Application of NK1.1 scavenger in preparation of medicine for treating and / or relieving acute retinal injury

Abstract

The invention relates to the field of ophthalmology, in particular to application of an NK1.1 scavenger (cloned PK136) in preparation of a medicine for treating and / or relieving acute retinal injury. The invention provides application of an NK1.1 scavenger in preparation of a medicine for treating and / or relieving acute retinal injury. According to the invention, emergency treatment can be carried out during acute retinal injury (such as traffic accidents and retinal detachment), retinal injury is relieved, sufficient time is provided for treatment methods such as operations, and injury is reduced to the minimum.

Description

Technical Field

[0001] This invention relates to the field of ophthalmology, and more particularly to the use of NK1.1 scavengers in the preparation of medicaments for treating and / or alleviating acute retinal injury. Background Technology

[0002] The retina is a highly differentiated neural tissue within the eye, crucial for its photosensitive function. Its structural integrity and functional stability are vital for visual function. Global Burden of Disease studies show that acute retinal injuries, such as blunt trauma, penetrating injuries, retinal detachment, and laser-induced damage, can cause retinal neuron apoptosis, inflammatory cell infiltration, and structural disorder, thereby affecting visual function and even leading to irreversible blindness. With an aging population, the number of patients with acute retinal injuries (including surgically induced retinal detachment and choroidal neovascularization (CNV)) continues to rise, drastically increasing the social and economic burden.

[0003] Although significant progress has been made in clinical interventions for the above-mentioned diseases (such as anti-inflammatory treatment (Dexamethasone), surgery (scleral buckling, vitrectomy, gas injection repositioning), laser and intraocular drug injection, anti-angiogenic treatment (Ranibizumab, Aflibercept), neurotrophic protection (N-acetylcysteine, BDNF, CNTF), etc., which can alleviate damage to a certain extent, the following limitations still exist: (1) Limited effect, such as the continued presence of neuronal apoptosis after surgical treatment (PPV, Scleral Buckling, etc.), resulting in unsatisfactory visual recovery; (2) Significant side effects: Although glucocorticoids have good anti-inflammatory effects, they can cause increased intraocular pressure and cataracts; (3) Lack of immunomodulatory target drugs: The treatment does not target specific cells (such as NK cells and macrophages) involved in the immune response during the damage process; (4) Inability to treat in a timely manner, such as the need to schedule surgical treatment, and while patients are waiting for surgery, the damage to retinal photoreceptor cells worsens as the retinal detachment time increases, making recovery more difficult. Therefore, there is an urgent need to develop new and efficient treatment strategies that can provide emergency treatment for acute retinal injuries (such as after a car accident or retinal detachment), protect the damaged retina, give ample time for surgical and other treatment methods, minimize damage, and improve vision recovery.

[0004] NK cells and ILC1 cells (type 1 innate lymphoid cells) both belong to the innate immune system and share a common surface marker, NK1.1 (also known as member 1 of the killer cell lectin-like receptor B subfamily, KLRB1, NKR-P1A, or CD161). They possess potent cytotoxic and immunomodulatory functions and have been applied in the treatment of various diseases, particularly in the fields of oncology and immunomodulation. NK cells possess the natural ability to kill tumor cells without prior sensitization. For acute myeloid leukemia (AL), CAR-NK cell (chimeric antigen receptor-modified NK cell) therapy has entered early clinical validation, demonstrating low adverse reactions and good safety. ILCs are an important component of the innate immune system, playing a crucial role in the early stages of the immune response against pathogens such as viruses, bacteria, and parasites. In the inflamed intestines of Crohn's disease patients, ILC1 cells aggregate at a high frequency, and these ILC1 cells play an important role in the pathogenesis of intestinal mucosal inflammation by producing IFN-γ. Furthermore, ILC1 plays an important role in diseases such as liver injury, kidney injury, brain injury, and necrotizing enterocolitis. In the pathogenesis of wet age-related macular degeneration, animal studies have reported that NK cells can inhibit retinal regrowth.

[0005] Currently, there are no reports on the roles of NK cells and ILC1 cells in the acute phase of acute retinal injury. Summary of the Invention

[0006] In view of this, the present invention provides the use of NK1.1 scavengers in the preparation of medicaments for treating and / or alleviating acute retinal injuries. The present invention enables emergency treatment in cases of acute retinal injury (such as car accidents or retinal detachment), alleviating retinal damage and allowing sufficient time for surgical or other treatment methods, thus minimizing the damage.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides the use of NK1.1 scavengers in the preparation of medicaments for treating and / or alleviating acute retinal injury.

[0009] In some embodiments of the present invention, in the above-described applications, the NK1.1 scavenger inhibits angiogenesis.

