Application of type III interferon in diagnosis and treatment of optic neurodegenerative diseases

Abstract

The invention relates to application of type III interferon (IFN-lambda) in diagnosis and treatment of optic neurodegenerative diseases. According to the invention, it is clear that the type III interferon can be used as a biomarker of optic nerve degeneration, it is explained that inhibition of the type III interferon signal channel is helpful to significantly improve the RGC survival rate, increase the GCC thickness and protect optic nerve axons, and the type III interferon can be used as a key target for treatment of optic nerve degeneration. In general, the invention provides an experimental basis and a theoretical basis for the research and development and clinical application of the Type III interferon (IFN-lambda) as a molecular marker for optic neurodegenerative disease diagnosis and a Type III interferon inhibitor for treating optic neurodegenerative diseases, and solves the problem that model achievements of rodents such as mice and the like are difficult to transform. A new convertible way is opened up for optic nerve degenerative change, and the method has important clinical application prospects and social value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of type III interferon in diagnosing optic nerve degenerative diseases and type III interferon inhibitor in preparing medicine for treating optic nerve degenerative diseases. Background Art

[0002] Glaucomatous optic neurodegeneration is the leading cause of irreversible blindness worldwide and is characterized by progressive lesions of the optic nerve composed of retinal ganglion cells (RGCs) and loss of RGCs, with or without elevated intraocular pressure. Optic neurodegeneration occurs insidiously and there is a lack of effective screening methods, and many cases are often not diagnosed until visual impairment is discovered in the late stages of the disease. Currently, elevated intraocular pressure is the only controllable risk factor for glaucoma, but optic neurodegeneration often continues to progress despite efforts to control intraocular pressure. This highlights the urgent need to develop markers to predict optic neurodegeneration and neuroprotective therapies that directly target the molecular mechanisms of optic neuropathies.

[0003] Previous studies generally established rodent models of optic nerve degeneration in mice and rats, revealing the molecular mechanisms of excitotoxicity, nutritional deficiency, inflammation and oxidative stress in optic nerve degeneration, and explored optic nerve protection therapies, including supplementation of neurotrophic factors such as CNTF and BDNF, dietary supplements such as nicotinamide and vitamin E, anti-inflammatory treatment through anti-complement C1q antibodies and anti-FAS, and the use of memantine, phenytoin sodium and other drugs that regulate nerve excitability. However, the clinical conversion efficiency based solely on rodents is often poor, and there is currently no direct neuroprotective therapy on the market. Analyzing the reasons, the main limitation is that there are large conservative differences between rodents and humans, especially in the anatomical structure of the eye and optic nerve head (ONH). The ONH of rodents lacks collagen cribriform plate structure and blood supply is also different. In addition, the RGC types and molecular programs of rodents show significant differences compared with primates. The above factors limit the translatability of their results to humans. Therefore, exploring molecular markers and targets closely related to human optic nerve degeneration and conducting subsequent drug development and clinical treatment targeting key pathways have become key issues that need to be urgently addressed. Summary of the invention

[0004] The purpose of the present invention is to solve the problems existing in the treatment of optic nerve degeneration in the prior art. Through cross-species multi-model analysis from rodents, non-human primates to humans, it is clarified that type III interferon and its downstream pathways are abnormally induced in the pathological environment of ischemia, and are directly related to the progression of optic nerve degeneration and poor prognosis. At the same time, through type III interferon signal inhibitors, its therapeutic effect on optic nerve degeneration is clarified. The present invention provides experimental basis and theoretical basis for the development of type III interferon as a biomarker for optic nerve degeneration, and the development of drug research and development and clinical application of type III interferon inhibitors for the treatment of optic nerve degeneration such as glaucoma.

[0005] In order to solve the above technical problems, the present invention is implemented through the following technical solutions.

[0006] The first aspect of the present invention provides the use of a type III interferon inhibitor in the preparation of a medicament for preventing and / or treating optic nerve degenerative diseases.

[0007] Preferably, the optic nerve degeneration is selected from one or more of glaucoma and ischemic retinal disease; more preferably, the optic nerve degeneration is selected from glaucoma.

[0008] Preferably, the type III interferon inhibitor is selected from one or more of a type III interferon neutralizing antibody, a gRNA designed based on a type III interferon gene, and a gRNA designed based on a type III interferon receptor gene.

[0009] Preferably, the type III interferon inhibitor is selected from IFN-λ2 / 3 Antibody (R&D Systems, MAB17892).

