Biomarkers for diagnosis of herpes simplex virus keratitis and use thereof

Abstract

This invention belongs to the field of molecular diagnostics, specifically relating to biomarkers for the diagnosis of herpes simplex keratitis (HSK) and their applications. This invention is the first to demonstrate that C1QA and FCERT1G are specific and reliable inflammation-related biomarkers for HSK. Diagnostic and assessment methods based on these biomarkers can provide crucial molecular evidence for the early detection, accurate diagnosis, objective severity grading, and personalized treatment of HSK. Simultaneously, this invention reveals the potential roles of FCERT1G and C1QA in HSK and provides new ideas for targeted therapy of this disease, particularly the application of goserelin as an inflammation modulator, offering a new direction for HSK treatment.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to biomarkers for the diagnosis of herpes simplex keratitis and their applications. Background Technology

[0002] Herpes simplex keratitis (HSK) is primarily caused by infection with herpes simplex virus type I (HSV-1). After initial infection, the virus remains latent in the trigeminal ganglion and may recur when the immune system weakens, leading to progressive worsening of corneal opacity, scarring, and nerve damage. This structural damage eventually results in irreversible vision loss, with patients often experiencing persistent blurred vision, glare, and photophobia, severely impairing their quality of life and work ability.

[0003] Currently, the clinical diagnosis of HSK primarily relies on slit-lamp examination and viral PCR testing. Slit-lamp examination can observe clinical manifestations of the cornea, such as ulceration, edema, and neovascularization; however, this method is highly subjective and struggles to accurately quantify the degree of inflammation. While viral PCR testing can confirm viral infection, it cannot reflect the immune inflammatory status and severity of the disease. More importantly, these traditional methods have the following limitations: first, they cannot objectively quantify the degree of local corneal immune inflammation; second, they are difficult to accurately assess the severity and activity of the disease; third, they lack molecular indicators for predicting disease progression and recurrence risk; and finally, they cannot provide precise guidance for individualized treatment.

[0004] Therefore, there is an urgent need in this field to discover a set of biomarkers that are highly specific and closely related to the immune inflammatory state of HSK, and to develop novel molecular diagnostic methods that can achieve early diagnosis, objective grading of severity, and monitoring of inflammatory activity, so as to fill the gaps in existing clinical diagnostic technologies and promote the development of HSK diagnosis and treatment towards precision medicine. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide biomarkers for the diagnosis of herpes simplex keratitis and their applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the invention provides the use of substances for detecting the FCERT1G gene and / or the C1QA gene in the preparation of diagnostic or auxiliary diagnostic products for herpes simplex keratitis.

[0008] A second aspect of the present invention provides the use of reagents targeting the FCERT1G gene and / or the C1QA gene in the preparation of drugs for the prevention or treatment of herpes simplex keratitis.

[0009] A third aspect of the present invention provides a method for designing or screening medicaments for the prevention or treatment of herpes simplex keratitis, the method comprising: selecting from a pool of compounds aspirated to be screened one or more of the following actions as medicaments for the prevention or treatment of herpes simplex keratitis: (1) inhibiting the expression of the FCERT1G gene and / or FCERT1G protein; (2) inhibiting the expression of the C1QA gene and / or C1QA protein; (3) inhibiting the activity of the FCERT1G protein; and (4) inhibiting the activity of the C1QA protein.

[0010] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention is the first to demonstrate that C1QA and FCERT1G are specific and reliable inflammation-related biomarkers for HSK. Diagnostic and assessment methods based on these biomarkers can provide crucial molecular evidence for the early detection, accurate diagnosis, objective severity grading, and personalized treatment of HSK. Simultaneously, this invention reveals the potential roles of FCERT1G and C1QA in HSK and provides new ideas for targeted therapy of this disease, particularly the application of goserelin as an inflammation modulator, offering a new direction for HSK treatment. Attached Figure Description

[0011] Figure 1 The results of differentially expressed genes and their pathway enrichment analysis between HSK and normal corneal tissues are shown. Among them, A and B are volcano plots and heatmaps of DEGs, showing significant transcriptional differences between the normal group and the HSK group; C is the Venn plot of differentially expressed immune-related genes (DEIRGs) obtained by taking the intersection of DEGs and IRGs; D and E are the GO enrichment results of DEIRGs, showing that they are mainly involved in biological processes such as stress response, inflammatory response and stimulation response regulation; F and G are KEGG pathway enrichment analysis, showing that DEIRGs are mainly enriched in immune-related pathways such as leishmaniasis, hepatitis C, and TNF signaling pathway.

