Application of metabolic marker in preparation of fungal keratitis product and screening method of metabolic marker

Abstract

The invention relates to the technical field of biomedicine, and aims to solve the problems of poor drug permeability, drug resistance, drug toxicity, high surgical risk, infection recurrence, donor tissue shortage and the like of prevention and treatment means of fungal keratitis. The invention provides application of a metabolic marker in preparation of a product for diagnosing, preventing or treating fungal keratitis and a screening method of the metabolic marker. The metabolic marker is at least one of adenosine, adenosine monophosphate and uric acid. In fungal cornea, adenosine and adenosine monophosphate are reduced, and uric acid is increased. According to the invention, three differential metabolites are screened based on the combination of non-targeted metabonomics and targeted mass spectrometry detection, and the provided purine metabolic pathway regulation and the level of the three metabolites can be used for diagnosis of fungal keratitis patients and research and development of new therapeutic targets.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of metabolic markers in the preparation of products for fungal keratitis and a screening method thereof. Background Art

[0002] Fungal keratitis (FK) is a serious, sight-threatening corneal infection caused by pathogenic fungi. Common pathogens include Alternaria alternata, Aspergillus fumigatus, Alternaria spp., and Candida albicans. Because fungal keratitis progresses rapidly and can lead to corneal perforation and vision loss, early diagnosis and effective treatment are crucial. Detection methods for fungal keratitis mainly include: 1. Traditional methods: corneal scraping microscopy, fungal culture, and histopathological examination; 2. Molecular methods: PCR and real-time quantitative PCR; 3. Immunological methods: enzyme-linked immunosorbent assay and immunofluorescence staining. However, existing detection methods still face challenges in terms of sensitivity, specificity, and timeliness. The main treatment options for fungal keratitis include medication and surgery. However, due to the unique anatomical and physiological characteristics of the cornea, the application of topical antifungal drugs faces numerous challenges, such as poor penetration, poor solubility, and low bioavailability. Furthermore, long-term use of these drugs can lead to drug resistance and toxicity. Surgical treatment typically involves corneal lesion excision and keratoplasty. However, these procedures are subject to numerous limitations, including recurrent infection, surgical risks, and a shortage of donor tissue. Therefore, exploring the underlying pathological features of fungal keratitis and identifying new therapeutic targets are crucial for optimizing its prevention and treatment strategies.

[0003] Multi-omics analysis techniques are primarily based on high-throughput technologies, including gene sequencing, microarray analysis, and mass spectrometry, which enable high-throughput detection and data analysis of samples to obtain multiple omics data. Within these data, the interactions and changes between different omics reveal specific pathways of gene expression regulation, metabolite levels, and interactions within an organism. Analysis of these data can reveal interactions between different omics and investigate disease pathogenesis and individual differences. Metabolite changes are an important foundation for an organism's phenotype, providing a more intuitive understanding of biological processes and their mechanisms, not only aiding in disease diagnosis but also potentially identifying potential new therapeutic targets. However, metabolomics-based analysis of metabolic markers for fungal keratitis remains elusive. Summary of the Invention

[0004] There are problems such as poor drug penetration, drug resistance, drug toxicity, high surgical risk, infection recurrence and donor tissue shortage in the prevention and treatment of fungal keratitis, and the application of metabolic markers in the preparation of products for diagnosing, preventing or treating fungal keratitis and the screening method thereof are provided. Based on non-targeted metabolomics and targeted mass spectrometry detection, three kinds of differential metabolites are screened out, and the level of the proposed regulation purine metabolic pathway and three metabolites can be used for the diagnosis of fungal keratitis patients and the research and development of new target for treatment.

[0005] The application is realized by the following technical solutions:

[0006] The first aspect of the application provides the application of metabolic markers in the preparation of fungal keratitis related products, and the metabolic markers are one or a combination of two or more of adenosine, adenosine monophosphate (AMP) and uric acid. Adenosine and adenosine monophosphate are down-regulated in the cornea infected by fungi, and uric acid is up-regulated.

[0007] The proposed metabolic markers can be used to prepare products for diagnosing, preventing or treating fungal keratitis. The detection products include but are not limited to in vitro detection products such as in vitro detection reagents and kits, and prevention products such as antifungal eye drops and eye cleaning products.

[0008] The product for treating fungal keratitis comprises a therapeutically effective dose of a purine metabolic pathway regulator and a pharmaceutically acceptable carrier, and the purine metabolic pathway regulator can reduce the level of uric acid in the purine metabolic pathway and increase the levels of adenosine and AMP. The dosage forms include but are not limited to eye drops, eye ointments, gels and the like.

