New application of 4F dimer polypeptide and lipids thereof

Abstract

The invention discloses a new application of a 4F dimer polypeptide and a lipid compound thereof, and particularly relates to an application of the 4F dimer polypeptide and the lipid compound thereof in preparation of a medicine for treating xerophthalmia. The application disclosed by the invention has the advantages that pHDL is innovatively found to have a very good effect on xerophthalmia, and the application has the advantage of synergistic treatment aiming at a multi-factor pathological mechanism of xerophthalmia. Therefore, compared with the existing single-mechanism medicine (such as cyclosporine eye drops only inhibiting inflammation), the multi-dimensional action mechanism of the pHDL accurately conforms to the complex pathological network of the xerophthalmia, and the curative effect coverage range of the refractory xerophthalmia is expected to be improved.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and in particular relates to new uses of 4F dimer polypeptide and liposomes thereof. Background Art

[0002] Dry eye disease (DED) is a complex condition characterized by multiple pathological mechanisms, including abnormal tear quantity and quality, ocular surface inflammation, neural imbalances, and environmental factors. The etiology of DED can include autoimmune diseases (such as Sjögren's syndrome), meibomian gland dysfunction (MGD), lacrimal gland hyposecretion, oxidative stress, and corneal epithelial barrier disruption. Currently, the global prevalence of DED is as high as 5%-50%, and is showing a significant upward trend due to the increased use of electronic devices, the aging population, and increasing environmental pollution. Existing treatments, such as artificial tears, topical anti-inflammatory drugs (such as cyclosporine A and glucocorticoids), or mucin secretion-stimulating agents (such as diquafosol sodium), can partially alleviate symptoms, but they primarily target a single pathological component and are unable to comprehensively address the multifactorial DED process. For example, while traditional artificial tears can temporarily lubricate the ocular surface, they have limitations such as poor tear film stability, high-viscosity formulations that can blur vision, and a lack of lipid layer repair capacity. Long-term use of anti-inflammatory drugs can cause corneal toxicity or systemic side effects. Given that a single-target treatment strategy is difficult to cover all the pathological aspects of DED, developing a multi-effect therapy that can simultaneously regulate inflammation, repair the ocular surface barrier, replenish tear film lipids and prolong ocular surface retention time has become an urgent need in the field of dry eye treatment.

[0003] High-density lipoprotein (HDL) is the smallest and densest of all plasma lipoproteins in the human body. It possesses core functions such as anti-inflammatory, antioxidant, and lipid transport regulation, making it a research hotspot for the treatment of cardiovascular diseases. Apolipoprotein AI (ApoA-I) is the most important structural and functional component of HDL. Its esters, formed by self-assembly with phospholipids, can mimic the various biological functions of native HDL. However, high-purity full-length ApoA-I is prohibitively expensive, requires complex production processes, and requires long production cycles, making it difficult to widely use in the laboratory or clinically. 4F peptides and their dimers are highly effective alternatives to full-length ApoA-I, offering low cost, easy large-scale production, and avoiding the immunogenicity of full-length ApoA-I. Using microfluidic technology, 4F peptide dimers were mixed and self-assembled with the neutral ionic phospholipid DMPC to form flat, disc-shaped HDL-mimicking products approximately 30 nanometers in diameter, termed pHDL. Compared to native HDL, pHDL retains the biological functions of native HDL while offering the unique advantages of low immunogenicity, high tissue permeability, and targeted delivery. DMPC, in particular, is highly similar to natural tear film lipids (such as phospholipids secreted by the meibomian glands) and can integrate into the tear film lipid layer, filling defective areas. Furthermore, by forming an ordered lipid bilayer, DMPC reduces tear evaporation, improves tear film stability, and alleviates the core pathophysiological hallmarks of dry eye—tear film hyperosmolarity and excessive evaporation—thus stabilizing and repairing the tear film lipid layer. Furthermore, as an electrically neutral phospholipid, DMPC avoids the potential irritation of cationic components to the corneal epithelium, making it suitable for long-term use. As an ApoA-I analog, 4F dimer peptide possesses multiple anti-inflammatory and antioxidant properties. It also leverages ApoA-I's reverse cholesterol transport function, participating in the regulation of meibomian gland lipid metabolism, improving lipid secretion quality, and reducing the incidence of obstructive dry eye.