[0010] In some embodiments of the present invention, in the above-described applications, the NK1.1 scavenger inhibits the area of ​​retinal neovascularization lesions.

[0011] In some embodiments of the present invention, in the above-described applications, the NK1.1 scavenger reduces the number of congenital lymphocytes.

[0012] In some embodiments of the present invention, in the above applications, the congenital lymphocytes include NK cells and / or ILC1 cells.

[0013] In some embodiments of the present invention, in the above-described applications, the NK1.1 scavenger reduces the infiltration of inflammatory cells.

[0014] In some embodiments of the present invention, in the above applications, the inflammatory cells include: Iba-1 + Microglia.

[0015] In some embodiments of the present invention, in the above-described applications, the NK1.1 scavenger inhibits apoptosis of photoreceptor cells.

[0016] In some embodiments of the present invention, in the above applications, the NK1.1 clearing agent is applied within 48 hours before retinal injury to 24 hours after injury.

[0017] In some embodiments of the present invention, in the above applications, the dosage of the NK1.1 scavenger is 0.405~2.025 mg / kg / time.

[0018] This invention provides an effective emergency measure to protect retinal function for ophthalmic emergencies or high-risk groups of retinal detachment (such as patients with high myopia).

[0019] (1) Innovative timing of treatment: a single dose is administered from 48 hours before retinal injury to immediately after injury (≤24 hours);

[0020] Scientific basis: Acute inflammatory responses caused by retinal damage have a destructive effect, while subsequent inflammatory responses (after 2 days) can promote retinal repair; NK1.1 + Cells infiltrate the retina 1 hour after injury, reaching a peak at 24 hours. Early blockade can stop the acute inflammatory response.

[0021] (2) Innovation in emergency treatment methods: Targeted clearance of NK1.1 + cell.

[0022] Scientific basis: We validated the targeted ablation of NK1.1 in two acute retinal injury models (laser injury and retinal detachment). + The cells can reduce retinal inflammatory cell infiltration and protect the retina, including reducing the area of ​​retinal neovascularization lesions caused by laser damage and the number of nerve cell apoptosis caused by retinal detachment. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0024] Figure 1 CD45 in RPE-choroid-scleral smear after retinal laser injury + NK1.1 + Cellular responses; mouse eyeballs were collected before laser injury and at 1h, 4h, and 24h after injury to prepare RPE / choroidal sclera slides for immunofluorescence staining; A shows the changes in CD45 (green) and NK1.1 (red) cells in the RPE-choroid-sclera slides before retinal injury and at 1h, 4h, and 24h after retinal injury; B shows the CD45 cell count in the lesion area of ​​the RPE-choroid-sclera slides at different time points before laser injury and at 1h, 4h, and 24h after injury. + NK1.1 + Cell number; n=20-24, expressed as mean ± SD, Kruskal-Wallis and Dunn multiple comparison tests. *** p<0.001, 1 h: 1 hour after laser damage; 4 h: 4 hours after laser damage; 24 h: 24 hours after laser damage;

[0025] Figure 2 This study describes the detection of ILC1, ILC2, and ILC3 cell responses in RPE-choroid-sclera smears after retinal laser injury. Specifically, AC represents the immunofluorescence assay of ILC cells in the lesion area of ​​the RPE-choroid-sclera smear 1 hour and 24 hours after retinal injury (A represents ILC1:T-bet). + (Red) CD3 - (Green); B is ILC2:GATA3 + (Red) CD3 - (Green); C represents ILC3: ROR-γ + (Red) CD3 - (Green) Changes; D is a bar chart showing the number of ILC1, ILC2, and ILC3 in the lesion area of ​​the RPE-choroid-sclera patch at different time points before and 1 hour and 24 hours after laser injury; E is a bar chart showing the number of ILC1, ILC2, and ILC3 in the RPE-choroid-sclera patch at the same time points before and 1 hour and 24 hours after laser injury; n=20-28, expressed as mean ± SD, Kruskal-Wallis and Dunn multiple comparison tests. * p<0.05, ** p<0.01, *** p<0.001, 1 h: 1 hour after laser damage; 24 h: 24 hours after laser damage;