[0009] The application provides the possibility for non-invasive detection of fungal keratitis and provides a new idea for the treatment method.

[0010] The fungal keratitis is caused by a single fungus such as fusarium keratitis, aspergillus keratitis or alternaria keratitis, or caused by infection of two or more fungi such as infection of at least one of fusarium, aspergillus and alternaria.

[0011] In the metabolites of the corneal tissue infected by alternaria, the levels of adenosine and uric acid are higher than those of the corneal tissue infected by fusarium and aspergillus, and the level of adenosine decreases with the progress of the fungus. The level of adenosine can be used to monitor the disease progression and treatment effect.

[0012] The second aspect of the application provides a screening method of metabolic markers for fungal keratitis, comprising the following steps:

[0013] (1) Establish a mouse model of fungal keratitis, collect the corneal tissue samples of mice with fungal keratitis and normal mice, analyze the metabolites in the samples by non-targeted metabolomics analysis means, identify the significantly different metabolites and the most down-regulated metabolic pathway, which is the purine metabolic pathway;

[0014] (2) From the metabolites of the purine metabolic pathway, screen out the downstream metabolites that can better reflect the metabolic difference for targeted quantitative mass spectrometry detection, determine the metabolites with statistical significance in the purine metabolic pathway as markers related to fungal keratitis lesions through targeted quantitative mass spectrometry detection and differential metabolite analysis means.

[0015] It also includes (3) clinical verification: collect the ocular corneal scraping tissue of patients diagnosed with fungal keratitis in the clinic, extract metabolites, and perform quantitative mass spectrometry detection, while taking the matrix lens taken from the clinic myopia surgery patients as the normal corneal tissue for comparative analysis.

[0016] The step (1) uses liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) for metabolite detection, processes the obtained data, annotates the metabolites, screens the differential metabolites, analyzes the pathways and interprets the results.

[0017] The step (2) screens 6 downstream metabolites with high confidence level from the 13 metabolites enriched in the purine metabolic pathway: adenosine, AMP, inosine, hypoxanthine, xanthine and uric acid, respectively, for targeted quantitative mass spectrometry detection of mouse corneas and human corneas, processes the mass spectrometry data, performs statistical analysis and interprets the results, and identifies the significantly different metabolites between normal and abnormal tissues.

[0018] In the mouse cornea: the detection limit of adenosine is less than 0.1 fg / mL, indicating that it has a significant advantage in exploring the purine metabolic pathway in the pathogenesis of fungal keratitis. It is helpful for early diagnosis and intervention.

[0019] Among the infected corneal tissues compared with normal tissues, there are 3 metabolic markers with the same trend in mouse corneas and human corneas: adenosine and monophosphate adenosine are down-regulated, and uric acid is up-regulated.

[0020] The present application has the following beneficial effects:

[0021] The present application proposes a marker screening based on metabolomics and targeted mass spectrometry detection in the field of fungal keratitis research. The screened metabolic markers can be used for the diagnosis of fungal keratitis patients and the research and development of new therapeutic targets. The present application reveals the important role of the purine metabolic pathway in fungal keratitis, providing important clues for the pathological mechanism research and potential therapeutic targets of fungal keratitis. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 2 shows the two-dimensional PCA scores of quality control samples in untargeted metabolomics. Triangles and squares represent fungal keratitis and normal corneal samples, respectively.

[0023] Figure 2 Volcano plot of differential metabolites between the FK group and the NC group, blue represents down-regulated metabolites, and red represents up-regulated metabolites.

[0024] Figure 3 The KEGG enriched TOP20 pathways that were significantly downregulated in the FK group compared with the NC group.

[0025] Figure 4 The levels of 13 differential metabolites enriched in the purine metabolic pathway between the FK group and the NC group.

[0026] Figure 5 Representative chromatograms of standard solutions of six differential metabolites.

[0027] Figure 6 Representative chromatograms of six differential metabolites in mouse samples.

[0028] Figure 7 Figure 2 is the content diagram of six differential metabolites in the cornea of ​​mice in the FK group and NC group.

[0029] Figure 8 Representative chromatograms of six differential metabolites in human corneal samples.

[0030] Figure 9 Figure 2 is the content diagram of six differential metabolites in human samples of the FK group and the NC group.

[0031] Figure 10 Figure 2 shows the content of six differential metabolites in corneal samples from patients with fungal keratitis infected with three different strains.

