Application of neuropeptide coded by Tac1 gene in preparation of medicine for preventing and / or treating ocular surface injury and keratitis

Abstract

The invention provides application of neuropeptide coded by a Tac1 gene in preparation of a medicine for preventing and / or treating ocular surface injury and keratitis, and belongs to the technical field of biological medicines. The invention provides application of neuropeptide coded by a Tac1 gene in preparation of a medicine for preventing and / or treating ocular surface injury related diseases and / or ocular inflammation. The neuropeptide coded by the Tac1 gene is a neuropeptide with an amino acid sequence shown as SEQ ID NO: 1 at the C end. The three neuropeptides coded by the Tac1 gene can inhibit inflammatory response and promote repair of ocular surface injury. Therefore, the invention provides a new target and a treatment strategy for clinical prevention and treatment of ocular surface diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to the use of a neuropeptide encoded by the Tac1 gene in preparing a drug for preventing and / or treating ocular surface damage and keratitis. Background Art

[0002] The cornea, the most densely innervated tissue in the human body, is primarily innervated by dense sensory nerve fibers originating from the trigeminal ganglion and a subset of autonomic nerves. These trigeminal nerve branches penetrate the peripheral corneal tissue and radially enter the Descemet's membrane, where they form the subbasal plexus. Ultimately, these nerve fibers terminate in the corneal epithelium, forming the corneal sensory nerve. This rich innervation not only allows the cornea to sense touch, pain, and temperature but also plays a key role in regulating tear secretion, the blink reflex, and wound healing. The Tac1 gene is widely expressed in the trigeminal nerve and encodes a family of tachykinins, including substance P (SP), neurokinin A (NKA), neuropeptide K (NPK), and neuropeptide γ (NPγ). TAC1 is located on human chromosome 7 and consists of seven exons and six introns, which can be alternatively spliced ​​into four different mRNA variants. SP has been shown to be involved in physiological processes such as neurogenic inflammation, wound healing, and pain perception, but the functions of other neuropeptides are less fully understood.

[0003] Due to external exposure, the cornea is susceptible to mechanical or chemical damage. The healing of the damaged cornea is a complex process that includes epithelial regeneration, inflammation, and cell proliferation in the early stages, and corneal stromal remodeling in the late stages. Among these events, balanced inflammation is essential for high-quality healing. If the injured cornea cannot heal completely and in a timely manner, the risk of microbial infection, corneal ulcers, and stromal opacity increases. Compared with synthetic drugs, natural products are often able to act on multiple molecular targets and signaling pathways, while having fewer side effects, and play a more comprehensive regulatory role in the treatment of inflammatory diseases. Therefore, finding natural anti-inflammatory ingredients is crucial for the treatment of wound healing in the body. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the use of neuropeptides encoded by the Tac1 gene in the preparation of drugs for preventing and / or treating ocular surface damage and keratitis. The Tac1 gene encodes neurokinin A (NKA), neuropeptide K (NPK) and neuropeptide γ (NPγ), which have the effect of inhibiting inflammation and promoting eye wound healing, providing new ideas for the treatment of eye diseases.

[0005] The present invention provides the use of a neuropeptide encoded by the Tac1 gene in the preparation of a medicament for preventing and / or treating ocular surface damage-related diseases and / or ocular inflammation. The neuropeptide encoded by the Tac1 gene is a neuropeptide having an amino acid sequence as shown in SEQ ID NO: 1 at its C-terminus.

[0006] Preferably, the neuropeptide encoded by the Tac1 gene includes at least one of the following: neurokinin A, neuropeptide K and neuropeptide γ.

[0007] Preferably, the amino acid sequence of neurokinin A is shown in SEQ ID NO: 2;

[0008] The amino acid sequence of the neuropeptide K is shown in SEQ ID NO: 3;

[0009] The amino acid sequence of the neuropeptide γ is shown in SEQ ID NO:4.

[0010] Preferably, the ocular surface damage includes corneal damage caused by physical factors and / or chemical factors.

[0011] Preferably, the ocular surface damage-related diseases include neurotrophic keratitis, corneal injury, corneal inflammation, wound healing, corneal inflammation and dry eye.

[0012] The present invention provides a drug for preventing and treating ocular surface damage or ocular inflammation-related diseases, wherein the active ingredient includes a neuropeptide encoded by the Tac1 gene;

[0013] The neuropeptide encoded by the Tac1 gene is a neuropeptide having an amino acid sequence as shown in SEQ ID NO: 1 at its C-terminus.

