Use of KGF-2 in treatment of dry eye diseases

Abstract

Provided in the present invention is the use of KGF-2 in the treatment of dry eye diseases. By means of constructing a KGF-2 heterozygous mouse model and a dry eye disease mouse model induced by scopolamine combined with a dry environment, it is proved that KGF-2 can improve basal tear secretion, prolong the breakup time of the tear film, and reduce corneal epithelial and corneal nerve damage in dry eye disease mice in a dose-dependent manner, and can also decrease the expression level of inflammatory factors such as MMP-3, MMP-9 and TNFα in the lacrimal gland, and can further improve the tear secretion function of the lacrimal gland.

Description

Application of KGF-2 in the treatment of dry eye Technical Field

[0001] The present invention relates to the field of biomedicine technology, in particular to keratinocyte growth factor (KGF-2), and in particular to the application of KGF-2 in treating dry eye. Background Art

[0002] Studies have shown that the incidence of dry eye in my country has reached 21% to 30%, and the age of the affected population is tending to be younger.

[0003] In recent years, with the increasing incidence of dry eye, the treatment of dry eye has become a hot topic of discussion. The treatment of dry eye in China mainly focuses on symptomatic and topical medication, such as artificial tears, corticosteroids, and immunosuppressants such as cyclosporine A. Artificial tears can be used to relieve symptoms, but they themselves have no therapeutic effect and can only work for a short time. Long-term use of drugs such as corticosteroids and cyclosporine A has toxic side effects on the ocular surface and cannot fundamentally achieve a therapeutic effect. Artificial tears are suitable for mild dry eye, while moderate to severe dry eye requires targeted etiological drugs. Marketed targeted medications primarily fall into two categories: anti-inflammatory therapies and mucin-stimulating therapies. Examples include Allergan's 0.05% cyclosporine A ophthalmic emulsion, Restasis (launched in 2003 and the first anti-inflammatory dry eye medication), and Novartis' Lifitegrast eye drops, Xiidra (a lymphocyte function-associated antigen-1 (LFA-1) antagonist). Both products utilize anti-inflammatory therapies to treat dry eye. Regardless of the etiology, conventional treatment for dry eye is based on correcting contributing factors and / or using artificial tear substitutes. While these can alleviate dry eye symptoms in the short term, they are not fundamentally effective, especially for moderate to severe dry eye. In severe cases, dry eye can lead to severe corneal damage. To treat moderate to severe dry eye, it is necessary to identify pharmacological targets for targeted treatment. In recent years, precise, personalized therapies have gained increasing recognition among physicians and patients. However, faced with the huge market demand and huge unmet clinical needs for dry eye, there is still a lack of new drugs independently developed by my country that target multiple causes of dry eye and can be used for both mild and moderate to severe dry eye. The development of safe and reliable new drugs targeting dry eye caused by multiple factors is also a hot topic in the international ophthalmology research field. Summary of the Invention

[0004] To address the shortcomings of the prior art, the present invention provides, in one aspect, the use of keratinocyte growth factor (KGF-2) in the preparation of a medicament for treating dry eye. Preferably, the present application also relates to the use of orthologs, derivatives, and fragments of keratinocyte growth factor (KGF-2) wherein at least one residue is substituted with a different amino acid residue, in the preparation of a medicament for treating dry eye.

[0005] According to a preferred embodiment, keratinocyte growth factor KGF-2 increases basal tear secretion.

[0006] According to a preferred embodiment, keratinocyte growth factor KGF-2 upregulates tear film breakup time.

[0007] According to a preferred embodiment, keratinocyte growth factor KGF-2 downregulates the expression levels of inflammatory factors such as MMP-9, MMP-3, TNF-α, IL-6, and IL-1β.

[0008] According to a preferred embodiment, keratinocyte growth factor KGF-2 downregulates the phosphorylation level of NF-κB.

[0009] According to a preferred embodiment, keratinocyte growth factor KGF-2 downregulates the degree of corneal sodium fluorescein staining.

[0010] According to a preferred embodiment, keratinocyte growth factor KGF-2 downregulates corneal sodium fluorescein staining score.

[0011] According to a preferred embodiment, keratinocyte growth factor KGF-2 increases corneal nerve sensitivity.

[0012] According to a preferred embodiment, keratinocyte growth factor KGF-2 downregulates the expression level of NF-κB P65.

[0013] Another aspect of the present invention provides use of keratinocyte growth factor KGF-2 in the preparation of a medicament for treating dry eye caused by meibomian gland abnormalities.

[0014] Another aspect of the present invention provides the use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating dry eye caused by meibomian gland abnormalities due to changes in immune cells.

[0015] On the other hand, the present invention also provides the use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating dry eye caused by meibomian gland dysplasia due to changes in dendritic cells and macrophages.

[0016] Another aspect of the present invention provides the use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating corneal damage.

[0017] Another aspect of the present invention provides the use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating corneal nerve damage.

[0018] Another aspect of the present invention provides use of keratinocyte growth factor KGF-2 in preparing a medicament for treating conjunctival damage.

[0019] Another aspect of the present invention provides use of keratinocyte growth factor KGF-2 in the preparation of a medicament for treating conjunctival nerve damage.

[0020] Another aspect of the present invention provides use of keratinocyte growth factor KGF-2 in the preparation of a medicament for treating Harderian gland damage.

[0021] Another aspect of the present invention provides the use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating dry eye caused by Sjögren's syndrome.

[0022] Another aspect of the present invention provides the use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating dry eye induced by scopolamine combined with dry environment.

[0023] According to a preferred embodiment, keratinocyte growth factor KGF-2 increases the basal tear secretion of subjects with dry eye induced by scopolamine combined with a dry environment.

[0024] According to a preferred embodiment, keratinocyte growth factor KGF-2 upregulates the tear film breakup time in subjects with dry eye induced by scopolamine combined with a dry environment.

[0025] According to a preferred embodiment, keratinocyte growth factor KGF-2 downregulates the expression levels of inflammatory factors such as MMP-9, MMP-3, TNF-α, IL-6, and IL-1β in subjects with dry eye induced by scopolamine combined with a dry environment.

[0026] According to a preferred embodiment, keratinocyte growth factor KGF-2 downregulates the phosphorylation level of NF-κB in subjects with dry eye induced by scopolamine combined with dry environment.

[0027] According to a preferred embodiment, keratinocyte growth factor KGF-2 downregulates the degree of corneal fluorescein sodium staining in subjects with dry eye induced by scopolamine combined with a dry environment.

[0028] According to a preferred embodiment, keratinocyte growth factor KGF-2 downregulates the corneal sodium fluorescein staining score of subjects with dry eye induced by scopolamine combined with dry environment.

[0029] Another aspect of the present invention provides that keratinocyte growth factor KGF-2 improves corneal nerve sensitivity in subjects with dry eye induced by scopolamine combined with dry environment.

[0030] Another aspect of the present invention provides the use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating corneal damage in subjects with dry eye induced by scopolamine combined with a dry environment.

[0031] Another aspect of the present invention provides the use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating corneal nerve damage in subjects with dry eye induced by scopolamine combined with a dry environment.

[0032] Another aspect of the present invention provides the use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating Harderian gland damage in subjects with dry eye induced by scopolamine combined with a dry environment.

[0033] Another aspect of the present invention provides the use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating conjunctival damage or conjunctival nerve damage in subjects with dry eye induced by scopolamine combined with a dry environment.

[0034] Another aspect of the present invention provides the use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating meibomian gland abnormalities induced by scopolamine combined with a dry environment.

[0035] On the other hand, the present invention also provides the use of keratinocyte growth factor KGF-2 in the preparation of a medicine for treating meibomian gland dysfunction caused by electric burns.

[0036] According to a preferred embodiment, keratinocyte growth factor KGF-2 increases basal tear secretion in subjects with meibomian gland dysfunction caused by electrical burns.

[0037] According to a preferred embodiment, keratinocyte growth factor KGF-2 upregulates tear breakup time in subjects with meibomian gland dysfunction caused by electrical burns.