[0012] Figure 2To reveal the role of immune cells in HSK through immune infiltration analysis and weighted gene co-expression network analysis (WGCNA), the following data are presented: A shows the overall characteristics of immune cell infiltration based on CIBERSORT; B compares the relative abundance of immune cells between the HSK group and the normal control group, showing a significant increase in memory B cells in the HSK group; C shows the relationship between the scale-free network fitting index and the soft threshold, determining β=14 as the optimal soft threshold; D is a dendrogram of gene co-expression modules constructed by WGCNA, with different colors representing different modules; E is a Pearson correlation heatmap between co-expression modules and immune cell infiltration, showing that black modules are significantly correlated with memory B cells and M2 macrophages, while light yellow modules are significantly correlated with CD8 T cells; F shows the significant positive correlation between gene significance (GS) and module membership (MM) in black and light yellow modules; G shows the 30 core genes obtained from the intersection of DEIRGs and hub genes.

[0013] Figure 3 To further screen and identify the functions of pivot genes, A shows a heatmap of expression correlation analysis among 30 pivot genes; B shows the protein interaction network constructed by DEIRGs; C shows the optimal lambda value (λ=0.14) determined by LASSO regression analysis and the corresponding gene screening results, identifying 5 candidate genes: FCER1G, C1QA, APOE, PTPN6, and SLC11A1; D shows the three core pivot genes screened by LASSO-Cox regression and PPI analysis: FCER1G, C1QA, and APOE; E shows the comparison of core gene expression levels between the HSK group and the normal control group, with significantly increased expression in the HSK group; F shows the ROC curves of core genes in the internal dataset, indicating that FCER1G, C1QA, and APOE have good diagnostic efficacy.

[0014] Figure 4 To identify the potential functions of diagnostic genes using sg-GSEA, pathway A was enriched in the APOE high-expression group, including angiogenesis, apical membrane surface, and inflammatory response; pathway B was enriched in the C1QA high-expression group, mainly involving complement activation, IL6 / JAK / STAT3 signaling, and interferon response; and pathway C was enriched in the FCERT1G high-expression group, involving allogeneic transplantation, Hedgehog signaling pathway, and inflammatory response. Enrichment analysis was performed on sg-GSEA based on samples divided into high-expression (≥50%) and low-expression (<50%) groups according to gene expression levels.

[0015] Figure 5Immunohistochemical expression characteristics of FCERT1G, C1QA, and APOE in HSK patient tissues are shown. In the figure, A represents the corneal tissue morphology of healthy controls and HSK patients as shown by HE staining. B to D represent the IHC staining results and mean optical density (AOD) analysis. The results showed that FCERT1G and C1QA were significantly highly expressed in HSK tissues (p<0.001), while APOE expression showed no significant difference. This suggests that FCERT1G and C1QA may play an important role in the pathogenesis of HSK.

[0016] Figure 6 Immunofluorescence expression characteristics of FCER1G, C1QA, and APOE in HSK tissues are shown. In A, there is no significant difference in APOE expression between the HSK group and the normal control group. In B and C, C1QA and FCER1G are significantly upregulated in HSK corneal tissues. The results suggest that C1QA and FCER1G may serve as potential diagnostic biomarkers for HSK.

[0017] Figure 7 The results validated the diagnostic efficacy of FCERT1G, C1QA, and APOE in normal individuals and HSK patients. In this study, A represents the ROC curve plotted based on the relative expression of APOE in normal individuals and HSK patients; B represents the ROC curve plotted based on the relative expression of C1QA in normal individuals and HSK patients; and C represents the ROC curve plotted based on the relative expression of FCERT1G in normal individuals and HSK patients.

[0018] Figure 8 The results of screening potential drugs to inhibit the development of inflammation in HSK are shown. In this diagram, A represents small molecule compounds related to inflammation in HSK, which were screened using the CMap database; B to F represent the molecular structures of the compounds.

[0019] Figure 9 The results show the molecular docking of Gosorelin with FCERT1G and C1QA proteins. In A, the docking score of Gosorelin with FCERT1G is -6.8 kcal / mol, showing strong binding affinity. In B, the docking score of Gosorelin with C1QA is -7.0 kcal / mol, indicating that the compound has a high binding affinity to the target and may have potential for treating herpes simplex keratitis. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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 pertains.

[0021] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0022] As mentioned above, there is currently no research on molecular markers for the diagnosis and treatment of herpes simplex keratitis in the prior art. Therefore, this invention provides biomarkers for the diagnosis of herpes simplex keratitis and their applications.

