Application of snail mucus extract in preparation of medicine for promoting corneal epithelium injury healing

Abstract

The invention discloses an application of a snail mucus gel purified product in preparation of a medicine for promoting corneal epithelium injury repair. The snail mucus extract is snail mucus gel purified product Z1 subtype (s-GAG Z1), and is obtained through cell and animal experiments: when the concentration of the s-GAG Z1 is 0.05 mg / mL, the cell proliferation speed is high; after the s-GAG Z1 is applied to a mouse, the cornea healing speed is higher / faster, the inflammatory cells are more / fewer, and the epithelium growth is better / better, which indicates that the s-GAG Z1 has the effects of promoting the proliferation of corneal limbal stem cells, promoting the regeneration of corneal epithelium cells and resisting inflammation in the corneal epithelium injury healing process, thereby accelerating the corneal injury healing speed. The therapeutic effect and mechanism of the s-GAG Z1 in the cornea are discussed for the first time, an experimental basis and a potential medicine are provided for clinically treating corneal ulcer, keratitis, xerophthalmia, diabetes-related keratopathy and other related corneal diseases, and toxic and side effects are avoided.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to application of a snail mucus extract in preparing a medicine for promoting the healing of corneal epithelial damage. Background Art

[0002] The cornea is located at the front of the eyeball. It is transparent, avascular, and in direct contact with the outside world. It is easily damaged by microorganisms, trauma, and physical and chemical stimulation, which can cause inflammation, thereby affecting corneal transparency and causing decreased vision. [1,2] Corneal blindness is reported to be the second leading cause of blindness after cataracts, and diseases affecting the cornea are the leading cause of blindness worldwide, second only to cataracts in overall importance. [3] Ocular trauma and corneal ulcers are important causes of corneal blindness that are often underestimated but may account for 1.5-2 million new cases of monocular blindness each year. [3] .

[0003] Currently, more than 10 million people worldwide are affected by corneal blindness. [4] Corneal trauma and disease can cause irreversible damage to the normal structure and physiological function of the cornea, and severe cases may even require corneal transplantation. However, donors are in short supply and the risk of rejection is an ever-present problem. Despite significant progress in recent years, the wound healing cascade remains complex and has not yet been fully addressed. [4] During the healing process of corneal trauma or ulcer, epithelial cell regeneration and inhibition of inflammation are the key to corneal repair. Delayed corneal wound healing or inflammation increases the possibility of visual impairment. Therefore, anti-inflammatory and rapid healing of corneal wounds are crucial. [5] .

[0004] Sutures and staplers are the main means of achieving wound closure or implant fixation. Over the past few decades, tissue sealants and adhesives have been developed as alternatives to sutures and staplers for closing wounds or incisions. [6] Bioadhesive materials can help stop bleeding and promote wound healing [7] , a study developed a bioadhesive using snail mucus gel (SMG) as raw material, which is composed of positively charged proteins and polyanionic glycosaminoglycans (GAG). In addition, GAG interacts with growth factors, morphogens, chemokines, extracellular matrix proteins and their bioactive fragments, receptors, lipoproteins and pathogens, and these interactions mediate their functions, from embryonic development to extracellular matrix assembly, and the regulation of cell signaling in various physiological and pathological contexts (such as angiogenesis, cancer, neurodegenerative diseases and infections). [7]Researchers are actively investigating the therapeutic potential of GAGs and their mimetics to treat many other diseases, such as cancer, inflammation, infection, wound healing, lung disease, Alzheimer's disease, etc. [8] .

[0005] At present, there are no reports on the treatment of corneal diseases with GAG. The present invention team aims to explore the effect and mechanism of snail glycosaminoglycan (s-GAG) Z1 subtype of snail mucus gel (SMG) extract on inflammation and epithelial regeneration during the healing process of dry eye and corneal epithelial injury, so as to provide a theoretical basis for the development of new methods for the treatment of corneal epithelial cell dysfunction diseases. Summary of the invention

[0006] The purpose of the present invention is to provide a use of a snail mucus extract in preparing a medicine for promoting the healing of corneal epithelial damage.