[0038] According to a preferred embodiment, keratinocyte growth factor KGF-2 downregulates the expression levels of inflammatory factors such as MMP-9, TNF-α, IL-6, and IL-1β in subjects with meibomian gland dysfunction caused by electrical burns.

[0039] According to a preferred embodiment, keratinocyte growth factor KGF-2 downregulates the phosphorylation level of NF-κB in subjects with meibomian gland dysfunction caused by electrical burns.

[0040] According to a preferred embodiment, keratinocyte growth factor KGF-2 downregulates the degree of corneal fluorescein sodium staining in subjects with meibomian gland dysfunction caused by electrical burns.

[0041] According to a preferred embodiment, keratinocyte growth factor KGF-2 downregulates corneal sodium fluorescein staining scores in subjects with meibomian gland dysfunction caused by electrical burns.

[0042] Another aspect of the present invention provides an external preparation, which includes keratinocyte growth factor KGF-2.

[0043] According to a preferred embodiment, the topical preparation is administered via ocular therapy.

[0044] According to a preferred embodiment, the external preparation can include eye drops, eye washes, eye drops, eye gels or lacrimal duct insertion / implants.

[0045] According to a preferred embodiment, the dosage regimen of the external preparation involved in the present application can be: eye drops 1 to 10 times a day, preferably 2 to 8 times a day, more preferably 3 to 6 times a day, and further preferably 5 times a day.

[0046] According to a preferred embodiment, the keratinocyte growth factor KGF-2 involved in the present application can be derived from recombinant keratinocyte growth factor KGF-2 engineered bacteria or plants into which the keratinocyte growth factor KGF-2 encoding gene is inserted using recombinant DNA technology.

[0047] Another aspect of the present invention provides a pharmaceutical composition, which includes keratinocyte growth factor KGF-2 and excipients used in eye drops.

[0048] According to a preferred embodiment, the adjuvant comprises an antiseptic with an antiseptic effect, such as chlorhexidine compounds, boric acid compounds, chlorite compounds, parahydroxybenzoate compounds, sorbic acid compounds, chlorobutanol, benzethonium chloride. Preferably, the boric acid compound can be boric acid and its salts. More preferably, the adjuvant comprises sodium borate, potassium tetraborate, potassium methylborate, ammonium borate or borax. Preferably, the chlorhexidine compound can be an organic acid salt (monocarboxylate, polycarboxylate) or an inorganic acid salt (hydrochloride, nitrate or phosphate) allowed as a drug. Preferably, the chlorite compound can be chlorite and its salts, such as sodium chlorite, potassium chlorite, calcium chlorite, magnesium chlorite. Preferably, the parahydroxybenzoate compound can be parahydroxybenzoate and its salts, such as ethyl parahydroxybenzoate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, isopropyl parahydroxybenzoate, butyl parahydroxybenzoate, isobutyl parahydroxybenzoate. Preferably, the sorbic acid compound may be sorbic acid and its salts, such as potassium sorbate.

[0049] According to a preferred embodiment, the excipient further comprises an isotonic agent, a buffer, a surfactant, a stabilizer or a pH adjuster within the range permitted by ophthalmic preparations. Preferably, the isotonic agent comprises sodium chloride, potassium chloride, or glycerol. The buffer comprises sodium phosphate, sodium acetate, or ε-aminocaproic acid. The surfactant comprises Tween 80, polyoxyethylene (40) stearate, or polyoxyethylene hydrogenated castor oil. The stabilizer comprises sodium citrate or sodium edetate. The pH adjuster comprises phosphate buffer.

[0050] Another aspect of the present invention provides a pharmaceutical composition, which comprises one of the following groups: a. a preparation of keratinocyte growth factor KGF-2 and T5122; b. a preparation of keratinocyte growth factor KGF-2 and LY29004.

[0051] Another aspect of the present invention provides a method of treatment, comprising applying the topical preparation of the present application to the eye of a human patient.

[0052] Another aspect of the present invention provides a method for treating dry eye with combined medication, comprising administering to the eyes of a human patient an effective amount of one of the following pharmaceutical compositions: a. a preparation of keratinocyte growth factor KGF-2 and T5122; b. a preparation of keratinocyte growth factor KGF-2 and LY29004.

[0053] Beneficial effects of the present invention:

[0054] The present invention constructs a KGF-2 heterozygous mouse model and a dry eye mouse model induced by scopolamine combined with a dry environment, and confirms that KGF-2 can improve the basal tear secretion of dry eye mice in a dose-dependent manner, prolong the tear film break-up time (T-BUT), and reduce corneal epithelial and corneal nerve damage; and KGF-2 can reduce the expression levels of inflammatory factors such as MMP-3, MMP-9 and TNFα in the lacrimal gland, and improve the tear secretion function of the lacrimal gland; in addition, KGF-2 can not only minimize the signs of dry eye in mice, but also repair damaged corneal nerves and reduce the inflammatory level of the corneal microenvironment.

[0055] In the study of KGF-2 heterozygous mouse model, the present invention explored the effect of KGF-2 on the meibomian gland. Through comprehensive phenotypic analysis and RNA-seq transcriptome analysis, the present invention innovatively found that during the development of KGF-2 heterozygous mice, the meibomian glands appeared delayed, shorter, thinner, and more disordered, and locked in the specific cell type as immune cells, thus concluding that KGF-2 may play an important role by regulating the immune microenvironment; In addition, the present invention used immunofluorescence experiments with immune cell-specific markers to clarify that the meibomian gland immune cells affected by KGF-2 are mainly Cd11b + / Cd11c- Dendritic cells. The above results indicate that KGF-2 can be well used in the treatment of moderate to severe dry eye and can play an important role in the treatment of multifactorial dry eye. Furthermore, the in vitro experiments provided by the present invention utilize different concentration gradients of KGF-2 for administration, further confirming the therapeutic effect of KGF-2 in dry eye.

[0056] This study explores the effects of KGF-2 on meibomian gland development and identifies key target cells, providing new insights into meibomian gland development and the treatment of dry eye-related diseases. It also demonstrates that KGF-2 has broad application prospects in the treatment of dry eye. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG1 shows the measurement results of the fluorescein sodium staining degree ( FIG1A ), fluorescein sodium staining score ( FIG1B ), basal tear secretion ( FIG1C ), and tissue pathology ( FIG1D ) of the scopolamine-treated mouse model in a dry environment provided by the present invention;

[0058] FIG2 is the immunohistochemical staining results ( FIG2A ) and statistical analysis results of the expression levels ( FIG2B ) of corneal inflammatory factors MMP-9, IL-6, and TNF-α in a mouse model of scopolamine combined with a dry environment provided by the present invention;

[0059] FIG3 shows the results of sodium fluorescein staining ( FIG3A ), the results of sodium fluorescein staining scoring ( FIG3B ), and the results of basal tear secretion ( FIG3C ) after administration of KGF-2 provided by the present invention;

[0060] FIG4 is a diagram showing the histopathological determination results of cornea, Harderian gland and conjunctiva provided by the present invention;

[0061] FIG5 is the immunofluorescence staining results ( FIG5A ) and statistical analysis results of the expression level ( FIG5B ) of the inflammatory factor MMP-9 after drug administration provided by the present invention;

[0062] FIG6 is the immunofluorescence staining results and statistical analysis results of the expression levels of the inflammatory factors TNF-α ( FIG6A and FIG6B ) and IL-6 ( FIG6C and FIG6D ) after the administration of the present invention;

[0063] FIG7 shows the basal tear secretion ( FIG7A and FIG7B ) and corneal injury results ( FIG7C ) of different groups of mice provided by the present invention;

[0064] FIG8 shows the results of sodium fluorescein staining ( FIG8A ), sodium fluorescein staining scoring ( FIG8B ), corneal tissue pathology ( FIG8C ), basal tear secretion ( FIG8D ), BUT ( FIG8E ), and corneal nerve sensory test ( FIG8F ) of mice induced by scopolamine combined with a dry environment provided by the present invention;

[0065] FIG9 is a staining diagram of KGF-2 repairing corneal superficial nerves provided by the present invention;

[0066] FIG10 is a result of detecting the levels of inflammatory factors TNFα, MMP-3, and MMP-9 in lacrimal gland tissue under KGF-2 treatment provided by the present invention;

[0067] FIG11 shows the effects of hypertonic stress conditions provided by the present invention on the activity of human corneal epithelial cells ( FIG11A and FIG11B ), and the effects of KGF-2 on the activity of human corneal epithelial cells ( FIG11C and FIG11D ).