[0023] In one typical embodiment of the present invention, the use of substances for detecting the FCERT1G gene and / or the C1QA gene in the preparation of diagnostic or auxiliary diagnostic products for herpes simplex keratitis is provided.

[0024] This invention, based on the HSK dataset GSE241715, integrates and analyzes IRGs, and uses various bioinformatics methods, including differential expression analysis, functional enrichment analysis, immune infiltration assessment, WGCNA co-expression network construction, PPI protein interaction analysis, and LASSO regression, to screen for diagnostic marker genes of HSK closely related to inflammatory responses. The analysis results show that FCER1G (Ensembl: ENSG00000158869), C1QA (Ensembl: ENSG00000173372), and APOE (Ensembl: ENSG00000130203) are significantly upregulated in HSK, potentially promoting inflammatory responses by activating the immune cascade. However, clinical sample validation results show that only FCER1G and C1QA are significantly highly expressed in HSK lesion tissues, indicating that these two may be key molecules related to inflammation in HSK.

[0025] Further research revealed that FCER1G and C1QA were highly expressed in HSK lesions. Receiver curves confirmed their high sensitivity and specificity, suggesting they could serve as biomarkers for the diagnosis or auxiliary diagnosis of HSK.

[0026] In some embodiments of the present invention, the substances used to detect the FCERT1G gene and / or C1QA gene include reagents for detecting the expression levels of the FCERT1G gene and / or C1QA gene by real-time quantitative PCR, in situ hybridization, and microarray technology.

[0027] In some embodiments of the present invention, the primer sequences for detecting the expression level of the FCERT1G gene by real-time quantitative PCR are shown in SEQ ID NO: 1-2; and the primer sequences for detecting the expression level of the C1QA gene by real-time quantitative PCR are shown in SEQ ID NO: 3-4.

[0028] In some embodiments of the present invention, the product includes a chip and a reagent kit.

[0029] In some embodiments of the present invention, the reagents for detecting the expression levels of the FCERT1G gene and / or C1QA gene in the chip include probes that specifically recognize the FCERT1G gene and / or C1QA gene.

[0030] In some embodiments of the present invention, the kit contains reagents for quantitatively analyzing the expression levels of the FCERT1G gene and / or the C1QA gene; preferably, the reagents are obtained by means of PCR, immunohistochemistry, or immunofluorescence.

[0031] In some embodiments of the present invention, the diagnosis or auxiliary diagnosis of herpes simplex keratitis includes the following steps: Collect samples from the subjects to be tested and control samples; The expression levels of the FCERT1G gene and / or C1QA gene in the test subject samples and control samples were detected and compared. If the expression levels of the FCERT1G gene and / or C1QA gene are elevated in the sample of the test subject, it indicates that the subject has HSK or is at high risk of HSK.

[0032] The control sample is derived from healthy individuals or healthy tissue from the test subject; the sample from the test subject can be a blood sample, body fluid, tissue, organ, or secretion. More specifically, the test sample includes corneal tissue from the test subject.

[0033] In another typical embodiment of the present invention, the use of reagents targeting the FCERT1G gene and / or the C1QA gene in the preparation of drugs for the prevention or treatment of herpes simplex keratitis is provided.

[0034] In some embodiments of the present invention, the reagents include RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNAs, and gene knockout substances targeting the FCERT1G and / or C1QA genes.

[0035] In some embodiments of the present invention, the reagent is a small molecule inhibitor targeting the FCERT1G gene and / or the C1QA gene. Preferably, the small molecule inhibitor is selected from at least one of BRD-K87226815, Cefadroxil, AMN-082, Famprofazone, or Gosorelin.

[0036] In some embodiments of the present invention, the reagent is a small molecule inhibitor of the FCERT1G gene and / or the C1QA gene, namely Gosorelin.

[0037] This invention validated the potential targeting effects of goserelin on FCE R1G and C1QA through molecular docking and immunohistochemical experiments. Goserelin is a synthetic GnRH agonist that regulates sex hormone levels in vivo by mimicking the action of gonadotropin-releasing hormone (GnRH). Its CAS number is 65807-02-5. Its chemical structure is as follows: .

[0038] Goserelin is a drug already approved for clinical treatment, but its application in ophthalmic diseases has not been fully explored. This invention, through molecular docking analysis, reveals that Goserelin is a potential targeted drug for HSK-related inflammation, exhibiting high binding affinity to FCER1G and C1QA, providing a new direction for further therapeutic research.

