Methods of corneal epithelial repair associated with hypertonic stress and uses thereof

Abstract

The application relates to the technical field of medicine, and discloses a method for repairing a high-osmotic stress related corneal epithelium and an application of Blebbistatin in the preparation of a medicine for promoting the repair of a high-osmotic stress related corneal epithelium and / or the reconstruction of a barrier function, and a pharmaceutical composition containing Blebbistatin and an ophthalmic excipient, wherein the composition is used on a high-osmotic stress corneal epithelium injury model, the application effectively improves the migration ability of damaged corneal cells under high-osmotic stress, accelerates the wound closure speed and re-epithelialization process, improves the epithelial integrity, improves the trans-epithelial resistance and reduces the epithelial permeability, restores the expression level of a tight connection related protein and the membrane positioning integrity, promotes the repair of a high-osmotic related corneal epithelium injury in vivo, improves the corneal transparency, and is suitable for the intervention of a dry eye related high-osmotic ocular surface disease state.

Description

Technical Field

[0001] This application relates to the field of medical technology, specifically to methods and applications for corneal epithelial repair related to hyperosmolar stress. Background Technology

[0002] Dry eye and other ocular surface diseases are often accompanied by increased tear osmotic pressure, creating a localized hyperosmolar ocular surface microenvironment. Hyperosmolar stress leads to dehydration and volume changes in corneal epithelial cells, inducing abnormal cytoskeleton rearrangement. This abnormality directly weakens the normal mechanical behavior of cells, significantly reducing their migration ability, and consequently causing delayed closure of corneal epithelial wounds, resulting in persistent epithelial defects. Existing conventional interventions often focus on simple lubrication or anti-inflammation, paying less attention to the negative impact of the hyperosmolar environment on the mechanical behavior of the cytoskeleton, making it difficult to effectively promote corneal epithelial cell migration and wound healing from the mechanical basis of improving cell contraction.

[0003] Hyperosmolar environments not only hinder cell migration but also disrupt the tight junction structures between corneal epithelial cells. Under pathological conditions, the expression levels of tight junction-related proteins such as ZO-1, Occludin, and Claudin decrease, and the membrane continuity at cell boundaries is disrupted. This molecular-level structural loss directly leads to a decrease in transepithelial electrical resistance of the corneal cell layer and an abnormally increased epithelial permeability, severely impairing the corneal physiological barrier function. Current technologies, when addressing hyperosmolar stress-related corneal epithelial defects, often struggle to simultaneously promote reepithelialization while effectively restoring tight junction-related proteins and fully rebuilding barrier function.

[0004] Regarding ocular surface administration, conventional topical ocular preparations have limitations when dealing with hyperosmolar pathological microenvironments such as dry eye. On one hand, conventional preparations lack osmotic pressure regulation designs tailored to the damaged ocular surface microenvironment, and direct application may exacerbate existing cellular dehydration. On the other hand, due to tear flushing and blinking, conventional solutions have a short physical retention time on the corneal surface. This pharmacokinetic deficiency makes it difficult for the drug to maintain an effective concentration and exert its interventional effect in the hyperosmolar pathological microenvironment. Consequently, its repair effect on corneal tissue morphology in in vivo applications is limited, and it is difficult to effectively improve the opacity and decreased transparency of damaged corneas. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this application provides a method for corneal epithelial repair related to hyperosmolar stress and its application. It solves the problems of cell migration obstruction and delayed wound closure caused by abnormal corneal epithelial cell cytoskeleton mechanics due to hyperosmolar ocular microenvironment, as well as the damage to corneal physiological barrier function caused by the disruption of tight junctions between cells. At the same time, it overcomes the technical defects of conventional ocular topical preparations, which have a short retention time on the damaged ocular surface and are difficult to exert an effective intervention effect continuously.

[0006] To achieve the above objectives, this application provides the following technical solution: According to the embodiments of the first aspect of this application, the use of Blebbistatin in the preparation of a medicament for promoting hyperosmolar stress-related corneal epithelial repair and / or barrier function reconstruction is proposed.