[0004] Therefore, how to apply pHDL in the treatment of dry eye is a problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of the present invention is to solve the above technical problems.

[0006] To achieve the above objectives, this application employs the following technical solution: The present invention provides novel uses of the 4F dimer polypeptide and its liposomes, including the use of the 4F dimer polypeptide and its liposomes in the preparation of a drug for treating dry eye disease. Dry eye disease (DED) is a complex disease characterized by multiple pathological mechanisms, including abnormal tear quality or quantity, ocular surface inflammation, neural imbalance, and environmental factors. The etiology of DED can include autoimmune diseases (such as Sjögren's syndrome), meibomian gland dysfunction (MGD), lacrimal gland hyposecretion, oxidative stress, and corneal epithelial barrier disruption. Currently, the global prevalence of DED is as high as 5%-50%, and is showing a significant upward trend with the increased use of electronic devices, the aging population, and worsening environmental pollution. Existing treatments, such as artificial tears, topical anti-inflammatory drugs (such as cyclosporine A and glucocorticoids), or mucin secretion-promoting agents (such as diquafosol sodium), can partially alleviate symptoms, but they often target a single pathological component and fail to comprehensively address the multifactorial DED process. For example, while traditional artificial tears can temporarily lubricate the ocular surface, they suffer from limitations such as poor tear film stability, high-viscosity formulas that can blur vision, and a lack of lipid layer repair capabilities. Long-term use of anti-inflammatory drugs can also cause corneal toxicity or systemic side effects. Given that single-target therapeutic strategies struggle to address all aspects of DED pathology, developing multi-action therapies that can simultaneously regulate inflammation, repair the ocular surface barrier, replenish tear film lipids, and prolong ocular surface retention is a pressing need in the dry eye treatment field.

[0007] High-density lipoprotein (HDL) is the smallest and densest of all plasma lipoproteins in the human body. It possesses core functions such as anti-inflammatory, antioxidant, and lipid transport regulation, making it a research hotspot for the treatment of cardiovascular diseases. Apolipoprotein AI (ApoA-I) is the most important structural and functional component of HDL. Its esters, formed by self-assembly with phospholipids, can mimic the various biological functions of native HDL. However, high-purity full-length ApoA-I is prohibitively expensive, requires complex production processes, and requires long production cycles, making it difficult to widely use in the laboratory or clinically. 4F peptides and their dimers are highly effective alternatives to full-length ApoA-I, offering low cost, easy large-scale production, and avoiding the immunogenicity of full-length ApoA-I. Using microfluidic technology, 4F peptide dimers were mixed and self-assembled with the neutral ionic phospholipid DMPC to form flat, disc-shaped HDL-mimicking products approximately 30 nanometers in diameter, termed pHDL. Compared to native HDL, pHDL retains the biological functions of native HDL while offering the unique advantages of low immunogenicity, high tissue permeability, and targeted delivery. Currently, pHDL is primarily used in cardiovascular applications. This innovative discovery demonstrates that pHDL also offers promising results in dry eye, for example, through its synergistic therapeutic advantages across the multifactorial pathogenesis of dry eye. Regarding anti-inflammatory and immunomodulatory properties, the 4F peptide can block the inflammatory cascade. Regarding oxidative stress protection, the phospholipid bilayer structure efficiently captures free radicals and, through the peroxidase activity of phosphatidylcholine, degrades oxidized lipids, repairing oxidative damage to the corneal epithelium. Regarding tear film lipid repair, DMPC can replenish the polar lipids missing in patients with meibomian gland dysfunction, improving tear film stability and osmotic balance. Therefore, compared to existing single-action drugs (such as cyclosporine eye drops, which only inhibit inflammation), pHDL's multi-dimensional mechanism of action precisely aligns with the complex pathological network of dry eye, potentially expanding its therapeutic reach in refractory dry eye.