[0010] Preferably, the type III interferon receptor gene is selected from Ifnlr1, and the gRNA sequence designed based on the type III interferon receptor gene is selected from SEQ ID NO: 1 (5'-CAACCAGGTAGGTGCTCCTT-3') and SEQ ID NO: 2 (5'-GGGGACGGAAATACCTGGTG-3').

[0011] The second aspect of the present invention provides the use of a reagent for detecting the expression level of type III interferon in the preparation of a product for auxiliary diagnosis of optic nerve degeneration.

[0012] Preferably, the reagent for detecting the expression level of type III interferon includes a reagent for detecting the level of type III interferon protein.

[0013] Preferably, the reagent for detecting type III interferon protein level is selected from one or more of IFN-λ2 / 3Antibody (Santa Cruz Biotechnology, Cat#sc-137151) and IFN-λ2 / 3Antibody (Santa Cruz Biotechnology, Cat#sc-365834).

[0014] Preferably, the optic nerve degeneration is selected from one or more of glaucoma and ischemic retinal disease; more preferably, the optic nerve degeneration is selected from glaucoma.

[0015] The third aspect of the present invention provides the use of a type III interferon inhibitor in the preparation of a drug for improving physiological indicators of optic nerve degeneration.

[0016] Preferably, the optic nerve degeneration-related disease is selected from one or more of glaucoma and ischemic retinal disease; more preferably, the optic nerve degeneration is selected from glaucoma.

[0017] Preferably, the type III interferon inhibitor is selected from one or more of a type III interferon neutralizing antibody, a gRNA designed based on a type III interferon gene, and a gRNA designed based on a type III interferon receptor gene.

[0018] Preferably, the type III interferon inhibitor is selected from IFN-λ2 / 3 Antibody (R&D Systems, MAB17892).

[0019] Preferably, the type III interferon receptor gene is selected from Ifnlr1, and the gRNA sequence designed based on the type III interferon receptor gene is selected from SEQ ID NO: 1 (5'-CAACCAGGTAGGTGCTCCTT-3') and SEQ ID NO: 2 (5'-GGGGACGGAAATACCTGGTG-3').

[0020] Preferably, the physiological index is selected from one or more of retinal ganglion cell survival rate, retinal ganglion cell complex thickness, and optic nerve axon protection.

[0021] A fourth aspect of the present invention provides a pharmaceutical composition for treating optic nerve degeneration and / or improving physiological indicators of optic nerve degeneration, comprising a type III interferon inhibitor and a pharmaceutically acceptable carrier.

[0022] Preferably, the optic nerve degeneration is selected from one or more of glaucoma and ischemic retinal disease; more preferably, the optic nerve degeneration is selected from glaucoma.

[0023] Preferably, the type III interferon inhibitor is selected from one or more of a type III interferon neutralizing antibody, a gRNA designed based on a type III interferon gene, and a gRNA designed based on a type III interferon receptor gene.

[0024] Preferably, the type III interferon inhibitor is selected from IFN-λ2 / 3 Antibody (R&D Systems, MAB17892).

[0025] Preferably, the type III interferon receptor gene is selected from Ifnlr1, and the gRNA sequence designed based on the type III interferon receptor gene is selected from SEQ ID NO: 1 (5'-CAACCAGGTAGGTGCTCCTT-3') and SEQ ID NO: 2 (5'-GGGGACGGAAATACCTGGTG-3').

[0026] Preferably, the physiological index is selected from one or more of retinal ganglion cell survival rate, retinal ganglion cell complex thickness, and optic nerve axon protection.

[0027] Preferably, the pharmaceutically acceptable carrier includes one or more of a solvent, a solubilizer, a surfactant, an antibacterial agent, an antioxidant, a chelating agent, a filler, a binder, a disintegrant, a lubricant, a flavoring agent, and a coloring agent.

[0028] It should be noted that, unless otherwise specified, the "type III interferon inhibitor" or similar expressions in the context of the present invention refer to preparations that can specifically antagonize or inhibit type III interferon and / or its receptor, including but not limited to small molecule inhibitors, monoclonal antibodies, polyclonal antibodies, genetic engineering vectors, etc., which can be obtained commercially or prepared by themselves. In order to fully display the relevant experiments and technical solutions, and to facilitate the understanding of those skilled in the art, the commercially available type III interferon neutralizing antibody (IFN-λ2 / 3Antibody, R&D Systems, MAB17892) is used in the context of the present invention for relevant experimental studies, but the inhibitor used is not used to limit the technical solution and protection scope of the present invention, and all preparations that can specifically antagonize or inhibit type III interferon and / or its receptor should be understood as the scope of the "inhibitor" mentioned in the present invention.

