Application of STAT3 agonist ML115 in preparation of medicine for preventing and / or treating corneal epithelium lesion

Abstract

The invention provides application of an STAT3 agonist ML115 in preparation of a medicine for preventing and / or treating corneal epithelium lesion, and belongs to the technical field of biological medicine preparation. The invention relates to an application of ML115 in preparation of a medicine for preventing and / or treating corneal epithelium lesion. Experiments show that diabetes can cause down-regulation of expression of STAT3 in corneal epithelium cells and delay healing of mouse corneal epithelium wounds, while addition of the STAT3 agonist ML115 can reverse the healing delay phenomenon of diabetic mouse corneal epithelium wounds and promote repair of the corneal epithelium wounds. Therefore, the technical scheme provided by the invention provides a new treatment means for repairing the corneal epithelium lesion.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceutical preparation technology, specifically relating to the use of the STAT3 agonist ML115 in the preparation of drugs for the prevention and / or treatment of corneal epithelial lesions. Background Technology

[0002] Corneal epithelial lesions manifest as ocular irritation symptoms such as redness, stinging, photophobia, tearing, foreign body sensation, and dryness; some patients may experience mild symptoms due to corneal nerve dysfunction. In addition, there are visual function-related symptoms such as blurred vision, fluctuating or decreased visual acuity. Corneal epithelial lesions can lead to complications such as corneal perforation, corneal scarring, and decreased vision. Diabetic patients are prone to primary corneal lesions, and the main pathogenesis includes: corneal neuropathy, abnormal growth factor expression, excessive inflammatory response, abnormal limbal stem cells, abnormal oxidative stress response, and abnormal accumulation of glucose metabolites (such as AGEs, advanced glycation end products).

[0003] Currently, drugs that promote corneal epithelial healing mainly include autologous serum eye drops, calf blood deproteinized ophthalmic gel, and anti-inflammatory and antibacterial drugs such as glucocorticoids; drugs that nourish nerves and promote nerve growth include neurotrophic factors. These drugs can be widely used to treat various types of corneal epithelial damage and promote the repair of corneal nerves. However, there is a lack of specific drugs for epithelial damage in diabetic patients, and some patients, due to the severity of their condition, can only ultimately use surgery to stop further corneal damage. Summary of the Invention

[0004] The purpose of this invention is to provide an application of ML115 in the preparation of drugs for the prevention and / or treatment of corneal epithelial lesions. ML115 promotes the repair of corneal epithelial wounds by increasing the expression level of STAT3 in limbal stem cells, thereby enhancing the proliferation and cloning ability of limbal stem cells.

[0005] This invention provides the use of ML115 in the preparation of medicaments for the prevention and / or treatment of corneal epithelial lesions.

[0006] Preferably, the corneal epithelial lesion includes at least one of the following: corneal epithelial lesions caused by diabetes, limbal lesions caused by physical or chemical means, and corneal epithelial lesions caused by autoimmune diseases.

[0007] Preferably, the corneal epithelial lesion includes delayed corneal epithelial healing.

[0008] Preferably, the use of the ML115 in combination with a STAT3 agonist in the preparation of a medicament for the prevention and / or treatment of corneal epithelial lesions.

[0009] Preferably, the drug comprises at least one of the following dosage forms: eye drops, eye ointment, gel, and conjunctival injection.

[0010] This invention provides a medicament for treating corneal epithelial lesions, the active ingredient of which includes ML115 or a combination of ML115 and a STAT3 agonist.

[0011] Preferably, the drug includes pharmaceutically acceptable excipients.

[0012] This invention provides the application of a reagent for detecting STAT3 expression in the preparation of diagnostic reagents or kits for corneal epithelial lesions caused by diabetes.

[0013] Preferably, the reagent for detecting STAT3 expression includes reagents for detecting STAT3 protein expression and / or reagents for detecting STAT3 gene expression.

[0014] Preferably, the reagents for detecting STAT3 protein expression include reagents for immunoblotting or immunofluorescence detection.

[0015] This invention provides the application of ML115 in the preparation of drugs for the prevention and / or treatment of corneal epithelial lesions. Experiments of this invention show that administration of ML115 can enhance the proliferation and cloning ability of human and mouse limbal stem cells, and can increase the expression of target genes or proteins in the cell cycle signaling pathway. Therefore, the technical solution provided by this invention not only expands the pharmaceutical application scope of ML115, but also provides a new therapeutic approach for the repair of corneal epithelial lesions.

