild to moderate dry eye, was 0.859.

[0020] Figure 5 The expression levels of Galectin-9 in dry eye cells and mouse models were as follows: (A. Increased Galectin-9 protein expression in human conjunctival epithelial cells after hyperosmolar treatment; B. Increased Galectin-9 expression in the conjunctiva of dry-eye mice induced by a dry environment; C. A dry-eye mouse model induced by a dry environment was constructed, and single-cell sequencing was performed on conjunctiva samples taken at different time points; E. Galectin-9 mRNA levels...) Lgalsl9) The expression level increases with drying time.

[0021] Figure 6 Galectin-9 inhibitors can alleviate corneal damage in dry-eye mice; (A. Slit-lamp photographs of mouse corneas stained with sodium fluorescein; B. Statistical analysis of mouse corneal staining scores). Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. 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.

[0023] Example 1: Tear proteomics data from dry eye patients ( Figure 1 &2) The specific clinical trial protocol included in the implementation of this invention is as follows: Figure 1 Referring to the 2020 Chinese Medical Association Ophthalmology Branch's "Chinese Expert Consensus on Dry Eye: Diagnosis" criteria, a healthy control group (12 cases) and dry eye patients (27 cases) who visited the Eye Hospital Affiliated to Wenzhou Medical University were included. Based on the dry eye grading criteria in the 2020 Chinese Medical Association Ophthalmology Branch's "Chinese Expert Consensus on Dry Eye: Diagnosis," the dry eye patients were divided into a mild dry eye group (14 cases) and a moderate to severe dry eye group (13 cases).

[0024] Subjective symptoms and various clinical signs of the eyes were assessed in the participants. Tear samples were collected from healthy volunteers and dry eye patients using Schirmer test strips, immediately placed in dry ice, and then transferred to a -80°C freezer for short-term storage. Tear proteins were extracted and analyzed using an Orbitrap Astral mass spectrometer (Thermo). Figure 1B shows that the expression level of Galectin-9 protein in the tear fluid of patients in the severe dry eye group was significantly higher than that in the healthy control group and the mild dry eye group. Figure 1 C indicates that the expression level of Galectin-9 in tears is positively correlated with the redness index and tear river height, and negatively correlated with the OSDI score.

[0025] Figure 2 Using dry eye grading as the state variable and tear Galectin-9 content as the test variable, ROC curves were plotted. Figure 2 As shown in D, the AUC for distinguishing healthy controls from dry eye patients based on Galectin-9 protein expression level was 0.823. Figure 2 E shows that the AUC (average value) for distinguishing between healthy controls and patients with severe dry eye based on Galectin-9 protein expression level was 0.859. These results suggest that using Galectin-9 protein expression level as a diagnostic biomarker for tear film in dry eye is highly accurate.

[0026] Example 2: Transcriptomic data of ocular surface flushing fluid in VDT ​​dry eye population ( Figure 3 &4) Figure 3 Video display terminal (VD)-related dry eye is a subtype of dry eye caused by prolonged use of electronic screens (such as computers, mobile phones, tablets, etc.), and is listed by the International Dry Eye Association (TFOS DEWS II) as an important type of lifestyle-related dry eye. This study included 9 healthy subjects who underwent a one-week washout (daily VDT use <3 hours), followed by a VDT intervention of more than 8 hours per day (daily VDT use >8 hours). Clinical symptoms and signs were assessed at these three time points (using the same procedure as described above), and ocular surface flushing fluid was collected, centrifuged, and subjected to RNA transcriptome sequencing. A total of 8 Galcetin protein family mRNAs were detected, among which… LGALSL9 Expression levels increased with increasing VDT time. Figure 3 B).

[0027] Figure 4 ROC curve for Galectin-9 mRNA content used in dry eye diagnosis. Figure 4 Using VDT stress time as the state variable and tear Galectin-9 mRNA content as the test variable, ROC curves were plotted. Figure 4 A shows that Galectin-9 mRNA ( LGALS9 The expression level had an AUC of 0W and 3W in VDT ​​(Volume Depression) of 0W and 0W, and an AUC of 0W and post-stress of 0W in VDT ​​(Volume Depression) of 078. Figure 4B). The above results suggest that using Galectin-9 mRNA expression level as a diagnostic marker for tear film in dry eye is highly accurate.

[0028] Example 3: Results of cell and mouse experiments ( Figure 5 &6) Figure 5 The primary cultured human cell model is subjected to hypertonic treatment (commonly used to simulate dry eye cell models). Figure 5 A. Western blotting analysis of Galectin-9 protein in the control and hyperosmolar treatment groups showed that Galectin-9 protein expression was significantly higher in the hyperosmolar treatment (HOP) group than in the control group (NC). A mouse dry eye model was constructed using an Intelligent Control Environment System (ICES) at different time points (0, 1W, 3W). Immunohistochemical staining revealed increased Galectin-9 expression in the conjunctiva of the dry eye group mice compared to the control group (5B). mouse conjunctiva organize Single-cell transcriptome sequencing (5B) revealed the presence of mRNAs from eight Galcetin protein families in mice. Lgals9 The expression level increased with increasing dry stress duration.

[0029] Figure 6 In this experiment, healthy female 8-10-week C57 mice were housed in a normal environment and an ICES dry environment, respectively. Each group of mice was randomly divided into two groups, injected with Galectin-9 neutralizing antibody and isotype IgG control, respectively. Images stained with sodium fluorescein showed that corneal damage was significantly reduced in the Galectin-9 antibody blocking group compared to the dry eye group. Figure 6 A), staining score statistics also showed that blocking Galectin-9 could effectively alleviate corneal damage in dry-eye mice ( Figure 6 B) in conclusion The above results indicate that Galectin-9 protein in the tear fluid of dry eye patients and Lgalsl9 Gene expression levels were significantly higher than in the healthy control group, and the ROC curve AUC > 0.7, indicating good diagnostic efficacy for dry eye. Its advantages are: this invention is the first to propose using tears as a test sample; by detecting the Galectin-9 content in tears, it is possible to determine whether a patient has dry eye and differentiate its severity. Compared with existing technologies, this method is simpler to operate, enables non-invasive detection and treatment, and has better results, showing great application potential.

Claims

1. The application of reagents for detecting biomarkers in the preparation of reagents for the diagnosis and subtyping of dry eye syndrome, characterized in that, The biomarker mentioned is Galectin-9 in body fluids.

2. The application according to claim 1, characterized in that, The typing test includes typing tests for aqueous-deficient dry eye and evaporative dry eye.

3. The application according to claim 1, characterized in that, The bodily fluid in question is tears.

4. The application according to claim 1, characterized in that, The biomarker mentioned is the expression of the Galectin-9 gene. LGALS9 / Lgals9 Or Galectin-9 protein LGALS9 / Lgals9.

5. The application according to claim 1, characterized in that, The reagents used to detect biomarkers are reagents for detecting the mRNA expression level and / or the protein expression level of Galectin-9.

6. The application according to claim 5, characterized in that, The reagents for detecting biomarkers include reagents required for LC / MS mass spectrometry, PCR, Western blot, and immunohistochemistry.

7. Application of Galectin-9 protein inhibitors in the preparation of drugs for treating dry eye syndrome.

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