[0007] In some embodiments, the corneal epithelial repair includes at least one of the following processes:

[0008] Corneal epithelial injury wound closure; Corneal epithelial cell migration; The process of re-epithelialization; Improve epithelial integrity.

[0009] In some embodiments, the barrier function reconstruction includes at least one of the following processes: Increase transepithelial resistance; Reduce epithelial permeability; Restoring the integrity of intercellular connections; Restore or enhance the expression levels of tight junction-related proteins and membrane localization integrity.

[0010] In some embodiments, the hyperosmolar stress-related state is at least one of corneal epithelial damage, epithelial barrier impairment, and dry eye-related ocular surface dysfunction caused by dry eye-related hyperosmolar ocular surface microenvironment.

[0011] According to an embodiment of the second aspect of this application, a pharmaceutical composition comprising Blebbistatin and a pharmaceutically acceptable excipient is provided.

[0012] In some embodiments, the pharmaceutical composition includes, but is not limited to, solution eye drops, suspension eye drops, ophthalmic gels, ointments, sustained-release formulations, or nanodelivery formulations.

[0013] In some embodiments, the excipients include ophthalmic carriers, excipients, buffer solutions, osmotic pressure regulators, thickeners, preservatives, or stabilizers.

[0014] In some embodiments, the pharmaceutical composition is used to promote the recovery and reconstruction of tight junction-associated proteins; the tight junction-associated proteins include one or more of ZO-1, Occludin, and Claudin.

[0015] According to an embodiment of the third aspect of this application, a method for hyperosmolar stress-related corneal epithelial repair is proposed, comprising the following steps: applying the pharmaceutical composition described in any one of the embodiments of the second aspect of this application to a hyperosmolar stress corneal epithelial injury model.

[0016] In some embodiments, the hyperosmolar stress corneal epithelial injury model includes dry eye-related hyperosmolar corneal epithelial injury, ocular surface barrier dysfunction, or other ocular surface disease states accompanied by abnormal local hyperosmolar environment.

[0017] This application has the following beneficial effects: 1. This application utilizes Blebbistatin as a myosin II inhibitor to regulate the contractile state and skeletal biomechanical behavior of corneal epithelial cells by inhibiting the activity of related ATPases. These technical features directly target cell dehydration and abnormal skeletal rearrangement caused by hyperosmolar stress, providing a biomechanical basis for cell migration. This effectively improves the situation of impaired corneal epithelial cell migration under hyperosmolar conditions, accelerates the closure speed of corneal epithelial wounds, and solves the problem of delayed healing of corneal epithelial defects under hyperosmolar conditions.

[0018] 2. This application promotes the expression of tight junction-related proteins such as ZO-1, Occludin, and Claudin through targeted intervention of the active ingredient Blebbistatin, and restores the continuity of the localization of these proteins in the cell boundary membrane. This molecular-level restoration directly increases the transepithelial electrical resistance of the corneal epithelial cell layer and reduces the elevated epithelial permeability under pathological conditions, thereby accelerating the process of cell re-epithelialization while simultaneously achieving the structural and functional reconstruction of the corneal physiological barrier.

[0019] 3. This application describes the formulation of Blebbistatin in combination with auxiliary ingredients such as osmotic pressure regulators and thickeners to create a topical formulation specifically for the ocular surface. The osmotic pressure regulator adapts the osmotic pressure of the drug solution to the damaged ocular surface microenvironment, while the thickener effectively prolongs the retention time of the active ingredient on the corneal surface. This formulation design, combined with the local administration route, ensures a continuous supply of the drug to the hyperosmolar pathological microenvironment, thereby improving corneal tissue morphology and enhancing the transparency of damaged corneas in in vivo applications. Attached Figure Description

[0020] Figure 1 These are images showing the results of corneal epithelial cell scratch repair under different treatment conditions in this application; Figure 2 The images show the results of corneal epithelial barrier function testing under different treatment conditions in this application. Figure 3 This is a diagram showing the expression and distribution of tight junction-related proteins under different treatment conditions in this application; Figure 4 This image shows the results of the Blebbistatin eye drops in this application promoting corneal epithelial repair and improving corneal transparency under hypertonic conditions. Figure 5This is a flowchart of the corneal epithelial barrier function repair and reconstruction method based on Blebbistatin in this application. Detailed Implementation

[0021] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0022] Please see Figures 1-5 This application provides a method for corneal epithelial repair related to hyperosmolar stress and its application.

