Use of hippo signaling pathway inhibitor in preparation of corneal endothelial repair drugs

Abstract

The application discloses application of a Hippo signal pathway inhibitor in preparation of a corneal endothelial repair drug, and the Hippo signal pathway inhibitor comprises at least one of an MST1 / 2 kinase inhibitor, a LATS1 / 2 inhibitor and a MAP4Ks inhibitor. Research results show that the Hippo signal pathway inhibitor promotes YAP nuclear entry by inhibiting YAP phosphorylation, thereby promoting corneal endothelial cell cycle progression, and further promoting corneal endothelial cell proliferation, and significantly improves corneal edema and corneal turbidity caused by corneal endothelial damage at an animal level, and has an obvious promoting effect on damage healing and function reconstruction of the corneal endothelial cells. Animal and cell experiments prove that the Hippo signal pathway inhibitor can significantly promote corneal endothelial cell regeneration.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the application of Hippo signaling pathway inhibitors in the preparation of corneal endothelial repair drugs. Background Technology

[0002] Corneal endothelial cells (CECs) play a crucial role in maintaining corneal transparency. Tight junctions (ZO-1) and adhesion junctions (N-cadherin) formed between CECs create a physical barrier between the aqueous humor and the stroma. This physical barrier, along with the active "fluid pump" function of the CECs, maintains the relative dehydration of the corneal stroma, thus preserving corneal transparency. Therefore, CECs play a key role in maintaining normal vision. Damage to the structure or function of the CECs, regardless of the cause, can lead to corneal edema, opacity, and in severe cases, vision loss.

[0003] The proliferative capacity of cell emulsions (CECs) varies significantly among different species. CECs from rabbits, pigs, and mice exhibit strong proliferative capacity both in vivo and in vitro, while CECs from cats, monkeys, and adult humans show virtually no mitotic activity in vivo and have very limited proliferative capacity in vitro. However, under the influence of strong mitotic-promoting factors, adult human CECs can enter and complete the cell cycle, possessing regenerative potential. Studies suggest that although adult human CECs possess mitotic potential, their cell cycle remains permanently locked in the G1 phase, preventing them from entering a complete cell cycle and completing proliferation.

[0004] Because adult corneal endothelial cells (CECs) cannot regenerate in the body, corneal endothelial diseases caused by corneal endothelial dysfunction have become one of the most serious causes of blindness in corneas. Currently, the most effective treatment for corneal endothelial dysfunction is corneal transplantation. However, due to the extreme scarcity of corneal donors, less than 1.5% of patients are eligible for corneal transplantation, which limits the widespread application of this therapy.

[0005] The Hippo signaling pathway is a highly conserved pathway composed of a series of protein kinases and transcriptional coactivators that regulate cell proliferation and apoptosis, playing a crucial role in embryonic development and tissue / organ growth. Recent studies have shown that the Hippo signaling pathway plays an important role in the development and regeneration of various tissues and organs, including the heart, liver, and muscles. However, the role of the Hippo signaling pathway in corneal endothelial cells remains unreported. Summary of the Invention

[0006] Based on the above issues, this application provides the application of Hippo signaling pathway inhibitors in the preparation of corneal endothelial repair drugs.

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

[0008] This invention provides the use of Hippo signaling pathway inhibitors (including pharmaceutically acceptable salts, isomers, solvates, and metabolites thereof) in the preparation of corneal endothelial repair drugs.

[0009] Optionally, the Hippo signaling pathway inhibitor includes at least one of MST1 / 2 kinase inhibitors, LATS1 / 2 inhibitors, and MAP4Ks inhibitors. The MST1 / 2 kinase inhibitors include, but are not limited to, XMU-MP-1; the LATS1 / 2 kinase inhibitors include, but are not limited to, LATS-IN-1; and the MAP4Ks inhibitors include, but are not limited to, DMX-5804.

[0010] This invention also provides the application of YAP gene overexpression promoters in the preparation of corneal endothelial repair drugs. The YAP gene overexpression promoters include the aforementioned Hippo signaling pathway inhibitors. Hippo signaling pathway inhibitors promote YAP nuclear translocation by inhibiting YAP phosphorylation. This invention reveals that YAP gene overexpression can promote corneal endothelial cell cycle progression and promote corneal endothelial cell proliferation.

[0011] The present invention also provides a corneal endothelial repair drug comprising at least one of the above-mentioned Hippo signaling pathway inhibitors or their pharmaceutically acceptable salts, isomers, solvates, and metabolites at a pharmacologically effective concentration.