[0008] Furthermore, the gene sequence of the 4F dimer polypeptide is shown in SEQ ID NO:1. The 4F dimer polypeptide and its lipid product are complexes of the 4F dimer polypeptide and phospholipids, with the molar ratio of the 4F dimer polypeptide to the phospholipid being 1:50-1:100. Traditional HDL analogs have large molecular weights and poor corneal penetration, making them difficult to meet the requirements of ocular surface drug delivery. In terms of drug molecule particle size, under normal physiological conditions, the intercellular gap is approximately 20-30 nanometers, and the barrier function is intact, preventing the penetration of pathogens and harmful substances. However, in dry eye, due to cell apoptosis, junction protein degradation, or edema, the gap can expand to more than 50 nanometers, leading to barrier damage and increased permeability. Microfluidic technology can be used to prepare pHDL nanoparticles with a particle size of approximately 30 nanometers, which can significantly improve their transcorneal penetration efficiency. In addition, its nanostructure can also increase the retention time on the ocular surface, avoiding the defect of traditional eye drops that are easily washed away by tears. In terms of charge, the use of neutral ionic phospholipids can reduce local irritation and, by integrating into the tear film lipid layer, fill defective areas and prolong retention time on the ocular surface. Therefore, pHDL eye drops are superior to traditional formulations in terms of bioavailability, local tolerance, and dosing frequency, without any safety risks.

[0009] Furthermore, pHDL's differentiated design offers opportunities for clinical translation. In terms of drug composition, the ApoA-I analog peptide 4F dimer (sequence: SEQ ID NO: 1) is combined with a specific ratio (molar ratio of 1:50-1:100) of natural neutral phospholipids, giving pHDL the dual benefits of anti-inflammatory and antioxidant properties, as well as tear film integration. Regarding the manufacturing process, the "one-step self-assembly microfluidic" process overcomes the low yield and batch-to-batch instability of traditional methods, enabling large-scale production.

[0010] Furthermore, when the 4F dimer polypeptide and its liposome are used to prepare a drug for treating dry eye, they also include drugs that can be encapsulated when used as drug carriers. The 4F dimer polypeptide and its liposome are prepared using physiological saline, with a 4F dimer polypeptide concentration of 5-20 mg / ml and a liposome concentration of 5-20 mg / ml.

[0011] In summary, by employing the above-mentioned technical solutions, the present invention achieves the following beneficial effects: The present invention innovatively discovered that pHDL is also highly effective for dry eye, offering the advantage of synergistic treatment targeting the multifactorial pathological mechanisms of dry eye. Therefore, compared to existing single-mechanism drugs (such as cyclosporine eye drops, which only inhibit inflammation), pHDL's multi-dimensional mechanism of action precisely aligns with the complex pathological network of dry eye, potentially expanding the scope of therapeutic efficacy for refractory dry eye. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The morphology of pHDL under transmission electron microscopy.

[0013] Figure 2 These are ophthalmological indicators of dry eye in mice after 3 days of atropine intervention; Middle: *** p <0.005 vs Blank.,# p <0.05, ## p <0.01, ### p <0.005 vs DES.

[0014] Figure 3 These are ophthalmic indicators of dry eye in mice after 7 days of atropine intervention; in the figure: *** p <0.005 and**** p <0.001 vs Blank.,# p <0.05, ### p <0.005 vs DES.

[0015] Figure 4 HE staining of the cornea and lacrimal gland in the mouse atropine dry eye model.

[0016] Figure 5 It is a safety indicator of the atropine dry eye model in mice.

[0017] Figure 6 These are the ophthalmological indicators of the mouse dry eye model after 5 days of intervention with benzalkonium chloride; in the figure, * p <0.05 and *** p <0.005 vs Blank. # p <0.05 and ### p <0.005 vs DES.

[0018] Figure 7 These are the ophthalmological indicators of the mouse model of dry eye after 10 days of intervention with benzalkonium chloride; in the figure, *** p <0.005 vs Blank. ### p <0.005 vs DES.

[0019] Figure 8 HE staining of the cornea and lacrimal gland in the benzalkonium chloride dry eye model of mice.

[0020] Figure 9 PAS staining of the conjunctiva in the benzalkonium chloride dry eye model in mice.