[0039] In another typical embodiment of the present invention, a product is provided, the active ingredient of which includes a substance for inhibiting the expression level of the FCERT1G gene and / or the C1QA gene, the product having any of the following functions: (1) inhibiting the expression of the FCERT1G gene and / or the FCERT1G protein; (2) inhibiting the expression of the C1QA gene and / or the C1QA protein; (3) inhibiting the activity of the FCERT1G protein; (4) inhibiting the activity of the C1QA protein.

[0040] The product may be a drug.

[0041] According to the present invention, the concept of "treatment" means any measure applicable to the treatment of herpes simplex keratitis-related diseases, or preventive treatment of such diseases or symptoms, or prevention of recurrence of such diseases, such as recurrence after the end of a treatment period or treatment of symptoms of an already occurring disease, or preemptive intervention to prevent, suppress or reduce the occurrence of such diseases or symptoms.

[0042] According to the present invention, the above-mentioned drug also includes at least one inactive pharmaceutical ingredient.

[0043] The inactive components of the drug can be pharmaceutically commonly used carriers, excipients, and diluents. Furthermore, according to conventional methods, it can be formulated into oral, topical, suppository, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays.

[0044] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0045] In another specific embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate and mineral oil, etc.

[0046] In another specific embodiment of the present invention, the drug of the present invention can be administered into the body by known means. For example, it can be delivered to the tissue of interest via intravenous systemic delivery or local injection. Administration can be selected via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via single or multiple doses. Those skilled in the art will understand that the actual dose to be administered in the present invention can vary considerably depending on a variety of factors, such as target cells, biological type or tissue, the general condition of the subject to be treated, route of administration, manner of administration, etc.

[0047] In another specific embodiment of the present invention, the drug can be administered to humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, etc.

[0048] In another typical embodiment of the present invention, a method for designing or screening drugs for the prevention or treatment of herpes simplex keratitis is provided, the method comprising: selecting from the compounds to be screened one or more of the following effects as drugs for the prevention or treatment of herpes simplex keratitis: (1) inhibiting the expression of the FCERT1G gene and / or FCERT1G protein; (2) inhibiting the expression of the C1QA gene and / or C1QA protein; (3) inhibiting the activity of the FCERT1G protein; (4) inhibiting the activity of the C1QA protein.

[0049] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments. Those skilled in the art can make various changes, modifications, and substitutions without departing from the spirit of the present invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described, unless otherwise specified, are all commercially available.

[0050] Example 1: Systematic screening and identification of inflammation-related biomarkers (1) Data Acquisition and Preprocessing: The dataset GSE241715 was publicly obtained from the Gene Expression Omnibus Database (GEO) of the National Center for Biotechnology Information (NCBI). This dataset contains raw data obtained from transcriptome sequencing of corneal tissues from 5 clinically diagnosed HSK patients, 7 healthy donors, and 5 keratoconus patients using the Illumina HiSeq platform. The healthy donors and keratoconus group served as normal controls. All sample collection and processing followed standard operating procedures to ensure data quality. The raw sequencing data were quality assessed using FastQC software, and low-quality sequences and adapter sequences were removed using Trimmomatic software. High-quality sequences were aligned to the human reference genome (GRCh38) using STAR software, and gene expression levels were calculated using the featureCounts tool and standardized using FPKM (Fragments Per Kilobase of transcript per Million mapped fragments) values ​​to ensure the accuracy and comparability of gene expression levels.

[0051] (2) Differentially expressed gene analysis: Differential expression analysis was performed using the "Limma" software package in the R language (v4.2.1) environment. First, the gene expression matrix was transformed by Voom to correct for heteroscedasticity caused by sequencing depth and ensure the validity of subsequent statistical analysis. The control group was set as HSK group versus normal control group, and the significance criterion was |log2 (fold change)|>2 and the false discovery rate (FDR) after Benjamini-Hochberg correction was <0.05. Through rigorous statistical analysis, a total of 1308 differentially expressed genes (DEGs) were identified, of which 633 genes were upregulated and 675 genes were downregulated. Among these differentially expressed genes, C1QA and FCER1G were upregulated most significantly in the HSK group and were located in the upper right of the volcano plot ( Figure 1 A and B) suggest that they are closely related to the pathological process of HSK. Taking the intersection of differentially expressed genes (DEGs) and tissue-associated genes (IRGs) yielded a total of 96 differentially expressed tissue-associated genes (DEORGs). Figure 1 C).

