Use of c-type lectin-like receptor-1 as a therapeutic marker for fungal keratitis

Abstract

The application discloses application of C-type lectin-like receptor-1 as a therapeutic marker in fungal keratitis, and provides a kit for evaluating a use time of a glucocorticoid drug in the treatment of fungal keratitis. The C-type lectin-like receptor-1 can be used as a biomarker to simply and safely judge the use time of the glucocorticoid drug in the fungal keratitis, and has important significance in guiding clinical decision-making.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of C-type lectin-like receptor-1 (CLEC-1) as a therapeutic biomarker for fungal keratitis. Background Technology

[0002] Fungal keratitis (FK) is a serious infectious eye disease worldwide. Currently, due to the lack of effective clinical treatments, it often leads to serious ocular complications, and in severe cases, can result in permanent vision loss. my country, a developing country primarily based on agriculture, is a high-incidence area for fungal keratitis, particularly due to corneal trauma from crops during farming, which is a major cause of the disease. Although many antifungal keratitis drugs have been used clinically in recent years, their efficacy is poor, and fungi are gradually developing resistance, especially to azole drugs. Due to the lack of effective treatments, many patients require corneal transplantation or even enucleation (removal of the eye).

[0003] Following fungal infection of the cornea, pattern recognition receptors in the cornea interact with pathogen-associated molecular patterns on the fungal surface, initiating an innate immune response to clear the fungal pathogen. However, glucocorticoids can inhibit the phagocytosis and processing of antigens by macrophage membranes and cellular immune processes. Various pathogenic factors of fungi can assist fungi in adhering to host cells, breaking down phospholipids in the cell membrane, thereby disrupting the cell membrane, allowing hyphae to penetrate host cells more effectively, and reducing neutrophil chemotaxis and macrophage phagocytic function. Indiscriminate use of glucocorticoids can lead to disease progression and, in severe cases, corneal perforation.

[0004] Once the corneal innate immune response is initiated, a large number of neutrophils and chemokines accumulate, leading to corneal edema and inflammatory infiltration, and even corneal ulceration or perforation. Therefore, an overly strong immune response, while controlling external infections, can damage corneal tissue, making it difficult for the cornea to regain transparency and causing serious impairment to the patient's vision. Recent studies have shown that using corticosteroids in the later stages of the disease can reduce corneal opacity and help maintain good corneal transparency. Therefore, determining the timing of corticosteroid use is crucial. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a therapeutic biomarker for fungal keratitis and its application.

[0006] The C-type lectin-like receptor family encodes the centromere portion of the natural killer (NK) gene complex on human chromosome 12 and can recognize multiple ligands and perform various functions. However, no literature has disclosed or indicated an association between the C-type lectin-like receptor family and fungal keratitis.

[0007] The applicant of this invention has demonstrated through in vivo and in vitro experiments that C-type lectin-like receptor-1 (CLEC-1), as a member of the C-type lectin-like receptor family, exhibits decreased expression during the acute inflammatory phase of fungal keratitis and increased expression during the recovery phase. Treatment of fungal keratitis with natamycin resulted in a decrease in CLEC-1 expression. Upregulation of CLEC-1 expression led to a decrease in myeloperoxidase (MPO) levels, reduced neutrophil recruitment, decreased IL-1β expression, and increased fungal burden.

[0008] This invention used quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence to detect the expression of CLEC-1 in the cornea of ​​patients with fungal keratitis. In vitro and in vivo experiments were then conducted in THP-1 macrophages and C57BL / 6 mouse models, with qRT-PCR, Western blot, and immunofluorescence used to detect CLEC-1 expression. The results showed that CLEC-1 expression decreased during the acute inflammatory phase of fungal keratitis and increased during the recovery phase. Treatment with natamycin for fungal keratitis resulted in a decrease in CLEC-1 expression. Using a viral vector to overexpress CLEC-1 in mouse cornea and THP-1 macrophages, experiments including slit-lamp photography, clinical scoring, colony-forming units (CFU), bioluminescence imaging, MPO detection, immunofluorescence staining, qRT-PCR, and Western blot confirmed that upregulating CLEC-1 expression led to decreased MPO levels, reduced neutrophil recruitment, decreased IL-1β expression, and increased fungal burden.

[0009] Based on the above research results, the first aspect of the present invention provides the application of C-type lectin-like receptor-1 as a therapeutic biomarker for fungal keratitis.

[0010] In the application provided in the first aspect of the invention, the fungal keratitis is Aspergillus fumigatus keratitis.

[0011] In the application provided in the first aspect of the invention, the therapeutic biomarker is used to indicate the timing of administration of glucocorticoid drugs.