[0016] This invention provides the application of a reagent for detecting STAT3 expression in the preparation of diagnostic reagents or kits for diabetic corneal epithelial lesions. Experiments demonstrated by this invention show that diabetes delays the healing time of corneal epithelial damage; simultaneously, transcriptomic analysis results indicate differential STAT3 expression in diabetic-induced corneal epithelial stem cells and normal corneal epithelial stem cells, with downregulated expression in diabetic-induced corneal epithelial stem cells. Furthermore, this invention verifies that the STAT3 expression agonist ML115 can effectively reverse the delayed healing phenomenon of human and mouse corneal epithelial wounds and promote corneal epithelial wound repair. Therefore, this invention provides the application of STAT3 expression as a target in the preparation of diagnostic reagents or kits for diabetic corneal epithelial lesions. Attached Figure Description

[0017] Figure 1 Results of corneal epithelial injury healing in normal and diabetic mice; Figure 2 Transcriptome data results from healthy individuals and diabetic patients; Figure 3The proliferation and cloning results of diabetic limbal stem cells after application of ML115 are shown in Figure A, which is the crystal violet staining result of the cell cloning assay; Figure B is the quantitative analysis result of the cloning assay; and Figure C is the proliferation assay result. Figure 4 Results of ML115 upregulating the expression of target genes in the cell cycle signaling pathway in HLECS; Figure 5 The results show the upregulation of target gene expression in the cell cycle signaling pathway in diabetic mice by ML115. A represents the results of fluorescein sodium staining of corneal epithelial wounds, B represents the statistical results of corneal epithelial wound area, C represents the results of immunoblotting, D represents the statistical results of target gene expression in the cell cycle signaling pathway, and E represents the results of immunofluorescence detection. Detailed Implementation

[0018] This invention provides the use of ML115 in the preparation of medicaments for the prevention and / or treatment of corneal epithelial lesions.

[0019] In this invention, the corneal epithelial lesion preferably includes at least one of the following: corneal epithelial lesions caused by diabetes, limbal lesions caused by physical or chemical means, and corneal epithelial lesions caused by autoimmune diseases. The corneal epithelial lesion preferably includes delayed corneal epithelial healing.

[0020] In this invention, diabetic corneal epithelial lesions mainly manifest as decreased or absent corneal sensation, superficial punctate keratosis, persistent corneal epithelial erosion, and delayed healing of epithelial wounds, which can develop into corneal ulcers. In one embodiment of this invention, diabetic corneal epithelial lesions are characterized by delayed healing of corneal epithelial wounds and downregulation of STAT3 expression.

[0021] In this invention, the physical factors in the physically caused limbal lesions preferably include at least one of the following: mechanical damage, temperature damage, pressure damage, and radiation damage. The chemical factors in the chemically caused limbal lesions preferably include at least one of the following: chemical substance deposition and staining, chemical eye irritation or burns, eye allergic reactions caused by chemical substances, and damage caused by chemical poisoning.

[0022] In this invention, the corneal epithelial lesions caused by autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, scleroderma, primary adrenal cortical atrophy, chronic thyroiditis, juvenile diabetes mellitus, hyperthyroidism, etc.

[0023] In this invention, ML115 is preferably used in combination with a STAT3 expression agonist in the preparation of a medicament for the prevention and / or treatment of corneal epithelial lesions. In one embodiment of this invention, experimental results demonstrate that STAT3 expression is downregulated in response to the occurrence of corneal epithelial lesions. In another embodiment of this invention, it is demonstrated that ML115, as a known STAT3 expression inhibitor, can reverse delayed healing of human and murine corneal epithelial wounds and promote the repair of corneal epithelial wounds.

[0024] In this invention, the drug preferably comprises at least one of the following dosage forms: eye drops, eye ointment, gel, and conjunctival injection. This invention does not impose any particular limitation on the preparation method of the drug; any drug preparation method well known in the art can be used. The concentration of ML115 in the drug is preferably 50 μM or higher, and can be 60 μM to 100 μM.

[0025] This invention provides a medicament for treating corneal epithelial lesions, the active ingredient of which includes ML115 or a combination of ML115 and a STAT3 expression agonist.

[0026] In this invention, the drug preferably includes pharmaceutically acceptable excipients. The drug preferably includes at least one of the following dosage forms: eye drops, eye ointment, gel, and conjunctival injection. The drug preferably also includes pharmaceutically acceptable excipients. The selection and addition of the excipients can be conventionally performed according to the preparation of different dosage forms.

[0027] This invention provides the application of a reagent for detecting STAT3 expression in the preparation of diagnostic reagents or kits for corneal epithelial lesions caused by diabetes.

[0028] In this invention, the reagent for detecting STAT3 expression preferably includes reagents for detecting STAT3 protein expression and / or reagents for detecting STAT3 gene expression. The reagent for detecting STAT3 gene expression preferably uses primers for RT-qPCR amplification and a PCR amplification premix. This invention does not impose any particular restrictions on the sequence of the primers, as long as they are primers capable of specifically amplifying the STAT3 gene. The reagent for detecting STAT3 protein expression preferably includes reagents for immunoblotting or immunofluorescence detection. The immunoblotting reagent preferably includes an anti-STAT3 protein antibody, an enzyme-labeled secondary antibody, and a substrate. The immunofluorescence detection reagent preferably includes an antibody labeled with a fluorescent dye.