[0052] (3) Functional enrichment and pathway analysis: To elucidate the biological functions of differentially expressed genes, the "clusterProfiler" R package was used for gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. A p-value < 0.05 and a q-value < 0.2 were set as significant enrichment criteria. Subsequently, multi-level biological function enrichment analysis was performed on DEIRGs. GO functional annotation results showed that both upregulated and downregulated DEIRGs were mainly involved in biological processes such as stress response, inflammatory response, and regulation of stimulus responses. After REVIGO redundancy removal, certain differences were found between the two types of genes at the cellular component (CC) and molecular function (MF) levels. Figure 1 D, E). KEGG analysis further showed that these genes were significantly enriched in pathways such as leishmaniasis and hepatitis C TNF (D, E). Figure 1 F, G).

[0053] (4) Weighted gene co-expression network analysis: To further elucidate the relationship between immune response and HSK occurrence, we constructed an immune infiltration feature map based on the GSE241715 dataset. Figure 2 A). The relative abundance of immune cells in HSK and control tissues was estimated using the CIBERSORT algorithm. The results showed that the proportion of memory B cells in the HSK group was significantly higher than that in the normal control group. In addition, although the proportion of M2 macrophages was relatively low in both tissue types, it was more abundant in HSK tissues, while resting CD4 memory T cells were more common in the control group. Figure 2 B). In the WGCNA analysis, based on the scale-free network characteristics, the soft threshold power β = 14 was selected as the optimal parameter ( Figure 2 C). Ten co-expression modules were identified in the data containing 16 samples and 10,804 genes, represented by different colors (C). Figure 2 D). Pearson correlation analysis was then used to explore the relationship between each module and immune-infiltrating cells. The results showed that the black module had the strongest correlation with memory B cells (Cor = 0.82, P = 6 × 10⁻⁶). -5 The black module was strongly correlated with M2 macrophages (Cor = 0.79, P = 7 × 10⁻⁶). -4 The light yellow module showed a strong correlation with CD8 T cells (Cor = 0.71, P = 9 × 10⁻⁶). -3 () Figure 2 E). Significant positive correlations were observed between GS (gene significance) and MM (module membership) within both the black and light yellow modules. Figure 2F). Therefore, these three key modules related to immune infiltration were selected for further analysis. Based on the screening criteria (GS>0.20 and MM>0.80), a total of 552 hub genes were obtained for subsequent studies. The intersection of these genes with DEIRGs identified 30 core genes: ACP5, APOE, APOL3, C1QA, C3, CAMK1D, CCN4, CD14, CSF1R, CYBB, ETS1, FCER1G, FCGR3A, FN1, FYN, HCK, ITGB2, PTPN6, RASGRP1, SLC11A1, TLR2, TNFAIP3, TNFRSF1B, TYROBP ( Figure 2 G).

[0054] (5) Further screening and functional identification of hub genes: The expression correlation among 30 hub genes was analyzed. Figure 3 A), the results showed that there were strong positive correlations among most genes. Subsequently, a protein-protein interaction network of these DEIRGs was constructed ( Figure 3 B), we performed LASSO analysis on these 16 genes, with an ideal lambda value set at 0.14, and finally identified 5 genes: FCER1G, C1QA, APOE, PTPN6, and SLC11A1 ( Figure 3 C). Through LASSO-Cox regression and PPI analysis, we identified FCER1G, C1QA, and APOE as important hub genes. Figure 3 D). In the HSK group, the expression levels of FCERT1G, C1QA, and APOE genes were significantly higher than those in the normal control group. Figure 3 E). These three sites may play an important role in the pathogenesis of HSK, and their predictive performance was subsequently validated. To evaluate the diagnostic efficacy of these core genes, we performed ROC curve validation on an internal dataset. The validation results showed that the AUC values ​​of FCER1G, C1QA, and APOE were all 0.95, suggesting that these three pivotal genes may serve as diagnostic indicators for HSK. Figure 3 F).

[0055] (6) Functional analysis of pivotal genes: To elucidate the potential biological functions of FCER1G, C1QA, and APOE in HSK, samples were divided into a high-expression group (≥50%) and a low-expression group (<50%) based on gene expression levels, and sg-GSEA analysis was performed. The results showed that in the APOE high-expression group, pathways such as angiogenesis, apical membrane surface, and inflammatory response were significantly enriched ( Figure 4A). In the C1QA high-expression group, the significantly enriched pathways mainly involved gene sets such as the "classical complement activation pathway", "IL-6 / JAK / STAT3 signaling pathway", and "marker interferon-γ response". Figure 4 B). In the FCERT1G high-expression group, it was mainly enriched in gene sets such as "marker allogeneic rejection", "marker Hedgehog signaling pathway", and "inflammatory response". Figure 4 C). The above results confirm, from a functional perspective, the central roles of C1QA and FCERT1G in driving HSK-specific inflammatory responses, further supporting their biological rationale as diagnostic biomarkers.