[0023] This application provides a method and drug application for corneal epithelial barrier function repair and reconstruction based on Blebbistatin. In pathological conditions such as dry eye, increased tear osmotic pressure disrupts the homeostasis of the ocular surface microenvironment, leading to corneal epithelial cell dehydration, abnormal cytoskeleton rearrangement, and consequently, impaired cell migration (delayed wound closure) and damage to the tight junction structure between cells (decreased barrier function).

[0024] The core concept of this application lies in applying Blebbistatin (a myosin II inhibitor) to the preparation of drugs that promote corneal epithelial repair and barrier function reconstruction related to hyperosmolar stress. Blebbistatin can effectively regulate the contractile state and skeletal mechanical behavior of cells under hyperosmolar conditions by inhibiting the activity of related ATPases, thereby counteracting the mechanical damage caused by hyperosmolar stress, providing a mechanical basis for corneal epithelial cell migration, and promoting the expression and membrane localization restoration of tight junction-related proteins (ZO-1, Occludin, Claudin).

[0025] To fully and completely disclose the above-mentioned drug applications and their technical effects, the embodiments of this application will be verified according to the following progressive logical line: S100: Establish an intervention pathway for hyperosmolar stress-related corneal epithelial injury; S200: Discloses the specific preparation process of a Blebbistatin pharmaceutical composition and its formulation adapted for ocular surface administration; S300: Construct an in vitro corneal epithelial injury model, and S400: Construct an in vitro epithelial barrier model, and verify the mechanisms at the molecular and cellular levels from the dimensions of cell migration ability, transepithelial resistance and protein expression; S500: By establishing an in vivo local hyperosmolar animal model, the actual efficacy of the local drug delivery regimen is verified using an in vivo assessment system; S600: Establish a targeted disease intervention strategy based on Blebbistatin.

[0026] The technical solution of this application will be described in detail below with reference to specific implementation steps: S100: Establish an intervention pathway for hyperosmolar stress-related corneal epithelial damage. Targeting the pathological state of corneal epithelium caused by a hyperosmolar ocular microenvironment, targeted interventions are used to simultaneously close corneal epithelial structural defects and restore physiological barrier function.

[0027] S110: Clarify the damaging mechanism of hyperosmolar environment on corneal epithelial tissue of the ocular surface. In disease states such as dry eye, increased tear osmotic pressure disrupts the homeostasis of the ocular surface microenvironment.

[0028] S111: Analysis of the effects of hyperosmolar stress on cell migration. Increased osmotic pressure leads to dehydration and volume changes in corneal epithelial cells, inducing abnormal cytoskeleton rearrangement. The aforementioned dehydration, volume changes, and abnormal cytoskeleton rearrangement result in decreased cell migration ability, which in turn causes delayed closure of corneal epithelial wounds, leading to persistent epithelial defects.

[0029] S112: Analysis of the damage to barrier function caused by hyperosmolar stress. The hyperosmolar environment causes discontinuity in the tight junction structure between corneal epithelial cells, leading to a decrease in transepithelial electrical resistance and an increase in epithelial permeability.

[0030] S120: Implement the Blebbistatin intervention strategy. Apply Blebbistatin as the active ingredient to damaged corneal epithelial tissue to improve the cellular repair environment by regulating the mechanical signal transduction process.

[0031] S121: Regulates cellular mechanical state. Blebbistatin regulates cell contraction by inhibiting the activity of myosin II-related ATPases, thereby improving the mechanical behavior of corneal epithelial cells under hyperosmolar conditions and providing a mechanical basis for corneal epithelial cell migration.