[0021] Figure 10It is a safety indicator of the benzalkonium chloride dry eye model in mice. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0023] Example: The present invention has discovered a new use for 4F dimer polypeptides and their lipids for preparing drugs for treating dry eye. The gene sequence of the 4F dimer polypeptide is shown in SEQ ID NO: 1. The 4F dimer polypeptides and their lipids are complexes of 4F dimer polypeptides and phospholipids. The molar ratio of 4F dimer polypeptide to phospholipid is 1:50. It should be noted that the molar ratio of 4F dimer polypeptide to phospholipid can also be 1:100, 1:60, 1:70, 1:80, or 1:90. These molar ratios are all acceptable. The 4F dimer polypeptides and their lipids are complexes of 4F dimer polypeptides and phospholipids, and the particle size is 30 nm. 4F dimer peptide and its lipid (pHDL) were prepared as follows: (1) Preparation of aqueous phase: 20 mg of L-4F peptide (DWFKAFYDKVAEKFKEAFPDWFKAFYDKVAEKFKEAF) with C-terminal amidation was accurately weighed and dissolved in sterile water. After thorough mixing, it was set aside. (2) Preparation of organic phase solution: 50 mg of dimyristoylphosphatidylcholine (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC) was accurately weighed and dissolved in anhydrous ethanol. After thorough vortex mixing, it was set aside. pHDL was synthesized at a flow rate of 1 mL / min for the organic phase and 5 mL / min for the aqueous phase. The synthesized nanoparticles were concentrated by centrifugation at 4000 × g for 15 minutes at 25°C using a 10 kD ultrafiltration tube. Then, ddH2O was added and centrifuged three times under the same centrifugation conditions to elute the residual ethanol. After concentration and washing, store at 4°C. It should be noted that the molar ratio of 4F dimer polypeptide to phospholipid can also be 1:100, 1:60, 1:70, 1:80, 1:90, and the effects are the same. When used as a drug for treating dry eye, it also includes drugs that can be encapsulated when used as a drug carrier. 4F dimer polypeptide and its lipids are prepared with physiological saline, and the concentration of 4F dimer polypeptide is 5-20 mg / ml, and the concentration of lipids is 5-20 mg / ml. The obtained F dimer polypeptide and its lipids (pHDL) are as follows Figure 1Transmission electron microscopy (TEM) was used to observe the morphology of pHDL: 10 μL of freshly prepared pHDL nanoparticles was pipetted onto a parafilm membrane to form a droplet. The front side of a carbon-supported copper grid was exposed to the droplet for 2 minutes, followed by contact with a 2% phosphotungstic acid droplet for 1 minute. Residual dye on the copper grid was then blotted with damp filter paper and placed face-up in a sample box to dry overnight. The next day, a blank support and emodin nanoparticles were observed using a transmission electron microscope at an accelerating voltage of 80 kV.

[0024] The following two models are used to illustrate that 4F dimer polypeptide and its lipid compounds can be used to treat dry eye.

[0025] 1. Efficacy Evaluation Part I: Atropine was used to block cholinergic M3 receptors and inhibit lacrimal gland secretion, simulating a model of aqueous-deficient DED. The ability of the 4F dimer peptide and pHDL to restore tear secretion was evaluated. A DED model was established using 1% atropine (1 mg / 100 mg) with a 5 μl eye drop volume per eye. Modeling was performed at 8:00 and 14:00 daily, and drug administration was performed at 11:00 and 17:00 daily. Over a 7-day period, atropine eye drops were used to establish DED, while 0.1% sodium hyaluronate eye drops were administered simultaneously as a positive agent. Treatment groups included low-dose (5 mg / mL), medium-dose (10 mg / mL), and high-dose (20 mg / mL) peptide, and low-dose (5 mg / mL), medium-dose (10 mg / mL), and high-dose (20 mg / mL) pHDL. On days 3 and 7 after modeling and drug administration, mice in each group underwent tear secretion test (SIT) and corneal fluorescein staining score (FLS). Samples were collected the day after the last test to assess drug safety and efficacy.