[0029] In this invention, the preferred method for using the diagnostic reagent or kit is to detect the STAT3 expression level in the sample. If the STAT3 expression level in the test sample is significantly downregulated compared to that of a reference sample, it indicates a higher risk of diabetic corneal epithelial lesions in the test sample; conversely, it indicates a lower risk. The reference sample is a medically diagnosed sample without diabetic corneal epithelial lesions.

[0030] The following detailed description, in conjunction with embodiments, illustrates the application of the STAT3 agonist ML115 provided by the present invention in the preparation of drugs for the prevention and / or treatment of corneal epithelial lesions. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 Diabetes-induced delayed healing of corneal epithelial damage in mice A diabetic mouse model was established using intraperitoneal injection of streptozotocin (STZ), with healthy mice serving as the control group (n=6 per group). Corneal epithelium was scraped from both the control and diabetic mice. At 24 and 36 hours post-scraping, the corneal epithelial wound defects were stained with sodium fluorescein, and the area of ​​epithelial defects was analyzed using ImageJ software.

[0032] See results Figure 1 The results showed that, compared with normal mice, diabetes caused delayed corneal wound healing, and there was a significant difference in corneal epithelial healing time.

[0033] Example 2 Transcriptome sequencing analysis of limbal stem cells derived from diabetes and limbal stem cells from healthy individuals was specifically commissioned to Qingdao Ouyi Biotechnology Co., Ltd.

[0034] See results Figure 2 Transcriptome sequencing results showed that the cell cycle signaling pathway was significantly downregulated in limbal stem cells derived from diabetic patients, and STAT3 expression was also downregulated in limbal stem cells from diabetic patients.

[0035] Example 3 The application of ML115 promotes the proliferation and cloning ability of limbal stem cells in diabetic patients. Cell cloning and proliferation experiments were conducted using limbal stem cells derived from diabetic patients, with exogenous addition of... ML115 The specific experimental steps are as follows: HLESCs were isolated from limbal tissue of type 2 diabetic patients: limbal epithelial tissue was placed in DMEM containing 15 mg / mL dispersin II (Roche, Indianapolis, USA) and incubated overnight at 4°C. The epithelial cells were then further incubated for 15 minutes at 37°C using 0.25% trypsin / 0.02% EDTA (Thermo Fisher Scientific, USA). Cells were resuspended in DMEM / F-12 medium (Invictus Company) supplemented with the following components: 10% fetal bovine serum (FBS, Keppel Corporation, Grand Island, NY, USA), insulin-transferrin-selenium (ITS, Invictus Company), 1% non-essential amino acids (Invictus Company), 0.1 nM cholera toxin (Sigma-Aldrich, USA), 2 nM 3-oxocholesterol (Sigma-Aldrich, USA), 2 nM 3,3',5-triiodo-L-thyroxine (T3; Sigma-Aldrich, USA), 0.4 nM dexamethasone (Wako Pure Chemical Industries, Osaka, Japan), 2 nM L-glutamine (Invictus Company), penicillin-streptomycin (Hyclon, Logan, Utah, USA), 10 ng / mL recombinant human epidermal growth factor (EGF; R&D Corporation, USA), and 10 μM Y27632 (MCE, Shanghai, China). Primary HLESCs were cultured on NIH 3T3 feeder layers that had been inactivated by mitomycin C treatment. When the cell confluence reached 80%-90%, the cells were passaged and used in experiments.

[0036] Cloning experiments: (1) Discard the old culture medium and wash the cells twice with PBS; (2) Add 1 ml of 1× trypsin to each well and digest for 15 min; (3) Add 2 ml of complete culture medium to each well to stop digestion; (4) Transfer the cell suspension to a 15ml centrifuge tube and centrifuge at 1200r for 3 minutes; (5) Gently aspirate the supernatant, collect the precipitated cells, add 1 ml of complete culture medium and mix well by pipetting; (6) Take 10 μL and inject it into the cell counting chamber, and insert it into the fully automated cell counter for counting; (7) Calculate the number of cells based on cell count, and inoculate them on the feeder layer using a 12-well plate with 1000 cells / 1ml of culture medium per well; (8) After continuous culture for 3-4 days, add ML115 (0.5µM) or solvent solution of equal concentration, and then observe the cloning status; (9) Continue culturing for 3-4 days. After observing that the clonal cluster has basically formed, remove the well plate, add 1× trypsin diluted 10 times to digest for 3 minutes, and then continuously pipette the cells until the feeder cells are completely detached.