[0068] FIG12 is a graph showing the effects of different concentrations of KGF-2 on the expression levels of MMP-9, IL-1β, and IL-6 in human corneal epithelial cells under hypertonic stress conditions in vitro provided by the present invention;

[0069] FIG13 is a graph showing the effect of different concentrations of KGF-2 on the expression level of NF-κB P65 in human corneal epithelial cells under hypertonic stress conditions in vitro provided by the present invention;

[0070] FIG14 is the identification results and transcription levels of KGF-2 heterozygous mice (HET) provided by the present invention;

[0071] FIG15 shows the results of dry eye-like ocular surface damage in KGF-2 heterozygous mice provided by the present invention;

[0072] FIG16 shows the results of meibomian gland morphological changes at different developmental time points provided by the present invention;

[0073] FIG17 is a volcano plot of differential expression provided by the present invention;

[0074] FIG18 is the KEGG signaling pathway enrichment analysis results provided by the present invention;

[0075] FIG19 is a GO enrichment analysis result provided by the present invention;

[0076] FIG20 is a heat map analysis result of representative genes of immune cells provided by the present invention;

[0077] FIG21 is the immune cell staining results of mouse meibomian gland tissue provided by the present invention;

[0078] FIG22 is the immune cell staining results of mouse meibomian gland tissue provided by the present invention;

[0079] FIG23 shows the results of meibomian gland developmental defects caused by pharmacological ablation of immune cells provided by the present invention;

[0080] FIG24 shows a model of meibomian gland dysfunction caused by electric burns and the results of meibum secretion induced by electric burns provided by the present invention;

[0081] FIG25 shows the corneal epithelial wound healing results after administration of KGF-2 provided by the present invention;

[0082] FIG26 shows the healing results of the eyelid conjunctival epithelial tissue after administration of KGF-2 provided by the present invention;

[0083] FIG. 27 shows the results of inflammatory infiltration of meibomian glands after administration of KGF-2 provided by the present invention. DETAILED DESCRIPTION

[0084] Keratinocyte Growth Factor-2 (KGF-2: Keratinocyte Growth Fatcor-2) is also known as fibroblast growth factor-10 (FGF-10: Fibroblast Growth Factor-10). A decrease in the copy number of the KGF-2 gene leads to abnormal development of the lacrimal gland and the occurrence of dry eyes. KGF-2 is highly specific to epithelial tissue. KGF-2 can promote the repair of corneal epithelial damage, maintain the integrity of the ocular surface barrier, repair the ocular surface immune network, improve the ocular surface inflammatory microenvironment, and reduce apoptosis of lacrimal glands, corneas, and conjunctival cells. KGF-2 can reduce the expression of inflammatory factors (including IL-1α, IL-6, and TNF-α, etc.). KGF-2 can regulate signal pathways and inhibit cell apoptosis. In addition, KGF-2 can induce sympathetic nerve activity and has a certain protective effect on nerves.

[0085] In this application, T5122 is Alofanib (RPT835), a highly potent and selective allosteric inhibitor of fibroblast growth factor receptor 2; LY29004 is a selective inhibitor of mitogen-stimulated phosphatidylinositol 3-kinase activity. Both T5122 and LY29004 are commercially available reagents in the prior art.

[0086] The subjects of the present invention are mice, but can also be other mammals (such as humans). In the following examples, healthy C57BL / 6 background KGF-2 heterozygous mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) and Cx3cr1 GFP / + Fluorescently labeled mice (Jackson Laboratory, USA, Strain#: 005582), clean grade. All experimental procedures strictly followed the "Regulations on the Administration of Laboratory Animals of the People's Republic of China" and the "Animal Experimentation Guide of Wenzhou Medical University".

[0087] In this invention, the length of the phenol red-soaked cotton thread represents basal tear secretion, and is uniformly represented as "basal tear secretion" in the accompanying figures. The normal control group of mice refers to wild-type mice, also represented by "WT" in the accompanying figures. KGF-2 heterozygous mice are represented by "HET" in the accompanying figures.

[0088] Figure 4 shows histopathological examinations of the cornea, Harderian gland, and conjunctiva of mice in the treatment groups (25 μg / mL KGF-2, 100 μg / mL KGF-2, and 200 μg / mL KGF-2) compared to the model group (model). The cornea, Harderian gland, and conjunctiva all showed varying degrees of recovery (positive changes in cell number, cell arrangement, and inflammatory infiltration). This suggests that KGF-2 can act on the cornea, Harderian gland, and conjunctival tissues, thereby alleviating dry eye symptoms, including eye dryness, burning sensation, foreign body sensation, and blurred vision.

[0089] Example 1

[0090] This example provides a study on the effect of KGF-2 in treating dry eye in mice.

[0091] 1. Establishment of a scopolamine-induced dry environment mouse model

[0092] From 0d to 7d, 100 μL of 5 mg / mL scopolamine hydrobromide solution was injected subcutaneously behind both ears. Indicators, including basal tear secretion and ocular surface fluorescein sodium staining, were measured on 0d, 1d, 4d, and 7d. Samples were collected 7d later, stained with HE, and pathological features were observed.

[0093] 2. Administer medication and measure indicators

[0094] Groups: normal control group (Control), 0.9% NaCl + dry eye group (represented by 0.9% NaCl group in Figures 1 to 3), dry eye + KGF-2 administration group (25 μg / mL), dry eye + KGF-2 administration group (100 μg / mL), dry eye + KGF-2 administration group (200 μg / mL).

[0095] Eye drops: Topical eye drops were administered at 9:00, 13:00, 16:00, and 19:00 daily, with a volume of 5 μL / eye / time. Scopolamine was injected behind the ear at 9:00, 13:00, and 19:00 for the mice in the treatment group.

[0096] On days 0, 3, 7, 10, and 14, basal tear secretion was measured, ocular surface was stained with sodium fluorescein, and photographs were taken. After day 14, samples were collected and stained with HE.

[0097] Basal tear secretion was measured using the Schirmer I test in the control, 0.9% NaCl, and model groups. Measurements were taken at fixed time points after modeling. A tear detection phenol red cotton thread (Tianjin Jingming New Technology Development Co., Ltd.) was placed in the lateral canthus of the mouse eye using microtweezers and removed after 60 seconds. The length of the phenol red cotton thread infiltrated was measured with a ruler. This was repeated three times, and the average value was calculated.

[0098] Scoring of ocular surface fluorescein sodium staining: Mice were anesthetized with 4% chloral hydrate. 1 μL of 0.5% sodium fluorescein solution was dripped into the conjunctival sac of the mouse, and the eyelids were closed. Excess fluorescein was then rinsed away with 0.9% saline, and photographs were taken under cobalt blue light using a slit lamp microscope. Finally, corneal sodium fluorescein staining was graded. The cornea was divided into four equal quadrants and scored separately, with all scores added together to obtain the final score. The scoring criteria for this example were: 0 points—no staining; 1 point—no more than 30 punctate staining; 2 points—more than 30 punctate staining but not diffuse; 3 points—severe diffuse staining but no patchy staining; 4 points—patchy staining.