[0014] Preferably, the neuropeptide encoded by the Tac1 gene includes at least one of the following: neurokinin A, neuropeptide K and neuropeptide γ.

[0015] Preferably, the concentration of the active ingredient is 100-200 μM;

[0016] Preferably, the dosage form of the drug includes eye drops or conjunctival injections.

[0017] The present invention provides a use of a Tac1 gene knockout reagent or a neurokinin A receptor NK2R antagonist in constructing an animal model of ocular inflammation caused by ocular surface damage. The Tac1 gene knockout reagent includes a reagent that lacks the C-terminal amino acid sequence shown in SEQ ID NO: 1 of the neuropeptide encoded by the Tac1 gene.

[0018] Preferably, the neurokinin A receptor NK2R antagonist is L-424720; the concentration of L-424720 is 100-200 μM.

[0019] The present invention provides the use of a neuropeptide encoded by the Tac1 gene in the preparation of a drug for preventing and / or treating ocular surface damage-related diseases and / or ocular inflammation, wherein the neuropeptide encoded by the Tac1 gene is a neuropeptide having an amino acid sequence as shown in SEQ ID NO: 1 at its C-terminus. The present invention constructs genetically engineered mice by means of Tac1 gene knockout, uses Tac1 gene knockout mice (Tac1-KO mice) as research subjects, and observes ocular surface lesions. The results show that over time, ocular surface lesions develop in Tac1-KO mice and continue to worsen. At the same time, a corneal epithelial injury model is constructed using Tac1-KO mice as subjects. The results show that the corneas of Tac1-KO mice are turbid, wound closure is significantly delayed, and central corneal thickness is significantly increased, indicating that the knockout of the Tac1 gene affects the healing rate of corneal wounds in mice. At the same time, it can be seen from transcriptome data that the knockout of the Tac1 gene also leads to a significant upregulation of inflammation-related pathways in mice under injury conditions, inducing a significant inflammatory response. By supplementing the C-terminus with a neuropeptide having the amino acid sequence shown in SEQ ID NO:1, the ocular surface inflammation and corneal wound healing difficulties in the Tac1-KO mice were effectively reversed, rapidly repairing ocular surface damage and reducing ocular surface inflammatory responses. Further research into the molecular mechanisms by which neuropeptides regulate inflammatory responses and repair corneal damage revealed that the neuropeptide inhibits corneal inflammation and promotes corneal wound healing after injury by activating the downstream receptor NK2R. This application provides new therapeutic strategies and approaches for diseases related to ocular surface damage and ocular inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figures 2 and 3 show the progression of ocular surface pathology in Tac1-KO mice over time, where A is a gross photograph of the ocular surface of WT and Tac1-KO mice under steady-state conditions, B is a graph showing the central corneal thickness of WT and Tac1-KO mice under steady-state conditions, C is a statistical analysis of corneal transparency in WT and Tac1-KO mice under steady-state conditions, and D is a statistical analysis of central corneal thickness in WT and Tac1-KO mice under steady-state conditions;

[0021] Figure 2 The results of gene identification of Tac1-KO mice are shown in Figure 1, where A is the gene sequence of four tachykinin peptides, SP, NKA, NPγ, and NPK, encoded by the Tac1 gene; and B is the mRNA structure and corresponding sequence of the peptide products.

[0022] Figure 3Figures 2 and 3 show the results of ocular surface inflammation and healing tests in Tac1-KO mice under injury conditions, where A shows the morphology of the cornea of ​​Tac1-KO mice after injury, and B shows the opacity score of the cornea of ​​Tac1-KO mice after injury. C shows the results of fluorescein staining of the ocular surface of Tac1-KO mice under injury conditions, D shows the results of delayed wound closure in Tac1-KO mice, E shows the results of central corneal thickness measurements in Tac1-KO mice and normal mice, and F shows the changes in central corneal thickness in Tac1-KO mice and normal mice.