[0032] Figure 11 Figure 2 shows the content of six differential metabolites in corneal samples from patients with fungal keratitis of different disease courses. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0035] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0036] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0037] Example 1 Non-targeted metabolomics detection

[0038] 1. Establishment of fungal keratitis mouse model: First, Fusarium solani (F. solani) strain AS 3.182 was inoculated on potato dextrose agar medium and cultured at 28°C for 72 hours. Subsequently, spores were scraped from the surface of the culture medium to prepare a fresh spore suspension. After counting using a cell counter, the suspension was diluted with PBS to 1×10 7 Colony forming units (CFU) / mL. Female C57BL / 6 mice aged 6-8 weeks were randomly divided into two groups: fungal keratitis group (FK group) and normal control group (NC group). In the FK group, a corneal epithelial scraper was used to scrape the epithelial tissue with a diameter of 2.5 mm in the center of the mouse cornea. A circular filter paper with a diameter of 2.5 mm was soaked in a suspension of F. solani spores (10 7 CFU / mL) were added, and then the filter paper was applied to the center of the cornea. The upper and lower eyelids were sutured with 7-0 sutures and the sutures were removed after 24 hours.

[0039] 2. Sample collection and metabolite extraction: The sample preparation steps are as follows: mouse corneal tissue extract (80% (v / v) methanol / water, 500.0 μL), L-2-chlorophenylalanine methanol solution (0.2 mg / mL, 4.0 μL) and 2 steel beads were added to a 1.5 mL centrifuge tube. Use a high-throughput tissue grinder to grind at 60 Hz for 120 seconds, followed by ultrasonic treatment in an ice water bath for 30 minutes. This operation was repeated twice, and the extracts were combined and centrifuged at 13,000 rpm for 10 minutes (4°C). The supernatant was transferred to a new 1.5 mL centrifuge tube and dried under vacuum. Subsequently, the dried sample was dissolved with 80% (v / v) methanol / water (200.0 μL) solution, centrifuged again at 20,000 rpm for 10 minutes, and the supernatant was collected for non-targeted metabolomics analysis.

[0040] 3. Liquid Chromatography-Mass Spectrometry (LC-MS / MS) Analysis: Chromatographic separation was performed using an Acquity UPLC I-Class system (Waters Corporation, Milford, USA) equipped with a Vion IMS QTOF mass spectrometer (Waters Corporation, Milford, USA). LC-MS / MS analysis used mobile phases A: 0.1% (v / v) formic acid in water; mobile phase B: 0.1% (v / v) formic acid in methanol / acetonitrile. Chromatographic conditions: column, 2.1 mm i.d. × 100 mm length, 1.7 μm C18 particles; injection volume, 1.0 μL; flow rate, 400 μL / min; column temperature, 45°C. Gradient elution program: 0-1 min, 1%-30% B; 1-2.5 min, 30%-60% B; 2.5-6.5 min, 60%-90% B; 6.5-8.5 min, 90%-100% B; 8.5-10.7 min, 100% B; 10.8-13 min, 100%-1% B. Electrospray ionization tandem mass spectrometry (ESI-MS / MS) experiments were performed in positive and negative ion modes (m / z range 50-1,000). Electrospray interface conditions included argon (99.999%) for collision-induced dissociation (CID) dissociation (DID) gas, scan time 0.2 s, cone voltage 40 V, capillary voltage 2.5 kV, ion source temperature 115°C, nebulizer gas flow rate 900 L / h, and nebulizer temperature 450°C.

[0041] 4. Results: Figure 1 As shown, the PCA graph shows that the individual data of the samples formed a tight cluster, indicating that the current method can well guarantee the data quality in metabolomics analysis, has high reliability, and supports the subsequent screening and analysis of differential metabolites. In the positive and negative ion modes of UPLC-QTOF-MS, a total of 3651 metabolites were identified in the corneas of the NC group and the FK group. The screening criteria for metabolites were VIP value > 1, fold change (FC) > 1 or < 1, and p value ≤ 0.05, and identification was performed by comparing the precise molecular weight and MS / MS fragments with the standard data, and a total of 268 significantly different metabolites were screened out. Among them, there were 148 upregulated metabolites in the FK group and 120 downregulated metabolites in the FK group. Volcano plot ( Figure 2 ) intuitively and clearly shows the magnitude of changes and statistical significance of metabolites between the NC group and the FK group. The significantly up-regulated or down-regulated metabolites are distributed on both sides of the volcano plot. In addition, KEGG enrichment analysis was performed on the metabolic pathways of the FK group compared with the NC group ( Figure 3 ). Among the enriched pathways downregulated in the FK group, the purine metabolism pathway was the most significantly downregulated. The significant downregulation of the purine metabolism pathway may be related to the pathophysiological state of the FK group.