[0026] Figure 3This study describes the detection of ILC1, ILC2, and ILC3 cell responses in choroid-RPE smears after retinal laser injury; where AC represents ILC cells in the lesion area of ​​the choroid-RPE smear 1 h and 24 h after retinal injury, as detected by immunofluorescence (A represents ILC1:T-bet). + (Red) CD3 - (Green); B is ILC2:GATA3 + (Red) CD3 - (Green); C represents ILC3: ROR-γ + (Red) CD3 - (Green) Changes; D is a bar chart showing the number of ILC1, ILC2, and ILC3 in the lesion area of ​​the choroid-RPE patch before laser injury and at different time points (1 hour and 24 hours after injury); E is a bar chart showing the number of ILC1, ILC2, and ILC3 in the choroid-RPE patch before laser injury and at the same time point (1 hour and 24 hours after injury); n=20-28, expressed as mean±SD, Kruskal-Wallis and Dunn multiple comparison tests. ** p<0.01, *** p<0.001, 1 h: 1 hour after laser damage; 24 h: 24 hours after laser damage;

[0027] Figure 4 Flow cytometry was used to verify the responses of ILC1, NK cells, ILC2, and ILC3 after retinal laser injury. Specifically: A shows the flow cytometry gating scheme for ILC and NK cells; B shows the changes in the numbers of ILC1, NK cells, ILC2, and ILC3 in the peripheral blood of mice with retinal laser injury before and at 1 h, 4 h, and 24 h after laser injury; C shows the changes in the numbers of ILC1, NK cells, ILC2, and ILC3 in the spleen of mice with retinal laser injury before and at 1 h, 4 h, and 24 h after laser injury. n=6, values ​​are expressed as mean ± SD, and Kruskal-Wallis and Dunn multiple comparison tests were used. * p<0.05, ** p<0.01, *** p<0.001. 1h: 1 hour after laser damage; 4h: 4 hours after laser damage; 24h: 24 hours after laser damage;

[0028] Figure 5This study demonstrates the inhibitory effects of anti-NK1.1 scavengers on peripheral blood ILC1, NK cells, ILC2, and ILC3. A is a flowchart showing the changes in peripheral blood ILC and NK cells before and at 1, 2, and 3 days after injection of anti-NK1.1 into healthy wild-type mice. B shows the changes in peripheral blood ILC and NK cells before and at 1, 2, and 3 days after injection in the anti-NK1.1 scavenger group and the IgG group. C shows the reduction rate of peripheral blood ILC and NK cells in the anti-NK1.1 scavenger group compared to the IgG group at 1, 2, and 3 days after injection. (n=5, values ​​are expressed as mean ± SD, Kruskal-Wallis and Dunn multiple comparison tests are used.) * p<0.05, ** p<0.01;

[0029] Figure 6 This study illustrates the effect of multiple injections of anti-NK1.1 on retinal wound healing and inflammation. A is a flowchart showing the injection of anti-NK1.1 (100 μg / mouse) at 2, 4, and 6 days after laser injury. Immunofluorescence staining was performed on 7 days after laser injury to detect changes in blood vessels and inflammatory cells in the lesion area. B shows RPE / choroidal confocal images of mice in the IgG and anti-NK1.1 scavenger groups, displaying Collagen IV (red), Iba-1 (green), and DAPI staining, with a scale bar of 50 μm. C is a bar chart showing Collagen IV in the lesion area of ​​mice in the IgG and anti-NK1.1 scavenger groups. + The area of ​​blood vessels; D is a bar chart showing the Iba-1 lesion area in mice of the IgG group and the anti-NK1.1 scavenger group. + The number of microglia, mean ± SD, n = 36-40 lesions per group, from 9-10 eyeballs (4-5 mice) in group B. * p<0.05, *** p<0.001, Mann-Whitney test, IgG: IgG injection control group, anti-NK1.1: anti-NK1.1 scavenger injection group;

[0030] Figure 7This study investigated the effects of a single injection of anti-NK1.1 scavenger before or immediately after retinal laser injury on retinal wound healing and inflammation. A is a flowchart showing the injection of anti-NK1.1 (100 μg / mouse) two days before laser injury, followed by immunofluorescence staining to detect changes in blood vessels and inflammatory cells in the lesion area seven days after laser injury. B shows RPE / choroidal confocal images of mice in the IgG and anti-NK1.1 scavenger groups, displaying Collagen IV (red), Iba-1 (red), and DAPI staining, with a scale bar of 50 μm. C is a bar chart showing Collagen IV in the lesion area of ​​mice in the IgG and anti-NK1.1 scavenger groups. + The area of ​​blood vessels; D is a bar chart showing the Iba-1 lesion area in mice of the IgG group and the anti-NK1.1 scavenger group. + The number of microglia; mean ± SD in C and D, n = 36-40 lesions in each group, from 9-10 eyeballs (4-5 mice) in B. * p<0.05, Mann-Whitney test; E is a flowchart, showing an immediate injection of anti-NK1.1 (100 μg / mouse) after laser injury, and immunofluorescence staining to detect changes in blood vessels and inflammatory cells in the lesion area 7 days after laser injury; F is a confocal image of RPE / choroid in mice of the IgG group and anti-NK1.1 scavenger group, showing Collagen IV (red), Iba-1 (red), and DAPI staining, scalebar = 50 μm; G is a bar chart showing Collagen IV in the lesion area of ​​mice of the IgG group and anti-NK1.1 scavenger group. + The area of ​​blood vessels; H represents the bar graph showing the Iba-1 lesion area in mice of the IgG group and the anti-NK1.1 scavenger group. + Number of microglia; mean ± SD in G and H, n = 36-40 lesions per group, from 9-10 eyeballs (4-5 mice) in B. * p<0.05, ** p<0.01, *** p<0.001, Mann-Whitney test; IgG: IgG injection control group, anti-NK1.1: anti-NK1.1 scavenger injection group;