[0099] Samples were collected and HE staining was performed 14 days later:

[0100] (1) Paraffin embedding. Under a slit lamp microscope, the corneas, Harderian glands, and conjunctiva of mice in different groups were removed and fixed in 4% paraformaldehyde overnight. The tissues were placed in embedding clips, labeled, and rinsed for about 6 hours, then transferred to 70% ethanol and stored overnight. The tissues and organs were dehydrated using an alcohol gradient, dehydrated with 80%, 85%, and 90% ethanol for 30 minutes each, and then transferred to 95% (I) ethanol, 95% (II) ethanol, 100% (I) ethanol, and 100% (II) ethanol. Dehydrate for 15 minutes; transfer the dehydrated tissue to xylene to make it transparent, and then make it transparent in xylene (I) and xylene (II) for 20 minutes each; wax immersion, the soft wax immersion time is 1.5 hours, and the hard wax immersion time is 1 hour; inject paraffin into the embedding box, transfer the tissue to the embedding box, continue to add paraffin, and after the paraffin is filled, solidify for a period of time, use tweezers to break the paraffin liquid film and slowly add paraffin here until the embedding box is filled, solidify again, and after it is completely solidified, take out the paraffin block and store it at 4°C.

[0101] (2) Sectioning. Fix the embedding cassette on the microtome and slice the tissue at a thickness of 5 μm.

[0102] (3) HE staining. Place the paraffin sections to be stained in a constant temperature oven at 60-65℃ for 5 hours, then dewax in xylene (I) and xylene (II) for 20 minutes each; hydrate with 100% ethanol for 5 minutes (twice), 95% ethanol, 90% ethanol, 80% ethanol, and 75% ethanol for 5 minutes each; wash 3 times, 5 minutes each; add appropriate amount of hematoxylin staining solution to the tissue and stain for 10 minutes; transfer the sections to purified water and soak for 5 minutes to wash off excess hematoxylin staining solution; transfer the sections to 0.5% eosin and stain for 30 seconds; soak in purified water for 1 minute to wash off excess eosin staining solution; dehydrate with 75%, 80%, 90%, 95%, and 100% ethanol for 1 minute each; transfer the dehydrated tissue to xylene for transparentization twice, 3 minutes each time; seal the slides with neutral resin and dry them at room temperature overnight.

[0103] 3. Test results

[0104] Figure 1 shows the results of fluorescein sodium staining, fluorescein sodium score, basal tear secretion, and tissue pathology of the mouse model of scopolamine combined with a dry environment. As the modeling time increases, the fluorescein sodium staining of the model mice deepens (Figure 1A). Figure 1B shows the fluorescein sodium staining score results, with the horizontal axis representing the number of days and the vertical axis representing the fluorescein sodium staining score. As the modeling time increases, the fluorescein sodium staining score increases, and the fluorescein sodium staining of the mice on the 7th day is the darkest (Figures 1A and 1B). The basal tear secretion of the mice gradually decreases compared with that of healthy mice, with the lowest basal tear secretion on the 7th day (Figure 1C). Compared with the normal control group (wild-type control group or Control) and the 0.9% NaCl group, the corneal epithelial cells of the model group (scopolamine combined with dry environment mouse model or Model) showed cell nuclei loss, tightly arranged extracellular matrix, abnormal cell morphology, and irregular arrangement. Inflammatory invasion of the Harderian glands was observed, mainly manifested by: a large number of cell nuclei in the cytoplasm and extracellular matrix of the model group mice; the number of conjunctival goblet cells was reduced, and the cells were irregularly arranged (Figure 1D). Goblet cells mainly synthesize and secrete mucin to form a mucosal barrier and play a protective role. The reduced number of conjunctival goblet cells indicates that mucin secretion is correspondingly affected.

[0105] Figure 2 shows the immunohistochemical staining results and statistical analysis of the expression levels of the corneal inflammatory factors MMP-9, IL-6, and TNF-α in a mouse model treated with scopolamine combined with a dry environment. Figure 2A shows that the tissue staining for MMP-9, IL-6, and TNF-α in the Model group deepened. Figure 2B shows the expression levels of the three inflammatory indicators MMP-9, IL-6, and TNF-α in different groups, with the horizontal axis representing the group and the vertical axis representing the expression level. Specifically, compared with the normal control group and the 0.9% NaCl group, the expression levels of the three inflammatory factors in the Model group increased, and the expression levels of MMP-9, IL-6, and TNF-α in the Model group were significantly higher than those in the 0.9% NaCl group.

[0106] Figure 3 shows the results of sodium fluorescein staining, sodium fluorescein staining scores, and basal tear secretion after KGF-2 administration. In Figure 3, 25 μg / mL refers to the dry eye group receiving KGF-2 (25 μg / mL), 100 μg / mL refers to the dry eye group receiving KGF-2 (100 μg / mL), and 200 μg / mL refers to the dry eye group receiving KGF-2 (200 μg / mL).

[0107] Figure 3A shows the results of sodium fluorescein staining after KGF-2 administration, which demonstrates that the degree of sodium fluorescein staining decreases with prolonged administration. Figure 3B shows the sodium fluorescein staining score, with time plotted on the abscissa and the sodium fluorescein staining score plotted on the ordinate. The sodium fluorescein staining score decreases with prolonged administration. On day 10 and beyond, the sodium fluorescein staining scores in the KGF-2 (25 μg / mL), KGF-2 (100 μg / mL), and KGF-2 (200 μg / mL) administration groups all decreased to varying degrees compared to the 0.9% NaCl group, with a gradual decrease in the number of fluorescein-stained spots ( Figure 3A ). The sodium fluorescein score in the KGF-2 (100 μg / mL) administration group decreased compared to the Saline, KGF-2 (25 μg / mL), and KGF-2 (200 μg / mL) administration groups, with statistically significant differences observed on day 10 and beyond ( Figure 3B ). *Compared with day 0, *P<0.05, **P<0.01, ***P<0.001.

[0108] During continuous KGF-2 treatment, basal tear secretion in mice gradually increased, and basal tear secretion in the KGF-2-treated groups (100 μg / mL) and (200 μg / mL) was greater than that in the KGF-2-treated group (25 μg / mL). Basal tear secretion improved in the saline group and in the three KGF-2 treatment groups at different concentrations. Within 14 days of treatment, the improvement in basal tear secretion in the 100 μg / mL and 200 μg / mL KGF-2 groups was statistically significant (Figure 3C).

[0109] Figure 4 shows the results of histopathological examination of the cornea, Harderian gland, and conjunctiva. Compared with the model group, the number of corneal epithelial cells in the drug-treated groups (25 μg / mL KGF-2, 100 μg / mL KGF-2, and 200 μg / mL KGF-2) increased, and the cells were arranged more neatly. The Harderian gland cells of the drug-treated groups (25 μg / mL KGF-2, 100 μg / mL KGF-2, and 200 μg / mL KGF-2) were neatly arranged, and the inflammatory infiltration was reduced. There was a small amount of inflammatory cell infiltration in the 0.9% NaCl group. There was an increase in conjunctival cells in the drug-treated groups (25 μg / mL KGF-2, 100 μg / mL KGF-2, and 200 μg / mL KGF-2), and the cells were neatly arranged. There was a small amount of cell loss in the model group + 0.9% NaCl group (referring to the dry eye model group treated with 0.9% NaCl).

[0110] Figure 5 shows immunofluorescence staining results and statistical analysis of the inflammatory factor MMP-9 expression after drug administration. Figures 5A and 5B show that after 14 days of drug administration, the expression of the inflammatory factor MMP-9 decreased in the 25 μg / mL KGF-2, 100 μg / mL KGF-2, and 200 μg / mL KGF-2 groups compared to the model group + 0.9% NaCl group. After 14 days of drug administration, the expression of the inflammatory factor MMP-9 in the 100 μg / mL KGF-2 group was significantly reduced compared to the model group + 0.9% NaCl group (*P < 0.05; **P < 0.01; ns: P > 0.05).

[0111] Figure 6 shows the immunofluorescence staining results and statistical analysis of the inflammatory factors TNF-α and IL-6 after treatment. Figure 6A shows the immunofluorescence staining results for TNF-α; Figure 6B shows the expression level of TNF-α. The results show that after 14 days of treatment, the expression level of the inflammatory factor TNF-α was reduced in the 25μg / mL KGF-2, 100μg / mL KGF-2, and 200μg / mL KGF-2 groups compared to the model group + 0.9% NaCl group. After 14 days of treatment, the expression level of the inflammatory factor TNF-α was significantly reduced in the 100μg / mL KGF-2 group compared to the model group + 0.9% NaCl group. Figure 6C shows the immunofluorescence staining results for IL-6; Figure 6D shows the expression level of IL-6. The results show that after 14 days of treatment, the expression level of the inflammatory factor IL-6 was significantly reduced in the 25μg / mL KGF-2 group compared to the model group + 0.9% NaCl group. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns: P>0.05.