[0026] 1.1 Relief of Dry Eye Symptoms (Figures 2 and 3): Evaluation was performed using tear production and tear film stability tests. The Schirmer test directly reflects lacrimal gland function and is a core indicator of tear secretion and aqueous deficiency. Tear production in each group of mice was measured using the phenol red cotton thread test. Mice were immobilized, and a phenol red cotton thread was grasped with ophthalmic microtweezers and placed in the conjunctival sac at the outer third of the lower eyelid. After 15 seconds, the thread was removed and the wetted length (i.e., the length of the thread that turned from yellow to red) was measured using a vernier caliper to the nearest 0.1 mm. The average value was calculated for both eyes. Secondly, tear film stability was assessed using corneal fluorescein staining. This corneal fluorescein staining score directly reflects the ocular epithelial barrier disruption and secondary inflammatory damage caused by dry eye, and is a key indicator for assessing disease progression and treatment efficacy. Immobilize the mouse and moisten a sodium fluorescein test strip with one drop of saline. Lightly touch the tip of the test strip to the inside of the mouse's lower eyelid and hold for a moment. Blink several times to evenly distribute the fluorescein. Observe corneal fluorescent staining under cobalt blue light using a slit lamp and assess the staining grade. Scoring is based on the cornea being divided into four quadrants, with no staining designated as 0 and staining graded as mild, moderate, or severe. A score of 1 indicates fewer than five spots of staining, 3 indicates clumpy staining or filaments, and 2 indicates an intermediate score, for a total score of 0 to 12.

[0027] 1.1.1 After 3 consecutive days of administration ( Figure 2 Compared with the blank control group, the tear secretion of mice in the DED model group was significantly reduced, and the corneal epithelium showed signs of damage. Compared with the model group, treatment with the positive drug sodium hyaluronate eye drops (SH) did not significantly improve tear secretion or corneal epithelial damage. Treatment with different doses of 4F dimer peptide showed a trend of improvement in tear secretion and corneal epithelial damage, with the high dose (20 mg / mL) having the most significant effect on corneal damage. Treatment with different doses of pHDL showed a significant improvement in both tear secretion and corneal epithelial damage in mice, with the high dose (20 mg / mL) significantly improving both.

[0028] 1.1.2 After 7 days of continuous administration ( Figure 3 Compared with the blank group, the tear film breakup time of mice in the model group was significantly shortened, and the corneal epithelial damage was severe. Treatment with the positive drug sodium hyaluronate eye drops (SH) did not significantly improve tear secretion or corneal epithelial damage compared with the model group. Treatment with different doses of 4F dimer peptide and pHDL showed an improvement trend in tear secretion, with the highest dose (20 mg / mL) being the most significant. Different doses of 4F dimer peptide and pHDL had significant effects on corneal damage, with the highest dose being the most effective.

[0029] 1.2 Improve ocular tissue pathology of dry eye ( Figure 4After anesthesia and sacrifice, the left eyeballs of the mice were removed and fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and serially sectioned to a thickness of approximately 4 μm. After staining and drying, the pathological morphological changes of the cornea and lacrimal gland tissue were observed under an optical microscope. Images were collected and statistically analyzed for corneal epithelial thickness. The results showed: Hematoxylin and eosin (HE) staining of the cornea and lacrimal gland of mice in each group is shown in Figure 4 The corneal histology of mice in the normal group was essentially normal. The corneal epithelial cells showed regular morphology, distinct layers, well-defined contours, uniform chromatin, and tight intercellular connections. The epithelial cells were neatly arranged in approximately 3-4 layers, with a smooth surface and no thickening. The stromal layer consisted of collagen fibers parallel to the cornea, with scattered fibroblasts densely distributed within. No inflammatory cell infiltration was observed. Compared with the normal group, the corneal epithelial surface in the model group was rough, with disorganized cells and epithelial cell shedding (indicated by the red arrow). The collagen fibers in the stromal layer were loosely arranged, and the intercellular connections were slightly loose (indicated by the blue arrow). Compared with the model group, the corneal epithelial cells in the SH group were more neatly arranged, but the cell membrane boundaries were blurred, and the cytoplasm was partially edematous with pale cytoplasm staining. Corneal damage improved slightly in all peptide dose groups, but the epithelial layer remained rough, with cell shedding, disorganized arrangement, and loose collagen fibers. The peptide 20 mg group showed more significant recovery. In the pHDL 5 mg group, the corneal epithelial surface was rough, with significant cell exfoliation and cytoplasmic edema; however, the stromal collagen fibers were more uniformly arranged. In the pHDL 10 mg and 20 mg groups, corneal epithelial damage recovered somewhat, with the pHDL 20 mg group showing more pronounced recovery, with a smooth corneal epithelial surface, regular cell morphology, and neatly arranged stromal layers.