[0031] Figure 8This study investigated the effect of a single injection of anti-NK1.1 scavenger immediately after retinal detachment injury on retinal wound healing and inflammation. A shows confocal images of frozen sections of mouse eyes before and 24 hours after retinal detachment injury, displaying CD45 (green), NK1.1 (red), and DAPI staining (scale bar = 50 μm). B shows a bar chart of CD45 in the mouse retina before and 24 hours after retinal detachment injury. + NK1.1 + The number of samples, n=4-5, is expressed as mean ± SD, and the Mann-Whitney test is performed. ** p<0.01, healthy, RD24h: 3 days after retinal detachment; C is a flowchart, an immediate injection of anti-NK1.1 (100ug / mouse) after retinal detachment injury, and immunofluorescence staining to detect changes in retinal Tunel apoptotic cells and inflammatory cells 3 days after retinal detachment; D is a confocal image of frozen retinal sections from mice in the IgG group and the anti-NK1.1 scavenger group showing Tunel (green) and DAPI staining, scale bar = 50um; E is a bar chart showing the ONL layer per mm in mice in the IgG group and the anti-NK1.1 scavenger group. 2 Number of apoptotic cells in photoreceptors; F is a confocal image of frozen retinal sections from mice in the IgG and anti-NK1.1 scavenger groups, showing CD45 (green), Iba-1 (red), and DAPI staining, scale bar = 50 μm; G is a bar chart showing CD45 in the retinas of mice in the IgG and anti-NK1.1 scavenger groups. + Cell count; H represents the bar chart showing retinal Iba-1 in mice of the IgG group and the anti-NK1.1 scavenger group. + Microglia number, Scale bar = 50 μm; mean ± SD in G and H, n = 5-6 mice per group; * p<0.05, ** p<0.01, Mann-Whitney test, IgG: IgG injection control group, anti-NK1.1: anti-NK1.1 scavenger injection group. Detailed Implementation

[0032] This invention discloses the use of NK1.1 scavengers in the preparation of medicaments for treating and / or alleviating acute retinal injury.

[0033] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0034] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0035] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0036] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0037] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0038] C57BL / 6J mice (6-8 weeks old) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China) and housed in standard laboratory facilities with a 12-hour light / dark cycle, with free access to food and water. This procedure was conducted in accordance with the Association for Vision and Ophthalmology (ARVO) statement on the use of animals in ophthalmological and vision research, and the protocol was approved by the Animal Welfare and Ethics Committee of Aier Eye Hospital (Reference No.: AEI20230048).

[0039] In Examples 1 to 8 and Effect Examples 1 to 3 of the present invention, the raw materials and reagents used can all be purchased from the market.

[0040] The present invention will be further illustrated below with reference to the embodiments:

[0041] Example 1: Induction of retinal damage

[0042] We used two retinal injury models: retinal laser burns and low-pressure induced retinal detachment (RD).

[0043] Retinal laser injury: Mice were anesthetized with isoflurane, and pupils were dilated with drops of 0.5% tropicamide and 0.5% phenylephrine (Santen Pharmaceutical Co., Ltd., Osaka, Japan). The ocular surface was moistened with sodium carboxymethyl cellulose (Alligator Pharmaceutical Co., Ltd., Dublin, Ireland). At least two laser ablations were performed around the optic nerve head using a Topcon photocoagulator (Topcon, Tokyo, Japan, wavelength 532 nm, power 100 mV, spot size 60 μm).