[0112] In summary, the results of basal tear secretion, histopathological results, and immunohistochemical staining to determine the expression of inflammatory factors (TNF-α, MMP-9, IL-6) preliminarily determined the role of KGF-2 in the treatment of dry eye.

[0113] Example 2

[0114] This example provides a study on the effect of KGF-2 in treating dry eye in mice.

[0115] 1. Establishment of a scopolamine-induced dry environment mouse model

[0116] The construction method is the same as that in Example 1, and the determination method of relevant indicators is the same as that in Example 1, which will not be repeated in this example.

[0117] 2. Administer medication and measure indicators

[0118] After the model was successfully established, the experimental animals were transferred to a conventional environment with appropriate relative humidity.

[0119] Randomly divided into: normal control group (Control), model group + 0.9% NaCl group (or expressed as dry eye + 0.9% NaCl group), dry eye + high concentration KGF-2 administration group (100 μg / mL), dry eye + medium concentration KGF-2 (50 μg / mL), dry eye + low concentration KGF-2 administration group (25 μg / mL).

[0120] Each group of mice received topical eye drops at fixed time points every day: 9, 15 and 21 o'clock, and the drops were continued for 14 days.

[0121] Over 14 days of treatment, basal tear secretion, tear breakup time (BUT), and ocular surface fluorescein sodium staining were measured and photographed. Corneal nerve sensitivity was assessed on days 0, 7, and 14. On day 14, the animals were euthanized and the corneas, lacrimal glands, meibomian glands, and conjunctiva were collected and subjected to hematoxylin and eosin staining (to detect cell apoptosis) and immunofluorescence staining for inflammatory markers such as TNFα, IL-1, IL-6, MMP-9, and MMP-3.

[0122] Sodium fluorescein staining is used to determine whether there are defects on the corneal surface. The area of ​​corneal epithelial defect is stained yellow-green, and it is also used to measure the tear film break-up time (BUT).

[0123] Tear film breakup time (TBT) was determined by intraperitoneal injection of an appropriate amount of chloral hydrate on days 0, 7, 14, 21, 28, and 35 of model establishment. After anesthesia, 1–2 μL of 0.05% sodium fluorescein in saline was dripped onto the mouse ocular surface. After blinking thoroughly to evenly distribute the sodium fluorescein on the mouse ocular surface, the time it took for the sodium fluorescein covering the mouse ocular surface to break up was observed using diffuse cobalt blue light using a slit lamp. The TBT was recorded. Three measurements were repeated for each mouse at each time point, and the average value was used as the TBT for that day.

[0124] Corneal nerve staining: On the 14th day of treatment, the mice were anesthetized and the eyeballs were removed. The corneas were fixed with 4% paraformaldehyde for 30 minutes, washed three times with 1% Triton X-100 (Triton) / PBS, and permeabilized for 15 minutes. The corneas were blocked with 10% goat serum (BSA) in 0.1% Triton / PBS overnight, and then incubated with 1% FBS containing β-III tubulin (1:200) overnight. After overnight incubation, the sections were washed three times with 0.1% Triton / PBS for 30 minutes, incubated with fluorescently labeled secondary antibodies (1:500) at 37°C for 1.5 hours, and then stained with the nuclear dye 4'6-diamindo-2-phenylindole (DAPI). After staining, the sections were washed three times with 0.1% Triton / PBS for 30 minutes. Under darkroom conditions, the corneas were unfolded in PBS solution, and an incision was made on each cornea to obtain a flower-shaped whole scaffold (four quadrants). The sections were sealed with anti-fluorescence quenching PVP sealing fluid and photographed with an upright fluorescence microscope.

[0125] 3. Test results

[0126] The results after ocular administration of the drug to mice in the normal control group (Control), model group + 0.9% NaCl group, dry eye group receiving high concentration KGF-2 (100 μg / mL), dry eye group receiving medium concentration KGF-2 (50 μg / mL), and dry eye group receiving low concentration KGF-2 (25 μg / mL) are shown in Figure 7. The results in Figures 7A and 7B show that as the administration time prolonged, the basal tear secretion of the dry eye group receiving low-concentration KGF-2 (25 μg / mL), dry eye group receiving medium-concentration KGF-2 (50 μg / mL), and dry eye group receiving high-concentration KGF-2 (100 μg / mL) gradually increased compared with the Saline group. At 14 days after administration, the basal tear secretion of the dry eye group receiving low-concentration KGF-2 (25 μg / mL), dry eye group receiving medium-concentration KGF-2 (50 μg / mL), and dry eye group receiving high-concentration KGF-2 (100 μg / mL) was close to that of the Control group.

[0127] As the concentration of KGF-2 increased, the basal tear secretion of mice increased, that is, KGF-2 increased the aqueous tear secretion of dry eye mice in a dose-dependent manner (Figures 7A and 7B). In addition, Figure 7C shows the corneal damage results of mice in different groups as the treatment time increased. The results showed that corneal epithelial damage was improved in the dry eye group receiving medium concentration KGF-2 (50 μg / mL) and the dry eye group receiving high concentration KGF-2 (100 μg / mL), and the improvement in the dry eye group receiving high concentration KGF-2 (100 μg / mL) was more significant (Figure 7C).

[0128] Figure 8 shows the results of sodium fluorescein staining, sodium fluorescein staining scores, corneal histopathology, basal tear secretion, BUT, and corneal nerve sensory testing in mice induced by scopolamine combined with a desiccation environment. Figures 8A-8C show that over time, sodium fluorescein staining deepened, the sodium fluorescein staining score increased, and corneal inflammatory infiltration increased in mice induced by scopolamine combined with a desiccation environment. Tear secretion (Figure 8D) and BUT continued to decline over time (Figure 8E). Corneal nerve sensory testing remained abnormal, with decreased nerve sensitivity over time (Figure 8F). Corneal epithelial keratinization lesions were observed 28 days later (Figures 8A-8C).

[0129] Figure 9 shows the results of KGF-2 repairing damaged corneal superficial nerve morphology and the external reflex function of the corneal nerve. The results in Figure 9 show that after KGF-2 treatment, compared with the control group, the superficial corneal nerve structure and nerve fiber structure of mice treated with only normal saline (0.9% NaCl) were abnormal, the number of brush-like nerve fibers decreased, and the "vortex" structure in the center of the nerve disappeared; the superficial corneal nerve network of mice treated with 50μg / mL and 100μg / mL of KGF-2 was restored, and the high dose (100μg / mL) of KGF-2 had a better therapeutic effect, and the "vortex" structure in the center of the corneal nerve was restored. The morphology of damaged superficial corneal nerves was improved, the density of corneal axons increased, the sensitivity of the corneal nerve increased, and the external reflex function of the corneal nerve was enhanced.

[0130] Figure 10 shows the results of KGF-2 treatment in lacrimal gland tissues, showing levels of inflammatory factors such as TNFα, MMP-3, and MMP-9. KGF-2 refers to KGF-2 alone, KGF-2+T5122 refers to combined treatment with KGF-2 and T5122, and KGF-2+LY29004 refers to combined treatment with KGF-2 and LY29004. The results in Figure 10 demonstrate that, compared to the control group, KGF-2 alone resulted in fuller and more densely packed lacrimal gland cells. The fuller and more densely packed lacrimal gland cells in the KGF-2+T5122 and KGF-2+LY29004 treatments were less dense than those in the KGF-2 alone group.

[0131] Example 3

[0132] This example provides an in vitro experiment of KGF-2 administration, and obtains the results of the effect of hyperosmotic stress on human corneal epithelial cells.

[0133] 1. Experimental Materials and Methods

[0134] (1) Screening of hypertonic stress conditions: MTT method was used to detect cell activity, and modeling time and osmotic pressure concentration were screened.