[0007] In order to achieve the purpose of the present invention, the present invention adopts the following technical solutions and steps:

[0008] The invention discloses an application of the snail mucus gel purification product in preparing a medicine for promoting the repair of corneal epithelial damage.

[0009] The snail mucus gel extract is the snail mucus gel extract Z1 subtype.

[0010] The concentration of the snail mucus gel purified material Z1 subtype in the medicine is 0.01-0.5 mg / mL.

[0011] Preferably, the concentration of the snail mucus gel extract Z1 subtype in the drug of the present invention is 0.05 mg / mL.

[0012] The medicine of the present invention is an external medicine for eyes or an eye injection.

[0013] The ocular topical medicines of the present invention include, but are not limited to, eye ointments, eye creams, eye gels, and eye drops.

[0014] The eye drops of the invention contain Z1 subtype of the snail mucus gel extract and phosphate buffer.

[0015] The phosphate buffer of the present invention can also be replaced by any one of physiological saline and HBSS buffer.

[0016] The solvent of the eye drops of the present invention further contains one or more of an isotonic agent, an antibacterial agent, a stabilizer, a viscosity enhancer, a solubilizing agent, and a protein protective agent.

[0017] The pH value of the eye drops of the present invention is 7-7.5.

[0018] The eye drops of the present invention adopt a conventional eye drops preparation method, wherein the snail mucus gel purified material Z1 subtype is dissolved in a solvent, mixed evenly, sterilized and filtered, and packaged to obtain the eye drops.

[0019] Beneficial effects of the present invention:

[0020] 1. The present invention is the first to discover the therapeutic effect of snail mucus extract s-GAG Z1 in the cornea, which can promote the healing of corneal epithelial damage and provide an experimental basis and potential drugs for the clinical treatment of corneal ulcers, keratitis and other related diseases.

[0021] 2. The cell experiments of the present invention have proved that s-GAG Z1 can effectively promote cell proliferation. When the concentration of s-GAG Z1 is 0.05 mg / mL, RT-qPCR shows that the expression of K14, Ki67, ABCG2, PAX6, and P63 mRNA increased, and the difference was statistically significant (P<0.01), while the Sprr1b gene representing abnormal differentiation basically did not change, and the difference was not statistically significant (P>0.05).

[0022] 3. The present invention has proved through animal experiments that s-GAG Z1 can effectively promote corneal epithelial wound healing, promote corneal epithelial growth and have anti-inflammatory effects:

[0023] (1) After the eye drops containing 0.2 mg / mL s-GAG Z1 were administered to treat corneal injuries in mice, the wound healing of the corneal epithelium was significantly promoted at 24 hours; at 36 hours, the corneal epithelial injuries of the mice were completely healed without scars or dysplasia during the healing process. The healing speed was fast and the effect was significant, indicating that s-GAG Z1 can effectively promote the wound healing of the corneal epithelium;

[0024] (2) HE staining of corneal sections showed that after treatment with s-GAG Z1 eye drops, the corneal epithelial layers were more numerous, the cell morphology was better, and the epithelial damage was repaired more completely;

[0025] (3) Effect of s-GAG Z1 on mRNA expression of corneal epithelial damage: RT-qPCR results showed that compared with the control group, the mRNA expressions of K12, PAX6, ABCG2, P63, and Ki67 in the EGF-treated group and the s-GAG Z1-treated group were significantly increased, and the difference was statistically significant (P<0.01). The mRNA expressions of Sprr1b, TNF-α, and IL-6 were significantly decreased, and the difference was statistically significant (P<0.01). There was no significant difference between the EGF-treated group and the s-GAG Z1-treated group (P>0.05), and there was no significant difference between the control group and the PBS group (P>0.05), indicating that s-GAG Z1 has the effect of promoting corneal epithelial growth without obvious abnormal differentiation and reducing inflammation.

[0026] (4) Effect of s-GAG Z1 on protein expression of corneal epithelial damage: Western blot results showed that compared with the control group, the expression of K12, PAX6, and SOD2 proteins in the EGF treatment group and the s-GAG Z1 treatment group was increased, and the difference was statistically significant (P<0.05). There was no significant difference between the EGF treatment group and the s-GAG Z1 treatment group (P>0.05), and there was no significant difference between the control group and the PBS group (P>0.05), indicating that s-GAG Z1 has the effect of promoting corneal epithelial growth and anti-inflammatory. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 :Structural formula of compound 1 Effects of different concentrations of s-GAG Z1 on HCE cell proliferation

[0028] Figure 2 :The effect of s-GAG Z1 on HCE scratches (A is the proliferation of HCE cells under the action of each group, B is the proliferation rate curve, C is the proliferation rate statistical analysis chart, (1) compared with the control group, P < 0.05.)