[0032] S122: Promotes molecular-level reconstruction. After acting on corneal epithelial cells, Blebbistatin promotes the expression of tight junction-related proteins and restores the continuity of membrane localization. These tight junction-related proteins specifically include ZO-1, Occludin, and Claudin.

[0033] Based on the aforementioned intervention pathway, this application further provides a preparation process for an ophthalmic pharmaceutical composition containing the active ingredient Blebbistatin. To ensure sufficient disclosure and achieve optimal ocular surface delivery efficacy, specific designs were made for drug compatibility and dosage forms.

[0034] S200: Preparation of ophthalmic pharmaceutical compositions containing the active ingredient Blebbistatin. The pharmaceutical composition consists of the active ingredient Blebbistatin, a pharmaceutically acceptable ophthalmic carrier, and auxiliary ingredients. Through dosage form design and manufacturing process, the retention time and stability of Blebbistatin on the ocular surface are improved.

[0035] S210: Determine the active ingredient of the drug. Blebbistatin was selected as the active ingredient, and it was weighed according to the preset drug concentration.

[0036] S220: Select a pharmaceutically acceptable ophthalmic carrier. The ophthalmic carrier, serving as a medium for the drug, is responsible for the dispersion or dissolution of the active ingredient and may include sterile water, buffer solutions, or other pharmaceutically acceptable solvent systems. Add the weighed Blebbistatin to the ophthalmic carrier and mix thoroughly. The specific stirring speed and time can be selected based on the solubility and dispersion of the active ingredient; the stirring process is a well-known technique in the art.

[0037] S230: Configuring excipients for regulating drug performance. Selected excipients are added to the mixture to regulate formulation performance; the specific mechanisms of action of these excipients include: S231: Use an osmotic pressure regulator. Because the target disease is accompanied by a hyperosmolar state of the ocular surface, the osmotic pressure regulator is used to adjust the osmotic pressure of the topical ocular preparation to a range that is compatible with the damaged ocular surface environment, so as to avoid aggravating cell dehydration.

[0038] S232: Select a pH adjuster. Used to ensure that the pH of topical ophthalmic preparations meets the physiological requirements of ocular surface medication, reducing the stimulation of the ocular surface nerves by the drug.

[0039] S233: Use a thickener. The addition of a thickener aims to increase the viscosity of the drug solution, thereby prolonging the residence time of the active ingredient Blebbistatin on the corneal surface, ensuring that the drug can continuously intervene in the cell migration inhibition process caused by hyperosmolar stress, and improving the physical stability of the drug system.

[0040] S234: Select preservatives and stabilizers. Used to prevent microbial contamination and degradation of active ingredients during storage and use.

[0041] S240: Perform aseptic filtration and dispensing to create a specific ophthalmic formulation. After mixing, perform aseptic filtration and dispense the solution. Based on specific clinical dosing requirements and retention time requirements, the formulation is as follows: S241: Preparation of eye drops. The active ingredient Blebbistatin, ophthalmic carrier, and excipients are combined to prepare a fluid formulation with good flowability. The eye drops can act rapidly on the damaged corneal epithelium and promote wound closure by improving cell migration ability.

[0042] S242: Preparation of ophthalmic gel. During the preparation process, the proportion of thickening materials is increased. Utilizing the semi-solid formulation characteristics of the gel, the retention time of the active ingredient Blebbistatin on the ocular surface is further enhanced, which is more conducive to the long-term recovery of tight junction-related proteins and the continuous reconstruction of barrier function.

[0043] S243: Prepare other formulations suitable for ocular surface administration. These include suspensions, ointments, sustained-release formulations, or nanodelivery formulations to meet the intervention needs for hyperosmolar ocular surface injuries of varying severity.

[0044] After completing the drug preparation, the intervention effect and efficacy indicators of Blebbistatin were further verified using an in vitro cell evaluation system.

[0045] S300: Evaluation of the promoting effect of Blebbistatin on hyperosmolar stress-related corneal epithelial repair.