[0023] Figure 4 Figure 3 is the expression result of inflammation-related pathways in transcriptome sequencing of Tac1-KO mice under injury conditions, where A is the KEGG pathway enrichment result showing that most upregulated pathways in Tac1-KO mice after corneal injury are immune-related, B is the hierarchical clustering heat map showing that most genes in the process of neutrophil function after corneal injury in Tac1-KO mice are upregulated, C is Cytoscape showing that most genes in the process of neutrophil function, such as chemotaxis, migration, activation, degranulation, and mediated cytotoxicity, are upregulated, and D is the immunofluorescence staining result of corneal neutrophils (Ly6G) in WT and Tac1-KO mice under injury conditions;

[0024] Figure 5 These are the results of the effects of PBS, SP, NKA, NPK and NPγ supplementation on ocular surface inflammation and healing in Tac1-KO mice under injury conditions, among which A is a gross photograph of the ocular surface after supplementation with the six reagents after corneal injury, B is a statistical graph of corneal transparency scores after supplementation with the six reagents after corneal injury, C is the wound healing after supplementation with the six reagents after corneal injury, D is a statistical analysis graph of wound healing area after supplementation with the six reagents after corneal injury; E is the central corneal thickness after supplementation with the six reagents after corneal injury; F is a statistical analysis graph of central corneal thickness after supplementation with the six reagents after corneal injury.

[0025] Figure 6 Figure 1 shows the effects of PBS, NKA, NPK, and NPγ supplementation on neutrophils in Tac1 KO mice under injury conditions. A shows the number of neutrophils (Ly6G) stained 24 hours after corneal injury supplementation with the four reagents. B and C show the infiltration and statistical results of CD11b+Ly6G cells 24 hours after corneal injury supplementation with the four reagents.

[0026] Figure 7Figure 3: Effects of ocular surface inflammation and vascular conditions after alkali burn in normal mice after supplementation with PBS, SP, NKA, NPK, and NPγ. A is a gross photo of the ocular surface after alkali burn with the six reagents. B is a statistical graph showing corneal transparency scores after alkali burn with the six reagents. C is a statistical graph showing corneal neovascularization scores after alkali burn with the six reagents. D is a graph showing central corneal thickness after alkali burn with the six reagents. E is a statistical graph showing central corneal thickness after alkali burn with the six reagents.

[0027] Figure 8 Figure 1 is the result of blocking the promoting effect of downstream receptor NK2R on corneal inflammation, where A is a gross photo of eyes inoculated with PBS or L-424720 after corneal injury, B is the corneal wound healing of eyes inoculated with PBS or L-424720 after corneal injury, and C is the CD45 and Ly6G cell staining image 72 hours after corneal injury. DETAILED DESCRIPTION

[0028] The present invention provides the use of a neuropeptide encoded by the Tac1 gene in the preparation of a medicament for preventing and / or treating ocular surface damage-related diseases and / or ocular inflammation, wherein the neuropeptide encoded by the Tac1 gene is a neuropeptide having an amino acid sequence as shown in SEQ ID NO: 1 (FVGLM) at its C-terminus.

[0029] In the present invention, the neuropeptide encoded by the Tac1 gene preferably includes at least one of the following: neurokinin A, neuropeptide K, and neuropeptide γ, more preferably neuropeptide K. The amino acid sequence of neurokinin A is preferably as shown in SEQ ID NO: 2 (HKTDSFVGLM). The amino acid sequence of neuropeptide K is preferably as shown in SEQ ID NO: 3 (DADSSIEKQVALLKHRKHSIQGHGYLAKKTDSFVGLM). The amino acid sequence of neuropeptide γ is preferably as shown in SEQ ID NO: 4 (HSIQGHGADKRHKTDSFVGLM).

[0030] In the present invention, the ocular surface damage preferably includes corneal damage caused by physical and / or chemical factors. The physical factors preferably include mechanical damage. The chemical factors preferably include acid-base corrosion. The ocular surface damage-related diseases preferably include neurotrophic keratitis, corneal injury, corneal inflammation, wound healing, corneal inflammation, and dry eye.

[0031] In an embodiment of the present invention, the sequence of the neuropeptide encoded by the Tac1 gene is changed by gene knockout, which makes the mouse ocular surface susceptible to lesions, slows wound healing under corneal damage conditions and causes ocular inflammatory response; and replenishing each of the above neuropeptides or a combination of neuropeptides in gene knockout mice under damage conditions can promote the healing of mouse corneal wounds, downregulate inflammatory response, reduce neovascularization, and neuropeptide K has the best effect.