[0029] Figure 3 :Effects of s-GAG Z1 on mRNA in HCE cells

[0030] Figure 4 :Application of s-GAG Z1 in corneal epithelial injury (A is the photographic record of the corneal injury healing process in each group after sodium fluorescein staining under a microscope; B is the healing rate curve; C is the healing rate statistical analysis chart, (1) compared with the control group, P>0.05; (2) compared with the control group, P<0.05; (3) compared with the control group, P<0.05; (2) compared with (3), P>0.05.)

[0031] Figure 5 : HE staining after corneal epithelial injury healing (A is the corneal HE staining of each group; B is the statistical chart of inflammatory cell counts; C is the statistical chart of corneal epithelial cell layers, (1) compared with the control group, P>0.05; (2) compared with the control group, P<0.05; (3) compared with the control group, P<0.05; (2) compared with (3), P>0.05.)

[0032] Figure 6:The expression of mRNA after corneal injury healing (relative expression of corneal K12, PAX6, ABCG2, P63, Ki67, Sprr1b, TNF-α, and IL-6 mRNA in each group; (1) compared with the control group, P>0.05; (2) compared with the control group, P<0.01; (3) compared with the control group, P<0.01; (2) compared with (3), P>0.05.)

[0033] Figure 7 : Protein expression after membrane injury healing (A is the K12, Pax6, and SOD2 protein band diagram, B is the relative expression of K12, Pax6, and SOD2 proteins; (1) compared with the control group, there was no significant difference; (2) compared with the control group, P<0.05; (3) compared with the control group, P<0.05; (2) compared with (3), there was no significant difference.) DETAILED DESCRIPTION

[0034] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. For those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

[0035] Example 1 Preparation of eye drops containing Z1 subtype of snail mucus gel extract

[0036] The snail mucus gel extract was dissolved in PBS to prepare eye drops containing the snail mucus gel extract Z1 subtype at a drug concentration of 0.05 mg / mL.

[0037] Example 2 Preparation of eye drops containing Z1 subtype of snail mucus gel extract

[0038] The snail mucus gel extract was dissolved in PBS to prepare eye drops containing the snail mucus gel extract Z1 subtype at a drug concentration of 0.01 mg / mL.

[0039] Example 3 Preparation of eye drops containing Z1 subtype of snail mucus gel extract

[0040] The snail mucus gel extract was dissolved in PBS to prepare eye drops containing the snail mucus gel extract Z1 subtype at a drug concentration of 0.1 mg / mL.

[0041] Example 4 Preparation of eye drops containing Z1 subtype of snail mucus gel extract

[0042] The snail mucus gel extract was dissolved in PBS to prepare eye drops containing the snail mucus gel extract Z1 subtype at a drug concentration of 0.5 mg / mL.

[0043] Example 5 Preparation of eye drops containing Z1 subtype of snail mucus gel extract

[0044] The snail mucus gel extract was dissolved in HBSS to prepare eye drops containing the snail mucus gel extract Z1 subtype at a drug concentration of 0.05 mg / mL.

[0045] Example 6 Preparation of eye drops containing the Z1 subtype of the snail mucus gel extract

[0046] The snail mucus gel extract was dissolved in physiological saline to prepare eye drops containing the snail mucus gel extract Z1 subtype at a drug concentration of 0.1 mg / mL.

[0047] Example 7 Preparation of ophthalmic gel containing Z1 subtype of snail mucus gel extract

[0048] The snail mucus gel extract was dissolved in PBS to prepare an ophthalmic gel containing the snail mucus gel extract Z1 subtype at a drug concentration of 0.05 mg / mL.

[0049] Example 8 Application of the Z1 subtype of the snail mucus gel extract in the preparation of a drug for promoting the repair of corneal epithelial damage

[0050] According to the preparation methods of Examples 1 to 6 above, the prepared eye drops were dripped into the conjunctival sac four times a day.