[0046] S310: Establish an in vitro corneal epithelial injury repair model.

[0047] S311: In vitro culture and maintenance of corneal epithelial cells. Human corneal epithelial cell lines or primary corneal epithelial cells are selected, and cell expansion is performed using a conventional culture system.

[0048] S312: Constructing a hyperosmolar stress model. A hyperosmolar stress model was established by adding solutes to the culture system to increase the osmotic pressure. This model was used to simulate the ocular surface microenvironment associated with diseases such as dry eye. For cell experiments, the isotonic group cultured normally served as the control group (Con group), while the hyperosmolar group with the osmotic pressure adjusted to 420 mOsm served as the hyperosmolar stress model (Hyper group).

[0049] S313: Establish an in vitro corneal epithelial injury model. Corneal epithelial cells were seeded into an experimental container. After the cells grew to a suitable degree of confluence to ensure the formation of a continuous monolayer structure, a scratching operation was performed on the cell layer surface using a sterile pipette tip to establish an in vitro injury model.

[0050] S320: Implement drug intervention and experimental grouping.

[0051] S321: Dissolve the active ingredient Blebbistatin to prepare a stock solution, and dilute it to the corresponding working concentration using culture medium during the experiment.

[0052] S322: Set up experimental groups. Specific groups include the isotonic group (Con group) with normal culture, the hypertonic group with an osmotic pressure of 420 mOsm (Hyper group), and the hypertonic group with 10 μM lebbistatin (Hyper+Bleb10μM group).

[0053] S330: Collect scratch healing data and perform quantitative analysis.

[0054] S331: Take photos at 0h and 24h after the scratch to record the changes in the wound.

[0055] S332: Scratch healing rate and remaining wound area were extracted as detection indicators. The scratch healing rate was obtained by calculating the reduction ratio of the wound area to the initial wound area at each time point.

[0056] S333: Results Analysis. Compared with the normal osmotic pressure control group, cell migration was significantly inhibited and wound closure was delayed in the hyperosmotic stress group; however, after treatment with Blebbistatin under hyperosmotic stress conditions, cell migration ability was improved and wound closure speed was significantly accelerated.

[0057] S400: To evaluate the effect of Blebbistatin on corneal epithelial barrier function reconstruction.

[0058] S410: Constructing a stable corneal epithelial cell barrier. Corneal epithelial cells are seeded into a culture system suitable for constructing a barrier model and cultured until a continuous cell layer is formed, ensuring the formation of a preliminary physical barrier structure between cells.

[0059] S420: Detects electrophysiological and physical permeability indicators of the epithelial barrier.

[0060] S421: Measurement of transepithelial resistance (TEER). The isotonic group (Con group), hyperosmolar group (Hyper group, 420 mOsm), and hyperosmolar group combined with Blebbistatin (Hyper + Bleb 10 μM group) were cultured and measured separately. The magnitude of transepithelial resistance directly reflects the density between corneal epithelial cells. Hyperosmolar stress treatment led to a decrease in transepithelial resistance; after intervention with Blebbistatin, the transepithelial resistance increased, indicating the recovery of epithelial barrier function.

[0061] S422: Detection of corneal epithelial permeability. The reduction in permeability was used to verify the improvement in corneal epithelial integrity, confirming that Blebbistatin enhances the physical barrier function of the epithelial barrier.

[0062] S430: Analyze the reconstruction of tight junction-related proteins.

[0063] S431: Analysis of the effects of hyperosmolar stress on proteins. Hyperosmolar stress led to a decrease in the expression levels of tight junction-related proteins such as ZO-1, Occludin, and Claudin, and their distribution on the cell membrane became discontinuous.

[0064] S432: Verification of the effect of Blebbistatin on protein restoration. After treatment with Blebbistatin (e.g., observed by ZO-1 staining), the expression levels of ZO-1, Occludin, and Claudin rebounded, and the continuity of membrane localization at cell boundaries was effectively restored, and the continuity of tight junctions between cells increased.