[0037] (10) Add 500 μl of crystal violet staining solution (Beyotime Biotechnology Co., Ltd., China) to each well, stain for 5 min, wash with PBS 3 times, then take a picture under white light and use ImageJ software to count the number of colonies.

[0038] Proliferation experiment: (1) Discard the old culture medium and wash the cells twice with PBS; (2) Add 1 ml of 1× trypsin to each well and digest for 15 min; (3) Add 2 ml of complete culture medium to each well to stop digestion; (4) Transfer the cell suspension to a 15ml centrifuge tube and centrifuge at 1200rpm for 3 minutes; (5) Gently aspirate the supernatant, collect the precipitated cells, add 1 ml of complete culture medium and mix well by pipetting; (6) Take 10 μl and inject it into the cell counting plate, and insert it into the fully automated cell counter for counting; (7) Calculate the number of cells based on the cell count, and seed the cells using a 96-well plate with 5000 cells / 100μl of culture medium per well; (8) After 12 hours of cell adhesion, replace with starvation medium without FBS; (9) After 12 hours, ML115 (0.5µM) was added, and the cell growth status was then observed; (10) When the control group cells reached 60%~70% fusion, the samples were collected and tested using the CCK8 kit (Beijing Bio-Sens Biotechnology Co., Ltd., China). 10 μl of CCK8 hypersensitive culture medium was added to each well and incubated at 37°C for 2 hours. (11) Two hours later, remove the well plate, remove the well plate cap, put it into a preheated microplate reader and measure the absorbance at a wavelength of 450 nm, and then perform standard data analysis.

[0039] See results Figure 3 Compared with the control group, the number of limbal stem cell clones in diabetic patients with added ML115 was significantly increased, and their proliferation capacity was also significantly enhanced. This indicates that exogenous addition of ML115 is beneficial in promoting the proliferation and cloning capacity of limbal stem cells in diabetic patients.

[0040] Example 4 The application of ML115 upregulated the expression of target genes in the cell cycle signaling pathway in HLECS. Human limbal stem cells provided by diabetic donors were cultured for 48 hours with 0.5 µM ML115. Cell proteins were extracted, and the expression levels of CCNB2, CDC20, and CDC25B proteins were detected by Western blot.

[0041] See results Figure 4 The results showed that, compared with untreated cells, the expression of target proteins in the cell cycle signaling pathway (CCNB2, CDC20, and CDC25B proteins) was upregulated after exogenous addition of ML115.

[0042] Example 5 The application of ML115 upregulated the expression of target genes in the cell cycle signaling pathway in diabetic mice. Corneal epithelium was scraped from diabetic mice, and the mice were cultured with or without topical ML115. At 24 and 36 hours post-scraping, corneal epithelial wound defects were stained with sodium fluorescein, and the area of ​​epithelial defects was analyzed using ImageJ software. Subsequently, corneal epithelial proteins were extracted, and the expression of target proteins (CCNB2, CDC20, and CDC25B proteins) in the cell cycle signaling pathway was detected using Western blot and immunofluorescence staining.

[0043] See results Figure 5 Fluorescein staining results showed that the addition of ML115 promoted the healing of corneal epithelial wounds. Western blot and immunofluorescence staining results showed that, compared with the control group, the expression of target genes in the cell cycle signaling pathway was upregulated in mice with exogenous ML115 addition.

[0044] 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. The use of ML115 in the preparation of drugs for the prevention and / or treatment of corneal epithelial lesions.

2. The application according to claim 1, characterized in that, The corneal epithelial lesions include at least one of the following: corneal epithelial lesions caused by diabetes, limbal lesions caused by physical or chemical means, and corneal epithelial lesions caused by autoimmune diseases.

3. The application according to claim 1, characterized in that, The corneal epithelial lesions include delayed corneal epithelial healing.

4. The application according to claim 1, characterized in that, The use of ML115 in combination with a STAT3 agonist in the preparation of medicaments for the prevention and / or treatment of corneal epithelial lesions.

5. The application according to claim 1 or 4, characterized in that, The drug includes at least one of the following dosage forms: eye drops, eye ointment, gel, and conjunctival injection.

6. A drug for treating corneal epithelial lesions, characterized in that, The active ingredients include ML115 or a combination of ML115 and a STAT3 agonist.

7. The drug according to claim 6, characterized in that, The drug includes pharmaceutically acceptable excipients.

8. The use of a reagent for detecting STAT3 in the preparation of diagnostic reagents or kits for diabetic corneal epithelial lesions.

9. The application according to claim 8, characterized in that, The reagents for detecting STAT3 expression include reagents for detecting STAT3 protein expression and / or reagents for detecting STAT3 gene expression.

10. The application according to claim 9, characterized in that, The reagents for detecting STAT3 protein expression include reagents for immunoblotting or immunofluorescence detection.

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