[0051] In order to verify the effectiveness of the present invention, the invention team conducted a series of experiments, as follows:

[0052] 1. Instruments, reagents and materials

[0053] 1.1 Main instruments

[0054] Electric corneal trephine (Lot: 050421A, Algerbrush II, USA), surgical microscope, slit lamp microscope (Zeiss GmbH, Germany), fully automatic microplate reader (Bio-Tek, ELx-800), high-speed centrifuge (Gene Company, 1730R), electrophoresis instrument (BioRad, PowerPacbasic), membrane transfer instrument (BioRad, Trans-boltturbo), PCR instrument (BioRad, 71007M), biological microscope (Guangzhou Mingmei, ML31), ultra-low temperature refrigerator (Thermo, FORMA 900 SERIES).

[0055] 1.2 Main drugs and reagents

[0056] The following antibodies were used in this study: ABCG2, Ki67, K12, K14, and Pax6 (Abcam). The internal reference antibody GAPDH and secondary antibodies were purchased from Wuhan Proteintech Group, Inc. Other reagents and consumables are as follows: snail mucus extract s-GAG Z1, recombinant human epidermal growth factor EGF (Invitrogen, USA), BCA protein assay kit (Shanghai Biotech Biotechnology Co., Ltd., catalog number p0012), double antibody (Thermo Fisher Scientific Inc.), RIPA lysis buffer (Beijing Biotech Institute of Biology), HE staining kit, TRIzol kit, etc.

[0057] 1.3 Animals

[0058] Specific pathogen free (SPF) grade C57 / BL6 (100) male mice aged 6 to 8 weeks, weighing (20±2) g, were purchased from SBC Biotechnology (Beijing) Co., Ltd. [SCXK (Beijing) 2019-0010]. After 1 week of adaptive feeding, the corresponding operations were performed. All animals were raised at a temperature of 22-25°C, a relative humidity of 50%-60%, and a 12h light / 12h dark cycle, and the mice were free to eat.

[0059] 1.4 Cells

[0060] The cells used in the experiment were human corneal epithelial cells (HCE) from Xiamen Eye Center. The cells were inoculated in F12 medium containing 6.4% fetal bovine serum, 10 mg / mL insulin, 10 ug / mL EGF, 100 U / mL penicillin and 100 μg / mL streptomycin and incubated in saturated humidity and 5% CO 2 , cultured in a 37°C incubator.

[0061] 2 Methods

[0062] 2.1 Cell experiments

[0063] 2.1.1 Determination of the optimal concentration of s-GAG Z1

[0064] Different concentrations (0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 mg / mL) of snail glycosaminoglycan (s-GAG) Z1 subtype (hereinafter referred to as s-GAG Z1) extracted from snail mucus gel (SMG) were added to HCE cells. After 24 hours, the cell proliferation and drug toxicity were determined by CCK-8 method to determine the optimal concentration of s-GAG Z1.

[0065] 2.1.2 Cell scratch assay

[0066] HCE were uniformly seeded in a six-well plate. When the cells grew to 95%, a 200 μL pipette tip was used to make a cross in the center of each well. The cells were washed with PBS to remove the scratched cells. The culture medium was replaced with fresh culture medium and culture medium containing 0.05 mg / mL s-GAG Z1. The culture medium was placed in a 37°C CO 2 The cells were cultured in an incubator, and the scratches were observed and photographed under an inverted microscope at 0, 12, 24, and 30 h.

[0067] 2.1.3 Real-time quantitative polymerase chain reaction (RT-qPCR) to detect the expression of K14, Ki67, P63, PAX6, ABCG2, Sprr1b and other mRNAs in cells

[0068] Take the cells collected under "2.1.2" and extract total RNA according to the instructions of the TRIzol kit. The concentration is measured by a micro-spectrophotometer. Then, the RNA is reverse transcribed into cDNA according to the instructions of the cDNA reverse transcription kit. Then, real-time fluorescence quantitative PCR is performed: pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 10sec, annealing / extension at 60℃ for 30sec, for a total of 40 cycles. The primer sequences of each gene are shown in Table 1. -ΔΔCt The relative expression levels of K14, Ki67, P63, PAX6, ABCG2, and Sprr1b mRNA in cells were calculated.