[0012] Furthermore, in the application provided in the first aspect of the invention, an increase in the expression of C-type lectin-like receptor-1 in vivo indicates the timing of administration of glucocorticoid drugs.

[0013] A second aspect of the invention provides a kit for assessing the timing of glucocorticoid administration during the treatment of fungal keratitis, the kit comprising a reagent for detecting the level of C-type lectin-like receptor-1 in the cornea.

[0014] In the kit provided in the second aspect of the present invention, the fungal keratitis is Aspergillus fumigatus keratitis.

[0015] Furthermore, in the kit provided by the second aspect of the present invention, the reagent is used to detect the level of C-type lectin-like receptor-1 based on quantitative polymerase chain reaction or immunoblotting technology. Attached Figure Description

[0016] Figure 1 This is a graph showing the results of CLEC-1 mRNA expression in normal cornea and human cornea infected with Aspergillus fumigatus keratitis detected by qRT-PCR in Example 1.

[0017] Figure 2 This is a diagram showing the results of CLEC-1 protein expression in normal cornea and human cornea infected with Aspergillus fumigatus keratitis, detected by immunofluorescence staining in Example 1.

[0018] Figure 3 These are slit-lamp microscope images taken 1 / 2, 1, 2, 3, 5, 7, 10, and 14 days after the establishment of the mouse model of Aspergillus fumigatus keratitis in Example 2.

[0019] Figure 4 This is a graph showing the results of qRT-PCR detection of CLEC-1 mRNA expression in the cornea of ​​mice 1 / 2, 1, 2, 3, 5, 7, 10, and 14 days after the establishment of the mouse model of Aspergillus fumigatus keratitis in Example 2.

[0020] Figure 5 This is a graph showing the results of Western blot analysis of CLEC-1 protein expression in the cornea of ​​mice after 1 / 2, 1, 2, 3, 5, 7, 10, and 14 days following the establishment of the Aspergillus fumigatus keratitis mouse model in Example 2.

[0021] Figure 6 This is a graph showing the results of CLEC-1 protein expression in the mouse cornea, detected by immunofluorescence staining, 5 days after the establishment of the mouse model of Aspergillus fumigatus keratitis in Example 2.

[0022] Figure 7 These are slit-lamp microscope images taken on days 1, 3, and 5 of the mouse group infected with Aspergillus fumigatus and the group treated with Aspergillus fumigatus combined with natamycin, as shown in Example 2.

[0023] Figure 8The corneal clinical scores at 3 and 5 days in the Aspergillus fumigatus infection group and the Aspergillus fumigatus infection combined with natamycin treatment group in Example 2;

[0024] Figure 9 This is a graph showing the results of qRT-PCR detection of CLEC-1 mRNA expression in mice infected with Aspergillus fumigatus and treated with Aspergillus fumigatus combined with natamycin in Example 2.

[0025] Figure 10 This is a graph showing the results of Western blot detection of CLEC-1 protein expression in mice infected with Aspergillus fumigatus and treated with Aspergillus fumigatus combined with natamycin in Example 2.

[0026] Figure 11 This is a photograph of EGFP expression in mouse cornea taken using a fluorescence microscope, as shown in Example 3.

[0027] Figure 12 This is a graph showing the results of qRT-PCR detection of CLEC-1 mRNA overexpression in Example 3;

[0028] Figure 13 This is a graph showing the results of Western blot detection of CLEC-1 protein overexpression in Example 3;

[0029] Figure 14 These are corneal photographs of control mice and CLEC-1 overexpression mice one day after infection with Aspergillus fumigatus keratitis in Example 3.

[0030] Figure 15 The clinical corneal scores of control mice and CLEC-1 overexpression mice one day after infection with Aspergillus fumigatus keratitis in Example 3;

[0031] Figure 16 This is a diagram showing the corneal CFU results of mice in the control group and the CLEC-1 overexpression group one day after infection with Aspergillus fumigatus keratitis in Example 3.

[0032] Figure 17 These are the average bioluminescence images of the corneas of control mice and CLEC-1 overexpression mice one day after infection with Aspergillus fumigatus keratitis in Example 3.

[0033] Figure 18 This is a diagram showing the corneal MPO results of mice in the control group and the CLEC-1 overexpression group one day after infection with Aspergillus fumigatus keratitis in Example 3.

[0034] Figure 19 This is a diagram showing the distribution of neutrophils in the cornea of ​​control mice and CLEC-1 overexpression mice one day after infection with Aspergillus fumigatus keratitis, as detected by immunofluorescence in Example 3.