[0029] As the newest member of the IFN family, type III interferon shares many downstream signaling features with type I interferon. The heterodimeric receptor complex of type III interferon is composed of IFNLR1 and interleukin 10 receptor β chain (IL-10Rβ). After the receptor binds to type III interferon, it promotes the formation and nuclear translocation of the ISGF3 transcription factor complex, stimulating the expression of a series of IFN-stimulated genes (ISG).

[0030] Type III interferon is essential for the antiviral defense of the epithelial barrier, but existing studies still know little about its function in the central nervous system such as the retina. The present invention successfully constructed and analyzed the single-cell transcriptional map of the retina of the experimental glaucoma crab-eating macaque model, and found that type III interferon was activated in neurons in the early stages of optic nerve degeneration, and the activation of its downstream type III interferon signaling pathway was accompanied by the progression of optic nerve degeneration and the death of RGCs; and the same phenomenon was also found in the mouse model of chronic high intraocular pressure optic nerve degeneration, the mouse model of ischemia-reperfusion optic nerve degeneration, and glaucoma patients, thus clarifying the potential of type III interferon as a diagnostic molecular marker for optic nerve degeneration. In animal experiments, the signal transduction of type III interferon receptor was inhibited by knocking out the type III interferon receptor gene and injecting type III interferon neutralizing antibodies into the vitreous cavity, which reduced the effects of RGC degeneration and RGC death in the optic nerve degeneration model.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention successfully screened out the type III interferon signaling pathway as a biomarker for early optic nerve degeneration through cross-species multi-model analysis. And by knocking out the type III interferon receptor gene Ifnlr1 and using type III interferon neutralizing antibodies to inhibit its activity, the key effects of type III interferon on glaucoma and other ischemic optic nerve degeneration and its related physiological indicators were studied in depth, and it was clarified that inhibiting type III interferon can significantly improve RGC survival rate, increase the thickness of retinal ganglion cell complex, and protect optic nerve axons, and can be used as a key target for the treatment of optic nerve degeneration in ischemic retinal diseases. At the same time, type III interferon is abnormally activated during optic nerve degeneration, and its expression level is significantly increased, which is significantly ahead of optic nerve degeneration; and the level of type III interferon in aqueous humor samples of glaucoma patients is also significantly increased, indicating that the level of type III interferon has a very significant directional effect on optic nerve degeneration, and can therefore be used for auxiliary diagnosis and early prevention of optic nerve degeneration. In general, the present invention provides experimental basis and theoretical foundation for the drug development and clinical application of type III interferon signaling pathway as a biomarker for early optic nerve degeneration and type III interferon inhibitors for the treatment of optic nerve degeneration in ischemic retinal diseases, solves the problem of difficulty in transforming the results of rodent models such as mice, opens up a new transformable pathway for optic nerve degeneration in ischemic retinal diseases, and has important clinical application prospects and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the induction of type III interferon found by single-cell profiling of the retina in a non-human primate glaucoma model.

[0034] Figure 2 Schematic diagram of the association between type III interferon pathway activation and optic nerve degeneration found in retinal RGC cell trajectory analysis of non-human primate glaucoma model.

[0035] Figure 3 Schematic diagram of the results of decreased RGC survival rate and reduced GCC thickness as optic nerve degeneration progresses over time in the magnetic bead chronic intraocular hypertension model.

[0036] Figure 4 This is a schematic diagram of the results of Western blot verification of the early production of type III interferon in optic nerve degeneration in the magnetic bead chronic intraocular hypertension model.

[0037] Figure 5 Schematic diagram of the results of reduced RGC survival rate in the retinal ischemia-reperfusion model.

[0038] Figure 6This is a schematic diagram of the results of Western blot validation of the early production of type III interferon in ischemic optic nerve degeneration in the retinal ischemia-reperfusion model.

[0039] Figure 7 Schematic diagram of Western blot verification of the production of type III interferon in mouse retinal explants under hypoxia induction.

[0040] Figure 8 Schematic diagram of immunofluorescence verification of the production of type III interferon in human retinal explants under hypoxia induction.

[0041] Fig. 9 Schematic diagram of ELISA testing of patient aqueous humor samples to verify elevated levels of type III interferon in glaucoma patients.

[0042] Fig.10 Schematic diagram of the results that knockout of type III interferon receptor Ifnlr1 increased RGC survival rate and GCC thickness in the magnetic bead chronic ocular hypertension model.

[0043] Fig.11 Schematic diagram of the results showing that knockout of type III interferon receptor Ifnlr1 increased RGC survival in the retinal ischemia-reperfusion model.

[0044] Fig.12 Schematic diagram of the results of type III interferon inhibitors increasing RGC survival rate and GCC thickness in the magnetic bead chronic ocular hypertension model.