[0012] In this invention, the dosage form of the drug is any pharmaceutically acceptable dosage form.

[0013] Preferably, the drug dosage form is an ophthalmic preparation, including eye drops, ophthalmic injections, ophthalmic ointments, or ophthalmic gels.

[0014] Preferably, the drug further contains pharmaceutically acceptable excipients, including any one or a combination of at least two of the following: carrier, diluent, binder, wetting agent, disintegrant, emulsifier, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0015] On the other hand, the present invention provides the use of the above-mentioned drug in the preparation of drugs or medical devices that promote the repair, growth, migration, survival, cell adhesion, proliferation and / or differentiation, cell cycle progression, corneal endothelial function regeneration, reconstruction and / or maturation of corneal endothelial cells.

[0016] On the other hand, the present invention provides the application of the above-mentioned drugs in the preparation of drugs or medical devices for corneal endothelial cells to resist oxidative damage, inflammation, and apoptosis, reverse corneal endothelial-mesenchymal transition, and maintain the homeostasis of the corneal endothelial cell microenvironment.

[0017] On the other hand, the present invention provides the application of the above-mentioned drugs in the preparation of drugs or medical devices for relieving or treating corneal endothelial damage, corneal endothelial loss, corneal endothelial lesions, corneal endothelial dysfunction, and corneal endothelial cell decompensation.

[0018] On the other hand, the present invention provides the use of the above-mentioned drug in the preparation of drugs or medical devices for treating or relieving symptoms such as pain, blurred vision, and corneal edema caused by corneal endothelial dysfunction.

[0019] On the other hand, the present invention provides the application of the above-mentioned drugs in the preparation of corneal endothelial grafts, artificial corneal endothelium and artificial full-thickness corneas or medical devices.

[0020] On the other hand, the present invention provides that the above-mentioned drug promotes the proliferation and functional repair of corneal endothelial cells by promoting the nuclear translocation of YAP protein in corneal endothelial cells.

[0021] In this invention, the above-mentioned drug is applicable to the eyes of mammals, including but not limited to humans, rabbits, mice, rats, guinea pigs, cats, dogs, cattle, sheep, pigs, and monkeys.

[0022] The present invention also provides a method for promoting corneal endothelial repair: inhibiting the Hippo signaling pathway and / or promoting YAP nuclear translocation, thereby promoting corneal endothelial cell regeneration.

[0023] In summary, the beneficial effects of this invention are as follows: This invention provides the application of Hippo signaling pathway inhibitors in the preparation of corneal endothelial repair drugs. Research results show that Hippo signaling pathway inhibitors, by inhibiting YAP phosphorylation and promoting YAP nuclear translocation, promote the corneal endothelial cell cycle and proliferation. Furthermore, in animal studies, they significantly improve corneal edema and corneal opacity caused by corneal endothelial injury, and have a significant promoting effect on the healing and functional reconstruction of corneal endothelial cells. Animal and cell experiments have confirmed that Hippo signaling pathway inhibitors can significantly promote corneal endothelial cell regeneration, providing a new target and approach for the treatment of corneal endothelial decompensation-related diseases, and possessing significant clinical application prospects and value. Attached Figure Description

[0024] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0025] Figure 1This study demonstrates how the Hippo signaling pathway inhibitor XMU-MP-1 promotes the proliferation of human corneal endothelial cells (HCECs). (A) Fluorescent staining results of proliferation markers Ki67 and EdU after HCECs were treated with XMU-MP-1 for 12 hours; (B) Statistical graph of the proportion of Ki67-positive cells in the XMU-MP-1 treatment group and the control group; (C) Statistical graph of the proportion of EdU-positive cells in the XMU-MP-1 treatment group and the control group; (D) Effect of different concentrations of XMU-MP-1 on the proliferation rate of HCECs observed by the CCK-8 assay. The scale bar in the figures is 100 μm.

[0026] Figure 2 The Hippo signaling pathway inhibitor XMU-MP-1 promotes cell cycle progression in HCECs. (A) Cell cycle distribution map analyzed by flow cytometry after HCECs were treated with XMU-MP-1 for 12 hours; (B) Cell cycle ratio map of the XMU-MP-1 treatment group and the control group; (CF) Changes in relative gene expression levels of CCND3, CCNE1, CCNA2, and E2F2 detected by real-time quantitative PCR after HCECs were treated with XMU-MP-1 for 12 hours.