[0042] Example 2 Targeted Quantitative Mass Spectrometry Detection of Corneal Tissues of Mice with Fungal Keratitis and Normal Mice

[0043] The establishment of the fungal keratitis mouse model, sample collection, and metabolite extraction methods were the same as above.

[0044] 1. Targeted Quantitative Mass Spectrometry Detection: An Acquity UPLC system (Waters Corporation, Milford, MA, USA) coupled to a TSQQuantum triple quadrupole tandem mass spectrometer (Thermo Fisher Scientific, San Jose, CA) was used to quantitatively detect differentially expressed metabolites (DEMs) from mice. Mobile phase A consisted of 0.1% (v / v) formic acid in water, and mobile phase B consisted of acetonitrile. LC conditions included: column, 100 mm × 0.3 mm ID, 1.7 μm C18 particles; injection volume, 5.0 μL; flow rate, 7.0 μL / min; column temperature, 30°C. Gradient elution program: 0–3.0 min, 5% to 80% B; 3.0–6.0 min, 80% B; 6.0–6.1 min, 80% to 10% B; 6.1–6.5 min, 10% B. Mass spectrometry detection was performed in multiple reaction monitoring (MRM) mode using an electrospray ionization (ESI) interface in positive and negative ion modes. Mass spectrometry parameters included a nebulizer temperature of 350°C, a nebulizer gas flow rate of 650 L / Hr, a cone gas flow rate of 150 L / Hr, and a nebulizer gas pressure of 7.0 bar. The LC-MS system and data acquisition were operated and processed using Xcalibur software.

[0045] 2. Screening of differential metabolites: The purine metabolic pathway ranked first among the pathways enriched by FK downregulation and was selected to study the differential metabolism of the corneas between the NC group and the FK group. Figure 4 As shown in Table 1, 13 metabolites were enriched in this pathway. Screening strategy: Downstream metabolites were selected because they are closer to the end of the metabolic pathway and can better reflect metabolic differences. Metabolites with high confidence levels were selected to ensure the reliability of the data. Finally, 6 downstream metabolites with high confidence levels were selected for further analysis. Among them, adenosine, AMP and hypoxanthine were significantly downregulated in FK-infected corneas compared with normal corneas, while hypoxanthine, xanthine and uric acid were significantly upregulated. The ESI conditions of the 6 differential metabolites (DEMs) are listed in Table 2.

[0046] Table 1 Thirteen differential metabolites enriched in the purine metabolic pathway

[0047]

[0048]

[0049] Table 2 ESI conditions for mass spectrometry quantitative analysis of 6 differential metabolites

[0050]

[0051] 3. Preparation of standard curve: The standard curves of 6 standard solutions (concentration range 0-400 ng / mL) showed strong linear relationships, and all regression coefficients (r 2 ) were all above 0.995 (Table 3). The limits of quantification (LOQs) for different analytes varied, with the lowest LOQ being 0.10 fg / mL and the highest being 5.0 ng / mL, indicating that the method had high sensitivity and reliability in the quantitative detection of the six differential metabolites in the corneas of the two groups.

[0052] 4. Comparison of corneal metabolite levels between FK and NC groups: The six differential metabolites screened above were quantitatively analyzed in the corneas of FK and NC groups. Representative chromatograms of standards and samples are shown in Figure 2. Figure 5 and Figure 6 As shown in Figure 2, 6 DEMs were detected in both groups. The levels of hypoxanthine, xanthine, and uric acid in the FK group were significantly higher than those in the NC group, while the levels of AMP and adenosine were significantly lower than those in the NC group. The differences between the two groups were statistically significant (P<0.05). Figure 7 However, no significant statistical difference was found between the NC and FK groups for inosine. Furthermore, in the corneas of both NC and FK mice, inosine levels were significantly higher than those of other metabolites. This suggests that inosine, with its high abundance and stability, may be a key molecule in corneal metabolism. The high levels and stability of inosine make it a potential internal reference for corneal metabolic status.

[0053] Table 3 Linear range, regression equation, and correlation coefficient (r 2 ), detection limit, quantification limit

[0054]

[0055] Example 3 Targeted Quantitative Mass Spectrometry Detection of Corneal Scrapes and Normal Corneal Tissue from Patients with Fungal Keratitis

[0056] 1. Clinical Sample Collection: Patients diagnosed with fungal keratitis (FK) by confocal microscopy, corneal scraping smear, and fungal culture were assigned to receive equal amounts of corneal scraping tissue from the same physician. The samples were transferred to 0.6 mL centrifuge tubes and stored at -80°C until use. Normal corneal tissue (NC) was also collected from stromal lenses removed from patients undergoing myopia surgery. These tissues were weighed to equal the amount of lesion tissue, placed in centrifuge tubes, and stored at -80°C until use. The specific method for quantitative mass spectrometry detection was the same as in Example 1.