[0045] Fig.13 Schematic diagram of the results of type III interferon inhibitors protecting optic nerve axons in the magnetic bead chronic ocular hypertension model. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0047] Unless otherwise specified, the reagents used in the present invention are all commercially available. The experimental methods used in the present invention, such as molecular experiments, cell experiments, animal experiments, etc., are all conventional methods and techniques in the art. For the conduct of animal experiments, the experimental protocols were approved by the Animal Experiment Ethics Committee of Zhongshan Eye Center of Sun Yat-sen University and followed its guidelines.

[0048] Representative results selected from the biological experiment repetitions are presented in the context figures, and the data are displayed as mean±SD or mean±SEM as specified in the figure. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism9.0 or SPSS22.0 software. Conventional medical statistical methods such as t-test, chi-square test, analysis of variance, rank sum test, etc. were used to compare the mean differences between two or more groups. p<0.05 is considered a significant difference. For the expression level of type III interferon in cells / in vivo, conventional methods in the art (such as PCR, Western blot, etc.) can be used for detection; and the present invention verifies the detection specificity of the relevant antibodies IFN-λ2 / 3Antibody (SantaCruz Biotechnology, Cat#sc-137151, Cat#sc-365834) and the like described in the present invention for the expression level of type III interferon through specific experiments. For the inhibition of type III interferon, the present invention proves that the listed neutralizing antibodies can specifically inhibit the activation of the type III interferon pathway to produce a therapeutic effect. In view of the fact that the detection and inhibition of specific gene / protein expression levels belong to conventional means in the art, and are not the main improvement direction of the present invention, the relevant test results are not particularly presented in the present invention, and those skilled in the art can perform detection and verification according to the experimental methods described in the present invention or other conventional methods in the prior art as needed. In addition, in addition to the relevant primer pairs and antibodies listed in the present invention, those skilled in the art can also design relevant primer pairs / antibodies, inhibitors according to the structure of type III interferon protein, or obtain commercial reagents from the market to complete the detection or inhibition of type III interferon levels, so the specific information such as primer pairs, antibodies, inhibitors, etc. listed in the context of the present invention does not constitute a limitation on the actual protection scope of the present invention.

[0049] Example 1 Retinal scRNA analysis of non-human primate glaucoma model revealed type III interferon production and pathway activation

[0050] First, the cynomolgus monkey glaucoma model was constructed by laser photocoagulation. The specific steps are as follows:

[0051] (1) Using a dedicated gonioscope and retinal photocoagulation laser, 360° laser photocoagulation was performed on the trabecular area of ​​each eye of each cynomolgus monkey under a slit lamp, forming 200-400 continuous adjacent laser spots with a diameter of approximately 50 μm.

[0052] (2) Set the laser power to 0.8-1 W and the exposure time to 1 second.

[0053] (3) After laser surgery, IOP was measured every 3-4 days. Animals that did not show an increase in IOP (above 30 mmHg) underwent repeated procedures 2 weeks later until IOP increased. Eyes treated with laser photocoagulation were considered glaucomatous eyes, untreated eyes were considered control eyes, and individuals who had not received laser treatment were considered blank control eyes.

[0054] Subsequently, retinal sampling, single cell sample preparation, and scRNA analysis were performed. The specific steps are as follows:

[0055] (1) After the observation period, the eyeball was enucleated under deep anesthesia, the eyeball was cut open along the corneal limbus under a microscope, the retina was isolated in 4°C Ames medium, and the macular area with a diameter of 6 mm and its surrounding tissues were isolated using a rapid hole punch.

[0056] (2) The obtained retinal samples were digested with 200 μL papain, 5 mg / mL DNase, and 80 μL hyaluronidase in Ames medium at 37°C for 50 min, and then the digestion was terminated with 10% fetal bovine serum.

[0057] (3) The retina was perforated into a single-cell suspension in Ames medium containing 0.04% BSA, and diluted with 0.04% BSA / PBS solution to a single-cell suspension of 1000-1800 cells / μL and loaded into a 10×Chromium single-cell chip (X2).

[0058] (4) Single-cell libraries were constructed using the Chromium 3'v3 platform (10× Genomics, Pleasanton, CA) according to the manufacturer's protocol. Approximately 10,000 single cells were loaded into each channel, followed by cell lysis, reverse transcription of RNA, amplification, and sample labeling.

[0059] (5) Use Illumina NovaSeq6000-S2 for library sequencing and scRNA analysis.

[0060] (6) Use scRNA data analysis methods such as Seurat and Monocle trajectory analysis to analyze the main signaling networks in the process of optic nerve degeneration in glaucomatous eyes.