[0069] Table 1 Primer sequences of each gene

[0070]

[0071]

[0072] 2.2 Animal experiments

[0073] 2.2.1 Animal modeling, grouping and drug administration

[0074] 40 C57 / BL6 mice were selected, and all diseases such as eye trauma and cataract were excluded under slit lamp. The epithelium with a central diameter of about 2.5 mm of the mouse cornea was scraped with an electric corneal epithelial scraper under a microscope to construct a corneal epithelial injury model, and then randomly divided into 4 groups (n=10): control negative control group (no treatment), phosphate buffered saline (PBS) drug group (PBS), epidermal growth factor (Epidermal Growth Factor, EGF) positive control group (recombinant human epidermal growth factor eye drops 30000IU (60ug) / m), s-GAG Z1 treatment group (0.2mg / mL s-GAG Z1, solvent PBS), each group was given corresponding drug eye drops, 4 times / day, for 2 days. Corneal fluorescence staining was performed and photographed under a microscope at 0, 12, 24, 36, and 48 hours, and the healing rate of corneal epithelial injury was calculated. The mouse cornea was obtained 2 days after injury: the eyeball sections were embedded and HE staining was performed to observe the corneal morphology; total RNA and protein were extracted, and the expression of mRNA and protein such as IL-6, TNF-α, K14, Ki67, P63, and SPRR1B was detected.

[0075] 2.2.2.2 Animal materials

[0076] Mice were killed by cervical dislocation, and the eyeballs were lifted with microtweezers in lateral position. The conjunctiva was cut along the upper and lower eye fornix with ophthalmic scissors, and the optic nerve was cut, paying attention to maintaining the integrity of the eyeball and corneal epithelium. One mouse eyeball was fixed with 10% paraformaldehyde in each group, and then embedded in paraffin sections. The cornea of ​​the remaining eyeballs was completely separated along the corneoscleral limbus under a microscope and stored in 1.5 ml EP tubes. Each tube contained 3-5 samples and was stored in a -80°C refrigerator for subsequent RT-qPCR and Western Blot experiments.

[0077] 2.2.2.3 Corneal morphology observation

[0078] Take the eyeball tissue fixed with 10% paraformaldehyde solution under "2.2.2.2", prepare paraffin sections (thickness 4 μm) according to routine operation, and then perform HE staining, and observe the corneal epithelium and the number of inflammatory cells under a microscope.

[0079] 2.2.2.4 RT-q PCR detection of IL-6, K14, Ki67, P63, PAX6, ABCG2, and SPRR1B mRNA expression

[0080] Take the corneal tissue collected under "2.2.2.2", extract total RNA according to the instructions of TRIzol kit, measure the concentration by micro-spectrophotometer, and then reverse transcribe RNA into cDNA according to the instructions of cDNA reverse transcription kit, and then perform real-time fluorescence quantitative PCR: pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 10sec, annealing / extension at 60℃ for 30sec, for a total of 40 cycles. The primer sequences of each gene are shown in Table 1. -ΔΔCt The relative expression levels of IL-6, K14, Ki67, P63, PAX6, ABCG2, and SPRR1B in corneal tissue were calculated.

[0081] 2.2.2.5 Western blot (WB) detection of K12, Pax6, and SOD2 protein expression

[0082] Take the corneal tissue collected under "2.2.2.2", add pre-cooled RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors, put it into a tissue grinder for full homogenization, take it out and continue to lyse it on ice for half an hour, and take the supernatant after centrifugation. Determine the protein concentration with the BCA kit, take an equal amount of sample and load it on the gel, perform electrophoresis, transfer the membrane, block it with 5% skim milk at room temperature for 1.5 hours, incubate the primary antibody at 4℃ overnight, incubate the secondary antibody at room temperature for 1 hour, and wash the membrane with TBST. Use a gel imaging system to expose the bands, and Image J software to determine the gray value.

[0083] 2.3 Statistical methods

[0084] GraphPad Prism 10.2 was used for statistical analysis and plotting. Data that met normal distribution were described as mean ± standard deviation (x ± s). One-way analysis of variance was used for comparison among multiple groups, and LSD-t test was used for comparison between two groups. P < 0.05 was considered statistically significant.