[0032] Based on the effects of the neuropeptide described in the above scheme in promoting the repair of ocular surface damage and reducing ocular surface inflammatory response, the present invention provides a drug for preventing and treating diseases related to ocular surface damage or ocular surface inflammation, the active ingredient of which includes a neuropeptide encoded by the Tac1 gene; the neuropeptide encoded by the Tac1 gene is a neuropeptide whose C-terminus has the amino acid sequence shown in SEQ ID NO: 1.

[0033] In the present invention, the neuropeptide encoded by the Tac1 gene preferably includes at least one of the following: neurokinin A, neuropeptide K, and neuropeptide γ. The sequence of the neuropeptide is the same as above and is not particularly limited here. The concentration of the active ingredient is preferably 100 to 200 μM. The dosage form of the drug preferably includes eye drops or conjunctival injections. The drug is prepared by selecting suitable excipients according to the type of dosage form. The dosage of the eye drops is 100 μM. The dosage of the conjunctival injection is 100 μM.

[0034] Given that knocking out the Tac1 gene and blocking the function of the neurokinin A receptor NK2R can effectively promote ocular surface inflammatory response and inhibit the repair of ocular surface damage, the present invention provides a reagent for knocking out the Tac1 gene or a neurokinin A receptor NK2R antagonist for use in constructing an animal model of ocular surface inflammation caused by ocular surface damage. The reagent for knocking out the Tac1 gene includes a reagent that lacks the amino acid sequence shown in SEQ ID NO: 1 at the C-terminus of the neuropeptide encoded by the Tac1 gene.

[0035] In the present invention, the animals preferably include commonly used animal models, such as mice, rats, rabbits, etc. The ocular surface damage preferably includes corneal damage caused by physical damage.

[0036] In the present invention, when the reagent is used to construct an animal model, the method of genetically engineering animals well known in the art can be used.

[0037] In the present invention, the neurokinin A receptor NK2R antagonist preferably includes L-424720. The concentration of L-424720 is preferably 100 to 200 μM. When using the neurokinin A receptor NK2R antagonist to establish an animal model, the neurokinin A receptor NK2R antagonist is preferably administered via eye drops to animals with corneal damage. When compared to the control group, a significant increase in CD45 and Ly6G-positive cells in the cornea indicates that NK2R plays an important role in anti-inflammatory effects.

[0038] The following describes in detail the use of the neuropeptide encoded by the Tac1 gene provided by the present invention in preparing drugs for preventing and / or treating ocular surface damage and keratitis in conjunction with the examples. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] Ocular surface pathogenesis in Tac1-KO mice over time under steady-state conditions

[0041] To investigate the association between Tac1 gene deficiency and ocular surface inflammation, the ocular surface of Tac1 knockout (Tac1-KO) mice and age-matched normal mice were observed for 21 months. Tac1-KO B6.Cg-Tac1tm1Bm / J mice (Tac1-KO mice, strain #004103) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Corneal transparency was scored on a scale of 0 to 4: 0 = completely transparent; 1 = slightly hazy, with the iris and pupil easily visible; 2 = slightly opaque, with the iris and pupil still detectable; 3 = opaque, with the pupil barely detectable; and 4 = completely opaque, with no pupil visible.

[0042] The results showed that at approximately 6 months of age, Tac1-KO mice exhibited ocular surface lesions that progressively worsened over time, ultimately leading to corneal neovascularization at approximately 15 months of age. Figure 1 In contrast, the normal mouse control group did not develop any ocular surface problems until approximately 18 months of age ( Figure 1 (A, C).

[0043] OCT results showed that at approximately 8 months, there was a significant difference in corneal thickness between normal mice and Tac1-KO mice, and this difference further increased over time ( Figure 1 (B, D).

[0044] Example 2

[0045] Gene identification of Tac1-KO mice

[0046] Tac1-KO mice were genotyped using a standard PCR method. The following steps were performed: Mouse tail tissue was removed and placed in an EP tube. 200 μL of 0.05 M NaOH was added to each tube and placed in a metal bath at 95°C for 30–40 min. Subsequently, 20 μL of 1 M Tris-10 mM EDTA (121.14) was added and mixed thoroughly. Each tube now contained mouse DNA. A 15 μL volume (7.5 μL of 2× Taq MasterMix, 0.5 μL of primer F (oIMR2032), 0.5 μL of primer R (oIMR2033), 3.5 μL of RNase-free water, and 3 μL of DNA) was placed in a PCR instrument and reacted using a cycling system. The reaction schedule is detailed in Table 1.