[0065] Based on the above in vitro mechanisms and physicochemical indicators, the comprehensive in vivo application effect was further verified through animal models.

[0066] S500: Verify in vivo application effects and dosing regimens using animal models.

[0067] S510: Construct an animal model of corneal epithelial injury and establish a locally hyperosmolar ocular surface microenvironment.

[0068] S511: Perform corneal epithelial damage procedures on the eyes of experimental animals to establish a basic corneal defect state.

[0069] S512: Creates a locally hypertonic ocular surface microenvironment through localized treatment. This local hypertonic environment simulates the pathogenic state associated with dry eye, prolonging the corneal epithelial repair time and leading to decreased corneal transparency.

[0070] S520: Conduct experimental grouping and local drug administration treatment.

[0071] S521: A control group (applied isotonic treatment as the baseline for repair), a hypertonic treatment group (maintained a continuously hypertonic local microenvironment), and a hypertonic treatment group combined with Blebbistatin eye drops were established. The specific subgroups were: a simple corneal injury group (Con group), a hypertonic group (Hyper group) receiving a solution with an osmotic pressure of 420 mOsm after corneal injury, and a hypertonic group (Hyper+Bleb50μM group) receiving a solution of 50 μM lebbistatin after corneal injury.

[0072] S522: Administer the medication via topical eye drops. The topical ophthalmic formulation containing Blebbistatin was applied directly to the damaged corneal surface of the treated animals; equal volumes of the ophthalmic carrier were instilled into the isotonic control group and the hypertonic treatment group to eliminate interference from the administration procedure itself. Continuous drug supply was ensured throughout the animal model's lifespan.

[0073] S530: Establish a comprehensive living body assessment system to evaluate the quality of restoration.

[0074] S531: Perform corneal fluorescein staining. Instill sodium fluorescein solution onto the ocular surface, observe and record the stained area and degree of staining on the corneal surface. A decrease in the stained area of ​​the corneal epithelium directly indicates the restoration of epithelial tissue integrity and an accelerated repair process.

[0075] S532: Perform slit-lamp observation. Use a slit-lamp microscope to observe the cornea in vivo and record the tissue morphology repair under different pathological backgrounds and medication conditions.

[0076] S533: Perform corneal transparency assessment. Compare the degree of corneal opacity in each group at preset time points. The results showed that hypertonic treatment reduced corneal transparency; after intervention with Blebbistatin eye drops, corneal transparency was significantly improved in the hypertonic treatment group.

[0077] S534: Based on the above indicators, the evaluation conclusion is drawn. This verifies that Blebbistatin eye drops achieve the technical effects of improving repair quality and enhancing the stability of the ocular surface microenvironment by accelerating the corneal epithelial repair process and improving corneal transparency.

[0078] Based on the comprehensive validation system described above, a clinical intervention strategy for the target disease using Blebbistatin-based drug compositions was finally established.

[0079] S600: Implement targeted disease interventions based on Blebbistatin.

[0080] S610: Implement interventions targeting hyperosmolar stress-related corneal epithelial repair. By improving cell migration inhibition caused by hyperosmolar stress, wound healing time is shortened; cell dynamics are regulated to promote cell migration to the damaged area to cover the defect, thereby restoring the physical continuity of the corneal surface.

[0081] S620: Implement interventions targeting barrier function reconstruction. By increasing transepithelial electrical resistance and reducing permeability of the corneal epithelium, abnormal permeation of water and substances is reduced; at the same time, the membrane localization integrity of tight junction-related proteins (ZO-1, Occludin, Claudin) at the cell boundary is restored.

[0082] S630: Clearly defines the specific disease state for intervention. The active ingredient, Blebbistatin, targets pathological changes caused by increased tear osmotic pressure, providing an intervention plan for structural repair and functional reconstruction; it is suitable for intervening in corneal epithelial damage, corneal epithelial barrier impairment, and other ocular surface diseases caused by dry eye-related hyperosmolar ocular microenvironment, as well as other ocular surface diseases accompanied by abnormal local hyperosmolar environments.