[0056] Example 2: Clinical Histological Validation of Biomarkers (1) Sample collection and ethics: This study collected corneal tissue specimens obtained during surgery at the Department of Corneal Diseases, Shandong Eye Hospital, including corneal samples from 3 normal donors and diseased corneal samples from 3 HSK patients for immunofluorescence staining and histochemistry. This study strictly followed the ethical principles of the Declaration of Helsinki, was approved by the Medical Ethics Committee of Shandong Eye Hospital, and obtained written informed consent from all participants.

[0057] (2) Immunohistochemical staining: Paraffin-embedded tissue was cut into 4 μm thick sections, dewaxed with xylene, and hydrated with gradient ethanol. The sections were then subjected to heat retrieval of the antigen in citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked for 30 minutes with 3% hydrogen peroxide methanol solution. Subsequently, the sections were incubated overnight at 4°C in a humidified chamber with rabbit anti-human FCE R1G polyclonal antibody (Proteintech, 1:50), rabbit anti-human C1QA polyclonal antibody (Abclonal, 1:50), and rabbit anti-human APOE polyclonal antibody (Abclonal, 1:50). After washing with PBS, the sections were incubated at room temperature for 30 minutes with HRP-labeled goat anti-rabbit IgG secondary antibody (Servicebio, 1:300). DAB staining, hematoxylin counterstaining, gradient ethanol dehydration, xylene clearing, and neutral resin mounting were performed. PBS was used as a negative control instead of the primary antibody to ensure the reliability of the experimental results.

[0058] (3) Immunofluorescence staining: After antigen retrieval, non-specific binding sites were blocked with 1% bovine serum albumin (BSA) for 30 minutes. The sections were then incubated overnight at 4°C with the primary antibody. After washing with PBS, the sections were incubated for 1 hour at room temperature in the dark with CY3-labeled goat anti-rabbit IgG secondary antibody (Servicebio, 1:300). Cell nuclei were counterstained with DAPI (1 μg / mL) for 5 minutes, and the sections were mounted with anti-fluorescence quenching mounting medium. All sections were observed and images were acquired under the same parameters using a laser confocal microscope (Leica TCS SP8) to ensure comparability of experimental results.

[0059] All sections were observed and images acquired under a laser confocal microscope with the same parameters. Results showed that C1QA and FCE R1G were weakly expressed or almost not expressed in normal corneal tissue; however, strong positive staining signals of C1QA and FCE R1G were observed in the stroma of the cornea of ​​HSK patients, especially in areas of inflammatory cell infiltration, mainly located in the cytoplasm of infiltrating macrophages and neutrophils. The mean optical density (AOD) of APOE in HSK tissue was not significantly different from that in the control group. Figure 5 B), the mean optical density (AOD) of FCE R1G and C1QA in HSK tissues was significantly higher than that of the control group (p<0.001, Figures C and D), but the AOD of APOE in HSK tissues was not significantly different from that of the control group. Figure 5 A, B).

[0060] Immunofluorescence results ( Figure 6 Further investigation confirmed that the fluorescence signal intensity of C1QA (red) and FCERT1G (green) was significantly enhanced in HSK corneal tissue, and they co-localized with DAPI-labeled cell nuclei. Semi-quantitative analysis of fluorescence intensity using ImageJ software (v1.53) showed that the mean fluorescence intensity of C1QA and FCERT1G in the HSK group was 3.2 times and 2.8 times that of the normal control group, respectively, with a highly statistically significant difference (p<0.001).

[0061] In summary, FCERT1G and C1QA were significantly overexpressed in HSK corneal tissue, while APOE expression showed no significant change, suggesting that the former two may play a key role in the pathogenesis of HSK and have potential diagnostic value.

[0062] Example 3: Validation of the diagnostic performance of biomarkers (1) Sample collection and ethics: This study collected corneal tissue specimens obtained during surgery at the Department of Corneal Diseases, Shandong Eye Hospital, including corneal samples from 13 normal donors and diseased corneal samples from 13 HSK patients. This study strictly followed the ethical principles of the Declaration of Helsinki, was approved by the Medical Ethics Committee of Shandong Eye Hospital, and obtained written informed consent from all participants.