[0035] Figure 20 This is a diagram showing the results of qRT-PCR detection of CLEC-1 mRNA expression in the corneas of normal mice, mice infected with Aspergillus fumigatus keratitis, CLEC-1 overexpressing mice, and mice with CLEC-1 overexpression combined with Aspergillus fumigatus keratitis in Example 3.

[0036] Figure 21 This is a graph showing the results of Western blot analysis of CLEC-1 protein expression in the corneas of normal mice, mice infected with Aspergillus fumigatus keratitis, CLEC-1 overexpressing mice, and mice with CLEC-1 overexpression combined with Aspergillus fumigatus keratitis in Example 3.

[0037] Figure 22 This is a diagram showing the results of qRT-PCR detection of CLEC-1 mRNA expression in normal THP-1 macrophages, THP-1 macrophages treated with Aspergillus fumigatus, THP-1 macrophages overexpressing CLEC-1, and THP-1 macrophages overexpressing CLEC-1 and treated with Aspergillus fumigatus in combination with Aspergillus fumigatus in Example 3.

[0038] Figure 23 This is a graph showing the results of Western blot analysis of normal THP-1 macrophages, THP-1 macrophages treated with Aspergillus fumigatus, THP-1 macrophages overexpressing CLEC-1 and THP-1 macrophages overexpressing CLEC-1 and treated with Aspergillus fumigatus in Example 3.

[0039] Figure 24 This refers to the corneal perforation rate of mice in Example 4 after their corneas were infected with Aspergillus fumigatus, compared to the hormone group treated before CLEC-1 elevation and the hormone group treated after CLEC-1 elevation, two days after medication.

[0040] Figure 25 The results for Example 4 show the corneal clinical scores of mice after their corneas were infected with Aspergillus fumigatus, in the group that did not use hormones and the group that used hormones after CLEC-1 levels increased, two days after medication. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the various embodiments of this invention will be described in detail below. However, those skilled in the art will understand that many technical details have been provided in the various embodiments of this invention to facilitate a better understanding of this application. However, the technical solutions claimed in the claims of this application can be implemented even without these technical details and with various variations and modifications based on the following embodiments.

[0042] This invention proposes using C-type lectin-like receptor-1 in patients with fungal keratitis as a biomarker for the use of glucocorticoid drugs, and assessing the timing of glucocorticoid drug use based on the level of C-type lectin-like receptor-1 in the cornea, so as to guide clinical treatment.

[0043] The experimental protocol and results established in the embodiments of this invention are as follows: Corneas from 6 patients with Aspergillus fumigatus keratitis and 6 healthy patients were collected for immunofluorescence and qRT-PCR detection. The specimens were confirmed by fungal culture and morphology. The purpose and methods of the study were explained in detail to the patients, and samples were collected after obtaining informed consent. The results showed that CLEC-1 expression was significantly increased in infected corneas compared to normal corneas. When the disease duration was less than 1 month, CLEC-1 mRNA expression showed no significant change, but when the disease duration was greater than 1 month, CLEC-1 mRNA expression was significantly increased.

[0044] In in vivo experiments, a mouse model of fungal keratitis was established using intrastromal injection. The mouse corneas were observed and photographed daily using a slit-lamp microscope. Corneal tissue was harvested at 1 / 2, 1, 2, 3, 5, 7, 10, and 14 days post-infection for Western blot and qRT-PCR detection. Eyeballs were harvested 5 days later for immunofluorescence staining. The degree of corneal infection was observed, and clinical scores were recorded. CLEC-1 expression was detected by qRT-PCR and Western blot on days 3 and 5. CLEC-1 overexpression in the mouse cornea was achieved through intrastromal injection of the CLEC-1 adeno-associated virus (AAV) vector. One eye from each mouse was randomly selected for subconjunctival injection of 5 μl of the 10¹¹vg vector. Two weeks later, enhanced green fluorescent protein was detected using fluorescence stereomicroscopy to determine the uniform distribution of the virus in the corneal stroma. CLEC-1 overexpression was detected by qRT-PCR and Western blot. Corneal tissue was harvested 1 day post-infection for MPO detection, immunofluorescence staining, Western blot, and qRT-PCR. The results showed that CLEC-1 expression decreased during the acute inflammatory phase of fungal keratitis and increased during the recovery phase. Treatment with natamycin for fungal keratitis resulted in a decrease in CLEC-1 expression. Upregulation of CLEC-1 expression led to decreased MPO levels, reduced neutrophil recruitment, decreased IL-1β expression, and increased fungal burden.