[0061] The experimental results are as follows Figure 1 shown. Figure 1 a is a schematic diagram of the changes in optic nerve fiber layer thickness (RNFLT) over time, which shows that the thickness of the nerve fiber layer has become thinner over time after glaucoma modeling, and is most obvious in the early Stage 1. Figure 1b is a schematic diagram of the expression of the top 10 activated genes in RGC cells in the early stage 1 of RNFLT reduction and the stable late stage 2, which shows that in the early stage of optic nerve degeneration, the main activated differentially expressed genes of RGC include interferon signaling pathway related genes (IFI6, IRF9, STAT1, ISG15, IFIT3, MX1, OPTN, etc.); Figure 1 c is a schematic diagram of the expression of genes related to type III interferon production in different retinal cell groups, which shows that high levels of type III interferon transcripts and TBK1 and IRF3 (key genes for IFN production) appeared in RGCs.

[0062] Figure 2 The results of the RGC trajectory analysis are shown in Figure 3. The activation of type III interferon in RGCs precedes the rapid increase in optic nerve degeneration, and the activation of the type III interferon pathway has a similar expression pattern with the progression of neurodegeneration and the pan-apoptotic signaling signature of cell death in RGCs.

[0063] Example 2 Study on the relationship between type III interferon signaling pathway activation and optic nerve degeneration in a rodent optic nerve degeneration model

[0064] First, establish a mouse magnetic bead chronic ocular hypertension (CHOT) glaucoma model. The specific steps are as follows:

[0065] (1) After the mice were anesthetized and the eyelids were disinfected, 0.5% proparacaine hydrochloride eye drops were applied topically for corneal anesthesia. After mydriasis was dilated with 1% tropicamide eye drops, magnetic beads were injected into the anterior chamber using a glass needle with a diameter of 100 μm. The magnetic beads were sucked into the anterior chamber at the contralateral corneal limbus using a magnet and evenly distributed in the chamber angle. After the operation, antibiotic eye ointment was applied topically to the cornea and the mice were rewarmed on a heating pad.

[0066] (2) After modeling, the intraocular pressure increase was monitored and recorded weekly. After anesthetizing the mice, they were immobilized with their heads horizontal. The intraocular pressure at the central position of the cornea was measured with a handheld tonometer. The average of the three measured values ​​was recorded as the intraocular pressure at that time, and the measurement time period was kept consistent.

[0067] At the same time, a mouse retinal ischemia-reperfusion (IR) model was established. The specific steps are as follows:

[0068] (1) The perfusion device is a 250 mL saline infusion bag and an infusion tube. The needle on the infusion tube is replaced with a 32G insulin needle and hung at a position 147 cm above the eye level of the mouse.

[0069] (2) After inducing general anesthesia in mice as described above and performing eyelid disinfection, corneal anesthesia and mydriasis, a 32G insulin needle was inserted into the anterior chamber, the infusion switch was turned on, the needle position was fixed, and the hydrostatic pressure of the liquid in the infusion bag at this height was connected to the eye to induce intraocular pressure to rise to about 120 mmHg, compressing the intraocular blood vessels to reduce blood perfusion and cause retinal ischemia, which lasted for 1 hour, after which the infusion switch was turned off, the needle was removed from the anterior chamber, and the intraocular pressure returned to normal and the intraocular blood vessels resumed perfusion. After surgery, topical antibiotic eye ointment was applied to the cornea and the mouse was rewarmed on a heating pad to wake up.

[0070] Subsequently, ischemic optic nerve degeneration detection was performed based on the above model, and the specific steps are as follows:

[0071] (1) Mice were killed after deep anesthesia, and their eyeballs were removed and fixed in 4% paraformaldehyde at room temperature for 1 hour.

[0072] (2) After dehydration through 10%, 20% and 30% sucrose gradient, the cells were embedded in OCT and frozen at -80°C overnight.

[0073] (3) Use a microtome to slice the eyeball at 10 μm. Only the eyeball slices containing the optic disc structure are selected and mounted on a glass slide. After drying, fluorescent staining sections are prepared.

[0074] (4) The optic nerve about 1 mm behind the eyeball was isolated and fixed in glutaraldehyde, dehydrated in ethanol, and then embedded in resin for semi-thin sections with a thickness of 1.5 μm. The sections were stained with toluidine blue and tested for optic nerve degeneration.

[0075] The detection indicators of optic nerve degeneration are as follows:

[0076] (1) RGC survival analysis: RGCs were quantified by counting RGCs. RGCs were labeled with the primary antibody Rbpms. Three non-adjacent sections or 12 random areas of retinal flat mounts were selected for each eye. RBPMS was analyzed using ImageJ. + RGCs were counted blindly. RGC survival rate was obtained by dividing the counts of the modeling group by the baseline counts of the control group.