[0135] ① Human corneal epithelial cells were treated and seeded in 96-well plates, with 1×10 4 / 100 μl, set 6 replicate wells for each time period and osmotic pressure concentration, and when the cell inoculation volume reached 80%, starve the cells with serum-free culture medium and culture them overnight.

[0136] ② Discard the old culture medium and add hypertonic solution with a concentration gradient of 336mOsM, 400mOsM, 450mOsM, 500mOsM, and 550mOsM, and culture for 6h, 9h, 18h, and 24h.

[0137] ③ Prepare 5 mg / mL MTT solution in advance. Weigh 250 mg of MTT powder using an analytical balance and dissolve it in 50 mL of PBS. Mix thoroughly to ensure complete dissolution. Filter with a 0.22 μm filter, wrap in tin foil, protect from light, and store at 4°C.

[0138] ④ Remove the old culture medium and add 20 μL of MTT solution under light-proof conditions; continue to culture in a constant temperature cell culture incubator for 4 hours.

[0139] ⑤ After 4 h, remove the MTT solution and add 120 μL of DMSO solution to each well. Oscillate on an oscillator for 5 min to completely dissolve the formazan crystals.

[0140] ⑥ Use a microplate reader to measure the OD value at 490nm.

[0141] (2) Preliminary screening of KGF-2 toxicity to human corneal epithelial cells

[0142] ①This step is the same as ① in the screening conditions for hypertonic stress.

[0143] ② Set up a control group of 336mOsM and a model group of 450mOsM+KGF-2 drug administration groups (0.2, 0.4, 1.6, 3.2, 6.4μg / ml); treat the cells with KGF-2 and culture them in an incubator for 24 hours.

[0144] ③This step is the same as ③ to ⑥ in the screening conditions for hypertonic stress.

[0145] (3) Screening of KGF-2 dosage concentration

[0146] ①This step is the same as ① in the screening conditions for hypertonic stress.

[0147] ② A control group of 336mOsM and a model group of 450mOsM were set up, and the model group of 450mOsM+KGF-2 treatment groups (0.05, 0.1, 0.4, 0.8, 1.6, 3.2μg / ml) were used. The cells were pretreated with different concentrations of KGF-2 for 1h, and then hypertonic culture medium was added and cultured for 24h.

[0148] ③This step is the same as ③ to ⑥ in the screening conditions for hypertonic stress.

[0149] The results are shown in Figure 11. Compared with the normal group, ****p≤0.001, ***p≤0.001, **p≤0.01, *p≤0.05. Figure 11A shows the results of changes in cell activity under different osmotic pressure concentrations and at different times. According to Figure 11A, it can be concluded that the 24-hour hypertonic stress modeling has a significant effect on human corneal epithelial cells, so the 24-hour modeling was selected. Figure 11B shows the results of changes in cell activity under different osmotic pressure conditions within 24 hours. According to Figure 11B, it can be concluded that the survival rate of HCEC cells exposed to an osmotic pressure gradient is reduced. Combined with existing research results, the modeling condition of 450mOsM was finally selected. Figure 11C shows the effect of KGF-2 on the activity of human corneal epithelial cells. The horizontal axis is the concentration of applied KGF-2, and the vertical axis is the cell survival rate. According to the results in Figure 11C, after 24 hours of culture with KGF-2 drugs at different concentration gradients, the cell survival rate at KGF-2 drug concentrations of 0.2 μg / mL and 0.4 μg / mL was no different from that of the normal control group (represented as 336 mOsM in this example). As the KGF-2 concentration increased (1.6 μg / mL, 3.2 μg / mL, 6.4 μg / mL), the cell survival rate decreased significantly, indicating that 0.2 μg / mL and 0.4 μg / mL KGF-2 drug culture had no toxicity to the cells (*P<0.05; **P<0.01; ***P<0.001).

[0150] Figure 11D shows the changes in cell viability when treated with different concentrations of KGF-2 at 450 mOsM osmotic pressure. The abscissa of Figure 11D represents KGF-2 concentration, and the ordinate represents cell survival rate. Based on the experimental results in Figure 13 , after treatment with varying KGF-2 concentrations, cell survival rates were highest at 0.1 μg / mL, 0.2 μg / mL, and 0.4 μg / mL, indicating that treatment efficacy was best achieved at these concentrations. Therefore, the initial KGF-2 dosages of 0.1 μg / mL, 0.2 μg / mL, and 0.4 μg / mL were selected (*P < 0.05; **P < 0.01; ***P < 0.001).

[0151] Figure 12 shows the effects of different concentrations of KGF-2 on the expression levels of inflammatory factors MMP-9, IL-1β, and IL-6 under in vitro hypertonic stress conditions. Figure 12A shows a western blot analysis of the effects of different concentrations of KGF-2 on the expression levels of inflammatory factors MMP-9, IL-1β, and IL-6 under in vitro hypertonic stress conditions. Figure 12B shows the changes in MMP-9, IL-1β, and IL-6 after administration. The results show that KGF-2 administration downregulated the expression levels of MMP-9, IL-1β, and IL-6, with the most significant downregulation observed in the 0.4 μg / mL KGF-2 group, which was significantly different from the model group. Compared with the normal control group, ****p≤0.001, ***p≤0.001, **p≤0.01, and *p≤0.05 were observed.

[0152] NF-κB is a central regulator of ocular surface inflammation and disease. Figure 13 shows the effects of different concentrations of KGF-2 on NF-κB p65 expression under in vitro hypertonic stress conditions. Figure 13A shows a western blot analysis of the effects of different concentrations of KGF-2 on NF-κB p65 expression under in vitro hypertonic stress conditions. Figure 13B shows the expression changes of the NF-κB subunit p65 after administration. The results show that under hypertonic stress conditions, administration of different concentrations of KGF-2 decreased NF-κB p65 expression with increasing doses. Compared with the normal control group, ****p≤0.001, ***p≤0.001, **p≤0.01, and *p≤0.05 were observed.

[0153] In summary, this study cultured human corneal epithelial cells, used hypertonic medium for modeling, and administered KGF-2 at different concentration gradients to further determine the role of KGF-2 in the treatment of dry eye.

[0154] Example 4

[0155] This example investigates the effect of KGF-2 knockout on ocular accessory organ dysplasia and dry eye in mice. The experimental procedure in this example does not alter the KGF-2 gene; rather, it reduces the number of copies of the KGF-2 gene, thereby reducing the expression level of the protein encoded by KGF-2.

[0156] 1. Preparation of KGF-2 Heterozygous Mice

[0157] Using the CRISPR Design Tool website (http: / / crispr.mit.edu / ), sgRNAs were designed for the intronic sequences flanking Exon 2 of the mouse KGF-2 gene. sgRNAs with strong specificity and low off-target rates were selected from the candidate sgRNA library. Two pairs of sgRNAs and a Cas9 expression vector were co-injected into the genomic target sequence of mouse fertilized eggs. The Cas9 expression vector binds to and cleaves double-stranded DNA, disrupting the integrity of the KGF-2 protein by removing Exon 2, thereby downregulating the KGF-2 gene copy number. The results are shown in Figure 14.

[0158] The second exon of KGF-2 in the mouse genome was removed using CRISPR / Cas9 technology, deleting a total of 2165bp (Figure 14A), and the genotype was identified by agarose gel electrophoresis. The identification results of wild-type mice and knockout mice (also represented as KGF-2 heterozygous mice in this embodiment, represented as HET in the figure) are shown in Figure 14B. The wild-type mouse band size is 2565bp, and the heterozygous mouse band size is 400bp. KGF-2 heterozygous mice were obtained after identification. By real-time fluorescence quantitative PCR, the KGF-2 transcription level of heterozygous mice was reduced (*p < 0.05), as shown in Figure 14C.

[0159] 2. Index determination

[0160] KGF-2 heterozygous mice and wild-type control mice were subjected to basal tear secretion testing, corneal sodium fluorescein staining and photography, and meibomian gland Oil Red O staining analysis. The contents of this example that are identical to the aforementioned methods for measuring various indicators are not repeated here.