[0045] In in vitro experiments, the lentiviral vector CLEC-1 was pretreated in THP-1 macrophages. After 24 hours of pretreatment, THP-1 macrophages were infected with Aspergillus fumigatus conidia for 16 hours. THP-1 macrophages were then collected for Western blot and qRT-PCR detection. The results were consistent with those in the mouse keratitis model; upregulation of CLEC-1 expression decreased MPO levels, reduced neutrophil recruitment, decreased IL-1β expression, and increased fungal load. Note: * indicates P < 0.05, ** indicates P < 0.01 in all figures.

[0046] Based on molecular biology detection techniques in this field, those skilled in the art can detect CLEC-1 mRNA or protein through polymerase chain reaction or antigen-antibody immunoreaction. For example, CLEC-1 protein can be detected using an antibody against CLEC-1 protein.

[0047] The amino acid sequence of the CLEC-1 protein is shown in SEQ ID NO.1 below:

[0048] MQAKYSSTRDMLDDDGDTTMSLHSQGSATTRHPEPRRTEHRAPSSTWRPVALTLLTLCLVLLIGLAALGLLFFQYYQLSNTGQDTISQMEERLGNTSQELQSLQVQNIKLAGSLQHVAEKLCRELYNKAGGYTRNMVPASASSESLRQLPHMGE SAAAHRCSPCTEQWKWHGDNCYQFYKDSKSWEDCKYFCLSENSTMLKINKQEDLEFAASQSYSEFFYSYWTGLLRPDSGKAWLWMDGTPFTSELFHIIIDVTSPRSRDCVAILNGMIFSKDCKELKRCVCERRAGMVKPESLHVPPETLGEGD.

[0049] Western blot assay: Mouse corneas were collected and lysed in protein lysis buffer (containing 200 μL RIPA lysis buffer + 2 μL PMSF + 2 μL phosphatase inhibitor) for 2 hours, with shaking every 15 minutes during lysis. The cell lysis buffer was centrifuged at 12,000 rpm for 5 minutes at 4°C to obtain the supernatant. Protein concentration was measured using a protein assay kit (BCA, Solarbio). Protein samples were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF, Millipore) membranes. The PVDF membranes were blocked with protein blocking buffer (Beyotime) at room temperature for 2-3 hours, then soaked in primary antibody dilution buffer and incubated overnight at 4°C. After removing the membranes, they were washed three times with PBST (1 mL Tween-20 in 1 L PBS) and incubated with secondary antibody dilution buffer for 1-2 hours. The primary antibody used in this study was anti-CLEC-1 antibody (1:1000, NBP2-27096; Novus Biologicals). The corresponding anti-rabbit antibody was used as the secondary antibody. After the reaction, the bands were developed using a developing agent (ECL reagent, Thermo Fisher Scientific).

[0050] PCR detection method: Tissues were isolated using RNAiso plus reagent (TaKaRa), and the RNA content and purity in the tissues were rapidly measured using a microplate reader (Eppendorf). Reverse transcription was performed according to the recommended procedure of the PrimeScript RT Reagent Kit (TaKaRa). Quantitative RT-PCR was performed using a 20 μL reaction system (2 μL cDNA diluted 1:12.5 with DEPC). The amplification conditions were as follows: 95 cycles of 95℃ for 30 seconds, 5℃ for 40 seconds, and then 60℃ for 30 seconds. β-actin was used as an internal control. The primers used in the PCR reaction system are shown in Table 1 below.

[0051] Table 1 Primer sequence list

[0052]

[0053] Example 1: Increased expression of CLEC-1 in the cornea of ​​patients with Aspergillus fumigatus keratitis

[0054] 1. Experimental Materials

[0055] 1.1 Collection of human corneal specimens

[0056] Corneal samples were collected from 6 patients with Aspergillus fumigatus keratitis and 6 healthy patients. Specimens were confirmed by fungal culture and morphology. The purpose and methods of the study were explained to the patients in detail, and samples were collected after obtaining informed consent. Patients with any form of immunosuppression or topical steroid treatment, or acute or chronic systemic diseases, were excluded.

[0057] 1.2 Experimental Fungi

[0058] Aspergillus fumigatus strain 3.0772 (China General Microbiological Culture Collection Center)

[0059] 2. Experimental Methods

[0060] Corneas from 6 patients with Aspergillus fumigatus keratitis and 6 healthy patients were collected for immunofluorescence and qRT-PCR detection.

[0061] 3. Experimental Results

[0062] Figure 1 This image shows the results of CLEC-1 mRNA expression in normal cornea and cornea of ​​a person infected with Aspergillus fumigatus keratitis, detected by qRT-PCR. Figure 2 This image shows the results of CLEC-1 protein expression in normal cornea and cornea of ​​a person infected with Aspergillus fumigatus keratitis, detected by immunofluorescence staining.