[0047] Table 1 Reaction system

[0048] 194℃ 5min 294℃ 30s 365℃ 30s-0.5℃ / cycle 468℃ 45s Repeat steps 2 to 4 for 10 cycles 594℃ 30s 660℃ 30s 772℃ 45s 872℃ 5min Repeat steps 6-8 for 28 cycles 910℃ Insulation

[0049] The primers for PCR amplification are as follows:

[0050] Tac1-F1: AGAATTTAAAGCTCTTTTGCC (SEQ ID NO: 5);

[0051] Tac1-R1: GCTCATCAGTATGTGACATAGAAA (SEQ ID NO: 6);

[0052] Tac1-F2: CTCCCCGGTAGACTTTGTGG (SEQ ID NO:7);

[0053] Tac1-R2: GGAAGGAGCAAGCATGCAGA (SEQ ID NO:8).

[0054] The gene sequences of the four tachykinins encoded by the Tac1 gene, SP, NKA, NPγ, and NPK ( Figure 2 Middle A). Figure 2 Center B shows the structure of the mouse Tac1 gene and the existence of messenger RNA splice variants, with the peptide products represented by horizontal stripes below the mRNA structure.

[0055] Example 3

[0056] Ocular surface inflammation and healing in Tac1-KO mice under injury conditions

[0057] The experimental animals were a model of physically injured mouse corneal epithelium. Mice were anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg / kg) and then topically anesthetized with lidocaine eye drops. A 3.00 mm trephine and epithelial scraper were then used to scrape the epithelium within a central 3.00 mm area of ​​the corneas of Tac1-KO mice and normal control mice. Ocular surface inflammation was observed using a slit lamp microscope at 24, 36, 48, 60, and 72 hours after modeling. Fluorescein staining was used to assess epithelial repair and thickness in the mice using optical coherence tomography (OCT).

[0058] Slit lamp examination images showed that the corneas of normal mice remained transparent after injury, while the corneas of Tac1-KO mice became cloudy 48 hours after injury ( Figure 3 (A, B).

[0059] Fluorescein staining was used to monitor the area of ​​corneal epithelial defect. Fluorescein staining results showed that wound closure was delayed in Tac1-KO mice compared with normal mice after corneal injury ( Figure 3 (C, D).

[0060] The method for fluorescein sodium staining is as follows: take three strips of fluorescein sodium ophthalmic test paper and dissolve them in an EP tube containing 1.5 mL of sterile saline, shake them evenly, and wrap them in aluminum foil for later use. Before taking pictures, drop 1-2 drops on the mouse eye surface, then rinse with saline and take pictures). In addition, OCT was used to evaluate the central corneal thickness (CCT). The method is: use an optical coherence tomography (OCT) scanner to take pictures of the anterior segment of the mouse eye, record the thickness data of the central cornea of ​​the mouse, and perform subsequent statistical analysis). About 48 hours after injury, a significant difference in CCT was observed between Tac1-KO mice and normal mice ( Figure 3 (E, F).

[0061] Example 4

[0062] Transcriptome sequencing of Tac1-KO mice under injury conditions revealed significant upregulation of inflammation-related pathways

[0063] To analyze the immune cells that play a major role in corneal inflammation, the physical injury mouse corneal epithelial model was successfully established as described above. Corneal tissue was collected 48 hours after surgery and sent to OE Biotech Co., Ltd. in Shanghai, China for subsequent RNA sequencing and biological analysis.

[0064] KEGG pathway enrichment showed that most upregulated pathways in Tac1-KO mice after corneal injury were immune-related ( Figure 4 Middle A).

[0065] The hierarchical clustering heat map provides relevant validation, and Tac1-KO mice have upregulated the expression of most genes in neutrophil-related pathways after corneal injury (see Figure 4Middle B).

[0066] By using Cytoscape to integrate gene expression data, the results showed that most genes in the immune-related pathways were upregulated in Tac1-KO mice after corneal injury, among which most genes involved in chemotaxis, migration, activation, degranulation, and mediated cytotoxicity in the process of neutrophil function were upregulated ( Figure 4 Middle C).

[0067] The hierarchical clustering heat map provides relevant validation, and the expression of most genes involved in neutrophil development in Tac1-KO mice after corneal injury is upregulated (see Figure 4 Middle D).