[0044] A low-tension-induced retinal detachment model was established in mice following a previously reported protocol. Mice were anesthetized with compound tropicamide pupil dilation. Isoflurane was added to the air anesthesia machine, and the mice were placed in an anesthesia chamber. After anesthesia, an expiratory mask was used to maintain the anesthesia, and sodium carboxymethyl cellulose was used to moisten the ocular surface. Under a stereomicroscope, the mouse's head was elevated with gauze, and the head position and microscope focus were adjusted to allow the operator to clearly see the fundus. A full-thickness corneal puncture was performed using a 30.5 gauge needle. The needle remained in place to release aqueous humor until complete retinal detachment was observed. Rapid retinal detachment was observed, and a large area of ​​retinal bulging and whitening was observed in the fundus. The needle was then withdrawn. The mice were given 0.01% atropine sulfate eye drops and ofloxacin eye ointment to reduce inflammation and infection. The sham-operated group received anesthesia and pupil dilation without laser ablation or corneal puncture. Place all mice in a recovery box, keep their eyes moist until they wake up, and then return them to their cages.

[0045] Example 2: Immunofluorescence staining of RPE / choroidal membrane planks

[0046] (1) Seven days after retinal laser injury, mouse eyeballs were collected and placed in 1.5 mL EP tubes containing 2% PFA. The tubes were placed on a shaker and shaken slowly. The tubes were fixed at room temperature for 2 hours, washed three times with PBS for 5 minutes each time, and then placed in 1.5 mL EP tubes containing sterile PBS and stored at 4°C until the next step.

[0047] (2) Under a microscope, remove the fixed mouse eyeball, remove the extraocular muscles with microscissors and forceps, remove the lens of the mouse eyeball, and separate the retina and RPE / choroid.

[0048] (3) Blocking and permeabilization: Place the RPE / choroid membrane in a 96-well plate, one sample per well, add 100ul of blocking and permeabilization solution (10% goat serum and 2% bovine serum albumin + 0.1% Tritonx-100) to each well, and incubate on a shaker at room temperature for 2 hours to permeate the cell membrane and block non-specific antigens.

[0049] (4) Primary antibody incubation: Rat anti-mouse CD45 (1:400), mouse anti-mouse NK1.1 (1:400, MCE), rat anti-mouse CD3 (1:100), rabbit anti-mouse ROR-γ (1:200, Invitrogen), rabbit anti-mouse GATA3 (1:200, Invitrogen), rabbit anti-mouse T-bet (1:50, Invitrogen), rabbit anti-mouse IBA-1 (1:200, Cat:ab178846, Abcam), rat anti-mouse Iba-1 (1:200), and rabbit anti-mouse Collagrn IV (1:200) were incubated overnight at 4°C with RPE / choroidal membrane substrate. After removal, 100 μL of PBS was added to each well, and the substrate was washed three times rapidly in a shaker for 5 min each time.

[0050] (5) Secondary antibody incubation: Then incubate with Alexa Fluor 488 goat anti-rat IgG (1:400, Cat: A11006, Invitrogen), Alexa Fluor 594 goat anti-mouse IgG (1:400, Alexa Fluor 594 goat anti-rabbit IgG (1:400, Cat: 2616076, Invitrogen) at room temperature for 2 hours.

[0051] (6) Nuclear staining: Incubate with DAPI (1:400, cat: C0060, Solarbio, Beijing, China) at room temperature for 5 min. Wash three times with PBS, 5 min each time.

[0052] (7) Under a microscope, cut the RPE / choroid into four evenly spaced pieces, taking care to avoid lesions.

[0053] (8) Covering: Place the RPE / choroid membrane on the glass slide and spread it out. Add an anti-fluorescence quenching agent, cover it with a coverslip, and then seal the slide with nail polish.

[0054] (9) Use a confocal microscope to examine the sample.

[0055] Example 3: Removal of the sclera

[0056] (1) Take out the immunofluorescence-stained RPE / choroidal slide, hold one corner with tweezers, and then scrape off the sclera with a knife in one go.

[0057] (2) Place the sclera and choroid / RPE in a dish with PBS, let the tissue spread out naturally, flatten the tissue, and distinguish the front and back sides, making sure the choroid side is facing up. Tilt the slide and immerse it in the PBS of the large dish, then drag the tissue onto the slide and make sure it is flat.

[0058] (3) Remove the slide from the water surface and blot dry with paper so that the tissue adheres fully to the slide. Add an anti-fluorescence quencher to the tissue and cover it with a coverslip.

[0059] (4) Use a confocal microscope to examine the sample.

[0060] Example 4: Preparation of frozen slices

[0061] Mouse eyeballs were collected 1 day and 3 days after retinal detachment injury and frozen sections were prepared.

[0062] (1) Sample collection: Mice were euthanized by anesthesia and their eyeballs were collected under sterile conditions using ophthalmic curved forceps.

[0063] (2) Fixation: The mouse eyeballs were immediately fixed with 4% PFA at room temperature for 4 hours.