[0077] (2) Analysis of retinal ganglion cell complex (GCC) thickness: The thickness of the retinal ganglion cell complex (GCC) was defined as the distance from the inner limiting membrane of the retina to the edge of the inner nuclear layer, including the RGC nerve fiber layer, ganglion cell layer, and inner plexiform layer of RGC dendrites. Thickness was measured in three different retinal regions: central (500 μm from the midpoint of the optic disc), middle (midpoint between the midpoint of the optic disc and the edge of the retina), and peripheral (150 μm from the edge of the retina). Three representative sections were randomly selected from each retina, and the average of the measurements of each three cross-sections was calculated using ImageJ software to ensure data reliability.

[0078] (3) Quantitative analysis of optic nerve axons: Five different 50 × 50 μm square areas were sampled from the peripheral area of ​​each nerve section and analyzed using ImageJ software. The average number of axons in each optic nerve was calculated and compared with the number per 250 square microns.

[0079] Furthermore, the level of type III interferon in the retina after modeling was detected by Western-blot, and the specific steps were as follows:

[0080] (1) Mouse eyeballs were collected at different time points. After the retina was separated on ice, RIPA lysis buffer (containing 1% PMSF and 1% phosphatase inhibitor) was added to fully lyse for half an hour, and the supernatant was collected by centrifugation.

[0081] (2) Determine the protein concentration using a BCA kit, add SDS loading buffer and denature at 95°C for 10 min to prepare the protein sample.

[0082] (3) After electrophoresis, transfer to the membrane, block the PVDF membrane in sequence, incubate with Ifnλ (type III interferon), β-Actin or Gapdh primary antibody and HRP secondary antibody, prepare fresh luminescent solution and develop with an exposure instrument.

[0083] (4) Quantitative analysis of the target protein was performed using ImageJ, and the relative expression level was obtained by normalization based on the internal reference of each sample, followed by statistical analysis.

[0084] The experimental results are as follows Figure 3-6 shown. Figure 3 The results showed that in the chronic ocular hypertension optic nerve degeneration mouse model (COHT), with the increase of intraocular pressure and the progression of the disease, the survival of RGCs was significantly reduced 4 weeks after COHT induction, and the thickness of GCC became thinner, indicating the occurrence of optic nerve degeneration. Figure 4 The results showed that in the mouse magnetic bead chronic ocular hypertension model, type III interferon was induced before significant GCC optic nerve degeneration as time progressed after modeling. Figure 5 The results showed that in the retinal ischemia-reperfusion mouse model (IR), RGC survival was significantly reduced 7 days after IR induction. Figure 6 The results showed that in the retinal ischemia-reperfusion mouse model, type III interferon was significantly induced and activated in the early stage of IR modeling.

[0085] In summary, in the mouse ischemic retinal optic nerve degeneration model, type III interferon is activated and occurs earlier than neurodegeneration. Therefore, the detection of type III interferon levels can be used for auxiliary diagnosis and early prevention of optic nerve degeneration.

[0086] Example 3 In vitro hypoxia induces retinal explants to produce type III interferon

[0087] First, retinal explants were prepared from adult mice. The specific steps are as follows:

[0088] (1) After the mice were killed by overdose of anesthesia, the eyeballs were removed and transferred to 4°C HBSS, and the retina was dissected and isolated.

[0089] (2) Keep the retina intact as much as possible and remove the vitreous body. Cut the retina into four flaps radially with the optic disc as the center. Then use a Pasteur tube to transfer the retina to a transparent PET membrane (0.4 μm pore size) in an insert cell culture incubator. Flatten the retina and ensure that the ganglion cell layer faces upward.

[0090] (3) Place the incubator in a 1.5 mL culture medium (containing 0.8 mM L-glutamine, 10% FBS, 2% B27 and 1% N 2 Neurobase-A medium) in a 6-well plate.

[0091] (4) Place the explant in a 37°C, 5% CO 2 Cultured in an incubator with medium changed once a day, the experiment began after 3 days of stability.

[0092] Subsequently, hypoxia was induced in the retinal explants in vitro, as follows:

[0093] (1) Explants in the oxygen deprivation group were exposed to 0.1% O 2 and 5% CO 2 The control group was still placed in 21% O 2 and 5% CO 2 incubator.

[0094] (2) After 3 hours, the explants of the experimental and control groups were taken out of the incubator, washed off the transparent PET membrane of the cell culture insert with 4°C PBS, and added to RIPA solution containing 1% PMSF and 1% phosphatase inhibitor.