[0030] Furthermore, in the lacrimal glands of mice in the normal group, the lacrimal gland tissue was composed of lobules of varying sizes, with alveoli of uniform size and regular morphology. The acinar cells were densely arranged, and the lacrimal epithelial cells were intact and columnar. The cytoplasm was rich in secretory vesicles, and alveolar mucus was visible in the acinar lumen. Compared with the normal group, the lacrimal gland cells in the model group were atrophic, irregularly arranged, and structurally damaged. The mucus content in the lumen was reduced, and the alveoli and lacrimal epithelium were of varying sizes and disordered arrangement (as indicated by the green arrows). Connective tissue was significantly swollen, and inflammatory cells were infiltrated in the interstitium. Compared with the model group, the lacrimal gland tissue damage was not significantly alleviated in the positive drug sodium hyaluronate eye drops (SH) group, and the lacrimal epithelial cells were atrophied. The peptide 5 mg and 10 mg groups showed no significant improvement, with the lacrimal glands showing incomplete structure, varying sizes, and inflammatory cell infiltration. The peptide 20 mg group showed slightly regularized lacrimal cell morphology. The arrangement of acini in the pHDL 5 mg group was disordered; the pathological damage of lacrimal gland tissue in the pHDL 10 mg and 20 mg groups was alleviated, the lacrimal gland structure was relatively complete, and the morphology of glandular epithelial cells was relatively regular.

[0031] 1.3 Security Figure 5 Serum biochemical analysis of mice following atropine-induced, 4F dimer peptide, and pHDL treatment revealed no liver or kidney damage. ALT: alanine aminotransferase; AST: aspartate aminotransferase; TP: total protein; ALB: albumin; LDH: lactate dehydrogenase; CK: creatine kinase; BUN: serum urea nitrogen; CR: serum creatinine.

[0032] 2. Efficacy Evaluation Part II: Benzalkonium chloride (BAC) was used to create a model that disrupts the ocular epithelial barrier, induces inflammation, and destabilizes the tear film, resulting in lipid layer defects, ocular surface inflammation, and epithelial damage, mimicking evaporative dry eye or mixed dry eye. 0.2% BAC was used to create the DED model. Modeling was performed at 8:00 AM and 2:00 PM daily, and drug administration was performed at 11:00 AM and 5:00 PM. After 9 days of BAC eye drops to establish the DED model, mice were treated with the active agent, 0.1% sodium hyaluronate eye drops, and treatment groups were treated with low (5 mg / mL), medium (10 mg / mL), and high (20 mg / mL) pHDL for 10 days. Tear breakup time (BUT) and corneal fluorescein staining score (FLS) were measured in each group. Samples were collected the day after testing to assess drug safety and efficacy.

[0033] 2.1 Relieve dry eye symptoms ( Figure 6 、 7 ): Evaluated by tear film breakup time observation and tear film stability test.