[0063] Figure 1 , Figure 2 This indicates that, compared to healthy corneas, the expression of CLEC-1 mRNA and protein was significantly increased in the corneas of individuals infected with Aspergillus fumigatus keratitis. Furthermore, the expression of CLEC-1 mRNA was significantly elevated when the disease duration exceeded one month.

[0064] Example 2: CLEC-1 expression decreased during the acute inflammatory phase of Aspergillus fumigatus keratitis and increased during the recovery phase. 1. Experimental materials

[0065] 1.1 Laboratory Animals

[0066] C57BL / 6 mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.)

[0067] 1.2 Experimental Fungi

[0068] Aspergillus fumigatus strain 3.0772 (China General Microbiological Culture Collection Center)

[0069] 2. Experimental Methods

[0070] 2.1 Fungal Preparation

[0071] Inoculate Aspergillus fumigatus strains onto Sabouraud dextrose agar and incubate at 37°C for 2-3 days. Scrape off hyphae and conidia and place them in 5 ml of sterile PBS to form a mixture. Filter the mixture through sterile gauze to remove hyphae. Centrifuge at 4500 rpm for 10 minutes at 4°C, discard the supernatant, and resuspend the precipitate in 5 ml of bacterial PBS to form a conidia suspension. Adjust the conidia concentration to 5 × 10⁻⁶ using sterile PBS. 6 cfu / ml.

[0072] 2.2 Model Mice

[0073] Eight-week-old healthy, clean-grade female C57BL / 6 mice were selected as research subjects. Before the experiment, slit-lamp examination was used to rule out eye diseases, and the right eye was selected as the experimental eye. All procedures performed on the experimental mice complied with the Chinese Ministry of Science and Technology's Guidelines on Humanized Treatment of Laboratory Animals (vGKFCZ-2006–398) and the principles and standards of the Association for Research in Vision and Ophthalmology (ARVO) regarding the use of animals in ophthalmological and vision research.

[0074] 2.3 Expression of CLEC-1 in mouse cornea

[0075] 2.3.1 Changes in CLEC-1 expression during the natural course of Aspergillus fumigatus keratitis in mice

[0076] C57BL / 6 mice were randomly divided into a control group and a group with Aspergillus fumigatus keratitis. After anesthetizing the mice with an intraperitoneal injection of 8% chloral hydrate, 2.5 μl of a 2.5 × 10⁻⁶ chloral solution was injected using a microsyringe. 6 A CFU / ml conidial suspension was injected into the corneal stroma of mice with Aspergillus fumigatus keratitis. After modeling, the mouse corneas were observed and photographed daily using a slit-lamp microscope. Corneal tissue was harvested at 1 / 2, 1, 2, 3, 5, 7, 10, and 14 days post-infection. CLEC-1 expression was detected by Western blot and qRT-PCR. Eyeballs were harvested on day 5 for immunofluorescence staining. 2.3.2 Changes in CLEC-1 expression in mice with Aspergillus fumigatus keratitis during treatment intervention.

[0077] C57BL / 6 mice were randomly divided into two groups, both of which developed a model of Aspergillus fumigatus keratitis. One group received no treatment after corneal infection with Aspergillus fumigatus, while the other group received natamycin treatment after corneal infection. After modeling, the mouse corneas were observed and photographed daily using a slit-lamp microscope, and clinical scores were recorded. On days 3 and 5, corneal tissue was harvested for qRT-PCR and Western blot analysis to detect CLEC-1 expression.

[0078] 3. Experimental Results

[0079] 3.1 Changes in CLEC-1 expression during the natural course of Aspergillus fumigatus keratitis in mice

[0080] Figure 3 These are slit-lamp microscope images taken 1 / 2, 1, 2, 3, 5, 7, 10, and 14 days after the establishment of the mouse model of Aspergillus fumigatus keratitis. Figure 4 This is a graph showing the results of CLEC-1 mRNA expression in the cornea of ​​mice detected by qRT-PCR at 1 / 2, 1, 2, 3, 5, 7, 10, and 14 days after the establishment of the mouse model of Aspergillus fumigatus keratitis. Figure 5 This is a graph showing the results of Western blot analysis of CLEC-1 protein expression in the cornea of ​​mice at 1 / 2, 1, 2, 3, 5, 7, 10, and 14 days after the establishment of the mouse model of Aspergillus fumigatus keratitis. Figure 6 This is a graph showing the results of CLEC-1 protein expression in the cornea of ​​mice five days after the establishment of the Aspergillus fumigatus keratitis mouse model, detected by immunofluorescence staining.