[0064] (3) Dehydration: The eyeball is dehydrated in 15% and 30% sucrose in sequence. The eyeball is dehydrated at room temperature in each concentration of sucrose until the tissue settles to the bottom. When changing the liquid, the eyeball is removed, the liquid on the surface of the eyeball is dried with absorbent paper, and then it is put into the next liquid.

[0065] (4) Embedding: Take out the tissue, absorb the moisture with paper, and embed it in OCT. Leave it at room temperature for 5 minutes to allow the embedding agent to fully fill the entire tissue.

[0066] (5) Preparation of frozen sections: The embedded tissue is prepared into frozen sections in a compound at the optimal cutting temperature.

[0067] (6) Sectioning: Cut the frozen tissue into 10 μm thick sections in a microtome and attach them to a glass slide to make frozen sections.

[0068] (7) Drying: Place the sliced ​​pieces on a clean workbench to dry, then put them into a slice box and store them in a -80℃ refrigerator for later use.

[0069] Example 5: Immunofluorescence staining of frozen sections

[0070] (1) Take out the frozen section and warm it at room temperature for 15 minutes. Add water to the dark box so that the water just covers the bottom of the box. Place the section in the dark box compartment to create a humid environment to prevent liquid evaporation during subsequent antibody incubation.

[0071] (2) Draw a circle around the tissue section sample with a histochemistry pen, making sure to draw it in one stroke, ensuring the histochemistry circle is complete, and that it does not touch the tissue section. Add PBS and incubate for 5 minutes to melt the embedding agent.

[0072] (3) Blocking and permeation: 10% goat serum and 2% bovine serum albumin, 0.1% Tritonx-100 dissolved in PBS, are added to the histochemistry zone of the sliced ​​sample to permeate the cell membrane and block non-specific antigens. The solution is left at room temperature for 1 hour.

[0073] (4) Primary antibody incubation: Incubate the slide samples with rabbit anti-mouse ZO-1 (1:300) or rabbit anti-mouse IBA-1 (1:200) at 4°C overnight. After removal, wash three times with PBS for 5 min each time.

[0074] (5) Incubation with secondary antibody: All operations after this step must be performed in the dark. Incubate with Alexa Fluor 594 goat anti-rabbit IgG (1:400) at room temperature for 2 hours. Wash three times with PBS, 5 minutes each time.

[0075] (6) staining the nucleus: incubate with DAPI (1:400) at room temperature for 5 min. Wash three times with PBS, 5 min each time.

[0076] (7) Mounting: Add anti-fluorescence quenching agent, cover with a coverslip, and then apply nail polish around the coverslip to seal the slide to prevent the anti-fluorescence quenching agent from overflowing. Let it dry at room temperature and collect it in a slide box away from light. Store at 4°C.

[0077] (8) Use a confocal microscope to examine the sample.

[0078] Example 6: Preparation of spleen cell suspension

[0079] (1) After euthanizing mice by intraperitoneal injection of an excessive amount of sodium pentobarbital, the mice were immersed in alcohol for 1 minute and then fixed with a sterile plate.

[0080] (2) Make a small incision in the mouse's abdomen with scissors to avoid damaging the abdominal cavity, and bluntly separate the mouse's fur from the abdominal tissue.

[0081] (3) Open the mouse's abdominal cavity with scissors, take out the spleen and quickly put it into a labeled EP tube containing PBS, and place it on ice.

[0082] (4) Place a 40 μm filter in a 6 cm dish, add the prepared culture medium, and place the spleen on the 40 μm filter.

[0083] (5) Gently grind the tissue with the syringe handle until there are no large pieces of tissue. Rinse the cells with culture medium into a dish and collect the cell suspension in a 15-minute centrifuge tube.

[0084] (6) Centrifuge the spleen cell suspension at 300g, 4℃, for 5 minutes, and discard the supernatant.

[0085] (7) Add 2 mL of 1×RBC lysis buffer, mix well, lyse at 4℃ for 2 min, add 5 mL of red blood cell termination solution to stop lysing, centrifuge at 300 g, 4℃ for 5 min, and discard the supernatant.

[0086] (8) Resuspend the cells in culture medium, count the cells using CountStar software, and take 1×10^6 cells to prepare for flow cytometry staining.

[0087] Example 7 Flow Cytometry Staining

[0088] (1) Add the prepared suspension cells to a flow cytometer, wash once with PBS, centrifuge at 4°C for about 4 minutes at 300g, and discard the supernatant.

[0089] (2) Add mouse Fc receptor blocker and incubate at room temperature for 5 minutes.

[0090] (3) Incubate blood and spleen samples with the antibody mixture shown in Table 1 at 4°C for 30 min in the dark.