[0063] (2) Sample processing: Surgically obtained corneal tissue specimens were immediately placed under RNase-free conditions after ex vivo and gently rinsed with sterile PBS to remove residual blood and impurities. The tissue samples were then minced, rapidly frozen in liquid nitrogen, and thoroughly ground before homogenization with lysis buffer. Total RNA extraction was performed using a commercially available total RNA extraction kit from Vazyme, strictly following the kit's instructions. Genomic DNA removal steps were followed according to the kit's procedure to avoid DNA contamination. The concentration and purity of the extracted RNA were determined using a spectrophotometer. Samples with an A260 / A280 ratio between 1.8 and 2.0 were used for subsequent experiments. RNA integrity was initially assessed using agarose gel electrophoresis.

[0064] (3) Reverse transcription was performed using the Novizan reverse transcription kit.

[0065] (4) Real-time quantitative PCR (qPCR): qPCR was performed using a detection system based on SYBR Green fluorescent dye. The reaction was performed using the SYBR Green qPCR amplification kit from Vazyme, and the reaction system was prepared strictly according to the kit instructions. Each reaction system included SYBR Green qPCR Master Mix, forward primers, reverse primers, cDNA template, and RNase-free water. All samples were prepared in triplicate. The target genes and primer sequences amplified were all human-derived. Specifically, APOE (Apolipoprotein E) Forward sequence was 5′-GGGTCGCTTTTGGGATT-3′, Reverse sequence was 5′-CAACTCCTTCATGGTCTCGTCC-3′; C1QA (Complement C1q A chain) Forward sequence was 5′-CGAGCACCAGACGGGAAGAAAG-3′ (SEQ ID NO:3), Reverse sequence was 5′-AGGTTCCCCCTGGTCTCCTTTA-3′ (SEQ ID NO:4); FCER1G (Fc epsilon receptor IgE gamma chain) Forward sequence was 5′-AGCAGTGGTCTTGCTCTTACT-3′ (SEQ ID NO:1), Reverse sequence was 5′-TGCCTTTCGCACTTGGATCTT-3′ (SEQ ID NO:2); and the internal reference gene GAPDH Forward sequence was... The reverse side was 5′-GTCTCCTCTGACTTCAACAGCG-3′, and the reverse side was 5′-ACCACCCTGTTGCTGTAGCCAA-3′. The qPCR reaction conditions were 95 °C pre-denaturation, followed by 40 cycles of denaturation, annealing, and extension. Melting curve analysis was performed after amplification to verify the specificity of the amplified products. Relative gene expression levels were calculated using 2^(1 / 2)^(1 / 2)^(1 / 2). ΔCt or 2^ The ΔΔCt method was used for calculation, and the results were normalized using the internal reference gene GAPDH.

[0066] (5) Receiver Operating Characteristic (ROC) curve analysis: ROC curve analysis was used to evaluate the predictive performance of APOE, C1QA, and FCER1G in the diagnosis of HSK. The results showed that the area under the ROC curve (AUC) of APOE was 0.55, indicating limited diagnostic ability in this study sample; in contrast, the AUCs of C1QA and FCER1G reached 0.83 and 0.92, respectively, suggesting that they could completely distinguish between HSK-affected corneas and normal corneas in this sample, demonstrating extremely high diagnostic accuracy. ROC curve analysis was calculated using qPCR expression data, and the diagnostic value of the genes was quantified by the area under the curve (AUC). Figure 7 (As shown). Based on these results, this study will focus on C1QA and FCER1G, and, with consistent validation at both the transcriptomic and protein levels, will use them as potential HSK biomarkers to provide experimental evidence for future clinical diagnosis and disease mechanism research.

[0067] Example 4: Screening for potential drugs to inhibit the development and progression of inflammation in HSK Upregulated and downregulated genes related to HSK inflammation were input into the CMap database to identify small molecule compounds that may have therapeutic effects on HSK. Cross-analysis results showed that the five compounds with the highest negative correlation scores, including BRD-K87226815, Cefadroxil, AMN-082, Famprofazone, and Gosorelin, were considered to have potential therapeutic value. Figure 8 A). The molecular structures of these compounds were taken from the PubChem database ( Figure 8 BF).