[0083] Example 1 Blebbistatin promotes the repair of corneal epithelial cell wounds under hyperosmolar stress conditions.

[0084] Human corneal epithelial cell lines or primary corneal epithelial cells were selected for in vitro culture using a standard corneal epithelial cell culture system. Blebbistatin was dissolved in a suitable solvent to prepare a stock solution, which was then diluted to the corresponding working concentration for the experiment. The experiment included a normal osmotic pressure control group (Con group, the isotonic group with normal culture), a hyperosmotic stress group (Hyper group, the hyperosmotic group with an osmotic pressure of 420 mOsm), and a hyperosmotic stress combined with Blebbistatin treatment group (Hyper+Bleb group, the 420 mOsm hyperosmotic group with 10 μM Blebbistatin added). If necessary, different concentrations of Blebbistatin gradient groups could be further set up to screen the effective concentration range. A hyperosmotic stress model was established by increasing the osmotic pressure of the culture system. After the cells grew to a suitable degree of confluence, an in vitro injury model was established by scratching with a sterile pipette tip. Subsequently, the cells were treated with the different conditions mentioned above, and wound changes were recorded at 0h and 24h time points. The scratch healing rate and remaining wound area were used as detection indicators. The results showed that... Figure 1 As shown, compared with the normal osmotic pressure control group, cell migration was significantly inhibited and wound closure was delayed in the hyperosmotic stress group. However, after treatment with Blebbistatin under hyperosmotic stress conditions, cell migration ability improved and wound closure speed was accelerated, suggesting that Blebbistatin can promote the repair process of corneal epithelial cells under hyperosmotic conditions. Therefore, Blebbistatin can be used as an effective active ingredient to promote the repair of hyperosmotic stress-related corneal epithelial damage.

[0085] Example 2 Blebbistatin improves corneal epithelial barrier function under hyperosmolar stress conditions.

[0086] Corneal epithelial cells were seeded into a culture system suitable for constructing a barrier model. After forming a continuous cell layer, they were treated with normal osmotic pressure (Con group), hyperosmotic stress (Hyper group, 420 mOsm), and hyperosmotic stress combined with Blebbistatin (Hyper+Bleb group, 420 mOsm + 10 μM Blebbistatin) to evaluate the effect of Blebbistatin on improving corneal epithelial barrier function impaired by hyperosmotic stress. Experimental indicators included transepithelial electrical resistance (TEER), expression levels of tight junction-related proteins, and the continuity of tight junction protein distribution. Results showed that after hyperosmotic stress treatment, the transepithelial electrical resistance of the cell layer decreased, indicating impaired epithelial barrier function. Simultaneously, the expression of tight junction-related proteins such as ZO-1, Occludin, and / or Claudin decreased or their membrane distribution became discontinuous. Figure 2 and Figure 3As shown, after treatment with Blebbistatin, transepithelial electrical resistance increased, and the expression of tight junction proteins (such as ZO-1) and membrane localization continuity were restored, indicating that Blebbistatin helps to rebuild corneal epithelial barrier function under hyperosmolar stress. Therefore, Blebbistatin can not only promote epithelial damage repair, but also improve barrier dysfunction caused by hyperosmolar stress.

[0087] Example 3 Blebbistatin ophthalmic pharmaceutical composition and its preparation.

[0088] An ophthalmic pharmaceutical composition comprising the active ingredient Blebbistatin and pharmaceutically acceptable excipients, wherein the excipients include an ophthalmic carrier (such as sterile water, buffer solution, or other pharmaceutically acceptable solvent system) and optional excipients (such as osmotic pressure regulators, pH regulators, thickeners, preservatives, and stabilizers). The composition can be prepared into formulations suitable for topical ocular surface administration, such as solution-type eye drops, suspension-type eye drops, ophthalmic gels, ointments, sustained-release formulations, or nano-delivery formulations. The preparation method involves adding Blebbistatin to an ophthalmic carrier and stirring until homogeneous. If necessary, the pH and osmotic pressure are adjusted, and the mixture is then sterilely filtered and dispensed to obtain the ophthalmic formulation. To improve ocular surface retention time and drug stability, thickeners may be further added or a sustained-release delivery strategy may be employed. The above formulation can be used for intervention in dry eye-related hyperosmolar corneal epithelial damage, ocular surface barrier dysfunction, or other ocular surface diseases accompanied by abnormal local hyperosmolar environments.