[0091] (4) Wash once with PBS, centrifuge at 4°C for about 4 minutes at 300g, and discard the supernatant.

[0092] (5) Add the live and dead dye (1:1000), incubate at room temperature for 10 min, wash once with PBS, centrifuge at 4°C for about 4 minutes at 300g, and discard the supernatant.

[0093] (6) Blocking: FC incubated at room temperature for 5 min.

[0094] (7) Cell membrane staining: Mix the cells with the antibody mixture shown in Table 1, stain the cell membrane at 4℃ for 30 min, wash once with 2 mL of stain buffer, centrifuge at 4℃ for about 4 minutes at 300 g, and discard the supernatant.

[0095] (8) Permeability test: Incubate with 1 mL perm / fix 1× (dilute 4×fix / perm with diluent buffer to 1×fix / perm) at 4°C for 50 min, wash once with 1 mL 1×perm / wash, then wash again with 2 mL 1×perm / wash, centrifuge at 4°C for about 4 minutes at 300g, and discard the supernatant.

[0096] (9) Nuclear staining: Add 100 μL 1×perm / wash and mix with the antibody mixture shown in Table 2. Incubate at 4°C for 1 h.

[0097] (10) Wash twice with 2mL 1×perm / wash, centrifuge at 4℃ for about 4 minutes at 300g, and discard the supernatant.

[0098] (11) Resuspend in 300 μL stian buffer and run on the machine.

[0099] Table 1 Antibody Mixture

[0100]

[0101] Table 2 Antibody Mixture

[0102]

[0103] Example 8. Determination of Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL)

[0104] (1) Take out frozen sections of the eyeball 3 days after retinal detachment injury and thaw them at room temperature for 5 minutes.

[0105] (2) Infiltrate frozen sections of mouse eyeballs with 0.5% Tritonx-100 (dissolved in PBS) at room temperature for 5 minutes, and wash with PBS 3 times for 5 minutes each time.

[0106] (3) Prepare the terminal deoxynucleotidyl transferase-mediated dUTP nickel end labeling (TUNEL) test solution according to the instructions, and mix thoroughly. Place the solution onto a glass slide, cover the ocular section sample, and incubate at 37°C in the dark for 60 minutes. Wash three times with PBS for 5 minutes each time, then incubate with DAPI at room temperature for 10 minutes, and wash three times with PBS for 5 minutes each time. Detect the sample fluorescence using a confocal microscope.

[0107] Example 1: Rapid changes in NK / ILC1 innate immune cells caused by acute retinal injury.

[0108] We collected eyeballs from mice with no retinal injury and at 1 hour, 4 hours, and 24 hours after acute retinal injury. Figure 1 We prepared RPE-choroid-sclera and choroid-RPE slides, and used immunofluorescence to detect changes in innate immune cells before retinal laser injury and at 1h, 4h, and 24h after injury. We found that CD45 was significantly reduced after retinal injury. + NK1.1 + Cells significantly increased 24 hours after retinal injury. Figure 1 ), T-bet + CD3- Innate lymphoid cells type 1 (ILC1) increased significantly 1 hour after retinal injury, GATA3 + CD3 - Innate lymphoid cells type 2 (ILC2) cells, ROR-γ + CD3 - (Innate lymphoid cells 3, ILC3) cells significantly increased 24 hours after retinal injury. Figure 2 AD). And 24 hours after retinal injury, T-bet + CD3 - ILC1 and GATA3 + CD3 - ILC2 was significantly more abundant than ROR-γ. + CD3 - ILC3 ( Figure 2 E). In choroid-RPE sheets, T-bet + CD3 - ILC1 cells significantly increased 24 hours after retinal injury. Figure 3 AD), and it was most abundant in the three types of ILC cells ( Figure 3 (E). Analysis of the number of infiltrated cells in different RPE-choroid-scleral patches after retinal injury revealed the presence of CD45 innate immune cells. + NK1.1 + (Including NK cells, NKT cells and T-bet cells) + CD3 - ILC1 cells had the most cells ( Figure 1 B), followed by GATA3 + CD3 - Natural lymphocyte type 2 ( Figure 2 (D).

[0109] To verify the reliability of this result, we used flow cytometry to detect immune cells in peripheral blood and spleen of mice with no retinal damage and at 1 h, 4 h, and 24 h after retinal damage. Figure 4 A) found that the proportion of NK cells and ILC1 cells among CD45 leukocytes in peripheral blood and spleen increased significantly 1 hour after retinal injury. Figure 4 (B and C). This further suggests that acute retinal injury triggers a rapid response from the local ocular microenvironment and circulating immune cells, particularly NK / ILC1 innate immune cells, exacerbating retinal damage.