[0081] like Figures 3-6 As shown, C57BL / 6 mice exhibited significant corneal opacity one day after infection with Aspergillus fumigatus keratitis, which persisted until day 14 post-infection. With increased neovascularization, the keratitis gradually improved. Compared to the control group, the corneal CLEC-1 protein level in the infected mice decreased at 12 hours post-infection and persisted until day 3. From day 5 onwards, CLEC-1 expression significantly increased and remained elevated until day 14 post-infection. CLEC-1 expression was decreased during the acute inflammatory phase of Aspergillus fumigatus keratitis in mice and increased during the recovery phase.

[0082] 3.2 Changes in CLEC-1 expression during treatment intervention in mice with Aspergillus fumigatus keratitis

[0083] Figure 7 These are slit-lamp micrographs taken on days 1, 3, and 5 in the mouse group infected with Aspergillus fumigatus and the group treated with Aspergillus fumigatus combined with natamycin. Figure 8 The corneal clinical scores at 3 and 5 days were compared between the mouse group infected with Aspergillus fumigatus and the group infected with Aspergillus fumigatus combined with natamycin treatment. Figure 9 This is a graph showing the results of qRT-PCR detection of CLEC-1 mRNA expression in mice infected with Aspergillus fumigatus and treated with Aspergillus fumigatus combined with natamycin. Figure 10 This is a graph showing the results of Western blot detection of CLEC-1 protein expression in mice infected with Aspergillus fumigatus and treated with Aspergillus fumigatus combined with natamycin.

[0084] like Figures 7-10 As shown, natamycin treatment of mice with Aspergillus fumigatus keratitis resulted in a decrease in clinical scores and upregulation of CLEC-1 expression.

[0085] Example 3: CLEC-1 overexpression reduces inflammatory response

[0086] 1. Experimental Materials

[0087] 1.1 Experimental Supplies

[0088] CLEC-1 adenovirus (AAV) vector (Genechem)

[0089] CLEC-1 lentiviral vector (Genechem)

[0090] 1.2 Experimental Cells

[0091] THP-1 macrophages (Wuhan, China)

[0092] 1.3 Experimental Fungi

[0093] Aspergillus fumigatus strain 3.0772 (China General Microbiological Culture Collection Center)

[0094] 2. Experimental Methods

[0095] 2.1 In vivo experiments

[0096] One eye from each mouse was randomly selected and injected subconjunctivally with 10¹¹ vg of AAV vector (5 μl). Two weeks later, enhanced green fluorescent protein (EGFP) was detected using fluorescence stereomicroscopy to determine the uniform distribution of the virus in the corneal stroma. Overexpression of CLEC-1 was detected by qRT-PCR and Western blot. Corneal tissue was harvested one day post-infection for MPO detection, immunofluorescence staining, Western blot, and qRT-PCR.

[0097] 2.2 In vitro experiments

[0098] Human corneal epithelial cells were cultured at 37°C in a 5% CO2 incubator until the cell density reached 80%. The CLEC-1 lentiviral vector was added to THP-1 macrophages for pretreatment for 24 hours. THP-1 macrophages were then infected with Aspergillus fumigatus conidia at a fold increase in infection (MOI) of 1. After 24 hours of treatment, THP-1 macrophages were collected for Western blot and qRT-PCR detection.

[0099] 3. Experimental Results

[0100] 3.1 In vivo experiments

[0101] Figure 11 This is a photograph of EGFP expression in mouse cornea taken using a fluorescence microscope; Figure 12 This is a graph showing the results of detecting CLEC-1 mRNA overexpression using qRT-PCR. Figure 13This is a graph showing the results of Western blot detection of CLEC-1 protein overexpression; Figure 14 These are corneal photographs of control mice and CLEC-1 overexpressing mice one day after infection with Aspergillus fumigatus keratitis. Figure 15 The clinical corneal scores of control mice and CLEC-1 overexpression mice one day after infection with Aspergillus fumigatus keratitis; Figure 16 The image shows the corneal CFU results of mice in the control group and the CLEC-1 overexpression group one day after infection with Aspergillus fumigatus keratitis. Figure 17 These are average bioluminescence images of the corneas of control mice and CLEC-1 overexpression mice one day after infection with Aspergillus fumigatus keratitis. Figure 18 The image shows the corneal MPO results of control mice and CLEC-1 overexpression mice one day after infection with Aspergillus fumigatus keratitis. Figure 19 This image shows the distribution of neutrophils in the cornea of ​​control mice and CLEC-1 overexpression mice one day after infection with Aspergillus fumigatus keratitis, detected by immunofluorescence assay. Figure 20 The image shows the results of detecting CLEC-1 mRNA expression in the cornea of ​​normal mice, mice infected with Aspergillus fumigatus keratitis, CLEC-1 overexpressing mice, and mice with CLEC-1 overexpression combined with Aspergillus fumigatus keratitis using qRT-PCR. Figure 21 This figure shows the results of Western blot analysis of CLEC-1 protein expression in the corneas of normal mice, mice infected with Aspergillus fumigatus keratitis, CLEC-1 overexpressing mice, and mice with CLEC-1 overexpression combined with Aspergillus fumigatus keratitis.