[0095] In parallel, human retina was subjected to the same explant preparation and hypoxia induction as described previously.

[0096] Finally, as described above, WB experiment or immunofluorescence experiment was used to test whether type III interferon was induced in hypoxia. The specific steps of immunofluorescence experiment were as follows:

[0097] (1) Add 4% paraformaldehyde to the cell culture insert and fix at room temperature for half an hour, then add blocking solution (PBST solution containing 5% BSA and 0.5% Triton) and block for 1 hour.

[0098] (2) Add primary antibody (IFNλ, 1:200) and incubate overnight. After washing, incubate with fluorescent secondary antibody (1:200) at room temperature. After washing, add DAPI to cover the slide and take pictures.

[0099] The experimental results are as follows Figure 7-8 The results showed that, compared with the control group, the mouse retinal explants induced the production of type III interferon after 3 hours of hypoxia (see Figure 7 Immunofluorescence showed that IFNλ signals were evident in human retinal explants, and most of them overlapped with RGC axons (see Figure 8 ).

[0100] In summary, this section simulated in vivo ischemia through an in vitro hypoxia model and verified that hypoxia can induce the production of type III interferon.

[0101] Example 4: Higher levels of type III interferon factors detected in aqueous humor of glaucoma patients

[0102] Clinical samples were collected from patients undergoing cataract surgery (control group) or glaucoma surgery at the Zhongshan Eye Center of Sun Yat-sen University. The patients gave informed consent and signed a consent form. For cataract patients, after disinfection and irrigation of the eyes with povidone-iodine, a 30° corneal knife was used to make a corneal tunnel at the main incision site (without piercing the cornea), and then a 1 mL needle tube connected to the posterior capsule needle was used to pierce the anterior chamber from the main incision site to extract aqueous humor. For glaucoma patients, aqueous humor was extracted from the anterior chamber before penetrating eyeball operations (for example, trabeculectomy was performed after the scleral flap was made and before trabeculectomy) to avoid the influence of intraocular operations on aqueous humor, and also to avoid the influence of surgical operations due to reduced aqueous humor and softening of the eyeball. The aqueous humor samples were immediately injected into a sterilized 1.5 mL EP tube, frozen in dry ice, and then transferred to a -80 ° C refrigerator for storage. The collected aqueous humor samples were tested for IFNλ factor (LXR-Multi DTH-37, Bio-Rad) within 1 month.

[0103] The experimental results are as follows Fig. 9 The results showed that compared with non-glaucoma patients, all glaucoma patients (Glau), and different types of glaucoma patients (primary open-angle glaucoma (POAG), primary angle-closure glaucoma (PACG)), the IFNλ factor level in aqueous humor samples was significantly increased.

[0104] Example 5 Effect of Inhibiting Type III Interferon Pathway on Optic Nerve Degeneration

[0105] First, we studied the effect of knockout of the interferon receptor Ifnlr1 on optic nerve degeneration. The specific steps are as follows:

[0106] (1) The type III interferon receptor Ifnlr1 knockout C57BL / 6J mice (Ifnlr1) constructed with the gene editing tool CRISPR-gRNA (gRNA1, the sequence is shown in SEQ ID NO: 1, 5'-CAACCAGGTAGGTGCTCCTT-3', gRNA2, the sequence is shown in SEQ ID NO: 2, 5'-GGGGACGGAAATACCTGGTG-3') were respectively - / - )(Jiangsu Jicui Pharmaceutical, strain number #T013985) and wild C57BL / 6J mice were used to establish the optic nerve degeneration model.

[0107] (2) The mouse magnetic bead chronic ocular hypertension glaucoma model and retinal ischemia-reperfusion model were established as described above. For the magnetic bead chronic ocular hypertension model, the intraocular pressure changes were monitored regularly as described above.

[0108] (3) As mentioned above, optic nerve degeneration was detected based on the above model, and the detection indicators were RGC survival analysis, GCC thickness analysis, and optic nerve axon quantitative analysis.

[0109] Test results such as Figure 10-11 shown. Fig.10 The results showed that in the induced chronic ocular hypertension mouse model, Ifnlr1 knockout promoted RGC survival and significantly inhibited GCC degeneration compared with wild-type C57BL / 6J. Fig.11 The results showed that in the retinal ischemia mouse model, the survival rate of Ifnlr1 knockout RGCs was higher than that of wild-type C57BL / 6J, suggesting that Ifnlr1 knockout can significantly improve optic nerve degeneration in glaucoma and ischemic retinopathy.