[0110] Example 2: NK1.1 remover significantly reduced retinal lesions and inflammation caused by retinal laser damage.

[0111] NK cells and ILC1 cells share the surface marker NK1.1, and clone PK136 can bind to NK1.1 molecules, simultaneously depleting both NK cells and ILC1 cells. First, we intraperitoneally injected healthy wild-type mice with an NK1.1 scavenger (antibody, clone PK136, 100 μg / mouse)... Figure 5 Before and at 1D, 2D, and 3D after injection, we used flow cytometry to detect peripheral blood immune cells. We found that the NK1.1 scavenger (antibody) significantly reduced the proportion of NK cells and ILC1 cells in peripheral blood 1 day after injection, and still had a strong inhibitory effect on NK cells and ILC1 cells in peripheral blood 2 days after injection. The scavenging effect weakened on the 3rd day after injection. Figure 5 (B and C).

[0112] To investigate the roles of NK / NKT cells and ILC1 cells in retinopathy, we observed the effects of depleting NK / NKT cells and ILC1 cells at different time points on retinopathy and inflammation caused by retinal injury. We administered NK1.1 depletion agents once at 2, 4, and 6 days after retinal laser injury, for a total of three injections. Figure 6 (A) Seven days after retinal laser injury, we found that compared with the IgG group, the NK1.1 scavenger group had Collagen IV lesions in the affected area. + Vascular area and Iba-1 + The number of infiltrating cells increased significantly ( Figure 6 The data from the BD study indicates that these cells have a protective effect against disease progression two days after retinal injury.

[0113] When we inject NK1.1 remover once 2 days before retinal laser injury ( Figure 7 Compared with the IgG group, the NK1.1 clearer group showed Collagen IV in the lesion area 7 days after retinal laser injury. + The vascular area was significantly reduced ( Figure 7 (B and C), Iba-1 + The number of microglia infiltrating was significantly reduced ( Figure 7 (D). In addition, an NK1.1 clearing agent was injected immediately after retinal laser injury ( Figure 7 Compared with the IgG group, the Collagen IV lesion area in the NK1.1 clearer group 7 days after retinal laser injury was significantly lower in the NK1.1 clearer group. + The vascular area was significantly reduced ( Figure 7 (FG), Iba-1 + The number of microglia infiltrating was significantly reduced ( Figure 7H).

[0114] The above studies suggest that clearing NK1.1+ cells immediately before or after retinal laser injury can significantly reduce retinal lesions caused by retinal laser injury and reduce inflammatory cell infiltration.

[0115] Example 3: NK1.1 remover significantly reduced retinal detachment-related lesions and inflammation.

[0116] To further clarify whether this phenomenon is specific to retinal laser damage, we investigated a mouse model of low intraocular pressure-induced retinal detachment (RD). Compared with healthy mice, RD model mice showed higher levels of CD45 in the retina 24 hours after retinal detachment. + NK1.1 + The number of cells increased significantly ( Figure 8 (A and B). Immediate intraperitoneal injection of anti-NK1.1 after retinal detachment ( Figure 8 After 3 days, compared with the IgG group, the NK1.1 scavenger group showed a significant decrease in TUNEL cells in the ONL layer and apoptotic cells. Figure 8 (D and E), NK1.1 scavenger group mice inner retina and subretinal Iba-1 + The number of microglia infiltrating was significantly reduced ( Figure 8 (F and G).

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of an NK1.1 depleting agent in the preparation of a medicament for treating and / or reducing retinal acute injury.

2. Use according to claim 1, wherein The NK1.1 depleting agent inhibits vascular hyperplasia.

3. Use according to claim 1 or 2, characterized in that, The NK1.1 depleting agent inhibits retinal neovascular lesion area.

4. Use according to any one of claims 1 to 3, characterized in that, The NK1.1 depleting agent reduces the number of innate lymphocytes.

5. The use according to claim 4, wherein the compound is ###0002### The innate lymphocytes include: NK cells and / or ILC1 cells.

6. Use according to any one of claims 1 to 5, characterized in that, The NK1.1 depleting agent reduces inflammatory cell infiltration.

7. Use according to claim 6, wherein The inflammatory cells include: Iba-1 + Microglia.

8. Use according to any one of claims 1 to 7, characterized in that, The NK1.1 depleting agent inhibits photoreceptor cell apoptosis.

9. Use according to any one of claims 1 to 8, characterized in that, The NK1.1 depleting agent is administered within 48 hours before retinal injury to 24 hours after injury.

10. Use according to any one of claims 1 to 9, wherein The NK1.1 depleting agent is administered at a dose of: 0.405 ~ 2.025 mg / kg / time.

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