[0102] like Figures 11-13 As shown, overexpression of CLEC-1 is effective. Figures 14-21 As shown, upregulation of CLEC-1 expression led to decreased levels of MPO and CFU in the cornea, reduced neutrophil recruitment, decreased IL-1β expression, increased fungal load, and reduced inflammatory response.

[0103] 3.2 In vitro experiments

[0104] Figure 22 This is a graph showing the results of qRT-PCR detection of CLEC-1 mRNA expression in normal THP-1 macrophages, THP-1 macrophages treated with Aspergillus fumigatus, THP-1 macrophages overexpressing CLEC-1 and THP-1 macrophages overexpressing CLEC-1 and treated with Aspergillus fumigatus. Figure 23 This figure shows the results of Western blot analysis of CLEC-1 protein expression in normal THP-1 macrophages, THP-1 macrophages treated with Aspergillus fumigatus, THP-1 macrophages overexpressing CLEC-1, and THP-1 macrophages overexpressing CLEC-1 and treated with Aspergillus fumigatus.

[0105] like Figure 22 , 23 As shown, in THP-1 macrophages, the expression of IL-1β mRNA and protein was downregulated after treatment with Aspergillus fumigatus.

[0106] Example 4: CLEC-1 for evaluating the timing of glucocorticoid administration in fungal keratitis. 1. Experimental materials

[0107] 1.1 Laboratory Animals

[0108] C57BL / 6 mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.)

[0109] 1.2 Experimental Fungi

[0110] Aspergillus fumigatus strain 3.0772 (China General Microbiological Culture Collection Center)

[0111] 2. Experimental Methods

[0112] To further confirm the effectiveness of CLEC-1 in evaluating the timing of glucocorticoid administration in mice with fungal keratitis, this invention plotted corneal perforation rate and clinical scoring. The specific steps are as follows: Forty mice with fungal keratitis were established using the same method. Twenty mice were given hormones before CLEC-1 elevation (10 mice were given medication on day 2, and 10 mice on day 3), and 20 mice were given hormones after CLEC-1 elevation (10 mice were given medication on day 5, and 10 mice on day 7). Two days after hormone administration, corneal perforation rate data were analyzed.

[0113] Twenty mice with fungal keratitis were established using the same method. Ten mice were in the non-hormone group, and ten mice were in the hormone group after CLEC-1 elevation (5 days). Two days after hormone administration, corneal clinical score data were analyzed.

[0114] like Figure 24 As shown, the corneal perforation rate decreased significantly after the use of glucocorticoids following an increase in CLEC-1 levels.

[0115] like Figure 25 As shown, the use of glucocorticoids after CLEC-1 elevation significantly reduced the corneal clinical score.

[0116] Example 5 Reagent Kit

[0117] Embodiments of the present invention also provide a kit for assessing the timing of glucocorticoid administration during the treatment of aspergillosis keratitis. Specifically, the kit includes reagents for detecting CLEC-1 levels in the cornea.

[0118] Specifically, this kit detects CLEC-1 levels based on quantitative polymerase chain reaction (PCR) or Western blotting. The method of use is briefly described below: Corneal epithelium is scraped from patients with fungal keratitis, and the expression level of CLEC-1 receptor mRNA or protein in the corneal epithelium is detected using PCR or Western blotting. The results are analyzed using a two-sample t-test. When the expression level of CLEC-1 shows a significant increase (P < 0.05), it is the appropriate time to apply glucocorticoid drugs (see the preceding section of this application). Figure 4 or Figure 5 ).

[0119] Originating from different individuals and species, CLEC-1 proteins exhibit certain sequence differences, but they are essentially homologous proteins and can all serve as markers for the use of glucocorticoids in fungal keratitis. Therefore, the CLEC-1 of this invention can be the protein shown in SEQ ID NO.1, or a protein with at least 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO.1. By detecting CLEC-1 homologous proteins, the timing of glucocorticoid administration in fungal keratitis can also be determined.