[0068] Molecular docking results of Goserelin with FCERT1G and C1QA proteins: Molecular docking is a structure-based drug design and screening method that aims to identify potential drug candidates by predicting the optimal binding conformation between small molecule compounds and target molecules. In this example, the crystal structures of two molecular targets, FCERT1G (number: P30273) and C1QA (number: P02745), were downloaded from the AlphaFold protein structure database. Using AutoDock Tools 1.57 software, molecular docking analysis was performed on five compounds that may have potential for treating herpes simplex keratitis with the above targets. The results showed that the docking scores of Goserelin were -6.8 kcal / mol and -7 kcal / mol, indicating that these two compounds have strong binding affinity to the targets. Figure 9 A, B).

[0069] Goserelin is a synthetic GnRH agonist that regulates sex hormone levels by mimicking the action of gonadotropin-releasing hormone (GnRH). It is commonly used to treat diseases such as prostate cancer, breast cancer, endometriosis, and premature puberty. Goserelin initially causes a transient increase in sex hormones; over time, due to sustained GnRH stimulation, it inhibits gonadotropin secretion, leading to a decrease in sex hormone levels and thus slowing or controlling the progression of related diseases. In addition, Goserelin is also used in assisted in vitro fertilization (IVF). While Goserelin is a drug approved for clinical treatment, its application in ophthalmic diseases has not been fully explored. Through molecular docking analysis, we discovered that Goserelin is a potential target for HSK-related inflammation, exhibiting high binding affinity for FCER1G and C1QA, which provides a new direction for further therapeutic research.

[0070] In summary, this invention, through systematic research and multi-dimensional validation, is the first to demonstrate that C1QA and FCERT1G are specific and reliable inflammation-related biomarkers for HSK. Diagnostic and assessment methods based on these biomarkers can provide crucial molecular evidence for the early detection, accurate diagnosis, objective severity grading, and personalized treatment of HSK, and have broad clinical application prospects. Simultaneously, this invention reveals the potential roles of FCERT1G and C1QA in HSK and provides new ideas for targeted therapy of this disease, particularly the application of goserelin as an inflammation modulator, offering a new direction for HSK treatment.

[0071] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of substances used to detect the FCER1G gene and / or C1QA gene in the preparation of diagnostic or auxiliary diagnostic products for herpes simplex keratitis.

2. The application as described in claim 1, characterized in that, The substances include reagents for detecting the expression levels of the FCERT1G gene and / or C1QA gene using real-time quantitative PCR, in situ hybridization, and microarray technology.

3. The application as described in claim 2, characterized in that, The primer sequences for detecting the expression level of the FCERT1G gene by real-time quantitative PCR are shown in SEQ ID NO: 1-2; the primer sequences for detecting the expression level of the C1QA gene by real-time quantitative PCR are shown in SEQ ID NO: 3-4.

4. The application as described in claim 1, characterized in that, The products include chips and reagent kits.

5. The application as described in claim 4, characterized in that, The reagents for detecting the expression levels of the FCER1G gene and / or C1QA gene in the chip include probes that specifically recognize the FCER1G gene and / or C1QA gene; the kit contains reagents for quantitatively analyzing the expression levels of the FCER1G gene and / or C1QA gene; preferably, the reagents are obtained by PCR, immunohistochemistry or immunofluorescence.

6. The application as described in claim 1, characterized in that, The diagnosis or auxiliary diagnosis of herpes simplex keratitis includes the following steps: Collect samples from the subjects to be tested and control samples; The expression levels of the FCERT1G gene and / or C1QA gene in the test subject samples and control samples were detected and compared. If the expression levels of the FCERT1G gene and / or C1QA gene are elevated in the sample of the test subject, it indicates that the subject has HSK or is at high risk of HSK.

7. The application as described in claim 6, characterized in that, The sample from the subject to be tested is a blood sample, body fluid, tissue, organ, or secretion; preferably, the sample to be tested is corneal tissue.

8. Application of reagents targeting the FCER1G gene and / or C1QA gene in the preparation of drugs for the prevention or treatment of herpes simplex keratitis.

9. The application as described in claim 8, characterized in that, The reagents include RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, and gene knockout substances targeting the FCERT1G and / or C1QA genes.

10. A method for designing or screening drugs for the prevention or treatment of herpes simplex keratitis, characterized in that, The method includes selecting from the compounds to be screened one or more of the following effects as a drug for the prevention or treatment of herpes simplex keratitis: (1) inhibiting the expression of the FCERT1G gene and / or FCERT1G protein; (2) inhibiting the expression of the C1QA gene and / or C1QA protein; (3) inhibiting the activity of the FCERT1G protein; (4) inhibiting the activity of the C1QA protein.

Images