[0085] 3 Results

[0086] 3.1 Cell experiments

[0087] 3.1.1 Determination of the optimal concentration of s-GAG Z1

[0088] First, to determine the effect of s-GAG Z1 on the proliferation of cultured HCE cells, we selected 8 concentrations of rhFGF5 (0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 mg / mL) to treat HCE cells.

[0089] The results of CCK8 experiment showed that after 24 hours of drug treatment, the cell growth rate of the drug group increased slightly with the increase of concentration, and the cell proliferation rate of the concentration of 0.05 mg / mL seemed to be faster than that of other groups, and the difference was statistically significant (P < 0.001). At a high concentration of 1 mg / mL, s-GAG Z1 began to show cytotoxicity ( Figure 1 ).

[0090] 3.1.2 Effect of the optimal concentration of s-GAG Z1 on HCE cell proliferation

[0091] The results of the cell scratch test showed that the proliferation rate of HCE cells in the 0.05 mg / mL s-GAG Z1 treatment group was faster than that in the blank control group (P<0.05) ( Figure 2 A, B, C). The cell morphology was observed under an inverted microscope. The morphology of HCE cells treated with s-GAG Z1 was consistent with that of untreated cells.

[0092] 3.1.3 Effect of s-GAG Z1 on mRNA expression in HCE cells

[0093] After HCE was treated with 0.05 mg / mL s-GAG Z1, RT-qPCR showed that the expression of K14, Ki67, ABCG2, PAX6, and P63 mRNA increased, with statistically significant differences (P<0.01), while the Sprr1b gene, which represents abnormal differentiation, remained basically unchanged, with no statistically significant difference (P>0.05) ( Figure 3 ). These results indicate that s-GAG Z1 has little effect on cell morphology but can effectively promote cell proliferation.

[0094] 3.2 Animal experiments

[0095] 3.2.1 Effect of s-GAG Z1 on corneal epithelial membrane damage in mice (comparison of healing conditions among each group).

[0096] The corneal injury of mice was treated with eye drops containing different drugs (1xPBS, recombinant human epidermal growth factor eye drops 30000IU (60ug) / mL, 0.2mg / mL s-GAG Z1), and the control group was not treated. After fluorescein sodium staining and observation under a microscope, EGF and s-GAG Z1 significantly promoted the wound healing of corneal epithelium at 24 hours. At 36 hours, the corneal epithelial injury of mice in the EGF and s-GAG Z1 treatment groups was completely healed, while the cornea of ​​mice in the control group and PBS group still had unhealed damaged parts. After using s-GAG Z1 eye drops, there was no scar formation or dysplasia in the healing process of the mouse cornea ( Figure 4A, B). The healing speed of the EGF treatment group and the s-GAG Z1 treatment group was faster than that of the control group, and the difference was statistically significant (P<0.05). There was no significant difference between the EGF treatment group and the s-GAG Z1 treatment group (P>0.05), and there was no significant difference between the control group and the PBS group (P>0.05) ( Figure 4 C) These results indicate that s-GAG Z1 can effectively promote corneal epithelial wound healing.

[0097] 3.2.2 Comparison of corneal morphology among groups after treatment

[0098] HE staining of corneal sections showed that the inflammatory infiltrating cells (black arrows) in the whole cornea of ​​the EGF treatment group and the s-GAG Z1 treatment group were reduced compared with the control group and the PBS group, and the difference was statistically significant (P<0.05); compared with the control group, the EGF treatment group and the s-GAG Z1 treatment group had more corneal epithelial layers, with better cell morphology and more complete epithelial damage repair; there was no significant difference between the EGF treatment group and the s-GAG Z1 treatment group (P>0.05), and there was no significant difference between the control group and the PBS group (P>0.05) ( Figure 5 A, B, C).