[0120] Given that the sequence of the CLEC-1 protein to be detected is known, those skilled in the art can easily obtain antibodies against the CLEC-1 protein, and any antibody against the CLEC-1 protein can be used to detect the CLEC-1 protein. Therefore, regardless of the structure or sequence of the antibody against the CLEC-1 protein, as long as it is used to detect the CLEC-1 protein and prepared into a kit that guides the application of corticosteroids in fungal keratitis, it falls within the scope of protection of this invention.

[0121] Techniques for detecting CLEC-1 protein through antigen-antibody immunization include, but are not limited to, immunoblotting. Other similar methods for detecting CLEC-1 protein are also within the scope of protection of this invention.

[0122] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention. SEQUENCE LISTING <110> Affiliated Hospital of Qingdao University <120> Application of C-type lectin-like receptor-1 as a therapeutic biomarker for fungal keratitis <130> 220239CN-CH-I <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 307 <212> PRT <213> Homo sapiens <400> 1 Met Gln Ala Lys Tyr Ser Ser Thr Arg Asp Met Leu Asp Asp Asp Gly 1 5 10 15 Asp Thr Thr Met Ser Leu His Ser Gln Gly Ser Ala Thr Thr Arg His 20 25 30 Pro Glu Pro Arg Arg Thr Glu His Arg Ala Pro Ser Ser Thr Trp Arg 35 40 45 Pro Val Ala Leu Thr Leu Leu Thr Leu Cys Leu Val Leu Leu Ile Gly 50 55 60 Leu Ala Ala Leu Gly Leu Leu Phe Phe Gln Tyr Tyr Gln Leu Ser Asn 65 70 75 80 Thr Gly Gln Asp Thr Ile Ser Gln Met Glu Glu Arg Leu Gly Asn Thr 85 90 95 Ser Gln Glu Leu Gln Ser Leu Gln Val Gln Asn Ile Lys Leu Ala Gly 100 105 110 Ser Leu Gln His Val Ala Glu Lys Leu Cys Arg Glu Leu Tyr Asn Lys 115 120 125 Ala Gly Gly Tyr Thr Arg Asn Met Val Pro Ala Ser Ala Ser Ser Glu 130 135 140 Ser Leu Arg Gln Leu Pro His Met Gly Glu Ser Ala Ala Ala His Arg 145 150 155 160 Cys Ser Pro Cys Thr Glu Gln Trp Lys Trp His Gly Asp Asn Cys Tyr 165 170 175 Gln Phe Tyr Lys Asp Ser Lys Ser Trp Glu Asp Cys Lys Tyr Phe Cys 180 185 190 Leu Ser Glu Asn Ser Thr Met Leu Lys Ile Asn Lys Gln Glu Asp Leu 195 200 205 Glu Phe Ala Ala Ser Gln Ser Tyr Ser Glu Phe Phe Tyr Ser Tyr Trp 210 215 220 Thr Gly Leu Leu Arg Pro Asp Ser Gly Lys Ala Trp Leu Trp Met Asp 225 230 235 240 Gly Thr Pro Phe Thr Ser Glu Leu Phe His Ile Ile Ile Asp Val Thr 245 250 255 Ser Pro Arg Ser Arg Asp Cys Val Ala Ile Leu Asn Gly Met Ile Phe 260 265 270 Ser Lys Asp Cys Lys Glu Leu Lys Arg Cys Val Cys Glu Arg Arg Ala 275 280 285 Gly Met Val Lys Pro Glu Ser Leu His Val Pro Pro Glu Thr Leu Gly 290 295 300 Glu Gly Asp 305 <210> 2 <211> 18 <212> DNA <213> Artificial sequence <400> 2 cgttgtgtct gtgagaga 18 <210> 3 <211> 18 <212> DNA <213> Artificial sequence <400> 3 ccttcgccta atgtttca 18 <210> 4 <211> twenty one <212> DNA <213> Artificial sequence <400> 4 tgtgaatact tctgcctggc t 21 <210> 5 <211> twenty three <212> DNA <213> Artificial sequence <400> 5 gagagccctg tccaataaga gta 23

Claims

1. The use of a reagent for detecting C-type lectin-like receptor-1 in the preparation of a product for assessing the timing of glucocorticoid administration in the treatment of aspergillosis keratitis, wherein increased expression of C-type lectin-like receptor-1 in vivo indicates the timing of glucocorticoid administration.

2. A kit for assessing the timing of glucocorticoid administration during the treatment of aspergillosis keratitis, characterized in that, It contains reagents for detecting the level of C-type lectin-like receptor-1 in the cornea.

3. The reagent kit according to claim 2, characterized in that, The reagent is used to detect the level of C-type lectin-like receptor-1 based on quantitative polymerase chain reaction or immunoblotting technology.

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