[0027] Figure 3 The Hippo signaling pathway inhibitor XMU-MP-1 promotes the activation of YAP protein in HCECs. (A) Western blot results showing the changes in phosphorylation levels of YAP protein in HCECs after 12 hours of XMU-MP-1 treatment; (B) Statistical graph of YAP phosphorylation levels in the XMU-MP-1 treated group and the control group; (C) Immunofluorescence staining localization results of YAP protein in HCECs after 12 hours of XMU-MP-1 treatment; (D) Statistical graph of the nucleocytoplasmic localization ratio of YAP in the XMU-MP-1 treated group and the control group. The scale bar in the figure is 20 μm.

[0028] Figure 4 The Hippo signaling pathway inhibitor XMU-MP-1 promotes the healing of corneal endothelial injury in mice. (A) Slit-lamp observation of corneal opacity changes after XMU-MP-1 treatment in a mouse model of corneal endothelial injury; (B) Statistical graph of corneal opacity scores in the XMU-MP-1 treatment group and the control group; (C) OCT assessment of corneal thickness changes after XMU-MP-1 treatment in a mouse model of corneal endothelial injury; (D) Statistical graph of corneal thickness in the XMU-MP-1 treatment group and the control group; (E) Alizarin red staining to observe the corneal endothelial healing in mice after XMU-MP-1 treatment.

[0029] Figure 5The Hippo signaling pathway inhibitor XMU-MP-1 promotes corneal endothelial proliferation and functional reconstruction in mice. (A) Immunofluorescence staining results of ZO-1 after XMU-MP-1 treatment in a mouse corneal endothelial injury model; (B) Na+ staining results after XMU-MP-1 treatment in a mouse corneal endothelial injury model. + -K + (C) Statistical graph of ZO-1 staining fluorescence intensity in the XMU-MP-1 treatment group and the control group; (D) Statistical graph of Na in the XMU-MP-1 treatment group and the control group. + -K + ATPase staining fluorescence intensity statistics; (E) EdU-positive cell count statistics in the XMU-MP-1 treatment group and control group; (F) DAPI-positive cell count statistics in the XMU-MP-1 treatment group and control group. Scale bars in the figures are 100 μm. Detailed Implementation

[0030] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0031] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0032] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0033] Example 1: Effect of Hippo signaling pathway inhibitor XMU-MP-1 on corneal endothelial cell (HCEC) proliferation.

[0034] (1) Immunofluorescence and EdU labeling staining: 1*10 5HCECs were seeded in 3.5 cm confocal dishes and cultured at 37°C for 24 hours. EdU (5-ethynyl-2'-deoxyuridine) was added to the culture medium to a final concentration of 20 μM, along with 250 nM XMU-MP-1 (an inhibitor of MST1 / 2, purchased from MCE, Cat#HY-100526). The control group received an equal volume of DMSO (dimethyl sulfoxide). After culturing for another 8 hours, the culture medium was removed, and cells were fixed for 10 minutes using cell fixative. The expression of the proliferation marker Ki67 was detected by immunofluorescence. EdU imaging was then performed using the Click-iTEdU Alexa Fluor 647 imaging kit, and images were acquired using confocal fluorescence microscopy. Positive cell counts were also performed.

[0035] (2) CCK-8 assay for cell proliferation: using 7*10 3 HCECs were seeded into 96-cell plates and cultured at 37°C for 24 hours. Afterward, the drug was applied, and the cells were divided into control and treatment groups with different concentrations of XMU-MP-1: 50 nM, 100 nM, 250 nM, 500 nM, 1000 nM, 2000 nM, 4000 nM, and 5000 nM. The control group was treated with an equal volume of DMSO. The drug or DMSO was added to the cell culture medium, and after culturing for another 24 hours, cell proliferation was detected using a CCK-8 assay kit.

[0036] The results of Example 1 are shown below. Figure 1 EdU and Ki67 staining results showed that the percentage of EdU and Ki67 positive cells in HCECs treated with XMU-MP-1 was significantly higher than that in the control group (1A-1C). CCK-8 results showed that XMU-MP-1 could promote the proliferation of HCECs to a certain extent (1D), confirming that the Hippo signaling pathway inhibitor XMU-MP-1 has a proliferative effect on corneal endothelial cells.

[0037] Example 2: Effects of the Hippo signaling pathway inhibitor XMU-MP-1 on the cell cycle progression of HCECs.