[0089] Example 4 Blebbistatin eye drops promote the repair of corneal epithelial damage under hyperosmolar conditions and improve corneal transparency.

[0090] To verify the efficacy of Blebbistatin in hyperosmolar corneal epithelial injury in vivo, an animal model of corneal epithelial injury was constructed. A hyperosmolar ocular surface microenvironment was created through local treatment. The experiment included an isotonic control group (Con group, corneal injury alone), a hyperosmolar treatment group (Hyper group, a hyperosmolar group treated with a 420 mOsm solution after corneal injury), and a hyperosmolar combined with Blebbistatin eye drops treatment group (Hyper+Bleb group, a hyperosmolar group treated with 50 μM Blebbistatin solution after corneal injury). The medication was administered via local eye drops. Repair progress under different treatment conditions was observed using corneal fluorescein staining, slit-lamp observation, and corneal transparency assessment. Results showed that... Figure 4As shown, hyperosmolar treatment can prolong the corneal epithelial repair time and reduce corneal transparency. After administering Blebbistatin eye drops under hyperosmolar conditions, the corneal epithelial repair process is accelerated, and corneal transparency is improved compared to the hyperosmolar treatment group. Although the repair effect has not yet recovered to the isotonic control level, compared to hyperosmolar treatment alone, Blebbistatin eye drops can improve the delayed corneal epithelial repair and decreased transparency under hyperosmolar conditions, indicating that it has potential clinical application value in the intervention of hyperosmolar corneal epithelial damage.

[0091] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The use of Blebbistatin in the preparation of drugs for promoting corneal epithelial repair and / or barrier function reconstruction in response to hyperosmolar stress.

2. The application according to claim 1, characterized in that, The corneal epithelial repair process includes at least one of the following procedures: Corneal epithelial injury wound closure; Corneal epithelial cell migration; The process of re-epithelialization; Improve epithelial integrity.

3. The application according to claim 1, characterized in that, The barrier function reconstruction includes at least one of the following processes: Increase transepithelial resistance; Reduce epithelial permeability; Restoring the integrity of intercellular connections; Restore or enhance the expression levels of tight junction-related proteins and membrane localization integrity.

4. The application according to claim 1, characterized in that, The hyperosmolar stress-related state refers to at least one of the following: corneal epithelial damage, epithelial barrier impairment, and dry eye-related ocular surface dysfunction caused by the dry eye-related hyperosmolar ocular surface microenvironment.

5. A pharmaceutical composition comprising Blebbistatin and a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical compositions include, but are not limited to, solution eye drops, suspension eye drops, ophthalmic gels, ointments, sustained-release formulations, or nanodelivery formulations.

7. The pharmaceutical composition according to claim 5, characterized in that, The excipients include ophthalmic carriers, excipients, buffer solutions, osmotic pressure regulators, thickeners, preservatives, or stabilizers.

8. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition is used to promote the expression of tight junction-associated proteins and / or the restoration and reconstruction of membrane localization continuity; the tight junction-associated proteins include one or more of ZO-1, Occludin, and Claudin.

9. A method for promoting corneal epithelial repair related to hyperosmolar stress, characterized in that, Includes the following steps: The pharmaceutical composition according to any one of claims 5-8 was applied to a hyperosmolar stress corneal epithelial injury model.

10. The method according to claim 9, characterized in that, The hyperosmolar stress corneal epithelial injury model includes dry eye-related hyperosmolar corneal epithelial injury, ocular surface barrier dysfunction, or other ocular surface disease states accompanied by abnormal local hyperosmolar environment.

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