[0034] Among them, the tear film break-up time (BUT) observation is used to test the tear film stability by measuring the tear film break-up time. The tear film break-up time can reflect: (1) lipid layer abnormality, that is, meibomian gland dysfunction (MGD) leads to insufficient lipid secretion, unable to form a stable tear film, and accelerated evaporation rate; (2) mucus layer defect, that is, goblet cell reduction (such as vitamin A deficiency, chronic inflammation) destroys the adhesion of the tear film; (3) tear hyperosmolarity, that is, the osmotic pressure increases after tear evaporation and concentration, further destroying the stability of the tear film, indicating abnormal meibomian gland function or tear film composition. Fix the mouse, wet the sodium fluorescein test paper with 1 drop of normal saline, and touch the tip of the test paper to the inner side of the mouse's lower eyelid for 10 seconds. Blink three times to evenly distribute the fluorescein. The time when the first black dry spot appears on the cornea is observed under the cobalt blue light of the slit lamp. The average value of both eyes is taken. Secondly, the tear film stability is tested by corneal fluorescein staining score. Corneal fluorescein staining score is used to directly reflect the destruction of the ocular epithelial barrier and secondary inflammatory damage caused by dry eye, and is a key indicator for evaluating disease progression and treatment efficacy. Tear film stability is tested by corneal fluorescein staining score. Fix the mouse, moisten the sodium fluorescein test paper with 1 drop of normal saline, touch the tip of the test paper to the inside of the mouse's lower eyelid and stay for a while, blink several times to evenly distribute the fluorescein, observe the corneal fluorescence staining under cobalt blue light with a slit lamp, and evaluate the staining grade. The scoring method is to divide the cornea into 4 quadrants, with no staining as 0 points, staining as light, moderate, and heavy, 1 point for less than 5 staining points, 3 points for the presence of blocky staining or filaments, and 2 points for something in between, for a total of 0-12 points.

[0035] 2.1.1 After 5 consecutive days of administration ( Figure 6 Compared with the blank control group, the tear film breakup time of mice in the DED model group was shortened and the corneal epithelial damage was severe. Compared with the model group, treatment with the positive drug sodium hyaluronate eye drops (SH) significantly improved the tear film breakup time and showed a trend of improvement in corneal damage. Among the different pHDL treatment groups, the tear film breakup time of mice in the medium-dose (10 mg / mL) and high-dose (20 mg / mL) groups was significantly prolonged, and corneal damage was alleviated.

[0036] 2.1.2 After 10 days of continuous administration ( Figure 7 Compared with the blank group, the tear film breakup time of mice in the model group was significantly shortened, and corneal epithelial damage was severe. Treatment with the positive drug sodium hyaluronate eye drops (SH) did not significantly improve tear film breakup time compared with the model group, but corneal damage was significantly improved. Among the different pHDL treatment groups, the tear film breakup time of mice treated with the medium dose (10 mg / mL) and high dose (20 mg / mL) groups was significantly prolonged, and corneal damage was significantly improved. Corneal damage was also significantly improved in mice treated with the low dose (5 mg / mL).

[0037] 2.2 Improvement of Ocular Histopathology in Dry Eye: After anesthesia and sacrifice, the left eyeballs of mice were removed, fixed in 4% paraformaldehyde solution for 24 hours, embedded in paraffin, and serially sectioned to a thickness of approximately 4 μm. After staining and drying, the pathological morphological changes of the cornea, lacrimal gland tissue, and conjunctiva were observed under an optical microscope. Images were collected, and corneal epithelial thickness was statistically analyzed. The results showed: 2.2.1 Hematoxylin and eosin (HE) staining of the cornea and lacrimal glands of mice in each group Figure 8 The corneal histology of mice in the normal group was essentially normal. The corneal epithelial cells showed regular morphology, distinct layers, well-defined contours, uniform chromatin, and tight intercellular connections. The epithelial cells were neatly arranged in approximately 3-4 layers, with a smooth surface and no thickening. The stromal layer consisted of collagen fibers parallel to the cornea, with scattered fibroblasts distributed in a dense arrangement. No inflammatory cell infiltration was observed. Compared with the normal group, the corneal epithelium in the BAC group was significantly thickened, with an increased number of cell layers and a rough corneal surface (as indicated by the red arrows). The corneal epithelial cells were partially edematous with pale cytoplasm. The collagen fibers in the stromal layer were loosely arranged, with slightly loose intercellular connections (as indicated by the blue arrows). Compared with the model group, the corneal epithelial thickening in the SHD group remained unchanged, with blurred cell membrane boundaries and disorganized arrangement. The collagen fibers in the stromal layer were loosely arranged. The corneal epithelium in the pHDL 5 mg group was slightly thinner, with a smoother surface but some epithelial cell desquamation. The collagen fibers in the stromal layer were more neatly arranged. The corneal epithelium recovered somewhat in the pHDL 10 mg group, with a thicker, smoother surface and neatly arranged stromal layers. The cornea recovered significantly in the pHDL 20 mg group, with regular, clear epithelial cell morphology and tightly arranged stromal layers.