[0068] For immunofluorescence staining of whole corneal tissue, corneal tissues of normal mice and Tac1-KO mice 48 hours after epithelial injury were collected and the corneas were fixed in 4% paraformaldehyde on ice for 1 hour, and permeabilized and blocked overnight at 4°C in phosphate buffer containing 0.3% Triton X-100 and 5% bovine serum albumin. The corneas were then incubated with neutrophil marker Ly6G (mouse source) antibody at 4°C for 12 hours, and the corneal staining results were observed using a Zeiss LSM880 inverted microscope. The immunofluorescence staining results also showed that compared with the control group, neutrophil infiltration occurred in the cornea of ​​Tac1-KO mice (see Figure 4 Middle D).

[0069] Example 5

[0070] Ocular surface inflammation and healing in Tac1-KO mice after epithelial scraping and supplementation with PBS, SP, NPK, NPγ and NKA

[0071] The mouse corneal epithelial physical injury model was established according to Example 3.

[0072] To determine the effects of PBS, SP, NKA, NPK, and NPγ on corneal inflammation and corneal wound healing after epithelial abrasion, PBS, SP, NKA, NPK, and NPγ were topically applied 4 times daily at a concentration of 100 μM immediately after injury 3 days after injury. Control animals were treated with an equal volume of PBS eye drops, and slit-lamp photography was performed.

[0073] The results showed that the cornea of ​​the control group gradually became turbid 48 hours after injury. After treatment with five drugs, there was no significant difference between the SP treatment group and the PBS group, with high corneal turbidity and severe inflammation. The corneal turbidity of mice in the NKA, NPK and NPγ groups was reduced. Specifically, NPK showed the most significant effect, followed by NPγ and NKA ( Figure 5 Fluorescence staining results showed that the wound area of ​​mice treated with NKA, NPK, and NPγ decreased faster after injury than that of the control group ( Figure 5(C, D).

[0074] In addition to slit-lamp photography, animals also underwent OCT testing. In control eyes, high reflectivity of the corneal stroma was observed, which was consistent with the increased corneal opacity shown by slit-lamp examination. At 72 hours, corneas treated with NKA (157.4 ± 31.71 μm), NPK (140.3 ± 20.13 μm), and NPγ (143.8 ± 17.09 μm) showed lower CCT than those of damaged mice treated with PBS (218.1 ± 35.37 μm), while SP (210 ± 54.57 μm) did not differ significantly from the control group ( Figure 5 (E, F).

[0075] Example 6

[0076] Changes in neutrophils by scraping epithelium from Tac1-KO mice and supplementing with PBS, NPK, NPγ and NKA The physical injury model of mouse corneal epithelium was established according to Example 3.

[0077] To determine the effects of PBS, NKA, NPK, and NPγ on neutrophil recruitment during corneal inflammation, Tac1-KO mice were topically applied 100 μM PBS, NKA, NPK, and NPγ four times daily three days after injury. C57BL / 6J mice were also used as negative controls after epithelial scraping and PBS application. Corneal tissue was collected 24 hours after topical application and subjected to corneal fluorescence staining.

[0078] The results showed that compared with the control group, the number of neutrophils in Tac1-KO mice treated with PBS was the largest, and spread from the limbus to the center of the cornea, followed by NKA. Among them, the neutrophil infiltration in Tac1-KO mice treated with NPK and NPγ was less, and the neutrophil infiltration was closer to the limbus ( Figure 6 Middle A).

[0079] To characterize the kinetics of CD11b+Ly6G+ cell infiltration, corneas were collected 24 h after topical application of drugs by scraping the epithelium and analyzed by flow cytometry. Compared with PBS-treated mice (16.29%±2.9%), mice treated with NKA (9.12%±4.18%), NPK (5.5%±3.03%), and NPγ (7.5%±2.61%) showed a decrease in CD11b+Ly6G+ infiltration ( Figure 6 (B, C).

[0080] Example 7

[0081] Ocular surface inflammation and vascular status after alkali burn in normal mice after supplementation with PBS, SP, NKA, NPK and NPγ

[0082] To further explore the relationship between PBS, SP, NKA, NPK, and NPγ and inflammation, this example established an alkaline burn model in mice. A 2 mm diameter paper soaked in 1 M NaOH was placed on the central cornea of ​​normal mice for 30 seconds. Subsequently, on days 1, 3, 5, and 7, we monitored ocular surface inflammation using a BQ900 slit lamp (Haag Streit) and assessed corneal opacity using the aforementioned grading and scoring method. CCT was also measured.