[0161] The method for meibomian gland oil red O staining is as follows: mice died suddenly at different time points in the experiment, the mouse eyelids were removed under a slit lamp microscope, fixed in 4% paraformaldehyde overnight; washed with PBS; washed with 60% isopropanol; stained with oil red O; washed with 60% isopropanol; and washed with PBS.

[0162] 3. Test results

[0163] Figure 15 shows the results of dry eye-like ocular surface damage in KGF-2 heterozygous mice. Compared with the basal tear secretion of normal control mice (WT), the basal tear secretion of KGF-2 heterozygous mice (HET) was significantly reduced, as shown in Figure 15A (***p<0.001). In addition, the basal tear secretion of normal control mice (WT) was about three times that of KGF-2 heterozygous mice (HET). Compared with normal control mice (WT), the degree of fluorescein staining in KGF-2 heterozygous mice (HET) was significantly increased, as shown in Figure 15B, indicating that KGF-2 heterozygous mice (HET) developed dry eye-like ocular surface damage. The experimental results of Figures 15C and 15D show that the ocular surface accessory organs of KGF-2 heterozygous mice (HET) are abnormally developed, as manifested by the absence of lacrimal glands in KGF-2 heterozygous mice (HET); the Harderian glands of KGF-2 heterozygous mice (HET) are smaller than those of normal control mice (WT). FIG15E shows the results of Oil Red O staining. Compared with the normal control group (WT), the meibomian glands of KGF-2 heterozygous mice were morphologically disordered, indicating that the development of the meibomian glands of KGF-2 heterozygous mice was affected.

[0164] Meibomian gland morphology at different developmental time points was examined and analyzed, and the results are shown in Figure 16. In Figure 16A, p5, p9, p14, p21, and p135 represent postnatal days 5, 9, 14, 21, and 135, respectively. The red arrows in Figure 16A indicate Oil Red O staining signals. The results showed that the Oil Red O staining signal first appeared at p9 in the normal control group (WT) mice, while no Oil Red O staining signal was detected at p9 in the KGF-2 heterozygous mice (HET). Oil Red O staining signals appeared at p14 in the KGF-2 heterozygous mice (HET), indicating that insufficient KGF-2 expression leads to delayed meibomian gland development. Figures 16B to 16E show the control results of meibomian gland length at postnatal days 9, 14, 21, and 135, respectively. In terms of changes in meibomian gland morphology, the length of the meibomian glands of KGF-2 heterozygous mice (HET) was significantly shorter than that of the normal control group mice (WT) (***p<0.001), and the meibomian glands of KGF-2 heterozygous mice (HET) were more slender and disorganized; until the adult stage at p135, the disordered morphology of the meibomian glands of KGF-2 heterozygous mice (HET) did not improve or alleviate, as shown in Figures 16A to 16E.

[0165] In summary, KGF-2 deficiency can lead to abnormal development of ocular surface accessory organs and obvious dry eye phenotype. KGF-2 deficiency leads to meibomian gland hypoplasia, and this process is an irreversible pathological developmental process.

[0166] Example 5

[0167] This example explores the effect of KGF-2 on meibomian gland development.

[0168] 1. RNA-Seq analysis of meibomian gland tissue was performed at the key time point (P21) of meibomian gland morphological changes

[0169] Transcriptome sequencing steps: According to the sudden death of mice at different time points in the experiment, the mouse eyelids were removed under a slit lamp microscope and placed in a 1.5mL centrifuge tube. The samples were labeled and placed in a -80°C freezer. After all samples were collected, they were packaged on dry ice and sent to the company for transcriptome sequencing. RNA was extracted and tested for purity, concentration, and integrity. After the samples passed the test, libraries were constructed. After the libraries passed the test, they were sequenced using the Illumina platform. Finally, gene expression and differential expression analysis were performed, using FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) as an indicator to measure transcript or gene expression levels.

[0170] 2. Test results

[0171] Figure 17 is a differential expression volcano plot, where each dot represents a gene. The horizontal axis represents the logarithm of the fold difference in expression between the two samples (log2FC), and the vertical axis represents the negative logarithm of the statistical significance of the change in gene expression (-log10(Pvalue)). Green dots represent downregulated differentially expressed genes (down), red dots represent upregulated differentially expressed genes (up), and gray dots represent non-differentially expressed genes (normal). As shown in Figure 17, 246 genes are upregulated, 299 genes are downregulated, and 14,654 genes have no significant expression differences in the meibomian gland tissue of KGF-2 heterozygous mice.

[0172] Figure 18 shows the results of the KEGG signaling pathway enrichment analysis. The horizontal axis of Figure 18 represents GeneRatio, which is the ratio of the genes of interest annotated in that entry to the total number of differentially expressed genes, and the vertical axis represents each pathway entry. Count represents the number of enriched genes, the size of the dot represents the number of differentially expressed genes annotated in that pathway, and the color of the dot represents the p-value of the hypergeometric test. The results in Figure 18 indicate that P21 immune-related molecular pathways are severely affected, such as the MAPK signaling pathway, the IL-17 signaling pathway, the TNF signaling pathway, and the estrogen signaling pathway.

[0173] pathway) etc.

[0174] Figure 19 shows the results of the GO enrichment analysis. In Figure 19, the horizontal axis represents the enrichment score, the vertical axis represents the GO category, the size of the dot represents the number of enriched genes, and the color of the dot represents the significance of the enrichment; the darker the color, the higher the significance. The results in Figure 19 indicate that immune-related processes rank highly, including dendritic cell-related processes, T cell-related processes, and macrophage-related processes.

[0175] Figure 20 shows the heatmap analysis results for representative immune cell genes. In Figure 20, blue or red indicates the lowest to highest Log2 (FPKM) expression levels. Different color blocks represent different immune cell types: green for dendritic cells, purple for macrophages, yellow for neutrophils, blue for mast cells, and pink for lymphocytes. As shown in Figure 20, the most significant changes in dendritic cell status are observed in KGF-2 heterozygous mice.

[0176] In summary, the most significant molecular pathways and cell type changes in KGF-2 heterozygous mice are related to immune pathways, and dendritic cells and macrophages are most likely to be the key target cells of KGF-2 in meibomian gland development.

[0177] Example 6

[0178] This example explores the changes in immune cells in KGF-2 heterozygous mice.

[0179] 1. Transfection of KGF-2 Mice with Cx3cr1 GFP / + mice to determine whether dendritic cells and macrophages are involved in the development of the meibomian gland.

[0180] 2. Test results

[0181] Tissue-resident dendritic cells and macrophages are myeloid immune cells, and they all express Cx3cr1. As shown in Figure 21, Cx3cr1 surrounding the alveoli in the meibomian glands of KGF-2 heterozygous mice + To distinguish dendritic cells from macrophages, the present example used Cx3cr1 GFP / + Fluorescently labeled mice were used as experimental subjects, and the expression of Cd11b, Cd11c, and F4 / 80 in the meibomian glands was detected. The results in Figures 21B and 21C show that the dendritic cell-specific marker Cd11b is expressed in the meibomian gland tissue, and the signals surrounding the alveoli are all localized to Cx3cr1-positive cells. However, some Cx3cr1-positive cells have no Cd11b signals, and almost no co-localization signals of Cd11c and Cx3cr1 in the meibomian gland alveoli were detected.

[0182] The results in Figure 22 show that very few F4 / 80 signals colocalize with Cx3cr1, suggesting that Cx3cr1-positive cells in mouse meibomian glands are rarely macrophages. F4 / 80-positive cells were rarely observed in meibomian gland tissue (Figure 22A), while Csf1r staining revealed that nearly all Cx3cr1-positive cells expressed Csf1r (Figure 22B).

[0183] In this embodiment, the optic nerve is used for Cd11b; the skin is used for Cd11c; the bladder is used for F4 / 80; and the optic nerve is used for Csf1r.

[0184] Example 7

[0185] This example explores the process of investigating whether pharmacological elimination of Cx3cr1-positive cells leads to meibomian gland developmental defects.