[0110] Further, the effects of type III interferon inhibitors on optic nerve degeneration were studied. The specific steps are as follows:

[0111] (1) Wild-type C57BL / 6J mice were used to establish a mouse magnetic bead chronic high intraocular pressure glaucoma model as described above, and changes in intraocular pressure were monitored regularly.

[0112] (2) After the chronic high intraocular pressure glaucoma model was successfully established, type III interferon neutralizing antibody (IFN-λ2 / 3Antibody, R&D Systems, MAB17892) was injected into the vitreous cavity once a week until the sample was collected at 8 weeks. The specific operation was as follows: after the mice were anesthetized and the eyelids were disinfected, surface anesthesia was performed, and 2 μg of type III interferon neutralizing antibody or isotype control was injected into the vitreous cavity of the model eye through a 33G needle of a micro-syringe. The injection process was to avoid damaging the lens and to inject slowly to avoid a sharp increase in intraocular pressure. After the operation was completed, antibiotic eye ointment was applied locally.

[0113] (3) As mentioned above, optic nerve degeneration was detected based on the above model, and the detection indicators were RGC survival analysis, GCC thickness analysis, and optic nerve axon quantitative analysis.

[0114] The experimental results are as follows Figure 12-13 shown. Fig.12 The results showed that compared with the control group injected with the same type control into the vitreous cavity, the mice in the type III interferon neutralizing antibody group had a higher RGC survival rate and thicker GCC thickness 8 weeks after inducing the chronic high intraocular pressure model. Fig.13 The results showed that the optic nerve axons in the type III interferon neutralizing antibody group were more quantitative, indicating that ischemic optic nerve degeneration was improved.

[0115] Based on the above, the present invention has clarified the direct correlation between abnormal induction and pathway activation of type III interferon and ischemic optic nerve degeneration and poor prognosis through single-cell RNA sequencing analysis of glaucoma cynomolgus monkey model, mouse optic nerve degeneration model and retinal explant, glaucoma patient aqueous humor sample detection and human retinal explant and other sample analysis. In the mouse optic nerve degeneration model - chronic high intraocular pressure glaucoma model and retinal ischemia reperfusion model, the key effects of type III interferon on ischemic optic nerve degeneration and its related physiological indicators were deeply studied by knocking out the type III interferon receptor gene Ifnlr1 by gene editing tool CRISPR-gRNA and inhibiting its activity by type III interferon neutralizing antibody, and it was clarified that inhibition of type III interferon helps to significantly improve RGC survival rate, increase GCC thickness, protect optic nerve axons, and improve optic nerve degeneration, and can be used as a key target for the treatment of optic nerve degeneration in ischemic retinal diseases. In general, the present invention provides experimental evidence and theoretical basis for the drug development and clinical application of type III interferon inhibitors in the treatment of optic nerve degeneration caused by ischemic retinal diseases, and has important clinical application prospects and social value.

[0116] The above specific implementation method part specifically introduces the analytical method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and ideas of the present invention, rather than limiting the relevant content. Without departing from the principle of the present invention, those skilled in the art may also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. Use of type III interferon inhibitors in the preparation of drugs for preventing and / or treating optic nerve degenerative diseases.

2. The use according to claim 1, characterized in that: The optic nerve degenerative lesions are selected from one or more of glaucoma and ischemic retinal diseases.

3. The use according to claim 1, characterized in that: The type III interferon inhibitor is selected from one or more of a type III interferon neutralizing antibody, a gRNA designed based on a type III interferon gene, and a gRNA designed based on a type III interferon receptor gene.

4. Application of type III interferon inhibitors in the preparation of drugs for improving physiological indicators of optic nerve degeneration.

5. The use according to claim 4, characterized in that: The optic nerve degenerative lesions are selected from one or more of glaucoma and ischemic retinal diseases.

6. The use according to claim 4, characterized in that: The type III interferon inhibitor is selected from one or more of a type III interferon neutralizing antibody, a gRNA designed based on a type III interferon gene, and a gRNA designed based on a type III interferon receptor gene.

7. The use according to claim 4, characterized in that: The physiological index is selected from one or more of the following: retinal ganglion cell survival rate, retinal ganglion cell complex thickness, and optic nerve axon protection.

8. Use of a reagent for detecting the expression level of type III interferon in the preparation of a product for auxiliary diagnosis of optic nerve degeneration.

9. The use according to claim 8, characterized in that: The reagent for detecting the expression level of type III interferon includes a reagent for detecting the level of type III interferon protein.

10. The use according to claim 8, characterized in that: The optic nerve degenerative lesions are selected from one or more of glaucoma and ischemic retinal diseases.

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