[0038] (1) Cell cycle detection: using 1*10 5HCECs were seeded into 6-well plates and cultured at 37°C for 24 hours. 250 nM XMU-MP-1 was added to the culture medium, while the control group received an equal volume of DMSO. The drug or DMSO was added to the cell culture medium, and the cells were cultured for another 12 hours. The culture medium was then removed, and the cells were digested with 0.25% trypsin-EDTA for 3 minutes. After digestion, the cells were centrifuged, the supernatant was discarded, and the cells were stained using a cell cycle assay kit before being analyzed by flow cytometry.

[0039] (2) Detection of relative expression levels of cell cycle-related genes: using 1*10 5 HCECs were seeded in 6-well plates and cultured at 37°C for 24 hours. 250 nM XMU-MP-1 was applied, while the control group was treated with an equal volume of DMSO. The drug or DMSO was added to the cell culture medium, and the cells were cultured for another 12 hours. RNA was extracted from the cells using an RNA extraction kit and reverse transcribed into cDNA using a reverse transcription kit in a PCR instrument. The relative gene expression levels of CCND3, CCNE1, CCNA2, and E2F2 were detected by real-time quantitative PCR.

[0040] The results of Example 2 are shown below. Figure 2 After XMU-MP-1 treatment, the proportion of HCECs in the G0 / G1 phase was significantly downregulated, significantly lower than that in the control group, while the proportion of cells in the G2 / M phase was significantly upregulated, significantly higher than that in the control group (2A-2B). Real-time quantitative PCR results showed that genes related to cell cycle regulation by the Hippo signaling pathway, such as CCND3, CCNE1, CCNA2, and E2F2, were all upregulated after XMU-MP-1 treatment (2C-2F). These results confirm that XMU-MP-1 can promote the cell cycle progression of human corneal endothelial cells, enabling cells to enter the G2 phase from the G1 phase.

[0041] Example 3: Effects of the Hippo signaling pathway inhibitor XMU-MP-1 on the phosphorylation level and nuclear localization of YAP in HCECs.

[0042] (1) YAP phosphorylation level detection: using 1*10 5 HCECs were seeded in 6-well plates and cultured at 37°C for 12 hours. 250 nM XMU-MP-1 was applied, while the control group was treated with an equal volume of DMSO. The drug or DMSO was added to the cell culture medium, and the cells were cultured for another 12 hours. Proteins were then extracted using protein lysis buffer, and the phosphorylation level of YAP was detected by Western blot.

[0043] (2) YAP nuclear localization detection: with 1*10 5 / Number of wells: HCECs were seeded in confocal dishes and cultured at 37°C for 12 hours. 250 nM XMU-MP-1 was applied, while the control group was treated with an equal volume of DMSO. The drug or DMSO was added to the cell culture medium, and the cells were cultured for another 12 hours. The cells were then fixed with tissue fixative for 10 minutes, and the nuclear localization of YAP was detected by immunofluorescence.

[0044] The results of Example 3 are shown below. Figure 3 Western blot results showed that XMU-MP-1 reduced the phosphorylation level of YAP in HCECs (3A-3B) and promoted YAP nuclear translocation. In HCECs treated with XMU-MP-1, YAP nuclear localization was significantly enhanced (3C-3D).

[0045] Example 4: Animal experiment.

[0046] Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, and their corneas were exposed to ultraviolet light at a dose of 240 J / cm². 2 An ultraviolet radiation model of corneal endothelial injury in mice was established by irradiating the central corneal diameter to a range of 2 mm. The animals were divided into two groups: a solvent control group and a 1 μM XMU-MP-1 treatment group, with 6 mice in each group. Starting from the first day after injury, the mice were treated with ocular drops of the drug or DMSO solution 4 times a day.

[0047] (1) Observation of corneal endothelial injury healing in mice: Slit-lamp photography was performed on mice on days 3, 5 and 8 after injury to assess corneal transparency and OCT to detect corneal thickness. On day 5 after injury, the cornea of ​​mice was taken for alizarin red staining to assess the corneal endothelial cell healing.

[0048] (2) EdU labeling of mouse corneal endothelial cells: Mice were injected intraperitoneally with 50 mg / kg of EdU once daily starting on day 1 post-injury. On day 8 post-injury, mouse corneas were harvested, fixed in tissue fixative for 30 minutes, and then the cells were imaged using the Click-iT EdU Alexa Fluor 647 imaging kit. Images were acquired using a confocal fluorescence microscope, and positive cells were counted.

[0049] (3) Observation of corneal endothelial cell function reconstruction in mice: On the 8th day after injury, mouse corneas were harvested and fixed in tissue fixative for 30 minutes. The corneal endothelial cell function markers ZO-1 and NA were detected by immunofluorescence. + -K + ATPase expression status.