[0099] 3.2.3 Effect of s-GAG Z1 on mRNA expression in corneal epithelial damage

[0100] RT-qPCR results showed that compared with the control group, the mRNA expressions of K12, PAX6, ABCG2, P63, and Ki67 in the EGF-treated group and the s-GAG Z1-treated group were significantly increased (P<0.01), and the mRNA expressions of Sprr1b, TNF-α, and IL-6 were significantly decreased (P<0.01). There was no significant difference between the EGF-treated group and the s-GAG Z1-treated group (P>0.05), and there was no significant difference between the control group and the PBS group (P>0.05). Figure 6 ). This suggests that s-GAGZ1 has the effect of promoting corneal epithelial growth without obvious abnormal differentiation and alleviating inflammation.

[0101] 3.2.4 Effect of s-GAG Z1 on protein expression in corneal epithelial damage

[0102] Western blot results showed that compared with the control group, the expression of K12, PAX6, and SOD2 proteins in the EGF treatment group and the s-GAG Z1 treatment group was increased, and the difference was statistically significant (P<0.05); there was no significant difference between the EGF treatment group and the s-GAG Z1 treatment group (P>0.05), and there was no significant difference between the control group and the PBS group (P>0.05) ( Figure 7 A, B). These results suggest that s-GAG Z1 has the effects of promoting corneal epithelial growth and anti-inflammatory.

[0103] In summary, s-GAG Z1 promotes the proliferation of limbal stem cells and maintains stemness during the healing process of corneal epithelial injury, promotes corneal epithelial cell regeneration, and has an anti-inflammatory effect, thereby accelerating the healing of corneal injury. This study is the first to explore the therapeutic effect and mechanism of s-GAG Z1 in the cornea, providing an experimental basis and potential drugs for the clinical treatment of corneal ulcers, keratitis and other related diseases.

[0104] Although the present invention has been described in detail above by means of general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0105] References:

[0106] [1]DELMONTE DW,KIM T.Anatomy and physiology of the cornea[J].JCataract Refract Surg,2011,37(3):588-598.

[0107] [2]MEEK KM,KNUPP C.Corneal structure and transparency[J].Prog RetinEye Res,2015,49:1-16.

[0108] [3]WHITCHER JP, SRINIVASAN M, UPADHYAY M P.Corneal blindness: a globalperspective[J].Bull World Health Organ, 2001,79(3):214-221.

[0109] [4]BARRIENTEZ B,NICHOLAS S E,WHELCHEL A,et al.Corneal injury:Clinicaland molecular aspects[J].Exp Eye Res,2019,186:107709.

[0110] [5]FORTINGO N,MELNYK S,SUTTON S H,et al.Innate Immune SystemActivation,Inflammation and Corneal Wound Healing[J].Int J Mol Sci,2022,23(23).

[0111] [6]TABOADAG M,YANG K,PEREIRAM J N,et al.Overcoming the translationalbarriers of tissue adhesives[J].Nature Reviews Materials,2020,5(4):310-329.

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[0113] [8]MORLA S.Glycosaminoglycans and Glycosaminoglycan Mimetics inCancer and Inflammation[J].Int J Mol Sci,2019,20(8).

Claims

1. Application of snail mucus gel extract in the preparation of drugs for promoting the repair of corneal epithelial damage.

2. The use according to claim 1, characterized in that: The snail mucus gel extract is the snail mucus gel extract Z1 subtype.

3. The use according to claim 1, characterized in that: The concentration of the snail mucus gel extract in the medicine is 0.01-0.5 mg / mL.

4. The use according to claim 3, characterized in that The concentration of the snail mucus gel extract in the medicine is 0.05 mg / mL.

5. The use according to claim 1, characterized in that: The medicine is an external eye medicine or an eye injection.

6. The use according to claim 5, characterized in that: The topical eye medications include, but are not limited to, eye ointments, eye creams, eye gels, and eye drops.

7. The use according to claim 6, characterized in that: The eye drops contain a snail mucus gel extract and a phosphate buffer.

8. The use according to claim 7, characterized in that: The phosphate buffer can also be replaced by any one of physiological saline and HBSS buffer.

9. The use according to claim 6, characterized in that: The solvent of the eye drops also contains one or more of an isotonic agent, an antibacterial agent, a stabilizer, a viscosity enhancer, a solubilizing agent, and a protein protective agent.

10. The use according to any one of claims 6 to 9, characterized in that: The pH of the eye drops is 7.0-7.5.

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