[0057] 2. Clinical information collection: A total of 18 corneal scraping samples were collected from FK patients. Confocal microscopy showed filamentous, highly reflective structures with typical characteristics of fungal hyphae. Corneal scraping smears and cultures showed positive for fungi, including 10 cases of Fusarium infection, 5 cases of Aspergillus infection, and 3 cases of Alternaria infection. Through targeted metabolomics analysis, differential metabolites (DEMs) analyzed in mouse samples were successfully identified in human corneal samples ( Figure 8 ).

[0058] 3. Comparison of corneal metabolite levels between FK and NC groups: The 6 differential metabolites screened above were quantitatively analyzed in the corneas of NC and FK groups. Figure 8 As shown. It can be seen that the six DEMs were detected in both groups. The level of uric acid in the FK group was significantly higher than that in the NC group, while the levels of adenosine and AMP were significantly lower than those in the NC group. The differences between the two groups were statistically significant (P<0.05). The trends of adenosine, AMP, and uric acid were consistent with those in the mouse cornea ( Figure 9 ).

[0059] 4. Comparison of differential metabolites between different infectious species: Although the DEMs levels among Fusarium, Aspergillus and Alternaria showed no significant differences, the levels of adenosine (or uric acid) in the cornea of ​​patients infected with Alternaria were significantly different from those of patients infected with Fusarium or Aspergillus ( Figure 10 ). Adenosine levels decreased significantly with the progression of FK, while the levels of other metabolites showed no significant differences between the two groups ( Figure 11 ).

[0060] The same differential metabolites were detected in both animal models and patient samples, suggesting that these metabolites may be effective biomarkers for fungal keratitis. The roles of these differential metabolites and their associated metabolic pathways in disease may be universal. The findings based on animal studies have high potential for clinical translation.

[0061] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. Use of a metabolic marker in the preparation of a product for diagnosing, preventing or treating fungal keratitis, characterized in that: The metabolic marker is at least one of adenosine, adenosine monophosphate and uric acid.

2. The use according to claim 1, characterized in that Adenosine and adenosine monophosphate are downregulated, and uric acid is upregulated in fungal cornea.

3. The use according to claim 1, characterized in that The fungal keratitis is keratitis caused by infection with at least one of Fusarium, Aspergillus and Alternaria.

4. The use according to claim 3, characterized in that Among the metabolites of corneal tissue infected with Alternaria, the levels of adenosine and uric acid were higher than those of corneal tissue infected with Fusarium and Aspergillus, and the adenosine level decreased with the progression of fungal infection.

5. The use according to claims 1 to 4, characterized in that: The product for treating fungal keratitis comprises a therapeutically effective dose of a purine metabolic pathway regulator and a pharmaceutically acceptable carrier. The purine metabolic pathway regulator can lower the uric acid level in the purine metabolic pathway and increase the adenosine and AMP levels.

6. The use according to claim 5, characterized in that The product for treating fungal keratitis is eye drops, eye ointment or corneal repair gel.

7. The use according to claims 1 to 4, characterized in that: The product for diagnosing fungal keratitis is an in vitro detection reagent or kit.

8. The method for screening metabolic markers according to claim 1, characterized in that: The method includes the following steps: (1) establishing a fungal keratitis mouse model, collecting corneal tissue samples from fungal keratitis mice and normal mice, analyzing the metabolites in the samples by non-targeted metabolomics analysis, identifying significantly different metabolites and the most significantly downregulated metabolic pathway, which is the purine metabolic pathway; (2) screening downstream metabolites that can better reflect metabolic differences from the metabolites of the purine metabolic pathway for targeted quantitative mass spectrometry detection, and identifying statistically significant metabolites in the purine metabolic pathway as metabolic markers associated with fungal keratitis lesions through targeted quantitative mass spectrometry detection and analysis of differential metabolites.

9. The screening method according to claim 8, characterized in that The step (1) uses liquid chromatography-mass spectrometry / mass spectrometry to detect metabolites, and processes the acquired data, annotates metabolites, screens differential metabolites, analyzes pathways, and interprets the results.

10. The screening method according to claim 8, characterized in that The method also includes step (3) clinical validation: collecting corneal scraping tissue from patients diagnosed with fungal keratitis in the clinic, and collecting normal corneal tissue from the stromal lens of patients undergoing myopia surgery as a control, extracting metabolites, and performing quantitative mass spectrometry detection.

Images