[0186] 1. Csf1r is a cell membrane receptor in immune cells. PLX3397 is commonly used to eliminate Csf1r-expressing immune cells in tissues. Therefore, PLX3397 was administered to female mice, and morphological changes in the meibomian glands of P14 and P21 pups were measured.

[0187] 2. Test results

[0188] As shown in Figure 23 , the meibomian glands in the PLX-1 and PLX-2-treated groups on P14 exhibited shorter, more slender glands compared to the control group. By P21, the morphology of the meibomian glands had partially recovered, as shown in Figures 23A, 23C, and 23D. Immunofluorescence staining results in Figure 23B indicate that Cd11b expression in the PLX-treated group was significantly lower than in the control group. PLX-14 expression was the lowest, while PLX-21 expression showed a slight recovery compared to PLX-14, but still lower than that in the control group. P21 mice exhibited a milder phenotype than P14 mice (Figures 23B and 23C). The degree of change in immunofluorescence staining signal correlated with the degree of meibomian gland pathology as assessed by Oil Red O at P14 and P21.

[0189] Example 8

[0190] This study provides an investigation of the KGF-2 treatment of an electrocautery-induced meibomian gland dysfunction model. The results are shown in Figures 24 to 27 .

[0191] 1. To explore whether KGF-2 can exert a therapeutic effect on meibomian gland disorders, this example uses an electric burn method to create a model ( FIG. 24A ), and then treats the electric burn model by administering the drug via eye drops.

[0192] 2. Test results

[0193] Figure 24 shows the model of meibomian gland dysfunction induced by electrical burns and the results of electrical burn-induced meibum secretion in the meibomian glands. 9.8, 9.14, 9.28, and 10.9 in Figure 24 indicate the observation dates, and 1W, 2W, 3W, and 4W indicate 1 week, 2 weeks, 3 weeks, and 4 weeks, respectively. The study found that, as time progressed (9.8 to 10.9) after electrical burns, meibum secretion became abnormal in the mice, with eyelid redness and swelling. Meibomian gland openings could not be observed under slit lamp conditions (Figure 24B, red arrows). Because the cornea was not touched during the burn, the corneal epithelial defects were considered to be the result of dry eye. Furthermore, corneal sodium fluorescein staining revealed that after burns, corneal sodium fluorescein staining deepened (Figures 24B and 24D), and basal tear secretion decreased (Figure 24C). This result may be due to abnormal meibum secretion and increased tear evaporation.

[0194] Figure 25 shows the results of corneal epithelial wound healing after KGF-2 administration. Figure 25 includes groups I (Control), II (Model), III (Model + 100 μg / mL KGF-2), and IV (Model + 500 μg / mL KGF-2). Group I consists of wild-type mice, Group II consists of electrically burned mice, Group III consists of electrically burned mice treated with 100 μg / mL KGF-2, and Group IV consists of electrically burned mice treated with 500 μg / mL KGF-2.

[0195] According to the results, as time went on, the degree of sodium fluorescein staining in the electrically burned mice treated with KGF-2 decreased, and the degree of reduction in sodium fluorescein staining in the mice treated with 500 μg / mL KGF-2 was greater than that in the mice treated with 100 μg / mL KGF-2 ( Figure 25A ). Compared with the Model group, the corneal sodium fluorescein staining scores of the mice treated with 100 μg / mL and 500 μg / mL KGF-2 were significantly decreased, and the decrease in corneal sodium fluorescein staining scores of the mice treated with 500 μg / mL KGF-2 was greater ( Figure 25B ).

[0196] In addition, as the treatment time prolonged, the basal tear secretion of mice treated with 100 μg / mL and 500 μg / mL KGF-2 increased significantly compared with the Model group, and the basal tear secretion of mice treated with 500 μg / mL KGF-2 increased to a greater extent ( Figure 25C ).

[0197] The above results indicate that after KGF-2 administration, the corneal sodium fluorescein score decreased, the basal tear secretion volume decreased, and the therapeutic effect may depend on the dose.

[0198] Figure 26 shows the results of eyelid conjunctival epithelial tissue healing after KGF-2 administration. HE staining revealed that the eyelid conjunctival epithelium was more densely arranged and orderly than the cells in the Model group, and the epithelial layer thickened after treatment (Figure 26A). In addition, protein immunoblotting (WB) results showed that Bax decreased in the groups treated with 100μg / mL and 500μg / mL KGF-2. The decrease in Bax indicates that KGF-2 can inhibit cell apoptosis and promote the recovery of meibomian gland tissue structure (Figures 26B and 26C).

[0199] Figure 27 shows the results of inflammatory infiltration of the meibomian glands after KGF-2 administration. Compared with the normal control group, the model group showed increased expression levels of matrix metalloproteinase-9 (MMP-9), phosphorylated NF-κB (p-NF-κB), tumor necrosis factor-α (TNF-α), and IL-6 (Figure 27B). Western blotting (WB) results showed that the expression levels of matrix metalloproteinase-9 (MMP-9), phosphorylated NF-κB (p-NF-κB), tumor necrosis factor-α (TNF-α), and IL-6 decreased in the groups treated with 100 μg / mL and 500 μg / mL KGF-2 (Figure 27A). Compared with the control group, the expression levels of MMP-9, p-NF-κB, TNF-α, and IL-6 were significantly reduced in the groups treated with 100 μg / mL and 500 μg / mL KGF-2 (Figure 27B).

Claims

1. Application of keratinocyte growth factor KGF-2 in the preparation of a drug for treating dry eye.

2. The use according to claim 1, characterized in that Keratinocyte growth factor KGF-2 has one or more of the following functions: Increase basal tear secretion; Upregulates tear film breakup time; Improve corneal nerve sensitivity; Down-regulate the expression levels of inflammatory factors such as MMP-9, MMP-3, TNF-α, IL-6, and IL-1β; Downregulate the phosphorylation level of NF-κB; downregulate the expression level of NF-κB P65; Down-regulate the degree of corneal fluorescein sodium staining; or Down-regulate corneal sodium fluorescein staining score.

3. The use according to claim 1 or 2, characterized in that The dry eye syndrome includes: Dry eyes caused by abnormalities in the meibomian glands; Dry eye with meibomian gland dysplasia caused by changes in immune cells; or Meibomian gland dysplasia in dry eye caused by changes in dendritic cells and macrophages.

4. Use of keratinocyte growth factor KGF-2 in the preparation of a medicament for treating corneal injury, corneal nerve injury, Harderian gland injury, conjunctival injury or conjunctival nerve injury.

5. Use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating dry eye caused by Sjögren's syndrome.

6. Application of keratinocyte growth factor KGF-2 in the preparation of drugs for dry eye induced by scopolamine combined with dry environment.

7. Use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating corneal damage, corneal nerve damage, meibomian gland abnormality, Harderian gland damage, conjunctival damage or conjunctival nerve damage in subjects with dry eye induced by scopolamine combined with a dry environment.

8. Application of keratinocyte growth factor KGF-2 in the preparation of drugs for treating meibomian gland dysfunction caused by electric burns.

9. Use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating dry eye caused by meibomian gland dysplasia due to changes in dendritic cells and macrophages.

10. Use of keratinocyte growth factor KGF-2 in the preparation of a drug for treating dry eye caused by meibomian gland dysplasia due to changes in immune cells.

11. A preparation for external use, characterized in that The topical preparation comprises keratinocyte growth factor KGF-2. Preferably, the topical preparation is administered via ocular treatment.

12. The external preparation according to claim 11, characterized in that The topical preparation can include eye drops, eye washes, eye drops, eye gels, or lacrimal duct insertions / implants.

13. A method of treatment, characterized in that Applying the external preparation according to claim 11 or 12 to the eye of a human patient.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises one of the following groups: a. Keratinocyte growth factor KGF-2 and T5122 preparation; b. Keratinocyte growth factor KGF-2 and LY29004 preparation.

15. A method for treating dry eye with combined medication, characterized in that: The method comprises administering to the eyes of a human patient an effective amount of one of the following pharmaceutical compositions: a. Keratinocyte growth factor KGF-2 and T5122 preparation; b. Keratinocyte growth factor KGF-2 and LY29004 preparation.

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