[0050] The results of Example 4 are shown below. Figures 4 to 5Mice treated with XMU-MP-1 eye drops four times daily showed complete corneal endothelial healing by day 5 post-injury, with significantly more regular cell morphology compared to the control group, which still exhibited noticeable defects and incomplete healing (4E). Slit-lamp imaging revealed that by day 8 post-injury, the corneas of XMU-MP-1-treated mice had returned to complete transparency, while the central corneal region of the control group still showed a cloudy area (4A-4B). OCT corneal tomography also showed that the corneal thickness of XMU-MP-1-treated mice was significantly lower than that of the control group, with no corneal edema (4C-4D), demonstrating that XMU-MP-1 can promote the regeneration of corneal endothelial cells in mice. Co-staining results of corneal endothelial EDU with ZO-1 and NA+-K+ATPase on day 8 post-injury showed that ZO-1 and NA+-K+ATPase were significantly higher in the corneal endothelial cells of XMU-MP-1-treated mice. + -K + The integrity of ATPase was significantly better in the control group, while local defects were still present, and intercellular connections and function had not been fully restored (5A-5B). Furthermore, the ZO-1 and NA levels in the XMU-MP-1 eye drop group were significantly higher. + -K + The fluorescence intensity of ATPase was significantly higher than that of the control group (5C-5D). At the same time, the number of EdU and DAPI positive cells in the XMU-MP-1 eye drop group was significantly higher than that in the control group (5E-5F). These results confirm that XMU-MP-1 can promote the in vivo regeneration and functional recovery of mouse corneal endothelial cells.

[0051] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. Application of Hippo signaling pathway inhibitors in the preparation of corneal endothelial repair drugs.

2. The application according to claim 1, characterized in that: The Hippo signaling pathway inhibitors include at least one of MST1 / 2 kinase inhibitors, LATS1 / 2 inhibitors, and MAP4Ks inhibitors.

3. The application according to claim 2, characterized in that: The MST1 / 2 kinase inhibitors include XMU-MP-1; the LATS1 / 2 kinase inhibitors include LATS-IN-1; and the MAP4Ks inhibitors include DMX-5804.

4. Application of YAP gene overexpression promoters in the preparation of corneal endothelial repair drugs.

5. A corneal endothelial repair drug, characterized in that, This includes at least one of a pharmacologically effective concentration of a Hippo signaling pathway inhibitor or a pharmaceutically acceptable salt, isomer, solvate, or metabolite thereof.

6. The corneal endothelial repair drug according to claim 5, characterized in that: The pharmacologically effective concentration is 0.1 nM-1 mM.

7. The corneal endothelial repair drug according to claim 5, characterized in that: The drug can be any pharmaceutically acceptable dosage form, preferably an ophthalmic preparation, including eye drops, ophthalmic injections, ophthalmic ointments, or ophthalmic gels.

8. The corneal endothelial repair drug according to claim 5, characterized in that: The drug also includes pharmaceutically acceptable excipients, including any one or a combination of at least two of the following: carrier, diluent, binder, wetting agent, disintegrant, emulsifier, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

9. The use of the corneal endothelial repair drug according to claim 5 in the preparation of drugs or medical devices that promote the repair, growth, migration, survival, cell adhesion, proliferation and / or differentiation, cell cycle progression, corneal endothelial function regeneration, reconstruction and / or maturation of corneal endothelial cells.

10. The application of the corneal endothelial repair drug according to claim 5 in the preparation of drugs or medical devices for corneal endothelial cell anti-oxidative damage, anti-inflammation, anti-apoptosis, reversal of corneal endothelial cell-mesenchymal transition, and maintenance of corneal endothelial cell microenvironment homeostasis.

11. The use of the corneal endothelial repair drug according to claim 5 in the preparation of drugs or medical devices for relieving or treating corneal endothelial damage, corneal endothelial loss, corneal endothelial lesions, corneal endothelial dysfunction, and corneal endothelial cell decompensation.

12. The use of the corneal endothelial repair drug according to claim 5 in the preparation of drugs or medical devices for treating or relieving symptoms such as pain, blurred vision, and corneal edema caused by corneal endothelial dysfunction.

13. The use of the corneal endothelial repair drug according to claim 5 in the preparation of corneal endothelial grafts, artificial corneal endothelium and artificial full-thickness corneas in pharmaceuticals or medical devices.

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