[0038] Furthermore, in the lacrimal glands of mice in the normal group, the lacrimal gland tissue was composed of lobules of varying sizes, with alveoli of uniform size and regular morphology. The acinar cells were densely arranged, the lacrimal epithelial cells were intact and columnar, the cytoplasm was rich in secretory vesicles, and alveolar mucus was visible in the acinar lumen. Compared with the normal group, the lacrimal gland cells in the BAC model group were atrophic, irregularly arranged, and structurally disrupted. The mucus content in the lumen was reduced, the alveoli and lacrimal epithelium were of varying sizes and disordered, the connective tissue was significantly swollen, and inflammatory cells infiltrated the interstitium. Compared with the model group, the lacrimal gland tissue damage in the SHD group was less severe, with slightly irregular alveolar arrangement but generally similar to that of the normal group, with no significant abnormalities. The lacrimal gland tissue damage in the pHDL 5 mg group was more severe, similar to that of the model group. The pathological damage in the pHDL 10 mg group was less severe, with intact lacrimal gland structure and smaller but more regular glandular epithelial cells. The pHDL 20 mg group showed significant improvement, with the lacrimal gland structure remaining relatively intact.

[0039] 2.2.2 Periodic Acid-Schiff stain (PAS) for improving dry eye Figure 9 . PAS staining of the conjunctiva of mice in each group showed that the conjunctival epithelial goblet cells were purple-red and round or oval. In the normal group, the number of goblet cells in the conjunctival fornix was large, the morphology was plump, and the distribution was uniform. Compared with the normal group, the number of goblet cells in the BAC model group was reduced, the distribution was sparse, and some areas were completely missing and the cell morphology became smaller, as shown by the purple arrows (the area where goblet cells were missing). Compared with the model group, the goblet cells in the SH group recovered slightly, but there were still many missing cells; there was improvement in all doses of pHDL, and varying numbers of goblet cells were visible. Among them, the pHDL 20 mg group had more cells, and the morphology was fuller and more uniform.

[0040] 2.3 Security Figure 10 Serum was collected from mice and assayed for ALT (alanine aminotransferase); AST (aspartate aminotransferase); TP (total protein); ALB (albumin); LDH (lactate dehydrogenase); CK (creatine kinase); BUN (serum urea nitrogen); and CR (serum creatinine). Serum biochemical analysis was performed after benzalkonium chloride modeling, 0.1% sodium hyaluronate eye drops (SH), and pHDL treatment. No liver or kidney damage was observed with either treatment. ALT (alanine aminotransferase); AST (aspartate aminotransferase); TP (total protein); ALB (albumin); LDH (lactate dehydrogenase); CK (creatine kinase); BUN (serum urea nitrogen); and CR (serum creatinine) were detected.

[0041] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with this patent can make slight changes or modifications to equivalent embodiments using the above-mentioned technical contents without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. A new use of 4F dimer polypeptide and its lipidated product, characterized in that: The 4F dimer polypeptide and its lipidate are used to prepare a drug for treating dry eye.

2. The use according to claim 1, characterized in that The gene sequence of the 4F dimer polypeptide is shown in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that The 4F dimer polypeptide and its lipidated product are complexes of the 4F dimer polypeptide and phospholipid.

4. The use according to claim 3, characterized in that The molar ratio of 4F dimer polypeptide to phospholipid is 1:50-1:

100.

5. The use according to claim 3, characterized in that The 4F dimer polypeptide and its lipid product are complexes of 4F dimer polypeptide and phospholipid, and the particle size is 30 nm.

6. The use according to claim 1, characterized in that When the 4F dimer polypeptide and its lipid compound are used to prepare a drug for treating dry eye, a pharmaceutically acceptable carrier is also included.

7. The use according to claim 1, characterized in that The 4F dimer polypeptide and its lipid compound are prepared with physiological saline, with the concentration of the 4F dimer polypeptide being 5-20 mg / ml and the concentration of the lipid compound being 5-20 mg / ml.

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