[0083] Slit lamp photographs showed that the corneal inflammatory response and corneal neovascularization were reduced in mice treated with NKA, NPK, and NPγ compared with those treated with PBS and SP on day 7 after alkali burn, and these were scored ( Figure 7 AC). Neovascularization scoring method: Scoring is performed on a scale of 0-3. 0 = no neovascularization; 1 = neovascularization at the limbus; 2 = neovascularization across the limbus and close to the center of the cornea; 3 = neovascularization across the center of the cornea. The mouse cornea was divided into four quadrants for scoring, and the corneal neovascularization assessment of each quadrant was based on a scale of 0-3. On day 7, corneas treated with NPK (168.5±40.57μm), NPγ (174.8±42.48μm), and NKA (185.3±44.68μm) showed lower CCT ( ) than those of damaged mice treated with PBS (281.0±59.83μm) and SP (265.8±71.44μm). Figure 7 D and E).

[0084] Example 8

[0085] During corneal injury repair, inhibition of NK2R exacerbates inflammatory response

[0086] The mouse corneal epithelial physical injury model was established according to Example 3.

[0087] To further explore the relationship between the Tac1-KO gene-encoded products NKA, NPK, and NPγ and the receptor NK2R, an epithelial scraping model was established using C57BL / 6J mice, and then a NK2R antagonist (L-424720) was topically applied at a concentration of 100 μM.

[0088] The results showed that compared with the PBS-treated control group, corneal transparency decreased after NK2R antagonist administration, and the central cornea became cloudy ( Figure 8 Middle A), and corneal wound healing is also slowed ( Figure 8 Middle B).

[0089] Then, on the third day after the antagonist eye drop treatment, the mice were euthanized and their corneas were dissected for immunofluorescence staining. The results showed that compared with the PBS-treated control group, the number of CD45 and Ly6G-positive cells in the cornea increased after NK2R antagonist treatment ( Figure 8 Middle C). The results suggest that blocking the downstream receptor NK2R can promote corneal inflammation and slow down corneal wound healing after injury.

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a neuropeptide encoded by the Tac1 gene in the preparation of a drug for preventing and / or treating ocular surface damage-related diseases and / or ocular inflammation, wherein the neuropeptide encoded by the Tac1 gene is a neuropeptide having an amino acid sequence as shown in SEQ ID NO: 1 at its C-terminus.

2. The application according to claim 1, characterized in that: The neuropeptide encoded by the Tac1 gene includes at least one of the following: neurokinin A, neuropeptide K and neuropeptide γ.

3. The application according to claim 2, characterized in that: The amino acid sequence of neurokinin A is shown in SEQ ID NO: 2; The amino acid sequence of the neuropeptide K is shown in SEQ ID NO: 3; The amino acid sequence of the neuropeptide γ is shown in SEQ ID NO:

4.

4. The use according to any one of claims 1 to 3, characterized in that: The ocular surface damage includes corneal damage caused by physical factors and / or chemical factors.

5. The use according to claim 4, characterized in that: The ocular surface damage-related diseases include neurotrophic keratitis, corneal injury, corneal inflammation, wound healing, corneal inflammation and dry eye.

6. A drug for preventing and treating ocular surface damage or ocular inflammation-related diseases, characterized in that: The active ingredients include the neuropeptide encoded by the Tac1 gene; The neuropeptide encoded by the Tac1 gene is a neuropeptide having an amino acid sequence as shown in SEQ ID NO: 1 at its C-terminus.

7. The drug according to claim 6, characterized in that: The neuropeptide encoded by the Tac1 gene includes at least one of the following: neurokinin A, neuropeptide K and neuropeptide γ.

8. The drug according to claim 6 or 7, characterized in that The concentration of the active ingredient is 100 to 200 μM; Preferably, the dosage form of the drug includes eye drops or conjunctival injections.

9. Use of a Tac1 gene knockout agent or a neurokinin A receptor NK2R antagonist in constructing an animal model of ocular inflammation caused by ocular surface damage, wherein the Tac1 gene knockout agent comprises an agent lacking the amino acid sequence shown in SEQ ID NO: 1 at the C-terminus of the neuropeptide encoded by the Tac1 gene.

10. The use according to claim 9, characterized in that: The neurokinin A receptor NK2R antagonist is L-424720; The concentration of L-424720 is 100-200 μM.

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